A commercial nutraceutical mix Metabolic Cell-Support (MC-S[TM]) inhibits proliferation of cancer cell lines in vitro.Background
Cancer is one of the leading causes of human disease related death in the world today (Gao 2005). The major methods for treating cancer include surgery, radiation, chemotherapy, and immunotherapy (Gao 2005, Gibbs 2000). Conventional cancer chemotherapy is used to kill or disable tumor cells while preserving the normal cells in the body by the application of synthetic compounds (Gao 2005, Fidler 2000). These agents have a narrow safety margin, and the therapy can fail due to drug resistance and dose limiting toxicities (Gao 2005). In Asia herbal preparations have been increasingly used for cancer therapy in an attempt to assist in the killing of tumor cells and to reduce the toxicity of combined chemotherapeutic agents (Gao 2005, Ho 2002, Tang 2003, Vickers 2002). Today many clinically effective anticancer drugs have been derived from natural sources, e.g. paclitaxel (Taxol) from Taxus brevifolia L. and vincristine (Oncovin) from Cantharanthus roseus G. Don (Pezzuto 1997). Plant and fungus extracts continue to offer a wide range of compounds with diverse structures and activity which will continue to occupy an important role in modern cancer treatments.
The medicinal properties of mushrooms and herbs have long been utilised in traditional medicine for healing and health. In the past decades scientific validation of the clinical efficacy of medicinal therapies has encouraged modern medicine to investigate the active ingredients of medicinal mushrooms and herbs. Unfortunately this approach may in some circumstances have resulted in products that have reduced functionality when compared with their natural sources (Borchers 2004). It is known that a range of active compounds found in medical mushrooms and herbs can stimulate the human immune system and/or inhibit tumor growth. As these compounds can vary in bioactivity and biospecificity, it may be possible to utilise a range of the compounds to symbiotically stimulate the immune system leading to a more efficacious response and inhibit tumor growth (Smith 2002).
The formulation of MC-S (Clinical Health Pty Ltd, Newcastle Australia) is based upon this principle and is composed of three medicinal mushrooms, Ganaderma lucidum, Lentinus edodes (mycelia) and Coriolus versicolor, a herb Astragalus membranaceous (from the root) and ascorbic acid. The individual components of this commercially available product have demonstrated immune stimulating potential and antitumor properties (Smith 2002, Monograph 2003).
Combined in its present formulation MC-S has been demonstrated to have a proliferative effect on PBLs in vitro (Clark 2007a) and reduced colds, flu and secondary infections in vivo (Clark 2007b).
Ganaderma Lucidum (lingzhi or reishi) is a highly regarded traditional Chinese medicine for enhancing the body's resistance to disease and consolidating the constitution of patients (Lin 2005). Studies on the polysaccharide extracts of Ganaderma lucidum, mainly in the form of (1?3)-[beta]-D-glucans, have demonstrated mitogenicity and activation of immune effector cells (Smith 2002, Zhou 2002) as well as a stimulating effect on the production of cytokines (Smith 2002, Goa 2004).
In vitro studies have also shown inhibitory effects on breast cancer cells (Sliva 2002, 2003, 2006, Hu 2002, Jiang 2006), prostate cancer cells (Sliva 2006, Sliva 2002, Sliva 2003, Jiang 2004, Stanley 2005), leukemia, lymphoma, myeloma cells (Sliva 2006, Muller 2006) and colon cancer cell lines (Sliva 2006, Hong 2004).
Lentinus edodes (Shiitake mushroom) is a common edible mushroom. The noted extracts, LEM (from Lentinus edodes mycelium) and Lentinan, are both proven immunomodulators augmenting activation and proliferation of peripheral mononuclear cells (Aoki 1984, Hobbs 2000). Lentinan shows no direct inhibition of growth on tumor cell lines in vitro (Ooi 2000).
Coriolus versicolor contains two bioactive polysaccharides that are chemically similar; polysaccharide peptide (PSP) and polysaccharide-K (PSK) also known as krestin. These differ by the presence of fucose on PSK and rhamnose and arabinose in PSP (Smith 2002, Kidd 2000). Both are potent immunomodulators and induce INF gamma and IL2 production (Kidd 2000, Tzianabos 2000).
Extracts of Coriolus versicolor have been shown to have inhibitory effects in vitro on gastric cancer, lung cancer, leukemia, lymphoma cell lines (Ooi 2000, Chu 2002, Xu 1999). Similar results were obtained by others using another leukemic cell line, liver cancer and stomach cancer cell lines (Chu 2002, Yang 1992, 1993, Dong 1997).
Finally extracted polysaccharides and saponins from Astragalus membranaceous have been shown both in vitro and in vivo to stimulate NK cell activity and PBL proliferation (Monograph 2003, Block 2003). Significant proliferation suppression was noted with macrophage like myeloid and lymphiod tumor cell lines (Cho 2007) and colon cancer cells (Tin 2007).
This study examines the immune modulating product MC-S to determine whether its herbal based formulation has potential effect on a diverse range of human cancer cell lines in vitro. Each of the cancer cell lines represents a cancer class common to man. The types of cancer covered in this study are breast, prostate, colon and melanoma all of which have a profound impact world wide.
Chemicals and reagents
Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, trypsin/EDTA, and L- glutamine 2mM were all Gibco, Invitrogen (Carlsbad California USA). Heat inactivated fetal bovine serum (FCS) was purchased from Sigma (St Louis USA).
Metabolic Cell-Support (MC-S)
MC-S (without ascorbic acid) (supplied by Clinical Health Pty Ltd, Newcastle Australia) was used in powdered form. The ascorbic acid was excluded from the in vitro testing due to its acidity and potential inhibitory effect on the tissue culture. Prior to testing a stock solution of MC-S was prepared in sterile water (100 000 [micro]g mL-1). The test material was then prepared by diluting this stock to a final concentration of 781.25[micro]g mL-1 in DMEM complete with 10% FCS and sterile filtering through a 0.22 micron filter (Sartorius, Hannover Germany). Inhibitory efficacy of this test material was studied using 1:4 dilutions to a minimum concentration of 0.0015 [micro]g mL-1 in DMEM complete with 10% FCS.
Four cancer cell lines were acquired for this study. Human melanoma cell line MM200 was kindly supplied by Oncology Immunology Newcastle Australia. Human breast cancer cell MCF7, human prostate cancer cell line DU145 and human colonic carcinoma cell line HT29 were all kindly supplied by Inter-K Newcastle Australia. All cell lines were seeded on to 85 cm2 culture flasks (Cellstar Greiner Bio-one) and raised in DMEM and 10% FCS (v/v) until 95% confluency then harvested with Trypsin/EDTA, washed 3 times with PBS before use in the proliferation assay at a concentration of 1x105 mL-1.
Cell proliferation assay
An amount of 50 [micro]L of each dilution of MC-S test solution was added to six wells of a 96 well flat bottom culture plate (Nunc, Roskilde Denmark). To three wells of each dilution set, 50 [micro]L of cancer cell suspension was added giving a final concentration of 5 x 103 cells per well. In the remaining three wells for each dilution set were added 50 [micro]L of DMEM complete media and 10% FCS (v/v) as a background control. Controls for this assay were non treated (positive control). Plates were incubated at 370C in a CO2 incubator for 48 or 72 hours. Proliferation was measured using CellTiter 96 AQueous Non-Radioactive Cell proliferation assay kit (Promega Corporation, Madison Wisconsin USA) as per manufacturers' instructions with absorbance measured at 490 nm using a Bio-Rad microplate reader model 680 (Bio-Rad Laboratories Hercules California USA). Absorbance data was calculated as a percentage of inhibition in comparison to non treated control. Results were graphed as mean [+ or -] SE of 3-4 separate assays.
Statistical analysis was performed using T-test analysis for two samples assuming equal variances, two tailed using means on percentage of decrease of each assay (Microsoft Excel). A p-value of less than 0.05 was considered to be significant.
The exposure of 4 cancer cell lines to varying doses of MC-S over a 48 hour and 72 hour period mediated an inhibitory response at most doses (Fig 1). The melanoma cell line MM200 (Fig 1A) after 48 hours displayed significant inhibition of proliferation at all doses with peak inhibition at a dose of 390.625 [micro]g/mL with 25.6 [+ or -] 1.3%. After a period of 72 hours incubation significant inhibition at all doses except 1.526 [micro]g/mL was observed peaking at 390.625 [micro]g/mL with 37.7 [+ or -] 2.7% inhibition. The prostate cancer cell line DU145 (Fig 1B) after 48 hours exhibited significant inhibition at all doses above 0.006 [micro]g/mL in a dose dependant manner peaking at 390.625 [micro]g/mL with 22.4 [+ or -] 2.1% inhibition. At 72 hours significant inhibition at all doses in a dose dependant manner from 0.381 [micro]g/mL peaking at 390.625 [micro]g/mL with 24.1 [+ or -] 3.2% inhibition.
The breast cancer cell line MCF7 (Fig 1C) after 48 hours displayed significant inhibition at all doses with peak inhibition of 19.7 [+ or -] 2.7% at 390.625 [micro]g/mL. After 72 hours significant inhibition at all doses except at 1.526 and 6.104 [micro]g/mL peaking at a dose of 24.414 [micro]g/mL with 34 [+ or -] 9.2% inhibition. The colorectal adenocarcinoma cell line HT29 (Fig 1D) after 48 hours displayed significant inhibition at all doses except 0.381 and 1.526 [micro]g/mL in a dose dependant manner peaking at 390.625 [micro]g/mL with 18.2 [+ or -] 3.9 % inhibition.
[FIGURE 1 OMITTED]
After 72 hours data displayed significant inhibition at all doses in a dose dependant manner again peaking at a dose of 390.625 [micro]g/mL with 27.5 [+ or -] 0.4% inhibition. Although there is an obvious increase in inhibition of proliferation from 48 hours to 72 hours incubation only a few doses showed statistically significant difference between the two time points. These are indicated on graphs (A) and (C) of figure 1. Results represent the mean of 3-4 experiments performed in triplicate. Graph (A) MM200 melanoma cell; line (B) DU145 prostate cancer cell line; (C) MCF7 breast cancer cell line; (D) HT29 human colonic carcinoma cell line.
These experiments demonstrated that MC-S inhibits proliferation of all four cancer cells lines exhibiting a statistically significant difference in most instances between non treated control and MC-S treated cells. These findings compare favourably with the results achieved for the individual medicinal components of MC-S shown in other studies. However most research is usually directed specifically at concentrates of the isolates extracted from the whole fungi and plant making it difficult for direct comparisons. The highest dose of MC-S used in these experiments was 390.63[micro]g/mL in culture medium.
The recommended human dose of MC-S ranges from two 900 mg tablets per day for long term use to six tablets per day for short term use, resulting in a daily dose of 1.8-5.4 g/day of the MC-S concentrate bearing in mind that each tablet contains 250 mg of ascorbic acid.
While it is impossible to correlate human doses with cell culture concentration in the absence of data on serum concentrations of MC-S, we can only speculate that the cell culture conditions may be comparable to human exposure (Hong 2004). In this study the concentration of the known active compounds would be relatively low when compared with its purified counterparts lentinan, PSP and PSK. This makes the results of the study more significant as it takes large quantities of mushroom or herb to gain useable amounts of active compounds.
Previously published data demonstrated MC-S at a comparable range of doses promoted PBL proliferation in vitro (Clark 2007a). This would suggest that MC-S is selective in its action by inhibiting cancer cell proliferation while enhancing PBL proliferation. As mentioned in the introduction isolates of the individual components of MC-S have yielded similar results.
Most of the major polysaccharides extracted from medicinal mushrooms have been subject to preclinical studies (Smith 2002, Sullivan 2006). The degree of safety testing on mushroom products is in general more advanced than that for most herbal products (Smith 2002, Sullivan 2006, Wasser 2000). Further to this, large scale clinical trials utilising Lentinan and other medicinal mushroom derivatives have not reported any serious adverse reactions or evidence of serious drug/drug interactions (Smith 2002, Sullivan 2006). In comparison to conventional chemotherapy, mushroom polysaccharides appear to be benign (Sullivan 2006).
The clinical efficacy of the individual components of MC-S has been established and utilised clinically in Asian countries. The Western world has only in recent years realised the potential of complementary medicines. There is however the need for scientific validation of potential clinical applications. Therapeutic drug agencies such as the TGA in Australia and the FDA in the USA monitor claims made of complementary therapies, but still a gap exists between accepted applications of whole or parts of plants and fungi, isolated active agents and combinations of both. Efficacy re-trialing is again required when these products are turned into pharmaceutical preparations. Unfortunately worldwide there are many products on the market which could be considered inadequately scientifically reviewed.
This study demonstrates that MC-S actively inhibits the growth of breast, prostate, melanoma and bowl cancer cells in vitro. In contrast historical data has shown MC-S to promote PBL proliferation in vitro, indicating that MC-S is selective in its action.
This selectivity suggests that MC-S may offer additional benefits to standard cancer therapies, although further mechanistic and clinical studies are needed to support these findings.
This study was undertaken at the University of Newcastle Australia. The authors would like to acknowledge that the funding for this study was provided by Metabolic Health Pty Ltd (Newcastle Australia).
The authors of this paper would like to acknowledge Dr Xu Dong Zhang, Immunology and Oncology Unit, Calvary Mater Newcastle Hospital, Newcastle Australia for his encouragement and valued advice.
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David Clark is a Technical Officer who has over 20 years of experience in Medical Research both with Hunter New England Health and with the University of Newcasle. His field of expertise is immunology. The areas of research covered in his career are transplant immunology, study of cancer cell lines, drug development, cell culture and flow cytometric procurement and analysis.
Dr Michelle Adams is a senor lecturer at the University of Newcastle, Faculty of Science and IT. She has been working for the University of Newcastle since 1998. Her primary areas are health (allied health), science and technology. Her field of expertise is probiotics, direct fed microbials, food microbiology, food poisoning and toxicity, food saftey and microbiology.
DA Clark *
Newcastle Innervation, Industry Development Centre, University Drive, Callaghan NSW 2308
School of Environmental and Life Sciences, Faculty of Science and IT, University of Newcastle Callaghan NSW 2308
* Corresponding author email email@example.com