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The multidrug resistance-associated protein (MRP) is over-expressed and functional in rat hepatoma cells
L Payen1, A Courtois, L Vernhet
1INSERM U456 Détoxication et Réparation Tissulaire, Faculté de Pharmacie, Rennes, France.
Abstract:
Expression of multidrug-resistance-associated protein (MRP), a drug efflux pump transporting a wide range of xenobiotics, including anti-cancer drugs and chemical carcinogens, and present at low levels in normal hepatocytes, was investigated in rat hepatoma cells. Northern-blot analysis allowed detection of high levels of MRP mRNA in rat diethylnitrosamine-induced hepatocarcinomas when compared with normal liver. Similarly, elevated expression of MRP transcripts were evidenced in 6 rat hepatoma cell lines of different origins, especially in HTC cells, that, in contrast, failed to express mRNA of the canalicular multispecific organic anion transporter (cMOAT), an efflux pump sharing numerous substrates with MRP. HTC cells were also found by Western blotting to display much higher amounts of MRP than those observed in normal hepatocytes. In contrast, the MRP gene copy number was similar both in hepatoma HTC cells and in hepatocytes, as assessed by Southern blotting. Analysis of MRP-related transport using 3 types of MRP substrates, namely, the fluorescent glutathione-bimane, the anionic dye calcein and the cationic anti-cancer drug vincristine, demonstrated that HTC cells displayed cellular efflux of these 3 compounds, an efflux strongly inhibited by MRP modulators such as indomethacin. These results indicate that MRP is over-expressed and functional in rat hepatoma cells and may therefore be included in the de-toxifying pathways that are altered during hepatocarcinogenesis and are thus thought to contribute to the known multidrug resistance of liver tumors.
Insights
Multidrug-resistance-associated protein (MRP) is overexpressed and functional in rat hepatoma cells. This suggests MRP contributes to multidrug resistance in liver tumors.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Multidrug-resistance-associated protein (MRP) is a drug efflux pump involved in xenobiotic transport.
- MRP is typically expressed at low levels in normal hepatocytes but is implicated in drug resistance.
- Hepatocarcinogenesis involves alterations in detoxification pathways, potentially including MRP expression.
Purpose of the Study:
- To investigate the expression and function of MRP in rat hepatoma cells.
- To determine if MRP is overexpressed in liver tumors compared to normal liver tissue.
- To assess the role of MRP in the multidrug resistance of hepatoma cells.
Main Methods:
- Northern-blot analysis to detect MRP mRNA levels.
- Western blotting to quantify MRP protein expression.
- Functional assays using MRP substrates (glutathione-bimane, calcein, vincristine) and inhibitors (indomethacin).
- Southern blotting to assess MRP gene copy number.
Main Results:
- High levels of MRP mRNA and protein were detected in rat hepatoma cells and cell lines compared to normal hepatocytes.
- HTC hepatoma cells showed elevated MRP expression but lacked canalicular multispecific organic anion transporter (cMOAT) mRNA.
- Hepatoma cells exhibited functional MRP-mediated efflux of substrates, inhibited by indomethacin.
- MRP gene copy number was similar in hepatoma cells and normal hepatocytes, indicating transcriptional upregulation.
Conclusions:
- MRP is significantly overexpressed and functionally active in rat hepatoma cells.
- Upregulated MRP contributes to altered detoxification pathways during hepatocarcinogenesis.
- MRP overexpression is a key factor in the multidrug resistance observed in liver tumors.