Study On The Drug Resistance Of Anlotinib In Liver Cancer

According to the relevant literature,liver cancer has ranked fourth in China and second in cancer incidence rate,which has caused serious threats to the health of our people.At the world level,the incidence rate of liver cancer is sixth among the common malignant tumors.Although more and more attention has been paid to hepatitis B patients around the worldwide,the early diagnosis of liver cancer is still not perfect due to economic conditions and regional reasons.Especially in the economically backward rural and remote mountainous areas,the patients did not start to see a doctor until they had clinical symptoms.At this time,most of the cancer had developed to the middle and late stage.Nowadays,the treatment of advanced liver cancer has always been a problem we are facing.Although there are multiple treatment options,it is difficult to achieve long-term survival of patients.The emergence of each new drug will cause great repercussions in the medical field,and there are many problems that need to be faced.The most important thing is that almost every chemotherapeutic drug will produce drug resistance in the long-term application.As a new type of small molecule enzyme kinase inhibitor developed in China,anlotinib can effectively inhibit VEGFR,PDGFR,FGFR,c-kit and other kinases,which have the effects of anti-tumor angiogenesis and anti-tumor growth.The clinical trials include non-small cell carcinoma,soft tissue sarcoma,gastric cancer,colorectal cancer,medullary thyroid cancer,differentiated thyroid cancer and esophageal squarmous cell carcinoma.At present,there are few reports about the effect of arotinib on HCC,especially the study on the drug resistance of anlotinib.In this study,we intend to study the possible mechanism of drug resistance of anluotinib in liver cancer,and provide theoretical basis for future clinical and experimental studies.This topic is studied from the following three parts.In the first part,the high-dose shock combined with continuous low-dose increasing of the concentration of anlotinib was used to culture drug-resistant strains of liver cancer,and CCK8 experiment was used to determine the successful culture of drug-resistant strains.In the second part,we compared the ability of metastasis and invasion between drug-resistant and normal hepatoma cell lines by Transwell experiment.In the third part,we use gene chip to explore the mechanism of drug resistance of arotinib in liver cancer.Part1.Establish some hepatoma cell lines resistant to anlotinibAim:To establish hepatoma cell resistant to anlotinibMethods:1.Hepatoma cell line was selected,and the drug-resistant hepatoma cell lines were cultured by high-dose shock combined with low-dose induction.2.CCK8 method was used to detect the half inhibition rate of anlotinib on hepatoma cells and verify the success culture of drug-resistant cell lines.ResultsThe half inhibition rate of normal hepatoma cells was 2.99umol/L,and that of drug-resistant cancer cells was 27.85 umol/L.The hepatoma cell line resistant to anlotinib was cultured successfully.Part2.Phenotypic changes of drug resistant hepatomaAim:To study on the phenotype of hepatoma cell lines resistant to anlotinib in vitro.Methods:1.Transwell method was used to detect the migration and invasion of hepatoma cell lines resistant to anlotinib.2.To detect the migration ability of drug-resistant hepatoma cell lines by cell scratch healing test.ResultsThe ability of invasion and metastasis of hepatoma cell line resistant to arotinib was stronger than that of normal hepatoma cell line.(P<0.5)。Part3.Study on the mechanism of drug resistance of hepatoma cellAim:To infer the possible existence of key RNA and signal pathway,according to study the gene expression difference between drug-resistant and normal hepatoma cell lines,so as to provide theoretical basis for further clinical experimentsMethods:1.Using gene chip to detect the gene expression difference between drug-resistant hepatoma cell line and normal hepatoma cell line.2.QRT PCR was used to verify the gene difference detected by the chipResults2.We screened the 10 genes with the greatest difference by reducing the scope.There are four related to drug resistance or tumor growth:BIRC2,BIRC7,ABCC2 and MAPK8.The expression of BIRC2,ABCC2,MAPK8 decreased(P<0.5)and BIRC7 increased(P<0.5).3.QRT PCR was used to verify the gene difference detected by the chip.Conclusions:1.The hepatoma cell line resistant to anlotinib was cultured successfully.2.The ability of invasion and migration of hepatoma cell lines becomes stronger after resistant to anlotinib in vitro.3.The result of qRT-PCR is consistent with that of gene chip.