Screening Of Curcumin Analogs For Autophagy Inhibitors And Study On Their Anti-tumor Effect Against Non-small Cell Lung Cancer

Curcumin analogues are a class of small molecules synthesized based on the modifications of curcumin,which have attracted much attention due to their biological activities such as anti-tumor,anti-inflammatory and anti-proliferation.Autophagy is self-clearing life phenomenon that widely existed in eukaryotes.It has important regulatory roles in the development of tumors.Discovery of new small-molecule modulators of autophagy as well as their potential uses as anti-cancer agents would be of great significance.Based on this,we preliminarily screened a series of curcumin analogues synthesized previously in our lab using U87 cells with stable expression of GFP-LC3 B.Then,we detected the levels of LC3 B and SQSTM1/p62,two markers of autophagy,using western blot analysis,and observed autophagic structures under transmission electron microscopy.Among all the compounds we examined,(3E,5E)-1-methyl-3-(3,4-dimethoxybenzylidene)-5-(3-indolemethylene)-piperidine-4-Ketone(CA-5f)and(3E,5E)-1-methyl-3-(4-hydroxybenzylidene)-5-(3-indolemethylene)-piperidin-4-one(CA-5g)showed emerging roles to effectively regulate autophagy.We further introduced autophagy regulators which modulating different stage of autophagy and used the tandem mRFP-GFP-LC3 plasmid to study the mechanism of CA-5f in autophagy regulation.Our results suggested that CA-5f inhibits autophagosome-lysosome fusion independent of the hydrolysis function or the pH of lysosome.Data from cell viability assays revealed that CA-5f has significant cytotoxicity on non-small cell lung cancer cell(NSCLC)lines including A549,H157,H358,H1650,and so on,but no or low toxicity on normal cell lines such as human umbilical vein endothelial cells(HUVECs),human bronchial epithelial cells(BEAS-2B),and renal tubular epithelial cells(HK-2)at the same concentrations.Moreover,in vivo experiments showed that mice have an excellent tolerance to CA-5f and CA-5f effectively inhibits the growth of A549 lung cancer xenografts.The results of Western blot,immunofluorescence and TUNEL(Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)analysis showed that CA-5f inhibits autophagic flow and induces apoptosis in vivo without affecting the levels of Cathepsin B(CTSB)and cathepsin D(CTSD),which are consistent with the results of in vitro experiments.The above experimental results unraveled that CA-5f,as a novel late-stage autophagy inhibitor,can effectively inhibit the growth of NSCLC.Besides,we identified another inhibitor of late-stage macroautophagy/autophagy,CA-5g,a small chemical molecule that also inhibits autophagy flow by preventing autophagosome-lysosome fusion.Syntaxin 17(STX17)localized on the autophagosome,through the recruitment of SNAP29 and lysosome-associated vesicle-associated membrane protein 8(VAMP8)interaction to induce the autophagosome fusion with lysosomes.Western blot and immunofluorescence results showed that CA-5g did not affect the expression of STX17,but CA-5g inhibits the recruitment of STX17 and SNAP29 to autophagosomes,which may be the molecular mechanism of CA-5g as a late-stage autophagy inhibitor.In summary,our research results show that CA-5f and CA-5g are novel late-stage autophagy inhibitors which inhibit autophagic flux by blocking autophagosome-lysosome fusion.Our study provides potent tools to study the molecular mechanisms of autophagy.At the same time,CA-5f,as a small molecule with no/low toxicity to normal cells and apparent toxicity to tumor cells,can provide potential lead compounds for drug discovery in clinical treatment of NSCLC.