Anti-hepatocarcinoma Effects And Molecular Mechanism Of Shikonin In Vitro And In Vivo

Objective:Hepatocellular carcinoma(HCC)is the fifth most common malignant tumor in the world and the second leading cause of cancer death in Asia.HCC accounts for 80%-90% of primary liver cancer,and the incidence of HCC increases by 3%-9% globally every year.More than 500,000 people are diagnosed with hepatocellular carcinoma each year in worldwide,and half of them occured in China.Early detection of HCC tumors can be treated by liver transplantation,surgical resection,and percutaneous radio frequency ablation.Liver transplantation can also significantly improve the survival rate of patients with HCC,but the application of this treatment is limited due to the lack of donor organs.Surgical resection and percutaneous ablation also showed relatively high response and survival,but long-term(10-year)survival rates were only 22%-35%.The survival rate of patients with advanced liver cancer is less than 12 months,therefore,it is urgent to develop new and effective drugs for clinical application.A lot of literatures have shown that shikonin can significantly inhibit the cellular proliferation of breast cancer,human squamous cell carcinoma,and human glioblastoma.In recent years,studies have found that shikonin can also inhibit the cellular proliferation and metastasis of hepatocellular carcinoma in vitro.In this paper,the cell model and tumor-bearing animal model were used to investigate the inhibitory effect of shikonin on the growth and metastasis of HCC and its molecular mechanism,which provided a theoretical and experimental basis for the research and development of shikonin,and also provided a new idea for the treatment of HCC.Methods:The antiproliferative effects of shikonin were detected by MTT assay.The anti-migration effects of shikonin were detected by Transwell chamber experiment and scratch test.The mRNA and proteins expression were detected by Real-Time PCR and Western blotting,respectively.Transfection was used to construct EGFR and SP1 overexpressing HCC,and then transwell chamber test and scratch test were used to detect whether shikonin could reverse the inhibitory effect of shikonin on HCC after overexpression of EGFR and SP1.Evaluation of the effect of shikonin on the growth of solid tumor and lung metastasis of HCC in mice with axillary transplantation tumor model and tail vein injection model.Result:1.MTT assay showed that shikonin inhibited the proliferation of Hep G2 cells and Huh-7 cells in a time and dose-dependent manner.2.Scratch test and Transwell chamber experiment showed that shikonin inhibited the migration of Hep G2 cells and Huh-7 cells.3.Fluorescence quantitative PCR experiments showed that shikonin inhibited the expression of EGFR mRNAin Hep G2 cells and Huh-7 cells.4.Protein immunoblotting experiments showed that shikonin inhibited the expression of p-EGFR,EGFR and SP1 proteins in Hep G2 cells and Huh-7 cells.5.The overexpressing cell linesof EGFR and SP1 were successfully constructed,respectively.MTT assay showed that overexpression of EGFR or SP1 could reverse the inhibitory effect of shikonin on the proliferation of Hep G2 cells and Huh-7 cells.6.Transwell chamber experiments and scratch experiments also demonstrated that overexpression of EGFR or SP1 could reverse the inhibitory effect of shikonin on migration and invasion of Hep G2 cells and Huh-7 cells.7.Shikonin significantly inhibits theH22 growth in subaxillary transplantation tumor model.8.Shikonin significantly inhibitslung metastasis in H22 lung metastasis model.Conclusions:1.Shikonin can inhibit the expression of EGFR,thereby inhibiting the proliferation and migration of hepatocellular carcinoma cells.2.Shikonin can inhibit the expression of SP1 and inhibit the proliferation and migration of the hepatocellular carcinoma cells.3.Shikonin is likely to inhibit the growth and metastasis of hepatocellular carcinoma cells by inhibiting the transcription of SP1-mediated EGFR.