Effect Of Hepatic Stellate Cells And Research On Liver Cancer Drug Resistance Mechanisms

Background and AimsChemotherapy is a method to damage and kill tumor cells by drugs. For HCC patients,chemotherapy is a major application in eradicating the microscopic residual and curing theadvanced HCC patients who are not feasible for surgery. In recent year, the antitumordrugs have been gotten a rapid development and new chemotherapy drugs are constantlydeveloped, which play a role to improve the effect of the treatment of patients with HCC.However, many unfavorable factors restricted the chemotherapeutic effect of liver cancer.In addition to plenty of adverse reactions, the effect of chemotherapy is limited as theresult of inevitable generation of drug resistance.Tumor microenvironment, which consisted with stromal cells， soluble cytokines，and extracellular matrix protein, is a newly-developing research field. The reciprocalcrosstalk between hepatoma cells and their surrounding tumor stromal cell determinate thebiological behaviors of tumor cells, including epithelial mesenchymal transition (EMT),cancer stem cells(CSCs), invasion, migratory and chemoresistance. As the main stromalcell in tumor stromal, hepatic stellate cells (HSCs) have a great impact on biologicalbehaviors of HCC by producing ECM, growth factor and cytokines. Studies haveconfirmed that hepatic stellate cells play an important role in the growth, invasion andmetastasis of HCC. It is not yet clear the influence of hepatic stellate cell onchemoresistance of hepatoma cells.Hypoxia, nutrient deficiency and some cytokines (HGF and TGF-β) can induce EMTof HCC. A growing number of studies have confirmed that1.tumor cells underwent EMTacquire CSC properties;2.Similar pathways can be seen in both EMT and CSC；3.Acquirement and maintenance of CSC properties are considered as a main mechanism oftumor chemoresistance. Therefore, the aims of the study are:1. The influence of Hepaticstellate cells on HCC chemoresistance；2. Whether hepatic stellate cells induced EMT ofHCC to obtain CSC properties；3. The molecular mechanism of HSCs on chemoresistanceof HCC. Methods and resultsPart1: The influence of Hepatic stellate cells on HCC chemoresistance Hep3B andSMMC7721cells were treatment with LX-2cells and chemotherapy drugs, and observedthe resistant phenotypes change of HCC cells. Cell proliferation activity was determinedby MTT assay, apoptosis rate was detected by PI/Annexin V-FITC double staining,Western Blot was used to test the level of activated apoptosis protein Caspase3and8. Invivo tests, we subcutaneously inoculated Hep3B cells or mixed inoculation Hep3B and LX-2cells in nude mice and observe their ability to form xenografts and the tumor growthunder the effect of chemotherapy drug.The experimental results showed that hepaticstellate cells reduced proliferation inhibition and apoptosis effect of chemotherapy drugs,and the results was verified in the animal experiment.Part2: Whether hepatic stellate cells induced EMT of HCC to obtain CSC properties.Real time PCR, Western Blot and cell immunofluorescence were used to analyze thechanges of EMT molecular markers E-cadherin and Vimentin; wound scratch andTranswell assays were used to detect the changes of cell invasive and migratory abilities.Real time PCR, Western Blot were used to analyzed the CSCs related transcription factorsBMI1and KLF4expression, cytometry flow technique to detect the expression of CD133and clone formation experiment to test cell clone form ability. Results showed that underthe effect of LX-2CM, the expression of E-cadherin was decreased, Vimentin wasupregulation in HCC cells, and invasive and migratory abilities were enhanced; the BMI1,KLF4and CD133expression of hepatoma cells were raised as well as the clone formatingability.Part3: Investigated a mechanism whereby HSCs modulate the chemoresistance ofhepatoma cells. By using Real time PCR and ELISA to detect HGF expression in HSCs,we found the hepatocyte growth factor high expression in hepatic stellate cells. We foundthat LX-2CM exposure could raise activated c-Met level, and HGF neutralizatingantibody effective antagonist the effect of LX-2CM. Meanwhile，HGF neutralizatingantibody application could effectively weaken the LX–CM effect on EMT、CSCproperties and chemoresistance.ConclusionHSCs synthesis and secrete HGF to activate HGF/c-Met signaling pathway of HCC.The activated HGF/c-Met signaling pathway induce EMT of HCC to acquire CSC properties，which may be involved in the chemoresistance of HCC.