The Role And Possible Mechanisms Of Nucleophosmin In Multidrug Resistance Of Hepatoma Cells

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and about half of the patients with liver cancer focused in China, where about 50,000 people die from liver cancer each year, ranking the second in malignant tumor mortality. Chemotherapy is one of the important ways in the comprehensive treatment for liver cancer, while the tumor multidrug resistance(MDR) is one of the main reasons for significant effects on chemotherapy and patients' survival. Nucleophosmin (NPM, also known as B23, numatrin,or NO38), was first identified as a nucleolar phosphoprotein expressed at high levels in the granular region of the nucleolus. NPM was soon thought to have a role in the regulation of cell growth, proliferation and transformation, based on the observation that its expression rapidly increases in response to mitogenic stimuli, and that increased amounts of the protein are detected inhighly proliferating and malignant cells. Another of the mechanisms through which NPM might suppress apoptosis is the functional inhibition of p53 and p14. The NPM protein is overexpressed in various tumours, and it has been proposed as a marker for gastric, colon, ovarian and prostate carcinomas. Polo-like kinase 1(Plkl) is a serine/threonine kinase.Many studies have shown that the Plkl has multiple functions during mitosis and,perhaps more importantly, has a significant role in ensuring the fidelity of checkpoint controls. with non-small-cell lung cancer. Multiple follow-on studies showed that determining Plkl expression levels has prognostic value for different cancers, including oropharyngeal carcinoma, melanoma, colon cancer and hepato-blastoma. Furthermore, some reports have indicated that Plkl expression is a reliable marker for identifying a high risk of metastasis.lt is reported that antisense inhibitors against Plkl may be considered as highly efficient promoters for the antineoplastic potential of taxanes, causing synergistic effects in breast cancer cells.Thus, we postulated that Plkl has the same role in MDR of HCC.Objective1.To investigate the expression of NPM in HepG2/ADM and study the the effects of NPM on multidrug resistance of HCC;2.To construct a recombinant plasmid carrying enhanced green fluorescent protein and human NPM gene and transfected into HepG2, with the aim of better elucidating the mechanisms of NPM in MDR of HCC;3.To investigate the expression of Plkl in HepG2/NPM,with the aim of better elucidating the mechanisms of Plkl in MDR of HCC.Methods:1.The expressions of NPM in HepG2/ADM and HepG2 were determined by real-time PCR and Western blot.2.The effects of NSC348884, which is a special inhibitor of NPM, on MDR of HepG2/ADM were examined by following perspects:(1) By CCK-8, the dose-response curve of NSC348884 on HepG2/ADM cell was drawn, and with the flow cytometry to detect the effect of NSC348884 with above-mentioned dose on Rh123 concentration in HepG2/ADM cell, then, the appropriate dose of NSC348884 was selected according to the above experimental results.(2) Cell proliferation were determined by Cell counting Kit-8(3) Rh 123 accumulation is used to detect the effect of NSC348884 with above-mentioned dose in HepG2/ADM cell, then, the appropriate dose of NSC348884 was selected according to the above experimental results.3.The recombinant plasmid pEGFP/NPM was transfected into HepG2 with lipofectamineTM2000. It was divided into two groups:HepG2 cell and HepG2/NPM cell group:(1)Detected cell proliferation by Cell counting Kit-8;(2)Potentiation of drug-induced apoptosis by NSC348884 was evaluated by using Annexin V/propidium iodide flow cytometry;(3)Caspase-3 activity assay was used to analyze apoptosis of cells.(4)The protein expression level of MDR-1,MRP-1 and LRP-1 were determined by Western blot;(5)The gene expression level of MDR-1,MRP-1 and LRP-1 were determined by real-timePCR.4.The effects of BI2536,which is a special inhibitor of Plkl, on MDR of HepG2/NPM were examined by following perspects: (1)The expressions of Plkl in HepG2/NPM and HepG2 were determined by real-time PCR and Western blot;(2)By CCK-8, the dose-response curve of BI2536 on HepG2/NPM cell was drawn, the appropriate dose of BI2536 was selected according to the above experimental results;(3)Detected cell proliferation by Cell counting Kit-8;(4)Potentiation of drug-induced apoptosis by BI2536 was evaluated by using Annexin V/ Propidium iodide flow cytometry;(5)The protein expression level of MDR-1,MRP-1 and LRP-1 were determined by Western blot;(6)The gene expression level of MDR-1 was determined by real time-PCR.Results:1.The levels of NPM in HepG2/ADM1.1 Compared with HepG2 cells,NPM was identified that show significantly high expression in HepG2/ADM cells by real-time PCR and Western blot analysis.1.2The results showed treatment with NSC348884 for 24h followed by ADM,5-Fu or DDP for 48 hours resulted in significant cell growth inhibition.2.Establishment of HepG2/NPMThe recombinant plasmid carrying enhanced green fluorescent protein and human NPM gene was successfully constructed and transfected into HepG2.The protein was secreted which confirmed by real-time PCR and Western blot.2.1 Measurement of MDR in HepG2/NPM2.1.1CCK-8 assay reveals that overexpression of NPM produced multidrug resistance (10.08 folds of ADM-resistance,7.16 folds of 5-Fu-resistance and 2.98 folds of DDP-resistance).2.1.2The combination of NSC348884 and 5-Fu resulted in a stronger apoptotic effect in comparison with either agent alone after 48h treatment by using Annexin V/propidium iodide flow cytometry (P<0.05).2.1.3To further test the ability of NPM on apoptosis, caspase-3 in both cell lines tested were evaluated.It is revealed that NPM caused a decrease in caspase-3 activity (P<0.05).2.1.4Effects of NPM on MDR-1,MRP-1 and LRP-1 expressionThe MDR-1 and MRP-1 protein level in HepG2/NPM was greatly increased as compared to that of HepG2(4.93±0.60 vs 1.12±0.21和2.49±0.35 vs 1.12±0.21, P<0.05). Whereas there was no significant change in LRP-1 protein level among them(1.13±0.09 vs 1.11±0.08, P>0.05).2.1.5Changes of MDR-1,MRP-1 and LRP-1 mRNA levelConsistent with the results of protein level,real-time PCR showed MDR-1 and MRP-1 expression mRNA wasere markedly increased in HepG2/NPM than in HepG2.3.The role of Plkl in MDR nduced by NPM of HepG23.1 Compared with HepG2 cells,Plkl was identified that show significantly high expression in HepG2/NPM cells by real-time PCR and Western blot analysis;3.2 the role of Plkl in MDR induced by NPM of HepG23.2.1 To all these three chemotherapy drugs-ADM, DDP and 5-Fu, HepG2/NPM cell showed resistance. Under the action of BI2536, the sensitivity of the drug-resistant cell HepG2/NPM enhanced to all the anticancer drugs, the IC5o decreased significantly (P<0.05).3.2.2The combination of BI2536 and 5-Fu resulted in a stronger apoptotic effect in comparison with either agent alone after 48h treatment by using Annexin V/propidium iodide flow cytometry (P<0.05).3.2.3Effects of NPM on MDR-1,MRP-1 and LRP-1 expressionOur results of western blot displayed the MDR-1 expression in HepG2/NPM was significantly up-regulated after BI2536 treatment. Whereas there was no significant change in MRP-1 and LRP-1 protein level.3.2.4Changes of MDR-1 mRNA levelTo determine whether the change in MDR-1 protein expression inHepG2/NPM is regulated at a transcriptional level, mRNA level of MDR-1 was analyzed by a real time-PCR assay. There were no significant changes in MDR-1 mRNA level among all groups. Conclusions1.Overexpression of NPM could induce MDR in HepG2 cells, indicating that NPM plays an important role in the drug-resistant phenotype of human hepatic cancer cells;2.The role of NPM in MDR of HCC is associated with MDR-1 and MRP-1;3.Plk1 is involved in MDR of HepG2 induced by NPM.