Clinical And Experimental Study On Antitumor Effect Of Apatinib Combined With Chemoradiotherapy On Cervical Cancer

Chapter 1.Clinical study of Efficacy and Safety of Apatinib combined with Chemoradiotherapy on Cervical Cancer.Objective:Apatinib mesylate is a tyrosine kinase inhibitor that highly selectively inhibits vascular endothelial growth factor receptor 2(VEGFR2).Apatinib has shown good safety and efficacy in several types of solid tumor.This study aimed to assess the clinical efficacy and safety of apatinib combined with concurrent chemo-brachytherapy(CCBT)in patients with recurrent and advanced cervical cancer.Methods:This randomized controlled trial was carried out on 59 patients with recurrent or untreated stage IVB(International Federation of Gynecology and Obstetrics staging,FIGO staging)cervical cancer,which were admitted to the Department of Gynecology and Oncology of Shandong Cancer Hospital and Institute from July 2016 to May 2018.All patients(n=59),who met the inclusion criteria,we put them into the following groups randomly:the apatinib group(n=30)and the control group(n=29).Patients with recurrent cervical cancer in the apatinib group were administered apatinib and carboplatin-paclitaxel as first-line chemotherapy.Patients with advanced cervical cancer were administered apatinib in combination with CCBT.In control group,patients with recurrent cervical cancer were treated with chemotherapy alone while patients with advanced cervical cancer received CCBT.Results:Among the 59 patients enrolled,7 patients were censored before the finally efficacy evaluation.52 cases were included in this analysis(28 in the apatinib group and 24 in the control group).The clinical characteristics such as age,ECOG score,body mass index,number of metastatic sites,degree of differentiation,pathological type,surgical history and treatment methods are similar in two groups.In this clinical trial,the median follow-up time for patients was 14.0 months.As of the deadline for follow-up on April 30,2019,there have been 27 deaths,accounting for 51.9%of the total number of cases.The efficacy evaluation suggested that the median total progression-free survival(PFS)in the apatinib group was significantly prolonged than that in the control group(10.1 months vs 6.4 months;P<0.01;HR,0.44,P<0.01).Stratifiedanalysis demonstrated that the median PFS of patients with apatinib combined with chemotherapy was notably longer than that of patients with chemotherapy alone(10.1 months vs 6.4 months,P<0.01).In patients with untreated stage IVB cervical cancer,the median PFS of apatinib combined with CCBT was remarkably higher than that of CCBT patients in the control group(10.3 months vs 6.1 months,P<0.01).The objective response rate(ORR)of the apatinib-treated group was obviously higher than that of the control group(64.3%vs 33.3%,P<0.05)with statistically significant.Multivariate analysis showed that bone metastasis(P<0.001),adenocarcinoma(P<0.0 5)and apatinib combined therapy(P<0.0 1)are independent prognostic factors of the disease.And patients with bone metastasis and adenocarcinoma might lead to the poor prognosis,including shortened PFS.Whereas apatinib combined treatment as a protective factor,had prolonged the patient's PFS.Safety analysis indicated that the incidence of proteinuria,hand-foot syndrome,mucositis,and hypertension was significantly higher in apatinib group than that in control(P<0.05).Reducing the dose of apatinib or being given symptomatic treatment,serious side effects of patients can be alleviated.Apatinib did not obviously aggravate other side effects of radiotherapy or chemotherapy.Conclusion:Apatinib combined with CCBT or chemotherapy has a good effect on patients with recurrent and advanced cervical cancer.The apatinib combined therapy may prolong the PFS and improve the ORR of patients,and with controllable side effects.Apatinib has a synergistic anti-tumor effect with Chemo-radiotherapy.Chapter 2.Inhibitory Effect of Apatinib Combined withRadiotherapy on Trans-plantation Tumor of Human Cervical Cancer SiHa and HeLa Cells in Rude Mice.Objective:Radiotherapy(RT)plays a very important role in the treatment of cervical cancer.Some patients are insensitive to radiotherapy,which leads to poor effect of radiotherapy.As a highly selective inhibitor of VEGFR2,apatinib has become a potential radiosensitizer because of its anti-angiogenic mechanism.However,the experimental study of apatinib combined with radiotherapy in the treatment of cervical cancer is rarely reported,and the mechanism is unclear.In this chapter,we will further verify the anti-tumor effect of apatinib and its influence on tumor cell radiosensitivity in vivo through establishing human cervical carcinoma SiHa and Hela Xenograft mice model,and further explore its possible molecular mechanism.Methods:Antitumor experiment was carried out using SiHa and Hela xenograft mice model.The animals are divided into 4 groups:?control group;?Apatinib group;?radiotherapy group;?Apatinib+radiotherapy group.The results were evaluated with the difference of each group by drawing tumor growth curve.The potential toxicity resulting from treatment was monitored by blood cell analysis and histopathological examination.At the end,the athymic mice were euthanized and specimens were taken from the tumor,heart,liver and kidney.Immunohistochemical analyses were performed on tumor tissue sections using an antibody specific for CD31 to measure the change of microvessel density count(MVD)with the treatment of apatinib,and the antibody specific for Ki-67 and PCNA to detect its effects on the proliferation of tumor cells,and for BCL-2 to detect its effects on tumor cell apoptosis.We analyzed the phosphorylation and key molecule expressions of VEGFR2 and JAK2/STAT3 signal pathway:VEGFR2/p-VEGFR2,JAK2/p-JAK2,STAT3/p-STAT3 and downstream radiotherapy sensitive molecules HIF-1? by western blotting to explore the effects of apatinib on the activation status of the pathway and cell radiosensitivity.Results:? The outcomes of blood cell analysis in cervical cancer xenografts mice model demonstrated that,the white blood cells were a little bit decreased in apatinib group and combination treatment group,the same results were met in red blood cells.but the difference was not statistically significant.No other types of hematological toxicities were found.?Notably,no histopathological changes were found in the heart,liver or kidney of mice? The growth curve of CC xenografts mice model demonstrated that,the growth of tumor in experimental groups were much slower than control,and the tumor in combinationgroup was the smallest.Growth rate and the weight of SiHa transplanted tumor finally were as follows:Control group(1.80±0.60g)>Apatinib group(0.97±0.17g)>Radiotherapy group(0.33±0.23g)>Apatinib+Radiotherapy group(0.32±0.08g).The growth rate and final weight of HeLa transplanted tumor were as follows:Control group(1.01±0.30g)>Radiotherapy group(0.74±0.32g)>Apatinib group(0.57±0.20g)>Apatinib+Radiotherapy group(0.48±0.26g).The tumor inhibition rate of Apatinib+Radiotherapy group was the highest among the experimental groups.The tumor inhibition rate of SiHa xenografts mice model in Apatinib+RT group(84.4±4.38%)was significantly higher than that in Apatinib group(46.24±9.52%)and RT group(71.03±12.85%).The same to HeLa xenografts mice model,tumor inhibition rate in Apatinib+RT group(73.04 ±6.04%)was also higher than that in Apatinib group(43.59±19.82%)and RT group(52.33±3.25%).?Immunohistochemical staining of CD31 indicates that Apatinib+RT group exerted a significant reduced in the microvessels density count(MVD)compared with that in the control group.Apatinib+RT group had the strongest inhibitory effect on angiogenesis(P<0.01).The expressions of PCNA,Ki-67 and BCL-2 in each group indicates that Apatinib and radiotherapy can both inhibit the expression of these molecules,and the expressions in Apatinib+RT group rank the lowest,(P<0.01),and has the strongest inhibitory effects on tumor proliferation.?The expression levels of different moleculars of SiHa xenografts tumor tissue were analyzed by Western blot.Apatinib inhibits the activity of VEGFR2 and JAK2/STAT3 signal pathway by dephosphorylation.And the expression levels of different moleculars of HeLa xenografts tumor tissue were consistent with the trend of SiHa xenografts.Conclusion:?The results confirms that apatinib can increase the sensitivity of radiotherapy on human cervical cancer SiHa and Hela xenografts in nude mice;?In SiHa and HeLa xenograft nude model,the VEGFR2 and JAK2/STAT3 signaling pathway exist and are highly activity.?The radiosensitizing effect of Apatinib on CC xenografts in mice can be achieved by inhibiting the activity of VEGFR2 and JAK2/STAT3 signal pathway,and then inhibiting the expression of downstream HIF-1 a and abnormal tumor angiogenesis.Chapter 3.Apatinib enhances Radiosensitivity of Cervical Cancer cell by affecting VEGFR2 and JAK/STAT3 signal pathway.Objective:Apatinib mesylate(YN968D1)highly selectively inhibits the vascular endothelial growth factor receptor2(VEGFR2)pathway.In the second part,we confirmed that apatinib can increase the radiosensitivity of cervical cancer cells(SiHa and Hela)xenograft mice model in vivo.And we found that the radiosensitivity is closely related to the VEGFR2 pathway and JAK2/STAT3 pathway.In the third part of the study,we established experiments in vitro using cervical cancer cell lines(SiHa and Hela).The mechanism of apatinib on the enhancement of radiosensitivity was further explored.Methods:(1)We conducted CCK-8 experiment to test the effect of apatinib on the proliferation of cervical cancer cells(SiHa and HeLa).(2)Colony formation assay was conducted,aimsed to detect the effects of apatinib on the radiosensitivity of cervical cancer cells.(3)Western blotting was conducted to detect the activation of VEGFR2 and JAK2/STAT3 signaling pathway,and the expression level of HIF-lain the downstream.The aims of this experiment are as follows:?To evaluate the effect of apatinib on the activation of the two relevant pathways and the expression of downstream molecules;?Testing the states of the two pathways and expression change of HIF-1?,is to compare the differences of apatinib combined RT group with simple RT group;?By useing specific STAT3 signaling pathway inhibitor,we observed changes of the effects of apatinib on the VEGFR2 and STAT3 pathway,as well as the changes in the downstream HIF-1? expression,so as to further confirm the mechanism of radiosensitivity for apatinib.Result:(1)CCK-8 experiment results showed that:Apatinib inhibited the proliferation of cervical cancer SiHa and HeLa cells,with IC50(699.4±112.9)?M and(115.8±3.3)?M,respectively.(2)The results of Colony formation assay suggested that the Apatinib and radiotherapy combined group had the strongest inhibitory effect on cell proliferation among the experimental groups,and the inhibitory effect increases with the increase of radiation dose.(3)Results of western blotting for SiHa cell are as follows:?Compared with the control group,the expressions of VEGFR2 and STAT3 in the apatinib group did not have significantly changes.But the expression of p-VEGFR2,p-STAT3 and downstream HIF-1? decreased significantly,showing a significant dephosphorylation trend;?Compared with RT group,the expressions of VEGFR2 and STAT3 in the Apatinib+RT group show no significant changes,but the Apatinib+RT has a significant decrease in the expression of p-STAT3,p-VEGFR2 and HIF-1?;?Compared with Apatinib group,the expressions of VEGFR2 and STAT3 in the Apatinib+S3i-201 group show no significant changes,but the Apatinib+S3i-201 has a significant decrease in the expression of p-STAT3,p-VEGFR2 and HIF-1?.The expression levels of different signal moleculars of VEGFR2 and JAK2/STAT3 signal pathway of HeLa cells were consistent with the trend of SiHa cells.Conclusion:For the radiosensitization mechanism of apatinib,we propose the hypothesis as follows:The formation of abnormal tumor blood vessels can lead to local hypoxia and reduce the radiosensitivity of tumor cells.At the same time,hypoxia can regulate the increase of hypoxia-inducible factor 1?(HIF1-?)and vascular endothelial growth factor(VEGF),which can further promote tumor angiogenesis and form a vicious circle.Apatinib inhibits the activities of key signal molecules in VEGFR2 and JAK2/STAT3 signal pathway in cervical cancer SiHa and HeLa cells through dephosphorylation mechanism,resulting in decreased transcription and expression of downstream HIF-1 a and VEGF,thus inhibiting tumor cell proliferation and effectively blocking tumor neovascularization,breaking the existing abnormal vascular system,restoring blood perfusion and oxygen supply,and finally increasing tumor cell radiosensitivity.Through this part of the experiment,both SiHa and Hela cells verified this conclusion.