| Objective: To study the multidrug resistance mechanism in primary hepaticcarcinoma, we established a multidrug resistant hepatocellular carcinoma cell line (HepG2/ADM) and investigated some of its biological behaviour. Recent developments of RNA interference (RNAi) technology make it possible to reverse MDR by suppressing MDR1 gene with RNAi, however, whether MDR could be reversed by RNAi-mediated MDR1 suppression in hepatoma cells remains unknown. We developed two short hairpin RNAs (shRNAs) targeting the MDR1 gene to observe the reversal efficiency of the two shRNAs on HepG2/ADM cells.Metheds: (1) The establishment of a MDR phenotype of HepG2 cells: Throughexposure to gradual increased concentration of ADM, the cell line HepG2 was induced to form a multidrug resistant sub-cell line (HepG2/ADM). The multidrug resistance of HepG2/ADM was tested by MTT assy. The distributions of cell cycle in HepG2 and HepG2/ADM cells, the drug concentration in HepG2 and HepG2/ADM cells and the variation of cell cycle in HepG2 and HepG2/ADM cells after incubation with ADM were analyzed by flow cytometry. We used real-time RT-PCR to detect the expression of MDR1 gene, LRP gene. MRP gene and BCRP gene in HepG2 and HepG2/ADM cells. Westem-blotting-Enhanced chemilluminesence (ECL) was used to detect the expression products of MDR1, MRP, LRP and BCRP in the two cell lines. (2) Some biological behavior differences between the two cell lines: using a transmission elctron microscope to study the morphologic differences between the multidrug resistance cell line HepG2/ADM and its mother cell line HepG2; real- time RT-PCR to check the peroxisome proliferator-activated receptor α (PPARα) mRNA and Twist mRNA levels in the two cell lines and western blotting to detect the PPARα protein and Twist protein levels in the two cell lines. (3)Two. different short hairpin RNAs (shRNAs) were designed and constructed into pGenSil-1 plasmid, respectively. They were then transfected into the highly adriamycin-resistant HepG2 hepatoma cell line (HepG2/ADM). The RNAi effect on MDR was evaluated by real-time PCR, cell cytotoxicity assay and Rhodamine123 (Rh123) efflux assy.Results: HepG2/ADM was resistant to many anti-tumor agents. Its ID50 of 8 kinds ofanti-tumor agents was 1.22-107.56 times higher than that of HepG2. The doubling time of HepG2/ADM was 2.05 times longer than HepG2. There was no difference in the distribution of cell cycle in the two cell lines. ADM had an obvious influence to the cell cycle of the two cell lines. After incubation with ADM, the cells were blocked in G2 phase, followed with a quantitive apoptosis. The expressions of MDR1 and BCRP were up regulated, and the MDR1 gene was up regulated significantly, but the expressions of LRP was of no difference, surprisingly, the MRP gene was slightly down regulated. The PPARα gene was down regulated in the HepG2/ADM cells, while the Twist gene was up regulated. The stable transfected clones showed varied degrees of reversal of MDR phenotype. Surprisingly, the MDR phenotype was completely reversed in two transfected clones.Conclusion: The HepG2/ADM cell line is a fine multidrug resistant model, whichhad all the basic biological characteristics that a multidrug resistant cell line should have. PPARα is related with the multidrug resistance phenomenon in the HepG2/ADM cells. The down regulation of PPARα may be one of the mechonisms in the forming of multidrug resistance in tumor cells. Compared with its mother cells, HepG2/ADM cells may be more tending to metastasis owing to the up regulation of Twist. MDR can be reversed by the shRNA-mediated RNAi in HepG2/ADM cells, which provides a valuable clue as to sensitizing multidrug-resistant hepatoma cells to anti-cancer drugs. |
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