The Effect And Mechanism Of Verteporfin Enhances The Sensitivity Of Lung Cancer Platinum-resistant Cells A549/DDP To Cisplatin

Background:The incidence of lung cancer and cancer-related mortality rank first in the world.Lung cancer is mainly non-small cell lung cancer(NSCLC),accounting for about 85%.Although targeted drugs have been widely used in the treatment of lung cancer,cisplatin(DDP)is still first-line chemotherapy drug for patients without EGFR mutations.However,a large number of clinical practices have shown that long-term application of cisplatin will lead to drug resistance,resulting in chemotherapy failure and tumor progression.Therefore,exploring safe and effective cisplatin chemotherapy sensitizers and probing their mechanism are extremely important to reversethe cisplatin resistance of NSCLCand improve the clinical efficacy.YAP(Yes-associated protein)is the core member of the highly conserved Hippo signaling pathway that controls the size of tissues and organs.Studies have shown that YAP is overexpressed in a variety of cancers and is associated with self-renewal,abnormal proliferation,migration and invasion of cancer stem cells and poor prognosis,with the function of oncogene.Recent studies have shown that overactivation of YAP is involved in driving platinum-based chemotherapy resistance.Inhibition of YAP can enhance the sensitivity of cervical cancer,oral squamous cell carcinoma and neuroblastoma to cisplatin.Therefore,targeted inhibition of YAP may become a new target to enhance the chemosensitivity of NSCLC to cisplatin.Autophagy means transportation of denatured,senescent,damaged organelles and proteins to lysosomes for degradation and recycling.Under normal circumstances,autophagy is widespread in eukaryotic cells and is essential for maintaining cell homeostasis.While in the process of tumorigenesis and development,autophagy is a double-edged sword,which may promote or suppress tumor.Recent studies have shown that autophagy may be a protective mechanism for cancer chemotherapy.Autophagy inhibitors combined with chemotherapeutic drugs have become a new research hotspot.Inhibition of autophagy can enhance the chemosensitivity of gastric,colorectal and ovarian cancer to cisplatin.So inhibition of autophagy may become a potential new strategy to reverse NSCLC cisplatin chemotherapy resistance.Verteporfin(VP)is a second-generation porphyrin photosensitizer approved by the FDA for the clinical treatment of macular degeneration.Recent studies have shown that VP plays an important role in anti-tumor and reversing tumor resistance:VP directly inhibits the expression of YAP,then inhibits the proliferation of ovarian cancer,invasion and metastasis of bladder cancer,induces apoptosis of pancreatic cancer cells and enhances the chemosensitivity of urothelial cells to cisplatin;VP enhances the chemosensitivity of pancreatic ductal adenocarcinoma to gemcitabine by inhibiting autophagy;VP enhances the chemosensitivity of hepatocellular carcinoma to sorafenib by damaging lysosomes,blocking autophagic flow.In addition,VP induces the formation of oligomers of p62 in colorectal cancer,while tumor cells are unable to clear macromolecular oligomers,resulting in tumor-selective protein toxicity.So,can VP enhance the chemosensitivity of lung cancer platinum-resistant cells A549/DDP to cisplatin?Does it reverse NSCLC cisplatin resistance by inhibiting YAP and autophagy?Therefore,this study aims to explore the effect and molecular mechanism of VP on cisplatin resistance in A549/DDP cells,and to provide laboratory basis for the clinical application of verteporfin as a cisplatin sensitizer in chemotherapy for NSCLC.Objective:1.Toclarify the effect of verteporfin on enhancing cisplatin chemosensitivity of lung cancer platinum-resistant cells A549/DDP;2.To explore the potential mechanism of verteporfin in enhancing cisplatin chemosensitivity of A549/DDP cells.Methods:1.MTS,Western Blot and Real-time PCR were used to detect the changes of cell proliferation,apoptosis-related proteins Cleaved-Caspase3,Cleaved-PARP and pro-apoptotic genes PUMA and BIM in A549/DDP cells treated with Verteporfin combined with cisplatin;2.Cell scratches and Transwell test were used to detect the changes of cell migration and invasion ability of A549/DDP cells treated with Verteporfin combined with cisplatin;3.Western Blot was used to detect the expression of EMT-related proteins E-cadherin,Vimentin and Snail in A549/DDP cells treated with Verteporfin;4.Western Blot was used to detect the expression of transporter ABCG2 and ABCG5 in A549/DDP cells treated with Verteporfin.5.Western Blotdetected the expression of YAP protein in A549 and A549/DDP cells,and the changes of YAP protein in A549/DDP cells treated with Verteporfin.Construction ofA549/DDP-shYAP cells,Real-time PCR,Western Blot and cellular immunofluorescence were used to detect the knockdown efficiency of YAP in A549/DDP cells;6.MTS and flow cytometry were used to measure the changes of cell proliferation and apoptosis of A549/DDP-shYAP cells treated with cisplatin;7.Cell scratches and Transwell test were used to measure the changesofcell migration and invasion abilityof A549/DDP-shYAP cells treated with cisplatin;8.Transient pYAP plasmid overexpressed YAP in A549 cells(A549-YAP),Western Blot verified the overexpression effect;9.Western Blotwas used to detectthe expression of EMT-related proteins in A549 and A549/DDP cells,and to detectthe expression of EMT-related proteins after overexpression of YAPin A549 cellsand YAP knockdown in A549/DDP cells;10.Western Blot detected the expression of ABCG2 and ABCG5 in A549 and A549/DDP cells,and the expression of transporter proteinsafter overexpression of YAPin A549 cells and YAP knockdown in A549/DDP cells;11.After transfection with GFP-LC3 plasmid,the number of LC3-GFP spots was observed in A549/DDP cells exposed toverteporfin or cisplatin;Western Blot detected the expression of autophagy-related proteins LC3,p62,Ub.Results:1.Compared with cisplatin alone,Verteporfin combined with cisplatin significantly inhibited cell proliferation(P<0.05),activated Caspase3,sheared PARP and promoted the mRNA expression of PUMA and BIM(P<0.05);2.Compared with cisplatin alone,Verteporfin combined with cisplatin significantly inhibited cell migration(P<0.05)and invasion;3.Western Blot results showed that the expression of E-cadherin was significantly increased,while the expression of Vimentin and Snail was significantly decreased in A549/DDP cells treated with Verteporfin;4.Western Blot results showed that the expression of ABCG2 and ABCG5 was significantly decreased in A549/DDP cells treated with Verteporfin.5.YAP was highly expressed in A549/DDP cells(P<0.05);Verteporfin significantly inhibited the expression of YAP protein in A549/DDP cells;Real-time PCR,Western Blot and cellular immunofluorescence showed that shYAP effectively inhibited the expression of YAP mRNA(P<0.05)and protein in A549/DDP cells.6.Knockdown of YAP promoted cisplatin-induced apoptosis(P<0.05);7.Knockdown of YAP increased the migration(P<0.05)and invasion inhibition of cisplatin;8.Western Blot results show that pYAP overexpression plasmid effectively up-regulated the expression of YAP protein in A549 cells;9.Compared with A549 cells,E-cadherin expression was significantly lower,while Vimentin and Snail expression were significantly higher in A549/DDP cells;E-cadherin expression decreased and Snail expression increased in A549 cells after overexpression of YAP.E-cadherin expression increased and Vimentin expression decreased in A549/DDP cells after YAP knockdown;10.Compared with A549 cells,ABCG2 and ABCG5 were significantly overexpressed in A549/DDP cells;ABCG2 and ABCG5 expression were increased after overexpression of YAP in A549 cells;ABCG2 and ABCG5 expression were reduced after YAP knockdown in A549/DDP cells.11.Cisplatin promoted the formation of autophagy in A549/DDP cells,which was significantly reduced after treatment with Verteporfin combined with cisplatin;Western Blot results showed that the expression of LC3 in A549/DDP cells increased after cisplatin treatment.The expression of LC3,p62 and Ub increased after treatment with Verteporfin,and the formation of polymer p62 oligomer was observed.Conclusion:1.Verteporfin can enhance the chemosensitivity of A549/DDP cells to cisplatin by inhibiting proliferation,migration,invasion and promoting apoptosis;2.Verteporfin may enhance the chemosensitivity of A549/DDP cells to cisplatin by inhibiting YAP and autophagy;3.Verteporfin may be a potential chemotherapeutic sensitizer for clinical treatment of NSCLC with cisplatin resistance.Regulation of YAP and autophagy may be a new strategy for reversing cisplatin resistance in NSCLC.