scholarly journals Inhibition of WIP1 Phosphatase in Multiple Myeloma Overcomes Bortezomib Resistance and Promotes Cell Death Via ER Stress-Induced Apoptotic JNK/c-Jun Signaling and Downregulation of Inhibitors of Apoptosis Proteins (IAPs)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1366-1366
Author(s):  
Katia Beider ◽  
Evgenia Rosenberg ◽  
Valeria Voevoda ◽  
Hanna Bitner ◽  
Yaarit Sirovsky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Stress Response ◽  
Er Stress ◽  
Cell Lines ◽  
Potential Role ◽  
Cyclin B1 ◽  
Parp Cleavage ◽  
Induced Apoptosis

Abstract Acquired or de novo resistance to the traditional and novel anti-multiple myeloma (MM) agents remains a major treatment obstacle, therefore novel therapies are in need. Wild-type p53-induced phosphatase 1 (WIP1) is an oncogenic serine/threonine phosphatase implicated in silencing of cellular responses to genotoxic stress. WIP1 overexpression was documented in various solid cancers in correlation with aggressive features and poor prognosis. Thus, we studied WIP1 in MM addressing its potential role in mediating resistance and aggressive phenotype. Increased expression of WIP1 was detected in MM cell lines (n=8) and primary samples (n=18) at both mRNA and protein level as compared with normal PBMCs (n=5). Furthermore, a positive correlation between WIP1 and CXCR4 levels (p<0.02, R2=0.5) was revealed. The latter is a well-known oncogenic receptor in MM. WIP1 expression levels were significantly up-regulated following bortezomib (Bort) treatment. Using MM cell lines with acquired resistance to Bort (RPMI8226BortRes and CAGBortRes), a higher induction of WIP1 upon Bort exposure could be demonstrated, suggesting a possible role for WIP1 in the acquisition of MM drug resistance to proteasome inhibitors. WIP1 was also upregulated in MM cells cultured on human BM stroma (BMSC) known to protect the tumor cells from Bort-induced apoptosis, further supporting its function in mediating resistance. GSK2830371 (GSK), a novel allosteric inhibitor of WIP1, significantly suppressed MM cells proliferation (p<0.01) and induced apoptosis, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization (ψm), caspase 3 and PARP cleavage, and DNA fragmentation. Moreover, combined treatment with GSK and Bort synergistically potentiated cell death in both Bort-sensitive and resistant MM cells and overcame BMSC protection (CI<0.5). The robust apoptosis induced by Bort/GSK treatment was accompanied by increased mitochondrial ROS accumulation, subsequent mitochondrial destabilization and extensive DNA damage. GSK treatment resulted in a reduction of WIP1 basal expression and abrogated WIP1 induction upon Bort treatment. Thus, we defined that GSK can regulate WIP1 expression in MM cells. To determine the molecular mechanism of Bort/GSK synergism we performed gene and protein expression analysis. Combination of both agents significantly reduced expression of anti-apoptotic proteins such as cIAP1, cIAP2, XIAP and Survivin. Previous studies indicate that maintaining IAPs expression is part of an adaptive unfolded protein response that promotes MM survival upon Bort-induced endoplasmic reticulum (ER) stress. Therefore, it is conceivable that targeting IAPs upon WIP1 inhibition may overcome protective responses, inducing unresolved ER stress and MM cell death. Indeed, we found that combination of Bort and GSK significantly enhanced ER stress, as indicated by increase in the pro-apoptotic factors ATF4, CHOP and GADD34. Concomitantly, mitosis-inducing factors Cyclin B1, CDK1 and PLK1 were prominently reduced upon Bort/GSK treatment. To assess the potential role of p53 activation in GSK-mediated effects, p53-stabilizing agents nutlin3a and PRIMA1 were applied in combination with WIP1 inhibition. We observed a significant (p<0.01) increase in the responsiveness of both p53WT and p53mut MM cells to GSK-mediated apoptosis. Consistently, combined GSK/Bort treatment upregulated p53 targets, including PUMA, NOXA, GADD45A and p21 genes. These data suggest that p53 may potentiate the WIP1 inhibition mediated stress induction. Finally, we assessed the signaling pathways that may be involved in WIP1 mediated cessation of stress response. GSK profoundly augmented Bort-induced phosphorylation of JNK and c-Jun, without affecting p38 phosphorylation. Accordingly, JNK inhibitor SP600125 successfully reverted both the apoptosis and the downregulation of IAPs induced by Bort/GSK treatment. Altogether, these results identify pro-apoptotic JNK/c-Jun signaling being preferential target of WIP1 in the process of dampening Bort-induced stress response. To conclude, we disclose the role of WIP1 in blunting stress response and promoting resistance to bortezomib. Collectively, WIP1 suppression prevents MM cell adaptation and recovery upon ER stress. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. Disclosures Peled: Cellect Biotherapeutics Ltd: Consultancy.

Targeting Proteinkinase C Alters ER-Stress and b-Catenin Signaling in Multiple Myeloma: Therapeutic Implications.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 258-258
Author(s):  
Marc S. Raab ◽  
Klaus Podar ◽  
Jing Zhang ◽  
Giovanni Tonon ◽  
Johannes H. Fruehauf ◽  
...  
Keyword(s):  
Cell Death ◽  
Stress Response ◽  
Growth Inhibition ◽  
Er Stress ◽  
Cell Lines ◽  
Wnt Pathway ◽  
Dependent Manner ◽  
P53 Family ◽  
Er Stress Response

Abstract We have previously shown that the novel orally available small molecule inhibitor of PKC enzastaurin (Eli Lilly and Company) inhibits MM cell growth, survival and angiogenesis both in vitro and in vivo. To date, however, the downstream effects contributing to growth inhibition and cell death remain to be determined. Here, we performed global gene expression profiling on enzastaurin treated MM cells and identified 200 Genes to be differentially regulated with a &gt; 2-fold cut off. Strikingly, two major groups of up-regulated probe sets were associated with either of two pathways - endoplasmatic reticulum (ER)-stress response or WNT-signaling. Importantly, MM cells, producing high levels of paraprotein, are highly susceptible to perturbation of ER function and protein folding. Moreover, PKC isoforms have been reported to directly regulate the canonical WNT pathway via phosphorylation of b-catenin (CAT), leading to its ubiquination and proteasomal degradation. Specifically, we fist evaluated the role of enzastaurin in mediating ER-stress in MM cells. The transcriptional up-regulation of genes involved in ER-stress (GADD153/CHOP, GADD34, ATF3), triggered by enzastaurin at 3h, was confirmed by western blot analysis, accompanied by induction of the molecular ER chaperone BiP/grp78, phosphorylation of eIF2a consistent with PERK activation, and up-regulation of p21. These events were preceded by an early (1h) increase of intracellular calcium levels, a hallmark of ER-stress, assessed by FLUO4 staining. These data suggest an important role of ER-stress response in the early growth inhibition of MM cells caused by enzastaurin. Second, we delineated effects of enzastaurin on WNT pathway in MM and other tumor cell lines. Upon enzastaurin treatment, CAT was dephosphorylated at Ser33, 37, 41 in a dose- and time-dependent manner in all cell lines tested (10 MM, 3 colon cancer, HeLa, as well as human embryonic kidney 293 cells). Consequently, accumulation of CAT occurred in both cytosolic and nuclear fractions of treated MM cells, associated with activated TOPflash LUC-reporter system, confirming nuclear transactivating activity. Specific inhibition of CAT by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a CAT-dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; CAT induction by enzastaurin led to p73 (but not p53) activation and was also abrogated by CAT-specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of CAT in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. Our studies therefore indicate that ER-stress response contributes to the immediate inhibition of proliferation by enzastaurin, followed by CAT accumulation leading to p73 activation, contributing to enzastaurin-mediated cell death. These findings provide a novel link between CAT and p53-family members. Moreover p73, which is only rarely mutated in human cancers, represents a novel therapeutic target in MM.

scholarly journals Activation of the Integrated Stress Response and ER Stress Protect from Fluorizoline-Induced Apoptosis in HEK293T and U2OS Cell Lines

2021 ◽  
Vol 22 (11) ◽  
pp. 6117
Author(s):  
José Saura-Esteller ◽  
Ismael Sánchez-Vera ◽  
Sonia Núñez-Vázquez ◽  
Ana M. Cosialls ◽  
Pau Gama-Pérez ◽  
...  
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Cell Lines ◽  
Integrated Stress Response ◽  
Protein Levels ◽  
Er Stress Response ◽  
Increased Sensitivity ◽  
U2os Cells ◽  
Induced Apoptosis

The prohibitin (PHB)-binding compound fluorizoline as well as PHB-downregulation activate the integrated stress response (ISR) in HEK293T and U2OS human cell lines. This activation is denoted by phosphorylation of eIF2α and increases in ATF4, ATF3, and CHOP protein levels. The blockage of the activation of the ISR by overexpression of GRP78, as well as an increase in IRE1 activity, indicate the presence of ER stress after fluorizoline treatment. The inhibition of the ER stress response in HEK293T and U2OS led to increased sensitivity to fluorizoline-induced apoptosis, indicating a pro-survival role of this pathway after fluorizoline treatment in these cell lines. Fluorizoline induced an increase in calcium concentration in the cytosol and the mitochondria. Finally, two different calcium chelators reduced fluorizoline-induced apoptosis in U2OS cells. Thus, we have found that fluorizoline causes increased ER stress and activation of the integrated stress response, which in HEK293T and U2OS cells are protective against fluorizoline-induced apoptosis.

VEGF Upregulates Mcl-1 Expression and Protects Multiple Myeloma Cells Against Starvation Induced-Apoptosis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 631-631
Author(s):  
Steven Le Gouill ◽  
Klaus Podar ◽  
Martine Amiot ◽  
Teru Hideshima ◽  
Dharminder Chauhan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Myeloid Cell ◽  
Fibroblast Cell ◽  
Transfected Cells ◽  
Murine Embryonic Fibroblast ◽  
Induced Apoptosis

Abstract Vascular endothelial growth factor (VEGF) induces proliferation of MM cells and induces interleukin-6 (IL-6) secretion in a paracrine loop involving MM cells and bone marrow stromal cells. In turn, IL-6 triggers multiple myeloma (MM) cell proliferation and also protects against apoptosis by upregulating Myeloid-cell-leukemia 1 (Mcl-1), a critical survival protein in MM cells. The goal of our study was to investigate the role of Mcl-1 in VEGF induced-proliferation and protection against apoptosis. Using two murine embryonic fibroblast cell lines as a model (a Mcl-1 deleted cell line and its wild type: Mcl-1Δ/null and Mcl-1wt/wt MEFs, respectively), we here demonstrate that deletion of Mcl-1 reduces fetal bovine serum (FBS), VEGF, and IL-6 induced-proliferation. In addition, we demonstrate that the percentage of cells in S phase is lower in Mcl-1Δ/null compared to Mcl-1wt/wt MEFs (21% (+/−1) versus 30% (+/− 3), respectively). Taken together, these results demonstrate that Mcl-1 is required to mediate VEGF, Il-6 and FBS-induced-proliferation and cell cycle progression. To highlight the key anti-apoptotic role of Mcl-1 in MM cells, humans MM1s cells were transfected with Mcl-1 siRNA. Specific inhibition of Mcl-1 was associated with decreased proliferation (42% and 61% decreases at 24 and 48 h, respectively) and induction of apoptosis (subG1 peak: 22% and 41% in Mcl-1 siRNA transfected cells versus 15% and 15 % in non-transfected cells at 24 and 48 h, respectively), confirming that Mcl-1 is critical for both proliferation and protection against apoptosis in MM cells. In 3 human MM cell lines (MM1s, U266 and MM1R) and MM patient cells we next showed that Mcl-1 protein expression, but not other bcl-2 family members, is upregulated by VEGF in a time and dose manner; and conversely that the pan-VEGF inhibitor GW654652, blocks VEGF induced-upregulation of Mcl-1. Furthermore using flow cytometry with a double staining (CD38-FITC and Apo 2.7-PE), we demonstrate that VEGF protects MM patient cells from FBS-starvation-induced-apoptosis: the percentage of apoptotic MM patient cells (CD38++ and Apo 2.7+) in non starved medium (RPMI 1640 supplemented with 10% FBS) was 15% versus 93% in starved medium (RPMI 1640 supplemented with FBS 2%), and 48% in starved medium supplemented with 25ng/ml VEGF. In conclusion, our study demonstrates that VEGF protects MM cells against apoptosis, and that VEGF-induced MM cell proliferation and survival is mediated via Mcl-1. these studies provide the preclinical framework for novel therapeutics targeting both Mcl-1 and/or VEGF to improve patient outcome in MM.

The mTOR Inhibitor Everolimus Overcomes CXCR4-Mediated Resistance to HDAC Inhibitor Panobinostat through Inhibition of p21 and Mitosis Regulators, Sensitizing MM Cells to DNA-Damaged Induced Apoptosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 891-891
Author(s):  
Katia Beider ◽  
Valeria Voevoda ◽  
Hanna Bitner ◽  
Evgenia Rosenberg ◽  
Hila Magen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Cell Lines ◽  
Hdac Inhibitor ◽  
Mtor Inhibitor ◽  
Mtor Inhibition ◽  
Induced Apoptosis

Abstract Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Despite the initial efficacious treatment, MM patients often become refractory to common anti-MM drugs, therefore novel therapies are in need. Pan-histone deacetylase (HDAC) inhibitor panobinostat exerts multiple cytotoxic actions in MM cells in vitro, and was approved for the treatment of relapsed/refractory MM in combination with bortezomib and dexamethasone. Although having promising anti-MM properties, panobinostat lacks therapeutic activity as monotherapy. The aim of the current study was to elucidate the mechanisms underlying MM resistance to panobinostat and to define strategies to overcome it. Results: Panobinostat at the low concentrations (IC50 5-30 nM) suppressed the viability in MM cell lines (n=7) and primary CD138+ cells from MM patients (n=8) in vitro. Sensitivity to panobinostat correlated with reduced expression of chemokine receptor CXCR4, while overexpression of CXCR4 or its ligand CXCL12 in RPMI8226 and CAG MM cell lines significantly (p<0.001) increased their resistance to panobinostat, pointing to the role of the CXCR4 axis in HDACi response. Notably, similar expression levels of class I HDACs (HDAC1-3) were detected in MM cells with either low or high CXCR4. Interaction with BM stromal cells that represent the source of CXCL12 also protected MM cells from panobinostat-induced apoptosis, further strengthening a role for CXCR4 downstream pathway. Decreased sensitivity to cytotoxic effect was concomitant with reduced histone (H3K9 and H4K8) acetylation in response to panobinostat treatment. In addition, resistance to HDACi was associated with the reversible G0/G1 cell growth arrest, whereas sensitivity was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators involved in CXCR4-mediated HDACi resistance revealed the pro-survival AKT/mTOR pathway to be regulated by both CXCR4 over-expression and interaction with BMSCs. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance and induced synergistic cell death of MM cell lines and primary MM cells, but not of normal mononuclear cells (CI<0.4). This effect was concurrent with the increase in DNA double strand breaks, histone H2AX phosphorylation, loss of Dψm, cytochrome c release, caspase 3 activation and PARP cleavage. The increase in DNA damage upon combinational treatment was not secondary to the apoptotic DNA fragmentation, as it occurred similarly when apoptosis onset was blocked by caspase inhibitor z-VAD-fmk. Kinetics studies also confirmed that panobinostat-induced DNA damage preceded apoptosis induction. Strikingly, combined panobinostat/everolimus treatment resulted in sustained DNA damage and irreversible suppression of MM cell proliferation accompanied by robust apoptosis, in contrast to the modest effects induced by single agent. Gene expression analysis revealed distinct genetic profiles of single versus combined exposures. Whereas panobinostat increased the expression of cell cycle inhibitors GADD45G and p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated DNA repair genes, including RAD21, Ku70, Ku80 and DNA-PKcs. Furthermore, combined treatment markedly decreased both mRNA and protein expression of anti-apoptotic factors survivin and BCL-XL, checkpoint regulator CHK1, and G2/M-specific factors PLK1, CDK1 and cyclin B1, therefore suppressing the DNA damage repair and inhibiting mitotic progression. Given the anti-apoptotic role of p21, the synergistic lethal effect of everolimus could be attributed to its ability to suppress p21 induction by panobinostat ensuing the shift in the DNA damage response toward apoptosis. Conclusions: Collectively, our findings indicate that CXCR4/CXCL12 activity promotes the resistance of MM cells to HDACi with panobinostat through mTOR activation. Inhibition of mTOR by everolimus synergizes with panobinostat by simultaneous suppression of p21, G2/M mitotic factors and DNA repair machinery, rendering MM cells incapable of repairing accumulated DNA damage and promoting their apoptosis. Our results unravel the mechanism responsible for strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel therapeutic strategy to eradicate MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals JNK1/2 regulates ER–mitochondrial Ca2+ cross-talk during IL-1β–mediated cell death in RINm5F and human primary β-cells

2013 ◽  
Vol 24 (12) ◽  
pp. 2058-2071 ◽  
Author(s):  
Gaurav Verma ◽  
Himanshi Bhatia ◽  
Malabika Datta
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Mitochondrial Permeability Transition ◽  
Apoptotic Cell ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Β Cells ◽  
Mediating Role ◽  
Induced Apoptosis

Elevated interleukin-1β (IL-1β) induces apoptosis in pancreatic β-cells through endoplasmic reticulum (ER) stress induction and subsequent c-jun-N-terminal kinase 1/2 (JNK1/2) activation. In earlier work we showed that JNK1/2 activation is initiated before ER stress and apoptotic induction in response to IL-1β. However, the detailed regulatory mechanisms are not completely understood. Because the ER is the organelle responsible for Ca2+ handling and storage, here we examine the effects of IL-1β on cellular Ca2+ movement and mitochondrial dysfunction and evaluate the role of JNK1/2. Our results show that in RINm5F cells and human primary β-cells, IL-1β alters mitochondrial membrane potential, mitochondrial permeability transition pore opening, ATP content, and reactive oxygen species production and these alterations are preceded by ER Ca2+ release via IP3R channels and mitochondrial Ca2+ uptake. All these events are prevented by JNK1/2 small interfering RNA (siRNA), indicating the mediating role of JNK1/2 in IL-1β–induced cellular alteration. This is accompanied by IL-1β–induced apoptosis, which is prevented by JNK1/2 siRNA and the IP3R inhibitor xestospongin C. This suggests a regulatory role of JNK1/2 in modulating the ER-mitochondrial-Ca2+ axis by IL-1β in apoptotic cell death.

Ascorbic acid enhances arsenic trioxide–induced cytotoxicity in multiple myeloma cells

Blood ◽  
2001 ◽  
Vol 98 (3) ◽  
pp. 805-813 ◽  
Author(s):  
Jennifer M. Grad ◽  
Nizar J. Bahlis ◽  
Isildinha Reis ◽  
Marc M. Oshiro ◽  
William S. Dalton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Ascorbic Acid ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Plasma Cells ◽  
Radical Scavenging ◽  
B Cell Malignancy ◽  
Drug Efflux Pumps ◽  
Induced Apoptosis

Abstract Multiple myeloma (MM) is a clonal B-cell malignancy characterized by slow-growing plasma cells in the bone marrow (BM). Patients with MM typically respond to initial chemotherapies; however, essentially all progress to a chemoresistant state. Factors that contribute to the chemorefractory phenotype include modulation of free radical scavenging, increased expression of drug efflux pumps, and changes in gene expression that allow escape from apoptotic signaling. Recent data indicate that arsenic trioxide (As2O3) induces remission of refractory acute promyelocytic leukemia and apoptosis of cell lines overexpressing Bcl-2 family members; therefore, it was hypothesized that chemorefractory MM cells would be sensitive to As2O3. As2O3 induced apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and U266/Bcl-xL. The addition of interleukin-6 had no effect on cell death. Glutathione (GSH) has been implicated as an inhibitor of As2O3-induced cell death either through conjugating As2O3 or by sequestering reactive oxygen induced by As2O3. Consistent with this possibility, increasing GSH levels with N-acetylcysteine attenuated As2O3 cytotoxicity. Decreases in GSH have been associated with ascorbic acid (AA) metabolism. Clinically relevant doses of AA decreased GSH levels and potentiated As2O3-mediated cell death of all 4 MM cell lines. Similar results were obtained in freshly isolated human MM cells. In contrast, normal BM cells displayed little sensitivity to As2O3 alone or in combination with AA. Together, these data suggest that As2O3 and AA may be effective antineoplastic agents in refractory MM and that AA might be a useful adjuvant in GSH-sensitive therapies.

scholarly journals Overcoming Resistance to Apoptosis in Multiple Myeloma By Simultaneous Inhibition of Bcl2 and IAP Families of Anti-Apoptotic Proteins

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2088-2088
Author(s):  
Marcus Gomez ◽  
Vijay G. Ramakrishnan ◽  
Vivek Prasad ◽  
Teresa K. Kimlinger ◽  
Utkarsh Painuly ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Er Stress ◽  
Cell Lines ◽  
Bcl2 Family ◽  
Er Stress Response ◽  
Simultaneous Inhibition ◽  
Apoptotic Proteins ◽  
Upr Pathway

Abstract Background: Multiple myeloma (MM) cells evade apoptosis through multiple mechanisms thus enabling it to evade therapy. The Bcl2 family of anti-apoptotic proteins is aberrantly expressed in MM cell lines and patient cells. Yet, pharmacological intervention of this family appears to have significant activity only in molecular subgroups of MM patients. This clearly suggests alternate mechanisms of overcoming apoptotic signals in MM cells in addition to the Bcl2 family, through proteins such as IAPs. We have previously shown that simultaneous inhibition of the three major IAP proteins, namely cIAP1, cIAP2 and XIAP is required to induce pronounced apoptosis in MM cells. However, IAP inhibition results in apoptosis in only some MM cell lines and patient cells. Given that levels of Bcl2 family proteins are unaffected by IAP inhibition, we hypothesized that combined inhibition of the IAP proteins using a SMAC mimetic LCL161 and the Bcl2 family proteins using a pan-Bcl2 inhibitor obatoclax (OBX) will lead to more pronounced and synergistic cell death in a broader subgroup of MM patients. Methods: LCL161 was synthesized by Novartis Inc. (Basel, Switzerland). OBX was purchased from Selleckchem (Houston, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drugs in order to study the cell signaling pathways and a Results: LCL161/OBX combination induced synergistic cytotoxicity and anti-proliferative effects on a broad range of human MM cell lines, including drug resistant cell lines like DOX40 and MM1R. Components of the bone marrow microenvironment including bone marrow stromal cells and tumor promoting cytokines (VEGF, IGF and IL6) were unable to protect MM cells from the effects of the drug combination. We saw a time dependent increase in apoptosis, with the combination inducing significantly more apoptosis than either of the single agents alone. Examining the mechanism of action of the drug combination showed clear inhibition of the IAP proteins, activation of caspases 9, 8, 3 and Bid by LCL161 and the combination and up regulation of the pro-apoptotic proteins Bim, Bid, Puma and Noxa and accumulation of LC3-II by OBX and the combination. Using chloroquine along with the OBX, we were able to demonstrate that OBX induced protective autophagy and the addition of LCL161 was able to overcome this protective effect induced after single agent OBX treatment. Since protective autophagy can be induced by the ER stress response, we then examined the expression levels of proteins involved in this pathway. We observed clear induction of ER stress mediated UPR pathway by both the drugs. However, LCL161 and OBX induced different branches of the UPR pathway. OBX activated the ATF6 and pErk/peif2α/ATF4 branches of the UPR, both of which have been implicated in cell survival during ER stress. ATF4 under irrecoverable ER stress can lead to increase in transcription of CHOP and cause apoptosis. We therefore examined levels of CHOP and observed no induction of CHOP post treatment with either of the drugs or the combination. LCL161, however differentially modulated the IRE1 branch of the UPR by down regulating Xbp-1 splicing, which is a pro survival activity of IREI and up regulating pJNK, which indicated a pro-apoptotic activity induced by IRE1 post irrecoverable ER stress This indicated that the ER stress induced apoptosis is triggered by LCL161, which might be important to overcome the ER induced protective effects induced by OBX. Conclusion: Taken together, our studies indicate that LCL161/OBX combination induces synergistic cell death through modulation of apoptosis, authophagy and the ER stress response. Disclosures No relevant conflicts of interest to declare.

Bim Is the Critical, and Bad and Bmf Are the Ancillary BH3-Only Proteins for Imatinib Mesylate-Induced Apoptosis of Bcr/Abl-Positive Leukemias.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2876-2876
Author(s):  
Junya Kuroda ◽  
Hamsa Puthalakath ◽  
Philippe Bouillet ◽  
Mark S. Cragg ◽  
Priscilla N. Kelly ◽  
...  
Keyword(s):  
Cell Death ◽  
Imatinib Mesylate ◽  
Cell Lines ◽  
Cell Killing ◽  
Leukemic Cells ◽  
Imatinib Treatment ◽  
Killing Activity ◽  
Induced Apoptosis ◽  
Transformed Cell Lines

Abstract Imatinib mesylate (imatinib) exerts the anti-Philadelphia-positive (Ph1+) leukemia activity both by the inhibition of cell proliferation and by the induction of apoptosis. Recent studies demonstrate that the induction of cell death is essential for eradication of Ph1+ leukemic clones in imatinib treatment; however, the molecular mechanisms have not yet been clearly described. By examining the effect of imatinib on parental K562 and subclones overexpressing either Bcl-2, Bcl-XL or a dominant interfering mutant of FADD/MORT1, which blocks death receptor apoptosis signalling, we found that imatinib triggers apoptosis exclusively via the Bcl-2 family-regulated intrinsic apoptotic pathway. We investigated the involvement of BH3-only proteins as apoptotic initiators in imatinib-induced cell death, because the cell life-or-death decision is arbitrated by the balance between pro-apoptotic BH3 only-proteins and anti-apoptotic Bcl-2 proteins. We found that imatinib treatment upregulated Bim in Ph1+ leukemic cell lines and bcr-c-abl transformed murine fetal liver cells (FLCs)-derived cell lines both by transcriptional and post-translational mechanisms. Imatinib also activated Bad through dephosphorylation and upregulated Bmf transcriptionally. To examine the role of Bim in imatinib-induced apoptosis, we examined the cell killing activity of imatinib in subclones of K562 and BV173 Ph1+ cells expressing abnormally reduced levels of Bim using stable RNA interference system. This revealed that the cell killing activity of imatinib largely dependent on Bim expression levels in these cell lines, although significant apoptosis was still evident. To further define the role of Bim, Bad and Bmf in imatinib-induced cell death, we examined the effect of imatinib on retrovirally bcr-c-abl transformed cell lines derived from FLCs from wild type C57BL/6, Bim-/-, Bad-/-, Bim-/-Bad-/- double KO and Bcl-2 transgenic fetuses. The bim-/-bcr-c-abl+ FLCs were shown to be more resistant to imatinib-induced cell death than wt.bcr-c-abl+ FLCs, however, bim-/-bcr-c-abl+ FLCs were eventually induced into cell death, indicating that Bim is not the only initiator of apoptosis. The bad-/-bcr-c-abl+ FLCs were also partially resistant to imatinib-induced cell death. Intriguingly, like in vav.bcl-2.bcr-c-abl+ FLCs, the cell death induction by imatinib (~5.0μM) was largely abrogated in bim-/-bad-/-bcr-c-abl+ FLCs, indicating that Bim collaborates with Bad for the apoptotic induction by imatinib. Importantly, we found that Bim was inducible by ex vivo imatinib treatment in primary Ph1+ leukemic cells only from clinically good responders but not from patients refractory to imatinib treatment. Collectively, these results demonstrate that Bim is the critical but not the only initiator required for imatinib-induced apoptosis of Bcr/Abl-positive hematopoietic cells; Bad and Bmf may be the ancillary BH3-only proteins in this process. Our results provide evidence for the therapeutic significance of regulation of BH3-only proteins, particularly Bim, for the eradication of Ph1+ leukemic cells.

OSU03012, a Novel Celecoxib Derivative, Induces Apoptosis in Multiple Myeloma Cells Independently of Caspase Activation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5075-5075
Author(s):  
Shuhong Zhang ◽  
Valerie L. White ◽  
Amy Johnson ◽  
Ching-Shih Chen ◽  
Sherif S. Farag
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Plasma Cells ◽  
Parp Cleavage ◽  
Caspase Inhibitor ◽  
High Concentration ◽  
Down Regulation ◽  
Vitro Effect

Abstract Multiple myeloma (MM) is a clonal disorder affecting terminally differentiated B cells, with the accumulation of plasma cells in the bone marrow. Previous studies showed that OSU03012 is a novel celecoxib derivative lacking cyclooxygenase-2 inhibitory activity that induces apoptosis in various types of cancer cells and is being developed as an anti-cancer therapy in the NCI Rapid Access to Intervention Therapy (RAID). Here, we examined the in vitro effect of OSU03012 in MM cell lines (U266, ARH-77, IM-9 and RPMI8226). Cytotoxicity data indicated that mean LC50 (lethal concentration 50%) of OSU03012 was 6.25±0.86 μM at 24 hours and 4.23±0.87 μM at 72 hours in these four cell lines. Using annexin V/PI (propidium iodide) flow cytometry assay, OSU03012 was shown to induce apoptosis in MM cells. OSU03012 activated caspases-8, -9, and -3, induced PARP (POLY ADP-RIBOSE Polymerase) cleavage, and reduced survivin and XIAP expression after 6 and 24 hour exposure. Although the caspase inhibitor Q-VD-OPH treatment strongly blocked OSU03012-induced PARP cleavage, it did not inhibit OSU03012-induced apoptosis of MM cells. The pan-caspase inhibitor z-VAD-fmk did not prevent OSU03012 mediated cell death. Cell death with OSU03012 treatment was associated with significant down-regulation of phospho-Akt. Several substrates of AKT, including phospho-GSK-3 beta (Ser9), phospho-FoxO1a (Ser256) and phospho-MDM2 (Ser166) were also down-regulated by OSU03012 drug. OSU03012 triggered both early (6h) and late (24h) down-regulation of cyclin D1 expression, but cyclin A and B1 expression was down-regulated only at 24h. There was no induction of p21 or p27 protein levels by OSU03012. After 24-hour exposure, low concentration (1–5 μM) OSU03012 arrested MM cell lines in the G1 phase of the cell cycle while high concentration (10 μM) OSU03012 induced G2 phase arrested. OSU03012 decreased both phospho-Stat3 (Ser727) and Stat3 expression. OSU03012 has on effect on phosphorylated MAP kinase kinase1/2 (pMEK1/2) but it decreased MEK1/2 expression at 24h. The expression levels of Bcl-2 family proteins, Bcl-2, Mcl-1, BAX, and BIM did not alter with OSU03012 treatment suggesting that Bcl-2 members may not play direct or significant roles in inducing cell death. Taken together, we conclude that OSU03012 is potently active against MM cells by predominantly caspase-independent mechanisms, and may involve downstream pathways consequent to phopho-Akt down-regulation. These studies provide preclinical rationale for investigating OSU03012 in the treatment of MM.

Displacement of Bim From Anti-Apoptotic Proteins Is the Primary Factor for Determining ABT-737 Activity in Multiple Myeloma Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2851-2851
Author(s):  
Alejo A Morales ◽  
Metin Kurtoglu ◽  
David Siefker ◽  
Shannon M Matulis ◽  
Delia M Gutman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Cell Types ◽  
Myeloma Cell ◽  
Resistant Cell ◽  
The Other ◽  
Multiple Myeloma Cell ◽  
Other Hand

Abstract Abstract 2851 Poster Board II-827 ABT-737 and its orally active analog ABT-263 are Bcl-2-family inhibitors that are currently in clinical trials for a variety of cancers including hematological malignancies such as multiple myeloma. Previously, we reported that the sensitivity of multiple myeloma cell lines to ABT-737 correlates with the interactions, but not the expression, of Bcl-2 proteins. Analysis of 6 multiple myeloma cell lines revealed that expression of Bcl-2 proteins did not correlate with sensitivity, however the sensitive cells (8226/S, MM.1S and KMS-11) have a substantial amount of their pro-apoptotic Bcl-2 protein, Bak, bound to Bcl-xL. On the other hand, in the insensitive cell lines (U266, KMS-11 and OPM2), Bak was found to be associated with Mcl-1, a family member that does not bind ABT-737 and thereby confers resistance to this drug. Furthermore, we also showed that release of the BH3-only protein Bim by ABT-737 from Bcl-xL and Bcl-2 also contributes to cell death in 8226/S and MM.1S. The purpose of the current study is to further investigate the role of Bim in ABT-737-induced cell death in the multiple myeloma lines. Similar to Bak, a substantial amount of Bim is bound to Bcl-xL and Bcl-2 in the ABT-737-sensitive cell lines, MM.1S and KMS-18, while in the insensitive cell lines, it is highly bound to Mcl-1. Surprisingly, in the ABT-737-sensitive 8226/S cells, Bim appears to bind to Mcl-1. However in these cells, ABT-737 treatment resulted in upregulation of Noxa, which is a BH3-only protein that binds Mcl-1 and can release Bim. Taken together these data suggest that although binding of Bim to Mcl-1 may confer resistance to ABT-737, in certain cell types this treatment could also induce Noxa expression that antagonizes Mcl-1-mediated resistance. Consistent with this hypothesis, Mcl-1 overexpression as well as knockdown of Noxa expression significantly protected 8226/S cells from ABT-737-induced cell death while they had no effect in MM.1S cells. To further demonstrate the role of Bim in ABT-737-induced cell death, ABT-resistant 8226/S, KMS-11, KMS-18 and U266 cell lines were generated. In the resistant cell lines of 8226/S and KMS-18, Bim is exclusively bound to Mcl-1, which was overexpressed as compared to the parental cells. Bak binding was not affected by acquisition of ABT-737 resistance. This result is in agreement with the findings that interaction of Bim and Mcl-1 confers resistance to ABT-737. On the other hand, in ABT-resistant U266 and KMS-11 cell lines, Bim expression was down-regulated while Mcl-1 levels were not changed. Thus, it appears that in cells where Bim is already bound to Mcl-1, further resistance is achieved by down-regulating the expression of this BH3-only protein. Overall, these results suggest that the complex interactions between Bcl-2 proteins need to be investigated in order to understand how multiple myeloma cells may respond to ABT-737 treatment. Disclosures: Boise: University of Chicago: Patents & Royalties.

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