Roles Of P15 On MDR Of Hepatoma And The Underlying Mechanisms

【Background】Multidrug resistance (MDR)of tumors is a major cause for failure of chemotherapy and death of tumor patients。Many results suggest that TGF-beta/Smads signal pathway effects MDR of tumor through two-ways regulation. In the early stage of tumorigenesis,TGF-beta induces tumor cell growth arrest;however, in the later stage of tumorigenesis,TGF-beta promotes tumor cell proliferation. Cyclin-dependent kinase inhibitor 2B ,also known as P15,Cdkn2b,INK4B,a member of INK4 protein family. P15 is a downstream Cell cycle inhibitor of TGF-beta/Smads signal pathway. TGF-beta inhibited cdk2 enzyme activity and increased p15(Ink4b) protein levels. The expression of p15 was found to be dramatically induced by TGF-beta, which suggested its role in the TGF beta induced growth inhibition。In this study, we investigate the roles and possible mechanisms of P15, a downstream Cell cycle inhibitor of TGF-beta/Smads signal pathway,on MDR of Hepatoma and the Underlying Mechanisms【Objective】⑴To study the differential expression of P15 in HepG2/CDDP kinetic drug fast model;⑵To study the roles of p15 in the phenotype of HepG2/CDDP kinetic drug fast model;⑶To explore the roles and possible mechanisms of P15 mediated MDR of hepatoma cells.【Methods】⑴To construct HepG2/CDDP kinetic drug fast model; ⑵To construct the sense expression vector of P15 by cloning the cDNAs of P15 into pcDNA3.1-B;Cells were divided into three groups, pCDNA3.1-p15 and pCDNA3.1-B were transfected into two groups respectively,another group is negative control grou,transfection was examined by Western blot analysis;⑶Differential expression of p15 was examined by Western blot analysis and reverse transcription-PCR Analysis;⑷Accumulation and detention of adriamycin in the transfected cells were examined by flow cytometry (FCM);⑸Using FCM, cell cycle distribution of the transfected cells was studied. The proliferous indexes (PI) were calculated.⑹To study the correlations between P15 and P-gp,MRP by Western blot analysis;⑺To study the expression of Bcl-2,Bax by Western blot analysis ;【Results】⑴HepG2/CDDP kinetic drug fast model was successfully constructed;Western blot analysis and reverse transcription-PCR Analysis⑵Detecting the expression of P15 in HepG2/CDDP kinetic drug fast model after cultivating cells induced by cisplatin 24h,48h,3d,5d,7d by Western blot analysis and reverse transcription-PCR Analysis,and the result shows that P15 expression levels decreased gradually,With MDR phenotype to enhance;⑶The sense expression vector of P15 was successfully constructed; By RT-PCR and Western blot verification, The transfection is successful. The sense expression vector of P15 upregulated the expression of P15;⑷As shown by the drug transport assay, P15 inhibit the drug transport by the membrane,and increase adriamycin accumulation and retention in intracellular adriamycin; ⑸Aftering the sense expression vector of P15 transfecting,cell proliferative activity was reduced;⑹The MRP, P-gp protein expression decreased after the sense expression vector of P15 transfected into HepG2/CDDP cells;⑺The Bcl-2 expression decreased, Bax expression increased, after the sense expression vector of P15 transfected into HepG2/CDDP cells;【Conclusions】1. HepG2/CDDP kinetic drug fast model was successfully constructed。2. P15 expression levels decreased gradually,With MDR phenotype to enhance;3. Up-regulated the expression of P15 can decrease the ability of the drug transport by the membrane, increase adriamycin accumulation and retention in intracellular adriamycin, inhibit MDR and cell proliferative activity.