The Study Of Wee1 Inhibitor AZD1775 Combined With Radiation In Small Cell Lung Cancer

Background:SCLC is the most aggressive malignant lung tumor.Majority SCLC patients eventually resistant to chemotherapy.In SCLC,the co-deletion of RB1 and P53 leads to an obvious defect in DNA repair ability in G1-S cell cycle checkpoint,which increases the dependence of cells on G2-M checkpoint when subjected to internal and external stress.The rational targeting of G2-M checkpoint is an effective treatment for SCLC.Tyrosine kinase Wee1 is a key kinase at the G2-M checkpoint.Studies have found that its inhibitors can selectively enhance the chemoradiotherapy sensitivity of P53 mutated SCLC,cervical cancer,etc.However,few studies have been studied on the application of AZD1775 in SCLC.Many studies have shown that P53 mutant cells are more sensitive to AZD1775,some other studies suggested that the antitumor effect of AZD1775 is not associated to P53 status.Objective:The aim of this study was to explore the clinicopathological and prognostic significance of Wee1 expression in SCLC.The radiotherapy sensitization of AZD1775 was analyzed in vitro and vivo.Methods : Immunohistochemistry,CCK-8,clonal formation assay,immune fluorescence staining,Annexin V-FITC/PI and PI were used to evaluate the effects of AZD1775.The P53-tet on H446 cell was transfected with lentivirus,and the difference of sensitization of AZD1775 on radiotherapy was observed after Dox inducing P53-tet on H446 cell.The SCLC mouse xenograft model was also applied.Results: 1.The overall positive rate of Wee1 in SCLC tissues was 63.89%.The patients with positive expression of Wee1 were more common in T2 stage and patients with lymph node metastasis and patients at TNM II and III stage.In the prognostic analysis,Wee1 expression was also an important factor affecting the prognosis,in addition to T stage,N stage and TNM stage.2.AZD1775 combined radiotherapy in SCLC cell line H446 and H69(with P53 mutations),or in H378 and H209 cells(without P53 mutation)can significantly inhibit cell proliferation,cloning formation,and promote cell apoptosis of SCLC.Moreover,the G2-M phase ratio of H69 and H446 cells in SCLC was significantly reduced after AZD1775 combined with radiotherapy compared with the radiotherapy alone group,which means that the application of AZD1775 can reverse the G2-M phase arrest caused by radiotherapy.The above sensitization effect of AZD1775 on radiotherapy was more remarkable in H446 and H69 cell lines which were with P53 mutation.3.Regardless of P53 mutation status,AZD1775 have obvious antitumor and radiotherapy sensitization effect for SCLC cells.In Dox induced P53-tet on H446 cell line,AZD1775 combined radiation decrease effect of the inhibiting proliferation,growth and promoting apoptosis significantly along with P53 overexpression.4.SCLC mouse xenograft model was applied to confirm the radiotherapy sensitization effect of AZD1775.AZD1775,radiotherapy and the combination of them inhibit the tumor growth curve of xenograft tumor.Immunohistochemical staining of tumors in different treatment groups showed that ?H2AX expression in AZD1775 and radiotherapy group was significantly higher than that in the control group,but ?H2AX expression was most significantly high in AZD1775 and radiotherapy group.Conclusion: 1.When it was compared with single agent,AZD1775 combined with radiotherapy can significantly inhibit the tumor progress of SCLC,and the application of AZD1775 can reverse the G2-M stage arrest caused by radiotherapy,so as to promote the therapeutic effect of radiotherapy.2.Regardless of P53 mutation status,AZD1775 had obvious sensitization effect on the radiotherapy of SCLC,but P53 overexpression decreased radiotherapy sensitization effect of AZD1775.3.In vivo,the combination of AZD1775 and radiotherapy can significantly inhibit tumor growth and tumor cell proliferation compared with the single treatment group.