Effect Of The Combination Of Disulfiram And Copper On Autophagy And Apoptosis In Non-small Cell Lung Cancer

Lung cancer is one of the leading cause of cancer-related mortality in China,Non-small cell lung cancer,as a common lung cancer,accounts for 80% of lung cancer-related deaths.The typical treatment for patients with advanced non-small cell lung cancer is standard chemotherapy which is Platinum combine with the third generation cytotoxic drugs,such as paclitaxel.However,the efficiency of standard chemotherapy is often hampered by the development of drug resistance in patients.Disulfiram (DSF) has been widely used as an anti-alcoholism drug in the clinic for over 60 years without severe side effects.Recently,different groups have found that the cytotoxicity of DSF depends on copper (Cu).Since it is a bivalent metal ion chelator,DSF has been considered to form a complex with Cu (DSF/Cu) which is more readily taken up by cells.Autophagy,a cellular survival mechanism,is thought to allow the recycling of cellular breakdown products when cancer cells are subjected to chemotherapy,thus decreasing drug-induced apoptosis.Our previous studies proved that DSF/Cu exerts increased anti-tumor effects on non-small cell lung cancer (NSCLC) xenograft models,and inhibits NSCLC recurrence driven by ALDH-positive cancer stem cells.Moreover,it has been found that the anticancer effect of DSF/Cu is mediated by the induction of apoptosis.As the above,autophagy and apoptosis have a close interaction in that they share similar effectors and regulators.So far,however,the role of autophagy in DSF/Cu treatment has not been reported.In this study,we explored whether DSF/Cu can induce autophagy in NSCLC,and to determine the relationship between autophagy and apoptosis.Firstly,we evaluated the effects of DSF/Cu on the proliferation of NSCLC cells (NCI-H1299,NCI-H460 and A549).Then,IC50 concentration of DSF with or without Cu for 24 h was determined.The result showed that DSF/Cu significantly inhibited cell proliferation as compared with DSF or Cu treatment alone.Moreover,DSF/Cu caused the majority of cells to shrink,round up,and display numerous vacuoles in the cytoplasm,which are typical morphological alterations induced by apoptosis and autophagy.These results indicated that DSF/Cu may induce apoptosis or autophagy.Secondly,we elucidate whether the cell death caused by DSF/Cu was due to apopotosis.The results of the Annexin V-FITC and PI double staining assay showed that DSF/Cu increased the proportion of apoptotic cells.The percentage of apoptotic cells in the DSF/Cu-treated group was 35.4% for A549,21.4% for H460,and 37.9% for H1299.The results of Western Blotting demonstrated that the active,cleavage forms of Caspase3,Caspase8 as well as PARP were significantly upregulated,and the expression of Bcl-2 was significantly downregulated after DSF/Cu treatment in all three cell lines as compared with control group.However,the pan-Caspase inhibitor z-VAD-fmk could suppressed caspase activation induced by DSF/Cu.The above data indicated that DSF/Cu induced-cell death and-increasing of Caspase3 activity was dependent on Caspase.Moreover,we elucidate whether DSF/Cu could induce autophagy in NSCLC cells using Western Blotting and immunofluorescence.The expression of LC3II was gradually increased and reached a peak at 12 h,which is a biomarker of autophagy.Besides,we detected a couple of autophagy markers as well,including ATG5 and p62.ATG5 was significantly upregulated after DSF/Cu treatment for 12 h, which was in accordance with the changes of LC3II.The level of p62 was notably decreased during this process.We also observed that LC3 co-localized with the lysosomal membrane protein LAMP2,and p62 expression was attenuated.Meanwhile,Western Blotting results showed that DSF/Cu can decrease PI3K,p-Akt,p-mTOR levels.The above data indicated that DSF/Cu may induce autophagy through PI3K/Akt/mTOR signaling pathway.Finally,we confirmed the relation between DSF/Cu-induced autophagy and apoptosis by using ATG5 knockdown or autophagy inhibitors,incluing 3-methyladenine (3-MA) and chloroquine.As we expected,the DSF/Cu-induced cleaved PARP was significantly upregulated after inhibiting autophagy.Similar phenomenon was also observed with the FITC/PI double staining,which indicated that inhibition of autophagy during DSF/Cu treatment could enhance apoptotic-promoting effect of DSF/Cu.In line with the in vitro findings,DSF/Cu inhibited H460 tumor growth in BALB/c nude mice.Moreover,coadministration with 3-MA potentiated DSF/Cu-induced anti-H460 xenograft activity in vivo.These results demonstrated that DSF/Cu-induced apoptosis was significantly enhanced after suppressing autophagy.In summary,autophagy inhibition could sensitize,to some extent,DSF/Cu-induced anti-NSCLC activity in vivo.All these work will provide preliminary experimental data and basis for the development of those anti-tumor compounds.