A Novel Hsp90 Inhibitor-SNX-2112 Mechanism Study For The Human Chronic Myeloid Leukemia Therapy

SNX-2112 is a novel screened Hsp90 inhibitor in recent years,which can specifically bind ATP pocket of Hsp90 families,inhibit the latter activity and induce their client proteins degradation resulting in cancer cell apoptosis,therefore currently SNX-2112 is considered to be a promising anti-cancer drug for exploring.However the molecular mechanism of the apoptosis effect of SNX-2112 is poorly understood.In vitro experiments using human chronic myeloid leukemia K562 cell line as a study model,we detected SNX-2112 effect on the proliferation,apoptosis and caspase families expression of K562 cells by employing MTT methods,Annexin-v/PI fluorescent staining,flow cytomertry,Westem blot analysis and etc,.Our results showed that 0.05-10μM SNX-2112 could reduce cells growth ration with IC₅₀=0.92μM and lead to apoptosis(the percentages covering from 10.6±2.1 to 75.8±7.4). Additionally,at 0.1-10μM concentrations,SNX-2112 activated pro-caspase-3,9 and PARP in a dose-dependent manner.Utilizing genomics expression profile chips we detected the total cDNA expression levels of K562 cells treated 1μM SNX-2112 for 48 h.By Gene Ontology analysis,we found 229 different genes related with mitochondrial function,particularly in metabolism groups with a notable significance(accounting for 62.15%of total selectors),which also provided evidence for mitochondrial dysfunction due to SNX-2112 action.2-D gel electrophoresis was performed to obtain K562 cells proteomics profiles induced by SNX-2112. MALDI TOF/TOF mass spectrometer identified 55 different protein dots,7 of which involve in mitochondrial impairment analyzed by function classification with bioinformatics.Next,we studied the possible mechanism of K562 apoptosis produced by SNX-2112.Together with the detected caspase activated pathway,transcriptomic and proteomic data,we speculated that SNX-2112 regulated K562 cells apoptosis mediated by the intrinsic pathway of mitochondrial impairment,which is further supported by JC-1 staining assay and Western Blot of cyto-c release. The two proteins,CRKL and Grb2,identified by proteomics study disclosed the up-stream signalinging partly originated from Akt signaling transduction pathway,tested by Western Blot and co-IP approaches. We tested the effect of SNX-2112 on NOD/SCID mice model inoculated with K562 cells.The implanted mice were treated with SNX-2112(6mg/kg,â…³).Simultaneously we also set 17-AAG and negative control groups,respectively.Mice survival time and expression of human chronic leukemia antigens(CD13 and CD33) were served as therapy criteria.SNX-2112 inhibited leukemia cells proliferation in vivo and prolonged the survival time of NOD/SCID mice inoculated with K562 tumor cells.In conclusion,our results demonstrated the therapeutic potential of SNX-2112 against human chronic myeloid leukemia and found its partial anti-cancer mechanism,all of which establish theory base for further researching Hsp90 inhibitors with self-owned intellectual property rights.