Phellinus Linteus Extract Induces Autophagy and Synergizes With 5-Fluorouracil to Inhibit Breast Cancer Cell Growth

Wednesday, 11/03/2015  |   Breast Cancer, Cancer  |  no comments

imagespic03

ABSTRACT
Phellinus linteus (PL) is a medicinal mushroom due to its several biological properties, including anticancer activity. However, the mechanisms of its anticancer effect remain to be elucidated. We evaluated the inhibitory effects of the ethanolic extract from the PL combined with 5-FU on MDA-MB-231 breast cancer cell line and to determine the mechanism of cell death. Individually, PL extract and 5-FU significantly inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner. PL extract (30 mg/mL) in combination with 5-FU (10 ?g/mL) synergistically inhibited MDA-MB-231 cells by 1.8-fold. PL did not induce apoptosis, as demonstrated by the DNA fragmentation assay, the sub-G1 population, and staining with annexin V-FITC and propidium iodide. The exposure of MDA-MB-231 cells to PL extracts resulted in several confirmed characteristics of autophagy, including the appearance of autophagic vacuoles revealed by monodansylcadaverine staining, the formation of acidic vesicular organelles, autophagosome membrane association of microtubule-associated protein light chain 3 (LC3) characterized by cleavage of LC3 and its punctuate redistribution, and ultrastructural observation of autophagic vacuoles by transmission electron microscopy. We concluded that PL extracts synergized with low doses of 5-FU to inhibit triple-negative breast cancer cell growth and demonstrated that PL extract can induce autophagy-related cell death.

INTRODUCTION
Metastasis is the main cause of therapeutic failure and death for breast cancer patients. To improve the prognosis of these patients, adjuvant chemotherapy is often used in a variety of clinical situations. However, the toxicity of these chemotherapeutic agents to normal tissues has been a major obstacle to successful cancer treatment. The antimetabolite 5-fluorouracil (5-FU) is a major chemotherapeutic agent for breast cancer treatment (1), and the mechanism of its cytotoxicity is the misincorporation of fluoronucleotides into DNA and RNA, thus inhibiting the normal function of these nucleic acids. Therefore, the development of less toxic and more effective anticancer drugs that can be used in combination with existing therapeutic agents may enhance treatment outcomes and reduce the toxicity of the existing drugs. Fungi have been intensively investigated as anticancer agents because they are able to modulate the immune responses against cancers with very low toxic potential (2) and thus represent a potentially important new source of anticancer agents.

Phellinus linteus (PL), a basidiomycete fungus, is commonly called Sangwhang in Taiwan and has gained significant recognition as a medicinal mushroom in traditional Oriental medicine (3). Studies have demonstrated that the extracts from the fruiting bodies or mycelium of PL not only stimulate immune function but also suppress tumor growth and metastasis in vitro (3–6). In vivo studies have also demonstrated that PL extracts can cause tumor regression (7). However, the mechanisms of its anticancer effect remain to be elucidated. Several studies have demonstrated that PL inhibits the metastasis of melanoma cells in mice through the regulation of urokinase-type plasminogen activator (8) and suppresses the growth of lung, prostate, and colon cancer cells by inducing cell cycle arrest and apoptosis (9–13). PL was also demonstrated to suppress the growth, angiogenesis, and invasion through the inhibition of AKT phosphorylation in breast cancer cells (14) and through the inhibition of Wnt/?-catenin signaling in colon cancer cells (15).

There are 3 reported cases of dramatically regressed cancers after treatment with PL, including 2 cases from Japan: 1 hormone-refractory prostate cancer with bone metastasis (16) and 1 hepatocellular carcinoma with multiple lung metastases (17); the third case occurred in Korea and was a hepatocellular carcinoma with skull metastasis (18). All of these cases suggested a linear relationship between the usage of PL and tumor regression. In addition, PL inhibited the growth of various prostate cancer cell lines without toxic effects on normal prostate epithelial cells (10) and reduced tumor growth and pulmonary metastasis without toxic effects in mice (6). Moreover, PL have been shown to synergize with doxorubicin in its noncytotoxic dose range to induce apoptosis in prostate and lung cancer cells (9,10), suggesting that PL can also function as an adjunct in cancer treatment to reduce the doses of conventional chemotherapeutic drugs and limit cytotoxicity.

Anticancer therapeutics activate several signal transduction pathways that regulate programmed cell death in cancer cells. Understanding the mechanisms of programmed cell death and designing specific therapeutic approaches to induce cell death in cancer cells are critical for cancer treatment (19). There are 2 morphologically distinct forms of programmed cell death: apoptosis and autophagic cell death. Traditional cancer therapies primarily aim to enhance apoptosis. However, cancer cells are often deficient in the induction of apoptosis, which results in resistance to most anticancer therapies (19,20). Thus, understanding the regulation and significance of the nonapoptotic form of programmed cell death in cancer therapy is critical to optimizing cancer therapy. Autophagic cell death is characterized by the massive degradation of essential organelles such as mitochondria. These intracellular contents are sequestered in a membrane-bound vesicle known as an autophagosome and then degraded following lysosomal fusion (21–23). Evidences indicate that autophagy plays a significant role in cancer initiation and progression. Nearly all therapeutic modalities currently used in cancer therapy, including cytotoxic chemotherapy, radiation, kinase inhibitors, and hormone therapy, can induce autophagy in cancer cells (21–23). Multiple studies have demonstrated that there is molecular cross-talk between autophagy and apoptosis (24), that the same stimulus can simultaneously induce both apoptosis and autophagy (25), and that 2 processes can be mutually exclusive, with each acting as a backup for the other (23).

PL has been shown to have anticancer effects in vitro and in vivo (3–7), but the underlying mechanism remained to be elucidated. PL was reported to induce apoptosis in various types of cancer, including colon, lung, prostate and melanoma cells (9–13,34). However, our study revealed that PL did not induce apoptosis detected by the DNA fragmentation assay, sub-G1 population, and annexin V-FITC/PI double staining in breast cancer cells.

Collins et al. showed that PL and the anti-cancer drug doxorubicin (Dox) did not induce apoptosis in prostate cancer cells at relatively low doses; however, the combination treatment with low doses of PL and Dox resulted in a synergistic effect on the induction of apoptosis (10). Guo et al. demonstrated that PL modulated cell cycle arrest at a low dose and induced apoptosis at a high dose in lung cancer cells (9). Taken together, the dose of PL used in this study may be the major reason for the differences from the previous studies. In addition, the mycelial species, extraction method (hot water vs. ethanolic), culture conditions and cell lines may affect the results.

Source
Lee W-y, Hsu K-F, Chiang T-A. Nutrition and Cancer. Volume 67, Issue 2, 2015. DOI: 10.1080/01635581.2015.989374
References
1. Wyatt MD and Wilson DM, III: Participation of DNA repair in the response to 5-fluorouracil. Cell Mol Life Sci 66, 788–799, 2009.
2. Sullivan R, Smith JE, and Rowan NJ: Medicinal mushrooms and cancer therapy: translating a traditional practice into Western medicine. Perspect Biol Med 49, 159–170, 2006.
3. Zhu T, Kim SH, and Chen CY: A medicinal mushroom: Phellinus linteus. Curr Med Chem 15, 1330–1335, 2008.
4. Kim GY, Lee JY, Lee JO, Ryu CH, Choi BT, : Partial characterization and immunostimulatory effect of a novel polysaccharide-protein complex extracted from Phellinus linteus. Biosci Biotechnol Biochem 70, 1218–1226, 2006.
5. Han SB, Lee CW, Kang JS, Yoon YD, Lee KH, : Acidic polysaccharide from Phellinus linteus inhibits melanoma cell metastasis by blocking cell adhesion and invasion. Int Immunopharmacol 6, 697–702, 2006.
6. Han SB, Lee CW, Jeon YJ, Hong ND, Yoo ID, : The inhibitory effect of polysaccharides isolated from Phellinus linteus on tumor growth and metastasis. Immunopharmacology 41, 157–164, 1999.
7. Tsuji T, Du W, Nishioka T, Chen L, Yamamoto D, : Phellinus linteus extract sensitizes advanced prostate cancer cells to apoptosis in athymic nude mice. PLoS One 5, e9885, 2010.
8. Lee HJ, Lee HJ, Lim ES, Ahn KS, Shim BS, : Cambodian Phellinus linteus inhibits experimental metastasis of melanoma cells in mice via regulation of urokinase type plasminogen activator. Biol Pharm Bull 28, 27–31, 2005.
9. Guo J, Zhu T, Collins L, Xiao ZX, Kim SH, : Modulation of lung cancer growth arrest and apoptosis by Phellinus Linteus. Mol Carcinog 46, 144–154, 2007.
10. Collins L, Zhu T, Guo J, Xiao ZJ, and Chen CY: Phellinus linteus sensitises apoptosis induced by doxorubicin in prostate cancer. Br J Cancer 95, 282–288, 2006.
11. Zhu T, Guo J, Collins L, Kelly J, Xiao ZJ, : Phellinus linteus activates different pathways to induce apoptosis in prostate cancer cells. Br J Cancer 96, 583–590, 2007.
12. Li G, Kim DH, Kim TD, Park BJ, Park HD, : Protein-bound polysaccharide from Phellinus linteus induces G2/M phase arrest and apoptosis in SW480 human colon cancer cells. Cancer Lett 216, 175–181, 2004.
13. Park HJ, Choi SY, Hong SM, Hwang SG, and Park DK: The ethyl acetate extract of Phellinus linteus grown on germinated brown rice induces G0/G1 cell cycle arrest and apoptosis in human colon carcinoma HT29 cells. Phytother Res 24, 1019–1026, 2010.
14. Sliva D, Jedinak A, Kawasaki J, Harvey K, and Slivova V: Phellinus linteus suppresses growth, angiogenesis and invasive behaviour of breast cancer cells through the inhibition of AKT signalling. Br J Cancer 98, 1348–1356, 2008.
15. Song KS, Li G, Kim JS, Jing K, Kim TD, : Protein-bound polysaccharide from Phellinus linteus inhibits tumor growth, invasion, and angiogenesis and alters Wnt/beta-catenin in SW480 human colon cancer cells. BMC Cancer 11, 307, 2011.
16. Shibata Y, Kurita S, Okugi H, and Yamanaka H: Dramatic remission of hormone refractory prostate cancer achieved with extract of the mushroom, Phellinus linteus. Urol Int 73, 188–190, 2004.
17. Nam SW, Han JY, Kim JI, Park SH, Cho SH, : Spontaneous regression of a large hepatocellular carcinoma with skull metastasis. J Gastroenterol Hepatol 20, 488–492, 2005. [CrossRef], [PubMed], [Web of Science ®]
18. Kojima H, Tanigawa N, Kariya S, Komemushi A, Shomura Y, : A case of spontaneous regression of hepatocellular carcinoma with multiple lung metastases. Radiat Med 24, 139–142, 2006.
19. Melet A, Song K, Bucur O, Jagani Z, Grassian AR, : Apoptotic pathways in tumor progression and therapy. Adv Exp Med Biol 615, 47–79, 2008.
20. Johnstone RW, Ruefli AA, and Lowe SW: Apoptosis: a link between cancer genetics and chemotherapy. Cell 108, 153–164, 2002.
21. Kondo Y and Kondo S: Autophagy and cancer therapy. Autophagy 2, 85–90, 2006.
22. Kung CP, Budina A, Balaburski G, Bergenstock MK, and Murphy M: Autophagy in tumor suppression and cancer therapy. Crit Rev Eukaryot Gene Expr 21, 71–100, 2011. [CrossRef], [PubMed], [Web of Science ®]
23. Maycotte P and Thorburn A: Autophagy and cancer therapy. Cancer Biol Ther 11, 127–137, 2011.
34. Park HJ, Han ES, and Park DK: The ethyl acetate extract of PGP (Phellinus linteus grown on Panax ginseng) suppresses B16F10 melanoma cell proliferation through inducing cellular differentiation and apoptosis. J Ethnopharmacol 132, 115–121, 2010.

Leave a Reply

You must be logged in to post a comment.