Studies On Molecular Mechanisms Driving Sofafenib Resistance In Hepatoma Stem-like Cells And Its Overcoming Solution

ObjectiveTo investigate the molecular mechanisms of intrinsic resistance to sorafenib in haptoma stem-like cells, and look for the possible ways to overcome it.MethodsUsing nonadherent spheroid culture system, haptoma stem-like cells were enriched and the following assays were performed.1. Using MTS assay to detect the intrinsic resistance to sorafenib in hepatoma stem-like cells.2. Using MTS assay to screen cell signaling pathway inhibitors that could overcome the resistance to sorafenib in hepatoma stem-like cells.3. Using MTS assay and Annexin V/FITC to assess the synergistic effect of MK2206, AKT inhibitor, combined with sorafenib in vitro.4. Using Western Blot analysis to determine the expression level of of AKT and ERK signaling pathways among hepatoma cells and hepatoma stem-like cells with or without drug treatments.5. Establishing hepatoma CSC xenografts model to evaluate the in vivo effects of sorafenib and MK2206combined treatment.6. Using immunohistochemistry and TUNEL assay to investigate the potential mechanisms of tumor growth retardation.Results1. Hepatoma stem-like cells confered intrinsic resistance to sorafenib.2. The anticancer effect of sorafenib could be significantly enhanced by MK2206. sorafenib-MK2206combination treatment induced more death rate and apoptotic rate than single drug treatment, valuated by MTS assay and apoptosis assay.3. Activation of AKT and ERK signaling pathway vary among hepatoma cell and hepatoma stem cells. pAKT (phosphorylated at Thr308) level in hepatoma stem-like cells was much higher than in PLC/PRF/5cells, while pAKT (phosphorylated at Ser473) was inactive in hepatoma stem-like cells. MK2206could inhibit both of them. pERK was down-regulated in hepatoma-stem like cells, but up-regulate by sorafenib or both drugs.4. Sorafenib and MK2206synergistically inhibited the growth of hepatoma CSC xenografts. After14days, mice receiving the combination treatment of sorafenib (20mg/kg) and MK2206(20mg/kg) showed a significantly reduced mean tumor volume (156±105mm3) compared with mice receiving sorafenib alone (405±125mm3), MK2206alone (947±236mm3), or control treatment (2582±268mm3).5. Sorafenib and MK.2206could inhibit the phosphonation of ERK and AKT separately. The combination of sorafenib and MK2206also inhibited the targets effectively. Furthermore, we observed an obvious reduction in tumor cell proliferation after sorafenib-MK2206combined treatment as analyzed by Ki67immunostaining. Likewise, blood vessel density decreased after combination treatment as examined by CD34staining in endothelial cells. The TUNEL assay showed that MK2206enhanced sorafenib-induced apoptosis significantly (P<0.01) from3.4%to18.1%in xenograft tumor tissue.ConclusionIntrinsic resistance to sorafenib is associated with weak ERK signaling and strong AKT signaling in hepatoma stem like cells. MK2206, a novel allosteric inhibitor of AKT could overcome the resistance to sorafenib of hepatoma stem-like cells both in vitro and in vivo.