Anti-cancer potential of a specific mixture of ... · Chondrosarcoma, a malignant tumor of cartilaginous origin, is the most common of the adult primary bone cancers. Forty percent

JOURNAL OF CELLULAR MEDICINE AND NATURAL HEALTH | 2017 1.

Journal of Cellular Medicine and Natural HealthM. W. RoomiJOURNAL

CM & NH Cellular Medicine & Natural Health

M. W. Roomi, Ph.D., B. Bhanap M.D., A. Niedzwiecki, Ph.D. and M. Rath, M.D.Dr. Rath Research Institute, CA, USA

Chondrosarcoma, a malignant tumor of cartilaginous origin, is the most common of the adult primary bone cancers. Forty percent of primary bone cancers are chondrosarcoma. However as chondrosarcoma typically does not respond to radiation and chemotherapy, surgery is the main treatment option. Cancer mortality usually results from the local and distant metastasis. Based on the poor prognosis of chondrosarcoma, we investigated effects of a phytonutrient mixture (PB) containing quercetin, CruciferexTM, curcumin, resveratrol and green tea extract on several parameters associated with the development of this type of cancer. The chondrosarcoma cells SW-1353 exposed to PB concentrations of 10, 25, 50, and 100 µg/ml were tested for cell proliferation/viability, morphology, matrix metalloproteinase (MMP) secretion, as well as their invasive and migration abilities. Cell proliferation was carried out by MTT assay, MMPs secretion by zymography, cell invasion by Matrigel assay, cellular migration by scratch test and morphology by H&E staining. The PB inhibited cell proliferation in dose-dependent manner starting at 25 µg/ml concentration. Zymography demonstrated two bands corresponding to MMP-2 and MMP-9 with enhanced MMP-9 expression upon PMA treatment. The PB inhibited expression of both MMPs in a dose-dependent manner in both PMA-stimulated and non-stimulated cells. Matrigel invasion was totally blocked at 25µg/ml. The H&E staining showed that the PB mixture at 25µg/ml to 100 µg/ml induced apoptotic changes in SW-1353 cells. Significant inhibition of important hallmarks of cancer progression by PB mixture suggests that this nutrient composition should be explored further as a therapeutic agent in chondrosarcoma.

Anti-cancer potential of a specific mixture of phytonutrients in chondrosarcoma SW-1358 cells

Abstract

Keywords Chondrosarcoma, phytonutrient mixture, MMP, Matrigel invasion, apoptosis

Correspondence toDr. Aleksandra Niedzwiecki, Dr. Rath Research Institute, 1260 Memorex Drive, Santa Clara, CA 95050, USA.

Email: [email protected]




IntroductionChondrosarcoma, a malignant tumor of cartilage

cells, mainly affects adults between 30-60 years of

age. According to American Cancer Society estimates,

chondrosarcoma has become the most common

primary bone cancer accounting for more than 40% of

the adult primary bone tumour cases (1). It develops

in the chondrocytes or cartilage cells, and mainly

affects the legs, upper arms, scapula, ribs and pelvic

bones. Although the exact etiology of chondrosarcoma

is not known, it is postulated that individuals with

certain genetic or chromosomal abnormalities are at

increased risk. Grade and stage are the most important

and independent prognostic factors for survival in

chondrosarcoma. Cancer mortality usually results from

tumor invasion and local or distant metastasis to vital

organs. High-grade chondrosarcoma has much poorer

prognosis with a calculated 38% of patients achieving

10-year survival (2). Recurrence is based on irregular

tumour margins and lesions greater than 10 cm.

Surgery is generally the main treatment option for

chondrosarcoma since chemotherapy or radiation

therapies are not very effective and are rarely given in

cases of extensive metastasis (3). The high metastatic

potential and poor outcomes associated with current

therapies call for development of more effective and

less toxic treatment alternatives.

The use of natural health products to prevent, inhibit

and reverse carcinogenesis has been gaining in

popularity since scientific evidence has demonstrated

that the high consumption of fruits and vegetables

is associated with a reduced risk of various cancers

(4,5). Different plant-derived polyphenols have been

reported to provide a wide range of anti-carcinogenic

properties, such as modulating cell proliferation, tumour

growth, angiogenesis, metastasis, inflammation and

inducing apoptosis. However, most studies have been

conducted with individual polyphenols, many of which

have poor bioavailability.

In our study, we used a combination of several

polyphenols in order to achieve pleiotropic effects

against chondrosarcoma cells and modulate

bioavailability of these natural compounds through

their interactions. The anti-cancer potential of a

mixture of phytobiologicals (PB) containing quercetin,

curcumin, green tea, cruciferex™, and resveratrol was

tested in human chondrosarcoma cells SW-1353 by

evaluating cell viability, MMPs secretion, cell invasion,

migration and morphology.

Methods and MaterialsCancer cell lines and cultureChondrosarcoma cells line SW-1353 came from

American Type Culture Collection (ATCC, Rockville,

MD), and grown in modified Dulbecco’s Eagle medium

supplemented with 10% fetal bovine serum, 100 U/

ml penicillin and 100 µg/ml streptomycin (Gibco BRL,

Long Island, NY) in 24-well tissue culture plates (Costar,

Cambridge, MA). Cells were incubated in 1 ml of media

at 37°C in a tissue culture incubator equilibrated with

95% air and 5% CO2. At near confluence, the cells were

treated with the PB dissolved in the media and tested

in triplicate: 0, 10, 25, 50, 100 µg/ml. Cells were also

treated with Phorbol Myristate Acetate (PMA) 100 ng/

ml to enhance MMP-9 expression. The plates were

then returned to the incubator.

Composition of the phytonutrient mixtureThe mixture of nutrients (PB) was composed of the

following in the amounts indicated: quercetin 400 mg;

CruciferexTM 400 mg; curcumin 300 mg; resveratrol

50 mg and standardized green tea leaves extract 300

mg, obtained from US Pharma Lab. The certificate




of analysis indicated the following characteristics of

green tea extract: total polyphenol 80%, catechins

60%, epigallocatechin gallate (EGCG) 35%, and caffeine

1.0%.

MTT AssayCell viability was evaluated by MTT assay, a colorimetric

assay based on the ability of viable cells to reduce a

soluble yellow tetrazolium salt [3-(4,5-dimethylthiazol-

2-yl) 2,5-diphenyl tetrazolium bromide] (MTT) to a

blue formazan crystal by mitochondrial succinate

dehydrogenase activity of viable cells. This test is a

good index of mitochondrial activity and thus of cell

viability. After 24h incubation, the cells were washed

with phosphate buffered saline (PBS) and 500 μl of

MTT (Sigma #M-2128); 0.5 mg/ml in media was added

to each well. After MTT addition (0.5mg/ml) the plates

were covered and returned to the 37o C incubator for

2h, the optimal time for formazan product formation.

Following incubation, the supernatant was carefully

removed from the wells, the formazan product was

dissolved in 1 ml dimethyl sulfoxide (DMSO), and

absorbance was measured at 570 nm in Bio Spec 1601,

Shimadzu spectrometer. The OD570 of the DMSO

solution in each well was considered to be proportional

to the number of cells. The OD570 of the control

(treatment without supplement) was considered as

100%.

Gelatinase ZymographyGelatinase zymography was performed in 10%

Novex Pre-Cast SDS Polyacrylamide Gel (Invitrogen

Corporation) in the presence of 0.1% gelatin under

non-reducing conditions. Culture media (20 µl)

were mixed with sample buffer and loaded for SDS-

PAGE with tris-glycine SDS buffer, as suggested by

the manufacturer (Novex). Samples were not boiled

before electrophoresis. Following electrophoresis,

the gels were washed twice in 2.5% Triton X-100 for

30 minutes at room temperature to remove the SDS.

The gels were then incubated at 37o C overnight in a

substrate buffer containing 50mM Tris-HCl and 10mM

CaCl2 at pH 8.0 and stained with 0.5% Coomassie Blue

R250 in 50% methanol and 10% glacial acetic acid for

30 minutes and destained. Upon renaturation of the

enzyme, the gelatinases digested the gelatin in the

gel, producing clear bands against an intensely stained

background. Protein standards were run concurrently

and approximate molecular weights were determined

by plotting the relative mobilities of known proteins.

Matrigel InvasionInvasion studies were conducted using Matrigel

(Becton Dickinson) inserts in 24 well plates. Suspended

in medium the SW-1353 were supplemented with

phytonutrients, as specified in the design of the

experiment and seeded on the insert in the well. The

plates with the inserts were then incubated in a culture

incubator equilibrated with 95% air and 5% CO2 for

24 hours. After incubation, the media from the wells

were withdrawn. The cells on the upper surface of the

inserts were gently scrubbed away with cotton swabs.

The cells that had penetrated the Matrigel membrane

and migrated onto the lower surface of the Matrigel

were stained with hematoxylin and eosin and visually

counted under the microscope.

Scratch TestA 2 mm-wide single uninterrupted scratch was made

on the culture plates of chondrosarcoma SW-1353

cells grown to confluence. Culture plates were washed

with PBS and incubated with PB in tested medium at

0, 10, 25, 50 and 100 µg/ml in triplicate at each dose

for 24 hours. Cells were washed, fixed and stained with

H&E and photomicrographs were taken.

Morphology: H&E stainingMorphology of cells cultured for 24h in test




concentrations of PB were evaluated by H&E staining and observed and photographed by microscopy.

Statistical AnalysisThe results were expressed as means +/- SD, as indicated in the results, for the groups. Data was analyzed by independent sample “t” test using MedCalc Software (Markakerke, Belgium).

ResultsCell proliferation/viability PB significantly inhibited proliferation of chondrosarcoma SW-1353 cells in a dose-dependent manner with 16.8% inhibition at 10 μg/ml, 30% at 25 μg/ml, 53% at 50 and 66% at100 μg/ml concentration, as shown in Figure 1.

Gelatinase zymographyAs shown in Figure 2, zymography indicated the presence of MMP-2 and MMP-9 in chondrosarcoma SW-1353 cells with strong induction of MMP-9 by the exposure of cells to PMA. MMP-2 and MMP-9 were inhibited by PB in a dose-dependent fashion in both PMA- stimulated and unstimulated SW-1353 cells. MMP-2 secretion was significantly blocked already at PB concentration of 25 µg/ml.

Matrigel invasionPB inhibited chondrosarcoma SW-1353 cell invasion through Matrigel by 84% at the concentration of 10 μg/ml, with virtual 100% inhibition at 25 µg/ml, as shown in Figure 3.

Figure 1A – Effect of PB on growth of chondrosarcoma SW 1353

cells: MTT assay 24h

0

20

40

60

80

100

120

Cell

Viab

ility

(% o

f con

trol

)

Control PB 10 μg/ml PB 25 μg/ml PB 50 μg/ml PB 100 μg/ml

Figure 2 – Effect of PB on MMP-2 and -9 in SW 1353 chondrosarcoma

cells: gelatinase zymography (Legend: 1 -Markers,

2- Control, 3-6 PB 10, 25, 50, 100 μg/ml)

MMP-9

MMP-2

1 2 3 4 5 6

Figure 3 - Effect of PB on Matrigel invasion of Chondrosarcoma SW 1353 cells

3A - Control 3B - PB-10 μg/ml 3C - PB-25 μg/ml 3D - PB-100 μg/ml




Scratch TestFigures 4A through 4G show that PB reduced cell migration of chondrosarcoma SW-1353 cells in a dose-response manner with a complete inhibition of cell migration at 50 µg/ml.

Figure 4 – Effect of PB on chondrosarcoma SW 1353 cell migration by scratch test: 4 A-Control, 4B-Just scartched, 4C- 24 hours after scratch,

4D-PB 10ugml, 4E- PB 25 μg/ml, 4F-PB 50 μg/ml, 4G- PB 100 μg/ml.

4A - No Scratch

4D - PB-10 μg/ml

4G - PB-100 μg/ml

4C - 24 hrs after

4F - PB-50 μg/ml

4B - Just scratch

4E - PB-25 μg/ml




Figure 5 – Effect of PB on morphology of chondrosarcoma SW 1353 cells: H&E staining

5A - Control

5C - PB-25 μg/ml 5E - PB-100 μg/ml

5B - PB-10 μg/ml

5D - PB-50 μg/ml

Morphology: H&E stainingH&E staining showed progressively increasing apoptotic changes in chondrosarcoma cells exposed to PB starting at 25 µg/ml up to 100 µg/ml as seen in Figures 5A through 5E.

DiscussionBased on available treatment options, the prognosis of chondrosarcoma is still poor. Therefore, in the search for an effective approach we investigated the cellular efficacy of a specific mixture of different polyphenols (PB) for their anti-cancer pleiotropic effects using chondrosarcoma cell line SW-1353.

Our results demonstrate that PB is effective in inhibiting several key mechanisms of cancer, including cell proliferation (up to 70% inhibition at a concentration of 100 µg/ml) and triggering apoptosis. In addition, using gel scratch test and Matrigel invasion assays, we observed a dose-dependent decrease in the secretion of MMPs largely associated with cancer aggressiveness, such as MMP2 and MMP9, and inhibition of cell migration. These critical steps in cancer invasion and metastasis were blocked by the PB.

In particular, enzymatic degradation of ECM is essential in facilitating tumour growth, invasion, angiogenesis and metastasis and controlling it is an important step in halting tumour progression. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade connective tissue and MMP-2 and MMP-9 in particular have been associated with cancer metastasis and tumour angiogenesis (6,7). These two enzymes play a key role in the degradation of collagen types I and IV, important components of the ECM and basic membranes (8). Enhanced expression of MMP enzymes has been associated with the malignancy potential of chondrosarcomas (8). Our studies demonstrated that exposure of cells to PB results in a significant inhibition of MMP-2 and MMP-9 expression both in unstimulated and PMA-stimulated chondrosarcoma cells.


Journal of Cellular Medicine and Natural HealthM. W. Roomi

PB was formulated by targeting critical physiological targets in cancer progression and metastasis using selected natural compounds of a known anti-cancer potential. Most of the studies tested these compounds individually in order to investigate their mechanisms of action. As such, curcumin (a derivative of turmeric), used for centuries to treat inflammatory conditions, has been reported to affect multiple metabolic targets in many cancers: leukemia, lymphoma, gastrointestinal cancers, genitourinary cancers, breast cancer, ovarian cancer, head and neck squamous cell carcinoma, lung cancer, melanoma, neurological cancers and sarcomas (9). It has been shown that curcumin can downregulate cancer cell proliferation, invasion (MMP-9 and adhesion molecules), angiogenesis (VEGF) and metastasis, as well as induce apoptosis (9). The anti-carcinogenic properties of CruciferexTM (a natural extract derived from cabbage, cauliflower, carrot and broccoli) include supporting detoxification and excretion of carcinogens, protection against oxidative stress, inhibition of cancer cell proliferation and increase in apoptosis resulting in inhibition of tumour growth (10). Green tea extract has been shown to modulate cancer cell growth, metastasis, angiogenesis, and other aspects of cancer progression (11-16). Resveratrol has been shown to inhibit cellular events associated with tumour initiation, promotion and progression, which is partially attributable to its antioxidant activities and inhibition of Cox-2 and protein kinase C, a key mediator of the tumour promotion stage of carcinogenesis (17). Among its anti-carcinogenic effects, resveratrol inhibits cell proliferation and induces apoptosis (17). Bishayee et al. (18) reported that resveratrol affects carcinogenesis by modulating signal transduction pathways that control cell division and growth, apoptosis, inflammation, angiogenesis and metastasis. The cancer preventative effects of quercetin include induction of cell cycle arrest, apoptosis and antioxidant functions (19). Induction of apoptosis by quercetin in cancer cells during different cell cycle stages without affecting normal cells has been documented in various cancers in vivo and in vitro (19). However, many of these polyphenol fractions show low bioavailability which could limit their anti-cancer efficacy in the whole body.

We applied a combination of these natural compounds in order to increase their anti-cancer cellular potential and modulate their bioavailability. As such, earlier studies showed that quercetin can increase the biological efficacy of green tea, curcumin, and resveratrol (20-22). In addition their synergistic and additive effects could allow for expanding anti-cancer mechanisms and as such increase overall efficacy against cancer (23). We demonstrated earlier that these micronutrients significantly affected anti-cancer mechanisms in other types of cancer cells, suggesting their universal, but not cancer specific, effects (24, 25).

ConclusionOur studies demonstrated that a mixture of specific natural phytobiological components (PB) could significantly inhibit key cancer mechanisms in chondrosarcoma cells, such as their invasive potential, cell proliferation, secretion of MMP-2 and -9, in addition to pro-apoptotic effects. These findings suggest that PB should be investigated further as an effective and safe prevention measure and treatment against chondrosarcoma.

AcknowledgmentsThe research study was funded by Dr. Rath Health Foundation (Santa Clara, CA), a non-profit organisation. We thank Ms. Cathy Flowers and Elizabeth Wells for proofreading assistance.

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Anti-cancer potential of a specific mixture of ... · Chondrosarcoma, a malignant tumor of cartilaginous origin, is the most common of the adult primary bone cancers. Forty percent