Study On The Effect Of Apatinib In The Treatment Of Advanced Hepatocellular Carcinoma And Its Drug Resistance Mechanism

Objectives:The prognosis of advanced hepatocellular carcinoma(HCC)remains clinically unsatisfying.The efficacy of apatinib in the treatment of advanced HCC and the mechanism of drug resistance to apatinib are not yet clear.This study aimed to investigate the efficacy of apatinib in advanced HCC and the mechanism of drug resistance.Methods:1.A clinical prospective study was performed to explore the efficacy of apatinib in the treatment of advanced HCC and drug-related toxicity,2.Tissue specimens from patients who were sensitive or resistant to apatinib were collected to perform next-generation sequencing,and screen the differences in gene expression between the two groups.3.A subcutaneous xenograft tumor model was established in nude mice,and mice were given apatinib(200mg/Kg)by intragastric administration every day to establish the apatinib resistant model in vivo.4.Western blot and immunohistochemical methods were performed to detect the expression of ITIH2 in subcutaneous tumors that were sensitive to apatinib and resistant to apatinib.5.A stable knock-down ITIH2 cell line was constructed in Huh7 cells by lentivirus infection.The effects of ITIH2 on the proliferation,migration,invasion and promoting angiogenesis of HCC cells were verified by CCK8 proliferation test,colony-forming assay,wound healing assay and transwell test in vitro.6.Western blot method was used to detect the expression levels of E-Cadherin,N-Cadherin,Vimentin,Snail and Twist to determine whether epithelial mesenchymal transformation occurred in tumor cells,and real-time quantitative PCR technology was used to detect the changes in m RNA level of the above genes.7.Co-IP test confirmed whether ITIH2 and E-Cadherin were bound to each other at the protein level.Next,the effect of ITIH2 on the degradation of E-Cadherin protein was verified by the addition of actinomycetes,a drug that inhibited protein synthesis.The ubiquitin overexpressed plasmid was transiently transferred into cells,and Co-IP test was performed to verify whether ITIH2 affected the degradation of E-Cadherin through ubiquitination degradation.Drugs that inhibit the degradation of different protein pathways,including MG132 inhibiting proteasomal ubiquitination degradation and chloroquine inhibiting lysosomal degradation,were added to explore in which pathway that ITIH2 inhibits the degradation of E-Cadherin.9.Gene enrichment analysis suggested that ITIH2 was related to the release of mitochondrial cytochrome C.Flow cytometry was used to determine the effect of ITIH2 on cell apoptosis,and the expression of Caspase3,Caspase8,Caspase9,Bcl-2 and Bax were detected by Western blot to further determine the effect of ITIH2 on tumor cell apoptosis.10.Cells were treated with PI3 K inhibitors to detect whether the PI3K-Akt pathway affected the apoptosis of Huh7 cells.11.The upstream and downstream relationship between E-Cadherin and PI3K-Akt was explored by knockdown of E-Cadherin in the control group and overexpression of E-Cadherin in the downregulated ITIH2 group.Results:1.Apatinib showed good clinical effects in patients with advanced HCC,and the toxicity was acceptable.2.Next-generation sequencing results suggested that ITIH2 was elevated in tumor specimens of drug-resistant patients.3.The level of ITIH2 was increased in the apatinib resistance model in vivo.4.In vitro experiments showed that the ability of cell clony formation,proliferation,migration,invasion and angiogenesis were significantly compromised after knockdown of ITIH2.5.E-Cadherin protein expression level decreased with the down-regulation of ITIH2,but N-Cadherin,Vimentin,Snail,Twist had no significant changes.RT-PCR indicated that E-Cadherin,N-Cadherin,Vimentin,Snail,Twist had no significant changes in m RAN level.6.Co-IP experiment verified that the interaction between ITIH2 and E-Cadherin at the protein level,and ITIH2 could inhibit the ubiquitin degradation of E-Cadherin lysosomal pathway after protein-protein interaction.7.Gene enrichment analysis indicated that ITIH2 was negatively correlated with the release of mitochondrial cytochrome C.In other words,it was negatively correlated with the apoptosis of mitochondrial pathway.Flow cytometry analysis confirmed that the apoptosis of down-regulated ITIH2 cell lines was increased,and Western blot confirmed that the down-regulated ITIH2 cell lines had an increased mitochondrial apoptosis pathway.8.PI3K-Akt pathway affected the apoptosis of Huh7 cells.9.The PI3K-Akt pathway was inhibited after down-regulation of E-Cadherin in Huh7 cells,and overexpression of E-Cadherin in down-regulated ITIH2 cells would activate the PI3K-Akt pathway and in turn inhibit cell apoptosis.Conclusion:1.Apatinib is a good choice for first-line treatment of advanced hepatocellular carcinoma.2.Overexpressed ITIH2 is associated with apatinib resistance.3.ITIH2 can affect the proliferation,migration,invasion and angiogenesis of hepatocellular carcinoma.4.The increase of ITIH2 can inhibite the ubiquitin degradation of the lysosomal pathway of E-Cadherin and further activate the PI3K-Akt pathway,thereby inhibiting the apoptosis of the mitochondrial pathway of HCC cells,and then inducing the resistance of HCC to apatinib.