MRP3 as a novel resistance factor for sorafenib in hepatocellular carcinoma
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Tetsu Tomonari1, Shunsaku Takeishi1, Tatsuya Taniguchi1, Takahiro Tanaka1, Hironori Tanaka1, Shota Fujimoto1, Tetsuo Kimura1, Koichi Okamoto1, Hiroshi Miyamoto1, Naoki Muguruma1, Tetsuji Takayama1
1Department of Gastroenterology and Oncology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima City, 770-8503, Japan
Tetsuji Takayama, e-mail: [email protected]
Keywords: hepatocellular carcinoma, sorafenib, resistance, transporter, MRP3
Received: September 30, 2015 Accepted: January 02, 2016 Published: January 12, 2016
The mechanism of resistance of hepatocellular carcinoma (HCC) to sorafenib is unknown and no useful predictive biomarker for sorafenib treatment has been reported. Accordingly, we established sorafenib-resistant HCC cells and investigated the underlying mechanism of resistance to sorafenib. Sorafenib-resistant cell lines were established from the HCC cell line PLC/PRF5 by cultivation under continuous exposure to increasing concentration of sorafenib. The IC50 values of the 2 resistant clones PLC/PRF5-R1 and PLC-PRF5-R2 were 9.2±0.47 μM (1.8-fold) and 25±5.1 μM (4.6-fold) respectively, which were significantly higher than that of parental PLC/PRF5 cells (5.4±0.17 μM) (p < 0.01 respectively), as determined by MTT assay. Western blot analysis of signal transduction-related proteins showed no significant differences in expression of AKT/pAKT, mTOR/pmTOR, or ERK/pERK between the 2 resistant clones versus parent cells, suggesting no activation of an alternative signal transduction pathway. Likewise, when expression of membrane transporter proteins was determined, there were no significant differences in expression levels of BSEP, MDR1, MRP2, BCRP, MRP4 and OCT1 between resistant clones and parent cells. However, the expression levels of MRP3 in the 2 resistant clones were significantly higher than that of parent cells. When MRP3 gene was knocked down by siRNA in PLC-PRF5-R2 cells, the sensitivity of the cells to sorafenib was restored. In the analysis of gene mutation, there was no mutation in the activation segment of Raf1 kinase in the resistant clones. Our data clearly demonstrate that the efflux transporter MRP3 plays an important role in resistance to sorafenib in HCC cells.
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