The Role Of Activated Notch1 In DNA Damage Induced By Cisplatin In Cervical Cancer

In China,the incidence of cervical cancer is the second highest among female reproductive system tumors.The problem of tolerance to chemotherapy for cervical cancer is still urgent to be solved in clinical practice.Therefore,it is necessary to further explore the mechanism of tolerance to chemotherapy drugs for cervical cancer.Studies have shown that abnormal expression of Notch1 is involved in the occurrence and development of multiple solid and non-solid tumors,and is associated with the tolerance to radiation and chemotherapy of various tumors.The Notch receptor regulates downstream targets by specifically binding to the corresponding ligands,and plays a role in promoting proliferation and multiple cell differentiation.This pathway plays a crucial role in regulating cell proliferation,apoptosis,and promoting or inhibiting human tumor formation.Notch has important nodes with various signal transduction pathways,including Ras,mTOR and NF-?B.Studies have confirmed that high expression of Notch receptor and its corresponding ligands are found in tumors such as lung cancer and colon cancer.At present,studies have shown that the abnormal expression of Notch1 is involved in the resistance of various tumors.Cisplatin is a first-line anticancer drug for the treatment of cervical cancer in the clinic,which plays an anticancer role by damaging cellular DNA.ATM protein is the key molecule that plays the main switch role in DNA damage response.Its main function is to initiate the repair pathway after DNA damage,and initiate the apoptosis program when the damage cannot be repaired in time.More importantly,studies have shown that Notch1 plays a direct negative regulatory role in DNA damage caused by radiation.Notch1 is able to bind directly to ATM proteins,thereby inhibiting ATM activation and disrupting ATM-mediated related signal transduction pathways.Here,we explored the mechanism by which Notch1 activation is involved in cisplatin-induced DNA damage in cervical cancer cells.We detected the expression of Notch1 and Ki67 proteins in cervical cancer tissues(66 cases)and normal cervical tissues(15 cases)by immunohistochemical techniques.Meanwhile,two types of cervical cancer cells,HeLa(adenocarcinoma)and SiHa(squamous cell carcinoma),were treated with cisplatin alone or combined with DAPT(Notch1 signaling pathway specific inhibitor)to detect DNA damage and apoptosis.To investigate the regulatory mechanism of inhibition of Notch1 activation on DNA damage and apoptosis induced by cisplatin in cervical cancer cells.Objective:To investigate the regulatory mechanism of inhibition of Notch1 activation on DNA damage and apoptosis induced by cisplatin in cervical cancer cells.Methods:1.Expression of Notch1 and Ki67 proteins was detected in cervical cancer tissues as well as normal cervical tissues by immunohistochemical techniques.2.The viability and proliferative capacity of HeLa and SiHa cells after treatment of cisplatin were tested by CCK-8 and colony formation assays.3.DNA damage induced by cisplatin in HeLa and SiHa cells were detected by flow cytometry and Western Blot.4.The expression of NICD in HeLa and SiHa cells after treatment of cisplatin was detected by Western Blot and laser confocal microscopy.5.The cell viability of HeLa and SiHa after incubation of DAPT was detected by CCK-8.The expression of NICD in HeLa and SiHa cells after incubation of DAPT was detected by Western Blot.The viability and proliferative capacity of HeLa and SiHa cells after co-incubation of DAPT and cisplatin were tested by CCK-8 and colony formation assays.6.DNA damage induced by cisplatin in combination with DAPT were detected by flow cytometry,TUNEL and Western Blot in HeLa and SiHa cells.DNA damage induced by cisplatin in combination with DAPT was detected by Western Blot at different time points(6h,12 h,24h)in SiHa cells.7.A nude mouse xenograft model was established by subcutaneous inoculation of SiHa cells.The effect of DAPT combined with cisplatin on tumor-bearing mice was observed by intraperitoneal administration.The expression of NICD and Ki67 protein in each group was detected by immunohistochemistry techniques.The apoptosis of cells in each group was detected by TUNEL.HE staining was performed on the heart,liver,spleen,lung and kidney of all nude mice.Results:1.The expression levels of Notch1 and Ki67 proteins are high in cervical cancer tissues,and the expression of the two proteins is positively correlated.2.With the doubling of cisplatin concentration,the viability and proliferation ability of HeLa and SiHa cells were significantly inhibited.3.With the doubling of the concentration of cisplatin in HeLa and SiHa cells,the expression of ?-H2 AX,p-ATM and p-CHK2 protein gradually increased.At relatively low concentrations,the expression level of the p-BRCA1 protein was up-regulated.As the concentration increases,its expression gradually reduced.The expression of p-P53,P53 and Cleaved-Caspase3 proteins gradually increased.The number of apoptotic cells gradually increased.4.Compared with the control group,the expression of NICD in HeLa and SiHa cells in the experimental group was up-regulated.5.Cell viability and expression of NICD protein were inhibited by DAPT in HeLa and SiHa cells.Compared with the control group,the DAPT group,and the cisplatin group,DAPT combined with cisplatin significantly inhibited the viability and proliferation of HeLa and SiHa cells.6.The expression levels of ?-H2 AX,p-ATM,p-CHK2,p-P53,Cleaved-Caspase3 and Cleaved-PARP proteins were up-regulated in the DAPT-conjugated cisplatin group compared with the control group and other groups.The expression of p-BRCA1 and P53 protein in the combined group was reduced compared to the cisplatin group.The number of apoptotic cells in the combined group was significantly increased.With the increase of the action time,the expression of NICD protein was significantly inhibited and the expression of ?-H2 AX,Cleaved-Caspase3,and Cleaved-PARP protein gradually increased in the combined group.7.Compared with the control group and other groups,the combined administration group had a smaller tumor volume and a lighter weight.The NICD protein expression was reduced in the combined group compared to the cisplatin group.The expression of the Ki67 protein in the combined group was reduced compared to the control group and the other groups.HE staining showed no abnormalities in heart,liver,spleen,lung and kidney tissues of nude mice.Conclusion:1.Notch1 is highly expressed in cervical cancer tissues compared to normal cervical tissues.The expression of Notch1 was positively correlated with the expression of Ki67 in cervical cancer tissues.2.Cisplatin can induce DNA damage as well as ATM-CHK2-P53-mediated apoptosis in cervical cancer cells.3.Inhibition of Notch1 activation enhances cisplatin-induced DNA damage as well as ATM-CHK2-P53-mediated apoptosis in cervical cancer cells.