Antitumor Effect Of Chk1 Inhibitor In Human Small Cell Lung Cancer Cell Lines And The Mechanism Of Acquired Resistance For Chk1 Inhibitor In SCLC

Background&Objective Small cell lung cancer(SCLC)accounts for about 20% of all lung cancers.It is one of the most malignant tumors with the highest degree of malignancy and the worst prognosis.The five-year overall survival rate is less than 5%.Small cell lung cancer has no clear target-driven oncogenes,is prone to recurrence and metastasis,and has a poor prognosis.Cell cycle checkpoint kinase 1(Chk1)arrests cell cycle arrested and give tumor cells enough time to promote DNA homologous recombination and damage repair under DNA damage response for chemotherapeutic drugs.It is considered to be one of the main causes of chemotherpeutic resistance.Chk1 inhibitor eliminates intra-S phase checkpoint during the replication can cause replication catastrophe and promotes tumor cells apoptosis;eliminate G2/M checkpoint arrest caused byDNA-toxic drugs and prompts cells mitosis entry carried with DNA damage,causing mitotic catastrophe and leading to cell death.Chk1 inhibitors in certain entities tumor clinical trials have also shown some inhibition of tumors,but the effect of Chk1 inhibitors on small cell lung cancer remains unclear,The mechanism of action remains unclear.Whether it has synergistic effect with cisplatin,whether it has a reversal effect on cisplatin-resistant small cell lung cancer,we also need to study.We hope to investigate the antitumor effect and promoting apoptosis effect of Chk1 inhibitor on small cell lung cancer cell lines on clinical pathologcial analysis,in vitro and in vivo.At the same time,the clinical application of Chk1 inhibitors is mainly due to the toxic side effects,tolerance and acquired resistance.We hope to explore the acquired resistant charactistic by establishing Chk1 inhibitor acquired resistant SCLCcell line and the regulation mechanism of cell characteristics and drug acquired resistance levels in drug resistance.Screening for drug-resistant hotspot proteins and the possibility of drug-resistant signalingpathways,providing potential new targets for the treatment of SCLC,and understanding the mechanism of acquired resistance regulation,providing a new horizon and strategies for reversing drug acquired resistance.Methods: Cell cycle checkpoint kinase 1 inhibitor prexasertib(LY2606368)and cisplatin were used to study the anti-tumor effect of different P53 and Rb gene status in small cell lung cancer cell lines.CellTitle Glo was used to detect cell viability and flow cytometry,Western blotting are using for detecting the changes of cell cycle in small cell lung cancer cells and protein changes for Caspase-dependent apoptosis pathway.Using siRNA to knockdown,and lentiviral transfection was used to enhance key protein expression.Using plasmid transfection up-regulated E2F1 expression,qRT-PCR and Western blot confirmed that E2F1 plays a role in the anti-tumor effect of Chk1 inhibitor.To establish a acquired resistant cell line of cisplatin,and to investigate the anti-tumor effect of cell cycle checkpoint kinase 1 and its combination with cisplatin on small cell lung cancer cell lines and cisplatin-resistant cell lines by in vitro and in vivo.The Chk1 inhibitor acquried resistant SCLC cell line was established,and the reverse phase protein assay(RPPA)was used to screen the hot-spot target protein compare to the parental cell line.The small cell lung cancer cell line with acquried resistance to the Chk1 inhibitor was established.By siRNA knockdown,the electroporation parental line with plasmid was used to increase the corresponding hot spot protein which obtained in RPPA screening.CellTitleGlo,Western blot and qRT-PCR were used to observe the changes of resistance to Chk1 inhibitor and the changes of related apoptosis proteins and cell cycle regulatory proteins.In the case of single-cell cloning and culture of acquired resistant cell lines,the use of CellTitleGlo,Western blot,qRT-PCR application to observe the relationship betweetn Wee1 gene copy number changes,mRNAexpression level,protein expression level of Wee1 and drug resistance.The relationship between Chk1,Wee1 expression levels and prognosis in surgically resected small cell lung cancer patients through immunohistochemistry for Wee1 and Chk1 and tissue microarray analysis(TMA).Results:Chk1 inhibitors showed significant anti-tumor effects in small cell lung cancer cell lines regardless p53 gene status,and had a significant synergistic effect with cisplatin.Chk1 inhibitor can eliminate the G1/S and G2/M phase cell cycle arrest caused by cisplatin treatment,and promoting the apoptosis obviously.The DNA damage marker γH2AX,DNA damage repair marker Cleaved PARP is obviously increased,and the marker for mitosis entry,pHH3 was significantly increased also.Western Blot results indicates the anti-tumor effect of Chk1 or combined with cisplatin on small cell lung cancer cell line was mainly by activating Caspase-dependant apoptosis pathway and down-regulating E2F1 expression.In vivo experiments confirmed that Chk1 inhibitor has anti-tumor effect on small cell lung cancer,and synergistic effect could obtain with cisplatin,and can reverse cisplatin resistance.The expression level of Wee1 is significantly correlated with the level of acquired resistance of Chk1.siRNA knockdown Wee1 expression or using Wee1 inhibitor can reverse acquired resistance for Chk1 inhibitor,eliminate G2/M transition arrest,and increase apoptosis.Wee1 plasmid transfection for parental cell lines may obtain resistance to some extent,The use of siRNA to down-regulate Wee1-regulated downstream proteins and kinases CDK1 and CDC25 C,both of which will confer acquired resistance for the Chk1 inhibitor to the parental cell lines.Single cell cloning experiments indicated that Wee1 DNA copy number,mRNA relative expression level and protein expression level were significantly positively correlated with acquried resistance ablities levels.The up-regulated proteins whichscreened and picked up by RPPA in the acquired resistant cell lines is mainly focus on apoptosis cell proliferation,and cell cycle-regulation.The phosphorylated protein of P38 MAPK is increased by more than 7 times after exposure of Chk1 inhibitor,siRNA or small molecule inhibitor to down-regulated p38 MAPK expression could reverse the drug resistance in Chk1 inhibitor acquired resistant small cell lung cancer cell lines.TMA results showed that Chk1 was positively correlated with E2F1 and Wee1 expression in small cell lung cancer patients.Wee1 expression was positively correlated with better prognosis of small cell lung cancer patients,and Chk1 expression was negatively correlated with prognosis.Conclusion: Chk1 inhibitors show significant antitumor effect in small cell lung cancer,especially with p53 mutant SCLC in vivo and in vitro.Chk1 inhibitor has synergistic effect with cisplatin.Chk1 inhibitor could overcome the Cisplatin acquired in small cell lung cancercell lines.Ch1 inhibitor mainly promotes cell apoptosis through Caspase-dependent signal pathway and down-regulation of E2F1.Wee1 overexpression plays an important role in the acquired drug resistance of Chk1 inhibitor.Wee1 DNA copy number,mRNA level and protein level are significantly correlated with Chk1 inhibitor acquired resistance.Bypass activation of the p38 MAPK signaling pathway may also play a role in the formation of Chk1 inhibitor acquired resistance.Chk1 and Wee1 could be one of the predictors of prognosis in small cell lung cancer.The combination of Chk1 inhibitors with Wee1 inhibitors may provide new therapeutic targets and therapeutic strategies for the treatment of small cell lung cancer.