It has been recently found that P-gp expression in the MDR1-transduced human breast cancer cell lines MCF-7/MDR and MDA-MB-231/MDR is positively regulated by the ERK pathway and blockade of the MEK-ERK-RSK pathway can suppress cell surface P-gp expression selleck kinase inhibitor by promoting its degradation[12]. In addition, there are several lines of evidence that modulation of ERK activation may reverse MDR in prostatic, gastric and hematopoietic cancers[13�C16]. However, there is little evidence that ERK activity is related to MDR of HCC. The aim of this research was to study the crucial kinases of ERK pathway, including expression and phosphorylation (activity) of ERK1 and ERK2 in MDR HCC cell lines, and to explore whether the relationship between MDR and ERK1/2 kinases involves specific molecular aspects of these cell lines.

Once they are well characterized, the ERK pathway might be exploited for overcoming MDR of HCC. MATERIALS AND METHODS Cell culture Human HCC cell lines, HepG2 and SMMC7721, were purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences. HepG2 was cultured with DMEM (HyClone, Logan, UT, USA) and SMMC7721 was cultured with RPMI-1640 (HyClone, Logan, UT, USA). Both media were supplemented with 10% calf serum (HyClone, Logan, UT, USA) and maintained at 37��C in a humidified atmosphere containing 50 mL/L CO2 and 950 mL/L air. Multidrug resistant human HCC cell lines, HepG2/adriamycin (ADM) and SMMC7721/ADM, were developed by our group. To develop the HepG2/ADM and SMMC7721/ADM cells, ADM (Pharm-sh Pharmaceutical Co.

, Ltd., Shanghai, China) was added respectively to HepG2 and SMMC7721 cells at a stepwise increasing concentration from 0.01 to 0.2 mg/L. Resistant cells were selected by removing the non-resistant dead cells. Multidrug resistance was maintained by culturing the cells with 0.2 mg/L ADM and MDR cells were named HepG2/ADM and SMMC7721/ADM. Measurement of cellular sensitivity to anticancer drugs MTT (Sigma-Aldrich, St. Louis, MO, USA) assay was used to determine drug sensitivity. Sensitivity of cultured HepG2/ADM and SMMC7721/ADM cells to anticancer drugs, including ADM, fluorouracil, cisplatin, cyclophosphamide, mitomycin and vincristine (Pharm-sh Pharmaceutical Co., Ltd., Shanghai, China), was detected, respectively[17]. IC50 value was assessed by probit regression analysis using SPSS11.

5 statistical software. Resistance index (RI) was calculated according to the formula: RI = IC50 for MDR cells IC50/IC50 for parental cells. Flow cytometric analysis of cell cycle distribution Cultured HepG2/ADM and SMMC7721/ADM cells and their parental cells were collected Cilengitide respectively through trypsinization, washed with ice-cold PBS, centrifuged at 500 �� g for 5 min at 4��C, washed twice with ice-cold PBS and fixed in 70% ethanol for 2 h at 4��C.