Chemical characterization and antitumor activities of polysaccharide extracted from Ganoderma lucidum

Abstract

Ganoderma lucidum polysaccharide (GLP) is a biologically active substance reported to possess anti-tumor ability. Nonetheless, the mechanisms of GLP-stimulated apoptosis are still unclear. This study aims to determine the inhibitory and apoptosis-inducing effects of GLP on HCT-116 cells. We found that GLP reduced cell viability on HCT-116 cells in a time- and dose-dependent manner, which in turn, induced cell apoptosis. The observed apoptosis was characterized by morphological changes, DNA fragmentation, mitochondrial membrane potential decrease, S phase population increase, and caspase-3 and -9 activation. Furthermore, inhibition of c-Jun N-terminal kinase (JNK) by SP600125 led to a dramatic decrease of the GLP-induced apoptosis. Western blot analysis unveiled that GLP up-regulated the expression of Bax/Bcl-2, caspase-3 and poly (ADP-ribose) polymerase (PARP). These results demonstrate that apoptosis stimulated by GLP in human colorectal cancer cells is associated with activation of mitochondrial and mitogen-activated protein kinase (MAPK) pathways.

Figure 1.

The UV spectra of polysaccharide fractions of Ganoderma lucidum.

Figure 2.

Infrared spectroscopy of Ganoderma lucidum polysaccharide (GLP).

Figure 3.

Cytotoxicity of GLP on HCT-116 cells: (A) GLP suppressed the cell viability of HCT-116. Inhibitory rate was measured by MTT method. Starch-incubated cells were applied as control. Data represent means ± SD of three independent experiments; and (B) Morphological changes in HCT-116 cells. After treatment with GLP, exfoliation of HCT-116 cells and naked areas were observed and captured under an inverted microscope (×100). The arrows (↑) show naked areas without cells.

Figure 4.

Effects of GLP on apoptosis in HCT-116 cells. (A) Apoptotic cells measured by Hoechst 33,258 staining after treatment with GLP for 48 h under a fluorescence microscope (×200). The arrows show cell fragments; (B) HCT-116 cells were exposed to the indicated concentrations of GLP for 24 h. DNA was isolated and examined on 1.2% agarose gel; (C) Mitochondrial membrane potential (ΔΨ_m) was monitored by microscopy and photographed at each concentration of GLP; and (D) quantitative evaluation. Data are presented as mean ± SD. a, p < 0.01 compared with control. b, c and d, p < 0.01 compared with 1.25, 2.5 and 5 mg/mL GLP treatment, respectively.

Figure 5.

Flow cytometry analysis of GLP-treated HCT-116 cells. Cells were incubated with GLP at various concentrations (1.25–10 mg/mL) for 24 h and then were harvested for quantifying apoptosis and cell cycle phase by flow cytometry (Propidium Iodide (PI) staining). Apoptosis and S phase cell populations of GLP-treated group are significantly higher than in the control. The blue peak = apoptosis; the first red peak = G1; the second red peak = G2; hatched peak = S.

Figure 6.

Effects of mitogen-activated protein kinase (MAPK) inhibitors on GLP-induced HCT-116 cell death. Cells were treated in the absence or presence of different MAPK-specific inhibitors, 1 h prior to the addition of GLP, and then incubated in 5 mg/mL GLP for 12 h. Cells were harvested to determine the percentage of viable cells as described in the Experimental section. A, p < 0.05 compared with only 5 mg/mL GLP treatment group. Data expressed as mean ± SD.

Figure 7.

GLP induces cell apoptosis via caspase-dependent mitochondrial pathways. (A) Activity of caspases in GLP treated HCT-116 cells. A, p < 0.05, and a, p < 0.01 compared with control. b, p < 0.01 compared with 2.5 mg/mL GLP treatment; (B) Western blot analyses of mitochondria pathway-related proteins. All bands were compared with the β-actin band and indicated as down-regulation (−) or up-regulation (+); and (C) Statistical analysis for western blot analysis.

Figure 7.

GLP induces cell apoptosis via caspase-dependent mitochondrial pathways. (A) Activity of caspases in GLP treated HCT-116 cells. A, p < 0.05, and a, p < 0.01 compared with control. b, p < 0.01 compared with 2.5 mg/mL GLP treatment; (B) Western blot analyses of mitochondria pathway-related proteins. All bands were compared with the β-actin band and indicated as down-regulation (−) or up-regulation (+); and (C) Statistical analysis for western blot analysis.