Polysaccharides extracted from the mushroom Grifola frondosa (GFP) are a potential anticancer agent. The objective of this study was to investigate the effect of GFP and vitamin C (VC) alone and in combination on the viability of human hepatocarcinoma SMMC-7721 and HepG2 cells. Studies designed to detect cell apoptosis and autophagy were also conducted to investigate the mechanism. Results from the cell viability assay indicated that a combination of GFP (0.2 or 0.25 mg/mL) and VC (0.3 mmol/L) (GFP/VC) led to 52.73 and 53.93% reduction in cell viability of SMMC-7721 and HepG2 cells separately after 24 h. Flow cytometric analysis indicated that GFP/VC treatment induced cell cycle arrest at the G2/M phase, and apoptosis occurred in approximately 43.62 and 42.46% of the SMMC-7721 and HepG2 cells separately. Moreover, results of Hoechst33258 and monodansylcadaverine staining, and transmission electron microscopy, showed that GFP/VC induced apoptosis and autophagy in SMMC-7721 and HepG2 cells. Western blot analysis showed changes in the expression of apoptosis-related proteins [upregulation of BAX and caspase-3, downregulation of Bcl-2, and activation of poly-(ADP-ribose)-polymerase] and autophagy protein markers (upregulation of beclin-1 and microtubule-associated protein 1A/1B light chain-3). We also demonstrated that the expression of both Akt and p-Akt was enhanced, suggesting the PI3K/Akt/mTOR pathway might not be involved in this process. Our study shows that the combined application of GFP and VC induced cell apoptosis and autophagy in vitro, and might have antitumor activity in vivo.
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Polysaccharides from Grifola frondosa
- IC50 :
50% inhibitory concentration
Dulbecco’s Modified Eagle’s Medium
Microtubule-associated protein 1 light chain 3
Phosphate buffered saline
Kessler SM, Barghash A, Laggai S, Helms V, Kiemer AK (2015) Hepatic hepcidin expression is decreased in cirrhosis and HCC. J Hepatol 62:977–979
Leake I (2014) Hepatocellular carcinoma: treatment potential of targeting Toll-like receptors in HCC. Nat Rev Gastroenterol Hepatol 11:518
Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. Cancer J Clin 55:74–108
Stefaniuk P, Cianciara J, Wiercinska-Drapalo A (2010) Present and future possibilities for early diagnosis of hepatocellular carcinoma. World J Gastroenterol 16:418–424
Rampone B, Schiavone B, Martino A, Viviano C, Confuorto G (2009) Current management strategy of hepatocellular carcinoma. World J Gastroenterol 15:3210–3216
Ivanov V, Roomi MW, Kalinovsky T, Niedzwiecki A, Rath M (2007) Anti-atherogenic effects of a mixture of ascorbic acid, lysine, proline, arginine, cysteine, and green tea phenolics in human aortic smooth muscle cells. J Cardiovasc Pharmacol 49:140–145
da Rocha AB, Lopes RM, Schwartsmann G (2001) Natural products in anticancer therapy. Curr Opin Pharmacol 1:364–369
Chua CW, Choo SP (2011) Targeted therapy in hepatocellular carcinoma. Int J Hepatol 2011:348297
Martin KR, Brophy SK (2010) Commonly consumed and specialty dietary mushrooms reduce cellular proliferation in MCF-7 human breast cancer cells. Exp Biol Med (Maywood) 235:1306–1314
Shomori K, Yamamoto M, Arifuku I, Teramachi K, Ito H (2009) Antitumor effects of a water-soluble extract from Maitake (Grifola frondosa) on human gastric cancer cell lines. Oncol Rep 22:615–620
Wang CL, Meng M, Liu SB, Hou LH, Cao XH (2013) A chemically sulfated polysaccharide from Grifola frondosa induces HepG2 cell apoptosis by notch1-NF-kB pathway. Carbohydr Polym 95:282–287
Soares R, Meireles M, Rocha A, Pirraco A, Obiol D, Alonso E, Joos G, Balogh G (2011) Maitake (D-fraction) mushroom extract induces a poptosis in breast cancer cells by BAK-1 gene activation. J Med Food 14:563–572
Lee S, Kim J, Jung S, Li C, Yang Y, Kim KI, Lim JS, Kim Y, Cheon CI, Lee MS (2015) SIAH1-induced p34SEI-1 polyubiquitination/degradation mediates p53 preferential vitamin C cytotoxicity. Int J Oncol 46:1377–1384
Leon IE, Di Virgilio AL, Porro V, Muglia CI, Naso LG, Williams PA, Bollati-Fogolin M, Etcheverry SB (2013) Antitumor properties of a vanadyl (IV) complex with the flavonoid chrysin [VO(chrysin)2 EtOH]2 in a human osteosarcoma model: the role of oxidative stress and apoptosis. Dalton Trans 42:11868–11880
Rajnikumar S, Sudharsan PT, Rajneesh P, Ahmad S, Kutiyanawalla A, Kolhe R, Bhattacharyya MH, Chutkan N, Hunter M, Hill WD, Hamrick M, Isales C, Fulzele S (2015) The crucial role of vitamin C and its transporter (SVCT2) in bone marrow stromal cell autophagy and apoptosis. Stem Cell Res 15:312–321
Lv L, Zhou Z, Huang X, Zhao Y, Zhang L, Shi Y, Sun M, Zhang J (2010) Inhibition of peptidyl-prolyl cis/trans isomerase Pin1 induces cell cycle arrest and apoptosis in vascular smooth muscle cells. Apoptosis 15(1):41–54
Yang YH, Chen K, Li B, Chen JW, Zheng XF, Wang YR, Jiang SD, Jiang LS (2013) Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway. Apoptosis 18(11):1363–1375
Shi S, Wang Q, Xu J, Jang JH, Padilla MT, Nyunoya T, Xing C, Zhang L, Lin Y (2015) Synergistic anticancer effect of cisplatin and Chal-24 combination through IAP and c-FLIPL degradation, Ripoptosome formation and autophagy-mediated apoptosis. Oncotarget 6:1640–1651
Biederbick A, Kern HF, Elsässer HP (1995) Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66:3–14
Mizushima N (2004) Methods for monitoring autophagy. Int J Biochem Cell Biol 36:2491–2502
Yip KW, Reed JC (2008) Bcl-2 family proteins and cancer. Oncogene 27:6398–6406
Soldani C, Scovassi AI (2002) Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update. Apoptosis 7:321–328
Miriam J Alter (2007) Epidemiology of hepatitis C virus infection. World J Gastroenterol 13:2436–2441
Wang XW, Hussain SP, Huo TI, Wu CG, Forgues M, Hofseth LJ, Brechot C, Harris CC (2002) Molecular pathogenesis of human hepatocellular carcinoma. Toxicology 181–182:43–47
Wang J, Xiong X, Xing Y, Liu Z, Jiang W, Huang J, Feng B (2013) Chinese herbal medicine for acute mountain sickness: a systematic review of randomized controlled trials. Evid Based Complement Altern Med 2013:732562
Zhou F, Hao YF, Chen Y, Wang T (2013) Chinese herbal medicine in treatment of polyhydramnios:a meta-analysis and systematic review. Chin Med Sci J 28:72–81
Soares R, Meireles M, Rocha A, Pirraco A, Obiol D, Alonso E, Joos G, Balogh G (2011) Maitake (D-fraction) mushroom extract induces apoptosis in breast cancer cells by BAK-1 gene activation. J Med Food 14:563–572
Shomori K, Yamamoto M, Arifuku I (2009) Antitumor effects of a water-soluble extract from Maitake (Grifola frondosa) on human gastriccancer cell lines. Oncol Rep 22:615–620
Valenti MT, Zanatta M, Donatelli L (2014) Ascorbic acid induces either differentiation or apoptosis in MG-63 osteosarcoma lineage. Anticancer Res 34:1617–1627
Rajnikumar S, Sudharsan PT, Rajneesh P (2015) The crucial role of vitamin C and its transporter (SVCT2) in bone marrow stromal cell autophagy and apoptosis. Stem Cell Res 15:312–321
Martin KR, Brophy SK (2010) Commonly consumed and specialty dietary mushrooms reduce cellular proliferation in MCF-7 human breast cancer cells. Exp Biol Med 235:1306–1314
Wang Y, Liu J, Qiu Y, Jin M, Chen X, Fan G, Wang R, Kong D (2016) ZSTK474, a specific class I phosphatidylinositol 3-kinase inhibitor, induces G1 arrest and autophagy in human breast cancer MCF-7 cells. Oncotarget 7:19897–19909
Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA, Malorni W, Nuñez G, Peter ME, Tschopp J, Yuan J, Piacentini M, Zhivotovsky B, Melino G (2009) Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 16:3–11
Goldar S, Khaniani MS, Derakhshan SM, Baradaran B (2015) Molecular mechanisms of apoptosis and roles in cancer development and treatment. Asian Pac J Cancer Prev 16:2129–2144
Chipuk JE, Bouchier-Hayes L, Green DR (2006) Mitochondrial outer membrane permeabilization during apoptosis: the innocent bystander scenario. Cell Death Differ 13:1396–1402
Czabotar PE, Lessene G, Strasser A, Adams JM (2014) Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol 15:49–63
Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, Levine B (1999) Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402:672–676
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728
Aoki H, Takada Y, Kondo S, Sawaya R, Aggarwal BB, Kondo Y (2007) Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways. Mol Pharmacol 72:29–39
Ellington AA, Berhow MA, Singletary KW (2006) Inhibition of Akt signaling and enhanced ERK1/2 activity are involved in induction of macroautophagy by triterpenoid B-group soyasaponins in colon cancer cells. Carcinogenesis 27:298–306
Qian WB, Liu JQ, Jin J, Ni WM, Xu WL (2007) Arsenic trioxide induces not only apoptosis but also autophagic cell death in leukemia cell lines via up-regulation of beclin-l. Leuk Res 31:329–339
Kanzawa T, Zhang L, Xiao L, Germano IM, Kondo Y, Kondo S (2005) Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein BNIP3. Oncogene 24:980–991
Duan WJ, Li QS, Xia MY, Tashiro S, Onodera S, Ikejima T (2011) Silibinin activated p53 and induced autophagic death in human fibrosarcoma HT1080 cells via reactive oxygen species-p38 and c-Jun N-terminal kinase pathways. Biol Pharm Bull 34:47–53
This work was supported by the Fundamental Research Funds for the Central Universities from Northwest Minzu University, China (Nos. 31920140070, 31920160009), The Science and Technology Project of Lanzhou City, China (No. 2016-3-37). And The National Natural Science Foundation of China (31560254; 81260442).
Conflict of interest
The authors declare that there are no conflicts of interest.
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Zhao, F., Zhao, J., Song, L. et al. The induction of apoptosis and autophagy in human hepatoma SMMC-7721 cells by combined treatment with vitamin C and polysaccharides extracted from Grifola frondosa . Apoptosis 22, 1461–1472 (2017). https://doi.org/10.1007/s10495-017-1421-z
- Grifola frondosa
- Vitamin C