MRP3 as a novel resistance factor for sorafenib in hepatocellular carcinoma

Abstract

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.

PharmGKB summary

“…In vitro and preclinical studies demonstrated that the hepatic uptake of sorafenib and its metabolites is mediated in part by organic cation transporter-1 (OCT1, encoded by gene SLC22A1 ) [32–36] and by organic anion transporting polypeptide 1B1 and 1B3 (OATP1B1 and OATP1B3, encoded by gene SLCO1B1, SLCO1B3 ) [34, 35, 37]. Sorafenib also showed moderate affinity for the efflux transporter P-glycoprotein (p-gp, encoded by gene ABCB1 ) and breast cancer resistance protein (BCRP, encoded by gene ABCG2 ) [38–43].…”

Poly-(ADP-Ribose) Polymerase-1 Promotes Prothrombin Gene Transcription and Produces Des-Gamma-Carboxy Prothrombin in Hepatocellular Carcinoma

“…Western blot analysis was performed using a rabbit anti-human PARP-1 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) as described previously [27]. …”

“…Cell viability was assessed by using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as described previously [27]. …”

Therapeutic Efficacy and Safety of Lenvatinib for Unresectable Hepatocellular Carcinoma Beyond Progression with Sorafenib

“…Establishment of the SOR-resistant PLC/PRF5 cell line (PLC/PRF5-R2) was performed as described previously. [23] Cells were grown in Dulbecco's modified Eagle's medium (Invitrogen Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS and 2 mM L-glutamine. Cell viability was assessed via a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as described previously.…”