| Background and purpose: Hsp90 functions to facilitate the folding of newly synthesized and denatured proteins. Hsp90 function is modulated through its interactions with cochaperones and the binding and hydrolysis of ATP. Hsp90 is a chaperone with over 100 identified client proteins. What makes Hsp90 especially promising as a target for anti-cancer drugs is that many of its client proteins are in proliferation signaling pathways, and these pathways are often important in many types of cancers. Both amino- and carboxyl-terminal domains of Hsp90 interact to cochaperones or ATP and then modulate chaperone activity. Numerous classes of Hsp90 inhibitors have recently been developed, such as the anasamysin geldanamycin (GA) and derivatives 17-AAG that bind to the N-terminal high-affinity ATP-binding domain of Hsp90. Other inhibitors have recently been shown to bind to the C-terminal dimerization domain of Hsp90, such as cisplatin and novobiocin( NB) , or modify Hsp90 postranslationally, such as histone deacetylase or proteasome inhibitors.NB is a member of the coumermycin family of antibiotics and is a well-established inhibitor of DNA gyrase. It is a well-studied antibiotic, whose pharmacokinetics and toxicity profile are clearly understood. Recent studies have shown that NB binds to a previously unrecognized ATP-binding site at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins.The bcr/abl fusion gene-encoded Bcr-Abl protein, which possesses higher activity of tyrosine kinase than that of normal p145c-abl, is known to be a resistant factor for apoptosis induced by chemotherapy drugs in chronic myelogenous leukemia ( CML) . Because Bcr-Abl is one of the client proteins of Hsp90, disruption of the chaperone functions of Hsp90 may reduce the protein level of Bcr-Abl, and therefore potentially retard several signal transduction pathways initiated by Bcr-Abl.In an effort to confirm the effects of NB on Bcr-Abl expressing CML cells, and the relationship between these effects and the molecular chaperone functions of heat shock protein 90 (Hsp90), we investigated the effects of NB on the proliferation viability and induction of apoptosis in CML cells, the expression of Bcr-Abl kinase protein, and the interaction between Hsp90 and Bcr-Abl kinase. Given both amino- and carboxyl-terminal domains inhibitors of Hsp90 modulate chaperone activity; the effects of combination of Hsp90 amino- terminal inhibitor GA and Hsp90 carboxyl- terminal inhibitor NB were also studied. If NB is able to inhibit the growth of CML cells by disruption of Hsp90 molecular chaperone functions, it may sensitive to STI-571 (Imatinib Mesylate,Gleevec)- resistant, Bcr-Abl -positive leukemia cells. Because resistance to Gleevec may develop is mainly due to overexpression or mutations in Bcr-Abl. To confirm this idea, the effects of NB on STI571– resistant K562 cells ( K562/G01) were identified. Induction of differentiation was also studied.Experimental approach: When CML cells were treated with NB or/and GA, the typical morphological changes of apoptosis were observed by AO/EB examined by fluorescence microscopy and Annexin V-FITC/PI examined by flow cytometer. In order to reveal the relationship between NB-induced apoptosis and Bcr-Abl, the amounts of Bcr-Abl protein were tested by Western blot. To clarify the mechanism of Bcr-Abl down-regulation, molecular chaperone functions of Hsp90 were measured by coimmunoprecipitation. Co-immunoprecipitation of Bcr-Abl and its molecular chaperones, the immunoprecipitate was then subjected to western blot analysis with anti-Abl, anti-Hsp90, anti-Hsp70, anti-P23, or anti-P60HopmAb. Proteasome inhibitor ALLnL was used to determine the down-regulation pathway of Bcr-Abl. The abundance of other client proteins in CML cells treated with NB was also determined by western blot. Real-Time RT-PCR was used to examine the effects of NB on the transcription of bcr/abl gene. To investigate the possible mechanisms of STI571 resistance, a resistant cell line, K562/G01, was used. The viability of K562/G01 cells were examined by MTT; the abundance of Bcr-Abl and p-Tyrosine was also determined by western blot. The molecular chaperone functions of Hsp90 in K562/G01 cells were also measured by coimmunoprecipitation. Differentiation of leukemia cells induced by NB was detected with NBT reduction test.Results: 1. NB was a potent inhibitor of proliferation of Bcr-Abl-expression cells with IC50 values of 0.5174 mM in HL-60/Bcr-Abl cells, with ectopic expression of p185 Bcr-Abl tyrosine kinase (TK), and 0.4353 mM in K562 cells, with endogenous expression of Bcr-Abl Bcr-Abl TK. As control, the IC50 values was 0.3546 mM in HL-60 cells.2. NB induced cytosolic accumulation of cytochrome c and activities of caspase-3 / 9 , triggering apoptosis of HL-60, HL-60/Bcr-Abl, and K562 cells. It seemed that combination of GA 5μM and NB 0.3 mM could increase this effect.3. At very low concentration of NB, differentiation was induced in NB4, HL-60, and K562 cells. NBT reducing assay revealed that the inhibition of proliferation by NB is associated with differentiation.4. An exposure of K562 or HL-60/Bcr-Abl cells to NB or GA produced down-regulation of intracellular Bcr-Abl protein levels, as well as reduced Akt and ERK kinases activity. GA was able to induce the expression of Hsp27 and Hsp70; in contrast, NB had no effect on the two proteins, while NB decreased the amounts of p60HOP.5. Although NB appears to bind to a site on Hsp90 that is different from the geldanamycin-binding site, it, like geldanamycin is able to interfere with the chaperone function of Hsp90. Similar to Raf-1, v-Src, and Her-2-neu, Bcr-Abl TK has chaperone association with Hsp90. By binding and inhibiting Hsp90, NB treatment decreased the binding of Bcr-Abl with Hsp90 and Hsp70 and induced the proteasomal degradation of Bcr-Abl, because cotreatment with proteasome inhibitor ALLnL increased Bcr-Abl levels in NP40-insoluble fraction. This is in contrast to the effects of GA that shifting the binding of Bcr-Abl from Hsp90 to Hsp70. Thus, NB, besides mimicking the biologic effects of GA, may have an additional negative impact on Hsp90 function.6. GA synergistically potentate NB–induced tumor cell death. NB→GA sequence treatment was able to potentate the effect of NB on tumor cells. GA→NB sequence treatment had no influence on the effect of GA on tumor cells.7. NB was sensitive to STI571– resistant K562/G01 cells, the the IC50 values was 0.3490 mM. Conclusions: These studies demonstrate for the first time the activity of NB against CML in vitro and suggest that destruction of Hsp90 chaperon function, resulting in disruption the combination of Bcr-Abl with Hsp90 and cochaperons, may be an important mechanism of NB-mediated effects on CML.
|
PDF Full Text Request