Ampelopsin promotes apoptosis of colon cancer hct116 cells by inhibiting autophagy / 肿瘤

ABSTRACT

Objective: To investigate the inhibitory effects of ampelopsin (AMP) on proliferation, autophagy and apoptosis to human colon cancer cells and its possible mechanism. To observe the function of autophagy in the apoptosis of colon cancer cells as well. Methods: The lentivirus infection method was adopted here to construct double fluorescence [green fluorescent protein (GFP) and red fluorescent protein (RFP)] labeled microtubule-associated protein light chain 3 (LC3) overexpressed recombinant HCT116-RFP-GFP-LC3 cells. The GFP and RFP labeled LC3 expression in HCT116-RFP-GFP-LC3 cells were observed by fluorescence microscope, and Western blotting was used to detect the expression of LC3 in HCT116-RFP-GFP-LC3 cells at the same time. The proliferation inhibition of recombination HCT116-RFP-GFP-LC3 cells and parental HCT116 cells treated with different concentrations of AMP (0, 50, 100, 175, 250, 350, 400 and 450 μg/mL) was detected by CCK-8 assay and the cell morphology was observed. The apoptotic rate of HCT116 cells treated with AMP (0, 25 50, and 75 μg/mL) was detected by FCM method, and the apoptotic-related proteins Bax, nuclear factor-kappa B (NF-κB) and Bcl-2 expression levels were detected by Western blotting. Autophagy induction was detected by counting fluorescence dots triggered by transformation of LC3to LC3Ⅱ visualized by laser confocal microscopy after HCT116-RFP-GFP-LC3 cells treated with AMP (0, 25 50, and 75 μg/mL) alone or combined with bafilomycin A1(Baf A1) (an autophagy inhibitor) (20 nmol/L). After the HCT116 cells were treated with AMP (25 μg/mL) alone or combined with Baf A1 (20 nmol/L), the apoptotic rate was detected by FCM method, and the expression level of autophagy-related proteins LC3Ⅱ and p62 as well as apoptotic-related proteins were detected by Western blotting. Results: RFP-GFP-LC3 could stably express in HCT116-RFP-GFP-LC3 cells, indicating that HCT116-RFP-GFP-LC3 recombinant cells were successfully constructed. The results of CCK-8 assay suggested that AMP could significantly inhibit the proliferation of recombinant and parental cells in a concentration-dependent manner. Meanwhile, there was no significant difference in cell morphology or cell proliferation inhibition rate between these two cells (P > 0.05). The apoptosis rate of HCT116 cells increased remarkably along with the enhancement of AMP concentration which means there is a concentration-dependent manner (all P < 0.05). The expression levels of pro-apoptotic protein Bax and NF-κB were increased, and the apoptosis inhibitory protein Bcl-2 was down-regulated (all P < 0.01). The confocal microscopy results showed that with the increase of AMP concentration, the numbers of autophagy points were significantly increased (P < 0.01), and the expression level of autophagy protein LC3Ⅱ was up-regulated, indicating that AMP could trigger autophagy on colon cancer cells. Comparing with AMP alone group, AMP combined with Baf A1 could increase the apoptosis rate of HCT116 significantly (P < 0.05), and the expression level of apoptotic protein Bax promoted remarkably (P < 0.05). The expression level of the apoptosis inhibitory protein Bcl-2 demoted as well. All these results suggest that autophagy blocking could promote AMP induced apoptosis. Conclusion: AMP can inhibit the proliferation of colon cancer cells HCT116, inducing autophagy and apoptosis; inhibition of autophagy can promote AMP-induced apoptosis on colon cancer cells.