Reversible Inhibition of Proliferation of Epithelial Cell ... · AGARICUS BISPORUS LECTIN INHIBITS EPITHELIAL CELL PROLIFERATION /~g/ml bovine serum albumin was added to each well

[CANCER RESEARCI153, 46274632, October 1, 1993]

Reversible Inhibition of Proliferation of Epithelial Cell Lines by Agaricus bisporus (Edible Mushroom) Lectin I

Lugang Yu, David G. Fernig, John A. Smith, Jeremy D. Milton, and Jonathan M. Rhodes 2

Departments of Medicine [L. Y, J. D. M., J. M. R.] and Biochemistry [D. G. E, J. A. S.], University of Liverpool, P. O. Box 147, Liverpool L69 3BX, United Kingdom

ABSTRACT

Galactosy113-1,3-N-acetyi galactosamine (Gal IB-1,3-GalNAc) (Thomsen Friedenreich antigen), the Class I core sequence in O-linked oligosaccharide chains, behaves as an oncofetal antigen showing increased expression in many epithelial malignancies. Previous work has shown that peanut agglutinin (PNA), a lectin that binds Gal 13-1,3-GalNAc, stimulates proliferation in HT-29 (human colon cancer) ceils and normal human colonic epithelium and this implies that cell surface glycoproteins which express Gal I~-I,3-GalNAc may play an important role in the regulation of epithelial cell proliferation. We have now studied the effect on epithelial cells of another dietary Gai l~-l,3-GalNAc-binding lectin, the edible mushroom Agaricus bisporus lectin (ABL). This differs from PNA in its ability to bind also to sialylated Gal 13-1,3-GaiNAc.

In contrast to PNA, ABL (25/xg/ml) inhibited incorporation of PH]- thymidine into DNA of HT29 colon cancer cells by 87% (95% confidence limit, 85-89%), Caco-2 colon cancer cells by 16% (95% confidence limit, 12--20%), MCF-7 breast cancer cells by 50% (95% confidence limit, 47- 52%), and Rama-27 rat mammary fibroblasts by 55% (95% confidence limit, 51-60%) when these cells were grown for 24 h in serum.free medium. When assessed by cell count, similar inhibition of proliferation of HT29 ceils by ABL was found. In the presence of 2% fetal calf serum (which contains the ABL-binding glycoprotein fetuin), the inhibitory effect of ABL on cell proliferation was still demonstrable but at increased ABL concentration (60 ~g/ml for 49% inhibition). Ten/~g/ml ABL completely abolished the stimulatory effect on [3H]thymidine incorporation of epidermal growth factor (100 pg/ml) and PNA (25 /tg/mi) and markedly inhibited the stimulatory effect of insulin (50 ng/ml).

ABL (0.2 mg/ml) caused no cytotoxicity to HT29, MCF-7, and Rama-27 cells as measured by trypan blue exclusion, and inhibition of proliferation in HT29 cells caused by 50/xg/ml ABL was reversible after removal of the iectin. Binding studies with 125I-labeled ABL suggested a single class of binding site with an apparent Kd value of (4.12 + 0.29) • 10 -7 M with (3.6 -+ 0.3) • 107 binding sites/ceil.

A. bisporus lectin is a reversible noncytotoxic inhibitor of epithelial cell proliferation which deserves study as a potential agent for cancer therapy.

I N T R O D U C T I O N

Colorectal cancer is the second commones t cause of cancer-related

death in the Western world and affects about 1 in 15 people at some

time in their lives (1). In the colon, as in other epithelia, changes in

surface carbohydrate expression are common in neoplasia and often

represent neo-expression of oncofetal antigens (2, 3). These changes

have often been identified in studies by ourselves (4) and others using enzyme-conjugated lectins as histochemical tools.

Until recently, very little has been known about the functional

significance of these changes in carbohydrate expression. There is increasing evidence that changes in carbohydrate expression may play

a key role in determining the metastatic behavior of tumor cells (5-9).

A m o n g the most commonly demonstrated abnormalities in malignant

and hyperplastic epithelia has been the increased expression of the

Received 5/18/93; accepted 7/27/93. The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a bursary to L. Y. from the Commission of the European Community.

2 To whom requests for reprints should be addressed, at the Department of Medicine, University of Liverpool, P. O. Box 147, Liverpool, L69 3BX United Kingdom.

blood group TF-antigen 3 which has the structure galactosyl 13-1,3-N-

acetyl-galactosamine or-. This structure, which is recognized by the

peanut lectin (PNA), occurs as the type I core structure in O-linked

oligosaccharide chains. In recent studies we have shown that peanut

lectin acts as a mitogen both for the colon carcinoma cell line HT29

and for normal human colonic explants; moreover, it is highly resis-

tant to digestion and can be extracted in an active form from feces

after ingestion of peanuts (10). We have therefore proposed the hy-

pothesis that Gal 13-1,3-GalNAc binding dietary lectins could act as

tumor promoters in the colon. We have now studied the effect of the

only other common dietary Gal 13-1,3-GalNAc binding lectin: the

edible mushroom ABL.

MATERIALS AND METHODS

Materials

All reagents were of analytical grade and purchased from BDH Chemicals Ltd. (Poole, United Kingdom) unless indicated otherwise. Culture medium, antibiotics, FCS, insulin, PBS, Nunc tissue-culture dishes and 24-multiwell plates were obtained from GIBCO Life Technologies Ltd. (Paisley, United Kingdom). [Methyl-3H]thymidine and carrier-free [12sI]-sodium iodide were obtained from Amersham International (Amersham, United Kingdom). EGF was purified from male mouse submaxillary glands (11). ABL, PNA, peroxidase conjugated PNA, bovine serum albumin, glucose, galactose, N-acetyl glucosamine, fucose, and N-acetyl galactosamine were obtained from Sigma Chemical Co. (Poole, United Kingdom). Galactosyl /3-1,3-N-acetyl-galactosamine was from Biocarb Chemicals (Lund, Sweden).

Cell Lines

The HT29 cell line, established from an adenocarcinoma from a 44-year-old female Caucasian (12) and Caco-2 cell lines, isolated from a primary colonic tumor in a 72-year-old Caucasian male (13), were obtained at unknown passage number from the European Cell Culture Collection at the Public Health Laboratory Service, Porton Down Wiltshire, United Kingdom. MCF-7 epithelial cell line, derived from a human malignant pleural effusion from a breast adenocarcinoma (14) and Rama-27 cell line, derived from the fast-sticking fraction of normal rat breast and defined as fibroblastic by its ability to differentiate toward the adipocyte phenotype (15), were from the Department of Biochemistry, University of Liverpool. Stock cultures in 9-cm diameter dishes were grown in monolayer and maintained at 37~ in a humidified atmosphere of 5% CO:J95% air. The cell lines were maintained in DMEM supplemented with 5% FCS, 100 units/ml of penicillin, 100/xg/ml streptomy- cin, 4 mM glutamine, and 50 ng/ml of insulin. The cells were routinely passaged when they had become 80% confluent at a 1:6 subculture ratio and narrow passage cell lines (passaged fewer than 10 times) were used for all studies.

Cell Proliferation Assays

[3H]Thymidine Incorporation. Cells were seeded at a density of 1.2-2.0 x 10a/well in 0.5 ml of DMEM containing 5% FCS in 24-well plates. After 48 h incubation at 37~ in a humidified atmosphere of 5% CO2/95% air, each well was washed twice with 0.5 ml PBS and then 0.5 ml DMEM containing 250

3 The abbreviations used are: TF antigen, Thomsen Friedenreich antigen; ABL, Agari- cus bisporus lectin; PNA, Arachis hypogaea (peanut) lectin; EGF, epithelial growth factor; Gal 13-1,3-GalNAc, galactose/3-1,3-N-acetyl-galactosamine; PBS, Dulbecco's phosphate buffered saline; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle's medium; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate.

4627

Research. on November 5, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

AGARICUS BISPORUS LECTIN INHIBITS EPITHELIAL CELL PROLIFERATION

/~g/ml bovine serum albumin was added to each well as described previously for short-term culture of rat mammary cell lines (16, 17). The cells were incubated at 37~ for a further 24 h in this medium. The lectin under test was then added to the cells for 24 h prior to a 1-h pulse with 0.5 /tCi/well [methyl3H]thymidine as described previously (10, 17). After 2 washes with PBS, the cells were precipitated by addition of 0.5 ml/well 5% trichloroacetic acid at 4~ The precipitate was washed once with 5% trichloroacetic acid at 4~ twice with 0.5 ml/well of 95% ethanol at 4~ followed by drying at room temperature, and then solubilized with 0.5 ml/well 0.2 M NaOH. 0.3 ml dissolved precipitate was added to 1 ml Optima Gold MV scintillation cocktail (Packard, Pangbourne, United Kingdom) and the cell-associated radioactivity was determined using a Packard scintillation counter.

These studies were performed in serum-free medium because of the possi- bility of interference by fetuin (which expresses sialyl-Gal/3-1, 3-GalNAc) in FCS. However, further studies were also carried out in the presence of 2% FCS. Studies were performed as described above except that the medium was replaced by 2% FCS in DMEM after the cells had been incubated for 24 h in DMEM containing bovine serum albumin.

Cell Counting. Cells were seeded and cultured as described above. At the end of the culture period, the cells were trypsinized with 0.5 ml 0.05% (w/v) trypsin in 1.6% (w/v) EDTA for 10 min and suspended after addition of 50/xl FCS, and 0.5 ml cell suspension was counted using a Coulter Counter (Luton, Beds, United Kingdom)

Reversibility of Lectin Effect and Viability of Lectin-treated Cells

Reversibility. Cells were seeded at 1.2-2.0 • 104/well in 5% FCS/DMEM in 24-well plates and after 48 h incubation at 37~ the medium was replaced with serum-free DMEM. The lectin was added after 24 h incubation in serum- free DMEM. Twenty-four h later the cells were washed twice with PBS containing 10 mM galactose and twice with PBS alone to remove ABE Cells were then cultured in 5% FCS/DMEM for an additional 5 days and sample wells were counted daily.

Cell Viability. Cells were seeded and cultured as described above. After 24 h of incubation with lectin in 24-well plates, the medium was removed and the cells were washed twice with PBS and the cell viability was determined by Trypan blue dye exclusion (18).

medium, 0.2 units/ml neuraminidase was added. Forty min later at 37~ cells were washed twice with PBS followed by binding medium as before. Fresh binding medium, 0.2 ml, containing 0.1 txg/ml of 125I-ABL was added into each well and PNA was added at a range of concentrations between 0 and 50 /zg/ml.

Membrane Preparation and Lectin Blotting

Confluent HT29 cells (10 s) were washed twice with PBS and scraped from the culture dishes and centrifuged at 1000 g. Two ml 20 mM Hepes-0.2 mM MgCI2, pH 7.0, was added to the pellet and the cell suspension was sonicated: twice for 30 s on ice using a MSE sonicator. EDTA was added to a concentration of 5 mM and the cells were sonicated twice more for 30 s followed by another centrifugation at 1000 g. After addition of 2% aprotinin (Sigma, St. Louis, MO) and 20 mg/ml of leupeptin (Sigma) the supernatant was centrifuged at 100,000 g for 60 min at 4~ The precipitate was resuspended in 1 ml of 20 mM Hepes, pH 7.0, and stored at -80~ until use. The protein content was tested by modified Lowry's method (22).

SDS-polyacrylamide gel electrophoresis was performed using a discontinu- ous buffer system (Tris-glycine, pH 8.0; 0.006% SDS) with 3% stacking and 5% running gels (23). The membrane preparation was mixed 1:1 with 0.125 M Tris buffer, pH 6.8, containing 2% sodium dodecyl sulfate, 5% 2-mercapto- ethanol, 0.01% bromophenol blue, and 10% glycerol and heated at 100~ for 5 min. A 25-/xl sample (50 /xg protein) was applied to each lane. After electrophoresis, the separated proteins were transferred to nitrocellulose (Schleicher & Schuell, Dassel, Germany) in a Bio-Rad blotting apparatus with Tris-glycine-methanol buffer for 17 h with 70 mA current at room temperature. The nitrocellulose sheet was blocked in 50 mM Tris/HCl and 150 mM NaCI (pH 7.5) containing 1% bovine serum albumin and 0.5% Tween-20 for 2 h and then incubated with peroxidase-conjugated lectin with or without fetuin or galactose on a roller at room temperature for 1 h. After 5 washes with Tris/HCl buffer, the peroxidase-conjugated lectin was visualized with 2-chloronaphthol (20 ml Tris/HCl buffer, 5 ml 3 mg/ml 2-chloronaphthol in methanol and 30 ~1 30% H 2 0 2 ) -

R E S U L T S

Preparation of 1251-ABL and 125-EGF

ABL and EGF were each radiolabeled with carrier-free 125I-sodium iodide (specific activity, 15.04 mCi 1251/~g) by a modification (17) of the Iodogen method (19). Briefly, 10/xl ABL, 1 mg/ml (5/xg of EGF), were dissolved in 40 p,l 0.5 M phosphate buffer, pH 7.4, and reacted for 10 min at room temperature with 5 / tg Iodogen in the presence of 0.2 mCi Na[125I]. The reaction was terminated by the addition of 5 t~l 100 mM sodium metabisulfite, 5 t~l 100 mM potassium iodide, and 50/xl PBS containing 0.2% gelatin and 0.02% azide. The iodinated protein was separated from free 125I by chromatography on Sephadex G-25. The specific activity of the radiolabeled ABL was 8.8/xCi//~g (labeled EGF was diluted to 5 txg/ml; specific activity, 18.8/xCi/ttg).

Binding Studies

Binding studies were performed as described previously (20). Briefly, confluent cells were released by trypsinisation and seeded at 2.5 x 104 cells/well in 24-well plates in DMEM containing 5% FCS. After 48 h, the cells were washed and the medium was changed to the serum-free medium followed by a further incubation for 24 h at 37~ Cells were rinsed twice with 0.5 ml/well PBS and once with 0.2 ml binding medium (DMEM containing 10 mM Hepes, pH 7.4, and 0.1% bovine serum albumin) at 4~ New binding medium 0.2 ml/ well containing 0.1 tzg/ml 125I-ABL was added followed immediately by cold ABL at the concentrations indicated in Fig. 9. Ceils were incubated at 4~ for 2 h. Preliminary studies of the time course of 125I-ABL binding had shown that a steady binding state was reached within 90 min (Fig. 9B). Unbound radioactivity was removed by washing twice at 4~ with 0.2 ml/well binding medium and then twice with 0.5 ml PBS. The washed cells were solubilized in 0.5 ml/well 0.2 M NaOH and the total cell-associated radioactivity was measured using a Cobra auto-gamma counter (Canberra Packard). The results were analyzed using the LIGAND programme of Munson and Rodbard (21).

The effect of neuraminidase treatment on competition between PNA and ABL binding by HT29 cells was studied. After 24 h incubation in serum-free

Effect of ABL on Proliferation of HT29, MCF-7, Caco-2, and Rama-27 Cells. A B L produced marked inhibition of thymidine in-

corporation in a dose-dependent manner in human colon cancer cells

HT29 and Caco-2, breast cancer cells MCF-7, and rat m a m m a r y

fibroblasts Rama-27. Fifty % inhibition was observed at 3 / t g / m l A B L

for HT29 cells, 25 ~g/ml for MCF-7, and 5 / zg /ml for Rama-27 cells

(Fig. 1). Twenty-f ive / tg/ml A B L produced 87% (95% confidence,

85-89%; n = 6 separate experiments, each performed in triplicate)

100 HT29

'~ 80

"~ ~ Rarna-27 ............ ][

, , ,o, '~ .E ~ ' /

0 i i i ! i i

0 10 20 30 40 50

ABL IJg/ml

Fig. 1. Dose response relationship for ABL inhibition of incorporation of [3H]thymidine into DNA by various cell lines. The ABL effect is expressed as percentage inhibition of the response obtained with medium alone. Values represent means • SD of triplicate determinations from one of five experiments.

4628



AGARICUS BISPORUS L E C T I N I N H I B I T S E P I T H E L I A L C E L L P R O L I F E R A T I O N

inhibition of thymidine incorporation for HT29 cells, 16% inhibition (12-20%) for Caco-2 cells, 50% inhibition (47-52% n = 5) for MCF-7, and 55% inhibition (51-60% n = 5) for Rama-27. In the presence of 2% FCS, ABL caused 46% inhibition of thymidine incorporation by HT29 cells at 60/xg/ml (Fig. 2).

Parallel experiments were performed in which HT29 cells were released by trypsinisation and counted to confirm that changes in thymidine incorporation were true reflections of altered cell proliferation. After 24 h incubation with 10 lxg/ml of ABL cell proliferation was almost totally inhibited (Fig. 3). A longer culture period was not possible using the serum-free medium. Further experiments showed that HT29 cell growth was inhibited 49% (45-53%; n = 3) by 60 /xg/ml ABL after cells were incubated with ABL for 3 days in the presence of 2% FCS (Fig. 4).

Inhibition of Proliferative Effect of EGF, PNA, and Insulin. In the presence of 10/~g/ml ABL, the normal stimulatory effect of EGF (100 pg/ml) and PNA (25 /xg/ml) was completely abolished (Fig. 5) and the stimulatory effect of insulin (50ng/ml) was markedly inhibited in a dose-dependent manner (Fig. 6). Further studies in the presence of EGF (100 pg/ml) and also in the presence of PNA (25 /~g/ml) showed the inhibitory effect of ABL to have a similar dose-response (data not shown).

e - , ~

, ~

E

-1-

O e -

.2

50

40

'~ 30

o i._

~ 2o ._~

10

i i i ! i i 1

0 10 20 30 40 50 60

A B L p g / m l

Fig. 2. Inhibition of [3H]thymidine incorporation of HT29 cells by ABL in the presence of 2% FCS. The effect is expressed as percentage inhibition of the response obtained with medium (3637 _-. 108 @m/well). Values represent means -4- SD of triplicate determinations from one of three similar experiments.

25

~= 20

,1o E 15 e,-

O 10 x

n 5 O

- - - a f t e r 3 days culture . . . .

--I--

'.,~ o 0 0 'E r ,--

Fig. 4. Effect of ABL on HT29 cell number after 3 days culture in the presence of 2% FCS. The results represent means +- SD of triplicate determinations.

6000

�9 - __~ o -I-

~, 5000 I , . ,

o

a.. 4000

"- ~. 3000 | E

'~. 2000 > , .

t , -

,ooo

-6 ~ ~ ~ + + -

m o r : Z ','- 0 0 t.~

'=-" 0 ~'- t%l v-

m

Fig. 5. Inhibition by ABL of the stimulatory effects of EGF and PNA on thymidine incorporation by HT29 cells. Values represent mean • SD of triplicate determinations from one of five similar experiments.

8.0

_ 7.0

~g 6.0

.~ 5.0 E =3 , - 4 . 0

m

"~ 3.0 O x 2.0

1.0 o

0.0

Time 0 hr 24 hours $

control 0 4 10 25 40

A B L p g / m l

Fig. 3. Effect of ABL on HT29 cell growth after 24 h culture in serum-free medium. The results represent means • SD of triplicate determinations from one of three similar experiments.

Reversibility and Lack of Cytotoxicity. HT29 cells incubated at 37~ for 24 h with ABL at 5, 10, and 50 txg/ml were able to proliferate again after removal of the lectin by 2 washes with 0.5 ml/well DMEM containing 10 mM galactose, and 2 washes with 0.5 ml/well of PBS (Fig. 7). Trypan blue dye exclusion analysis of cells incubated at 37~ for 24 h in the presence and absence of ABL at 5, 10, 50, 100, and 200 Ixg,/ml showed >95% viability as did the control cells. MCF-7 and Rama-27 cells were also cultured for 24 h in the presence of ABL 200 txg/ml and showed >95% viability by trypan blue exclusion.

Inhibition of ABL Effect by Gal ~]-I,3-GaiNAc. Gal/3-1,3-Gal- NAc, 0.5 mM, blocked 90% (85-94%; n = 4) of the inhibitory effect of ABL (Fig. 8), showing that ABL inhibition was mediated through its sugar-specific binding site. a-L-fucose, D-galactose, N-acetyl-D- glucosamine, N-acetyl-o-galactosamine, and o-lactose at 50 mM all failed to block ABL inhibition.

Effect of Neuraminidase. Since ABL can bind both Gal /3-1,3- GalNAc (TF-antigen) and sialyl Gal/3-1,3-GalNAc whereas PNA will not bind sialyl Gal/3-1,3-GalNAc (24), the effect of prior neuraminidase treatment of HT29 cells on the inhibitory effect of ABL and the

4629



AGARICUS BISI'ORUS L E C T I N I N I [ I B I T S E P I T H E L I A L C E L L P R O L I F E R A T I O N

stimulatory effect of PNA was investigated. Cells were incubated with

0.2 units/ml neuraminidase at 37~ for 40 rain, which removes greater

than 95% of the cell membrane-associa ted sialic acid while maintain- o

ing cell viability (25). This resulted in no significant difference to the '~

inhibition of D N A synthesis caused by A B L or the stimulation caused o.

by PNA as compared with control cells (Table 1).

Cell B i n d i n g Studies . 12SI-ABL binding to HT29 cells was best .__.o

fitted by a single binding site model with a Kd of (4.13 ___ 0.29) •

10 -7, corresponding to (3.6 + 0.3) • 10 7 receptors/cell (Fig. 9A ). The :~

t ime course of ]251-ABL binding at 4~ is shown in Fig. 9B. "~

P N A Interaction with 12SI-ABL Binding. P N A did not compete ,- 4--'

with A B L for binding sites on normal HT29 cells, but, if the cells were

treated with neuraminidase first, P N A competed partially with A B L

binding as shown in Fig. 10. This indicates that sialyl Gal /3-1,3-

GalNAc (cryptic TF-antigen) is expressed in addition to the TF anti-

gen itself in HT29 cells.

L a c k of Effect of ABL on EGF Binding. Competi t ive studies

using 125I-EGF showed that under conditions in which A B L inhibited

the proliferative effect of EGF, the binding of EGF was unaffected

even when high concentrat ions of A B L were used (Fig. 11).

4000 . . . . . . Insulin 50ng/ml . . . . . .

8

3000

t - 0 ~ == 2000

= IOOO

0 I Control 0 4 10 25 40

ABL p g / m l

Fig. 6. Inhibition by ABL of the stimulatory effect of insulin on thymidine incorporation by HT29 ceils. Control cells were cultured at the same time and for the same duration of 24 h but in the absence of insulin and ABL. Values represent means • SD of triplicate determinations from one of three similar experiments.

20

�9 Control .~-- �9 ABL 5pg/ml ~ [

A ABL 10Ng/ml 3= -= , , . , o . _

.O ~ j � 9 E =" 10 / ) ~ - - T,'- -- lectin r~,emoved /~//// ~ . . - " ~ "~ in moved . - ' " ~ ..'""""

| . .~.. . . '""'" O_ . �9

0 | I I I I I I

0 1 2 3 4 5 6

Days after addition of l e c t i n

Fig. 7. Reversibility of the ABL inhibitory effect after removal of lectins. Results represent the means • SD of triplicate determinations from one of 2 experiments.

4000

3000

m m

2000 E

" 0

1000

0 :1

ABL lOpg/ml

.__ , . = ~ ~ ~ ~ "" ~ o . -_ ~ oo o z z . . z

Fig. 8. Counteraction of ABL-mediated inhibition of thymidine incorporation by HT29 cells by mono- and disaccharides. After 24 h incubation in serum-free medium, the cells were incubated in new serum-free medium containing different mono- and disaccharides. ABL was used at 10/xg/ml. All monosaccharides were tested at 50 mM and Gal /3-1,3- GalNAc at 0.5 mM. The results represents mean • SD of triplicate determinations from one of three similar experiments.

Table 1 Effect of neuraminidase treatment of liT29 cells on their proliferative response to lectins and EGF

HT29 cells were incubated with 0.2 units/ml of neuraminidase at 37~ for 40 min, washed twice with PBS and once with serum-free medium, and cultured for 24 h before addition of [3H]thymidine for 1 h"

Neuraminidase treated Untreated

Inhibition Inhibition dpm (%) dpm (%)

Control 3270 • 584 ABL 5 Ixg/ml 794 • 99 76 ABL 10/~g/ml 596 • 64 81

EGF 100 pg/ml 4724 +__ 206 +ABL 10 ~g/ml 602 -+ 30 87

PNA 25 p~g/ml 4300 • 177 +ABL 10/xg/ml 732 • 88 82

3924 • 685 820 • 117 79 615 • 70 84

5726 • 167 612 • 31 89

5369 • 163 673 • 65 87

a The results represent means - SD of triplicate determinations from one of the three similar experiments.

Lec t in Blot t ing. SDS-polyacry lamide gel electrophoresis fol lowed

by lectin blotting demonstra ted that the HT29 membrane preparation

contained at least 12 glycoproteins which bind to A B L and PNA. No

definite difference in the binding patterns of the two lectins could be

observed (Fig. 12).

D I S C U S S I O N

This study shows that Agar i cus bisporus lectin causes dose-depen-

dent inhibition of proliferation of HT29 human colorectal carc inoma

cells, Caco-2 human colorectal cancer cells, human breast cancer

MCF-7 cells, and rat m a m m a r y fibroblast Rama-27 cells. The effect

on HT29 cells is reversible, is not associated with cytotoxicity, and

seems to be both potent and nonspecif ic as shown by the inhibition of

the growth o f cells st imulated by diverse factors including peanut

lectin, epidermal growth factor, and insulin.

Inhibition of proliferation of mi togen st imulated lymphocytes by

A B L has been reported previously (26) but in contrast A B L has been

shown to stimulate vascular smooth muscle and endothelial cell pro-

liferation (27). However, both these studies were performed in the

presence of serum, which contains glycoproteins which express sialyl

4630



AGARICUS BISPORUS LECq ' IN I N H I B I T S E P I T H E L I A L C E L L P R O L I F E R A T I O N

"v 8.0 == o

Fig. 9. Binding of 125I-ABL to HT29 cells, a, --~ ~ 6.0 effect of increasing lectin concentration b, time ~ x ~ course of binding (125I-ABL 0.1 p,g/ml). The data -- o 4.0 presented in a represent the means _+ SD of qua- m ,.- druplicate determinations from one of four similar experiments. ~"

~., 2.0

10.0 a

Kd. 4.13x10"rM

0.0 t i !

1,0 10,0 100.0 400.0

ABL {pg/ml}

10.0

8.0

6.0

4.0

2.0

, 0 . 0

T b ~ ~

0 20 40 60 80 100 120

Time [Mini

t-

O

E m o o x

_ J ! 110 o

E

14.0 -I / �9 untreated

12.0

10.0

6.0 , , , , , ,

0 10 20 30 40 50

PNA pg/ml

Fig. 10. Effects of neuraminidase treatment on the competition between PNA and ABL binding to HT29 cells. Values represent means • SD of triplicate determinations from one of three experiments.

(data not shown) in which 2% FCS could completely abolish the agglutinating effect of up to 12.5/xg/ml ABL on normal human red blood cells.

Lectins such as phytohemagglutinin and wheat germ agglutinin are well known for their toxic effects on intestinal epithelial cells (29, 30) and Vicia faba lectin inhibits thymidine incorporation in Caco-2 cells (31) but there has been no previous demonstration of a lectin that has such a profound inhibitory effect on proliferation as ABL without apparent cytotoxicity. This lack of cytotoxcicity suggests that it must be acting by a different mechanism than the irreversible mechanism by which ricin and abrin inhibit protein synthesis by inhibiting the ribosome-dependent GTPase activity (32).

We found that 50 mM L-fucose, o-galactose, N-acetyl-o-glucosamine, N-acetyl-o-galactose, and o-lactose all failed to block ABL

KD

10.0 -~ 191

"Io 8.0

o .o E

~- 6.0

u,.O ~o 4"0 uJ

m

2.0

0.0

- i ili

I I I - - I

0 1.0 10.0 100.0 400.0

ABL (pg/ml] Fig. 11. Lack of effect of ABL on tzSI-EGF binding; 0.2 ml per well of binding medium

containing 13 ng/ml of lz~I-EGF was used. Cells were incubated for 2 h at 4"C with 125I-EGF and ABL before cell-associated radioactivity was determined. The results represent the means +_ SD of quadruplicate determinations from 1 of 2 similar experiments.

Gal/3-1,3-GalNAc, the binding site for ABL (e.g., fetuin in fetal calf serum; Ref. 28) and this makes interpretation difficult. Inhibition by ABL of HT29 cell proliferation was nevertheless observed in our study in the presence of 2% FCS but required an approximately 20-fold increase in lectin concentration to demonstrate an equivalent inhibitory effect. This is in keeping with the results of experiments

4631

117

91.8-

57.8

40.8

a b c d e Fig. 12. Lectin blot of HT29 cell membrane preparation, a, blotted with 2.5 /xg/ml

peroxidase as control; b, 2.5 p,g/ml of peroxidase-ABL; c, 2.5/zg/ml peroxidase-ABL in the presence of 4 mg/ml fetuin; d, 2.5 p~g/ml peroxidase-PNA; e, 2.5 ~g/ml peroxidase- PNA in the presence of 0.4 M galactose.



AGARICUS BISPORUS LECTIN INHIBITS EPITHELIAL CEIJ, PROIJFERATION

inhibi t ion. These results are cons is tent wi th those repor ted by Presant

and Korn fe ld (33) w h o f o u n d that whereas G a l - G a l N A c - b e a r i n g gly-

copro te ins could produce m a r k e d inhibi t ion o f red cell agglu t ina t ion

by A B L , s ingle sugars p roduced on ly a sl ight effect but di f fer f rom

those repor ted by Greene et al. (26) w h o found that 50 mM, D-

ga lac tose , N-ace ty l -D-ga lac tosamine , L-rhamnose, and D-lactose cou ld

b lock the inhib i tory effect o f A B L on l y m p h o c y t e s .

It is in t r iguing that b lo t t ing s tudies s h o w e d no obv ious d i f fe rence

b e t w e e n the b ind ing prof i le o f the two lect ins to an H T 2 9 m e m b r a n e

prepara t ion a l though they have opposi te ef fects on pro l i fe ra t ion o f the

cell line. It is l ikely t hough that each lectin wil l be b ind ing to a

d i f ferent rage o f o l igosacchar ide cha ins on the g lycopro te ins . Pretreat-

m e n t o f cel ls wi th neu ramin idase p roduced no s igni f icant change in

the effect o f ei ther lectin. This sugges ts that the b ind ing o f A B L but

not P N A to s ia lyl G a l / 3 - 1 , 3 - G a l N A c is not the main reason for their

d i f ferent effects.

A B L inhibits pro l i fe ra t ion o f a w ide range o f cells whe the r un-

s t imula ted or s t imula ted by a wide range o f g rowth factors inc lud ing

FCS, EGF, insul in, and PNA, w h i c h sugges ts that A B L has a ve ry

genera l ef fect on cell g rowth , perhaps inhib i t ing a part o f the intra-

ce l lu lar s ignal l ing p a t h w a y or nutr ient uptake. It has been s h o w n that

l ec t in -b ind ing sites are f o u n d not on ly on the cy top l a smic m e m b r a n e

but also on the cis ternal surface o f the nuc lear enve lope , the mi to-

chondr ia l outer m e m b r a n e , the rough e n d o p l a s m i c re t iculum, and the

Golg i appara tus (34). In our hands , cy tochemica l s tudies wi th peroxi-

dase con juga t ed A B L and P N A have con f i rmed the s ta in ing o f H T 2 9

cells on the nuc lear enve lope as wel l as on the cell m e m b r a n e (data not

shown) . It is l ikely that A B L has to be in ternal ized for its ant iprol i f-

era t ion effect to occur. Lec t in - induced changes in m e t a b o l i s m in

Caco-2 cells have been s h o w n to be l inked wi th in ternal iza t ion o f

lect in (29, 35), w h i c h lends indirect suppor t for this idea.

These s tudies g ive fur ther suppor t to the hypo thes i s that lect ins

wh ich interact wi th epi thel ia l cell sur face g lycopro te ins wh ich express

G a l / 3 - 1 , 3 - G a l N A c (TF-an t igen) m a y have ve ry impor tan t effects on

pro l i fe ra t ion and that neo-express ion o f TF on ma l ignan t and hyper-

plast ic cel ls m a y have a crucial role in de t e rmin ing their rate o f

prol i fera t ion. The poten t and revers ible inhib i tory effect o f the A.

b i sporus lect in on pro l i fe ra t ion in a range o f cell l ines sugges ts that it

deserves s tudy as a potent ia l an t icancer agent.

ACKNOWLEDGMENTS

We are very grateful to Dr. Elizabeth G. H. Rhodes, Department of Hae- matology, for her helpful advice and Brian Getty, Department of Medical Microbiology, University of Liverpool, for the generous supply of HT29 and Caco-2 cell lines.

REFERENCES

1. Soybel, D. I., Bliss, D. E, and Wells, S. A. Colon rectal carcinoma, current problems in cancer. Cancer (Phila.), 61: 236-257, 1987.

2. Feizi, T. Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are oncodevelopmental antigens. Nature (Lond.), 314: 53-57, 1985.

3. Barr, N., Taylor, C. R., and Young, T. Are pancarcinoma T and Tn differentiation antigens? Cancer (Phila.), 64: 834-841, 1989.

4. Rhodes, J. M., Black, R. R., and Savage, A. Glycoprotein abnormalities in colonic carcinomata, adenomata and hyperplastic polyps shown by lectin peroxidase histo- chemistry. J. Clin. Pathol., 39: 1331-1334, 1986.

5. Nicolson, G. L. Cancer metastasis, organ colonization and cell-surface properties of malignant cells. Biochim. Biophys. Acta, 695: 113-176, 1982.

6. Schirrmacher, V. Cancer metastasis: experimental approaches, strategies. Adv. Cancer Res., 43: 1-71, 1985.

7. Roos, E. Cellular adhesion, invasion and metastasis. Biochim. Biophys. Acta, 738: 263-284, 1984.

8. Raz, A., and Lotan, R. Endogenous galactoside-binding lectins: a new class of functional turnout cell surface molecules related to metastasis. Cancer Metastasis Rev., 6: 433-452, 1987.

9. Yuan, M., Itzkowitz, S. H., Boland, C. R., Tomita, J. T., Palekar, A., Bennington, J. L., Trump, B. E, and Kim, Y. S. Comparison of T-antigen expression in normal and malignant human colonic tissue using lectin and antibody immunohistochemistry. Cancer Res., 46: 4841-4847, 1986.

10. Ryder, S. D., Smith, J. A., and Rhodes, J. M. Peanut lectin: a mitogen for normal human epithelium and human HT29 colorectal cancer cells. J. Natl. Cancer Inst., 84: 1410-1416, 1992.

11. Smith, J. A., Ham, J., Winslow, D. E, O'Hare, M. J., and Rudland, E S. The use of high-performance liquid chromatography in the isolation and characterization of mouse and rat epidermal growth factors and examination of apparent heterogeneity. J. Chromatogr., 305: 295-308, 1984.

12. Marshall, C. J., Franks, L. M., and Carbonell, A. W. Markers of neoplastic transfor- mation in epithelial cell lines derived from human carcinomas. J. Natl. Cancer Inst., 58: 1743-1747, 1977.

13. Fogh, J., Wright, W. C., and Loveless, J. D. Absence of HeLa cell contamination in 169 cell lines derived from human tumors. J. Natl. Cancer Inst., 58: 20%214, 1977.

14. Soule, H. D., Vazquez, J., Long, A., Albert, S., and Brennan, M. A human cell line from a pleural effusion derived from a breast carcinoma. J. Natl. Cancer Inst., 51: 140%1413, 1973.

15. Rudland, P. S., Twiston Davis, A. C., and Tsao, S. W. Rat mammary preadipocytes in culture produce atrophic agent for mammary epithelia--prostaglandin E2. J. Cell. Physiol., 120:364-376, 1984.

16. Smith, J. A., Winslow, D. E, and Rudland, E S. Different growth factors stimulate cell division of rat mammary epithelial, myoepithelial and stromal cell lines in culture. J. Cell. Physiol., 119: 320-326, 1984.

17. Fernig, D. G., Smith, J. A., and Rudland, E S. Appearance of basic fibroblast growth factor receptors upon differentiation of rat mammary epithelial to myoepithelial-like cells in culture. J. Cell. Physiol., 142: 108-116, 1990.

18. Freshney, R. I. Culture of Animal Cells--A Manual of Basic Technique, pp. 208-209. New York: Alan R. Liss, Inc., 1983.

19. Wiley, H. S., and Cunningham, D. D. The endocytotic rate constant, a cellular parameter for quantitating receptor-mediated endocytosis. J. Biol. Chem., 257." 4222- 4229, 1982.

20. Fernig, D. G., Rudland, E S., and Smith, J. A. Modulation of bFGF action by low-affinity receptors in rat mammary myoepithelial-like cells in culture. Growth Factors, 7: 27-39, 1992.

21. Munson, P. J., and Rodbard, D. A versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem., 107." 220-239, 1980.

22. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall R. J. Protein determination with the Folin-Phenol reagent. J. Biol. Chem., 193: 265-275, 1951.

23. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1971.

24. Novogrodsky, A., Lotan, R., and Sharon, N. Peanut agglutinin, a new mitogen that binds to galactosyl sites exposed after neuraminidase treatment. J. Immunol., 115" 1243-1248, 1975.

25. Bresalier, R. S., Rockwell, R. W., Dahiya, R., Duh, Q. Y., and Kim, Y. S. Cell surface siaioprotein alterations in metastatic murine colon cancer cell lines selected in an animal model for colon cancer metastasis. Cancer Res., 50: 1299-1307, 1990.

26. Greene, W. C., Fleisher, T. A., and Waldmann, T. A. Suppression of human T and B lymphocyte activation by Agaricus bisporus lectin. J. Immunol., 126: 580-586, 1981.

27. Sanford, G. S., and Hooker, S. H. Stimulation of vascular cell proliferation by /3-galactoside specific lectins. FASEB J., 4: 28-34, 1990.

28. Nilsson, B., Norden, N. E., and Svensson, S. Structural studies on the carbohydrate portion of fetuin. J. Biol. Chem., 254: 4545-4553, 1979.

29. Hendriks, H. G. C. J. M., Kik, M. J. L., Koninkx, J. E J. G., van den Ingh, T. S. G. A. M., and Mouwen, J. M. V. M. Binding of kidney bean (Phaseolus vulgaris) isolectins to differentiated human colon carcinoma Caco-2 cells and their effect on cellular metabolism. Gut, 32: 196-201, 1991.

30. Pusztai, A. P. The nutritional toxicity of PHA. Plant Lectins, pp. 111-113. Cambridge: Cambridge University Press, 1991.

31. Koninkx, J. E J. G., Hendriks, H. G. C. J. M., Van Rossum, J. M. A., van den Ingh, T. S. G. A. M., and Mouwen, J. M. V. M. Interaction of legume lectins with the cellular metabolism of differentiated Caco-2 cells. Gastroenterology, 102: 1516- 1523, 1992.

32. Olsnes, S., Refsnes K., Christensen, B. T., and Pihl, A. Studies on the structure and properties of the lectins from Abrus precatorius and Ricinus communis. Biochim. Biophys. Acta, 405: 1-10, 1975.

33. Presant, C. A., and Kornfeld, S. Characterization of the cell surface receptor for the Agaricus bisporus hemagglutinin. J. Biol. Chem., 247: 6837-6945, 1972.

34. Lis, H., and Sharon, N. Application of lectins. In: I. E. Liener, N. Sharon, and I. J. Goldstein (eds.), The Lectins: Properties, Functions and Applications in Biology and Medicine, pp. 293-370. London: Academic Press, Ltd., 1986.

35. King, T. P., Pusztai, A., Grant, G., and Slater, P. Immunogold localization of ingested kidney bean (Phaseolus vulgaris) lectins in epithelial cells of the rat small intestine. J. Histochem., 18: 413-420, 1986.

4632



1993;53:4627-4632. Cancer Res Lugang Yu, David G. Fernig, John A. Smith, et al.

(Edible Mushroom) LectinAgaricus bisporusReversible Inhibition of Proliferation of Epithelial Cell Lines by

Updated version

http://cancerres.aacrjournals.org/content/53/19/4627

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/53/19/4627To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Documents

Reversible Inhibition of Proliferation of Epithelial Cell ... · AGARICUS BISPORUS LECTIN INHIBITS EPITHELIAL CELL PROLIFERATION /~g/ml bovine serum albumin was added to each well