Proteasome,Sorcin

Drug resistance in cancer is a major obstacle to successful chemotherapies.Imatinib-Resistance in leukemia has greatly limited the clinical usage of Imatinib,the first small molecular tyrosine kinase inhibitor that has been successfully used to treat chronic myelogenous leukemia(CML),acute lymphoblastic leukemia(ALL),etc.Development of effective reversal agents is heavily dependent on our understanding of the various molecules/pathways involved in the emergence of drug resistance.Establishing drug-resistant cell model is the first step toward this goal.In present study,we established an Imatinib-resistant leukemia cell line by exposure of K562 parental cells to stepwise increasing concentrations of Imatinib,named as K562/G01 which resistant rate to Imatinib is more than 40.These cells are resistant to Imatinib-induced apoptosis,whereas remains sensitive to a panel of MDR agents and non-MDR agents,such as VP16,HHT and CDDP.Comparing with the parental cells K562, K562/G01 cells have distinct change in the number of mitochondria and mitochondria crista.Also,the level of mRNA and protein,tyrosins kinase activity and number of gene copy of BCR/ABL are increased.These results suggest that increase of the activity of RCR/ABL is the main course of drug-resistance of K562/G01 cells.So far,the mechanism of drug resistance that has been well studied and widely validated is over-expression of Pgp,a member of ABC superfamily encoded by mdr1 gene.However,it is still unclear if it is involved in Imatinib-resistance.In present study,we found that the Pgp level is highly expressed in K562/G01,while it is undetectable in its parental cells K562.This indicates that the expression of Pgp can be induced by Imatinib.When treating cells with Imatinib,the accumulation level of this drug in K562/A02 cell is lower than that in K562 and K562/G01 cells;Moreover,the resistant rate of K562/A02 to Imatinib is 2-3.All these results suggest that Pgp can efflux Imatinib at certain degree if any,but it is not the major contributor to the Imatinib resistant.The accumulation test of DOX and Rho123,the subtracts of Pgp,data showed that the level of these two compounds in K562/G01 cells is same as that in K562 cell;Moreover,this accumulation is not sensitive to inhibitor of Pgp,VPL.These suggest that the Pgp in K562/G01 cell is not functional. Treating cells with Imatibin to inhibit the activity of BCR/ABL or/and adding external ATP could restore the efflux activity of Pgp in K562/G01 cell.Thus,it is likely that lacking of enough intracellular ATP that is held by Tyrosine Kinase BCR/ABL is the course of de-function of Pgp in K562/G01 cells.In order to find new protein targets associated with drug resistance and further explore the mechanism,we employed proteomics technique to establish the protein profile of both drug resistant and drug sensitive cells.First we compared the protein profile of K562/G01 and its parental cell K562 and found 10 proteins which expression levels have g-fold or more difference.One of them is Proteasome which expression level in K562/G01 cell is three-fold higher than that in K562 cells.We demonstrated in this study that proteasome can cause cell tolerate to the apoptosis induced by Imatinib through degrading IkB and consequently active the NFkB pathway.We also found that proteasone is involved in the cell resistance to Imatinib by regulating the stability of BCR/ABL and Cyclin B1 proteins.And then,we compared protein profile of K562/A02 which possesses MDR phenomenon with its parental cells K562 and found 14 proteins which expression levels have two-fold or more difference.One of them is Sorcin that expression level is about ten-fold increase in K562/A02 cells.We demonstrated that Sorcin is involved in MDR by regulating the expression of apoptosis related proteins,Bcl-2 and Bax,expression of Pgp and the level of the intercellular Ca²⁺.From the results described above we can concluded that Sorcin and proteasome are likely not only markers of diagnosis of drug resistance and prognosis,but also new targets for developing new reversal agents.