Tripeptidyl Peptidase II Promotes Maturation of Caspase-1 in Shigella flexneri-Induced Macrophage Apoptosis

ABSTRACT The invasive enteropathogenic bacterium Shigella flexneri activates apoptosis in macrophages.Shigella-induced apoptosis requires caspase-1. We demonstrate here that tripeptidyl peptidase II (TPPII), a cytoplasmic, high-molecular-weight protease, participates in the apoptotic pathway triggered by Shigella. The TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) andclasto-lactacystin β-lactone (lactacystin), an inhibitor of both TPPII and the proteasome, protected macrophages fromShigella-induced apoptosis. AAF-cmk was more potent than lactacystin and irreversibly blocked Shigella-induced apoptosis by 95% at a concentration of 1 μM. Conversely, peptide aldehyde and peptide vinylsulfone proteasome inhibitors had little effect on Shigella-mediated cytotoxicity. Both AAF-cmk and lactacystin prevented the maturation of pro-caspase-1 and its substrate pro-interleukin 1β in Shigella-infected macrophages, indicating that TPPII is upstream of caspase-1. Neither of these compounds directly inhibited caspase-1. AAF-cmk and lactacystin did not impair macrophage phagocytosis or the ability of Shigellato escape the macrophage phagosome. TPPII was also found to be involved in apoptosis induced by ATP and the protein kinase inhibitor staurosporine. We propose that TPPII participates in apoptotic pathways.

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L-securinine inhibits cell growth and metastasis of human androgen-independent prostate cancer DU145 cells via regulating mitochondrial and AGTR1/MEK/ERK/STAT3/PAX2 apoptotic pathways

Bioscience Reports ◽

10.1042/bsr20190469 ◽

2019 ◽

Vol 39 (5) ◽

Cited By ~ 5

Author(s):

Dongxu Zhang ◽

Houxian Liu ◽

Binbin Yang ◽

Jiasheng Hu ◽

Yue Cheng

Keyword(s):

Prostate Cancer ◽

Protein Expression ◽

Annexin V ◽

Inhibitory Effect ◽

Apoptotic Pathway ◽

Growth Inhibitory ◽

Du145 Cells ◽

Apoptotic Pathways ◽

Induced Apoptosis ◽

Androgen Independent

Abstract The present study aims to evaluate the anticancer effect of L-securinine on androgen-independent prostate cancer (AIPC) DU145 cells. L-securinine (2.5, 5, and 10 μM) treatment for 24, 48 and 72 h displayed strong growth inhibitory effect on DU145 cells in a concentration and time-dependent fashion but has less toxicity toward normal androgen-dependent LNCaP cells. Hoechst 332582 staining of DU145 cells and Annexin V-FITC/ PI dual-labeling followed by flow cytometry assay identified that this growth inhibition by L-securinine would be due to the induction of apoptosis. Moreover Transwell assay revealed that L-securinine significantly inhibited the cell migration/invasion ability of DU145 cells. Furthermore, results of western blotting showed that the involvement of mitochondrial apoptotic pathway in the L-securinine-induced apoptosis of DU145 cell, as evidenced by an increase in the protein expression of Bax, cleaved caspase-9, cleaved caspase-3, cytosolic cytochrome c, and cleaved PARP, together with a unchanged cleaved caspase-8 and decreased Bcl-2 protein expression. Also, L-securinine-induced antimetastatic activity in DU145 cells was associated with decreased protein expression of MMP-2 and MMP-9 and concurrent reduction of VEGF. In addition, further studies revealed that L-securinine may inhibit the protein expression of AGTR1, p-MEK1/2, p-ERK1/2, p-STAT3, PAX2, and p-PAX2, while the expression of ERK1/2, MEK1/2, and STAT3 protein retains intact. These findings suggest that L-securinine may be a promising chemopreventive agent against AIPC.

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Apaf-1 and caspase-9 are required for cytokine withdrawal-induced apoptosis of mast cells but dispensable for their functional and clonogenic death

Blood ◽

10.1182/blood-2005-05-2160 ◽

2006 ◽

Vol 107 (5) ◽

pp. 1872-1877 ◽

Cited By ~ 21

Author(s):

Vanessa S. Marsden ◽

Thomas Kaufmann ◽

Lorraine A. O'Reilly ◽

Jerry M. Adams ◽

Andreas Strasser

Keyword(s):

Mast Cells ◽

Fetal Liver ◽

Wild Type ◽

Caspase 9 ◽

Receptor Stimulation ◽

Apoptotic Pathway ◽

Apoptotic Pathways ◽

Induced Apoptosis ◽

Cytokine Deprivation

Cytokines promote survival of mast cells by inhibiting apoptotic pathways regulated by the Bcl-2 protein family. We previously showed that lymphocyte apoptosis can proceed via a Bcl-2-inhibitable pathway independent of the canonical initiator caspase, caspase-9, and its adaptor, Apaf-1. Here we report that mast cells lacking caspase-9 or Apaf-1 are refractory to apoptosis after cytotoxic insults but still lose effector function and ability to proliferate. In response to cytokine deprivation or DNA damage, fetal liver-derived mast cells lacking Apaf-1 or caspase-9 failed to undergo apoptosis. Nevertheless, the cytokine-starved cells were not functionally alive, because, unlike those overexpressing Bcl-2, they could not degranulate on Fcϵ receptor stimulation or resume proliferation on re-addition of cytokine. Furthermore, mast cells lacking Apaf-1 or caspase-9 had no survival advantage over wild-type counterparts in vivo. These results indicate that the Apaf-1/caspase-9-independent apoptotic pathway observed in lymphocytes is ineffective in cytokine-deprived mast cells. However, although Apaf-1 and caspase-9 are essential for mast cell apoptosis, neither is required for the functional or clonogenic death of the cells, which may be due to mitochondrial dysfunction.

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Gabapentin is neuroprotective through glutamate receptor-independent mechanisms in staurosporine-induced apoptosis of cultured rat cerebellar neurons

Translational Neuroscience ◽

10.2478/s13380-013-0139-9 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 1

Author(s):

Bogdan Popescu ◽

Maria Ţuineag ◽

Radu Stoica

Keyword(s):

Glutamate Receptors ◽

Glutamate Receptor ◽

Ampa Receptors ◽

Kinase Inhibitor ◽

Neuronal Cell ◽

Morphological Aspect ◽

Cerebellar Granule Neuron ◽

Neuronal Cell Adhesion Molecule ◽

Apoptotic Pathways ◽

Induced Apoptosis

AbstractThe anticonvulsants that are currently available modulate the activity of neuronal receptors and ion channels, which are equally involved in apoptotic pathways. We investigated the hypothesis that gabapentin (GP), an anticonvulsant without effect on glutamate receptors acting as GABA analog, has neuroprotective properties. For comparison, we chose topiramate (TPM), which has been reported to be neuroprotective via AMPA receptors blockade. For this purpose, we used rat cerebellar granule neuron (CGN) cultures and we triggered apoptosis independent of glutamate receptors with staurosporine, a broad-spectrum protein kinase inhibitor. GP at therapeutic range concentration significantly increased cell viability in CGN cultures maintained in physiological KCl concentration and reversed apoptosis induced by staurosporine. Blockade of NMDA or AMPA receptors by MK801 or NBQX, respectively, did not alter GP neuroprotection, which was reversed instead by GABA. In contrast, protective effect of TPM on STS-treated CGN cultures was annihilated by NBQX, and not altered by MK801 or GABA. Treatments with neuroprotective concentrations of GP or TPM did not modify the expression of neuronal cell adhesion molecule or synaptophysin or the morphological aspect of neuronal endings. In summary, we report that GP is neuroprotective through glutamate-receptor independent mechanisms and without alteration of neuronal plasticity markers, which makes it a possible candidate for clinical neuroprotection trials.

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Proteasome Inhibiters Up-Regulate TRAIL/Apo2L and Its Receptors Significantly Contributing to Proteasome Inhibitor-Induced Apoptosis in Primary Chronic Lymphocytic Leukemia (CLL) Cells.

Blood ◽

10.1182/blood.v104.11.2810.2810 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2810-2810

Author(s):

Albert F. Kabore ◽

Kristin McCrea ◽

James B. Johnston ◽

Spencer B. Gibson

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Proteasome Inhibitor ◽

Substantial Reduction ◽

Proteasome Inhibitors ◽

Lymphocytic Leukemia ◽

Apoptotic Pathway ◽

Protein Levels ◽

Trail Receptors ◽

Induced Apoptosis ◽

Primary Chronic Lymphocytic Leukemia

Abstract The proteasome inhibitor, bortezomib has recently emerged as a new therapeutic treatment for refractory multiple myeloma and is presently being evaluated for other hematological malignancies either alone or in combination with other antitumor agents. Proteasome inhibitors cause the accumulation of many proteins but the precise mechanism responsible for their antitumor effect is unclear. In the present study, we have determined that cytotoxic effect the proteasome inhibitor MG-132 in primary chronic lymphocytic leukemia (CLL) cells is through the activation of the TRAIL (tumor necrosis factor-related apoptosis inducing ligand) apoptotic pathway. MG-132 induced apoptosis in approximately 70% of primary CLL cells as measured by annexin V staining. Addition of DR4:Fc that prevents TRAIL ligation with its receptors decreased the amount of MG-132 induced apoptosis by approximately 40% suggesting MG-132 caused activation of the TRAIL apoptotic pathway. MG-132 also up-regulated both the mRNA and protein levels of TRAIL and protein levels of TRAIL receptors DR4 and DR5. This upregulation correlated with activation of caspase 8 and cleavage of pro-apoptotic Bcl-2 family member Bid. Moreover, MG-132 treatment also induced a substantial reduction in the FLICE-like inhibitory protein (c-FLIP) protein levels. In contrast to CLL cells, proteasome inhibitors failed to activate the TRAIL apoptotic pathway in normal B-cells. This indicates that proteasome inhibitors are inducing apoptosis in primary CLL cells through activation of the TRAIL apoptotic signaling pathway through up-regulation of TRAIL and its cognate receptors and reduced FLIP expression. Thus, proteasome inhibitors may have a therapeutic role in CLL, either when used alone or in combination with TRAIL or antibodies against DR4/DR5.

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Arsenic Trioxide Induced p53-Dependant Apoptotic Signals in a Temperature Sensitive (Ts) p53 Mutant, BRK Cell Line.

Blood ◽

10.1182/blood.v104.11.4318.4318 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4318-4318

Author(s):

Cagla Akay ◽

Yair Gazitt

Keyword(s):

Arsenic Trioxide ◽

Mitochondrial Membrane ◽

Temperature Sensitive ◽

G1 Arrest ◽

Mutant P53 ◽

Apoptotic Pathway ◽

Apoptotic Pathways ◽

Induced Apoptosis ◽

Extrinsic Apoptotic Pathways ◽

In Cells

Abstract Mutations in the p53 gene are the most common genetic abnormality in human cancers and are the cause for drug resistance. Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia (APL) and is being tested in phase II studies in various types of cancers. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging the intrinsic apoptotic pathway in cells expressing w.t. p53 whereas in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. To further establish the differential effect of ATO in relation to the p53 status we studied the effect of temperature shift in temperature sensitive (Ts)-p53 expressing baby rat kidney (BRK) cells. We studied by Western immunoblotting the activation of the intrinsic and the extrinsic apoptotic pathways in ATO-induced apoptosis of BRK cells cultured at 32°C (w.t. p53 phenotype) and 37°C (mutant p53 phenotype). As expected, at 32°C, we observed a G1 arrest through activation of p21. We also observed depolarization of mitochondrial membrane; the release of cytochrome C and activation of caspase-9 and apoptosis as measured by Annexin V. We also observed release of SMAC at 32°C. In contrast, at 37°C we observed a G2/M arrest with no activation of p21 with activation of the extrinsic apoptotic pathway through early induction of TRAIL and TRAIL receptor- R2, activation of caspase-8, activation of BID, degradation of FLIP, rapid depolarization of mitochondrial membrane and release of AIF from mitochondria to the cytosol. We also demonstrate by flow cytometry and confocal imaging translocation of AIF to the nucleus in ATO-induced apoptosis at 37°C but not at 32°C. These results further substantiate our p53 model (Akay and Gazitt, Cell Cycle2:258–268, 2003).

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Induction of RKIP and Inhibition of the Transcription Repressor YY1 in B-NHL by the Novel Proteasome Inhibitor NPI-0052 and Sensitization to TRAIL-Induced Apoptosis.

Blood ◽

10.1182/blood.v108.11.2391.2391 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2391-2391

Author(s):

Eriko Suzuki ◽

Sara Huerta-Yepez ◽

Stavroula Baritaki ◽

Michael Palladino ◽

Benjamin Bonavida

Keyword(s):

Monoclonal Antibodies ◽

Tumor Cells ◽

Proteasome Inhibitor ◽

Kinase Inhibitor ◽

Proteasome Inhibitors ◽

Annexin V ◽

Underlying Mechanism ◽

Significant Inhibition ◽

The Novel ◽

Induced Apoptosis

Abstract Proteasome inhibitors have been shown to exert multiple effects including direct cytotoxic activity in sensitive cancer cells. In addition, proteasome inhibitors have recently been used therapeutically for certain cancers such as multiple myeloma and mantle cell lymphoma. We have recently reported that the novel proteasome inhibitor, NPI-0052 (Nereus Pharmaceuticals), can sensitize drug-resistant B-NHL tumor cells to apoptosis by various chemotherapeutic drugs. Also, NPI-0052 inhibits the NF-κB survival pathway and inhibition of NF-κB sensitizes tumor cells to TRAIL-induced apoptosis. We hypothesized that inhibition of NF-κB by NPI-0052 may also lead to sensitization of TRAIL-resistant B-NHL cells to TRAIL-mediated apoptosis. Human Burkitt’s lymphoma,Ramos cells, were treated with various concentrations of NPI-0052 (0–10ng/ml for 24h) and were then treated with recombinant human TRAIL (0–100ng/ml for 24h). The cells were examined for apoptosis by Annexin V/PI. The combination treatment resulted in significant potentiation of cytotoxicity and synergy in apoptosis was achieved. We have then examined a potential underlying mechanism of NPI-0052-mediated sensitization. The transcription repressor YY1 was recently shown to negatively regulate the transcription of the TRAIL death receptor DR5 and thus overexpression of YY1 is a resistant factor for TRAIL-induced apoptosis. Hence, treatment of Ramos cells with NPI-0052 resulted in significant inhibition of YY1 expression and upregulation of surface DR5 expression. We have also examined the effect of NPI-0052 on RKIP (Raf-kinase inhibitor protein) (Yeung et al., Molecular Cellular Biology; 21:7207, 2001) expression. Since both NPI-0052 and RKIP inhibit the NF-κB pathway, we hypothesized that treatment of Ramos cells with NPI-0052 may result in significant upregulation of RKIP expression. The findings, indeed, demonstrate that NPI-0052 significantly induced RKIP expression in Ramos cells. Overall, the findings show, for the first time, that NPI-0052 sensitizes B-NHL tumor cells to TRAIL-mediated apoptosis. Sensitization by NPI-0052 was correlated with both the induction of RKIP expression and inhibition of YY1 and upregulation of DR5 expression. Currently, TRAIL or agonist monoclonal antibodies against DR4 or DR5 are being examined clinically for anti-tumor activity. Therefore, our findings suggest the potential application of the combination of NPI-0052 and TRAIL or agonistic monoclonal antibodies against DR4 or DR5 in the treatment of tumor cells that are resistant to drugs and TRAIL.

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Molecular Mechanism by Which Rituximab Sensitizes B-NHL Cells to TRAIL Apoptosis: Induction of RKIP and DR5 Expression Via Inhibition of NF-Kb, Snail, and YY1.

Blood ◽

10.1182/blood.v112.11.1596.1596 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1596-1596

Author(s):

Mario I. Vega ◽

Melisa Martinez-Paniagua ◽

Sara Huerta-Yepez ◽

Yeung Kam ◽

Stavroula Baritaki ◽

...

Keyword(s):

Tumor Cells ◽

Cell Lines ◽

Kinase Inhibitor ◽

Proteasome Inhibitors ◽

Underlying Mechanism ◽

Inhibitor Protein ◽

Rt Pcr ◽

Yin Yang 1 ◽

Induced Apoptosis

Abstract There have been significant advances in the treatment of patients with B-NHL using combination of rituximab and CHOP. However, a subset of patients does not initially respond or develop resistance to further treatments; hence, the need for alternative therapies to overcome resistance. TRAIL and agonist DR4/DR5 monoclonal antibodies have been examined clinically against a variety of tumors in Phase I/II. However, the majority of B-NHL derived from patients and cell lines are resistant to TRAIL-induced apoptosis. Recent findings demonstrated that treatment of TRAIL-resistant-B-NHL with rituximab sensitizes the tumor cells to TRAIL apoptosis. The underlying mechanism of rituximab-induced sensitization to TRAIL, however, is not clear. We have recently reported that treatment of tumor cells with sensitizing agents (example CDDP, proteasome inhibitors) resulted in the reversal of resistance to TRAIL via induction of Raf-1 kinase inhibitor protein (RKIP) and demonstrated the pivotal role of RKIP in the regulation of tumor cell sensitivity to TRAIL. Hence, since rituximab induces the expression of RKIP in B-NHL, we determined the role of RKIP induction by rituximab in the sensitization of B-NHL to TRAIL apoptosis. Various B-NHL cell lines were used as models for study. Treatment of B-NHL cells with rituximab (20 ng/ml) and TRAIL (5–10 ng/ml) resulted in significant potentiation of apoptosis and synergy was achieved. Rituximab induced the expression of RKIP as determined by RT-PCR and western concomitantly with inhibition of NF-kB. The inhibition of NF-kB resulted in upregulation of RKIP expression and was mediated, in large part, by inhibition of the transcription repressor Snail (downstream of NF-kB). Further, RKIP-induced inhibition of NF-kB by rituximab resulted in downstream inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) and concomitantly with the upregulation of DR5 expression. The role of RKIP induction by rituximab in the upregulation of DR5 and sensitization to TRAIL apoptosis was corroborated by the use of cells over expressing RKIP which were sensitive to TRAIL apoptosis in the absence of rituximab. Our findings reveal a novel mechanism of rituximab-induced sensitization of B-NHL to TRAIL apoptosis via inhibition of NF-kB and Snail and upregulation of RKIP and DR-5. The combination of rituximab and TRAIL may be effective in the treatment of B-NHL. Further, our studies suggest that agents other than rituximab that can induce RKIP can reverse resistance to TRAIL in B-NHL that are unresponsive to rituximab treatment.

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Apoptosis Triggered by Myc-Induced Suppression of Bcl-XL or Bcl-2 Is Bypassed during Lymphomagenesis

Molecular and Cellular Biology ◽

10.1128/mcb.21.15.5063-5070.2001 ◽

2001 ◽

Vol 21 (15) ◽

pp. 5063-5070 ◽

Cited By ~ 135

Author(s):

Christine M. Eischen ◽

David Woo ◽

Martine F. Roussel ◽

John L. Cleveland

Keyword(s):

Protein Synthesis ◽

Transgenic Mice ◽

Tumor Cells ◽

De Novo ◽

Apoptotic Pathway ◽

Antiapoptotic Protein ◽

Protein Levels ◽

Apoptotic Pathways ◽

Induced Apoptosis ◽

De Novo Protein Synthesis

ABSTRACT Enforced Bcl-2 expression inhibits Myc-induced apoptosis and cooperates with Myc in transformation. Here we report that the synergy between Bcl-2 and Myc in transforming hematopoietic cells in fact reflects a Myc-induced pathway that selectively suppresses the expression of the Bcl-XL or Bcl-2 antiapoptotic protein. Myc activation suppresses Bcl-XL RNA and protein levels in cultures of primary myeloid and lymphoid progenitors, and Bcl-XL and Bcl-2 expression is inhibited by Myc in precancerous B cells from Eμ-myc transgenic mice. The suppression of bcl-X RNA levels by Myc requires de novo protein synthesis, indicating that repression is indirect. Importantly, the suppression of Bcl-2 or Bcl-XL by Myc is corrupted during Myc-induced tumorigenesis, as Bcl-2 and/or Bcl-XLlevels are markedly elevated in over one-half of all lymphomas arising in Eμ-myc transgenic mice. Bcl-2 and/or Bcl-XL overexpression did not correlate with loss of ARF or p53 function in tumor cells, indicating that these two apoptotic pathways are inactivated independently. Therefore, the suppression of Bcl-XL or Bcl-2 expression represents a physiological Myc-induced apoptotic pathway that is frequently bypassed during lymphomagenesis.

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FLT3-ITD Confers Resistance to Bortezomib By Protecting the mTOR/4EBP1 Pathway through Activation of STAT5 and Induction of Pim-1 Expression

Blood ◽

10.1182/blood.v126.23.1270.1270 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1270-1270

Author(s):

Ayako Nogami ◽

Keigo Okada ◽

Gaku Oshikawa ◽

Shinya Ishida ◽

Hiroki Akiyama ◽

...

Keyword(s):

Molecular Mechanisms ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Expression Level ◽

Apoptotic Pathway ◽

Expression Levels ◽

Combined Use ◽

Significant Difference ◽

Induced Apoptosis ◽

Novel Therapeutic Strategies

Abstract FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in AML, with the former strongly associated with a poor prognosis. We have recently revealed that FLT3-ITD confers resistance to the PI3K/AKT pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in AML. The proteasomal inhibitor bortezomib (BZM) has recently been reported as a promising agent for treatment of AML. We examine here the molecular mechanisms involved in induction of apoptosis by BZM in hematopoietic cells, including AML cells, and evaluate the effects of FLT3-ITD and TKD on these mechanisms and on the sensitivity of cells to BZM-induced apoptosis. We first comparatively examined the effect of BZM on survival of hematopoietic 32D cells and human leukemic UT7 cells driven by FLT3-ITD (32D/ITD and UT7/ITD) or FLT3-D835Y (32D/TKD and UT7/TKD). BZM induced activation of Bax, decline in mitochondrial membrane potential, and activation of caspase-9, thus leading to apoptosis, more conspicuously in cells driven by FLT3-ITD than FLT3-TKD. These results indicate that FLT3-ITD, as compared with FLT3-TKD, conferred resistance to apoptosis induced by BZM through the intrinsic pathway in these cells. To elucidate the molecular mechanisms involved in BZM-induced apoptosis in these cells, we next examined the effect of BZM on expression levels of FLT3 in these cells as well as in human leukemic MV4-11 cells expressing FLT3-ITD. Intriguingly, treatment of these cells with BZM conspicuously reduced expression levels of FLT3. It was further reveled that BZM further facilitated the decline in FLT3-ITD expression in MV4-11 cells treated with actinomycin D to shut down the transcriptional activity. Furthermore, BZM significantly retarded the recovery of FLT3-ITD expression in MV4-11 cells washed out from the translation inhibitor cycloheximide. These results suggest that BZM may downregulate the FLT3-ITD expression mainly at the translational level. However, we did not observe any significant difference in extent of the BZM-induced decline in expression levels between FLT3-ITD and FLT3-TKD. We next examined the effect of BZM on the mTOR/4EBP1 pathway, which we have shown to play important roles in regulation of apoptosis downstream of FLT3-ITD. It was found that BZM downregulated this pathway more significantly in 32D/TKD cells than in 32D/ITD cells. Because we have also previously found that STAT5 activated robustly by FLT3-ITD plays an important role in modulation of the mTOR/4EBP1 pathway, we examined the effects of BZM on 32D/TKD cells forced to express the constitutively activated STAT5 mutant, STAT5A1*6. As expected, STAT5A1*6 conferred resistance to BZM-induced downregulation of the mTOR/4EBP1 pathway as well as apoptosis in 32D/TKD cells. Consistent with this, the STAT5 inhibitor pimozide, clinically in use for neuropsychiatric disorders, abrogated the resistance of 32D/ITD, UT7/ITD, and MV4-11 cells to BZM-induced inhibition of the mTOR/4EBP1 pathway and apoptosis. We finally examined the possible involvement of the STAT5 target gene product Pim-1 in acquisition of resistance to BZM by cells expressing FLT3-ITD. We fist confirmed that Pim-1 was expressed at a higher level in 32D/ITD cells than in 32D/TKD cells and that STAT5A1*6 increased the expression level of Pim-1 in 32D/TKD cells. We then examined the effects of a specific Pim kinase inhibitor, AZD-1208, and found that it synergistically downregulated the mTOR/4EBP1 pathway and induced apoptosis with BZM in 32D/ITD cells as well as 32D/TKD cells expressing STAT5A1*6. We also examined the effects of a BET bromodomain inhibitor, JQ1, which has recently been shown to inhibit the STAT5 activity and to reduce specifically the expression level of Pim-1 as well as c-Myc in MV4-11 cells. As expected, pretreatment of 32D/ITD or MV4-11 cells with JQ1 conspicuously sensitized these cells to BZM-induced apoptosis. These results suggest that BZM downregulates FLT3 expression and the mTOR/4EBP1 pathway to activate the intrinsic apoptotic pathway and that robust STAT5 activation by FLT3-ITD confers resistance to BZM on AML cells through protection of the mTOR/4EBP1 pathway at least partly by inducing Pim-1 expression. The present study may contribute to development of novel therapeutic strategies against FLT3-ITD-positive AML by combined use of BZM and the STAT5/Pim-1 pathway inhibitors. Disclosures No relevant conflicts of interest to declare.

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