THE EFFECTS OF CALCIUM, SHORT CHAIN FArrY ACIDS AND · 2020. 4. 7. · Chapter 6: The effects of calcium concentration, short chain fatty acids and mammalian lignans on intracellular

THE EFFECTS OF CALCIUM, SHORT CHAIN FArrY ACIDS AND

MAMMALIAN LIGNANS ON CALCIUM TRANSPORT,

INTRACELLULAR ca2+ AND lNT RACELLULAR pH

IN THE HUMAN COLON TUMOR CELLS HCT-15

Zhi-Jian Liu

A Thesis submitted in confomity with the requirements for the Degree of Master of Science

Graduate Department of Nutritional Sciences University of Toronto

@ Copyright by Zhi-Jian Liu 1999

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THE EFFECTS OF CALCIUM, SHORT CHAIN FArrY AClDS AND MAMMALlAN LIGNANS ON CALCIUM TRANSPORT, INTRACELLULAR ca2*

AND INTRACELLULAR pH IN HUMAN COLON TUMOR CELL HCT-15

Master of Science, 1999 Uii-Jian Liu

Department of Nutritional Sciences University of Totonto

The objective of this study was to determine in vitro in human colon turnor cells

HCT-15 the effects of calcium, short chain fatty acids (SCFA) acetate, propionate and

butyrate and lignans secoisolariciresinot (SECO), enterodiol (ED) and enterolactone (EL)

on calcium transport, intracellular ca2+ and intracellular pH, whiai are thought to be

associated with inhibition of cell proliferation and induction of apoptosis. Transcellular

calcium transport was inueased by calcium, acetate, propionate, the mixture of SCFA or

the mixture of €0 and EL. lntracellular Ca2' was increased with increasing calcium

concentration, and further increased by calcium combined with EL or SECO. lntracellular

pH was decreased by SCFA, EL, or low extracellular pH. The results suggest that these

changes in intracellular ca2+ and intracellular pH may in part be responsible for the

previously observed effect of calcium, SCFA and lignans on cell proliferation and

apoptosis.

TABLE OF CONTENTS

Abstract

Table of contents

Acknowledgements

List of Table

List of Figure

List of Appendices

List of Abbreviation

Chapter 1 : introduction

Chapter 2: Liratute Review

Colon catcinogenesis: imbalance of celf proliferation

and differentiation-apoptosis.

2.1.1. Balance between cell proliferation and differentiation-apoptosis

2.1 -2. toss of balance during cancer development

2.1.3. lmbalance caused by mutations

Chemoprevention

2.2.1. Chemoprevention

2.2.2. Chemoprevention through nutritional manipulation

2.2.3. Dietary fiber

2.2.4. Lignans

2.2.5. Calcium

Effects of ~ a " . SCFA and mammalian lignans

2.3.1. ca2'

2.3.2. Short da in fatty acids

2.3.3. Mammalian lignans

Interactive effeds of ce2'. SCFA and rnarnmalian lignans

2.4.1. ca2' and SCFA

2.4.2. Mammalian lignans. ca2' andlor SCFA

Calcium absorption and transport

2.5.1. Calcium absorption in the colon

ii

iii

vii

viii

ix

X

xi

1

4

5

5

5

6

8

8

9

9

10

11

12

12

13

14

17

17

15

19

20

2.5.2. Paracellular and transcellular transport

2.5.3. Cell rnonolayer model

2.5.4. Measurernent of calcium concentration

2.6. lntracellular ca2+ and intracellular pH

2.6.1. Intracellular Ca2'

2.6.2. lntracellular pH

2.6.3. Measurement of [Ca21 and pHi

2.7. Mitochondrion-mediated apoptosis and intracellular ca2* and

intracellular pH

2.7.1. Pie-mitochondrion phase

2.7.2. Mitochondrion phase

2.7.3. Post-mitochondrion phase

2.8. Summary

Chaper 3: Hypothesis, objectives and overview of expenmental design

and methodology

3.1. Hypothesis

3.2. Overall hypothesis

3.3. Specific hypothesis

3.4. Overview of experirnental design

3.5. General method

Chapter 4: The effecti of calcium concentration, short chah fatty acids

and mammalian lignans on calcium transport in human colon

tumor cells

4.1 . Introduction

4.2. Materials and Methods

4.2.1 . Materials

4.2.2. Experimental design

4.2.3. Cell culture

4.2.4. Monolayer cell membrane

4.2.5. Calcium transport

4.2.6. Calcium determination

4.2.7. Statistics

4.3. Results

4.3.1 . Effect of calcium concentration

4.3.2. Effect of acetate, pmpionate, butyrate and their mixture

4.3.3. Effect of mammalian lignans

4.3.4. Effect of mammalian lignans and SCFA

4.4. Discussion

Chapter 5: The effects of calcium concentration, short chain fatty acids

and mammalian lignans on intracellular Ca2+ in human colon

tumor cells

5.1. Introduction


5.2.1. Materials

5.2.2. Experimental design

5.2.3. Tissue culture

5.2.4. Iso-tonic detecting solutions

5.2.5. Dye loading

5.2.6. Fluorescence determination

5.2.7. [ c a r i calculation

5.2.8. Cell viability

5.2.9. Statistics

5.3. Results

5.3.1. Effect of calcium concentration

5.3.2. Effect of acetate, propionate, butyrate and their mixture

5.3.3. Effect of mammalian lignans

5.3.4. Compan'ng the effect of enteroiactone with and without calcium

5.4. Discussion

Chapter 6: The effects of calcium concentration, short chain fatty acids

and mammalian lignans on intracellular pH in human colon

tumor cells.

6.1. Introduction


6.2.1. Materials

6.2.2. Gcperimental design


6.2.4. lsotonic detecting solutions

6.2.5. Dye loading


6.2.7. Fluorescence-pHi calibration


6.2.9. Statistics

6.3. Results

6.3.1. Cafibration of pHi and fluorescence ration

6.3.2. Effect of acetate, propionate, butyrate and their mixture

6.3.3. Effect of SCFA on pHi at different pHe

6.3.4. Effect of calcium concentration on pHi

6.3.5. Effect of mammalian lignans on pHi

6.3.6. Effect of marnmalian lignans and SCFA

6.4. Discussion

Chapter 7: General Discussion and Conclusion

7.1. Calcium concentration

7.2. Short chain fatty acids

7.3. Lignans

7.4. Interactive effects

7.5. Significance and future work

7.6. Conclusion

Chapter 7: References

Appendices

ACKNOLEDGEMENTS

I would like to express my sincere thanks and appreciation to Dr. Lilian U.

Thompson for her guidance, advice, support, kindness and patience. 1 have gained great

improvement in both expenence and inspiration from working and studying in her

laboratory. 1 would also like to sincerely thank rny advisory cornmittee, Or. W. R. Bruce

and Dr. T. M. S. Wolever for all their suggestions, guidance and help, and Dr. David E.

lsenman for the use of his spectrofluommeter and for technical instruction.

For their great encouragement, friendship and help in experimental technique and

language improvement, I would like to express my gratitude to: M. Jenab, J. Chen, H.

Skariah, S. Rickard, J. Tou, W. Ward, M. Starvo, M. Chen, F. Cheung. and Y. Yuan in Dr.

Thompson's Lab, and my dear brother and sister in Christ Kham and Ming Ho.

finally, I would like to Say that without endless love and guidance of Lord Jesus

Christ in my life I would never be able to finish this thesis. May His name be honored and

glonfied.

vii

LIST OF TABLES

Table 2.1.

Table 4.1.

Table 4.2.

Table 5.1.

Table 6.1.

Table 6.2.

Table 6.3.

Table 6.4.

Table 7.1

Comparison of different methods for measurements of intracellular Ca2' and intracellular pH

Treatments used in calcium transport experiment

Summary for the effects on calcium transport

Treatments used in intracellular Ca2' experiments

Treatments used in intracellular pH experiments

Preparation of gradient solutions of different pH

Effect of calcium combined with different SCFA on pHi

Summary of effects on intracellular pH

General Summary

viii

LIST OF FIGURES

Figure 2.1.

Figure 2.2.

Figure 2.3.

Figure 2.4.

Figure 2.5.

Figure 4.1.

Figure 4.2,

Figure 4.3.

Figure 4.4.

Figure 4.5.

Figure 4.6.

Figure 4.7.

Figure 5.1.

Figure 5.2.

Figure 5.3.

Figure 5.4.

Figure 6.1.

Figure 6.2.

Figure 6.3.

Figure 6.4.

Figure 6.5.

Figure 6.6.

Figure 6.7.

Figure 6.8.

A genetic mode1 of colon cancer

lnterrelationship between absorption of SCFA and movement of H4 CO2 and electrolytes through the digest tract epithelium

Interaction of ~ a " , SCFA and mammalian lignans

Transcellular calcium transport mechanism

Thme phases of apopstosis and the role of [ca2c]i and pHi

A cell monolayer model developed in Costar Transwell

Calibration of calcium concentration

Calcium concentrations on total calcium transport

Calcium concentration on transcellular and paracellular transport

Effects of SCFA on calcium transport

Effects of mammalian lignans on calcium transport

Effect of mammalian lignans and SCFA on calcium transport

Effect of calcium concentrations on [ca27i

Effect of SCFA on [Ca27i

Effect of lignans on [Ca2+]i

Effect of enterolactone with or without calcium on [Ca2Ti

Calibration for pHi

Effect of different concentrations SCFA on pHi

Tirne-dependent effect of Acetate on pHi

Effect of the mixture of SCFA and pHe on pHi

Effect of calcium on pHi at different pHe

Effect of calcium on pHi at SCFA solutions

Effects of lignans on pHi with or without SCFA.

Effects of SCFA, enterolactone and their combination on pHi

LIST OF APPENDICES

Appendix 1

Appendix 2

Appendix 3

Appendix 4

Appendix 5

Appendix 6

Appendix 7

Appendix 8

Appendix 9

Appendixl O

Appendix? 1

Appendixl2

Appendixl3

Effect of calcium concentration on calcium transport

Effect of SCFA on calcium transpon

Effects of mammalian lignans witMMtout SCFA on calcium transport

Effect of calcium concentrations on [ca2']i

Effect of SCFA on [ca2']i

Eflect of lignans on [Ca2']i

Interactive effect of EL with or without calcium on [ca27i

pHi Calibration at excitation wavelength, 488, 51 5 and 535nm

Effect of acetate, propionate and butyrate on pHi

Effect of SCFA and pHe on pHi

Effect of calcium on pHi at different pHe

Effect of lignans with or without SCFA on pHi

Cornparison effect of SCFA, enterolactone and their combination on pHi

ANT adenine nudeotide translocator

AOM azoxymethane

ATP adenosin triphosphate

[ca27i inttacellular ca2' level

CAMP cyclic adenosin monophosphate

DAG diacylgl ycerol

AYrn mitochondrion inner transmembrane potential

€0 enterodiol

EGTA

EL

FBS

Fura-2AM

HBSS

IP3

PBS

PH^

ethylene glycol-bis@-aminoethy1ether)-N, N, N',N',-tetraacetic acid

enterolactone

fetal bovine senrm

Fura-2 acetoxymethyl ester

Hank's balanced sodium solution

inositol triphosphate

phosphate buffered saline

extracellular pH

pHi intracellular pH

PKC pmtein kinase C

PTP penneability transition pore

ROS reactive oxygen species

SCFA short main fatty acids

SDG secoisolariciresinol diglycoside

SHBG sex hormone binding globulin

SNARF-1 AM carboxyl-SNARF-l acetoxymethyl ester

CHAPTERONE

INTRODUCTION

1. INTRODUCTION

Colon cancer is one of the most common cancers in North America, with an

incidence rate of 25-35 per 100,000 people, a value much greater than that in China or

Japan (Amencan Cancer Society, 1995). However, the incidence rate in Chinese and

Japanese descendants in North America is much higher than those in China and Japan,

and close to Caucasian level (U.S. National Cancer Institute, 1986). The westernization

in dietary habit is assumed to be a major reason (Boyle, 1998). High fat and low fiber in

a Western diet were believed to be risk factors, while low fat, high calcium and high fiber

in an Eastern diet were recognized to be protedive (Reddy 1995; Shike 1999). All these

suggest that it is possible to lower colon cancer incidence rate through dietary change.

Several studies have shown that high fiber or high calcium intake is associated

with decreased risk of colon cancer (Reddy 1995; Garland et al 1985; Lipkin and

Newrnark 1995). Undigested in the small intestine, soluble fiber is femented by the

bacteria in the colon to produce short chain fatty acids (SCFA), mainly acetate,

propionate and butyrate (Cumming and Englyst 1987). During fiber fermentation, fiber-

bound phytochernicals, such as plant lignans secoisolanciresinol diglucoside (SDG) and

matairesinol, are liberated and converted into the mammalian lignans enterolactone (EL)

and enterodiol (ED) (Setchell et al 1981). Minerais, such as ~ a " , bind to dietary fiber,

which decreases their absorption in the small intestine (Van Soest 1984), However,

bound minerals are released and absorbed when the fiber is femented in the colon

(Trinidad et al 1996b). Thus, calcium absorption was shifted from the small intestine to

the colon (Trinidad et al 1996b). Evidentîy, SCFA, calcium and mammalian lignans rnay

accumulate in the colon lumen after fiber fermentation. The accumulation of these

chernicals may cause biological funcüons which potentially are related to the protective

effect against colon cancer.

Individually, SCFA, calcium and mammalian lignans have been shown to be

protective against colon cancer. SCFA, particularîy butyrate, has Men show to have

many pmtective effects against colon tumor cells, induding arresting growth, inducing

differentiation, inducing apoptosis and d o m regulating oncogene expression (Nakano et

al 1997; Archer et al 1998; Cumming 1995). The protective effect of calcium

supptementation is highly associated with vitamin D (Kleibeuker et al 1994; Wargovich et

al 1995), suggesting a potential role of calcium transport and intracellular Ca2*.

Supplementation with either flaxseed or SDG significantly inhibited cell proliferation,

multiplicity and size of azoxymetftane-induced aberrant crypt foci in rats, which was

attributed to the mammalian lignans produced from SDG (Jenab and Thompson 1996).

In previous in w'tm studies, SCFA, calcium and mammalian lignans individually

significantly inhibited cell proliferation and induced apoptosis in the human colon tumor

cell line HCT-15 (Skatiah and Thompson 1998). Greater effects were observed when

cells were treated with these chemicals in combination (Skanah and Thompson 1998). A

better understanding of the mechanism behind these observed effects is important and

necessary to irnplement these observed effects for lowering colon cancer risk.

In this study, it was hypothesized that high calcium concentration, SCFA and

mammalian lignans increase calcium transport, and subsequently increase intracellular

ca2' ([ca27i) and dwease intracellular pH (pHi) in human colon tumor cells. Both high

[ca27i and Iow pHi then boost mitochondnon-mediated apoptosis (Ichas and Marat

1998) and consequently decrease cell proliferation. Therefore, calcium wmbined with

soluble fiber andior lignans may serve as a chemoprevention strategy against colon

cancer. The overall objective of this study was to determine the influence of calcium

concentration, SCFA and mammalian lignans on calcium transport. [Ca2Ti and pHi in the

human colon tumor cell line HCT-15.

CHAPTER TWO

LITERATURE REVJEW

2. UTERATURE REVlEW

2.1. Colon carcinogenesis: imbalance of cell proliferation and difilerentiation-

apoptosis

2.1.1. Balance between cell ~roliferation and d i f f e~n t i a t i ~n -a~o~ to~ i~

The colonic epithelium consists of a single layer of columnar epithelial cells,

punctuated by tubular structures called aypts. In normal tissue, cell proliferation and

differentiation-apoptosis maintain a balance spatially and temporally along the crypt.

Proliferative cells are found only at the bottom or lower part of the crypts. Cells move

upwards towards the surface during their division. Meanwhile, they gradually lose their

proliferative capability and differentiate into mature, functioning cells. Within 4-5 days,

mature cells in the top of crypt die and are sloughed off. New cells pushing up from the

bottom substitute them (Kune 1996).

2.1.2. Loss of the balance durina cancer develooment

lmbalance of cell pmliferation and differentiation-apoptosis is the major hallmark

(Fearon and Vogelstein 1990) during the threestage colon cancer development, i-e.

initiation, promotion and progression (Farber 1982a 8 b). In the initiation stage, through

DNA replication, DNA damage caused by extracellular factors can be fixed into genomic

mutation, which can be camed forward to progeny cells (Farber, 1982a). While most of

the mutations are subue, some mutations allow initiated cells to gain a growth

advantage. In the promotion stage, the proliferation of initiated cells is promoted, which

leads to the development of a neoplastic cfone. Hyperproliferation also results in initiated

cells being more susceptible to another mutation, subsequently developing a subclone in

a similar way (known as 'cloning se1ection")Fearon & Vogelstein 1990; Vogelstein et al

1988). Hyperproliferation is the most cornmon characteristic in different precursor lesions

of colon cancer, including aberrant crypts, polyps and adenoma (Hamilton 1996). In the

progression stage, the emergence of a subdone with capabilities of autonomous,

invasive and metastatic growth is the culmination of the 'cloning selection' (Vogelstein et

al 1988).

On the other hand, the transition from a normal cell to a malignant cell is a multi-

step process in which the tumor cell becornes less and less differentiated (Jass 1993).

Aberrant cell differentiation may play a uucial and central role in the tumorigenic

process. The critical event is that mutant cells may eventually lose the ability to undergo

apoptosis (Kikuchi-yanoshita et al 1992). which allows senescent or DNA-darnaged cells

to die and maintain physiological homeostasis. Loss of the capability for apoptosis will

allow DNA-damaged cells to accumulate mutations and accelerate the carcinogenic

process (Barinaga 1997). Thus, the imbalance between cell proliferation and

differentiation-apoptosis worsens in the later stage of cancer development.

2.1 -3. lmbalance caused bv mutations

Colonic carcinogenesis is a stepwise process driven by different mutations

(Figure 2.1) (Fearon and Vogelstein 1990; Reale and Fearon 1997), which are classified

into two main types. One is called 'gain-in-function" mutation, a mutation in a single

allele which activates or amplifies oncogenes and proto-onmgenes, such as K-ras

mutation, c-myc and bd-2 amplifications in colonic carcinogenesis. The other is called

uloss-of-function" mutation, mutations in both alleles which inactivate turnor suppressor

genes (Weinberg 1989), such as Apc and p53 mutations in colon cancer (Spirio et al

1993; Kikuchi-yanoshita R et al 1992). m i l e amplification of omyc and mutations of K-

ras and Apc increase ceIl proliferation (Vogelstein et al 1988; Smith et al 1993; Su et al

1993), overexpression of Bd-2 and p53 mutation allows tumor cells to avoid apoptosis

(Hague et al 1994; Greenblatt et al 1994). Thus, these gene mutations are directly

associated with the imbalance beWeen cell proliferation and differentiation-apoptosis.

l Apc loss of f i n c î i o ~

O - O B - B . p53 loss -- O

Figure 2.1. A genetic mode1 of colon cancer (Adapted from Fearon and Vogelstein 1990)

Although diagnosis and treatment of cancers have been greatly improved in the

past 25 years, mortalities and incidences of most cancers have not decreased.

Moreover, advanced cancer still remains incurable (Kosary 1995). The understanding of

stepwise development of cancer is important for chemoptevention, an active intervention

to arrest or reverse carcinogenic process in order ta lawer both incidence and mortality

(Kellof et al 1994; Greenwald 1996). Even when genetic damage already exists, the

following are possible ways to arrest or reverse cancer development (Hong 8 Spom

1997).

i) lnhibit cell proliferation. Cell proliferation is promoted by the expression of

cyclooxygenase (Cox-2) in colon cancer development. Different Cox-2 inhibitors have

been successfully used to suppress colon tumor formation experimentally and clinically

through inhibition of cell proliferation (Kelloff et al 1994; Lipkin et al 1999).

ii) lnduce differentiation. Abemncy of differentiation during colon cancer

development offers a defined target for intervention. By resuming nomal differentiation,

cell proliferation can be amested with certain agents (Greaves 1986; Hong and Spom

1997). For example, butyrate treatment can induce differentiation of Hi29 cells

(Augenlicht et al 1995; Augenlicht 1989).

iii) lnduce apoptosis. Some mutations can block apoptotic pathways, such as p53

mutation and bci-2 over-expression (Barinage 1997). In cell lines with p53 mutation or

bcl-2 over-expression, butyrate treatment can resume blocked apoptotic pathways

(Hauge et al 1994; Archer et al 1998).

2.2.2. Chernoorevention throuah nutritional manioulation.

The role of diet in colon cancer n'sk in Western countries is estimated to be about

50-90% (Kune 1996; Potter 1996; Lipkin et al 1999). Westernkation of dietary habit is

believed to be a primary factor for increasing colon cancer incidence rates in Japan and

other developing countries, and in Chinese and Japanese descendants in North Arnerica

(Boyle 1 998). Some foods and dietary factors, such as vegetables, fruits, grains, calcium

and fiber, and diets low in meat, animal fat and total energy, appear to be protective

against colon cancer; others, such as high saturated fat, r d meat and high energy

intake, are believed to be risk factors (Kune 1996). These pmvide a foundation for

chemoprevention through nutritional manipulation. Although the mechanisrns for many of

these dietary factors still remain unciear, many interesting findings on dietary fiber,

calcium and phytochemical lignans provide a better understanding of how they fundon.

2.2.3. Dietaw fiber

The protective effect of dietary fiber against colon cancer was supported in many

studies, done in different populations, using different fiber sources and biomarkers,

(Lipkin et al 1999; Freernan HJ 1999; Wjnands et al 1999; Jenab and Thompson 1998).

The biological funçtions of fiber are detennined by its physico-chemical properties.

Solubility in the aqueous phase is a very important one (Anderson 1985; Lupton and

Turner). Colonic miwflora can degrade soluble fibers rapidly and completely. In

cantrast, insoluble fiber is not easy to break down (Ham's et al 1993). Consequently, the

bioiogical functions of soluble and insoluble fiber differ in the colon lumen.

Insoluble fiber maintains water holding, cation binding and bile absorption

properties in the colon (Harris et al 1993; Anderson et al, 1994). The water-holding

capability increases fecal volume and accelerates fecal passage, so that the

concentration of carcinogens and the time they are in contact with colonic epithelia are

decreased (Anderson 1985). Some fiber components a n reduce the absorption of

organic substances, such as bile acids, that promote colon cancer (Anderson 1985).

When soluble fiber is fennented in the colon, microflora metaboiize it to short

chain fatty acids (SCFA), mainly acetate, propionate and butyrate (Lupton and Kurh

1993). SCFA, partiwlarly butyrate, can con- the imbalance between cell proliferation

and differentiation-apoptosis, which will be discussed later. lncreased SCFA production

decreases lumen pH which, in tum, decreases the synthesis of secondary bile acids,

which sewe as promoters of tumor cell gr& (Lupton and Kurtz 1993). Any fiber-

entrapped ca2' is released upon fiber fermentation (Van Soest, 1984; Trinidad et al

1996b). This inueases ca2+ in the colon lumen, which may then reduce the growth of

cancer cells. Fiber-bound phytochemicals, such as lignans, are released as well and

wnverted into their biologically active compounds (Adlerceutz and Mazur 1997). Thus,

soluble fiber may have more profound inhibitory effects on colonic carcinogenesis than

insoluble fiber.

2.2.4. Limans

Plant lignans exist in many plant foods, such as grains, vegetables and fruits,

which have been associated with a reduction of isk of colon cancer (Thompson et al

1996; Potter and Steinmeitz 1996). Precursor plant lignans, secoisotariciresinol

diglycoside (SDG) and matairesinol, are converted into mammalian lignans,

enterofactone (EL) and enterodiol (ED), by colonic microflora (Borriello et al, 1985;

Thompson et al 1991). Some of the mamrnalian lignans escape absorption and remain

in the colon lumen, M i l e some are absorbed and undergo entero-hepatic circulation

(Adlercreutz and Mazur, 1997). After consumption of 5% flaxseed diet, the lignan

concentration in the colon lumen may mach as high as 665 ph4 (Sung et al 1998).

Administration of flaxseed or SûG significantly lowered the incidence, number

and size of mammary tumors in animal studies (Rickard and Thompson, 1997). Lignans

may also be protecüve against colon cancer. In epidemiological studies, higher urinary

fignan secretions were obsewed in subjeds consuming die& that may lower the nsk of

colon cancer (0.8. vegetarian) (Adlemutz et al 1986). and in areas with low colon

cancer incidence (Korpela 1988). Animal studies also supported the hypothesis that

phytoesttogens may reduce colon cancer. Diets supplemented with flaxseed or SDG

inhibited the formation of aberrant crypt foci, prewrsor lesions of colon cancer, in

azoxymethane (A0M)-treated rats (Serraino et al, 1992; Jenab and Thompson, 1996).

The protecüve effect of calcium against colon cancer was proposed a long time

ago (Newmark et al 1984; Garland et al 1985). However, only modest or no protecüve

effect was concluded from many case-control, cohort and intervention studies using

calcium supplement to control colon cancer (Baron et al 1999; Lipkin et al 1999;

Martinez and Willett 1998). The anti-tumorigenic effect of calcium is more consistent in

animal experiments on colon cancer (Pence 1993). The suggested mechanisms include

that the binding of calcium with unabsorbed free fatty acids and unconjugated bile acids

forms insoluble products in the colon lumen. Thus, calcium teduces the cancer

promoting effect of free fatty acids and unconjugated bile acids (Lupton et al 1996;

Alberts et al 1996). In support of this, calcium showed stronger effect in animals fed with

high fat diet (Pence and Buddingh1988), and in population with Western high fat diet

(Newmark et al 1984; Hyman et al 1998).

Of particular interest is the interactive effect of calcium and vitamin D3 in colon

cancer. Vitamin D also can prevent colon cancer in chemically induced rat color!

carcinogenesis (Belleli et al 1992). Calcium wmbined with vitamin Dj significantly

decreased azoxymethane (AOM) or 1,2-dimethylhydrazine-induced aberrant crypt foci

and K-ras mutation, while calcium in the absence of 1 ,25(OH)t-& had no effect (Pence

and Buddingh 1988; Lior et al, 1991). In human invention studies, the combination of

calcium and vitamin D also decreased the risk of colon cancer (Bostick et al 1993;

Martinez and Willett 1998). The interactive effect of vitamin D and calcium in

biochemistry and physiology. especially ca2' absorption and ca2' signaling, may be

highly related to their intefaciive effect in colon cancer (Lipkin et al 1999).

2.3. Effects of SCFA, ~ a " and rnammalian lignans

2.3.1. ca2+

ca2+ perfonns a number of functions in the colon epithelium i-e. cell cycle

promotion, differentiation ûiggenng, and killing (Whitfield 1995). In different cell cultures,

cell proliferation is optimal at an extemal ca2+ concentration of 0.1 mM and is stopped at

0.8-2.0mM (Friedman 1991). In vivo, mucins secreted by goblet cells, like sialomucin and

sulfomucin, bind Ca2' and form a gradient of ca2+ concentration along the colonic crypt.

High Ca2+ concentration in the upper part of uypt induces differentiation and apoptosis,

while low concentration in the lower part of crypt stimulates cell proliferation (Whitfield

1995).

A 'ca2+ sensof was suggested (Whitfield 1995) to respond to extemal ca2+

change. lncreased calcium concentration resulted in induction of differentiation

promoting CAMP-dependent protein kinase II and tumor growth factor P, and down

regulation of proliferation-promoting CAMP-dependent protein kinase I, tumor growth

factor a and c-myc and c-myb (Whitfield, 1995). Gama et al (1997) identified a Za2'

sensoi'. which responds to extracellular ca2' concentration and increases intracellular

Ca2' as a second messenger. However, many colon tumor cells lose their sensiüvity to

extemai ca2' mit f ie ld 1995).

Colon cancer incidence is dramatically higher than srnall intestine cancer

incidences. Potten (1992) suggested that calcium sensitive cells are located at the

bottom of the colonic crypt and middle of the small intestinal crypt. Thus, the epithelial

cells in the small intestine are more sensitive to ~a~+-rnadiated apoptosis than those in

the colon. On the other hand, most dietary calcium is absorbed in the srnall intestine,

with only a srnall portion of calcium absorption taking place in the colon. The question is

whether the difference in cancer incidence is related to the difference in calcium

absorption.

2.3.2. Short chain fatty acids

SCFA, particularly butyrate, has multiple effects on colonic cells and colon tumor

cells. With butyrate in cell culture, cell proliferation was inhibited and cells were arrested

at G1 phase (d'Anna et al 7980; Heedt et al 1997). Butyrate down regulated oncogenes

including bcl-2, c-myc and c-fos (Toscani et al 1988; Archer et al 1998). Butyrate can

resume apoptotic pathway in cell lines h o s e normal apoptotic pathway is blocked by

p53 mutation (Nakano et al 1997; Litvak et al 1998). Butyrate also induces cell

morphological change (Bretton and Pennypacker 1989) and expression of funcüonal

proteins (Augenlicht 1989). However, in vivo study has show that SCFA prornote

normal cell praliferation (Sakata 1995), which may enhance the carcinogenic initiation of

normal colon cells (Lupton 1995).

In intestines, SCFA are absorbed easily mainly ttirough passive diffusion

(Bugaut, 1987; Ruppin et al 1980). Some non-ionized compounds are lipid-soluble and

easily diffuse across a lipid cell membrane, but not their water-soluble ionized

compounds (Shanker 1959; Westergaard and Diestchy 1974). Although more than 99%

of SCFA are ionized at pH 7.0 (Weast, 1980). SCFA can be rapidly absorbed at neutral

pH (Vemay, 1987) by two mechanisms (Figure 2.2). One is HCWSCFK exchange

(Mascolo et al 1991; Binder and Mehta 1989), which exists in the colon and is analogous

to the anion wunter transport system Cr-HCW exchange (Bugaut 1987). The other

mechanism is direct diffusion of the protonated SCFA. The proton, recycling across the

luminal membrane by the Na+-H' antiporter, allows a high rate transport of SCFA

(Schultz, 1981). Similarly, the K+-H4 antiporter (Buguat 1987) and the ca2+-H' antiporter

(Lutz and Scharrer 1991) were found in different segments of the digestive tract.

Once the protonated SCFA diffuse into cytoplasm, they are immediately

dissociated into the ionized SCFA and H* (Englehart, 7995). Most absorbed SCFA are

transported to the liver for metabolism. Some SCFA are metabolized in the epithelium.

They are converted into acyl-CoA, which then may be used for lipogenesis, ketogenesis

and gluconeogenesis (Stevens, 1970; Awad et al 1991). The complete oxidation to CO2

is often a preponderant catabolic pathway (Stevens, 1970). Butyrate is the most favored

energy source for colonic cells, and glycolysis is decreased in the presence of butyrate.

In contrast, propionate and acetate are absorbed into the blood Stream (Cummings and

Englyst 1987).

2.3.3. Mammalian limans

The major mamrnalian lignans are ED (2.3, bis[(3-hydroxylphenyl) methyl]

butane 1.4 diol) and EL (trans 2,3, bis((3-h ydroxylphenyl) methyl] butyolactone) with

structures similar to p-estradiol and diethylstilbesterol (Sathyamoorthy et al 1994;

Setchell and Adlemutz 1988). An anti-tumongenic effect of lignans has been observed

in animal studies or suggested by epidemiological studies (Thompson et al 1996;

Adlercrutz and Mazur 1997).

Metabolites & ATP

Figure 2.2. lnterrelationships between absorption of SCFA and movements of

hydrogen ion, CO2 and electrolytes through the digestive tract epithelium.

(Adapted from Bugaut 1987)

Several mechanisms may be involved in the anti-tumorigenic effect of

rnammalian lignans: a) competition with estrogen for estrogen receptors (Mousavi and

Adlercnitz 1992); b) stimulation of hypothalamic feedback regulation of estrogen

(Cassidy et al 1995); c) inhibition of enzymes involved in estmgen bio-synthesis

(Adlercreutz et al 1990); d) increase of plasma sex hormone binding globulin (SHBG)

synthesis, which, in tum, decreases plasma free estrogen (Adlercreutz et al 1987). Thus

lignans may decrease estrogenic effect by reducing the availability of free estrogen for

sensitive tissues and estrogen receptors (Mousavi and Adlercreutz f 992).

Lignans also have non-hormone-related mechanism for anti-carcinogenic effects:

a) they have anti-oxidative effect (Yuan et al 1999). b) EL exerts a cytotoxic effect in

some tumor cell lines, which has been associated with the inhibition of non-sodium or

potassium dependent membrane ATPases (Hirano et al 1990). A phytoestrogen

genistein can inhibit receptor tyrosine kinase activity and dom-regulates proliferation

signais. Estrogen, 1 .2S(0H)r4, and other steroid chamicals can stimulate Ca2+ release

from intemal store and influx fom extemal source, which may intenupt intracellular Ca2'

signal (Wali et al 1990; Peter et al 1998; Biossonnette et al, 1994; Morley et al 1992).

With structural similarity to the above, mammalian Iignans may also have these two

effects.

Mammalian lignans have been shown to affect colon cancer cells, including

reduction of proliferation (Sung et al, 1998) and dom-fegulation of oncogene omyc

(Henith 1995). Estrogen receptors were expressed in normal mucosa, adenornatous

polyp and cancer cells in the colon (Xu and Thomas 1994). with low expression in the

epithelial cells, but a high level in the stromal cells (Waliszewski et al 1997). In a cell-cell

interaction pattern, the estrogen-receptor complex up-regulates growth factors in the

stromal cells, which, in tum, serve as a paracrine to stimulate epithelial cell growth.

mus, the effect of mammalian lignans in the colon rnay also be associated with estmgen

receptors.

2.4. Interactive effects of Ca", SCFA and mammalian lignan

2.4.1. ca2' and SCFA

Butyrate and ca2+ have sirnilar affects. induding dom-mgulation of oncogenes

c-myc, bcl-2 and K-ras, inhibition of cell pmliferation and induction of apoptosis. Greater

effects were found when colon tumor cells were treated with them in combination

(Skariah and Thompson, 1998).

The interactive effect also was obsewed in the absorption of ca2' and SCFA.

The presence of SCFA increased calcium absorption in the colon of rats (Lu& and

Scharrer 1991). In colorectal infusion studies in healthy human subjects, calcium

disappearance was increased with increasing SCFA concentration (Trinidad et al,

1996a). SCFA absorption was also efihanced in the presence of calcium in a similar

colorectal infusion study volever et al 1996). Possible rnechanisrns involve either

uptake of Ca2' and expelling H' (Lub and Scharrer 1991). or formation of a Ca-SCFA

cornplex, which decreases the electronic charge of ca2' and easily ovemornes the cell

membrane bamer (Marshall 1976).

As a consequence of SCFA and ca2' absorption (Figure 2.3). intracellular ca2'

may be inueased and intracellular pH may be deueased. lnuease of intracellular ca2'

inhibits basolateral Na'/H+ antiporter, which may further decrease intracellular pH

(Semrad and Chang 1987). Decreased intracellular pH may also augment intracellular

ca2' increase, because inueasing [ca2+Ji induced by some agonists showed stronger

SCFA-H

SCFA-H

Figure 2.3. The interaction of Ca2+. SCFA and mammalian lignans (1 represents H'c~*'

exchange; 2 Ca2+ channels; 3 Endoplasmic reticulum; 4 Mitochondrion). SCFA increases

ca2' entry by forming penneable SCFA-C~~* complex and dnhancing H+/c~" exchange.

SCFA-ca2' complex also increases SCFA uptake. Mammalian lignans may affect ca2'

entry through these possible mechanisms: stimulating ca2' channel, indirectly or directly,

stimulating internai ca2' stores (mitochondrion and mdoplasmic reticulum). The final

results may inctease cytosolic Ca2' and H'.

effect in low pHi (Nitshke et al 1997). Meanwhile, inaeased intracellular ca2+ can

enhance H* uptake into the nucleus to promote apoptotic process (Nicotera et al, 1989).

2.4.2. Mammalian limans. ca2+. andlor SCFA

Some steroid hormones, such as 1,25(OH)& and estrogen, show interactive

effect with butyrate and ca2+(Yoneda et al 1984). First, as previously reviewed, steroid

hormones stimulate a cell membrane receptor, which consequently increases

intracellubr ca2+ through extraceIIular ca2' entq (Peter et al, 1998; Bissonnette et al

1994). Second, calcitriol enhances the butyrate-induced differentiation of human colon

tumor cells (Tanaka et al 1990). Similar results are expected in the case of mammalian

Iignans since they have steroid structure similar to estrogen and 1, 25(OH)rD3.

The influence on PKC acüvity and intracellular ca2+ may be the common

mechanism in the biological effect of combined ~ a " , SCFA and mamrnalian lignans.

Butyrate can selectively affect the incorporation of long-main fatty acids into colonic cell

membranes. Changes in membrane lipids, in tum, affect the activity of PKC (Awad et al,

1991). 1, 25 (OH)rvitamin and ca2+ induce apoptosis UIrough the activation of PKC

and increasing intracellular Ca2' (Bissonnette et al 1994).

2.5. Calcium absorption and transport

Calcium is present as relatively insoluble salts in foods and dietary supplements

(calcium carbonate). However, it is absorbed only in its ionized fonn, and ca2+ must be

released from salts in the acidic lumen. Thus in the alkaline intestinal lumen, formation of

ca2' cornplex limits calcium availability (Sheikh et al, 1987; Bronner and Pansu 1999).

2.5.1. Calcium absomtion in aie colon

Most calcium absorption occurs in the small intestine, and only a very small

amount of calcium is believed to be absorbed in the colon (Allen and Wood, 1994).

However, presewed function of the colon in calcium absorption plays an important role

in patients suffering from malabsorption in the small intestine (Hylander et al 1980). A

robust calcium transport capacity was obsewed in different segments of the colon, which

also was inducible by 1, 25(OH)& (Karbach 1990). With a high fiber intake, fiber-

bound Cah readies the colon lumen, and is teleased after fiber fermentation (Trinidad et

al, 1996b). the presence of acetate and propionate enhanced ca2+ absorption in the

colon (Trinidad et al, 1996a). The protective effect of calcium on colon carcinogenesis

may be associated with calcium absorption as teviewed in section 2.3.1.

2.5.2. Paracellular and transcellular transport

Calcium absorption occurs ttirough two transport processes: transcellular and

paracellular. Paracellular transport is passive, unsaturated and concentration dependent,

and is the major mechanism for increasing calcium absorption when calcium intake is

increased (Allen and Wood, 1994).

In contrast, transcellular transport (Figure 2.4) is saturable, energy consuming

and responsible for the uptake of calcium at low luminal concentration. It indudes caZ'

entry at bmsh border membrane, intracellular ca2+ movement and CI'' extrusion at the

basolateral membrane (Bronner, 1987). Calcium channels, regulated by membrane

voltage and receptor activation, predominantly determine calcium influx (Benham and

Tsien, 1986). lntracellular ca2' movement is the ntelimiting step in the transcellular

transport (Bronner, 1990). Ca2' is expelled by a ca2'-~a' exchange or an ATP-

dependent ca2' pump at the basolateral membrane (Allen and Wood, 1994). Changes in

transcellular calcium transport may increase intracellular Ca2'.

Voltago dependent cbrnncl

Trans- membrane diffusion

Eodoplasmic reticulum

Calcium binding proteins

4 Mitochondrion Encrgy-

N~+/K+ ATPas

K car+ Exchange Ca2*

Figure 2.4. Transcellular calcium transport mechanism.

2.5.3. Calcium transwrt in colon tumor cells and the monolaver cell model

Colon tumor cefl lines are able to retain some characteristics of differentiated

normal colonic cells (Augenlicht 1989), even with Vull differentiation" characteristics in

some specific functions, Iike transport (Augenlicht 1989). With these characteristics, a

transport model of colonic epithelium was developed using a monolayer of colon tumor

cells placed in the insert wells of Transwell plate (Hidalgo et al 1989). Under the

electronic microscope, these cells underwent a certain level of differentiation and polarity

after a culture period, with brush borders in apical membrane, intercellular junctures and

alkaline phosphatase polar distribution (Hidalgo et al 1989; Augenlichet 1989).

Measurernents of transepithelial electrical resistance and transport of different molecular

size water-soluble indicators suggested that this represented a good model for transport

studies (Hidalgo et al, 1989).

This model has been used in studies on the effect of vitamin D on calcium

transport (Giuliano and Wood 1991). Compared to other models, such as everted sacs,

intestinal rings, brush border membrane vesicles, intestinal loops and vaswlar perfused

intestine, this model can more diredy detect transepithelial transport (Hidalgo et al

1989).

2.5.4. Measurement of calcium concentration

Methods for the measurement of calcium concentration in calcium transport

studies include ~ a " isotope counting (Bissonnette et al 1994). atomic absorption

spectrophotornetry (Trinidad 1996) and the use of a ca2' probe. ca4 is lirnited by the

need to obtain isotope permission, and potential influence of the isotope on cell

funcüons. Atomic absorption spedrophotometry requires a special treatment and a large

sample volume. In contrast, calcium probe allows direct detection of calcium

concentration in the transport solution without wmplicated sample treatment. The

fluorescence probe Fura-2 can al- be used to detemine intracetlular ca2' (Section

2.6.3.), and thus is commonly used for the type of study we proposed.

2.6. lntncellular ~ a * and intncellular pH

2.6.1. lntracellular ca2'

Many stimuli, such as estrogen and 1,25(OH)rvitamin D3, may increase

intracellular Ca2+ as a second messenger (Wali et al 1990; Peter et al 1998;

Biossonnette et al, 1994). The interaction of these chernicals and their receptors at the

plasma membrane leads to activation of phospholipase, which, in tuml hydrolyses

phosphatidylinositol, a membrane phospholipid, to generate inositol 1,4,5,-triphosphate

(IP,) and diacylglyceml (DAG) (Berridge 1987). IP3 triggen ca2+ release from the

endoplasmic reticufum into the cytoplasm (Wasserman 1990). lncreased intracellular

ca2+, in tum, can directly or indirectly stimulate ca2+ release from mitochondnon or ca2'

entry from the extracellular environment (Putney 1997).

lncreased intracellular ca2+. as a very important second messenger, is involved

in the regulation of many uitical intracellular processes (Henning et al 1980). ca2+

signals decode into biological functions through at least three different pathways. First,

inueased intracellular ca2+ activate PKCs, which. in tum, catalyze Me phosphorylation

of signal proteins involved in multiple biological functions. Second, intrscellular ca2' has

a number of direct effet%, e.g. ca2+ diredy activates phosphodiesterase. which

hydrolyzes cydic AMP (CAMP) to regulate CAMP signals. Third. ca2* binds with

calmodulin to become a motif of many different macromolecules or enzymes

(Rasmussen, 1986).

In quiescent cells, intracellular ca2+ is maintained at a very low concentration of

about 100nM (approximately 1/10,000 of the extracellular concentration)(Rasrnussen

f988). As soon as its biological fundion is finisheû, increased intracellular ca2+ is

cleared out of cytoplasm. Ca2' pump either expels ca2* out of the cell, or ca2' is taken

up into mitochondrion and endoplasmic retiwlum. The increased ca2+ concentration

then goes back to stationary level (Rasmussen 1986). Any cause of sustained increase

in intracellular ca2+ will cause cell death (reviewed in Section 2.7.). Colon tumor cells

have a lower intracellular Ca2* level than normal tissue both with and without calcium

(Edelstein et al 1991). which cause tumor cells to avoid apoptosis.

2.6.2. lntracellular OH

Maintenance of the homeostasis of intracellular pH (pHi) is vital to most cells

because most cellular functions are pH sensitive (Nuccitelli and Helple 1982). Cells

maintain the homeostasis of intracellular H' through complex ion transport systems,

such as HCO-&ï exchangers, lactate-H* symporten and Na'-H' antiporters (Madshus

1988). The HCO+Cr exchanger is one of the most common ion transport system

existing in most cells.

Tumor cells in a hypoxic environment mainly depend on anaerobic glycolysis for

energy, h i c h produce 5-7 times more protons than aerobic glycolysis per ATP

produced (Owen 1996). The Na'-H' antiporter is vety important for tumor growth when

the bicarbonate exchangers are insuffident to regulate acidification caused by anaerobic

glycolysis (Rotin et al 1989). Tumor cells, through changes in H'-Na' antiporter and

HCO+CI- exchange, expel H' efficiently and maintain their cytoplasm alkaline (Kraus

and Wolf 1996, Bischof et al, 1996). Acid extrusion, combined with microcirculatory

inadequacy in the surrounding tissue, causes extracellular microenvironment

acidification, which promotes tumor cell invasion, hampers surrounding tissue growth

and inhibits immune system function (Kraus and Wolf, 1996). Moreover, many anti-

cancer agents are weakly alkaline (Litman et al, 1998). The low penneability of their

ionized state in an acidic microenvironment makes it difficuît for them to enter cells; the

high permeability of their unionized state in an alkaline cytoplasm makes it easy for them

to be expelled (Gerweck and Seetharaman, 7996). Such a situation is closely related to

multi-drug resistance of tumor cells (Litman et al, 1998; Wadkin and Roepe 1997).

Decreased intracellular pH has been documented in a number of studies that

exposed cells to vanous apoptosis-inducing stimuli (Gotüieb et al 1996). lntracellular

addification triggers activation of acidic deoxyribonuclease II, which causes DNA

cleavage and apoptosis (Bany and Eastman 1993). When cells are treated with an

ionosphere to make their membranes permeable to H4, low extracellular pH (pHe)

directly induces apoptosis (Bischof et al 1996a). Using a similar method, clamping

intracellular pH at pH 6.75 for 24 hours or pH 6 for 6 hours significantly induced

apoptosis (Boyle et al, 1997). Reduction of intracellular pH was suggested as a possible

mechanism for killing cells in acidic regions of solid tumor (Newell and Tannock 1989).

Reduced pHi was achieved by using the proton ionophore carbonyl cyanide 3-

cholophenylhydrazone and nigericin (Newell et al 1992) and the Na+-H* antiporter

inhibitor amiloride and its derivatives in experimental treatments (Maidom et al 1993).

The stress-activated protein kinase pathway has been suggested as one mechanism for

acidification-induced cell death (Zanke et al 1998). Litman's study suggested that

decreasing pHi is a possible way to overcome rnultidrug resistance in tumor cells

(Litman et al 1998).

2.6.3. Measurements of [ca2*li and pHi

Many methods have been used to measure [ca2?i and pHi. The use of ion

probes appears to be more reliable, more precise and more sensitive, and causes less

interruption to nomal cell functions.

Of many ca2+ ion probes, Fura-2. with increased brightness of fluorescence. is

the most frequently used. F irot, the inueased brightness allows ca2* measurement with

low intracellular Fura-2 loading. Thus, the effect of ion probes on buffering of cytosolic

free ca2' can be avoided. The bright fluorescence also ovemmes the interference of

cell autofluorescence. Second. Fura-2 shows better selecüvity for ca2' than other

divalent cations, such as ~e*+, Mg2+, Zn" and ~ n " (about 42 to 510-fold). Third, the

influence of pH within physiological range is vety srnaW. Fourth, Ath the binding of Ca2'.

Fura-2 changes the excitation wavelength from 340nm to 380nm, not just the amplitude

of the fluorescent peak. By using the ratio of different excitations, the biases from Fura-2

concentration, path length and sensitivity of instrument can be avoided. Moreover, its

membrane-permeable ester derivative, Fura-ZAM, allows Fura-2 to accumulate inside

the cell after cytosolic esterases split off the ester group. After washing away

extracellular Fum-2AMI Fura-2 trapped inside the cell will only respond to change in

intracellular ca2+ (Grynkiewiu et al 1985).

Similady, the H* probe SNARF-1 has bright fluorescence and high selectivity for

H'. Upon binding to H', SNARF-1 shifts its emission wavelength from 575 to 635nrn,

thus allowing the use of the wavelength ratio to estimate pH change. Its membrane-

permeable ester denvative, SNARF-1AM, also makes rneasurernent of intracellular pH

possible (Barry and Eastman 1993).

Different methods have been established to measure the fluorescence change,

mainly by flow cytometry (Barry et al 1993), fluorescence microscopy (Williams and

Fay 1990) and spectrofluorometry (Edelstein et al 1991). Each method has its own

advantages and disadvantages, which can be summahzed in Table 2.1. Fluorescence

microscopy provides the best method of measurement because it measures attactied

growing cells more precisely and rapidly responding to signal changes (even in

milliseconds and individual cells). Hawever, it requires more complicated equipment and

working conditions. It measures only about 200 cells (maximum) which may result in

Table 2.1. Cornparison of different methods for the measurements of intracellular ca2'

and intracellular pH

Cell numbers t-- Detecting system 7

1 envimnment;

requirement

Flow cytometry

Individual cell

Cell suspension

One

Low concentration

Not for Fura-2

Medium;

flow cytometer

Fluorescence Spectrofluorometry

microscopy

About 200 10-15X106 f

About 200 cells 1 All cells together

Attached cells Cell suspension I

Multiple Multiple

High concentration Low concentration

High; LOW;

fluorescence 1 spectrofluorometer

microscope, digital

data systern, dark work

station I

large variation between repeated measurements, and it also requires a high

concentration of probe which may buffer intracellular ion. Flow cytometry can precisely

rneasure ion changes occurring in individual cells. However, it only detects permanent

changes at one time point due to the stability of flow cytometer, and the excitation

wavelength of argon lasers prevents the use of Fura-2 in the flow cytometer. Although

spectrofluorometry only detects the total changes in intracellular ion in a cell suspension,

it allows the measurement at different time points with a low probe concentration, gives

highly repeatable results, and has a low requirernent for instrument and working

environment.

2.7. Mitochondrion-mediated Apoptosis and [ ~ a T i and pHi

Apoptosis, programmed cell death, is a physiological process critical for organ

development, tissue homeostasis and elimination of defective or potentially dangerous

cells (Dragovich et al 1998; Wyllie 1992). The apoptotic cell is characten'zed by cell

dehydration, with decreased cell sire, and condensation of cytoplasm and chromatin.

Cellular membrane fragments occasionally surround functional organelles in a process

called "blebbing" (Darrykiewicz, 1995). The remnants are subsequently phagocytised by

surrounding cells, without a concomitant inflammation (Dragovich et al 1998).

Mitochondria have been found to play a central role in control of apoptosis (Susin et al

1 W8), with the process divided into three phases, the pre-mitochondrion, mitochondrion

and post-mitochondion phases (Figure 2.5.) (Susin et al, 1998).

A rnitochondrion consists of two membranes, a smooth and porous outer

membrane and a tight, selectively penneable and folded inner membrane. Cristae, made

up of folded inner membranes, provide enonnous surface area for attachment of

enzymes involved in energy release and ATP formation. The space inside the inner

membrane (called matrix) accumulates very high concentrations of Ca2+ and anions. The

\( Apoptosomo

Figure 2.5. The three phases of apoptosis and the role of [ca27i and pHi (Adapted from

Suun et al 1998). Fas= Fas ligand; ROS= readve oxygen species; Bax= Bax protein;

AlF= apoptosis inducing factor; DFF= DNA fragmentation factor.

respiratory chain, located on inner membrane, produces protons and expels them into

the inter-membrane space. H' extrusion results in an inner transmembrane potential

(AYm), usually 120-1 7OmV (Macho et al 1996; Kroemer et al 1997). AYm functions as a

versatile force for various tasks in the life and death of cells (Zoratü and Szabo 1 995).

2.7.1. Pre-rnitochondrion hase

The pre-mitochondrion phase can involve many different apoptotic pathways

resulting in the formation of stimuli for mitochondna. a). So-called death factors, such as

Fas, tumor necrosis factors (TNFs), and transfom growth factor+ (TGF-P), activate their

receptors causing activation of caspase-8 (Murio 1998) or production of ceramide (Susin

et al 1998). b). Stresses, such as radiation and hyperthemia, activate stress-activated

protein kinase (SAPK). SAPK in tum either induces ceramide production (Susin et al

1998). or activates wild type p53, which in turn induces acüvation of Bax,

hyperprodudion of reactive oxygen species (ROS), and expression of p21dlh"' and Fas

(Susin et al 1998). c). Glucocorticoid induces ROS or ceremide through activation of its

receptor. Ceremide, ROSI Bax and upstream caspases can stimulate the mitoctrondnon

phase of apoptosis.

lncreased intracellular ca2' and deueased intracellular pH are very important

stimuli in the pre-mitochondrion phase of apoptosis. Increasing intracellular Ca2+ to 10fl

is sufficient to directly induce the mitochondrion phase (Zorrati and Szabo 1995;

Zamzami et al 1998). lncreased intracellular ca2* also is a very important m e d i a t o r of

apoptosis, which may be related to the pemeability transition of mitochondfion

membrane (Zamzami et al 1998). BcJ-2 overexpression and permeability transition

inhibitor enhance the tolennce to increased intracellular ca2' (Murphy et al 1996).

Decrease in intracellular pH can directiy acüvate the SAPK pathway to induœ apoptosis

(Zanke et al 4995).

2.7.2. Mitochondrion phase

During the mitochondrion phase of apoptosis, mitochondria undergo a

pemeability transition of the inner membrane and disruption of AYm before any sign of

apoptosis (Susin et al 1998). The pemeabilty transition is due to opening modulation of

the pemeability transition pore (PTP). a mitochondrial megachannel regulated by ca2+.

pH, AYm, redox and cell volume (Zoratti and Szabo 1995).

In live cells, PTP usually can be opened at the low-conductance state, allowing

molecules srnaller than 3ôûDa to be released (Susin et al 1998). The opening usually is

triggered by an intracellular ca2* signal and regulated by the pH in the mitochondrion

maûix (Ichas and Mazat 1998). This process is recoverable and functions as

amplification of the intraceMar ca2' signal. Mitochondria retain their integrity during this

process (Ichas and Mazat 1998).

However, some stimuli, such as a slow and continued increase in intracellular

ca2', can shift PTP opening fmm low- to high-conductance state, allowing molecules

smaller than 1500Da to be released. It is not regulated by mitochondrion matrix pH, and

the redistribution of H' collapses AYm irreversibly (Mas et al 1997). A concentration

gradient of big molecules (Le. protein) in the matrix still allows water entry into matrix,

causing high amplitude swelling and unfolding of cristae. Eventually, the outer

membrane is ruptured (Ichas and Mazat 1998). lmpaired mitochondrion functions

indude interruption of the respiratory chain, ATP depletion and cessation of CaZ+

pumping. The most important effect is the telease of ca2+, H' and other molecules to

tum on the post-mitochondrion phase of apoptosis.

Stimuli from the pre-mitochondrion phase of apoptosis, such as ceremide, ROSI

Bax and upstream caspases, mainly target at pemeability transition (Susin et al 1998).

lncreasing oxidative stress not only depletes reductive glutathione, NADPHz and NADH2,

but also increases nitric oxide and Ca2'. Mitochondrie are both a souria and target of

these factors (Richter et al 1996). In addition, depletion of ADP and ATP also are stimuli

for the mitochondrion phase of apoptosis (Ritchter et al 1996). Lower intracellular pH

also hampers the regulation of matrix pH and shifts PTP opening from the low

conductance state to the high one, eventually, inducing apoptosis (Ichas and Mazat

1 998).

2.7.3. Post-mitochondrion hase

AYm collapse causes uncoupling of the respiratory chain, and cessation of ATP

production. Meanwhile, the mitochondrion starts to augment superoxide anions.

Consequently, reductive glutathione, NADPH, NADH and ATP are depleted (Susin et al

1998). As a result of rupture of the outer membrane, ca2+ and H' are released and

increase intracellular Ca2' and cytoplasmic acidification (Macho et al 1997; Susin et al

1998). lncreasing [Ca21i acüvates ca2'-dependent endonuclease (type 1) and

decreasing pHi acüvates endonuclease (type H). Bath of these result in DNA digestion

as the end result of apoptotic signaling (Nicotera et al 1994; Barry and Eastman 1993).

Moreover, mitochondria liberate two different factors to initiate the post-

mitochondrion phase of apoptosis. One is 50kD apoptosis-inducing factor (AIF), which is

sufficient to activate caspase-3 and endonudeases (Susin et al 1996). The other is

cytochrome c, a respiratory protein also known as Apaf-2. Apaf-2, together with Apaf-1

(a mitochondrion outer membrane protein), Apaf-3 (a cytosol protein) and ATP, form a

protein cornplex called nApoptosome". "Apoptosome" proteolytically acüvates caspase-3.

Activated caspase-3 in tum cleaves and activates DNA fragmentation factor (DFF),

which in tum activates endonudeases (Susin et al 1996).

2.8. Summary

Previous studies have shown that calcium, SCFA and lignans individually can

influence colon cancer risk. Since they are commonly found in the colon, their interactive

effects on the growth and apoptosis of human colon tumor cells such as HCT-15 were

detennined in vitro, The results showed that their effects are synergistic in reducing the

colon tumor cell proliferation and inducing apoptosis. However, the mechanism whereby

these take place is not dear and need further investigation. Literature data suggest that

they may be related to the extent whereby intncellular ca2' and H' are affected by their

interactions. Their influence on mitochondrion-mediated apoptosis may also be involved.

The potential interactions among apoptosis, (Ca2Ti and pHi are summarired in Figure

2.5.

CHAPT ER THREE

HYPOTHESIS, OBJECTIVES AND OVERVIEW OF

EXPERIMENTAL DESIGN AND METHODOLOGY

3. HYPOTHESIS, OBJECTIVES AND OVERVIEW OF

EXPERIMENTAL DESIGN AND METHODOLOGY

3.1. Hypothesis

Calcium concentration, short chain fatty acids and mammalian lignans wîll

increase the calcium transport and intracellular Ca2' and deaease the intmcellular pH in

the human colon tumor cell HCT-15. These effects in tum may be responsible for the

previously observed effects of calcium, SCFA and mammalian lignans on inhibition of

cell proliferation and induction of apoptosis in the hurnan colon tumor cell HCT-15.

3.2. Overall objectives

To determine the influence of calcium concentration, SCFA and mammalian

lignans alone or in combination on calcium transport, intracellular ca2+ and intracellular

pH in the human colon tumor cell HCT-15.

3.3. Specific objectives

1) To detemine the effect of calcium concentration, SCFA andlor mammalian lignans on

paracellular, transcellular and total calcium transport in a monolayer cell transport model

of colon tumor cell HCT-15.

2) To detennine the effect of different calcium concentrations afone, or in combination

with SCFA or mammalian lignans on intracellular Ca2' in the suspension of Fura-2-

loaded colon tumor cells HCT-15.

3) To determine the effect of calcium, SCFA and mammalian lignans alone or in

combination on intracellular pH in the suspension of SNARF-1-loaded colon tumor cells

HCT-15.

3.4. Ovewiew of experimentat design

In this thesis, three independent experiments were designed to determine the

effects of calcium, SCFA and mammalian Iignans alone and in combination on calcium

transport, intracellular ca2+ and intracellular pH in human colon tumor cells HCT-15. In

study l(Chapter 4), HCT-15 cells were culturd in Transwell insert wells to develop a

monolayer ceIl model for calcium transport. The change in transport velocity and/or

percent of transported calcium were measured in treatrnent solutions containing different

concentrations of calcium, or calcium combined with SCFA andor mammalian tignans.

By adding treatment solutions in either inner (apical membrane side) or outer

(basolateral membrane side) cornpartment separated by ceil monolayer, it was possible

to measure paracellular, transcellular and total calcium transports.

In study 2 (Chapter 5). HCT-15 cells were loaded with ~a"-sensitive probe Fura-

2AM. After isolation, the Fura-2-loaded cells were suspended in treatrnent solutions

containing different concentrations of calcium, SCFA and mamrnalian lignans alone or in

combination. By measurement of the change of fluorescence intensity at S05nm with the

altemating excitation et 340nm and 380nm wavelength, the intracellular Ca2' can be

estimated.

In study 3 (Chapter 6), the pH sensitive probe SNARF-1AM was loaded in HCT-

15 cells. After isolation, cells were suspended in treatment solutions containing different

concentrations of calcium, SCFA and mammalian lignans alone or in combination. The

influence on pHi was measured by detection of the change in the fluorescence

intensities at 57Snm and 635nm with the excitation at 535nrn.

3.5. General mahods

3.5.1. Cells and Tissue culture

HCT-15 cells, which were used in a previous study on cell proliferation and

apoptosis (Skariah and Thompson 1998), were purchased fmm American Type Tissue

Culture (Rockville, MD). This cell Iine was developed from human colon

adenocarcinorna. Cells were cultumd and maintained in RPMIl640 (Life Technology Int,

Burlington, ON) supplemented with 10% fetal bovine serum, in 95% relative humidity

and 5% CO2 at 37°C. Subconfluent cells were used in the experirnents.

3.5.2. Trypsin treatment

Subconfluent cells were treated with 0.25% trypsin-EDTA solution (3-5ml per 75

cm2 flask) for 5 minutes. EDTA chelates ca2+ to dissociate intenellular juncüons. mainly

tight junction, adheren junction and desmosome. Thus, trypsin only needs to digest very

few intercellular junctions to maintain cefl integrity. By rinsing, the residual EDTA are

then washed out to prevent their influence on calcium treatments. The effect of trypsin

was tenninated with 10% FBS containing medium. Then, celfs were agitated by pipetting

to individualize them. Compared to other methods of isolating cells, this method caused

less ceIl damage. In study 1 (Chapter4), isolated cells were seeded into the insert wells

of Transwell for the monolayer culture. In Studies 2 and 3 (Chapters 5 and 6), isolated

cells were directly used for rneasurement of intracellular ca2* and intracellular pH. Two

cuvettes of cells were used for baseline detedion throughout the experimental period.

After the experiment, cells were stained with trypan blue to check cell viability. Trypsin

treatment did not influence cell viability. membrane integrity, intracellular ca2+ and

intracellular pH in the studies.

CHAPTER FOUR

THE EFFECT OF CALCIUM, SHORT CHAIN FArrY ACIDS AND

MAMMALIAN LIGNANS ON CALCIUM TRANSPORT

4. THE EFFECT OF CALCIUM, SHORT CHAIN FAITY AClOS AND

MAMMALIAN LJGNANS ON CALIUM TRANSPORT

4.1. Introduction

When fibre is fennented by the colonic microflora, a large amount of SCFA,

mainly acetate, propionate and butyrate, are produced (Cummings and Englyst 1987).

Many natural, fibre-bound phytochemicals, such as SDG, are released and converted

into rnammalian Iignans, primariiy EL and ED (Setchell and Adlercreutz 1988). Fibre

loses its cation binding capacity during fermentation, and calcium ions (ca2+). which bind

to fibre in the small intestine, are released in the colonic lumen (Trinidad 1986b). CaZ',

SCFA and mammalian lignans individually have biological funcüons in the colonic

epithelium (WhitfÏeld, 1995; Cummings 1995; Setchell and Adlercreutz 1988). However,

there is very limited information on their interactive effects when they accumulate in the

colonic lumen during fermentation.

As well as being a source of energy for the colonic epithelial cells, SCFA

prornote water and electtolyte absorption in the colon (Roediger 1982). Calcium

absorption is significantly increased in the presence of SCFA (Trinidad et al, 1996a), and

based on similar resuits in rats, Lutz and Scharrer (1991) assumed that ca2* entry is

increased by H*-ca2' exchange in the edonic epithelial cells. The formation of a Ca-

SCFA cornplex, which increases transmembrane permeability, is another hypothetical

mechanism (Marshall 1976). However, the cellular and molecular details behind these

effects are mainly unknown.

There may be a possible relationship between calcium absorption in the colon

and the protedive effect of calcium against colon cancer. Cancer incidence rate is

dramatically higher in the colon than in the small intestine, and the reason rnay be

related to the fact that calcium absorption in the colon is much less than in the small

intestine (Sedion 2.3.1). The protective role of calcium absorption against colon cancer

was demonstrated by studies which showed that the absence of 1, SIS(0H)rVitamin D3

annihilated the protective effect of calcium against colonic carcinogenesis in animal

experiments (Pence and Budding 1988; Lior et al 1991). Patients with a long-terni

administration of calcium antagonist ( ~ a " channel blocker) increase the risk of colon

cancer (Hardell et al 1996; Olson et al 1997), suggesting the risk of inhibition of calcium

absorption in the colon. However, the direct association between calcium absorption and

colon cancer is hardly known.

Calcium absorption in the gastrointestinal tract occurs via two transport

processes: transcellular and paracellular (Bronner and Pansu 1999). Paracellular

transport is concentration dependent and responsible for the increasing calcium

absorption when intake is increased (Allen and Wood, 1994). On the other hand,

transcellullar transport is satunble and energy wnsuming. It indudes ca2' entry at the

brush border membrane, intracellular Ca2' movement and extrusion at the basolateral

membrane (Bronner, 1987). lntracellular movement of ca2' is the rate-limiting step in

transcellular transport (Bronner, 1990). Factors affecting transcellular calcium transport

may change intracellular ~ a " level.

Previously. it has been shown that ca2', SCFA or marnmalian lignens can inhibit

cell proliferation and induce the apoptosis of the human colon tumor cell HCT-15

(Skariah and Thompson 1998). Synergistic effect on proliferation inhibition and

apoptosis induction was observed when these chemicals were cornbined. My hypothesis

is that the effect of these chemicals on calcium transport and consequently intracellular

CaZ' is responsible for these effects. Thus, this study was designed to investigate the

influence of calcium concentration, SCFA and mammalian lignans on calcium transport

(paracellular, transcellular and total) in the human colon tumor cell HCT-15.


4.2.1. Materials

All chemicals were purchased from Sigma Canada (Mississauga, ON), unless

otherwise indicated.

Cells were cultured in Transwell (Corning Inc., NY) insert wells for two weeks to

fom a monolayer. After n'nsing with phosphate buffered saline (PBS), transfer buffers

containing different treatment concentrations of calcium, SCFA and/or rnammalian

lignans (ED or EL) (Table 4.1) were added to one of the two compartments separated by

the cell monolayer. After 30 minutes incubation, sarnples were collected from the other

cornpartment and calcium contents were measured using ca2' sensitive fluorescent

indicator Fura-2. The velocities and percent of ttanscellular, paracellular and total

calcium transport were calculated. The details follow.

4.2.3. Celf Culture

The human colon tumor cell line HCT-15 was purchased from Arnerican Type

Tissue Collection (Rockville, MD). The cells were grown and maintained at 37°C 95%

humidity and 5% COz, in the medium RPM11640 (Life Technologies Int., Burlington, ON)

which was supplemented with 10% FBS. Exponentially gWng cells were harvested

after 0.25% trypsin-EDTA solution treatment for 5 minutes. After neutralization with FBS-

wntaining medium, cells were agitated and individualized by repeating the pipetting 5-10

times. This method for individualizing cells caused less influence in cell viability and the

integrity of cell membrane. The individualized cells were seeded in Transwell insert wells

(descnbed later) at a density of 3 x 1 0 ~ . The cells in Transwells were cultured for two

weeks, the fint week (before confluence) in 10% RPM11640 with medium change every

other day, the second week (after confluence) in 1% RPM11640 witti medium change

every day (Giuliano and Wood 1991).

Table 4.1. Treatrnents used in calcium transport experiment

Experirnent

1. Calcium concentration

2. Short chain fatty acids

3. Mammalian lignans

-- - -

4. Mammalian lignans and SCFA

0,2 , 5, 10, 15 and 20

Ac: 10 or 20

Pr 10 or 20

Bu: 5, t O or 20

SCFA: 10 or 20

SCFA: 10

~- ~ -

Mammalian lignans (M)

ED: 100

EL: 100

ED+EL: 100+100

ED: 100

EL: 100

ED+EL: 100+1W

Ac= acetate; Pi- propionate; Bu= butyrate; SCFA= mixture of acetate, propionate and

butyrate at 3:2:1 ratio; ED= enterodiol; EL= enterolactone.

4.2.4. Monolaver cell Membrane

The in vitro calcium transport model in cell monolayer was based on the method

of Giuliano et al (1991). The apparatus, 12-well Costar Transwell plate 3462 (Corning

Costar Co., Cambridge MA), was composed of cluster wells and insert wells. Two

compartments were separated by a porous membrane (pore size 3.OpM and growth

area 1 .0cm2) (Figure 4.1). The membrane was coated with a thin layer of collagen IV

( l ~ ~ g l c m ~ ) , a major protein component of basement membrane in Me gastrointestinal

epithelia- Cells were seeded on the top of collagen layer and cultured for two weeks.

Colon tumor cells underwent a certain degree of differentiation to fonn a cell monolayer.

Differentiated cells grew side by side with the brush border on the top and the basal

membrane contacting the surface of collagen layer (Augenlidit et al 1995; Hidalgo et al,

1989). Thus, transcellular calcium transport can only go in one direction from brush

border membrane to basal membrane, white paracellular transport can go in either

direction depending on calcium concentration (Bronner 1987).

4.2.5. Calcium Transoort

Hank's balanced sodium solution, containing 140mM NaCI, 5.8mM KCI, 0.34mM

Na2HP04, 0.44mM KH2P04, 0.8mM MgSO,, 20mM HEPE, 25mM Glucose and 4mM

glutamine, pH 7.4, was used as transport buffer (Giuliano et al, 1991). Various

concentrations of CaClz andlor sodium SCFA (Table 3.1) isotonically substituted NaCI in

the transport buffer. The wells of two-week growing cell monolayer were rinsed twice

with 37°C preheated PBS, containing 138rnM NaCI, 8mM Na2HP04, 2.7mM KCI and

1 SmM KH2P04, and then treated as follows:

ca2+ solution

lnsert well

--__-_--------------------

Collagen IV 1 opg/m2

1111111111111111111llll \

T Cluster well

Membrane with 3vM pore

Sample 6 f

- Cell monolayer

-) Sample A

solution

Figure 4.1. A mode1 of monolayer cell membrane developed in Costar Transwell. A.

outward transport (paracellular transport + transcellular transport) and B. inward

transport (paracellular transport).

i) Outward transmrt (total transport, including transcellular and paracellular

transport): Calcium solution was added to the insert well (upper cornpartment), and the

calcium free buffer to the cluster well (bottom cornpartment). After 30 minutes, sample

was collected from the cluster well and analyzed for calcium concentration. The calcium

in the cluster well represented the total calcium transported by both paracellular and

transcellular pathways.

ii) lnward transport (only paracellular transport): Calcium solution was added to

the cluster well, and the calcium free buffer to the insert well. After 30 minutes, sample

was collected from the insert well and analyzed for calcium concentration. The calcium

in the insert well represented only the paracellular transport.

iii) Transcellular transport: This is detemined as outward (total) transport minus

inward (paracellular) transport in this study (Bronner 1987).

4.2.6. Calcium Determination

The calcium sensitive dye Fura-2 (Calbiochern, San Diego, CA) was used to

detemine calcium concentration (Grynkiewiu et al 1985) using the Spex 2

spectrofluorometer (Spex Industrial, Edison, NJ). In detecting solutions, 50pJ 5fl Fura-2

(final concentration OS*) and 50pl 5mM EGTA (final concentration 0.5mM) were

added to different volumes of sample. Transport buffer was then added to reach a final

volume of 500pl. After agitation, two dissociation balances can be reached as follows:

While free Fura-2 gives out an emission pike at 50Snrn under the excitation of 380nrn.

ca2'-bound Fu-2 gives out the same emission at the excitation of 340nm. The

fluorescent intensities are in proportion to the different States of Fura-2. The ratios of the

fluorescence intensities at 340nm and 380nm were used to calculate ca2' concentration

(Grynkiewict 1985). Wthin a range of concentration. free Ca2' is in proportion to total

calcium content. The fluorescence ratio and calcium concentration calibration is

presented in Figure 3.2. Calcium concerrtrations were calculated according to the

equation of calibration curve. The velocity of calcium transport was calculated as the

nanomoles of calcium transported in 30 minutes through 1 un2 cell membrane.

4.2.7. Statistics

Data are presented as means I standard error of means of at least two

experiments, each done in duplicate. One-way ANOVA was used for multiple

cornparison, and Tukey test or Student-Newman-Keuls test were used for pairwise

cornparison. p<O.OS was regarded as significant.

4.3. Resuit

4.3.i. Ex~eriment 1 : Effect of calcium concentration

The velocity of total calcium transport (outward transport) significantly increased

with increasing calcium concentration. in a polynomial pattern (Y- = -0.33~~ + 7 . 0 9 ~ ~

+ 1 V . 3 1 X - 48.1 1, RZ=l.OO, p<O.Ol) (Figure 4.3 and Appendix 1). The percent of

calcium transport was significantly increased when calcium concentration increased from

2mM to SmM, flattened from 5mM to lSmM, and deueased from 15mM to 2OmM

(peO.05).

The velocity of paracellular transport significantly increased with increasing

calcium c4ncentmtion. and the relationship was linear (Y- =62.92X + 46.14; ~~=0 .99 ,

p<O.OOl) (Figure 4.4a and Appendix 1). The percent of calcium transport was about 15%

and did not change with calcium concentration.

Figure 4.2. Calcium calibration with fluorescence ratio. R is the ratio of the fluorescent

intensities at the 340nm and 380nm excitation. Rmin is the ratio with 20mM EGTA

solution (ca2' free). Rmax is the ratio with 2OmM calcium solution (ca2' saturated).

O 5 40 15 20 25

Calcium concentration (mM)

Figure 4.3. Velocity and percent of total calcium transport at different calcium

concentrations. Values are means I standard error of means. Means with different

letters in the same line are significantly different (peO.05).

The velocity of transcellular transport vs. calcium concentration showed an S-

shaped polynomial cuwe (Y- = -0.38~~ + 9.28x2 +27.07X -44.13, ~~=0.99. pe0.01)

(Figure 4.4b and Appendixl). The velocities significantly increased with increasing

calcium concentration from 2mM to 15mM (p<0.05); there was no difference between

15mM and 20mM calcium. The percent of calcium transport in 5, 10, 15 and 20mM

calcium solutions did not differ from each other, but all were higher compared to that in

2mM calcium solution (p40.05).

4.3.2. Ex~erirnent 2: Effects of acetate. ~ro~ionate. butyrate and theit mixture

Different concentrations of acetate (10 or 2OmM), propionate (10 or 20mM),

butyrate (5, 10 or 2OmM) and their mixture (tO or 20mM) had no effect on paracellular

transport (Figure 4.5 and Appendix 2). Transcellular calcium transport was significantly

increased by 20mM acetate (p<0.05), 10 or 20mM propionate (p<O.OS), with 20mM

propionate more significant than 1 OmM propionate (p<0.05), mi le 1 OmM acetate and 5,

10 or 20mM butyrate had no effects. The 1OmM and 20mM mixture of SCFA (acetate,

propionate and butyrate in ratio of 3 2 1 ) significantiy increased transcellular calcium

transport (peO.05). The total calcium transport significantly increased only when 1OmM

calcium was combined with 20mM propionate (peO.05).

4.3.3. Ex~erirnent 3: Effect of mammalian limans

Either individually or in combination, EL (1OOpM) and ED (100fl) did not affect

the paracellular and total calcium transport (Figure 4.6; Appendix 3). The combination of

1 OO@ ED and 100pM EL significantly increased transcellular transport (p<O.OS), but

individually, they have no effect.

Transœlluiar transport

Figure 4.4. Velocity and percent of paracellular and transcellular transport at different

calcium concentrations. Values are means + standard error of means. Means with

different letters in the same line are significantly different (pe0.05).

Total trrns port Parace llular transport Trans ce Ilular transport

Figure 4.6. Effect of mammalian lignans on calcium transport. Values are means I

standard error of means. Means with different letten in the same group are significantiy

different (pc0.05). ED= enterodiol; EL= enterolactone.

4.3.4. Ex~erirnent 4: Effed of mammalian limans and SCFA

The combination of mammalian lignans (100fl ED+fOOpM EL) and 1OmM

SCFA significantly inhibited transcellular transport and total calcium transport (pe0.05)

(Figure 4.7; Appendix 3), although the SCFA mixture alone and the combination of ED

and EL alone enhanced the transcellular and total calcium transport (peO.05). There

were no effects on paracellular transport.

4.4. Discussion

The results of this study are summarized in Table 4.2.

The first experiment showed that increasing calcium concentration increased the

velocities of paracellular transport, but not the percent of calcium transport (Figure 4.4).

Paracellular calcium transport is an unsaturated and passive diffusion, and is regarded

as the major mechanism involved in increasing calcium absorption with increasing

calcium intake (Allen and Woods 1994). Although extracellular ca2+ regulates

paracellular bamers of junctional proteins, tight junction, adherens junction and

desmosome (Collares-Buzato et al 1994; Denker and Nigam 1998), the percent of the

paracellualr transport was roughly unchanged.

The transcellular calcium transport showed a sigmoid shape curve with

increasing calcium concentration in this study (Figure 4.4). Both velocity and percent of

calcium transpoR were negative until calcium concentration reached 5mM. The velocity

lineady increased when calcium concentration increased from 5mM to ISmM, and flatted

when the concentration increased from 1SmM to 20mM. The percent of transported

calcium was barely unchanged when calcium concentration increased from 5mM to

20mM.

Total transport Parace Ilular Transcellular transport transport

Figure 4.7. Effect of mammalian lignans and short chah fatty aQds on calcium

transport. Values are means I standard error of means. Means with different letters in

the same group are significantiy different (pe0.05). ED=enterodiol; EL=enterolactone,

SCFA= a mixture of short chain fatty acids.

Table 4.2. Summary of the effects of calcium concentration, short chain fatty acids and

mammalian lignans on calcium transport

Calcium

(2-20 mM)

Acetate*

Propionate*

(1 0 or 20 mM)

Butyrate*

(5, 10 or 20 mM)

SCFA*

(10 or 20 mM)

ED*

(1 O0 jlM)

EL*

Paracellular 1 Transcellular

with 10 mM calcium; SCFA= the mixture of short chain fatty acids; ED= enterodiol;

EL= enterolactone. NS- no significant effed;m significant increase; =ignificant

decrease.

Transcellular transport is usually regarded as the mechanism for uptake of

calcium from a low calcium lumen (Bronner 1990; Allen and Wood 1994), but there was

not transcellular transport in low calcium concentration (2mM) in this study. As reviewed

in Sedion 2.5.2. the pmcess of transcellular cslcium transport indudes ca2' entry,

intracellular ca2' movement and ca2' extrusion. Ca2+ entry is mainly through three

ways, transrnembrane diffusion, agonist-dependent channel and voltage-dependent

channel. lncreased extemal calcium concentration may increase transrnembrane

diffusion. Although transmembrane diffusion only accounts for a very small amount of

calcium entry, it can change transmembrane voltage to open voltage-dependent channel

which, in tum. increases Ca2+ entry. The other possible mechanism is that extemal ca2'

activates of %a2' sensof, which triggen ca2' release from endogenous reservoir and

ca2' channel opening (Whitfield 1995; Gama et al 1997). Whitfield (1 995) suggested that

transforrned coionic cells decreased sensitivity of their %a2* sensor". Intracellular ca2'

level is lower in transformed cells than in normal tissue regardless of the extemal

calcium concentration (Edelstein et al 1991 ). suggesting decreasing sensitivity of 'ca2'

sensor". The negative transcellular calcium transport in low calcium concentration (2mM)

may also be caused by the decreased sensitivity of HCT-15 cells.

Intracellular ca2' movement is the rate-limiting step of transcellular calcium

transport. It depends on the availability of calcium binding protein 9KD calbindin

(Bronner, 1990). The ca2' that enters the cells binds with calbindin and is carried to

basolateml membrane to be pumped out of cell. lncreased extemal calcium

concentration may increase ca2' entry through al1 the above presuibed mechanism.

Before calbindins are saturated, inaeased ca2' entry may continually increase the

amount of Ca2' that moves to basolatenl membrane and is pumped out of the ceIl. That

is why transcellular calcium transport lineady increased when calcium concentration

increased from SmM to 15mM. When ca2' entry increases to such a Ievel that calbindins

are saturated, the amount of ca2' that moves to the basolateral membrane and is

pumped out is maximired. Further inuease in ca2' entry does not increase the

transcellular calcium transport. Thus, the velocity of transcellular calcium transport was

fi attened when calcium concentration increased from 15mM to 20mM.

The second expenment showed that acetate and propionate, but not butyrate,

enhanced transcellular calcium transport. One possible mechanism is that the highty

Iipid soluble protonated SCFA easily diffuse through cytoplasm membrane, and

irnmediately dissociate into H' and SCFK (Bugaut 1987). Some of the protons recycle

across the luminal membrane by the Na+-H' antiporter, allowing a high rate transport of

protonized SCFA to continue (Schultz 1981). Similady, ca2+-~' exchange has been

shown to occur in colonic epithelia (Lu& and Scharrer 1991). SCFA absorption results in

H' accumulation subsequently adivating the Ca2*-H' exchange and enhancing calcium

transport (Lu& and Scharter 1991). The other potential mechanism involves the

formation of a Ca-SCFA complex with diminished elec(rical charge of ca2+. The complex

then could easily pass the cell membrane banier (Marshall 1976).

Acetate, propionate and butyrate have different physico-chernical characteristics

With increasing carbon chain, SCFA increase lipid solubility and decrease acidity.

Acetate dissociates easily into anion, which then may bind ca2' and fom a stable Ca-Ac

complex (Nancollas 1956; Trinidad et al 1 996). Therefore, the increased transceliular

calcium transport by acetate may occur partly by the diffusion of the less charged Ca-Ac

corn plex.

Propionate may also fom a complex with ca2', but its complex is kss stable

than Ca-Ac (Cannan and Kibrich 1938). On the other hand, propionate is easy to

protonate to pmpionic acid with high lipid salubility allowing it to diffuse through cell

membrane faster than acetate (Dawson et al 1964; Saunders 1991). H+ accumulation

from propionic acid dissociation rnay enhance Ca2'-~* exchange. Both two mechanisms

rnay take place; thus propionate showed the strongest effect in enhancing transceliular

calcium transport.

Butyrate has the longest carbon chain of the three SCFA, and thus has the

highest lipid solubility. However, butyrate has the weakest acidity and tends to be

protonated to butyric acid. The Ca-Bu cornplex rnay not form. After butyric acid diffuses

into cells, it rnay remain in the protonized state and gets metabolized. Butyrate is one of

the energy sources for the colonic epithelial cells (Roeduger 1982). Therefore, both of

the above mentioned mechanisms rnay not happen in butyrate, and butyrate has no

effect on transcellular calcium transport.

In al1 experiments, the HBSS solution contains 4mM glutamine and 25mM

glucose, which are more than necessary energy source for the tumor cells. Therefore,

the substitution of different SCFA or other compounds for NaCl will not affect the energy

metabolism of the tumor cells.

The third experiment showed that the combination of ED and EL, but not ED or

EL alone, enhanced transcellular calcium transport. Their effect rnay be similar to

compounds with similar molecular structure such as estrogen and 1, 25(OH)2Vit-D3.

These compounds bind an unknown membrane receptor to activate phospholipase C,

which produces inositol triphosphate (NP& IPa stimulates ca2' release from endoplasmic

reticulurn, which further intensifies ca2' release from rnitochondrion and endoplasmic

reticulum (Peter et a1 1998). The depletion of intemal ca2' reservoir and inueased

intracellular ca2+ can stimulate ca2' channel to open (Putney 1997). Therefore,

transcellular transport was increased.

The reason that the combination of ED and EL enhanced ttanscellular transport

but not the individual compound rnay be related to the dose. i.e. EL or ED alone was

used at 100pM, mi le the combination was at 2 W N . However, the reason wtiy

transcellular calcium transport was inhibited by the combination of mammalian Iignans

and SCFA is still unclear, and the following studies on intracellular ca2+ and intracellular

pH rnay provide some explanation.

In conclusion, high calcium concentration and its combination with SCFA and

marnmalian iignans enhanced calcium transport in human colon tumor cells, mainly

through a transcellular transport mechanism. Since intracellular ca2' movement is a

rate-limiting step whid, restricts ca2+ exit, intracellular Ca2+ may be increased when ca2+

entry is larger than ca2' exit.

CHAPTER FIVE

THE EFFECT OF CALCIUM, SHORT CHAIN FATTY AClDS AND

MAMMALIAN LIONANS ON INTRACELLULAR CALCIUM

5. THE EFFECT OF CALCIUM, SHORT CHAJN FATTY AClDS AND

MAMMALIAN LIGNANS ON INTRACELLULAR CALCIUM

S. 1. Introduction

lntracellular ca2' is one of the principal second messangers controlling a number

of cellular processes (Henning et al, 1980). As reviewed in Section 2.7, increased

intracellular ca2+ level ([ca27i) play a crucial role in the three phases of mitochondrion-

mediated apoptosis. lnueased [ca27i can directly stimulate mitochondrion or enhance

other stimuli in the pre-mitochondrion phase, and trigger mitochondrion inner membrane

pemeability transition in mitochondrion phase (Susin et al, 1998; lchas and Mazat

1998). In the post-mitochondrion phase, inueased [Ca21i adivates endonudease to

digest DNA, which is a decisive event of apoptosis (Nicotera et al, 1994).

Calcium plays a complicated role in cell proliferation, differentiation and death in

colonic cells (Whitfield, 1995). Low [ca2']i in colon tumor cells was suggested to be a

major reason that colon tumor cells can escape apoptosis and differentiation (Edelstein

et al, 1991). lnueasing [Ca27i, through different mechanisms to inuease Ca2' entry, is

associated with apoptosis induction by different agents (Fawthrop et al 1991). In colon

tumor cells, 1,25(OH)r&-induced apoptosis has been associated as well with

increasing [ca27i (Bissonnette et al 1995). ln a previous study (Skaflah and Thompson

1998), high calcium concentration, short chain fatty acids (SCFA) and mammalian

lignans, individually or in combination, induced apoptosis and inhibited cell proliferation

in human colon tumor cell HCT-15. Transcellular transport in HCT-15 cells, shown in our

first study (Chapter 4), was increased by calcium concentration, SCFA and mammalian

lignans. The objective of this study was to detennine whether [ca27i was inueased by

this increased transcellular transport.

5.2. Methods and Materials

5.2.1. Matetials

A11 reagents were purchased frorn Sigma Canada (Mississauga, ON), unless


5.2.2. Ex~erimental desian

Exponentially growing hurnan colon tumor cells HCT-15 were used in this study.

Cells were incubated with lipid soluble Fura-2AM, which then diffused through cell

membrane and deesterified into water soluble Fura-2 inside the cells. Fura-2, trapped in

cytoplasm because of its water solubility, fundons as a free calcium ion indicator to

reflect [ca27i change. The Fum-2-loaded cells were isolated with trypsin, and subjected

to different treatments in several experiments (Table 4.1). The fluorescence intensities of

Ca2+-bound Fura-2 (excited at 340nm) and free Fura-2 (excited at 380nM) were

measured in a spectrofluorometer. The ratio of 340nm and 380nm intensities was used

to calculate [ca2c]i.

5.2.3.Tissue culture

Human colon adenocarcinorna cells HCT-15 (purchased from American Type

Culture Collection, Rockville, MD) were cultured in medium RPMl 1640 (supplemented

with 10% fetal bovine senim (FBS), 100UI penicillin, 100ug streptomycin and 5pg

gentamicin) at 37%. in 95% humidity and 5% CO2. After a 10-day culture, sub-confluent

cells were used in this study.

5.2.4. Iso-osmotic detectin~ solution

Hank's balanced sodium solution (HBSS) was used as control solution. CaClz

and sodium SCFA isotonically substituted NaCl in HBSS in order to prepare different

treatment solutions (Table 4.1). Lignans (100rnM) were directly added into treatment

solutions to reach a required final concentration. All solutions were adjusted to pH 7.4.

Table 5.1. Treatmenb used in intracellular cs2' experiments'

1. Calcium concentrations

2. Short chain fatty acids

3. Lignans

4. EL and calcium O (with or without 1mM EGTA), 2, 5 or 10

Ac: 10, 20 or 30

Pr: 10 or 20

Bu: 5, 10 or 20

SCFA: 10, 20 or 30

Lignans

(PM)

ED: 200

EL: 200

ED+EL: 100+100

SECO: 200

EL: 200

' Ac= acetate, Pr= propionate; Bu= butyrate; SCFA= mixture of short chain fatty acids at

3 Ac: 2 Pr: 1 Bu; ED= enterodiol; EL= enterolactone; SECO= secoisolariciresinoI;

EGTA= ethylene glycol-bis(P-aminoethyi etherEN,N, N', N',- tetraacetic acid.

5.2.5. Dve loadinp

The cells were incubated in HBSS containing 2pM FU-2AM (Calbiochern, San

Diego, CA) at 37% for 40 minutes. Then, cells were treated with 0.25% Trypsin-EDTA

solution (3-Sml par 75 cm2 Rask) at 3T0C for five minutes; FBS-containing medium was

used to terminate the trypsin effect. After agitation by repeated pipetting to individualize

cells, cells were rinsed with phosphate balance solution (PBS) and centn'fuged (1 000rpm

for three minutes, Accuspin FR^, Beckman, Fullerton, California) three times to get rid

off residual Fura-2AM. Cells then were suspended in HBSS at a density of

approximately 7 . 5 ~ l d cells/ml. Cell suspensions were transferred into 1 cm fluorescent

cuvettes (lxlxScrn, polyester, allow 240-7Wnm wavelength, VWR Canada,

Mississauga, ON), 2ml per cuvette. Cells were stored in a dak box at room temperature

for at least 30 minutes. Although loaded dye is stable for more than 5 houn, the

following treatrnenl and fluorescence detenination were done within 3 hours.


The method of Edelstein et al (1991) was used to detect [ca27i and measure

fluorescent intensities. After storage for more than 30 minutes, cells settled at the

cuvette bottom. Before fluorescence detemination, supernatant was removed and the

cell pellet was re-suspended in 3ml treatrnent solutions (pre-heated to 37°C) to a final

cell density of 5x10' cellslml. The Spex 2X spectnim fluorescence meter (Spex

industrial Inc., Edison, New Jersey) was used to measure fluorescence intensities.

Sarnples were altemately excited at 340nm and 380nm, and their fluorescent intensities

(emission at 505nm) were rneasured at 2, 5, 10, 20 and 30 minutes while the micro-

stirring system maintained the cells in suspension.

In experiment 4, cells were suspended in different solutions, 0, 2, 5 or 1OmM

CaClz or 1mM EGTA. In solutions with no added calcium, there still contains 2-7pM

~ a " , as an impurity from different chernicals used. Supplementation with 1mM EGTA

Gan chelate al1 these cab. Fluorescence intensities in diffemt detecting solutions were

detected for 1 O minutes (three times at 2, 5 and 10 minutes) as a baseline. After that, 6pl

1OOmM EL was added into the cell suspensions (final concentration 200pM). The

intensities were detected wntinuously for another 20 minutes (four times at 2, 5, 10 and

20 minutes).

5.2.7. iCa27i calculation

[ca2Yi was calwlated from the ratio of intensities at 340nm1380nm according to

Grynkiewicz equation (Grynkiewia et al, 1985) as follows:

[CaH]i= K& (R-Rmin)/(Rmax- RJ

Where, Kd is the dissociation constant, which is 205 for Fura-2 (Grynkiewicz et al, 1985);

j3 is the ratio of 380nm fluorescent intensities at calcium free state and at calcium

saturated state (Bissonnette, 1994);

R is the ratio of 340nml380nm intensities in different treatment solutions;

Rmin is the minimal ratio of intensities at 340nrn/380nm;

Rmax is the maximal ratio of intensities at 340nrn/380nm.

For detemination of p, Rmin and Rmax, cells were treated with SOpM digitonin

(Calbiochem, San Diego, CA) for five minutes to make cell membrane penneable to

ca2'. Thus, intracellular Ca2+ can be equilibrated to extracellular Ca2' after cells were

incubated for a certain period of time. Rmin, Me ratio at ca2+ free state, was the ratio of

intensities at 340nmî380nm in the presence of 20mM EGTA. Rmax, Vie ratio at ca2'

saturated state, was the ratio in the presence of 10mM CaC12 (Edelstein et al, 1991).

5.2.8. Ceff viability

At the end of each experiment, cells were stained with 0.5% trypan blue for 5

minutes, and dead cells counted under inverted microscope. If more than 10% celis

were trypan blue stained, the data were rejected.

5.2.9. Statistics

The presented values are means I standard error from at least two experiments,

each done in duplicate. One-way ANOVA analysis was used to detemine significance. If

there is a significant difference (p<0.05), Tukey test was used for pairwise comparison.

5.3. Result

5.3.1. Experiment 1 : Effect of calcium concentration

There were no significant differences between different time points within the

same calcium concentration (Appendk 4). But at the same time point, intracellular caZ4

level was significantly increased with increasing calcium concentration (pcO.001)

(Figure 5.1 and Appendix 4). [ca27i at 2, 5.10, 15 and 20mM extemal ca2+ were

significantly (pe0.05) higher than that at OmM Ca". [ca2']i at 10, 15 and 20mM caZ+

were significantly higher that that at 2mM ~ a " , and [ca27i at 15 and 20mM were higher

than that at 5mM Ca2' (pc0.05). There were no significant differences between 2 and

SmM, between 5 and IOmM, and among 10,15 and 20mM.

5.3.2. Emeriment 2: Effects of SCFA

The addition to calcium of different concentrations of acetate, propionate,

butyrate or their mixture did not change [Ca27i (Figure 5.2 and Appendix 5).

I 1

5 10 15

Extncellular concentration (mM)

Figure 5.1. Effect of calcium concentration on intracellular CI''. Values are means I

standard emr of means at 20minutes. Means with different letters are significantly

different (pe0.05)

Acetate Propionate Butyrate SCFA

O 0 rn 1 OCa abne i 5SCFA+lOCa r 10SCFA +lOCa 20SCFA+IOCa m 30SCFA+lOCa

Figure 5.2. Effect of acetate. propionate, butyrate and their mixture on [ca2Yi. The

concentration of SCFA and calcium is in mM. Values are means and standard error of

means at 20 minutes.

5.3.3. Exmiment 3: Effects of mammalian limans

The supplementation of calcium (1OmM) with ED (2WpM) did not change the

[ca2']i (Figure 5.3). but EL (200pM). SEC0 (2WpM) and the mixture of ED (100pM) and

EL(100fl) significantly (p4.05) increased the [ca27i (Figure 5.3 and Appendix 6 ).

The effect of EL was the highest.

5.3.4. Ex~eriment 4: interactive effect of enterolactone and calcium

There were no significant changes during the baseline detemination (Figure

5.4). In O rnM calcium solution, a significant inaease in the [ca2?i (p~0.05) was recorded

immediately after adding EL reaching a final concentration of 200pM. EL showed similar

effect even in the presence of IrnM EGTA solution so that al1 extemal ca2* ions are

chelated. The effect of EL was not significant at 2 and SmM calcium solutions, although

an increasing trend was observed, but #vas significant at 1OmM calcium solution (Figure

5.4 and Appendix 7).

O control Ca control ED SEC0 EL ED+EL

Figure 5.3. Effect of lignans on intracellular ~ a " . The values are means I standard e m r

of means at 20 minutes. Means with different letten are significantly different (p<0.05).

(O control represents OmM Ca solution; Ca control=lOmM calcium solution, €O= 200w

ED + 10mM Ca; SECO= 2WpM SECO +1OmM Ca; EL= 200pNl EL+ 10mM Ca, ED+EL=

100pM ED + 100pM EL +lOmM Ca.)

1 add enterolactone

O 10 20 30 Tirne (min)

Figure 5.4. The affect of enterolactone on [ca27l with and without calcium at different

tirnepoints. Values are means and the standard error of means. Means with different

letters are significantiy different (pe0.05)

5.4. Discussion

This study showed that increasing calcium concentration can significantly

increase [caz7i (Figure 5.1). Two different mechanisms may be involved in this effect.

One mechanism is that Ca2+ entry through transmembrane diffusion is increased with

increasing calcium concentration (Bronner 1987). Ca2' entry ocairs through three

pathways, transmembrane diffusion, voltagedependent channel and agonist-dependent

channel (Bronner 1990). Although transmembrane diffusion only accounts for very low

amount of caZ' entry, it can overcome transmembrane voltage to open voltage-

dependent ca2' channel and allow large amount of ca2' entry (Bronner 1987). The

other mechanism is activation of the 'ca2+ sensoi' (Whitfield 1995) (Section 2.3.1).

Gama et al (1997) nported that a cell membrane protein functions as a Ta2' sensof.

which is activated by extemal calcium concentration and cause intracellular ca2'

increase.

lncreased [ca27i is a uitical step for apoptosis and cell differentiation. [ca27i in

human colon tumor cells is significantly lower than in normal cells, which is suggested to

be a possible reason M y colon tumor cells lose capability to differentiation and

apoptosis (Edelstein et al 1991 ; Bemdge 1987). In this study, the increase in extemal

calcium concentration resulted in an increase in [Ca2*]i in colon tumor cells. Thus,

apoptosis and differentiation in tumor cells rnay be induced by increasing calcium

wncentration.

[ca2*]i was not changed in the presence of acetate, propionate. butyrate and

their mixture in this study, although in our first study (Chapter 4), the presence of

acetate, propionate and the mixture of acetate and propionate significantly increased

transcellular transport. There are two rnechanisms whereby SCFA may enhance ca2'

entry; CaZ'-H' exchange and formation of C~~'-SCFA complex (Lue and Scharrer 1991 ;

Marshall 1976). The results suggest that ca2' enûy caused by these two mechanisms is

not larger than the capacity of tumor cells to extrude ca2'. ca2' that enters cells

immediately combines with calcium binding proteins, mainly 9 KD calbindin. lntracellular

ca2+ is unchanged unless ca2* binding proteins are saturated (Bronner 1990).

In this study. [ca2)i was increased in the presence of 2WpM SECO. 2 0 0 N EL

and 1OOpM ED + 1 WpM EL, but not 2WpM ED, suggesting that only SEC0 and EL had

an effect on inueasing [ca2?i. As discussed in Chapter 4, the possible mechanism

involved in this effect could be activation of an unknown membrane receptor by lignans,

which then triggers ca2+ release fmrn intracellular ca2+ reservoir and open the Ca2'

channels to allow extemal Ca2' entry into cells. The reason that different lignans showed

different effects in intracellular ca2* is discussed in Section 7.3.

In experiment 4. [~a) i was increased with the addition of 200pM EL, aven after

extemal ca2' was chelated by 1 mM EGTA. A greatet effed was observed when 200w

EL was added 1OmM calcium solution. However, the effect of EL addition was not

significant in 2mM and 5mM calcium solutions. The results strongly suggest that EL

functions in a pathway different from that of calcium concentration. Direct stimulation of

Ca2' release from intracellular ca2' reservoir by EL may be possible.

As previously mentioned, intracellular Ca2* is a very important second

rnessenger, mediating many biological processes. It also is a causal factor and a result

of mitochondrion-mediated apoptosis. This study showed that [ca27i was increased by

calcium concentration, and the lignans EL and SECO. Therefore, the result may at least

partially explain the previously observed effects of calcium and EL on inhibition of cell

proliferation and induction of apoptosis (Skariah and Thompson 1998). However, the

mechanism behind the effect of SCFA on proliferation and apoptosis needs further

investigation.

CHAPTER SIX

THE EFFECT OF CALCIUM, SHORT CHAIN FAlTY AClDS AND

MAMMALIAN LlGNANS ON INTRACELLULAR pH

6. THE EFFECT OF SHORT CHAIN FArrV ACIDS, CALCIUM AND

MAMMALIAN LIGNANS ON INTRACELLULAR pH

6.1. Introduction

Maintenance of the homeostasis of intracellular pH (pHi) is vital ?O most cells

because most cellular functions are pH sensitive (Nuccitelli and Helple 1982). In a

hypoxic environment, tumor cells mainly depend on anaerobic glycolysis for energy,

which produce 5-7 times more protons per ATP produced than aerobic glycolysis (Owen

1996). However, turnor cells still can rnaintain their cytoplasm alkaline by expelling H',

through changes in n'-Na' antiporter and HCO+Cr exchange (Kraus and Wolf 1996.

Bischof et al, 1996). Acid extrusion, combined with microcirculatory inadequacy in the

surrounding tissue, causes extracellular microenvironment audification, which promotes

tumor cell invasion, hampers surrounding tissue growth and inhibits immune system

function (Kraus & Wolf, 1996). Alkaline cytoplasm and acidic microenvironment are

closely related to multi-drug resistance of tumor cells because many anti-cancer agents

are weakly alkaline (Litman et al, 1998, Wdkin and Roepe 1997). Lower permeability of

the ionized state of these anti-cancer agents in acidic microenvironment make them

difficult to enter cells while high pemeability of their unionized state in alkaline

cytoplasm make them easy to be expelled (Gerweck and Seetharaman, 1996).

Reduction of intracellular pH (pHi) has been suggested as a possible mechanism

for killing cells in the acidic regions of solid tumor (Newell and Tannock t 989). Reduced

pHi can be achieved by using proton ionosphere carbonyl cyanide 3-

cholophenylhydrazone and nigenun (Newell et al 1992) and Na'-H* antiporter inhibitor

amiloride and its derivatives (Maidom et al 1993). The stress-activated protein kinase

pathway has been suggested as one mechanism for acidification-induced cell death

(Zanke et al 1998). Cytoplasmic addification was found to be a critical step of apoptosis

(Barry and Eastman 1993), and equilibrating pHi through adjustrnent of extracellular pH

@He) and ionosphere can directly induce apoptosis (Bischof et al, 1996). The study of

Litman et al (1998) suggested that decreasing pHi is a possible way to overcome

multidrug resistance in tumor cells. However, the methods or agents applied to decrease

pHi are still tentative.

Of interest to us is the potential effect of SCFA on decreasing pHi in colon tumor

cells. SCFA are believed to be absorbed mainly in their protonated fom, and then

dissociate into proton and SCFA anion irnrnediately after diffusion through cytoplasrnic

membrane (Binder and Mehta 1989). Although some protons cyde through the Na+-H*

antiporter to protonire SCFA and facilitate absorption (Binder and Mehta 1989), sorne

protons may also accumulate in the cytoplasm and cause acidification. An SCFA--HCO$

exchange is another mechanisrn of SCFA absorption (Mascolo et al 1991). Extrusion of

HCO; can also cause cytoplasmic acidification. Thus, the SCFA, acetate, propionate

and butyrate, have been shown to dewease pHi in normal colonic crypt (Diener et al

1993).

Previous studies have shown a synergistic effect of CaZ+, SCFA and mammalian

lignans in the inhibition of proliferation and the induction of apoptosis in the human colon

tumor cells HCT-15 (Skariah and Thompson 1998). The objective of this study was to

determine the effect of ca2', SCFA and marnmalian lignans on pHi and its possible

relationship to the proliferation and apoptosis of human colon tumor cells.

6.2. Materials and Method

6.2.1. Materials

All reagents were purchased from Sigma Canada (Mississauga, On), unless

otheNvise indicated.

6.2.2. Exmrimental desian

Exponentially growing hurnan colon turnor cells HCT-15 were used in this study.

Cells were inwbated with lipid soluble SNARF-IAM, which diffused through ceIl

membrane and was deesterified into water soluble SNARF-1 inside the cells. SNARF-1,

trapped in cytoplasm because of its water solubility, functions as a free hydrogen ion

indicator to reflect [HTi change. After isolation, SNARF-1-loaded cells were suspended

in different treatrnent solutions in six experiments (Table 5.1). The fluorescence ratio of

H'-bound SNARF-1 (emission at 575nm) and free SNARF-1 (emission at 635nm) was

used to calculate pHi.


Human colon adenocarcinorna cells HCT-15 (purchased from American Type

Culture Collection, Rockville, MD) were cultured at 37% in 95% humidity and 5% C02, in

medium RPMl 1640 suppiemented with 10% fetal bovine senim (FBS), 100 unitsiml

penicillin, 100 (~glml streptomycin and 5 pglml gentamicin. After te-day culture, sub-

confluent cells were used in this study.

6.2.4. lsotonic treatrnent solutions

Hanks balanced sodium solution (HBSS, containing 140mM NaCI, S.8mM KCI,

20mM HEPE, 25mM Glucose, 0.34mM K2HP04, 0.44mM NaH2P04, 4mM glutamine and

1mM pyruvate) was used as a basic detecting solution. Since osmosis may influence

pHi, all detecting solutions were required to have similar osmolarity. Thus, SCFA and

CaClt isotonically substituted NaCl in the HBSS. Sewisolanciresinol (SECO), enterodiol

(ED), enterolactone (EL) or the mixture of ED and EL were direcüy added into HBSS to

the required final concentrations. The solutions were adjusted to pH 7.4, unless


Table 6.1 : Treatments used in intracellular pH expenments'

1. Calibration t-i 6.0,6.4,6.8, 7.0, 7.4 and 8.0

2. Short chain fatty acids 1 Ac: 0, 10,20, 30,

50 and 100

Pr: 0, 10,20 and 30.

Bu: 0,5,1O and 20

6.0, 6.4,6.8, 7.0, 7.4 and 8.0

SCFA: 0,50 and 140

4. Calcium

5. Ca & short chain fatty acids

0, 10 and 20 6.0, 6.4,6.8, 7.0, 7.4 and 8.0

Ac: O, 10, 20, 30. and 50

Pr: O, 10.20 and 30.

Bu: O, 5.10 and 20

SCFA: O, 10,20, 30, 50, 100

O and 10

SCFA: O and 100 ED: 200

EL: 200

SECO: 200

ED+EL: 100+100

a Ac= acetate; Pr=propionate; Bu= butyrate; SCFA= mixture of 3 Ac: 2Pr. 1 Bu; ED=

enterodiol; EL= enterolactone; SECO= secoisolariciresinol.

6.2.5. Dve loading

Exponentially growing cells were treated with 0.25% Trypsin-EDTA solution (3-

Sml per flask) for 5 minutes at 37%, and the trypsin effect was teminated by addition of

10% FBS medium. After rinsing and centrifuging (1000rpm for three minutes, Accuspin

FR^, Beckman, Fullerton, Califomia) with phosphate buffered solution (PBS), the

cell pellet was re-suspended in HBSS supplemented with 5pM SNARF-1AM (Probe Co.

OR), in a density of 10-1 5 X 106 cells/rnl. The cell suspension was incubated at 37OC in

95% humidity and 5% COz for 30 minutes. Then, celfs were rinsed three times with PBS

to remove the extracellular residual SNARF-IAM, and are suspended in HBSS again at

a density of approximately 7.5~10' cells/ml. The cell suspension (2mVcuvette) was

transferred into lm fluorescent cuvette (Ixlx5cm, polyester, allows 240-700nm

wavelength, VWR Canada, Mississauga, ON), and stored in a dark box at room

temperature for 30 minutes to allow cells to recover from the above stated treatments.

Although SNARF-1 is stable for more than 5 hours, the following treatments and

fluorescence detemination were done within 3 hours.


After storage for 30 minutes, cells seffled to the bottom. Following removal of

supernatant, cell pellet was re-suspended in 3ml treatment solutions (pre-heated to

37'~) to a density of SXIO~WII~/~I. ~ h e Spex w spednim fluorescence meter (Spex

industrial Inc. Edison, NJ) was used to determine fluorescence intensities. The

fluorescent intensities were measured at different time points, 2, 5, 10, 20 and 30

minutes, while a micmstimng system was applied to maintain cells in suspension. At the

excitation of 535nm. H*-bound SNARF-1 gives out an emission peak at 57Snm and H'

free SNARF-1 at 635nm. The ratio of intensities at 575nm and 635nm was used to

calibrate pHi.

6.2.7. FluorescenceoHi calibration

The method of Wefsh and Al-Rubeai (1994) was used to calibrate the pHi and

fluorescent intensity. Briefiy, phosphate buffet solutions at pH 6.0,6.4,6.8,7.0, 7.4 and

8.0 were prepared as in Table 5.2. pH was confirmed using a pH meter. SNARF-1-

loaded cells were suspended in different pH solutions. Nigiricin (Biomed Co., CA) was

added to reach a final concentration of 2pM, to make the cell membrane penneable to

H'. After 5 minutes incubation at 37% to equilibrate cytoplasmic and extraceflufar H'

concentration, the cell suspensions were excited at wavelengths of 488, 514 or 535nm,

and emission detected at 575 and 635nm.


At the end of each experiment, cells were treated with 0.5% trypan blue for 5

minutes to test for dead cells. If more than 10% cells were stained, the data were

rejected.

6.2.9. Statistics

Data are presented as means i standard error of means from at least two

experiments, each done in duplicate. One-way ANOVA test was used for multiple

comparisons, and Tukey test for painvise cornparison. PcO.05 was regarded as

statistically significant.

Table 6.2. Preparation of gradient solutions of different pHa

Solution B (ml)

a For each pH, solutions A and B are mixed and diluted to a final volume of 50ml.

Containing: 135mM KH2P04, 20mM NaCI, 1mM MgC12, 1mM CaClz and 10mM glucose.

Containing: 135mM K2HP04, 20mM NaCI, 1mM MgCI2, ImM CaC12 and 1 OmM glucose (ml)

6.3. Resuits

6.3.1. Exneriment 1 : Calibration of D H ~ and fi uorescence ratio

At the excitation wavelength of 488nm, 514nm and 535nrn, the fluorescence ratio

635nm/575nm changed with increasing pHi (Figure 6.1), which was made equal to pHe

by treatment of H* ionosphere nigirin. At the excitation of 535nm. the fluorescence ratio

was the most sensitive to the change of pHe (Figure 5.1 and Appendix 8); therefore, it

was selected as the standard calibration for al1 later experiments. For ease of use, the

ratio was expressed as 575nrn/635nm1 since it has a linear correlation with pHi (Figure

5.1 b). The equation is as follows:

Where, x is the ratio of fluorescence intensities at 575nrn to 635nrn.

6.3.2. Exmriment 2: Effect of acetate. nro~ionate and butvrate

A doseresponse effect of acetate on pHi was obsewed at different time points,

and pHi values at 10 minutes were chosen to represent the effect of different treatments

(Figure 6.2 and Appendix 9). pHi was significantly decreased by 30, 50 and 100mM

acetate, while 10 and 20mM had no effects (pc0.05) compared to control. pHi at 100mM

acetate was significantly lower than that at I O and 2OmM acetate, and the differences,

among 30, 50 and 1OOrnM acetate or among 10,20, 30 and SOmM, were not statistically

significant.

Butyrate (10 or 2OmM) and propionate (20 or 30mM) significantly decreased pHi,

but 5mM butyrate and 1OmM propionate had no effects ( ~ ~ 0 . 0 5 ) compared to control

(Figure 6.2 and Appendbc 9). There were no significant differences among different

concentrations of propionate or butyrate. There were no signifiant differences among

acetate, propionate and butyrate at the same concentration.

Figure 6.1. Calibration curve of pHi. a. At different excitation wavelengths. b. At

excitation wavelength 535nm, the fluorescence ratio 575/635nm.

O OmM CI 5rnM 0 1OmM 12OmM

30mM 5OmM 1 OOmM

Acetate P mpionate

Figure 6.2. Effect of acetate, propionate and butyrate on intracellular pH (pHi). Values

are means f standard error of means. Means with different letters in the same fatty acids

are significantly different (pe0.05).

A pHi %me-dependent recovery" (Le. pHi retum to near neutral range during the

detemination) was observed in the treatrnents with acetate, propionate, butyrate or their

mixture. The dassic pattern curve is presented in Figure 6.3. In the HBSS control (OmM

acetate), the recorded pHi started at 6.955M.015 (2 min.), and ended at 7.177H.021. At

the lower acetate concentrations (1 0 and 20mM), the pHi at 2 minutes were 6.816M.022

and 6.855a.041 respectively, which were significantly lower (p<0.05) than that in the

HBSS control. pHi gradually recovered and rose to 7.1 33fl.012 and 7.1 12M.017 at 30

minutes, values which were not different from that in the control. However, at high

concentrations (30, 50 and l WmM) of acetate, the recorded pHi started at 6.773H.028,

6.75M.047 and 6.703a.054, and ended at 7.083M.017, 7.071M.040 and

7.01 3H.032 re~pe~ve ly , which were al1 significantly lower than thos8 in HBSS control.

6.3.3. Ex~eriment 3: Effect of SCFA at different extracellular pH f~He)

pHi was significantly decreased (pe0.05) by 140mM mixture of acetate,

propionate and butyrate (SCFA) at al1 extracellular pH (pHe), ranging from 6 to 8. SCFA

(50mM) had significant (pc0.05) effect at acidic pH, 6.0 to 7.0, but had no effect at

alkaline pH, 7.4 and 8.0. The effect of 140mM SCFA on pHi was significantly (p<O.OS)

stronger than 50mM SCFA at acidic pHe (Figure 5.4 and Appendix 8.10).

A pHi "neutral maintenance" (Le. cells function to maintain pHi neutral regardless

of extemal pH change) was observed when cells were suspended in different pHe HBSS

solution (Figure 6.4b and AppendixlO). When pHe changed from 6.0 to 8.0, pHi was

maintained within a near neutral range (6.47 to 7.45). The pHi 'neutral maintenance"

was impaired by 50 and 14OrnM SCFA. At acidic extemal pH 6.0, pHi was significantly

decreased to 6.032 * 0.005 at 50mM SCFA (pc 0.05 wmpared to HBSS control), and to

5.853 i 0.032 at 140mM SCFA (p < 0.05, compared to HBSS control and SOmM SCFA).

Figure 6.3. A "tirnedependent recovery" in the effect of acetate on imtracellular pH (pHi). Values are means + standard emr of means.

OmM SCFA: y = 0.5034~ + 3.4543 M e / - /

R.2.r.0.394.7 ........................... .............................. ........................

Figure 6.4. Effect of the mixture of SCFA (3 acetate: 2 propionate: 1 butyrate) on

intracellular pH (pHi) at different extemal pH (pHe) solutions. a. pHi at different pHe and

concentrations of SCFA solutions. Means m'th different letters within the same pHe (a, b

and c), or at the same SCFA concentration (x, y and z) are significantly different. b. The

relationship between pHe and pHi. Changes in pHi 'neutral maintenance" wete caused

by 50 and 140mM SCFA

6.3.4. Ex~eriment 4: Effect of different concentrations of Ca

The presence of 1OmM and 2OmM Ca alone did not significantly change pHi at

different pHe, although a linear correlationship between pHe and pHi was also observed,

Le. OmM Ca. ~10 .994; 1OmM Ca. ~k0.999; 2OmM Ca. ~h0 .998 (Figure 6.5 and

Appendix 11).

6.3.5. Ex~eriment 5: Effect of Ca concentration and SCFA

pHi was significantly decreased (~~0 .05 ) when cells were treated with 1OOmM

SCFA combined with 1OmM Ca cornpared to that with 1WmM SCFA alone (Figure 6.6

and Table 6.3). Ca (IOmM) did not have an effect on pHi when combined with lower

concentrations of SCFA (0, 10, 30 or 50mM) (Figure 6.6 and Appendix 12). The effects

of acetate (0, 10, 30 or SOmM), propionate (10,20 or 30mM) or butyrate (5, 10 or 20rnM)

were not changed by the supplernentation of O, 10 or 20 mM CaCli (Table 6.3).

6.3.6. Ex~eriment 6: Effect of mammalian limans and SCFA.

pHi was significantiy deueased pHi ( ~ ~ 0 . 0 5 ) by 200uM EL and the mixture of

lOOpM EL and 100pM ED, but not by 200vM ED or 200pM SECO, with or without

100mM SCFA (Figure 6.7 and Appendix 12). The EL effect on pHi was different from the

SCFA effect. The E t effect on pHi was not changed during the different tirne-points,

h i l e SCFA effect was overcorne in a time-dependent manner (Figure 6.8 and

Appendixl3). Compared to their individual effects, a greater effect (pe0.05) was

observed when 200pM EL was cornbined with 100mM SCFA at early time point (2min)

(Figure 6.8 and Appendix13).

Figure 6.5. Effect of calcium on intracellular pH (pHi) at different extemal pH (pHe). Values are means f standard emr of rneôns. There are no significant differences.

-

O without Ca i with 1 OmM Ca

...........................................................................................................................................

- ---.-- ........ a ................

- ...... ............. .... b

- ...... -...... ..... -- ..........

1 I

OmM 1OmM 30mM 5ûmM 1WmM

SCFA concentration (M)

Figure 6.6. Effect of calcium and short chain fatty acids (SCFA) on intracellular pH (pHi).

Values are means t standard error of means. Means with different letters are

significantiy different (peO.05).

Table 6. 3. Effect of calcium combined with different short chah fatty acids on pHia

Propionate (mM)

O 1 7.185kO.027 7.174M.028 7.21 4M.034

I -

I Omhî Ca 1 OmM Ca -

20mM Cu

a Values are means + standard enar of rneans of pHi at 10 minutes. Means with different

letters are significantiy different (p<0.05).

20 7.055k0.019 [ 7.037M.004 7.122H.025

SCFA (3 acetate: 2 pmpionate: 1 butyrate) (mM)

Acetate (mM)

O

O

10

30

7. 16M.015

7.108H.031

7.03W.034

6.947s. 075

7.212M.029

50 1 7 -02710.095

7.121I0.007

7.025H.004

7.036$rO.û61

7.12OM.033

7.09OM.016

6.993M.021

6.99SO.007 6. 93410.026

Figure 6.7. Effect of lignans on intracellular pH @Hi) with or without IOOmM short chain

fatty acids (SCFA). Values are means I standard error of means. Means with different

letters within the group (a, b) or between groups (x, y) are significantly different (pc0.05).

ED= enterodiol; EL= enterolactone; SECO= secoisolariciresinol.

HBSS SCFA EL EL+SCFA

Figure 6.8. Effect of short chah fatty acids (SCFA), enterolactone (EL) and EL+ SCFA at

different time points. Values are means I standard enor of means. Means with different

letters in the same treatrnent are significantly different (pe0.05).

6.4. Discussion

The results of different treatments are summarized in Table 6.4. This study has

shown that acetate, propionate, butyrate and their mixture can significantly decrease

pHi. Acidic piie can also significantly decrease pHi, and the greatest effect was

observed when SCFA was combined with acidic pHe.

SCFA can be absorbed through two pathways: passive diffusion of protonated

SCFA or through HCO'rSCFA' exchange (Bugaut 1987). The lipid cell membrane is a

bamer for water-soluble SCFA' anion, but lipid soluble. unionized SCFA can easily

diffuse across cell membrane (Westergaard and Diestchy 1974). Once the protonated

SCFA diffuses into cytoplasm. it immediately dissociates into SCFA- and H'(Englehart

1995). The decreasing pHi seen in this study indicates the accumulation of H* during

SCFA absorption. Stronger effect was observed at higher SCFA concentration and lower

pHe. These results indicate that SCFA-induced cytoplasmic acidification is dependent on

the availabilities of both proton and SCFA. On the other hand, HCO+SCFA' exchange

also may decrease pHi by pumping out HC03.

A "tirnedependent recovery" of acidified cytoplasm was observed in the different

SCFA treatments. Cells maintain the pHi neutral through cornplex ion transport systems,

such as HCOGCI' exchange and Na'-H' antiporter (Madshus et al 1988; Rotin et al

1989). Once pHi is decreased. Na+-H' antiporter is activated to recover the pHi changes

(Rotin et al 1989). Moreover, ion transport system, as well as buffer systems in

cytoplasm, can neutralize pHi changes. Thus, a pHi 'neutral maintenance" was also

observed in different pHe treatment. However, M e n high concentration of SCFA was

combined with acidic pHe, both "tirnedependent recovsry" and "neutral maintenance"

were overcome. Therefore, sustained cytoplasrn acidification can be induced to initiate

events such as apoptosis.

Table 6.4. Summary of effects on intracellular pH

Decreasing pHs

SECO

pHi

a Ac: 30, 50 or 100 mM

Pr: 20 or 30 mM

Bu: 10 or 20 mM

SCF& 50 or 140 mM

Calcium + pHe

:pH= 6-û,50 or 140 mM)

SCFA + Calcium :IO0 mM + 10 mM)

4 SCFA + EL&" 200 PM)

11 SCFA + ED 8*b

200 pM) 1

SCFA + SECO 4b 200 FM)

1

NS

pH= 7.4; with 1mM calcium; pHe= extemal pH; SCFA= short chain fatty acids; ED=

enterodiof; EL= enterolactone; SECO= secoisolariciresinol; asignificant deaaase;

NS= no significance; double arrow indicate a greater effect.

Calcium alone did not show an effect on pHi at different pHe. A greater decrease

in pHi was obsewed when IOmM Ca was combined wïth 1OOmM SCFA, but no effects

were observed when calcium was combined with the lower concentration of different

SCFA. Na'/H' exchange, as a maintenance mechanisrn of pHi, may have been inhibited

by inaeased [ca2Yi (Friis and Johansen 1996).

Acidification was induced by EL, but not ED and SECO, suggesting that the

effect of lignans on pHi is related to molecular structure. The EL effect did not show

"tirne-dependent recovev, which is different from the SCFA effect. A greater effect was

obsewed when EL was combined with SCFA (Figure 6.8). Thus, different mechanisms

of EL effect may be suggested: one is the inhibition of ceIl pHi homeostatic system and

the other is stimulating H' release from endogenous reservoir. When EL was combined

with SCFA, a 3me-dependent recovery" was still obsewed (Figure 6.8), suggesting that

celf pHi homeostatic system was not impaired by EL. Moreover, there is no reason to

believe that EL can cause H* influx from alkaline extracellular environment (pHe 7.4).

Thus, the reasonable expianation may be that EL continuously stimulates H' release

from endogenous source, possibly the mitochondria.

As previously mentioned, homeostasis of pHi is wcia l to many cellular functions.

Tumor cells showed strong capability to expel H' while they huease H' generation. The

findings that decreased pHi induced apoptosis are supported by different mechanisms.

This study showed that cytoplasmic acidification was induced by SCFA, mammalian

lignans and micro-environmental pH. Thus, the induced cytoplasmic acidification may be

a major mechanisrn involved in the effect of SCFA and mammalian lignans, but may not

calcium, on cell proliferation and apoptosis in human colon tumor cells, which were

obsewed in previous studies in out labofatory (Skariah and Thompson 1998).

CHAPTER SEVEN

GENERAL DISCUSSION AND CONCLUSIONS

7. GENERAL DlSCUSSlON AND CONCLUSION

The effects of calcium, SCFA and mammalian Iignans on calcium transport,

intracellular ca2' and intracellular pH are summarized in Table 7.1.

7.1. Calcium concentration

This study has shown that increasing calcium concentration can significantly

increase intracellular ca2'. paracellular and transcellular calcium transport. Paracellular

calcium transport is a passive diffusion through intercellular gap, wtiich is dose-

dependent and unsaturated. Transcellular transport indudes Ca2' entry through brush-

border membrane, intracellular Ca2' movement and ca2' exit at basolateral membrane.

lntracellular ca2' movement is a rate-limiting step (Bronner and Pansu 1999). lnueasing

calcium concentration can inuease ca2' entry and thus intracellular ca2+, through either

increasing trans-membrane diffusion or acüvating "Ca2' sensof to open ca2' channels.

Before intracellular ca2* movement is saturated, inueasing ca2' entry results in

increasing transcellular transport. After saturation is achieved, increasing entry may not

increase transcellular transport, and intracellular ca2+ may inuease. In this study,

transcellular transport was saturated at 15mM and intracellular Ca2' flattened at 10rnM.

The difference suggests that increasing intracellular ca2+ can modulate caZ+ channel

opening (Petersen et al 1999).

Intervention studies only modulate or no protective effect of calcium

supplementation on colon cancer (Mobarhan 1999; Baron et al 1999; Lipkin et al 1999).

Supplementation with vitamin pj showed stronger protective effect against colon cancer,

either with calcium or without calcium (Kleibeuker 1994; Martinez ME and Willett 1998;

Lipkin et al 1999). The study of Bissonette et al (1994) showed that inueased [Ca2']i

was associated with 1 ,25(OH)rVit kinduced apoptosis in human colon turnor cells.

Table 6.1. Sumrnary of the effects of calcium concentration, short chain fatty acids and

mammalian lignans on calcium transport, intracellular ca2' and intracellular pHa

Ca Transport

Paracellular

Calcium

concentration

Acetate

Propionate

Butyrate

SCFA= mixture of 3 acetate: 2 propionate: 1 butyrate; ED= enterodiol; EL=

enterolactone; t rignificant increare; 1 signifiant deuease; ft or U means

greater effect was observed.

[Ca2yi has been show to be involved in al1 three phases mitochondrionniediated

apoptosis (Susin et al 1998). These suggest that increased calcium absorption or

intracellular ca2' may be one of the mechanisms behind the protective effect of calcium

supplementation. Calcium, from food or from supplement, is usually in the fom of

comparable insoluble salts, but calcium is absorbed only in its ionized state (Bronner

1990). Moreover, dissociation of calcium is slow even in acidic environment and alkaline

environment in the intestines prevent calcium from ionization. The formation of ca2'

complex with other food components also hampers calcium absorption (Allen and Wood

1994). Thus, the calcium supplement used in the past intervention studies may not

always increase intracellular ca2'. Whether or not intracellular ca2' was increased may

partly explain the inconsistent results on the colon cancer protective effect in calcium

intervention studies.

7.2. Short chain fatty acids

This study has shown that SCFA can significantly decrease pHi, and acetate and

propionate can inuease transcellular ca2' transport. However, SCFA had no significant

effect on intracellular ca2'. Pmbably, SCFA absorption caused H' accumulation in the

cytoplasm and pHi decrease, which in tum enhanced Ca2'-H' exchange. The formation

of Ca-Ac cornplex may also enhan- CI'' entry. Therefore, transcellular ca2' transport

was increased. Before intracellular Ca2+ movement is saturated, intracellular Ca2' may

not further increase.

The decreased pHi was also observed in this study 4 t h lower pHe. A greater

effect was observed when lower pHe was combined with SCFA. The SCFA-induced

decrease in pHi was recovered to neutral by Na'-H' exchange in a time-dependent

rnanner. However, the combination of SCFA and low pHe produced a more permanent

effect in decreasing pHi. The results suggest that both acidification of colon lumen and

accumulation of SCFA dunng fiber fermentation are important for the induction of

cytoplasm acidification.

7.3. Lignans

EL significantly inueased the [caz7i and decreased the pHi while SECO only

increased the [Ca27i. Although €0 cornbinecl Ath EL increased the [ca2']i, decreased

the pHi and increased the transcellular calcium transport, €0 alone had no effects.

The results suggest that the effects of lignans depend on their molecular

structure. ED, EL and SECO share structure similarity with estrogen and 1, 25(OH)r

vitamin Da. Estrogen and 1, 2S(OH)?vitamin can activate a membrane receptor to

acüvate IP-dependent ca2* rekase in picomolar level. In this study, the minimum

concentration of EL to effecüvely inaease [Ca2+Ji is about 50-100pM. suggesting that

lignans rnay be only partial agonist to the receptor. Zhu (1996) suggested that the

activation of receptor by partial agonists is concentration-dependent; they require high

concentration, unlike full agonists which activate receptor at low concentration. I

speculate that partial binding of EL and SECO to the receptor can activate the receptor,

mi le the partial binding of ED cannot. Due to the partial occupation of binding site, ED

may enhance the effect of other partial agonists, but may inhibit the effect of full agonist.

Although the mechanism is still unclear, results suggest that EL induces ca2' and

H+ release from endogenous reservoir, like mitochondrion and endoplasmic reticulum.

This is based on the fact that EL increased the [Ca2qi even when extemal ca2' was

depleted with 1mM EGTA, and also since the pHi decreased by EL occurred in alkaline

pHe and decreased pHi was not recovered to neutral unlike that caused by SCFA. With

ca2' release from endogenous reservoir, ca2' channel may have been opened to

replenish the resewoir, and ca2* pump may have enhanced the pumping of ca2+ out of

the cells. Therefore, transcellular transport was increased by EL but more so by the

combination of ED + EL which was treated at a higher concentration.

Many experimental or clinical studies have used different drugs to decrease pHi

in order to indum tumor WH apoptosis (Kraus and Wolf 1996; Maidon et al 1993, Newell

and Tannock 1989) (Section 2.6.2). Othem used estrogen and 1,25(OH)rVitamin & to

increase [ca27i and thus to induce apoptosis (Bissonnette et al 1994; Peter et al 1998).

EL has been shown both to increase [Ca2']i and decrease pii i in this study. Thus, EL.

produced from plant lignans by colonic microflora, may have potential as a

chemopreventive agent against colon cancer.

7.4. Interactive effects

A greater effect on deueasing pHi was observed when SCFA was wmbined with

EL than when either one was used alone. EL combined with higher calcium

concentration also resulted in a greater effed on increasing intracellular ca2' Vian when

either one was given alone. However, when SCFA was combined with ED+EL,

transcellular Ca2' transport was inhibited. The reason for this may involve changes in

pHi and [Ca2+]i. ca2' is involved in many biological functions. Many biological funcüons,

such as calcium transport, are pH sensitive. Some agonists, which are able to increase

[ca27i, have stronger effed in the presence of low pHi (Nitshke et al 1997). suggesting

that ca2+ pump is pH sensitive. The inhibition of transcellular transport may therefore be

associated with deueesing ca2' pump upacity in low pHi.

On the other hand, Na'IH' exchange. as a maintenance rnechanism of pHi, can

be inhibited by increased [ca27i (Friis and Johansen 1996; Madshus and Chang 1988).

Both decreased pHi and increased [Ca2']i can induce permeability transition of

mitochondrial inner membrane to induce apoptosis (Section 2.7.). Depletion of ATP

production is an early phenomena accompanying pemieability transition (Susin et al

1998). Because pumping ca2+ out of the cell depends on ATP for energy (Bmnner

1990). depletion of ATP production will automatically lower the capacity of Ca2' pump.

Thus, decrease in transcellular calcium transport was obsewed when cells were treated

with calcium combined with lignans and SCFA.

7.5. Significance and Future Work

A previous study (Skariah and Thompson 1998) has shown that calcium, SCFA

and mammalian lignan can induce apoptosis and inhibite cell proliferation in aie human

colon tumor cells HCT-15. The effects were synergistic when calcium was combined

with SCFA andlor mammalian lignans. In this study, [ca27i was inaeased in HCT-15

cells by increased calcium concentration, EL and SEC0 H i l e lntracellular pH was

decreased by SCFA and EL. ûecreasing pHi and increasing [Ca23i can induce

apoptosis as reviewed in Section 2.7. Thus, the effects of calcium, SCFA and

mammalian lignans in intracellular ca2' and intracellular pH provide possible

mechanisms for their effects on cell proliferation and apoptosis in HCT-15 cells.

Fiber fermentation in the colon lumen causes accumulation of SCFA and

lowering of pHe (Lupton and Kurb 1993; Lupton and Turner 1999). ca2' bound to fiber

in the small intestine c m be released when fiber is fermented (Trinidad et al 1996a).

lncreased water content and the colon lumen addification also can irnprove calcium

ionization (Lupton and Ku- 1993). Plant lignans are converted into their active

components, i. e. €0 and EL in the wlon (Adlercreutz and Mazur 1997). The greater

effects of calcium, SCFA andlor mammalian lignans in decreasing pHi and increasing

[ca27i suggest that the intake of calcium, soluble fiber and plant lignans in combination

may produce strong protedve effects against colon cancer. Results also suggest that

chemoprevention through dietary manipulation may in part be achieved through a

change in dietary habit or intake of forrnulated foods containing these food components.

This study, however, mainly facused on short time in vit10 effect of calcium,

SCFA and mammalian lignans on orrly one human colon tumor cell line, Le. HCT-15. It is

not known if the same effect can be observed in the other transfonned cells although

similar treatments on Caco-2 cells and LST174 cells also resulted in decreased cell

proliferation and induced apoptosis (Skariah and Thornpson unpubrished data). It is

necessary and important to validate these observed effects in other cell lines and in vivo

in animal model and then in humans. Although previous animal studies (Jenab and

Thompson 1997) have shown that SDG and flaxseed can lower colon cancer risk, the in

vivo effects of the combination of the calcium, soluble fiber and lignans in the induction

of apoptosis, inhibition of cell proliferation, induction of cytoplasrnic acidification and

inuease of intracellular Ca2' are basically unknown.

The dose of ~ a " , SCFA and mammalian lignans used in this study is very high

and not available to tissues other than the colonic epithelium. It will be of interest to

detemine whether calcium, SCFA and mammalian lignans may also serve as

chemopreventive agents in others tissues in a lower dose and longer treatrnent pefiod.

EL showed more profound effects in HCT-15 cells than calcium and SCFA in this

study. Its effect in decreasing pHi can ovetcome 'neuttal maintenance" and "tirne-

dependent recovery" mechanism in tumor cells. Its effect in increasing [ca2']i can occur

without extemal ca2+. Further study on its mechanism, such as its influence on

mitochondnon, n'-Na+ antiportor, ca2' channel and ca2' pump, is necessary for better

understanding of the use of EL as potential chemopreventive agent.

7.6. Conclusion

i. In the cell monolayer calcium transport model, calcium transport (transcellular

and paracellular) of human colon tumor cells HCT-15 was significantiy increased with

increasing calcium concentration from OmM to 1SmM. Only transcellular transport was

increased by acetate (20mM), propionate (30 and 2OmM) and EL (1OOpM) + ED

(lOOph4). ED (IOOpM), EL (1OOpM) and butyrate (5, 10,20mM) alone had no effect.

ii. In the suspension of Fura-24oaded HCT-15 cells, increasing calcium

concentration significantly inaeased [ca27i. [Ca2Ti also was significantiy increased by

200w EL, but different concentrations of SCFA and 200pM ED had no effect. A greater

effect was observed when EL was combined with calcium

iii. In the suspension of SNARF-1-loaded HCT-15 cells, decreased pHi was

observed in the presence of different concentrations of SCFA, acidific pile and 200pM

EL. There were greater effects when SCFA was cornbined with lower pHe or EL.

Calcium and €0 had no effects.

Overall, in human colon tumor cells HCT-15, calcium concentration, SCFA and

lignans alone and in combination can influence calcium transport, intracellular ca2+ and

intracellular pH, which in tum may be responsible for their effects in decreasing cell

proliferation and inducing apoptosis obsewed in a previous study.

CHAPTER ElGHT

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APPENDICES

Appendix 1. Effect of calcium concentration on calcium transport.

Calcium

mM

Paracellular Total

Velociîy % of Ca transport Velocity % of Ci tnnsport Vetocity % of Ca tnnsport nmoucm2/30min nmoucm2/30min nrnoücm2130min

Values are means f standard error of means. Means with different letters in the same column are significantly different (P<O.OS).

% of calcium transport is the percentage of calcium that was transported in total available calcium.

Appendix 2. Effects of short chain fatty acids (SCFA) on calcium transport

I l Paracellular 1 Ttanrcellular Total 1 1 Vebciîy 1 % of control

I O ~ M ca~c~um + proplonate I I 1 I I I

I OmM 1 7a6.4î15.0 1 100f2.12 1 553.3î43.1 1 100f7.78' ( 1259.0I57.2 1 100t4.54a 1

Velocity 1 % of control

10mM calcium + d a t e

Velocity 1 % of controf

OmM

1OmM

2OmM

I O ~ M crlclum + t h mixture ot SCFA I I I

366.M. 33

436.3î163.0

523.2+37.2

1 OmM 1 481.5I29,7 1 OOîû. 16 301.8f 70.9 lO0f23.Sa 1 783.3î100.6 100k12,84'

7 t2.6î74.6

658.9f 8.2

673.922.0

1 07Q.Ok82.0

1095.3I171.3

1197.0î88.8

100fz.00'

1 1 9.08144.48a

142.77f 19.97~

1OmM calclum + butyraîe

Values are means f standarû emr of means. Means with different lette6 in the same column are significantly different (PeO.05). % of control are the percentage of velocities in treatment group compared to control group.

100f 10.47

82.47î1. 15

94.5713.09

lûûf7.59

101.51f15.88' ,

1 10.0418.23~

OmM

SmM

1OmM

301.8î70.9

301.8S3.3

295.1 130. 2

481 S e Q . 7

418.614.4

417.1k51.7

100123.Sa

100.01&20.96a

97.7911 0.0O8

783.31100.6

720.4177.6

712.2I21.5

100I6.16

86.93I2.98

96,63f 10.73

1 0M12.84'

91.07f8,91'

90.82fZ. 74'

Appendix 4. Effect of calcium concentrations on intracellular ca2+ at different time points

1 Omin.

OmM

2mM

SmM

10mM

1 SmM

2OmM

Values are means î standard emr of the means at different time points. Means in the same column with different letters are

significantiy different (p< 0.05).

Zmin.

110.9k7.4'

218.0k21.0~

250.2122.6~

27 4.3114.4~

316.9fl2.od

309.5116.2~

Smin.

1 1 0.4k8.5'

209.4k1 7.5b

241.911 8SbC

274,611 5.6*

320.2114.2~

314.3116.6~

Appendix 5. Effect of short chah fatty acids on intracellular ~ a "

The values are means I standard etror of the means at 20minutes. The supplementation of 10mM calcium with different

concentration of acetate, propionate, butyrate and their mixture did not affect [ca2']i. SCFA= 3 acetate: 2 propionate: 1 butyrate.

Ca Cl&nM)

Short chah fatry acids

(mM)

, Acetate

Pmpionate

Butyrate

l

SCFA

O

O

1 1 0.8fs.2

82.4k4.9

82.2~1.6

129.1î3.5

I O

O

214.114.8

234.215. 5

221.7*14.1

206.8k11.5

10

5

-O-

---

260.2115.0

--O

10

10

21 1.6I20.0

242.9113.6

2753113.4

195.9I11.5

10

20

230.3Q0.5

240.6k4.7

294.5I27.7

190.6I5.6

1

10

30

226.7115.7

--

-o.

183,8*8,8

Appendix 6. Effed of lignans on intracellular ca2' .-

Treatment

HBSS control

lOmM calcium alone

1OmM calcium + 200ph4 SEC0

lOmM calcium + 200phl ED

1OmM calcium + 200pM EL 3 4 6 , 2 ~ . 1 ~

IOmM calcium + 100pM ED + 1 W f l EL 277.118.5~

Values are means f standard error of means at the tirne point of 20 minutes. Means with different letten are

significantly different (p<0.05). ED= enterodiol; EL= enterolactone; SECO= secoisolanciresinol

f: r'.

a z C i-i

W 2 z Co

E r

4 'm. e4

Appendix 8. lntracellular pH calibntion at different excitation wavelengths (488nm, 51 5nm and 535nm).

Values are means f standard error of means of the fluorescence ratio of the emission at 575nm to 636nm. pHi=

intracellular pH; pile= extemal pH; x in the equation represents the ratio of fluorescence intensities.

Appendix 10. Effect of different concentrations of the mixture of short chain fatty acids on pHi

Equation

Values are pHi means and the standard error of means. Means with different letters (x-z) within a row (different

concentrations of SCFA at the same pHe) and different letten (a-f) within a column (diffarent pHe at the same SCÇA

concentration) are significantly different ( ~ 4 . 0 5 ) .

Values are means f standard enor of means at 10 minutes. Means with different letten within a column (a and b) or

within a row (x and y) are significantly different (p~0.05). €O= enterodiol; EL= enteroladone; SCFA= 3 acetate : 2

propionate : 1 butyrate.

Appendix 12. Effect of lignans with or without 1OOmM short chain fatty acids on intracellular pH

Control

200pM SEC0 - 2ûû~M ED

2OOpM EL

200pM ML

Without SCFA

7.205f0.009aa

7.21 1 kû.017"

7.2û4?9.û42a'

6.984f0.017~*"

7.003HI.033~~

IOOmM SCFA

7 .09710.004'bv

7,076iû.035aJ

7.043f0.026'"

6.966f0.024~~

6.971î0.060~~

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THE EFFECTS OF CALCIUM, SHORT CHAIN FArrY ACIDS AND · 2020. 4. 7. · Chapter 6: The effects of calcium concentration, short chain fatty acids and mammalian lignans on intracellular