Abstract
BackgroundHepatocellular 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.MethodsThe 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.ResultsBoth 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.ConclusionThis 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.
Highlights
Hepatocellular carcinoma (HCC) exhibits strong intrinsic and acquired drug resistance which is the main obstacle to chemotherapy
GW5074 inhibited HCC cell growth and Raf1 expression To determine the role of Raf1 inhibition on HCC cell growth and drug resistance, HCC cells were treated with the Raf1 kinase inhibitor GW5074 (5 μM–20 μM)
We further examined the effects of GW5074 on mitogen activated protein kinase (MAPK) pathway and protein expression of Multidrug resistance-associated protein 1 (MRP1) and Multidrug resistanceassociated protein 3 (MRP3) in HCC cells
Summary
Hepatocellular carcinoma (HCC) exhibits strong intrinsic and acquired drug resistance which is the main obstacle to chemotherapy. 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. A high intrinsic and acquired drug resistance in HCC is mainly responsible for this failure of the systemic chemotherapy [12]. ABC proteins contribute to the major form of drug resistance by increasing the efflux of anticancer drugs out of cancer cells [14]. Our previous analysis revealed that, among these ABC proteins, MRP1 and MRP3 were overexpressed in HCC tissue and may contribute to the high intrinsic drug resistance [15]. Inhibition of MRP1 and MRP3 might reverse multidrug resistance and improve chemotherapeutic efficiency in HCC
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