Study On The Behavior Of ERK1/2Signal Pathway In Reversing Multidrug Resistance Of Hepatocellular Carcinoma Cells

Objective1. To induce poly multidrug resistant (MDR) cell models from human hepatocellular carcinoma (HCC) cell lines by chemotherapy drugs and detect resistance of the developed HCC MDR cells.2. To detect the effects of ERK1/2pathway inhibitors sorafenib and PD98059on resistance of HCC drug-resistant cells, thus to explore if down-regulating ERK1/ERK2could reverse resistance of HCC MDR cells.3. To determine the effect of classic reversal agent Cyclosporine A (CsA) on the ERK1/2expression level. Preliminarily explore the relationship between ERK1/2and reverse of MDR.Methods1. Human hepatocarcinoma cell lines SMMC7721and BEL7402were exposed to high concentration of ADM transiently. CellTiter-Glo luminescent cell viability assay was used to evaluate drug sensitivity. And then the resistance index was calculated to evaluate drug resistance of the MDR models.2. Detect the toxicity of sorafenib, PD98059and CsA to MDR hepatocellular carcinoma cell models by MTT assay.3. Western blot assay was used to detect expression levels of ERK1/ERK2in HCC MDR cells after treatment of sorafenib, PD98059and CsA.4. SMMC7721/ADM and BEL7402/ADM cells were treated by ADM combined with sorafenib, PD98059or CsA, then CellTiter-Glo luminescent cell viability assay was used to detect cell viability and reversal fold was calculated.Results1. Compared to parent cells, the induced MDR cells showed less sensitivity to ADM. The IC50of ADM for SMMC7721/ADM was16.44times higher than that of SMMC7721cell lines. The IC50of ADM for BEL7402/ADM was20.34times higher than that of BEL7402cell lines.2. After treatment with PD98059or sorafenib, expression of pERK1/2decreased dose-dependently. After treatment with5μM PD98059, the IC50of ADM for SMMC7721/ADM and BEL7402/ADM cells reduced to0.8±0.056μg/ml and1.583±0.284μg/ml, respectively. After treatment with2.5μM sorafenib, the IC50of ADM for SMMC7721/ADM and BEL7402/ADM cells reduced to0.264±0.049μg/ml and1.099±0.135μg/ml, respectively.3. When combined with4μg/ml CsA, the IC50of ADM in SMMC7721/ADM and BEL7402/ADM cells reduced to0.349±0.023μg/ml and0.427±0.039μg/ml, respectively. CsA increased expression levels of pERK1/2without affecting the total ERK1/2levels.Conclusions1. SMMC7721/ADM and BEL7402/ADM showed a stable drug resistance.2. Inhibiting ERK1/2pathway activity can indeed reverse the MDR of HCC cells.3. Down-regulation of ERK1/2pathway activity was not be involved in reversal function of CsA, which indicated that inhibiting the ERK1/2pathway activity is not an unique way to reverse the MDR of HCC cells.