BET Protein Inhibitor JQ1 Enhanced The Proliferation Inhibition Effect Of Sorafenib On Hepatocellular Carcinoma Cells And Its Mechanism

Background Hepatocellular carcinoma(HCC)is a common clinical malignant tumor,ranking5 th among malignant tumors and 3rd among tumor-related deaths worldwide.HCC is the main histological subtype of liver cancer,accounting for90% of primary liver cancers,Genetic and Epigenetic changes,Chronic Hepatitis B virus or Hepatitis C virus infection,Aflatoxin exposure,smoking,Obesity and iabetes are major risk factors for liver cancer.The cause of poor prognosis of liver cancer is mainly due to the high incidence of tumor recurrence and metastasis.At present,transplantation is the most effective method for treating HCC,but more than 70% of advanced cases are not suitable for transplantation due to tumor burden or poor liver function.Chemotherapy is mainly used in patients who cannot tolerate advanced surgery.Sorafenib,a multi-targeted kinase inhibitor,is a first-line targeted drug approved by the FDA,but it can only provide 2.8-month survival benefits for patients with advanced liver cancer.The response and drug resistance make its therapeutic effect unsatisfactory.Therefore,efforts to find effective drugs and optimal treatment methods are the focus of current research.BRD4 is called bromodomain-containing protein 4,which is a member of the BET family.The BET bromodomain and extra-terminal domain contain four proteins,namely BRD2,BRD3,BRD4 and BRDT.BET has a conservative modular structure: it includes two N-terminal tandem BRD effect modules(BD1and BD2),an additional terminal group(ET),several conservative regions(A,B,SEED regions)and C-Terminal region(CTM).Bromine-containing protein 4(BRD4)is the most important member of the BET family and is deeply involved in the regulation of epigenetics.BRD4 protein can bind acetylated histones throughout the cell cycle through its bromodomain,and recruit different transcription regulators to regulate target gene expression.In addition,BRD4 protein can interact with RNA polymerase II(Pol II)Positive transcription elongation factor(P-TEFb)binds to regulate the transcription process of oncogenes(such as MYC gene.et.al),thereby regulating cell proliferation and apoptosis.Studies have shown that the imbalance of BRD4 expression is related to the formation of various cancer diseases such as lung cancer,hematological tumors,breast cancer,colon cancer and liver cancer.These findings suggest that BRD4 plays an important role in tumorigenesis and development,and targeting BRD4 may be a promising cancer treatment strategy.JQ1 is a small molecule inhibitor of BET protein that can competitively bind to acetylated lysine residues of the BET bromodomain,thereby blocking the function of the bromodomain-containing protein(BRD).JQ1 has been observed in many cancers(such as multiple myeloma,acute myeloid leukemia,breast cancer,etc.)with promising anti-proliferative effects and clinical significance.This study observed JQ1 combined with sorafenib on liver cancer cell lines Inhibition of proliferation,and exploring its possible mechanism,provide theoretical basis for clinical treatment of liver cancer.Objective By using JQ1 and sorafenib separately and in combination for liver cancer cell Hep G2 and Bel-7402,observe whether JQ1 enhances the proliferation Inhibition,cell cycle inhibition,apoptosis inhibition,and expression of apoptotic proteins Bcl-2,BIM,and oncogene c-MYC of Hep G2 and Bel-7402 at different time periods.To elucidate whether the BRD4 inhibitor JQ1 can increase the proliferation inhibition of sorafenib in liver cancer cells.Methods(1)Five concentrations of JQ1(0.25μM,0.5μM,1μM,2μM,5μM)and five concentrations of sorafenib(1.25μM,2.5μM,5μM,10μM,20μM)were used to treat Hep G2 and Bel-7402 respectively,The MTT method was used to detect the proliferation inhibition rate of different concentrations of drugs on liver cancer cells,draw a concentration-inhibition rate curve,calculate the IC50 values of JQ1 and sorafenib on two types of liver cancer cells,and select the drug concentration required for subsequent experiments.(2)Select the appropriate concentration of JQ1,sorafenib and the combination of the two for Hep G2 and Bel-7402.After 24 h and 48 h,the MTT method was used to detect the proliferation inhibition rate of each group of drugs on liver cancer cells,calculate the combined drug index CI of the two drugs.(3)JQ1 and Sorafenib were used in respectively and in combination with Hep G2 and Bel-7402.Flow cytometry was used to detect the effects of each group on the cell cycle of two liver cancer cells.(4)JQ1 and Sorafenib were used in respectively and in combination with Hep G2 and Bel-7402.Flow cytometry was used to detect the effects of each group on the apoptosis rate of two liver cancer cells.(5)JQ1 and Sorafenib were used in respectively and in combination with Hep G2 and Bel-7402.Western-Blot was used to detect the expression of apoptotic proteins Bcl-2,BIM,and oncogene c-MYC.Results(1)The MTT experiment results showed that JQ and Sorafenib inhibited the proliferation of Hep G2 and Bel-7402 in a dose-dependent manner.In Hep G2,IC50 of JQ1 is 3.00±0.95(μmol / L),IC50 of sorafenib is 11.43±2.60(μmol / L);in Bel-7402,IC50 of JQ1 is 2.60 ± 0.65(μmol / L),the IC50 of sorafenib is 11.68±2.19(μmol / L).Compared with JQ1 and sorafenib alone,the combined effect of JQ1 and sorafenib on cell proliferation inhibition rate increased significantly,JQ1 can increase the proliferation inhibition of sorafenib on liver cancer cells,the combined index CI shows two drugs are synergistic.(2)Flow cell cycle experiments showed that in Hep G2 and Bel-7402,compared with the single effect of JQ1 and sorafenib,the G1 phase cells of liver cancer cells increased after the combined action of JQ1 and sorafenib,while the S-phase liver cancer cells decreased.JQ1 could increase the cycle inhibition of sorafenib on liver cancer cells.(3)Flow cytometry apoptosis experiment showed that in Hep G2 and Bel-7402,compared with the single action of JQ1 and sorafenib,the combined action of JQ1 and sorafenib significantly increased the apoptosis rate of liver cancer cells.JQ1 could increase the apoptosis effect of sorafenib in liver cancer cells. (4)Western Blot showed that in Hep G2 and Bel-7402,JQ1 could increase the inhibition of sorafenib on apoptotic bcl-2 protein and oncogene c-myc protein,as well as promote the expression of apoptotic BIM protein.Conclusions This result indicates that a certain concentration of JQ1 can enhance the proliferation inhibition and apoptosis induction effects of sorafenib on liver cancer cell lines.The mechanism might be related to the change of cell cycle distribution,down-regulation of the expression of Bcl-2 and c-MYC,and up-regulation of the expression of BIM protein.