MEK inhibition induced downregulation of MRP1 and MRP3 expression in experimental hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) exhibits strong intrinsic and acquired drug resistance which is the main obstacle to chemotherapy. Overexpression of ATP binding cassette (ABC) proteins correlates with activation of mitogen activated protein kinase (MAPK) pathway in HCC. Here, we systematically investigated the inhibition of MAPK pathway and its role in regulating HCC cell growth as well as ABC proteins MRP1 and MRP3 expression.

Methods: The Raf1 kinase inhibitor (GW5074) and different MEK inhibitors (U0126 and AZD6244) were used to treat HCC cells to identify their effects on HCC cell growth and ABC proteins expression in vitro. Cell viability tests were performed after the treatment of MAPK pathway inhibitors and in combination with gemcitabine or doxorubicin. Western blot was applied to assess the changes of MAPK pathway and protein expression of MRP1 and MRP3. Flow cytometry was used to measure intracellular doxorubicin accumulation after the treatment of MEK inhibitors.

Results: Both Raf1 inhibitor (GW5074) and MEK inhibitors (U0126 and AZD6244) suppressed HCC cell growth in a dose dependent manner. Pre-treatment of MEK inhibitor U0126 or AZD6244 sensitized HCC cells to gemcitabine or doxorubicin based chemotherapy. Raf1 inhibitor GW5074 had no effect on MRP1 and MRP3 protein expression. Treatment of gemcitabine or doxorubicin activated phosphorylated ERK and induced the upregulation of MRP1 and MRP3. MEK inhibitors U0126 and AZD6244 deactivated phosphorylated ERK, decreased endogenous MRP1 expression, reversed gemcitabine or doxorubicin induced MRP1 and MRP3 upregulation, and increased the intracellular doxorubicin accumulation.

Conclusion: This study provides evidence that MEK inhibitors sensitize HCC cells to chemotherapy by increasing intracellular chemodrug accumulation. MEK inhibirors U0126 and AZD6244 reduced MRP1 as well as MRP3 expression, and may contribute partially to the sensitization. The combination of MEK inhibitor and conventional chemotherapy may offer new therapeutic option for the treatment of resistant HCC.

Figure 1

Role of GW5074 on HCC cell growth and ABC proteins expression. A: HepG2 and Huh7 were treated with three different concentrations (5 μM, 10 μM and 20 μM) of Raf1 kinase inhibitor GW5074. After 48 hours treatment, cell viability test was performed to measure the relative viability. * P<0.05 compared with control group. B: HepG2 and Huh7 cells were treated with three different concentrations (5 μM, 10 μM and 20 μM) of GW5074 for 48 hours, and then cells were lysed and western blot was performed.

Figure 2

AZD6244 inhibited HCC cell growth and enhanced chemosensitivity. A: HepG2 and Huh7 were treated with four different concentrations (5 μM, 10 μM, 15 μM and 20 μM) of MEK inhibitor U0126 and AZD6244. After 48 hours treatment, cell viability test was performed to measure the relative viability. * P<0.05 compared with control group. B: HepG2 and Huh7 cells were treated with U0126 (10 μM) and AZD6244 (10 μM) for 24 hours, followed by gemcitabine (50 μg/ml, 100 μg/ml, 150 μg/ml and 200 μg/ml) or doxorubicin (0.15 μg/ml, 0.30 μg/ml, 0.45 μg/ml and 0.6 μg/ml) for another 48 hours. Cell viability test was performed to measure the relative viability. *: synergistic effect was detected.

Figure 3

MEK inhibitors reversed MRP1 and MRP3 expression. A: HepG2 and Huh7 were treated with four different concentrations (2.5 μM, 5 μM, 10 μM and 20 μM) of U0126. After 48 hours of U0126 treatment, the cells were lysed and western blot was performed. B: HepG2 and Huh7 were treated with four different concentrations (1 μM, 2 μM, 5 μM and 10 μM) of AZD6244. After 48 hours treatment, the cells were lysed and western blot was performed. C: HepG2 and Huh7 were treated with gemcitabine (114 μg/ml) or doxorubicin (0.6 μg/ml) for 48 hours, and followed by another 24 hours of U0126 (10 μM) treatment. Then the cells were lysed and western blot was performed. D: HepG2 and Huh7 were treated with gemcitabine (114 μg/ml) or doxorubicin (0.6 μg/ml) for 48 hours, and followed by another 48 hours of AZD6244 (5 μM) treatment. Then cells were lysed and western blot was performed.

Figure 4

U0126 and AZD6244 increased intracellular doxorubicin accumulation. A: HepG2 or Huh7 cells were treated with U0126 or AZD6244 for 48 hours, followed by incubation of doxorubicin (6 μg/ml) for 2 hours. Then the cells were trypsinized and resuspended in PBS followed by FACS analysis. B: Statistical analysis of FACS results. * P<0.05 compared with control group.

Figure 5

Hypothesized mechanism of involvement of the EGF pathway in the regulation of ABC protein expression.