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value of 0.05 was considered statistically significant. Results NiPT decreases viability of both Bcr-Abl wild-type and Bcr-Abl-T315I cells Numerous 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-T315I cell lines KBM5R and BaF3-p210-T315I, were treated with numerous concentrations of NiPT for 48?h. study, we investigated the effect of NiPT, a novel proteasomal deubiquitinase inhibitor, on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl. Methods Cell viability was examined by MTS assay and trypan blue exclusion staining assay in KBM5, KBM5R, K562, BaF3-p210-WT, BaF3-p210-T315I cells, and CML patients bone marrow samples treated with NiPT. Cell apoptosis in CML cells was detected with Annexin V-FITC/PI and rhodamine-123 staining followed by fluorescence microscopy and circulation cytometry and with western blot analyses for apoptosis-associated proteins. Expression levels of Bcr-Abl in CML cells were analyzed by using western blotting and real-time PCR. The 20S proteasome peptidase activity was measured using specific fluorogenic substrate. Active-site-directed labeling of proteasomal DUBs, as well as the phosphorylation of USP14 was utilized for evaluating the inhibition of the DUBs activity by NiPT. Mouse xenograft models of KBM5 and KBM5R cells were analyzed, and Bcr-Abl-related proteins and protein biomarkers related to proliferation, differentiation, and adhesion in tumor tissues were detected by western blots and/or immunohistological analyses. Results NiPT induced apoptosis in CML cells and inhibited the growth of IM-resistant Bcr-Abl-T315I xenografts in nude mice. Mechanistically, NiPT induced decreases in Bcr-Abl proteins, which were associated with downregulation of Bcr-Abl transcription and with the cleavage of Bcr-Abl protein by activated caspases. NiPT-induced ubiquitin proteasome system inhibition induced caspase activation in both IM-resistant and IM-sensitive CML cells, and the caspase activation was required for NiPT-induced Bcr-Abl downregulation and apoptotic cell death. Conclusions These findings support that NiPT can overcome IM resistance through both Bcr-Abl-dependent and Bcr-Abl-independent mechanisms, providing potentially a new option for CML treatment. is the smallest diameter and is the diameter perpendicular to test was used to compare the differences between variables. value of 0.05 was considered statistically significant. Results NiPT decreases viability of both Bcr-Abl wild-type and Bcr-Abl-T315I cells Numerous CML cell lines, including IM-sensitive Bcr-Abl wild-type cell NECA lines KBM5, BaF3-p210-WT, and K562, as well as IM-resistant Bcr-Abl-T315I cell lines KBM5R and BaF3-p210-T315I, were treated with numerous concentrations of NiPT for 48?h. A marked dose-dependent decrease in viability of all CML cell lines was observed in response to treatment with NiPT (Fig.?1a), with 50% inhibitory concentration (IC50) values of 0.14, 0.17, 0.3, 0.16, and 0.98?M in KBM5, KBM5R, BaF3-p210-WT, BaF3-p210-T315I, and K562 cells, respectively. Open in a separate windows Fig. 1 NiPT inhibits cell viability in CML cell lines. a, b NiPT decreases the NECA viability of both IM-sensitive and IM-resistant CML cell lines. KBM5, KBM5R, K562, BaF3-p210-WT, and BaF3-p210-T315I cells were exposed to NiPT in various concentrations for 48?h, and then were subject to MTS assay. Cell viability was also examined by trypan blue exclusion staining assay. All CML cells were exposed to NiPT followed by trypan blue staining. symbolize data from three repeats. Mean??SD (either inhibiting Bcr-Abl expression or interfering other mechanisms [28, 29, 32], but the mechanism is far from being fully understood. Recently, we have reported that NiPT is able to inhibit the activity of 19S proteasome-associated DUBs USP14 and UCHL5 but not the 20S proteasome [27]. We confirmed that NiPT induces cell apoptosis and overcomes IM-resistance in CML cells through both Bcr-Abl-dependent and Bcr-Abl-independent mechanisms. On the one hand, NiPT inhibits the transcription of the Bcr-Abl gene, and future studies need to investigate the possibility that NiPT activates caspases which cleaves RNA pol II leading to decrease of Bcr-Abl mRNA. Around the other, NiPT-induced caspase activation cleaves Bcr-Abl (Fig.?4cCf), thus leading to Bcr-Abl downregulation and cell proliferation inhibition. Here, we proposed a novel pathway that NiPT-mediated UPS inhibition and caspase activation are responsible for the downregulation of Bcr-Abl protein, which contributes to overcoming IM-resistance by NiPT. Like in other cancer cells, NiPT induced UPS inhibition in both Bcr-Abl wild-type and T315I cell lines, as well as in main mononuclear malignancy cells derived from CML patients including IM-resistant or IM-sensitive in vitro. It was further confirmed that NiPT also inhibited USP function in. These findings suggest for the first time that NiPT may have clinical benefit for patients with CML, particularly in those suffering from imatinib-resistance and the recurrent forms of this disease, providing great importance in the future clinical CML therapy. Notably, we previously reported the anti-tumor effects of NiPT in other types of tumor models, such Pde2a as lung malignancy (A549), hepatocarcinoma (SMMC-7721), and myeloma (U266) [27]. Annexin V-FITC/PI and rhodamine-123 staining followed by fluorescence microscopy and circulation cytometry and with western blot analyses for apoptosis-associated proteins. Expression levels of Bcr-Abl in CML cells were analyzed by using western blotting and real-time PCR. The 20S proteasome peptidase activity NECA was measured using specific fluorogenic substrate. Active-site-directed labeling of proteasomal DUBs, as well as the phosphorylation of USP14 was utilized for evaluating the inhibition of the DUBs activity by NiPT. Mouse xenograft models of KBM5 and KBM5R cells were analyzed, and Bcr-Abl-related proteins and protein biomarkers related to proliferation, differentiation, and adhesion in tumor tissues were detected by western blots and/or immunohistological analyses. Results NiPT induced apoptosis in CML cells and inhibited the growth of IM-resistant Bcr-Abl-T315I xenografts in nude mice. Mechanistically, NiPT induced decreases in Bcr-Abl proteins, which were associated with downregulation of Bcr-Abl transcription and with the cleavage of Bcr-Abl protein by activated caspases. NiPT-induced ubiquitin proteasome system inhibition induced caspase activation in both IM-resistant and IM-sensitive CML cells, and the caspase activation was required for NiPT-induced Bcr-Abl downregulation and apoptotic cell death. Conclusions These findings support that NiPT can overcome IM resistance through both Bcr-Abl-dependent and Bcr-Abl-independent mechanisms, providing potentially a new option for CML treatment. is the smallest diameter and is the diameter perpendicular to test was used to compare the differences between variables. value of 0.05 was considered statistically significant. Results NiPT decreases viability of both Bcr-Abl wild-type and Bcr-Abl-T315I cells Various 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-T315I cell lines KBM5R and BaF3-p210-T315I, were treated with various concentrations of NiPT for 48?h. A marked dose-dependent decrease in viability of all CML cell lines was observed in response to treatment with NiPT (Fig.?1a), with 50% inhibitory concentration (IC50) values of 0.14, 0.17, 0.3, 0.16, and 0.98?M in KBM5, KBM5R, BaF3-p210-WT, BaF3-p210-T315I, and K562 cells, respectively. Open in a separate window Fig. 1 NiPT inhibits cell viability in CML cell lines. a, b NiPT decreases the viability of both IM-sensitive and IM-resistant CML cell lines. KBM5, KBM5R, K562, BaF3-p210-WT, and BaF3-p210-T315I cells were exposed to NiPT in various concentrations for 48?h, and then were subject to MTS assay. Cell viability was also examined by trypan blue exclusion staining assay. All CML cells were exposed to NiPT followed by trypan blue staining. represent data from three repeats. Mean??SD (either inhibiting Bcr-Abl expression or interfering other mechanisms [28, 29, 32], but the mechanism is far from being fully understood. Recently, we have reported that NiPT is able to inhibit the activity of 19S proteasome-associated DUBs USP14 and UCHL5 but not the 20S proteasome [27]. We confirmed that NiPT induces cell apoptosis and overcomes IM-resistance in CML cells through both Bcr-Abl-dependent and Bcr-Abl-independent mechanisms. On the one hand, NiPT inhibits the transcription of the Bcr-Abl gene, and future studies need to investigate the possibility that NiPT activates caspases which cleaves RNA pol II leading to decrease of Bcr-Abl mRNA. On the other, NiPT-induced caspase activation cleaves Bcr-Abl (Fig.?4cCf), thus leading to Bcr-Abl downregulation and cell proliferation inhibition. Here, we proposed a novel pathway that NiPT-mediated UPS inhibition and caspase activation are responsible for the downregulation of Bcr-Abl protein, which contributes to overcoming IM-resistance by NiPT. Like in other cancer cells, NiPT induced UPS inhibition in both Bcr-Abl wild-type and T315I cell lines, as well as in primary mononuclear cancer cells derived from CML patients including IM-resistant or IM-sensitive in vitro. It was further confirmed that NiPT also inhibited USP function in the xenografted tumor model bearing wild-type- and T315I-Bcr-Abl genes in vivo. We have reported that proteasome inhibition induced ER-stress response plays an important role in caspase activation and cell apoptosis [42]. The induced ER-stress response accumulates Ca2+ in cytoplasm to alter mitochondrial membrane permeability, leading to the release of cytochrome C and AIF, followed by caspase activation and apoptosis. Here, we also found that NiPT time-dependently induced the ER-stress response pathway, while the release of cytochrome C and AIF was induced and the anti-apoptotic proteins like Bcl-2, XIAP, and Mcl-1 were dramatically decreased. The released apoptotic factors bind with caspase-9 forming a protein complex, inducing caspase activation (Fig.?2). After caspase activation, on the one hand, it induced PARP cleavage and apoptosis; on the other hand, it directly cleaved Bcr-Abl protein which has been confirmed in our work (Fig.?4e, ?,ff). Bcr-Abl is a.