Relevance of organic cation transporter OCT1 (SLC22A1) in Diethylnitrosamine-initiated and Phenobarbital-promoted hepatocellular carcinoma in OCT3- knockout mice

Background: Organic cation transporters (OCT) are responsible for the uptake and intracellular inactivation of a broad spectrum of endogenous substrates. OCTs became pharmaceutically interesting, because they are determinants of the cytotoxicity of platin derivates and the transport activity has been shown to correlate with the sensitivity of tumors towards tyrosine kinase inhibitors. Recent data have shown a down-regulation of OCT1 (SLC22A1) and OCT3 (SLC22A3) in human hepatocellular carcinoma (HCC) and human cholangiocarcinoma (CCC), associated with tumor progression and a worse patient survival. The OCT1 down-regulation in HCC may affect the ability of sorafenib and platin derivates to reach active intracellular concentrations in these tumors.

Methods: HCCs were induced in OCT3-knockout (OCT3-/-) and wildtype (WT) mice with Diethylnitrosamine (DEN) and Phenobarbital. Tumor characteristics were assembled after 10 months. Fibrosis and inflammation were quantified by Sirius Red staining and leukocyte infiltration (Hematoxylin and Eosin, H&E) respectively. Steatosis was measured by quantification of lipid stain (Oil Red O). For proliferation and apoptosis analysis, Ki67- and Caspase 3- staining were assessed immunohistochemically. SLC22A1 mRNA expression was measured in HCC and corresponding non-neoplastic tumor surrounding tissue (TST) by real time PCR in OCT3-/- and WT mice. Protein expression was determined by western blot analysis. Finally mRNA expression of potential regulatory factors (HNF4α, PPAR-α and c-Myc) were investigated by real time PCR.

Results: Tumor size (p < 0.01) and quantity (p = 0.001) were enhanced in OCT3-/-mice in comparison to WT mice after 10 months of DEN/Phenobarbital treatment. Tumors in OCT3-/-mice showed significant higher steatosis than their WT littermates (p < 0.001). Immunohistochemistry showed significantly increased Ki67- (p ≤0.01) and Caspase 3- (p = 0.002) staining in HCCs in OCT3-/- vs. WT mice, suggesting enhanced proliferation and apoptosis. Tumors in OCT3-/- mice showed more fibrosis (p = 0.004) and more inflammation (p = 0.047) than HCCs in WT mice. Real time PCR indicated an up-regulation of SLC22A1 in untreated OCT3-/- mice (p = 0.002). SLC22A1 mRNA and OCT1 protein expression were down-regulated in HCC compared to TST in OCT3-/- mice (p = 0.001) after 10 months. HNF4α and PPAR-α mRNA expression were up-regulated in untreated OCT3-/- mice compared to their WT littermates and down-regulated in HCC vs. TST in OCT3-/- mice. No significant changes could be found in c-Myc mRNA expression.

Conclusion: OCT1 is up-regulated OCT3-/- mice. The down-regulation of OCT1 in HCC in OCT3-/- mice is associated with tumor progression. The expression of OCT1 might be regulated via HNF4α and PPAR-α in OCT3-/- mice.

Corresponding author: Knapstein, Johanna

E-Mail: johanna.knapstein@unimedizin-mainz.de