The Effect Of Apatinib On Esophageal Squamous Cell Carcinoma And Its Mechanism

BackgroundEsophageal carcinoma(ESCA)is one of the most common digestive tract malignant tumors in the world.Esophageal squamous cell carcinoma(ESCC)is the main pathological type in China.ESCC in its early stages often affects its functions slightly,without obvious symptoms.When showing clinical manifestations,most of the patients are in the advanced stage,and have missed the chance of surgical treatment.Chemotherapy is an essential method to treat advanced ESCC.However,the clinical efficacy of chemotherapy is limited,and the overall survival(OS)of patients with first-line treatment is only 5-10 months.Recurrence,metastasis and resistance to current chemotherapy are still the primary causes of death in ESCC patients.At present,targeted drugs have been widely used in the treatment of a variety of malignant tumors,with good therapeutic effects and less side effects than radiotherapy and chemotherapy.However,there are fewer targeted drugs for esophageal cancer,and the mechanism of it has not been fully elucidated.Apatinib is a small molecule tyrosine kinase inhibitor of vascular endothelial growth factor receptor-2(VEGFR2).It competes for the ATP binding site of VEGFR2 with high selectivity,blocks downstream signal transduction and inhibits the production of tyrosine kinase,thereby inhibiting the process of angiogenesis.A large number of studies have shown that VEGFR2 is closely related to the occurrence and development of malignant tumors.Apatinib inhibits tumor growth and metastasis,and has a synergistic anti-tumor effect with chemotherapeutic drugs to prolong patient’s survival.Studies have shown that the AKT/mTOR signaling pathway plays an important role in cancer progression.It regulates the growth and metastasis of cancer cells,including ESCC cells.Studies have confirmed that apatinib promoted tumor cell apoptosis and autophagy by inhibiting the AKT/mTOR signaling pathway.In addition,apatinib inhibits the progression of cancer through suppressing the invasion,metastasis and epithelial-mesenchymal transition(EMT)process of cancer cells.At present,there are a few studies on the treatment of ESCC with apatinib,mainly focusing on the competition of apatinib for the ATP binding site of VEGFR2 to inhibit angiogenesis,thereby exerting an anti-tumor effect.There is a lack of apatinib inhibiting the esophagus research on the mechanism of carcinogenesis and its synergy with cytotoxic drugs.In this study,we observed the effect of apatinib combined with docetaxel on the progression of esophageal cancer in patients with ESCC.Analyzed the aberrant expression of VEGFR2 in esophageal cancer and adjacent tissues in multiple databases,and its relationship with prognosis.The expression level of VEGFR2 in 99 cases of ESCC patients with esophageal squamous cell carcinoma tissues and 40 cases of adjacent normal tissues was detected,and its clinical significance was discussed.The effects of apatinib on the proliferation,apoptosis,migration and invasion of ESCC cells were explored in vitro.Western blot was used to detect the effects of apatinib on apoptosis,metastasis,EMT-related proteins and key proteins in the Akt/mTOR pathway,and its mechanism of action was analyzed.The effects of apatinib combined with cytotoxic drugs on ESCC cell proliferation,migration,invasion and angiogenesis were further explored in vitro.Western blot was used to detect the synergistic effect of apatinib and cytotoxic drugs on the related protein expression of apoptosis,metastasis and EMT.Establish the nude mice xenograft model of ESCC,and explore the effect of apatinib combined with cytotoxic drugs on tumor growth.Tunel assay was used to detect the rate of apoptosis in tumor tissues,and immunohistochemistry(IHC)staining was used to detect the expression of Ki67 proteins in tumor tissues.This research is mainly divided into the following four parts:Part 1.The expression level of VEGFR2 in human esophageal squamous cell carcinoma and normal esophageal tissues and its clinical significanceObjectiveIHC staining is used to detect the expression level of VEGFR2 protein in tumor tissues and adjacent normal tissues in ESCC patients,and explore the clinical significance of abnormal expression of VEGFR2.Methods1.The public database provided by GEPIA website was used to analyze the expression difference of VEGFR2 gene in a variety of malignant tumor tissues and normal tissues adjacent to cancer.Analyze the difference in the expression level of VEGFR2 gene in esophageal cancer tissue and normal esophageal tissue,analyze the relationship between VEGFR2 gene expression level and the clinical stage and prognosis of patients with esophageal cancer.2.IHC staining was used to detect the expression level of VEGFR2 in 99 cases of ESCC patients with esophageal squamous cell carcinoma tissues and 40 cases of adjacent normal tissues.3.Collected clinical data of 99 ESCC patients and analyzed the relationship between VEGFR2 expression level and clinicopathological characteristics and prognosis.Results1.The results of the GEPIA website database analysis showed that the expression of VEGFR2 gene in renal clear cell carcinoma(KIRC)cancer tissue was significantly higher than that in adjacent normal tissues.In the endometrial carcinoma of the uterus(UCEC),the expression level of VEGFR2 gene in tumor tissues was significantly lower than that in adjacent normal tissues.The expression level of VEGFR2 gene in esophageal cancer tissue was higher than its expression level in normal esophageal tissue,but the difference was not statistically significant(P>0.05).The expression level of VEGFR2 gene was correlated with the clinical stage of esophageal cancer(P<0.05).Analysis of survival data found that the OS of patients with esophageal cancer with high VEGFR2 gene expression was lower than that of patients with esophageal cancer with low VEGFR2 gene expression(P<0.05).2.IHC was used to detect the expression level of VEGFR2 in tumor tissues and adjacent normal tissues of ESCC patients.The results showed that the expression level of VEGFR2 in esophageal squamous cell carcinoma tissues was higher than that in adjacent normal tissues(P<0.0001).3.Analyzed the relationship between the expression level of VEGFR2 and clinicopathological characteristics and prognosis.The results showed that the expression of VEGFR2 was positively correlated with the depth of invasion,poor differentiation and lymph node metastasis(P<0.05).ESCC patients with high VEGFR2 expression had worse overall survival than patients with low VEGFR2 expression(P<0.0001).ConclusionThe expression of VEGFR2 in esophageal cancer tissues is significantly higher than that in adjacent normal tissues.The level of VEGFR2 is positively correlated with the depth of invasion and lymph node metastasis,and negatively correlated with differentiation and prognosis.VEGFR2 can be used as a potential target for ESCC therapy.Part 2.The effect and mechanism of apatinib on esophageal squamous cell carcinoma cells in vitroObjectiveThe effects of apatinib on the proliferation,apoptosis,migration,invasion and EMT of ESCC cells in vitro,and the effects of apatinib on the AKT/mTOR pathway in vitro will be explored.Methods1.CCK8 kit was used to detect the effect of different concentrations of apatinib on the cell proliferation of ESCC cells at different times.2.Detect the effect of apatinib on the apoptosis of ESCC cells by Annexin V/PI kit.3.The wound healing assay was used to detect the effect of apatinib on the migration ability of ESCC cells.4.Transwell assay was used to detect the effect of apatinib on the invasion ability of ESCC cells.5.Western blot was used to detect the expression the level of VEGFR2 protein in ESCC cells(KYSE450,EC1).Western blot was used to detect the changes of Bcl2,MMP9,E-cadherin,vimentin,AKT,p-AKT,S6 and p-S6 protein expression levels in the in ESCC cells treated with apatinib.Western blot was also used to detect the changes in AKT,p-AKT,S6 and p-S6 protein expression levels in ESCC cells treated with apatinib combined with AKT pathway inhibitor/mTOR pathway inhibitor.Results1.CCK8 assay results showed that apatinib significantly inhibited the cell proliferation ability of KYSE450 and EC1 cells(P<0.001),and this inhibition ability was time-and dose-dependent.2.The results of Annexin V/PI assay showed that apatinib promoted the apoptosis of ESCC cells(P<0.001).3.The results of the wound healing assay showed that apatinib can inhibit the migration ability of ESCC cells(P<0.001).4.Transwell results showed that apatinib can inhibit the invasion ability of ESCC cells(P<0.001).5.Western blot results show that the expression level of VEGFR2 protein in KYSE450 cells is higher than that in EC1 cells.The protein expression levels of Bcl2,MMP9 and vimentin decreased,and the protein expression levels of E-cadherin increased after ESCC cells were treated with apatinib.The expression levels of p-AKT and p-S6 proteins in the AKT/mTOR pathway decreased after ESCC cells were treated with apatinib.The level of p-AKT and p-S6 protein expression was down-regulated in ESCC cells treated with Akt pathway inhibitor or Apatinib,and Apatinib enhanced the effect of AKT pathway inhibitor.The p-S6 protein expression was down-regulated in ESCC cells treated with mTOR pathway inhibitor or Apatinib,and Apatinib enhanced the effect of mTOR pathway inhibitor.Conclusion1.Apatinib inhibits the proliferation,migration and invasion of ESCC cells,promotes apoptosis.Apatinib has a stronger inhibitory effect on the cell proliferation of VEGFR2 high-expressing ESCC cells.Apatinib inhibits the level of Bcl2,MMP9 and vimentin protein expression,enhances the level of E-cadherin protein expression in ESCC cells.2.Apatinib inhibits AKT/mTOR pathway in esophageal cancer cells.When apatinib is combined with AKT/mTOR pathway inhibitor to treat esophageal cancer cells,it can enhance its inhibitory effect on AKT/mTOR pathway by inhibiting the expression of p-AKT and p-S6 protein.Part 3.The anti-tumor effect and mechanism of apatinib combined with cytotoxic drugs on esophageal squamous cell carcinoma in vitroObjectiveThe effects of apatinib combined with cytotoxic drugs on the proliferation,migration,invasion and EMT of ESCC cells in vitro will be explored.Methods1.CCK8 kit was used to detect the effects of different concentrations of cytotoxic drugs(5-fluorouracil,paclitaxel,cisplatin)on the cell proliferation of ESCC cells.Then the cells were divided into 8 groups:control group,5-fluorouracil,paclitaxel,cisplatin,apatinib group,apatinib+5-fluorouracil,apatinib+paclitaxel,apatinib+cisplatin,CCK8 kit was used to detect the effect of apatinib combined with cytotoxic drugs on the proliferation of ESCC cells.2.The wound healing assay was used to detect the effect of apatinib combined with cytotoxic drugs on the migration of ESCC cells.3.Transwell assay was used to detect the effect of apatinib combined with cytotoxic drugs on the invasiveness of ESCC cells.4.Western blot was used to detect the changes in the expression levels of Bcl2,MMP9,E-cadherin and vimentin proteins in ESCC cells treated with apatinib combined with cytotoxic drugs.Results1.The results of CCK8 assay showed that the proliferation rates of cytotoxic drugs in ESCC cells were lower than those of the untreated control group with the increase of drug dose and treatment time(P<0.001).Besides,the combination of Apatinib with each cytotoxic drug displayed synergistic inhibition effects on the proliferation of ESCC cells compared with each treatment alone(P<0.001).2.The results of the the wound healing assay showed that the inhibitory effect on cell migration of ESCC cells treated with apatinib and cytotoxic drugs was stronger than that of the single-drug treatment group(P<0.01).3.Transwell results showed that the inhibitory effect on cell invasion of ESCC cells treated with apatinib and cytotoxic drugs was stronger than that of the single-drug treatment group(P<0.05).4.Western blot results showed that compared with the single-drug treatment groups,the expression levels of Bcl2,MMP9,and vimentin protein in the ESCC cells were significantly reduced,and the expression level of E-cadherin protein significantly increased in the apatinib combined with cytotoxic drugs treatment groups.ConclusionApatinib can enhance the inhibitory effects of cytotoxic drugs on the proliferation,migration and invasion of ESCC cells.Compared with the single-drug treatment groups,the expression levels of Bcl2,MMP9,and vimentin protein in the ESCC cells were significantly reduced,and the expression level of E-cadherin protein significantly increased in the apatinib combined with cytotoxic drugs treatment groups.Part 4.The anti-tumor effect of apatinib combined with cytotoxic drugs on esophageal squamous cell carcinoma in vivoObjectiveA subcutaneous xenograft model of ESCC in nude mice is constructed to detect the effects of apatinib single agent and combined cytotoxic drugs on the weight and volume of esophageal tumors,as well as on tumor apoptosis,proliferation.Methods1.ESCC cells were used to establish the nude mice xenograft model of ESCC.All mice were randomly divided into 8 groups:control group,5-fluorouracil,paclitaxel,cisplatin,apatinib group,apatinib+5-fluorouracil,apatinib+paclitaxel,apatinib+cisplatin.Regularly observe and measure the weight status of each group of mice and the volume of tumors.Take out the tumors and weigh the tumors.2.Tunel detects cell apoptosis of tumor tissues.3.IHC staining was used to detected the expression of Ki67 in tumor tissues.Results1.The results showed that apatinib inhibited the growth of esophageal cancer tumors in mice.The results showed that each agent alone inhibited tumor growth compared with the control group.The combination of Apatinib with each cytotoxic drug displayed synergistic inhibition effects on the growth of tumors compared with each treatment alone.Additionally,apatinib combined with cisplatin has the best anti-tumor effect(P<0.05).2.Tunel results showed that apoptosis rates of esophageal tumor increased after each agent treatment compared with control group,which were significantly enhanced in two agent treatment groups compared with each agent treatment groups.3.Immunohistochemistry results showed that the Ki67 protein level was significantly reduced in the combination of Apatinib and each cytotoxic drug groups compared with that in each agent groups.Conclusion1.Apatinib inhibits the growth of tumors in the nude mice xenograft model of ESCC,and enhances the anti-tumor activity of cytotoxic drugs.The anti-tumor effect is better on the tumors when combined with cisplatin.2.Apatinib and cytotoxic drugs can promote tumor cell apoptosis,inhibit tumor cell proliferation.Apatinib and cytotoxic drugs have a synergistic anti-esophageal squamous cell carcinoma effect when they are used in combination.