The Radio- and Chemo-sensitization Effects of Phytochemicals from Antrodia cinnamomea Mycelia on MCF-7 Breast Tumor Cells

• Main Authors: Dong Meng-Han, 董孟涵
• Other Authors: Chyau Charng-Cherng
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/07670904751232589496

碩士 === 弘光科技大學 === 生物科技研究所 === 101 === Antrodia cinnamomea (AC, also named Antrodia camphorata, Taiwanofungus camphoratus and Ganoderma comphoratum), “niu-chang-chih” or “niu-chang-ku” in Chinese, is an exclusive fungus parasitic on the rot wood of the endemic species Cinnamomum micranthum. The liquid submerged culture has been recognized as the most potential method for producing and replacing the rare sources of wild growing fruiting bodies. However, the secondary metabolites of the mycelia from submerged liquid cultures are quite different from those of the wild fruiting bodies due to the different cultivation methods. According to the Health Annual Report, breast cancer has become the first leading cause of death from cancer among women in Taiwan. The purpose of this study was to evaluate the effects of mycelia extracts and/or polysaccharides from the liquid culture of A. cinnamomea in combined with the chemotherapy or radiotherapy on the proliferative activity of MCF-7 breast cancer cell line. In the study, total of three parts of experiments are conducted to examine the bioactivities of A. cinnamomea mycelia, in which the prepared extracts from secondary metabolites of mycelia are conducted in the first and second part of studies. In Part I, the solvent extraction and SephadexTM LH-20 column fractionation were used to prepare extracts from secondary metabolites of mycelia. While the liquid-liquid partition and Amberlite XAD-7 column adsorption methods for fractionating the extracts with high quantities of triterpenoids were conducted in the Part II experiment. The prepared extracts from these two experiments were applied to evaluate the anticancer activities on breast cancer cell line MCF-7. Additionally, the huge amounts of polysaccharides are enriched in the mycelia. In which, the isolated polysaccharides AC-2 performed by the method of “alkali extraction and acid precipitation” has been found with significant anti-inflammatory effects. In hence, the Part III experiment was to examine the anticancer activities of polysaccharides AC-2 in combining with the chemotherapy drug Doxorubicin on breast cancer for underlying the inhibition mechanisms. Results in the Part I indicate that camphorataimides A and C, antrocinnamomin C, dehydrosulphurenic acid and sulphurenic acid identified by LC/MS/MS analysis were the major components in the sub-fraction 4, which was the highest concentrations of triterpenoids and polyphenolics found in the seven fractions of mycelia extracts. Under the concentrations of 34.6 and 53.4 g/mL of sub-fraction 4, significant inhibition activities up to 29.65 and 37.95 %, respectively were shown in the cell viability when combining treatment with radiotherapy (15 Gy) on MCF-7 cells. The cell death appeared in a late growth phase was found from the cell apoptosis and necrosis by the flow cytometric analysis with the cell apoptosis as the major cause. Western blot analysis reveal that the effects of extract in combining treatment with radiotherapy significantly promote the expressions of pro-apoptotic proteins, such as p53 (202.22 %), p21 (272.75 %) and Bax (197.84 %). In the Part II work, the Amberlite XAD-7 eluate (identified sesamin, camphorataimide C and antroquinonol as the major components) from mycelia extracts presented significant inhibition activity on cancer stem cells with marked effects in low dosage of IC25 (24.4 g/mL) and high dosage of IC50 (61.8 g/mL). In a low dosage IC25 (24.4 µg/mL) of Amberlite XAD-7 eluate and combining treatment with doxorubicin on MCF-7 cells resulted in an inhibition rate of 40.91% and caused a G2/M phase arrest in cell cycle. The apoptosis occurred in early growth phase when a low dosage used, while a higher dosage resulted in an increasing of cell apoptosis in the late growth phase. The combined treatment reduced the anti-apoptotic Bcl-2 protein (61.25 %) expression, and increased expressions of pro-apoptotic proteins such as p53 (219.5 % ), p21 ( 110.65 % ) and Bax ( 155.47 % ). In the Part III study, the combined treatment with AC-2 (1.2 µg/mL) and ionizing radiation (0  6 Gy), resulted in the decreasing of cell proliferation and clonogenic survival in a dose-responsive manner. The cell apoptosis was simultaneously proved by the flow cytometry. By western blot analysis, the expression of apoptotic regulatory molecules were induced through the down-regulation expression of anti-apoptotic Bcl-2 protein (14.74 %), and the up-regulation of pro-apoptotic protein, including p53 (271.04 %), p21 (314.32 %) and Bax (213.51 %). The cell apoptosis was further confirmed by TUNEL staining assay in DNA fragmentation. The observation of the fluorescence from the labeling of polysaccharides AC-2 through the fluorescein isothiocyanate method revealed that the accumulation of polysaccharides AC-2 on MCF-7 cell membranes was time responsive. Furthermore, the inhibition expression of integrin 1 64.05% and apoptotic protein caspase 9 significant increase 181.91% induced by polysaccharides AC-2 was presented in the western blot analysis. Thus, it was concluded that AC-2 could sensitize the effect of radiation in breast carcinoma partly from the regulating effects on cell membrane protein which involved in cell cycle and apoptosis. It is concluded that the apoptosis of MCF-7 cells were enhanced by the combination of doxorubicin or radiotherapy regardless of the sample from secondary metabolites (small molecules) or polysaccharides (macromolecules) according to the above three studies. In which, the choice of polysaccharide AC-2 in combination of radiotherapy might be a new approach for developing effective complementary therapy on breast cancer.