The Inhibition Of Apatinib On Human Lung Cancer Cells And Nude Mice Transplantation Tumors And Induction Of Vascular Normalization

Lung cancer is the most common malignant tumor in China,and most patients are advanced lung cancer,with poor treatment results and high mortality.With the application of current new treatment options,the overall survival of lung cancer patients has significantly improved,but The 5-year survival rate of patients with advanced lung cancer is still low,and new treatments are still needed to further improve the survival time of patients with advanced lung cancer.Folkman proposed that tumor growth requires the formation of new blood vessels,so it inhibits neovascularization as an important means of treating tumors,anti-angiogenic therapy has become a hot topic in the field of treatment of patients with advanced lung cancer,and new anti-angiogenic molecularly targeted drugs have continuously appeared,and their efficacy and safety have also been obtained clinically.Now anti-vascular targeted therapy has become a new treatment for advanced lung cancer.Bevacizumab is the first anti-angiogenic drug approved by FDA for the treatment of advanced lung cancer.Bevacizumab is a recombinant humanized monoclonal antibody that blocks its biological activity by binding to VEGF anti-angiogenic effect.However,bevacizumab cannot be used as a single drug for lung cancer.Studies have confirmed that bevacizumab combined with chemotherapy can improve the survival of patients with advanced non-small cell lung cancer treated with traditional chemotherapy to a certain extent.Apatinib is the latest drugs that inhibit tumor angiogenesis is a small molecule tyrosine kinase inhibitor.By selectively inhibiting the tyrosine kinase activity of VEGFR-2,blocking the signaling pathway and inhibiting tumor angiogenesis.Therefore,it has an inhibitory effect on tumor growth.At present,apatinib is mainly used in the third-line or higher treatment of advanced gastric cancer and there is less research on lung cancer,especially the lack of randomized controlled clinical trials of patients with advanced lung cancer,the clinical evidence is not sufficient.Moreover,apatinib has less basic research on lung cancer cells and nude mice transplant tumors,and has the advantages of apatinib treatment.It is not clear whether apatinib combined chemotherapy and targeted drug treatment can improve the efficacy of patients with advanced lung cancer and which combination method can achieve the best treatment effect This study focuses on the above issues,mainly including the following three part of the content:Part ⅠApatinib inhibits proliferation of different lung cancer cells by promoting apoptosis and inducing cell cycle arrest in vitro and vivoObject:Observe the inhibitory effect of apatinib on five lung cancer cells in vitro and vivo.It is not clear whether apatinib has an inhibitory effect on lung cancer cells in vitro and whether the inhibitory effect on different lung cancer cells is different.The predominant population of patinib in the treatment of lung cancer,and preliminary revealed the mechanism of action of apatinib in inhibiting the proliferation of lung cancer cells.Contents and methods:CCK-8 method was used to determine the inhibitory effect and the IC50 values of five different lung cancer cells to apatinib.Monoclonal test was used to detect the proliferation of five lung cancer cells to apatinib in vitro.Western blot and qPCR were used to detect VEGFR2 of five lung cancer cell lines in protein and gene expression levels,and further selected A549 cells with low expression of VEGRFR-2 and H1975 and H446 cells with high expression of VEGFR2 as the representative of the next experiment.Flow cytometry was used to determine the changes in cell cycle;Tunel and Flow cytometry was used to determine its effects on apoptosis.Western blot was used to detect the expression of c-Caspase3 and PARP apoptosis-related proteins,and c-Caspase3 was detected by cellular immunofluorescence to indicate its apoptosis mechanism.Three kind nude mice xenograft model with lung cancer cell lines were constructed.We observed the effects of different doses of apatinib on the growth of xenografts in nude mice,and the expression of CD31,Ki-67 and c-caspase3 by immunohistochemistry.We measured body weight of nude mice,and measured serum ALT/AST,BUN/Cr,LDH-L/CK and GLU at the same time by biochemical methods,and determine FT3/FT4 by ELISA;And we observed the pathological change of heart,liver,kidney,pancreas and thyroid in order to observe the side effects of apatinib.At the same time,western blot and immunofluorescence were used to detect the expression of LC3-Ⅱ after applying apatinib to H1975 and H446 cells.Changes of IC50 of two kinds of lung cancer cells after phagocytic inhibitor(chloroquine)were established,and a nude mouse xenograft model of the above two kinds of lung cancer cells was constructed to observe whether apatinib combined with chloroquine could increase the anti-tumor effect.Results:Apatin inhibited H1975 and H446 cells that high expressed VEGFR2 significantly stronger than the other three lung cancer cells,and its IC50 value was significantly lower than the other three lung cancer cells.With the increase in concentration,the number of colonies formed was significantly reduced H1975 and H446 cells(P<0.05).Apatinib mainly blocked cells in the G2 phase,which was significantly different from the control group(P<0.05).It was showed that with the increase of apatinib concentration,the proportion of apoptotic cells increased significantly,which was statistically different from the control group(P<0.05).The expressions of c-caspase3 and c-PARP increased significantly(P<0.05).The application of apatinib in A549 cells with low expression of VEGFR2 did not have the above-mentioned changes.For nude mice xenograft models constructed by H1975 and H446 cells,as the dose of apatinib was increased,the inhibitory effect on the growth of xenografts was more obviously compared with the control group,there were significant statistical differences(P<0.05).The expressions of CD31 and Ki67 were significantly reduced compared with the control group,and the expression of c-caspase3 was significantly increased,all with significant statistical differences(P<0.05).In the model of transplanted tumors,only apatinib in the high-dose group can see a significant tumor growth inhibition effect.There were no significant differences in serum ALT/AST,BUN/Cr,and LDH-L/CK levels(P>0.05).GLU in nude mice in the high-dose group was significantly higher than that in the control group,and FT3/FT4 levels were significantly reduced(P<0.05);The HE staining of other organs showed no significant pathological changes after application of apatinib,but electron microscopy showed that mitochondria,endoplasmic and other microstructure has different degrees of damage.After applying apatinib in vitro and vivo,the expression of autophagy-expressing protein LC3-Ⅱ in H1975 and H446 cells can be increased,which is significantly different from the control group(P<0.05).Combined with autophagy inhibitors can reduce the IC50 of apatinib on two lung cancer cells,there is a significant statistical difference(P<0.05),combined with autophagy inhibition after administration,the inhibitory effect on nude mice transplanted tumor was significantly stronger than that of apatinib monotherapy(P<0.05).Conclusion:Apatinib can effectively inhibit the proliferation of lung cancer cells that highly express VEGFR-2,and can induce apoptosis through the Caspase3/PARP pathway;it can effectively inhibit the growth of transplanted tumors and reduce the microvascular density of transplanted tumors.The side effects of the drug on other organs are mainly ultrastructural damage,which is more significant for thyroid and pancreatic damage,and serum related indicators have changed to some extent.Apatinib can induce autophagy in the above two types of cells.With the increase of drug concentration,autophagy is more obvious.Autophagy can induce drug resistance to a certain extent and reduce the drug effect.It can be effective when combined with autophagy inhibitors can improve the inhibitory effect of apatinib on transplanted tumors.Part ⅡThe effect of apatinib combined with chemotherapy or targeted therapy on non-small cell lung cancer in vitro and vivoObject:Apatinib is a small-molecule tyrosine kinase inhibitor that selectively acts on VEGR2.It has shown good therapeutic effects in a variety of malignancies,but single-agent treatment is fast and the overall effectiveness is limited.In this study,we explored the difference and mechanism of the inhibitory effect of apatinib combined with in vitro and vivo on non-small cell lung cancer,and observed whether the combination of anti-angiogenic drug with chemotherapy or targeted treatment drugs can effectively improve the therapeutic effect of chemotherapy or targeted drugs as a single drug,and preliminary explore its mechanism of action,and observe the safety of combined drugs.Contents and methods:In vitro the effect of apatinib on the proliferation of three human non-small cell lung cancer cells(A549,HCC827and H1975)was examined using the CCK8 test,and apatinib was detected the inhibition of three kinds of lung cancer cells monotherapy and combined the pemetrexed,icotinib,and osimertinib,respectively.The IC50 of three kinds of lung cancer cells after apatinib single and combined treatment were calculated in order to observe the synergistic inhibitory effect.The effect of apatinib was verified by three nude mouse xenograft models of non-small cell lung cancer in vivo.Nude xenograft models of each type of lung cancer cells were divided into 4 groups(Control group,apatinib monotherapy group,pemetrexed or icotinib or osimertinib monotherapy group,apatinib combined with pemetrexed or icotinib or osimertinib group).For 21 days,drawing the growth curve of the transplanted tumor,and observing whether apatinib combined with chemotherapy and targeted drugs can improve its tumor suppressing effect.Immunohistochemical staining of the transplanted tumor was used to analyze the microvessel density of and the the expression of ki67 in transplanted tumor tissue.Western blot analysis the expression of p-AKT,p-mTOR,and p-ERK related signal pathway in transplanted tumor groups in each group.By observing the body weight of nude mice in each group,and observing the structure of important organs such as heart,brain,liver,and kidney by HE staining and immunohistochemical staining in microvessel density changes,to explore the safety of apatinib combined targete and chemotherapy treatment.Results:The inhibitory effect of apatinib on three types of non-small cell lung cancer cells increased with increasing drug concentration,of which H1975 cells were the most sensitive to apatinib,and the IC50 values of apatinib on three types of lung cancer cells were 44.59±3.95μM,39.88±3.77μM,16.49±1.48μM,and there was a statistically significant difference(P<0.05).The combined drug group had a stronger proliferation inhibitory effect than the single drug group at the same concentration.In A549 cells,The synergy index CI of apatinib combined with pemetrexed CI=0.61±0.07,in HCC827 cells,apatinib combined with icotinib CI=0.53±0.05,in H1975 cells,apatinib combined with osimertinib CI=0.60±0.05,all have a synergistic effect.Compared with the control group,apatinib monotherapy,chemotherapy and targeted drug monotherapy all have a certain transplantation effect on nude mice transplant tumors.The inhibitory effect of transplantation tumors was best in apatinib combined with pemetrexed,icotinib or osimertinib.The microvessel density and Ki67 protein expression in the transplantation tumor tissues of the combined group were significantly reduced,which were significantly statistical differences from other groups(P<0.05);It was the most obvious down regulation on the phosphorylation of signal pathway-related proteins AKT,mTOR,and ERK in transplanted tumor tissues in the combined groups,and there was a statistically significant difference compared with other groups(P<0.05).Among the three nude mice xenograft models of lung cancer,the inhibitory effect on H1975 cell lines and nude xenograft were the strongest.There was no significant statistical differences on the weight of nude mice in each group,and no abnormalities in HE pathology in heart,brain,liver,and kidney tissues,and no significant difference in heart and brain microvascular density among the groups.There was significant reduction of microvascular density in apatinib and combined groups.And there were no significant difference between apatinib montherapy and combined group on the microvascular density in liver and kidney tissues.Conclusion:Apatinib monotherapy has a certain inhibitory effect on non-small cell lung cancer cells,which is most obvious for H1975 lung cancer cell line.Apatinib combined with chemotherapy and targeted drugs can effectively increase its inhibitory effect on lung cancer cells.The IC50 values of three kinds of lung cancer cells is significantly reduced,and there is a significant synergistic inhibitory effect.Apatinib combined with chemotherapy and targeted drugs can significantly increase the inhibitory effect of the xenograft tumors in nude mice and reduce the microvessel density and cell proliferation in transplanted tumor tissue,and effectively reduce the levels of signal pathway-related protein phosphorylation.Apatinib combined with chemotherapy and targeted drugs are highly safe and do not increase toxic side effects.Part ⅢExperimental study of the vascular normalization window for tumours treated with apatinib and the efficacy of sequential chemotherapy with apatinib in lung cancer-bearing mice and patientsObject:The growth of tumors depends on new blood vessels,which play an important role in tumor proliferation,invasion and metastasis.In the tumor vascular system,the structure of new blood vessels is disordered,the shape is abnormal and the structure of the blood vessel wall is incomplete,resulting the increased interstitial fluid pressure and blood flow resistance.These lead to changes in the local microenvironment of tumor tissues,mainly manifested by hypoxia and acidosis,which further affect the efficacy of chemotherapeutic drugs,leading to poor efficacy of chemotherapeutic drugs.Studies show that some anti-angiogenesis drugs can normalize the abnormal state,thereby more effectively transporting oxygen and drugs to tumor cells through vascular normalization,improving the efficacy of chemotherapy drugs.Apatinib is a specific VEGFR2 inhibitors can block the VEGF/VEGFR2 signaling pathway to inhibit tumor neovascularization,and control tumor growth.However,it is unclear whether apatinib can induce vascular normalization in tumor tissues.The time and dose of apatinib which induced vascular normalization are also unclear,so it is necessary to conduct research on the apatinib induced vascular normalization to determine the dose and time.At the same time,observe the effect of sequential systemic chemotherapy on lung cancer cell transplantation tumors and patients after applying apatinib which induced vascular normalization,and further optimizing the administration strategy of anti-angiogenic drugs combined with chemotherapy to improve the efficacy of combined treatment and prolong the overall survival of patients with non-small cell lung cancer.Contents and methods:In this study,we selected human non-small cell lung cancer cell A549 for research,cultured in vitro,and constructed a nude mice xenograft model.After tumor formation,60 tumor-bearing nude mice were selected for vascular normalization studies.Administered saline,apatinib 60mg/kg and 120 mg/kg for intervention management and continuous intervention for 10 days.Before dosing and on the 1st,3rd,7th and 10th days after administration,four nude mice were sacrificed in each group.Immunohistochemistry was used to detect the expression of HIF-1α,α-SMA,and college-Ⅳ at different time points in the transplanted tumor tissues.Western blot was used to detect the expression level of MMP-2 and MMP-9 protein,and the ultrastructure of tumor blood vessels in each group of tumor tissues was observed by electron microscopy,and the above-mentioned indicators were combined to determine the dose and time of apatinib to induce blood vessel normalization in transplantation tumors.Then 32 tumor-bearing nude mice were randomly divided into four groups according to the random number table,and each group of eight,which were the control group,pemetrexed monotherapy group,apatinib and pemetrexed simultaneous treatment group,apatinib sequential pemetrexed group.The treatment group refers to the administration of apatinib for a certain period of time to induce vascular normalization,and then the intraperitoneal injection of pemetrexed at the same dose during vascular normalization,where the dose and time of apatinib required for vascular normalization are based on the The results of the one-step study were designed for experiments.Nude mice were treated for 14 consecutive days and tumor suppression curves were drawn.Immunohistochemistry and western blot were used to detect the expression levels of related proteins in transplanted tumor tissues.At the same time,20 patients with advanced non-small cell lung cancer were selected,and apatinib sequential chemotherapy was used as a third-line chemotherapy regimen to observe its clinical efficacy and safety.Results:After treatment with apatinib in the low-dose group,HIF-1α decreased significantly on the 3rd and 7th days of treatment,and the levels of α-SMA and typeⅣ collagen increased significantly and there was a statistical difference between the two groups(P<0.05).However,in the high-dose apatinib group,there were no significant differences in HIF-1α,α-SMA,and Ⅳ collagen expression between groups at each time point(P>0.05).During 3-7 days of low-dose apatinib treatment,the expression levels of MMP-2 and MMP-9 in the transplanted tumor tissues decreased significantly(P<0.05),and at other times or in the high-dose group,the expression levels were no significant changes(P>0.05).On the 3rd and 7th days of low-dose apatinib treatment,the vascular structure in the transplanted tumor tissue was complete and regular,and the pericyte structure was observed,and the vascular basement membrane structure was intact and no obvious gap was observed.However,at other time points and in the high-dose group,the vascular structure in the xenograft tissue was still disordered,no mature pericyte structure was observed,and the vascular basement membrane was incomplete.From the xenograft inhibition curve,compared with the control group,the three groups showed significant inhibition of the growth of transplanted tumors.The tumor volume was significantly different from the control group and was lower sequential treatment with apatinib and pemetrexed produced the best inhibitory effect,which was significantly different from simultaneous treatment(P<0.05).There was no significant difference in body weight of nude mice in each group,indicating that all intervention methods are safer.In the four groups of transplanted tumor tissues,the expression of Ki67 in the transplanted tumor tissue of the apatinib sequential pemetrexed group was significantly reduced,while the expression of caspase3 was significantly increased compared with the other three groups(P<0.05).In the analysis of related signal pathway factors,the expression levels of p-AKT,p-mTOR,p-MEK and p-ERK in the transplanted tumor tissue of the sequential group were significantly lower than those of the other groups(P<0.05).The response of 20 patients with advanced non-small cell lung cancer treated with sequential apatinib chemotherapy is as follows:CR(n=0),PR(n=3),SD(n=14),and PD(n=3);The ORR and DCR were 15.0%and 85.0%respectively.The median PFS was 4.85 months and the median OS was 10.2 months.Patients experienced a grade 3/4 toxicity rate of 25%.In any patient no grade 4/4 toxicity was observed.Conclusion:The application of low-dose apatinib for 3-7 days can induce the normalization of tumor blood vessels in transplanted tumor tissues to a certain extent.In this time window,combined chemotherapy can effectively inhibit the growth of transplanted tumors in nude mice and can improve the efficacy of chemotherapy from the perspective of clinical treatment effect,low-dose apatinib sequential systemic chemotherapy has a certain effect on patients with advanced non-small cell lung cancer,which can prolong the patient’s survival period to a certain extent,and has higher safety.