The Study Of The Inhibitory Effect Of Ibrutinib And Its Derivative On The Proliferation Of Hepatocellular Carcinoma Cells And The Potential Mechanism

Objective:Liver cancer is a serious threat to human health diseases,liver cancer patients account for more than 5%of cancer patients worldwide,it is reported that the incidence of liver cancer in the next decade will continue to rise,and the incidence will peak in 2030.At present,the effective methods for the treatment of liver cancer clinically are surgical resection and liver transplantation.However,due to its high recurrence rate and metastasis rate,the treatment of liver cancer faces a huge challenge.The molecular targeted drugs,have become a research hot spot in recent years.Molecular targeted drugs,designed using the molecular biology differences,including signal transduction,cell cycle,cell fusion,swallowing and metabolism of different characteristics between tumor cells and normal cells to inhibit the growth and proliferation of tumor cells,with high efficacy and less side effects.Targeting tyrosine kinase inhibitors is a highly specific treatment for malignant tumors.BTK is a key kinase in the B cell signaling pathway and plays an important regulatory role in the maturation and differentiation of B cells and is associated with a variety of B cell immune disorders.Targeting tyrosine kinase inhibitors is a highly specific treatment for different malignancies.Initially,it was found that different receptors and non-receptor tyrosine kinases are crucial in this process during the maturation of B cells and the associated tumorigenesis.Many clinical trials on this pathway are under way.B cell receptor?BCR?complexes and downstream signaling pathways play an important role in the growth and differentiation of B cells.Therefore,the research on different inhibitors of BCR pathway has a wide range of perspectives.The spleen tyrosine Syk inhibitors,PI3K inhibitors,and Bruton's tyrosine kinase?BTK?inhibitors have yielded good clinical results.Ibrutinib is a small molecule BTK inhibitor developed by Pharmacyclics Company.Its oral absorption is rapid with a half-life of 2-3 hours.1-2 hours after taking,the plasma drug concentration reached its highest.Ibrutinib is a broad-spectrum kinase inhibitor that covalently binds to BKT.The current study shows that ibrutinib is mainly used in B-cell associated malignancies.Ibrutinib was first approved by the FDA for the treatment of mantle cell lymphoma in 2013 and subsequently approved for the treatment of chronic lymphocytic leukemia and primary macroglobulinemia.Ibrutinib remains the only BTK inhibitor approach for treatment of multiple B cell tumors so far.Studies have shown that ibrutinib not only has an effect on B cell-related malignancies,but also inhibits some solid tumors,such as lung cancer,breast cancer,glioma,prostate cancer,ovarian cancer,gastric carcinoma and pancreatic cancer.However,the effect of ibrutinib on liver cancer cells and its mechanism has not yet been reported at home and abroad.Our aim of this subject is to study the inhibitory effect of Ibrutinib on liver cancer cells and to explore its possible mechanism.Methods:The hepatocellular carcinoma cells HepG2,Bel-7402 and normal liver cells L02 were cultured.First,MTT assay was used to detect the inhibitory effect of ibrutinib and its derivative on the proliferation of HepG2,Bel-7402 and L02 cells.PI single staining method was detect the Cell cycle distribution.Annexin-V/PI double staining method was used to detect the effect of—ibrutinib and derivative on the apoptosis of hepatocellular carcinoma cells.The effect of ibrutinib and its derivative on the expression of apoptosis-related proteins in hepatocellular carcinoma cells was detected by Western blot.Results:1.MTT results showed that different concentrations of ibrutinib and derivative inhibited the HepG2 and Bel-7402 liver cancer cell growth significantly,and showed a time and concentration dependent manner.Although both drugs have almost the same IC₅₀ values for hepatocellular carcinoma cells,derivative are more selective for normal liver cells than ibrutinib.2.PI results showed that ibrutinib and derivative can cause S phase block of hepatocellular carcinoma cells.3.Flow cytometry results showed that different concentrations of ibrutinib and derivative can cause apoptosis of hepatocellular carcinoma cells.4.Western blot results showed that ibrutinib and derivative reduced the expression of p-BTK in hepatocellular carcinoma cells dose-dependently.5.Western blot results showed that ibrutinib and its derivative could decrease the expression of pro-caspase 3 and Bcl-2 protein in hepatocellular carcinoma cells in a dose-dependent manner.Conclusion:1.Ibrutinib and its derivative inhibited the proliferation of hepatocellular carcinoma cells in a dose and time-dependent manner.2.Ibrutinib and its derivative inhibited the proliferation of hepatocellular carcinoma cells through the cell cycle arrest and apoptosis induction.