| Background Cervical cancer is the most prevalent malignant tumor of female reproduction system,followed behind breast cancer in incidence rate among female malignant tumors in our country.At present,its main treatment is a comprehensive treatment of surgery,chemotherapy and radiotherapy.However,radiation resistance often leads to local recurrence and distant metastasis,leading to treatment failure.Therefore,studying the molecular mechanism of cervical cancer radiotherapy resistance,screening potential molecular targets and effective radiation sensitizing drugs,is essential to improve the radiosensitization of cervical cancer.Recombinant human endostatin is a biological inhibitor that inhibits vascular endothelial cell migration and angiogenesis.Recombinant human endostatin in combination with radiotherapy showed a synthetic effect in the treatment of oral squamous cell carcinoma and esophageal cancer,but the effect and specific mechanism of the combination therapy in the treatment of cervical cancer are still unclear.Objective To explore the effect of recombinant human endostatin on the radiosensitivity for cervical cancer and its possible molecular mechanism,with the intention of offering a safe and effective treatment option for cervical cancer patients.Methods CCK-8 assay was employed to explore the inhibitory effect of graded concentrations of recombinant human endostatin and its combined radiotherapy on Hela,Siha and HUVEC cells.Plate cloning experiment was used to detect the effect of recombinant human endostatin combined with radiotherapy on the cloning ability of Hela and Siha cells.In addition,the apoptosis and cycle distribution of Hela,Siha cells and HUVEC cells by recombinant human endostatin combined with radiotherapy were detected by flow cytometry.The tube formation assay was used to explore the effect of combined radiotherapy on the tubule formation of HUVEC cells.In addition,the expression of γ-H2 AX in HUVEC cells after combined treatment was detected by cellular immunofluorescence,and it was confirmed that recombinant human endostatin can repair DNA damage after radiotherapy.To further identify the mechanism of sensitization of recombinant human endostatin to radiotherapy in HUVEC cells,the VEGFR and its downstream signaling pathway activation were measured by Western blot.Establish a subcutaneous transplantation tumor model of cervical cancer in nude mice to explore the inhibitory effect of recombinant human endostatin combined with radiotherapy on tumor growth.Immunohistochemical was used to explore the changes of microvessel density(MVD)and pericyte coverage(αSMA)in tumor tissues of each group,and ELISA assay was employed to detect the VEGF-A expression in serum of each group.Results The gradient concentration of recombinant human endostatin showd inhibitory on HUVEC cells at 24 h after treatment,and there was a statistical difference at 200 μg/m L.It showed no inhibitory on Hela and Siha cells.In addition,recombinant human endostatin further increased the inhibitory effect of radiotherapy on HUVEC cells(P<0.05).The results of the plate cloning assay showed that the combination of recombinant human endostatin and radiotherapy did not show the synergistic effect of radiosensitization on cervical cancer cells.The results of flow cytometry showed that recombinant human endostatin promoted radiotherapy to HUVEC cell apoptosis and G2/M phase arrest(P<0.05),but did not show obvious promoting effect on Hela and Siha cells.The results of tube formation experiments showed that recombinant human endostatin combined with radiotherapy can significantly inhibit the formation of HUVEC cells(P<0.05).Immunofluorescence assay show that compared with the control group,radiotherapy can significantly promote the formation of intracellular γH2AX focus(P<0.05).Compared with radiotherapy alone,the treatment of recombinant human endostatin combined with radiotherapy at 6 h,the formation of γH2AX focus in HUVEC cells was inhibited,indicating that recombinant human endostatin can inhibit the formation of γH2AX focus induced by radiotherapy(P<0.05).Western blot results show that recombinant human endostatin can inhibit the phosphorylation of VEGFR2/PI3K/AKT/DNA-PK in HUVEC cells(P<0.05),but has no significant effect on the expression of HIF-1α in the cells.Nude mouse xenograft tumor model assay showed that compared with the control group,the volume and weight of nude mice treated with recombinant human endostatin did not change,while the nude mice treated with radiotherapy and recombinant human endostatin combined with radiotherapy could significantly inhibit the volume and weight of nude mice(P<0.05).In addition,compared with radiotherapy treatment alone,the volume and weight of nude mice in the recombinant human endostatin combined with radiotherapy group were mildly reduced,and no significant statistical difference was seen(P>0.05).Immunohistochemical results showed that,compared with the control group,recombinant human endostatin,radiotherapy and recombinant human endostatin combined with radiotherapy can inhibit the microvessel density and pericyte coverage in tumor tissues(P<0.05);and Compared with radiotherapy alone,the microvessel density in the tumor tissue was further reduced after the treatment of recombinant human endostatin combined with radiotherapy(P<0.05),but there was no significant change in pericyte coverage.ELISA results indicated that there was no significant difference in VEGF-A expression in the serum of recombinant human endostatin and radiotherapy alone treated groups as compared to the control group,while the combination of recombinant human endostatin and radiotherapy could significantly inhibit the expression of serum VEGF-A(P <0.05).Conclusion Recombinant human endostatin and it combined radiotherapy can inhibit the proliferation of HUVEC cell,promote radiotherapy-induced HUVEC cell apoptosis and G2/M phase block.In addition,it further inhibits the tubule formation ability and DNA damage repair of HUVEC cell induced by radiotherapy.In the nude mouse xenograft model of cervical cancer,Recombinant human endostatin can slightly promote the antitumor effect of radiotherapy,and the combined treatment can significantly inhibit the microvessel density in tumor tissues and the expression of VEGF-A in serum,but has no effect on the coverage of pericytes.The above results indicate that recombinant human endostatin combined with radiotherapy can inhibit DNA damage and repair of vascular endothelial cells and promote endothelial cell apoptosis through the VEGF/VEGFR pathway,thereby reducing the microvessel density in tumor tissues. |
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