Endostar Combined With Concurrent Chemoradiotherapy In Treating Cervical Cancer:a Clinical And Basic Research

B ackgroundA large number of studies have shown that cisplatin-based concurrent chemoradiotherapy(CCRT)results in superior local control rate and survival for locally advanced cervical cancer(LACC)over radiation therapy(RT)alone.However,approximately 30%of patients will eventually experience treatment failure.The potential of adding other chemotherapeutics to CCRT is limited.Therefore,to explore novel targeted agents whose toxicity profiles don’t overlap with cisplatin is becoming a future direction.As an anti-angiogenesis agent,Endostar plays a critical role in tumor growth,metastasis,and recurrence by activating its receptorVEGFR(vascular endothelial growth factor receptor).Studies on various types of tumors including head-and-neck cancer,lung cancer,and esophagus cancer have confirmed its safety and effectiveness when it is combined with RT and/or chemotherapy.In cervical cancer,the addition of Endostar to CCRT can significantly inhibit tumor cell activity,lower the risk of metastasis to lymph node,and prevent tumor angiogenesis,shown by a study on xenograft model in mice.However,in the era of precision RT marked by intensity-modulated radiation therapy(IMRT)and three-dimensional brachytherapy,the efficacy of the combination of Endostar and CCRT is largely unknown.Endostar has been proven to be able to inhibit proliferation and migration of vascular endothelial cell,leading to the inhibition of new angiogenesis.It can also suppress tumor proliferation or metastasis by cutting off the nutrition supply to the tumor.RT itself has a strong inhibitory effect on vascular endothelial cells,wherase Endostar acts on vascular endothelial cells to block the binding of vascular endothelial growth factor(VEGF)to its receptor,which increases the damage of endothelial cells induced by radiation.Therefore,when combined with RT,Endostar may have a more potent effect on anti-angiogenesis.VEGF as one of the most critical factors to promote angiogenesis,its receptor-VEGFR is considered to be closely associated with the process of angiogenesis and is the main target of anti-angiogenesis inhibitors.The combination of VEGF and VEGFR triggers a series of changes in endothelial cells and promotes tumor angiogenesis,infiltration and metastasis.By down-regulating VEGF expression,Endostar affects the buildup of peritoneal fluid,angiogenesis,and metastasis.However,the specific molecular mechanism in this process is largely unknown.In addition,to the best of our knowledge,no literature report so far has been published regarding the functional impact of the combination of Endostar and chemoradiotherapy on cervical cancer.In this research,we aimed to evaluate the efficacy and toxicities of Endostar in combination with CCRT versus CCRT alone in treating LACC.We also assessed the proliferation of cancer cells and tumor formation in nude mice when they were treated with this combination,and explored the potential molecular mechanism in regulating angiogenesis in cervical cancer.PartⅠ A Prospective,Randomized Trial on Endostar Combined with Concurrent Chemoradiotherapy for Locally Advanced Cervical CancerPurposeTo evaluate the efficacy and toxicities of Endostar in combination with CCRT versus CCRT alone in treating LACC.Methods and materialsIn this prospective,randomized controlled,single-institute trial,patients with LACC were randomly assigned in a 2:1 ratio to receive CCRT plus Endostar(CCRT+E arm)or CCRT alone(CCRT arm).All patients received pelvic IMRT and three-dimensional brachytherapy with high dose rate after the complete of external RT.Weekly cisplatin(40 mg/m2)was administered concurrently with IMRT for 5 cycles.In addition,patients in the CCRT+E arm also received continuous infusion with Endostar for 2 cycles,starting on the first day of IMRT.Endostar was repeated every 3 weeks.The expression of VEGFR-2 in the tumor tissues was examed by immunohistochemistry.The primary endopoints were progression-free survival(PFS)and acute toxicities;the secondary endpoints were overall survival(OS),distant metastasis-free survival(DMFS)and locoregional recurrence-free survival(LRRFS);the exploratory endpoints were the correlation between VEGFR-2 expression and clinicopathological factors and the impact of VEGFR-2 expression on survival.Results1.Patient baseline characteristics:One-hundred and sixteen patients were enrolled,including 78 in the CCRT+E arm and 38 in the CCRT arm.The median age was 55 years old.According to the International Federation of Gynecologists and Obstetrics(FIGO)Staging System(version 2014),there were 112 patients(96.5%)with stage IIA or above.Fifty-nine(50.9%)and 9 patients(7.8%)had pelvic lymph node and para-aortic lymph node metastases,respectively.No significant differences in age,performance status,stage,histological type,pelvic lymph node metastasis,and para-aortic lymph node metastasis were found between the two arms.One patient in the CCRT arm and 2 patients in the CCRT+E arm left the study before it initiated.The intention-to-treat(ITT)population was 78 patients for the CCRT+E arm and 38 patients for the CCRT arm.2.Treatment compliance:Overall,patient compliance to the planned treatment was well maintained.All patients completed the planned IMRT and brachytherapy.Sixty-eight patients(85.9%)in the CCRT+E arm completed≥4 cycles of chemotherapy,42 of whom(53.8%)completed 5 cycles;thirty-one patients(81.6%)in the CCRT arm completed≥4 cycles of chemotherapy,17 of whom(44.7%)completed 5 cycles.Sixty-seven patients(87.2%)in the CCRT+E arm completed 2 cycles of Endostar.3.Survival analysis:An improvement tendency in PFS was observed in the CCRT+E arm,but it didn’t meet the study endpoint.The 1-and 2-year PFS in the CCRT+E arm and in the CCRT arm were 91.4%vs.82.1%and 80.8%vs.63.5%(p=0.091),respectively.Significant differences in DMFS were found between the two arms,with 1-and 2-year DMFS of 92.7%vs.81.1%and 86.0%vs.65.1%(p=0.031),respectively.With regard to OS and LRRFS,there were no significant differences between the two arms.4.Multivariate analysis:Stage,overall treatment time,cycles of chemotherapy,para-aortic lymph node metastasis,and expression of VEGFR-2 were independent factors for PFS.5.Subgroup analysis:On subgroup analysis,the CCRT+E arm had a favorable PFS over the CCRT arm for ages ≥60 years old,PS score of 1,stage,histological type,OTT>56 days,cycles of chemotherapy>3,pelvic lymph node metastasis,para-aortic lymph node metastasis,and positive expression of VEGFR-2,whereas the CCRT arm had a favorable PFS for ages<60 years old,and negative VEGFR-2 expression.6.The impact of VEGFR-2 expression on long-term outcome for the ITT population:Overall,70.5%and 73.7%of patients had positive VEGFR-2 expression in the CCRT+E arm and in the CCRT arm,respectively.However,the expression of VEGFR-2 had no significant impact on LRRFS and DMFS.7.The impact of VEGFR-2 expression on long-term outcome within each arm:In the CCRT+E arm,patients with positive VEGFR-2 expression had significantly longer PFS,OS,LRRFS,and DMFS,compared with those with negative VEGFR-2 expression.In the CCRT arm,although patients with positive VEGFR-2 expression had relatively lower PFS,OS,LRRFS,and DMFS in general,compared with those with negative VEGFR-2 expression,no statistical significance was found.8.Inter-arm comparison of long-term outcome according to VEGFR-2 expression:Patients in the CCRT+E arm had significantly longer PFS,OS,and DMFS than those in the CCRT arm when VEGFR-2 expression was positive.However,no significant difference was found in LRRFS between the two arms.No significant differences were found in PFS,OS,LRRFS,and DMFS between the two arms when VEGFR-2 expression was negative.9.Acute and late toxicities:Patients in both arms had similar acute and late toxicity profile.The most frequently seen acute toxicities included bone marrow suppression,nausea,diarrhea,and fatigue,manifested mainly as grade Ⅰ-Ⅱ.The most commonly observed late toxicities were injuries to skin and subcutaneous tissue,leg edema and pain,irradiation enteritis,irradiation cystitis,and uronephrosis.The incidences in the CCRT+E arm and in the CCRT arm ranged from 3.9%to 10.5%and from 2.7%to 13.5%,respectively.Conclusions1.For patients with LACC,the addition of Endostar to CCRT resulted in a good treatment compliance with manageable toxicities.2.CCRT plus Endostar significantly improved DMFS but not PFS over CCRT alone.3.For patients with positive VEGFR-2 expression,the addition of Endostar to CCRT could significantly improve PFS,OR,LRRFS,and DMFS.Patients with positive VEGFR-2 expression and treated with CCRT alone had slightly lower PFS,OS,LRRFS,and DMFS than those with negative VEGFR-2 expression,but no statistical significance was found.4.For patients with negative VEGFR-2 expression,CCRT with or without Endostar had no significant impact on PFS,OS,LRRFS,and DMFS.Part Ⅱ The Molecular Mechanism of Endostar Incorporated into Concurrent Chemoradiotherapy in Regulating Angiogenesis via VEGFR-2/VEGF Pathway in Cervical CancerPurposeTo investigate the effect of Endostar combined with chemoradiotherapy on cervical cancer cells and xenograft tumors in nude mice and its molecular mechanism of regulating angiogenesis.Methods and materialsVEGFR-2 overexpressed or silenced lentiviral vectors was constructed separately and then verified in Siha and Hela cells.Groups were set as the following:Control group(blank control),CCRT group(concurrent chemoradiotherapy),CCRT+E group(concurrent chemoradiotherapy plus Endostar),CCRT+VEGFR2+E group(VEGFR-2 overexpressed and transfected prior to concurrent chemoradiotherapy plus Endostar),CCRT+KD-VEGFR2+E group(VEGFR-2 silenced and transfected prior to concurrent chemoradiotherapy plus Endostar),CCRT+NC+E group(empty vector transfected prior to concurrent chemoradiotherapy plus Endostar).Hela and Siha cervical cancer cells and Huvec vascular endothelial cells were treated according to the study protocol.Proliferation and apoptosis of Hela and Siha cells were detected.The changes of angiogenesis ability of Huvec vascular endothelial cells were evaluated.The effect of Endostar combined with concurrent chemoradiotherapy on tumor proliferation in vivo after overexpression or silencing of VEGFR-2 in nude mice xenograft model was analyzed.Immunofluorescence was used to detect the changes in VEGF expression and protein nucleocytoplasmic localization in Hela cervical cancer cells treated with different concentration of Endostar.The protein half-life of VEGF and VEGFR-2 was measured.The protein interaction between VEGF and VEGFR-2 was verified for Hela cervical cancer cells after treatment with Endostar.Results1.In Hela and Siha cells,mRNA in the VEGFR-2-overexpressed group was significantly increased,compared with the negative vector control group(p<0.001),and the silencing efficiency of the VEGFR-2-silenced group was above 50%(p<0.001).The VEGFR-2 protein expression in the VEGFR-2 overexpressed group was significantly increased,compared with the negative vector control group(p<0.05),whereas the VEGFR-2 protein in the VEGFR-2 silenced group was significantly decreased(p<0.05).VEGFR-2 was successfully overexpressed or silenced in both cells,which were eligible for subsequent experiments.2.In Hela and Siha cells,the CCRT and CCRT+E groups significantly inhibited cell proliferation,compared with the control group(p<0.01);compared with the CCRT+NC+E group,the CCRT+VEGFR2+E group significantly inhibited cell proliferation(p<0.05),whereas the ability of CCRT+KD-VEGFR2+E group to inhibit cell proliferation was lower than before(p<0.05).3.In Hela and Siha cells,compared with the control group,the apoptosis in the CCRT and CCRT+E groups was significantly increased(p<0.01).Compared with the CCRT+NC+E group,significantly increased apoptosis was found in the CCRT+VEGFR2+E group(p<0.01),whereas the cell apoptosis in the CCRT+KD-VEGFR2+E group was decreased(p<0.05).4.In Huvec cells,compared with the control group,the CCRT and CCRT+E groups significantly inhibited the angiogenesis of vascular endothelial cells(p<0.01),with the most noticeable changes found in the CCRT+E group.Compared with the CCRT+NC+E group,after VEGFR-2 was overexpressed,the CCRT+VEGFR2+E group inhibited the angiogenesis of vascular endothelial cells more significantly(p<0.01).After VEGFR-2 silencing,the ability of the CCRT+KD-VEGFR2+E group to inhibit angiogenesis was weakened(p<0.05).Compared with the control group,the CCRT and CCRT+E groups significantly inhibited the expression of CD31(p<0.01),but the CCRT+E group inhibited the expression more significantly.Compared with the CCRT+NC+E group,the CCRT+VEGFR2+E group inhibited the expression of CD31 more significantly(p<0.01),whereas the ability of the CCRT+KDVEGFR2+E group to inhibit the expression of CD31 was weakened(p<0.05).5.Tumorigenicity assay in nude mice showed that compared with the control group,both the CCRT and CCRT+E groups significantly inhibited the tumor volume(p<0.01).Compared with the CCRT+NC+E group,the CCRT+VEGFR2+E group significantly inhibited the tumor volume(p<0.05),whereas the ability of the CCRT+KD-VEGFR2+E group to inhibit tumor volume was lower than before(p<0.05).Compared with the control group,both the CCRT group and the CCRT+E group significantly inhibited the expression of CD31(p<0.05,p<0.001,respectively).Compared with the CCRT+NC+E group,the CCRT+VEGFR2+E group significantly inhibited the expression of CD31(p<0.05),whereas the CCRT+KD-VEGFR2+E group was less able to inhibit the expression of CD31 than the CCRT+VEGFR2+E group.6.The expression of VEGF was significantly decreased with the increase of Endostar concentration after treating Hela cells with different concentrations(p<0.05).The results of the protein half-life experiment showed that compared with the control group,VEGF protein was significantly decreased at 4 h and 8 h(p<0.05).After the separation of nucleus and cytoplasm,with the increase of Endostar concentration,the expression of VEGF in the nucleus was significantly decreased,whereas its expression in the cytoplasm was significantly increased(p<0.05).7.The results of COIP experiment showed that VEGF and VEGFR-2 were detected in the Input samples.The VEGFR-2 protein was detected both in the control group and in the Endostar-treated group when an IP experiment was performed by using VEGFR-2 antibody.The VEGF protein was detected in the control group but not in the Endostar-treated group.The pulldown experiment showed that the marker proteins(i.e.,Flag and GST)of VEGF and VEGFR-2 were detected in the Input samples.VEGFR-2 protein was detected both in the control group and in the Endostar-treated group when Flag antibody marked with VEGFR-2 was used for IP experiments.VEGF protein was detected in the control group but not in the Endostar-treated group.ConclusionsWe found that in this study,VEGFR-2 enhanced the inhibition of cervical cancer cells in vitro treated with Endostar in combination with chemoradiotherapy.Endostar inhibited the expression of VEGF by promoting the cytoplasmic transfer of VEGF and by shortening its protein half-life,and inhibited vascularization by regulating the interaction between VEGF and VEGFR-2.The findings are expected to provide a new theory and a new strategy for cervical cancer treated with Endostar and concurrent chemoradiotherapy.