Exploration Of Sorafenib Resistance Related Pathway In Liver Cancer Stem Cells

ObjectiveTo test inhibition efficacy of sorafenib to liver cancer stem like cells and investigatethe key gene and signaling pathway involved in sorafenib resisitance of liver cancerstem like cells, and to explore possible ways to reverse the drug resistance, so as to provetheoretical basis for liver cancer stem cell targeted hepatocellular carcinoma treatment andnewclinical guidance for treating sorafenib resistant hepatocellular carcinoma.MethodsBased on the serum free culturing system established by our laboratory, liver cancerstem like cells Huh7C and Hep3B C are cultured and enriched for the following research:1. Determining sorafenib efficacy to liver cancer stem like cells and correspondingnormal hepatoma cells using morphology observation and CCK8kit, and comparing thecells’ sensitivity to sorafenib treatment.2. Detecting cell cycle and apoptosis influenced by sorafenib treatment using PIsingle staining and Annexin V/PI double staining ing flowcytometry.3. Deep sequencing gene expression profile of liver cancer stem like cells andcorresponding hepatoma cells, screening significant genes and comparing with sorafenibtarget genes in GeneGO database so as to gain the candidate genes affecting liver cancerstem like cells specific resistance to sorafenib, and then mapping the candidate genes backto KEGG database to predict pathway related to liver cancer stem cells specific resistanceto sorafenib.4. Verifying the sorafenib key target gene expression level in liver cancer cell likecells using RT PCR, Western Blot and immunofluofescence.5. Checking inhibition efficacy of molecular inhibitors combined with sorafenib toliver cancer stem like cells. Verifying the mechanism of combinant treatment affectingpredicted pathway.Results1. Sorafenib shows different inhibition efficacy to liver cancer stem like cells andcorresponding hepatoma cells. In the effective dug concentration range, liver cancerstem like cells Huh7C and Hep3B C are more resistant to sorafenib than thecorresponding hepatoma cell lines Huh7and Hep3B.After sorafenib treatment, liver cancerstem like cells can still maintain complete cell ball growth morphology and permeable cell state, while hepatoma cells are already difficult to sustain adherent growth and show somecells shrinkage.2. Sorafenib has obvious effect of inhibitingmitosis and inducing apoptosis tohepatoma cells and thus achieves its treating effect to liver cancer; while under the similardrug condition sorafenib exhibits limited impact on mitosis and apoptosis of liver cancerstem like cells and hardly possible to completely eliminate liver cancer stem like cells.3. Deep sequencing results of expression profiles for liver cancer stem like cells areanalysed to screen differentially expressed genes(FoldChange>2or FoldChange<0.5).1012genes are screened and compared with7genes related to sorafenib potency fromGeneGO database. Hence4genes named B RAF, C KIT, RAF1and PDGFRB areconcluded as sorafenib resistance related candidate genes for liver cancer stem likecells.The candidate genes are returned to KEGG database, and MEK pathway areconcluded as sorafenib resistance related pathway of liver cancer stem like cells.4. RNA level and protein level detected PDGFRB expression of liver cancerstem like cells is lower than that of corresponding hepatoma cells, which result agrees withthat from deep sequencing.5. MEK pathway in hepatoma cells is susceptible to sorafenib treatment and ERK1/2expression is negatively correlated with sorafenib concentration. ERK1/2expressionlevel is lower and the MEK pathway is rather stable in liver cancer stem like cells.Combination treatment of MEK pathway inhibitor U0126and sorafenib can obviouslyinhibit vitality of liver cancer stem like cells and reverse the sorafenib resistance.ConclusionCompared with hepatoma cells, liver cancer stem like cells show stronger resistanceto sorafenib. MEK pathway in hepatoma cells is susceptible to sorafenib treatment andERK1/2expression is decreased with the drug concentration increasing. Among thesorafenib related genes, PDGFRB is specific down regulated in liver cancer stem likecells and hence sorafenib inhibition to MEK pathway through PDGFRB is limited. Soliver cancer stem like cells can maintain stable MEK pathway and ensure the basic celllife activities. MEK pathway inhibitor U0126combined with sorafenib can effectivelyinhibit the vitality of liver cancer stem like cells and reverse sorafenib resistance.