Induction Effect to Apoptosis by Maitake Polysaccharide: Synergistic Effect of Its Combination With Vitamin C in Neuroglioma Cell

Abstract

Polysaccharide extracted from the Maitake mushroom (MP) is considered as a potential anticancer agent. The present study was performed to investigate the cytotoxic effects of MP and vitamin C (VC) alone and in combination on the viability of human neuroglioma M059 K cells in vitro. A combination of MP (1.0 mg/mL) and VC (0.4 mmol/L) led to a 53.10% reduction in cell viability and this treatment induced cell cycle arrest at the G2/M phase, and apoptosis occurred in 38.54% of the cells. Results of Hoechst 33258 staining and Western blot showed apoptotic cells appeared and 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). Moreover, the activities of caspase-3, caspase-8, and caspase-9 were enhanced in M059 K cells. The inhibiting effect of combined treatment with MP and VC on M059 K cells indicates the mechanism of anticancer activity involved induction of cell apoptosis.

Keywords: Maitake; antitumor; apoptosis; neuroglioma; vitamin C.

Figure 1.

Effects of Maitake polysaccharide (MP) or vitamin C (VC) on M059 K cell viability. Cells were treated with different concentrations of MP (0.1-2.0 mg/mL) (A) or VC (0.1-1.0 mmol/L) (B) for 48 hours. Cell inhibition (absorbance at 490 nm) was determined by a MTT assay and compared with control.

Figure 2.

Synergy effect of the combinations of Maitake polysaccharide (MP) and vitamin C (VC) to inhibit cell proliferation in M059 K cells. (A) MTT assay of combined application with different concentrations MP and VC. (B) Cell inhibition rate for without drugs, 1.0 mg/mL MP, 0.4 mmol/L VC, and MP/VC. Data are expressed as mean ± SD of 3 separate experiments (*P < .5; **P < .01 compared with control).

Figure 3.

Effect of Maitake polysaccharide (MP) or/and vitamin C (VC) on M059 K cell apoptosis. (A) Flow cytometry (FCM) analysis: cell apoptosis was induced by control (without treatment), MP (1.0 mg/mL), VC (0.4 mmol/L), and MP/VC after 48 hours using Annexin V-FITC/propidium iodide staining. The cells are characterized as early apoptosis (bottom right quadrant), late apoptosis (top right quadrant), necrotic (top left quadrant), and healthy cells (bottom left quadrant) based on the FCM results, and the total apoptosis rate is summarized in a bar chart on the right. (B) Histogram of corresponding apoptotic ratios. Data are expressed as mean ± SD of 3 separate experiments. *P < .05; **P < .01 compared with control.

Figure 4.

Effect of Maitake polysaccharide (MP) or/and vitamin C (VC) on cell cycle in M059 K cells. (A) Diagram of cell cycle analyzed by flow cytometry (FCM). Cells were treated with control, Maitake polysaccharide (MP) (1.0 mg/mL), VC (0.4 mmol/L), and MP/VC for 48 hours. (B) Histogram of cell cycle. The results shown are mean ± SD of 3 separate experiments, *P < .05; **P < .01 compared with control.

Figure 5.

Detection of apoptosis in M059 K cells after Maitake polysaccharide (MP) or/and vitamin C (VC) treated by fluorescent staining. After cells were treated with control, MP (1.0 mg/mL), VC (0.4 mmol/L), and MP/VC for 48 hours. Hoechst 33258 staining was performed, the stained M059 K cells were immediately examined by fluorescence microscope. (A) Control group. (B) MP treatment group. (C) VC treatment group. (D-F) Apoptotic cells were stained by Hoechst 33258 dye, the morphological changes of nucleus (nuclear tear, nuclear fragmentation, nuclear hyperchromatism, and nuclear enrichment) were indicated by arrows.

Figure 6.

Effect of Maitake polysaccharide (MP) or/and vitamin C (VC) on expression of apoptosis-related proteins in M059 K cells. Cells were treated with control, MP (1.0 mg/mL), VC (0.4 mmol/L), and MP/VC for 48 hours. (A) Expressions of Bax, Bcl-2, cleaved caspase-3, and PARP were analyzed with Western blotting. In addition, β-actin is shown as a protein loading control. (B) Histogram of Western blotting results. Data are expressed as mean ± SD of 3 separate experiments. ^# P < .05; ^## P < .01 compared with control.

Figure 7.

Activation of caspase-3, caspase-8, and caspase-9 in M059 K cells treated with Maitake polysaccharide (MP), vitamin C (VC), and MP/VC after 48 hours. Caspase-3 activity significantly increased in cells treated with VC and MP/VC. Caspase-8 and caspase-9 activity significantly increased in cells treated with MP/VC. ^* P < .05 compared with control.