The induction of apoptosis and autophagy in human hepatoma SMMC-7721 cells by combined treatment with vitamin C and polysaccharides extracted from Grifola frondosa

Abstract

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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Abbreviations

GFP:

Polysaccharides from Grifola frondosa

HCC:

Hepatocellular carcinoma

IC50 :

50% inhibitory concentration

MTT:

3-(4,5-Dimethythiazol-2-yl)-2,5-diphe-nyl-tetrazolium bromide

DMEM:

Dulbecco’s Modified Eagle’s Medium

LC3:

Microtubule-associated protein 1 light chain 3

MDC:

Monodansylcadaverine

VC:

Vitamin C

PBS:

Phosphate buffered saline

PI3K:

Phosphatidylinositide 3-kinase

PARP:

Poly-(ADP-ribose)-polymerase

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Acknowledgements

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).

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Correspondence to Ke-Hu Yang.

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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

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Keywords

  • Anticancer
  • Apoptosis
  • Autophagy
  • Grifola frondosa
  • Vitamin C
  • Hepatoma