| Relationship between Na+/K+-ATPaseα3 and theAnti-tumor Effect of Bufalin on HepatocellularCarcinoma Cell LinesObjectiveTo study the roles and potential mechanism of Na+/K+-ATPase in the anti-tumor effect of Bufalin on hepatocellular carcinoma cell lines.Methods1.Human hepatocellular carcinoma cell lines PLC/PRF/5, HepG2 and SMMC7721 were treated with Bufalin. Cells growth inhibition was analyzed by WST(water-soluble tetrazolium salt) assay.2.The mRNA expression of Na+/K+-ATPase isoformα1,α2,α3,α4 in human hepatocellular carcinoma cell lines PLC/PRF/5, HepG2 and SMMC7721 were detected by RT-PCR. The protein expression of Na+/K+-ATPase isoformα1 andα3 were detected by Western Blot.3.The Na+/K+-ATPase isoformα3 of PLC/PRF/5 was silenced by the StealthTMRNAi method. The expression of Na+/K+-ATPase isoformα3 of PLC/PRF/5 and PLC/PRF/5 after silenced were identified by RT-PCR.4.The changes of drug sensitivity to Bufalin on PLC/PRF/5 and HepG2 after Na+/K+-ATPase isoformα3 RNAi silenced were analyzed by WST assay.Results1. The liver cancer cell lines were treated with various concentration Bufalin for 24h, the OD (optical density) value of each well was detected by CCK-8. The IC50 of bufalin in HepG2, PLC/PRF/5 and SMMC7721 were 182.3nM, 52.2nM and 97.74nM respectively.2. The expression of mRNA for Na+/K+-ATPase isoformα1,α2,α3 andα4 was detected in all liver cancer cell lines. The median expression values of Na+/K+-ATPaseα1,α2,α3 andα4 in HepG2, PLC/PRF/5 and SMMC7721 were 2.78, 1.76, 1.72; 1.28, 0.92, 0.84; 0.45, 1.79, 0.88 and 0.79, 0.64, 0.47 respectively. The response to Bufalin was related to expression of Na+/K+-ATPase isoformα3.3. Protein expressions were analysed by Western blot assays. The expression of Na+/K+-ATPase al andα3 in HepG2, PLC/PRF/5 and SMMC7721 were 1.86, 1.46, 1.64 and 1.73, 2.94,1.91 respectively.4.Three interference sequences of Na+/K+-ATPase isoform a3 were transfected to PLC/PRF/5 by Stealth RNAi techniques. The Na+/K+-ATPase isoformα3 mRNA expression in PLC/PRF/5/siRNA1, PLC/PRF/5/siRNA2 and PLC/PRF/5/siRNA3 were 41.8%, 10.5% and 74.1% compare to control cells.5.PLC/PRF/5/lipofect and PLC/PRF/5/siRNA2 were treated with various concentration of Bufalin for 48 hours. The IC50 of PLC/PRF/5/siRNA2 cells and PLC/PRF/5/lipofect cells were 42.10nM and 29.29nM respectively. The test result in HepG2 that transfected by interference sequences was similar to the PLC/PRF/5. IC50 of HepG2/siRNA2 and HepG2/lipofect were 200.10 nM and 84.53 nM.Conclution1. Bufalin could effectively inhibit the growth of liver cancer cells in vitro.2. The inhibition correlated with the expression of Na+/K+-ATPase isoformα3 expression. Na+/K+ -ATPase isoform a3 is the target of bufalin in liver cancer cells. The inhibition of Bufalin decreased when the expression of Na+/K+-ATPase isoformα3 was down-regulated by silenced sequences.3. The response of different liver cancer cells to Bufalin were related to different expression of Na+/K+-ATPase isoformα3. The detection of Na+/K+-ATPase isoformα3 expression in liver cancer tissue can be the predictor of sensitivity in patients treats by drugs that include Bufalin. |
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