In Vitro And In Vivo Study Of Nickel Pyrithione Overcome Imatinib Resistance In Chronic Myeloid Leukemia

Acquired Imatinib(IM)resistance is frequently characterized by Bcr-Abl mutations that affect IM binding and kinase inhibition in patients with chronic myelogenous leukemia(CML).Bcr-Abl T315 I mutation is the predominant mechanism of the acquired resistance to IM.Therefore it is urgent to search for additional approaches and targeting strategies to overcome IM-resistance.Nickel pyrithione(NiPT)potently inhibits the ubiquitin proteasome system via targeting the 19 S proteasome-associated deubiquitinases(UCHL5 and USP14),without effecting on the 20 S proteasome as we have recently reported.Here we further report that(i)NiPT not only induces apoptosis in Bcr-Abl wild-type and Bcr-Abl-T315 I mutation cells including the primary mononuclear cells from CML patients clinically resistant to IM,but also inhibits the growth of IM-resistant Bcr-Abl-T315 I xenografts in vivo;(ii)NiPT induces Bcr-Abl decrease through downregulation of Bcr-Abl mRNA and Bcr-Abl protein expression mediated by proteasome inhibition-induced caspase activation;(iii)19S proteasome-associated deubiquitinases inhibition is required for NiPT-induced caspase activation and apoptosis.These findings support that NiPT overcomes IM resistance through both Bcr-Abl-dependent and-independent mechanisms,providing great clinical significance for CML treatment.Here we studied the specific mechanism of NiPT for overcoming Imatinib-resistance,and carry out the following trials: 1.NiPT decreases viability of both Bcr-Abl wild type and Bcr-Abl T315 I cellsVarious human CML cell lines,including IM-sensitive Bcr-Abl wild type cell lines KBM5,BaF3-p210-WT and K562,as well as IM-resistant Bcr-Abl T315 I cell lines KBM5 R and BaF3-p210-T315 I,were treated with various concentrations of NiPT for 48 hours.A marked dose-dependent decrease in viability of all CML cell lines was observed in response to treatment with NiPT,with 50% inhibitory concentration(IC50)values of 0.14,0.17,0.3,0.16,and 0.98 ?M in KBM5,KBM5 R,BaF3-p210-WT,BaF3-p210-T315 I and K562 cells,respectively.The effect of NiPT on cell viability was further examined in CML cell lines by trypan blue exclusion staining assay.All CML cells were exposed to NiPT followed by trypan blue staining,a time-and dose-dependent inhibition of cell viability were observed,reflecting its antileukemia activity in CML cells.2.NiPT induces apoptosis in both Bcr-Abl-WT and Bcr-Abl-T315 I cellsWe next assessed the ability of NiPT in cell death induction in Bcr-Abl wild type and T315 I mutant cell lines.KBM5,KBM5 R,K562,BaF3-p210-WT and BaF3-p210-T315 I cells were exposed to different doses of NiPT,a time-dependent increasing proportion of cell death was observed by recording the number of Annexin V/PI positive cells under an inverted fluorescence microscope.The 24-hours time point images were shown and similar results were obtained by flow cytometry analysis,supporting that NiPT induced apoptotic cell death.3.NiPT-induced apoptosis is associated with caspase activation and decreased expression of anti-apoptotic proteins in CML cellsTo understand the mechanism of action underlying NiPT-induced CML cell death,both Bcr-Abl wild type and Bcr-Abl T315 I cell lines were treated with various concentrations of NiPT for different durations,followed by measurement of apoptosis-associated proteins.NiPT markedly increased the cleavage of PARP,a hallmark of apoptosis.In accordance with these findings,our data showed that NiPT activates caspase 3,caspase 8 and caspase 9 in both dose-and time-dependent manner.It is a widely accepted concept that mitochondrion is the regulating center of apoptosis.We next examined whether NiPT affects the integrity of mitochondrial membranes.Our result showed that the integrity of mitochondrial membranes was decreased in all CML cell lines after the treatment with NiPT.Release of cytochrome C and apoptosis induce factor(AIF)from mitochondrial to the cytoplasm has been recognized as the early signs of apoptosis.We therefore determined whether NiPT triggers the mitochondrial pathway.Cytochrome C and AIF release elevated at earlier time points,indicating that NiPT can activate the mitochondrial apoptosis pathway in CML cells.To further investigate the mechanism of NiPT-induced apoptosis,the expression of other apoptosis-related proteins was detected.Results showed that there was a marked decline of anti-apoptotic proteins Mcl-1,XIAP,Bcl-2 and survivin.4.NiPT-induced proteasome inhibition is associated with ER-stress and cell apoptosis in CML cellsIt is well established that inhibition of DUBs or the proteasome causes accumulation of ubiquitinated proteins.Like in other cancer cells,we previously reported,we found that NiPT dose-and time-dependently induced accumulation of ubiquitinated proteins(Ubs)and proteasome substrate protein p27 in all CML cell lines we detected before the emergence of PARP cleavage.Specifically,NiPT treatment did not alter the proteasome peptidases in KBM5 and KBM5 R cells either.The proteasome inhibitor bortezomib served as a positive control for chymotrypsin-like activity inhibitor.In accordance with our previous report that NiPT potently inhibits the ubiquitin-proteasome system via targeting the 19 S proteasome-associated DUBs,NiPT treatment was able to compete with HA-UbVS for binding USP14 of DUB lysate from KBM5 R cells.Furthermore,the phosphorylation of USP14(S241)was substantially reduced in NiPT-treated CML cells,also indicative of suppression of the DUB activity of USP14 by NiPT treatment.Accumulation of ubiquitylated proteins induces unfolded protein response(UPR)and apoptosis.Examination of the effect of NiPT on endoplasmic reticulum(ER)stress response showed that treatment of both KBM5 and KBM5 R cells with NiPT activates PERK-mediated ER stress signaling,evidenced by significant induction of p-eIF2 a.Together,these results suggest that NiPT-mediated proteasome inhibition plays an important role in NiPT-induced ER stress response and caspase activation in CML cells.5.NiPT downregulates Bcr-Abl protein and inhibits its downstream signalingWe also found that NiPT downregulated the total and phosphorylation levels of Bcr-Abl protein in both Bcr-Abl-WT and Bcr-Abl-T315 I cells in a dose-and time-dependent fashion.Further examination displayed that NiPT affected the expression of Bcr-Abl downstream target proteins.The phosphorylation of STAT5,Akt and Crkl was also significantly decreased in a dose-and time-dependent manner,occurring earlier than the down-regulation of their total forms.Interestingly,NiPT treatment also stimulated ERK phosphorylation,which may have something to do with the inhibition of the catalytic process of autophagy in leukemia cells,similar to the function of bortezomib reported previously.6.Bcr-Abl downregulation results from diminished gene expression and caspase-dependent cleavageTo address the mechanism underlying the NiPT-mediated Bcr-Abl protein downregulation in CML cells,we analyzed the expression of Bcr-Abl at the transcriptional level.KBM5 and KBM5 R cells were treated with increasing concentrations of NiPT for 6 hours.RT-PCR revealed the mRNA level of Bcr-Abl was decreased to some extent in both cells.The degree of mRNA reduction is apparently less than the reduction of the corresponding protein levels but the downregulation of mRNA by NiPT likely contributes to the decrease of the Bcr-Abl proteins.To further address this issue,RNA polymerase II(RNA pol II)was analyzed.The phosphorylation of RNA pol II was diminished to a certain extent in both KBM5 and KBM5 R cells by NiPT treatment.Together,NiPT leads to inhibition of RNA polymerase followed by downregulation of Bcr-Abl mRNA and protein levels,regardless of mutation status of the Bcr-Abl gene.We and others have reported that Bcr-Abl could be cleaved by caspase activation.To gain insight into the mechanism of NiPT-induced Bcr-Abl decline,we studied its dependence on the caspase activation.Specifically,we observed that pan-caspase inhibitor z-VAD-fmk mostly recovered NiPT-mediated cell death,decreases of Bcr-Abl as well as its downstream proteins to a certain extent but not ubiquitinated protein accumulation.These results demonstrate that caspase activation triggered by NiPT-induced proteasome inhibition is required for the downregulation of Bcr-Abl and its downstream events.7.Ex vivo effect of NiPT on primary monocytes from patients with CMLThe above results clearly indicated that NiPT-mediated proteasome inhibition and cytotoxicity is effective in both IM-sensitive and IM-resistant CML cells.We next evaluated the ex vivo anti-neoplastic effect of NiPT on bone marrow mononuclear cells from 12 patients with CML(4 patients are IM-resistant).NiPT decreased the cell viability of primary monocytes from CML patients with IC50 values around 0.58 to 0.82 ?M,while for the peripheral mononuclear cells from three healthy volunteers the IC50 values were more than 3.0 ?M as we reported recently.It was further found that NiPT treatment at doses from 0.25 to 1.0 ?M for 36 hours resulted in significant apoptosis in all the monocytes from 12 CML patients as detected by Annexin V/PI double staining.NiPT treatment also significantly induced the accumulation of ubiquitinated proteins and proteasome substrate protein I?B-?,reduced the levels of pro-caspase-3 and pro-caspase-9,Mcl-1,and induced PARP cleavage in the primary monocytes.These results are consistent with the in vitro inhibitory effect of Ni PT on both Bcr-Abl-WT and Bcr-Abl-T315 I cell lines,suggesting the potential use of NiPT for treatment of CML patients.8.NiPT inhibits the growth of Bcr-Abl-WT and-T315 I mutant xenografts in nude miceWe next evaluate the effect of NiPT in vivo using a nude mouse xenograft model.KBM5 and KMB5 R cells were inoculated subcutaneously in nude mice.A marked reduction in tumor growth was noted in NiPT-treated mice versus mice receiving vehicle alone while body weight remained relatively stable in each group.Protein levels of Bcr-Abl and its downstream targets Akt and STAT5 were significantly decreased in the NiPT-treated tumors while the phosphorylation of ERK was highly accumulated consistent with the in vitro effect of NiPT.The ubiquitinated proteins and proteasome substrates p27 were highly accumulated in NiPT-treated tumor tissues versus the control-treated models,indicating that NiPT inhibits proteasome function in both IM-sensitive and IM-resistant xenograft.The protein biomarkers related to proliferation,differentiation and adhesion,such as Ki67,CD11b/c and CXCR4 were also downregulated by the NiPT-treatment in both KBM5 and KBM5 R models.Together,these results demonstrate that NiPT inhibits xenografted Bcr-Abl-WT and Bcr-Abl-T315 I harboring cells in vivo.Conclusion: NiPT induces Bcr-Abl decrease in Bcr-Abl wild-type and Bcr-Abl-T315 I mutation cells through downregulation of Bcr-Abl transcription and Bcr-Abl protein cleavage mediated by proteasome inhibition-induced caspase activation.Meanwhile,19 S proteasome-associated DUB inhibition plays an important role in NiPT-induced caspase activation and apoptosis.We here propose an alternative strategy to overcome IM resistance by enhancing Bcr-Abl downregulation,which should have great clinical significance in IM-resistant cancer therapy.