Effect And Mechanism Of Autophagy Of Colorectal Cancer Cells Induced By Licochalcone Ain Its Anticancer Effect

Objective:To clarify the effect of Licochalcone A(LA)on colorectal cancer by in vitro cellular assays and biological techniques.Methods:MTT assay,cell clone formation experiment and EdU staining were used to detect the effects of LA at different concentrations and different time on the proliferation of colorectal cancer cells(SW620,HCT116,HT29)and normal human colonic epithelial cells.The effect of LA on SW620 cell cycle was detected by PI staining and flow cytometry.Annexin V and PI staining were used to detect the changes of apoptosis after SW620 was treated with LA and inhibitors(autophagy inhibitor CQ and ERK inhibitor U0126).Western blot and immunofluorescence were used to detect the expression of related proteins(MAPKs,autophagy marker protein,apoptosis-related protein and heat shock protein)after SW620 was treated with LA and inhibitors(CQ,U0126 and Hsp70 inhibitor MKT-077).The mRNA expression of heat shock protein and c-FLIPL was detected by real-time quantitative RT-PCR.Results:①LA inhibited the proliferation of colorectal cancer cells SW620,HCT116 and HT29 in dose and time depend manner,but had little effect on NCM460 of normal cells.②LA blocked SW620 in G2/M phase.③LA induced apoptosis of SW620 cells by inhibiting c-FLIPL.④LA induced LC3Ⅱ accumulation in SW620 cells,while autophagy inhibitor CQ enhanced apoptosis and up-regulated cl-caspase-3 and cl-PARP.⑤LA enhanced Hsp70,while CQ inhibited Hsp70.⑥MKT-077,an inhibitor of Hsp70,could inhibit c-FLIPL and up-regulate cl-caspase-3 and cl-PARP,and inducing apoptosis.⑦LA could significantly induce the activation of ERK and p38,but had no significant effect on JNK.⑧U0126,an inhibitor of ERK,promoted LA-induced apoptosis,while SB203580,an inhibitor of p38,had no significant effect on it.⑨U0126 could induce apoptosis by inhibiting c-FLIPL and up-regulating cl-caspase-3 and cl-PARP.⑩U0126 inhibited LA-induced LC3II accumulation.Conclusion:LA was a promising anti-colorectal cancer drug.However,LA increased the expression of Hsp70 through ERK-mediated autophagy to maintain the continuous expression of c-FLIPL,thereby inhibiting apoptosis.Therefore,inhibition of Hsp70 was an effective strategy to enhance the anti-colorectal cancer effect of LA.