Protein expression and function of organic anion transporters in short-term and long-term cultures of Huh7 human hepatoma cells

Abstract

Human-derived hepatic cell lines are a valuable alternative to primary hepatocytes for drug metabolism, transport and toxicity studies. However, their relevance for investigations of drug-drug and drug-organic anion (e.g., bile acid, steroid hormone) interactions at the transporter level remains to be established. The aim of the present study was to determine the suitability of the Huh7 cell line for transporter-dependent experiments. Huh7 cells were cultured for 1 to 4 weeks and subsequently were analyzed for protein expression, localization and activity of solute carrier (SLC) and ATP-binding cassette (ABC) transporters involved in organic anion transport using liquid chromatography-tandem mass spectroscopy, immunocytochemistry, and model substrates [³H]taurocholate (TCA), [³H]dehydroepiandrosterone sulfate (DHEAS) and 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) diacetate. The extended 4-week culture resulted in a phenotype resembling primary hepatocytes and differentiated HepaRG cells: cuboidal hepatocyte-like cells with elongated bile canaliculi-like structures were surrounded by epithelium-like cells. Protein expression of OSTα, OSTβ and OATP1B3 increased over time. Moreover, the uptake of the SLC probe substrate DHEAS was higher in 4-week than in 1-week Huh7 cultures. NTCP, OATP1B1, BSEP and MRP3 were barely or not detectable in Huh7 cells. OATP2B1, MRP2 and MRP4 protein expression remained at similar levels over the four weeks of culture. The activity of MRP2 and the formation of bile canaliculi-like structures were confirmed by accumulation of CDF in the intercellular compartments. Results indicate that along with morphological maturation, transporters responsible for alternative bile acid secretion pathways are expressed and active in long-term cultures of Huh7 cells, suggesting that differentiated Huh7 cells may be suitable for studying the function and regulation of these organic anion transporters.

Keywords: ATP-binding cassette proteins; Hepatocytes; Huh7 cell line; Mass spectrometry; Membrane transport proteins; Phenotype; Solute carrier proteins.

Figure 1.

Morphology of representative Huh7 cells maintained in culture for 7 days (A) or 28 days (B, C, D). Filamentous actin (F-actin) was stained with Alexa Fluor^® 594-phalloidin (red). Hepatocyte-like cells and epithelium-like cells are indicated by “h” and “e”, respectively. Accumulation of F-actin suggests the formation of bile canaliculi-like structures on the canalicular plasma membranes, indicated by a triangle.

Figure 2.

Transporter protein expression in Huh7 cells and human hepatocytes measured by LC-MS/MS. Data represent mean ± SD (n=3-4 independent experiments, except n=2 for OATP2B1 Huh7-1W and Huh7-2W, and MRP4 Huh7-4W). When protein expression was detected in less than one-half of the samples in a group, the group is expressed as not detectable (ND). ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001, significantly different from Huh7 cells cultured for 1 week (Huh7-1W).

Figure 3.

Expression and localization of SLC transporters in representative Huh7 cells. Huh7 cells were cultured for 1 or 4 weeks, followed by immunostaining of transport proteins (green) with either anti-NTCP, anti-OSTα, anti-OSTβ, anti-OATP1B or anti-OATP2B1, and Alexa Fluor^® 488-labeled secondary antibodies. Human hepatocytes were cultured in sandwich configuration for 5 days followed by immunostaining. Nuclei were visualized with DAPI (blue).

Figure 4.

Co-localization of transporters and filamentous actin (F-actin) in representative Huh7 cells cultured for 4 weeks. Transporters (green) were detected with anti-OATP1B, anti-OATP2B1, anti-OSTα, anti-OSTβ, anti-MRP2 and anti-MRP4 together with Alexa Fluor^® 488-labeled secondary antibodies. Actin filaments and nuclei were visualized with Alexa Fluor^® 594-labeled phalloidin (red) and DAPI (blue), respectively.

Figure 5.

Expression and localization of ABC transporters in representative Huh7 cells. Huh7 cells were cultured for 1 or 4 weeks, followed by immunostaining of transport proteins (green) with either anti-BSEP, anti-MRP2, anti-MRP3 or anti-MRP4, and Alexa Fluor^® 488-labeled secondary antibodies. Human hepatocytes were cultured in sandwich configuration for 5 days followed by immunostaining. Nuclei were visualized with DAPI (blue).

Figure 6.

Cellular uptake of TCA (A) and DHEAS (B) in 1-week and 4-week cultures of Huh7 cells. Cell cultures were treated with extracellular fluid (ECF-Na⁺) or with extracellular fluid where sodium chloride was replaced with choline chloride (ECF-Chol⁺) supplemented with TCA (10 μM and 300 nCi/mL) or DHEAS (2.5 μM and 300 nCi/mL) for 2 min, followed by washes, lysis and scintillation counting. Data represent mean ± SD (n=3 in triplicate). ns, not significantly different; *, p<0.05; ***, p<0.001 significantly different from ECF Na⁺ buffer; †, p<0.05; ††, p<0.01; †††, p<0.001 significantly different from 1-week culture.

Figure 7.

Disposition of 5 (6)-carboxy-2',7'-dichlorofluorescein (CDF) (green) in 1-week and 4-week cultures of Huh7 cells. Huh7 cell cultures were treated for 10 min with 2 μM CDF diacetate, followed by washes with HBSS and confocal microscopy.