Effects Of Bortezomib On The Expression Of SHIP Gene In K562 Cells

Objective: Chronic myelogenous leukemia (CML) is a kind of acquired malignancy which is caused by clonal proliferation after mutation in multiple potential stem cells. The discovery and study of Ph chromosome and fusion gene of bcr/abl contribute a lot to the diagnosis and therapy of CML.The disease arises as a consequence of a rare mutational event resulting in a reciprocal translocation between the long arms of chromosomes 9 and 22. This creates the chimeric oncogene BCR-ABL with the protein product BCR-ABL, a tyrosine kinase with constitutive activity. BCR-ABL is responsible for the pathogenesis of CML[1-2].The current recommended first-line therapy for patients with chronic phase (CP) CML is imatinib mesylate (IM), The majority of newly diagnosed patients with CP CML will achieve a complete cytogenetic response (CCyR) with IM[3].However, it is accepted that IM induces a state of minimal residual disease (MRD) rather than cure.Evidence for this is provided from clinical studies in which the number of patients sustaining a CCyR (63%) is fewer than the number achieving MDR (82%).responding patients who discontinue IM are likely to suffer recrudescence of disease[4].It is likely that CML HSCs are the source of MRD.CML HSCs are relatively resistant to IM[5].In addition to MRD, IM resistance is well documented with mechanisms including BCR-ABL mutations[6-9].More potent TKIs have been developed and include dasatinib and nilotinib and other TKIs,These drugs show clinical benefit in IM-resistant CML[10-11]. However, key problems remain: the primitive CML HSC is insensitive in vitro to dasatinib, nilotinib, and other TKIs[12-14]; and none of the TKIs inhibit the T315I BCR-ABL mutation[15]. It is particularly important for us to seek new methods of treatment in the current.Bortezomib,which is the first proteasome inhibitor to enter clinical trials,has been widely used for patients with refractory and relapsed multiple myeloma[16].In vitro studies, bortezomib had shown a good effect on a variety of malignant tumors cells[17],but the mechanism is not clear.My study aims to test the expression level of SHIP in K562 cells treated by bortezomib and 5-azacytidine, observation the changes of cell proliferation and apoptosis in order to research the function and clinical significance of SHIP gene in the pathogenesis of CML,and this will provide a theoretical basis for the treatment of CML.Methods: The object was divided into 12 group as the density of bortezomib and 5-azacytidine. The time was divided into three phases:24h,48h,72h. To detect the change of corrected index: (1) The cell proliferating activity was assessed with MTT assay; (2) Cell apoptosis rate was examined by Flow Cytometry(FCM); (3)The expression of SHIP mRNA was detected by Reverse Transcription -Polymerase Chain Reaction(RT-PCR).Results:1 MTT assay displayed that: both single drug and combination group can inhibit K562 cells , and the effect was related to the concentration and acting time of drug ,and presented dose-dependent and time-dependent relationship. The suppressant effect of combination group obviously outweigh the single group.2 FCM analysis showed that: both single drug and combination group can induce apoptosis of K562 cells , and the effect was related to the concentration and acting time of drug ,and presented dose-dependent and time-dependent relationship. The suppressant effect of combination group obviously outweigh the single group.3 RT-PCR disclosed that the expression of SHIP gene was significantly increased by bortezomib or 5-azacytidine.Conclusion:Bortezomib alone and in combination with 5-azacytidine can inhibite the proliferation of K562 cells and increase apoptotic cells, in which increase of the expression of SHIP mRNA is the possible mechanism.