Requirement of Caspase-8 Versus Caspase-9 during Apoptosis in Multiple Myeloma Cells Induced by Bortezomib- or a Novel Proteasome Inhibitor NPI-0052.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3378-3378 ◽  
Author(s):  
Dharminder Chauhan ◽  
Laurence Catley ◽  
Mugdha Velankar ◽  
Anthony Letai ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibition ◽  
Proteolytic Processing ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Transfected Cells ◽  
Viable Cells ◽  
Vector Versus ◽  
Induced Apoptosis

Abstract Proteasome inhibition is an effective therapy for the treatment of relapsed/refractory multiple myeloma (MM); however, the sequence of events leading to apoptosis following proteasome inhibition is unclear. Here we defined the requirement for caspase-8 versus caspase-9 during Bortezomib (VelcadeTM)- or novel proteasome inhibitor NPI-0052-induced apoptosis in MM cells. Incubation of MM.1S cells with pan-caspase inhibitor (Z-VAD-FMK) markedly abrogates both NPI-0052- and Bortezomib-induced apoptosis. Inhibition of caspase-8 (IETD-FMK) led to a significant decrease in NPI-0052-triggered cell death, whereas inhibition of caspase-9 (LEHD-FMK) only moderately blocked NPI-0052-triggered decreased viability in MM.1S cells (P < 0.005). In contrast, Bortezomib-induced decrease in viability is equally blocked by either caspase-8 or caspase-9 inhibitors (P < 0.005). These biochemical data were further examined by genetic studies using dominant-negative (DN) strategies. Treatment of DN-caspase-8-transfected MM cells with NPI-0052 (IC50: 7 nM) markedly increases survival compared to cells transfected with DN-caspase-9. In contrast, treatment of either DN-caspase-8 or DN caspase-9-tranfected MM.1S cells with Bortezomib (IC50: 5 nM) increases survival to a similar extent. The proteolytic processing of pro-caspase-8 is mediated by Fas Associated Death-Domain (FADD) protein, and we next examined blockade of FADD with DN-FADD to further confirm the role of caspase-8. DN-FADD significantly attenuated NPI-0052-induced cytotoxicity compared to empty vector-transfected MM.1S cells (42 ± 2.0% viable cells in vector- versus 76 ± 5.1% viable cells in DN-FADD-transfected cells; P < 0.05). Importantly, treatment of DN-FADD-transfected MM.1S cells with Bortezomib results in only 16% increase in survival compared to vector-transfected cells (39 ± 2.4% viable cells in vector- versus 55 ± 4.1% viable cells in DN-FADD-transfected cells; P < 0.05). These data, coupled with caspase-8 or caspase-9 inhibition studies, suggest that NPI-0052 relies more on FADD-caspase-8 signaling than does Bortezomib, confirming a differential mechanism of action of NPI-0052 versus Bortezomib in MM cells. To further address this issue, we examined alterations in Bax, a proapoptotic protein which translocates from cytosol to mitochondria during apoptosis, inhibits Bcl-2, and facilitates release of cyto-c and activation of caspase-9. NPI-0052 induces little, if any, increase in Bax levels in mitochondria, whereas Bortezomib triggers a significant accumulation of Bax in mitochondria. Experiments using Bax wild type (WT) or knockout mouse embryonic fibroblast (MEFs) show that NPI-0052 decreases viability in both Bax (WT) and Bax (knock-out) MEFs, whereas deletion of Bax confers significant resistance to Bortezomib. These findings suggest a differential requirement for Bax during NPI-0052- versus Bortezomib-induced apoptosis. Collectively, our data suggest that 1) NPI-0052-induced MM cell apoptosis is predominantly mediated by caspase-8; and 2) Bortezomib-induced apoptosis requires both caspase-8 and caspase-9 activation. These data provide rationale for combining agents based on differential signaling cascades to amplify apoptosis and enhance anti-tumor activity.

scholarly journals Combination of novel proteasome inhibitor NPI-0052 and lenalidomide trigger in vitro and in vivo synergistic cytotoxicity in multiple myeloma

Blood ◽  
2010 ◽  
Vol 115 (4) ◽  
pp. 834-845 ◽  
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Bryan Ciccarelli ◽  
Paul G. Richardson ◽  
Michael A. Palladino ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitor ◽  
Tumor Model ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Model Studies ◽  
Synergistic Cytotoxicity ◽  
Induced Apoptosis

Abstract Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 is distinct from bortezomib (Velcade) and, importantly, triggers apoptosis in multiple myeloma (MM) cells resistant to bortezomib. Here we demonstrate that combining NPI-0052 and lenalidomide (Revlimid) induces synergistic anti-MM activity in vitro using MM-cell lines or patient MM cells. NPI-0052 plus lenalidomide-induced apoptosis is associated with (1) activation of caspase-8, caspase-9, caspase-12, caspase-3, and poly(ADP) ribose polymerase; (2) activation of BH-3 protein BIM; (3) translocation of BIM to endoplasmic reticulum; (4) inhibition of migration of MM cells and angiogenesis; and (5) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities. Importantly, blockade of BIM using siRNA significantly abrogates NPI-0052 plus lenalidomide-induced apoptosis. Furthermore, studies using biochemical inhibitors of caspase-8 versus caspase-9 demonstrate that NPI-0052 plus lenalidomide-triggered apoptosis is primarily dependent on caspase-8 signaling. In animal tumor model studies, low-dose combination of NPI-0052 and lenalidomide is well tolerated, significantly inhibits tumor growth, and prolongs survival. Taken together, our study provides the preclinical rationale for clinical protocols evaluating lenalidomide together with NPI-0052 to improve patient outcome in MM.

Targeting MEK/MAPK Signal Transduction Module Potentiates Arsenic Trioxide (ATO)-Induced Apoptosis in Multiple Myeloma Cells through Multiple Signaling Pathways.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1517-1517
Author(s):  
Paolo Lunghi ◽  
Nicola Giuliani ◽  
Laura Mazzera ◽  
Francesca Morandi ◽  
Luigi Salvatore ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Arsenic Trioxide ◽  
Combined Treatment ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Down Regulation ◽  
P53 Pathway ◽  
Myeloma Cells ◽  
Trail Receptors ◽  
Induced Apoptosis

Abstract Multiple Myeloma (MM) cells are extremely resistant to apoptosis and currently new potential drug combinations are under investigation. We have shown that the combined treatment with the MEK1/2 inhibitor PD184352 (PD) and Arsenic Trioxide (ATO) resulted in the synergistic (Combination Index <1.0) induction of apoptosis in 7 human myeloma cell lines (HMCLs: XG1, XG6, OPM2, JJN3, RPMI, H929, Sultan) analyzed, irrespective of their p53 status. The combined treatment was also a highly potent inducer of apoptosis and mitochondrial damage in the majority of the primary multiple myeloma (MM) cell samples ex vivo analyzed at different disease stage (9 out of 12). Growth factors, IL-6 or insulin-like growth factor 1 (IGF-1), or a co-culture system with bone marrow stromal cells (BMSCs) failed to confer resistance to this combination regimen. The combination PD/ATO had a minimal effect on normal B cells in vitro. By investigating the molecular mechanisms involved in MM cells PD/ATO-induced apoptosis, we found that co-treatment with PD strikingly elevated the (DR4+DR5)/(DcR1+DcR2) tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors ratio, caspase-8 activation, Bid fragmentation, mitochondrial depolarization and caspase-9 activation of ATO-treated HMCLs that do not have a functional p53 pathway. In HMCLs carrying a functional p53 pathway, the treatment with PD greatly enhanced the ATO-induced p53 accumulation (two fold increase) and p73, a p53 paralogue, cooperated with p53 in the pro-apoptotic p53/p73 target genes up regulation, caspase-9, -3 activation and apoptosis induction; in these HMCLs the selective down-regulation of p53 or p73 demonstrated that both have a biological relevance in PD/ATO-induced caspase-3 activation, PARP fragmentation and apoptosis. In HMCLs carrying a functional p53 the extrinsic caspase-8 mediated pathway was partially activated by PD/ATO treatment. We also demonstrated that, in MM cells carrying or not a functional p53 pathway, the combined treatment PD/ATO increased the level of the pro-apoptotic Bim (PD-mediated) and decreased its neutralizing anti-apoptotic protein Mcl-1 (ATO-mediated). The selective down-regulation of Bim significantly diminished caspase-8/-9/-3 cleavage/activation, PARP fragmentation and apoptosis of PD/ATO-treated MM cells, thereby indicating that Bim can play an important role not only in the intrinsic mitochondrial programmed cell death but also in the extrinsic caspase-8 mediated pathway. Accordingly, a physical interaction between Bim and DR4/DR5 TRAIL receptors in PD/ATO-treated MM cells carrying a non functional p53 was found by coimmunoprecipitation and Western blot studies. Our experiments have enlightened some relevant mechanisms that explain the apoptotic response of myeloma cells to ATO plus MEK inhibitor combination.

Combination of a Novel Proteasome Inhibitor NPI-0052 and Lenalidomide Trigger in Vivo Synergistic Cytotoxicity in Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3662-3662 ◽  
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Mohan Brahmandam ◽  
Giada Bianchi ◽  
Klaus Podar ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Low Dose ◽  
Caspase 3 ◽  
Caspase 8 ◽  
Caspase 9

Abstract Background: Our previous study demonstrated that a novel proteasome inhibitor NPI-0052 is distinct from bortezomib in its chemical structure, effects on proteasome activities, and mechanisms of action, and importantly, triggers apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib therapies. These preclinical data provided the basis for the ongoing phase-I clinical trial of NPI-0052 in relapsed/refractory MM patients. Recently, a Phase-1/2 clinical trial of bortezomib with Lenalidomide and low dose dexamethasone demonstrated safety and remarkable efficacy in newly diagnosed MM patients. Given that the combination of bortezomib with Lenalidomide has proven a successful treatment strategy, coupled with our findings that NPI-0052 is a potent proteasome inhibitor, we determined whether combining NPI-0052 with Lenalidomide triggered synergistic/additive anti-MM activity. Material and Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, and INA-6 human MM cell lines, as well as purified tumor cells from patients relapsing after prior therapies including Lenalidomide or bortezomib. Informed consent was obtained from all patients in accordance with the Helsinki protocol. Cell viability and apoptosis assays were performed using MTT and Annexin V staining. In vitro angiogenesis was assessed by Matrigel capillary-like tube structure formation assay. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, BIM, p-JNK or tubulin. In vitro and in vivo proteasome activity assays were performed using fluorogenic peptide substrates. All animal studies were approved by the DFCI Institutional Animal Care and Use Committee. CB-17 SCID male mice (n = 30; 5 mice/EA group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum free RPMI-1640 medium. When tumors were measurable (~150 mm3) three weeks after MM cell injection, mice were treated with oral doses of vehicle alone, NPI-0052 (0.15 mg/kg), Lenalidomide (2.5 mg/kg), Lenalidomide (5.0 mg/kg), NPI-0052 (0.15 mg/kg) plus Lenalidomide (2.5 mg/kg) or NPI-0052 (0.15 mg/kg) plus Lenalidomide (5.0 mg/kg) on a twice weekly schedule for NPI-0052 and four consecutive days weekly for Lenalidomide for four weeks. Statistical significance of differences observed in NPI-0052, Lenalidomide or NPI-0052 plus Lenalidomide-treated mice was determined using a Student t test. Isobologram analysis was performed using “CalcuSyn” software program. A combination index < 1.0 indicates synergism. Results: Combining NPI-0052 and Lenalidomide induces synergistic/additive anti-MM activity in vitro using MM cell lines (P<0.005, n=3, CI < 1) or patient CD138-positive MM cells (5 patients, P< 0.004). NPI- 0052 plus Lenalidomide-induced synergistic apoptosis is associated with: activation of caspase-8, caspase-9, caspase-3, and PARP; induction of c-Jun-NH2-terminal kinase; activation of BH-3 protein BIM; inhibition of migration of MM cells and angiogenesis; suppression of chymotrypsin-like, caspase-like and trypsin-like proteolytic activities in an additive manner; and inhibition of NF-kappa B signaling. Importantly, blockade of BIM using siRNA significantly abrogates NPI-0052 plus Lenalidomide-induced apoptosis (61 ± 7.1% decrease in cell death; P < 0.003, n=2). Furthermore, studies using biochemical inhibitors of caspase-8 versus caspase-9 demonstrate that NPI-0052 plus Lenalidomide-triggered apoptosis is primarily dependent on caspase-8 signaling. In animal tumor model studies, low dose combination NPI-0052 (0.15 mg/kg) and Lenalidomide (2.5 or 5.0 mg/kg) is well tolerated, significantly inhibits tumor growth (P < 0.03), and prolongs survival (4–5 months in mice receiving combined drugs, P = 0.001). Immununohistochemistry analysis of MM tumors excised from NPI-0052 plus Lenalidomide-treated mice showed growth inhibition (Ki-67), apoptosis (TUNEL assay, caspae-3 activation), a decrease in associated angiogenesis (Factor VIII and VEGF receptor), and additive inhibition of proteasome activity. Taken together, our study provides the preclinical rationale for clinical protocols evaluating Lenalidomide together with NPI-0052 to improve patient outcome in MM.

The triterpenoid CDDO induces apoptosis in refractory CLL B cells

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2965-2972 ◽  
Author(s):  
Irene M. Pedersen ◽  
Shinichi Kitada ◽  
Aaron Schimmer ◽  
Youngsoo Kim ◽  
Juan M. Zapata ◽  
...  
Keyword(s):  
B Cells ◽  
Anticancer Agents ◽  
Fas Ligand ◽  
Proteolytic Processing ◽  
Caspase 8 ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Apoptosis Pathway ◽  
Apoptosis Protein ◽  
Induced Apoptosis

Chronic lymphocytic leukemia (CLL) cells develop chemo-resistance over time. Most anticancer agents function through induction of apoptosis, and therefore resistance against these agents is likely to be caused by selection for CLL cells with defects in the particular apoptosis pathway that is triggered by these drugs. Anticancer agents that function through alternative apoptotic pathways might therefore be useful in treating chemo-resistant CLL. Triterpenoids represent a class of naturally occurring and synthetic compounds with demonstrated antitumor activity. We examined the effects of CDDO (triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid) on CLL B cells in vitro. CDDO induced apoptosis in a dose-dependent manner in all (n = 30) CLL samples tested, including previously untreated and chemo-resistant CLL specimens. CDDO induced rapid proteolytic processing of caspase-8, but not caspase-9, in CLL B cells, suggesting activation of a mitochondria-independent pathway. CDDO-induced apoptosis of CLL B cells was blocked by cytokine response modifier A (CrmA), a suppressor of caspase-8, but not by X-linked inhibitor of apoptosis protein–baculovirus IAP repeat–3 (XIAP-BIR3), a fragment of XIAP, which selectively inhibits caspase-9. Examination of CDDO effects on expression of several apoptosis-relevant genes demonstrated significant reductions in the levels of caspase-8 homolog Fas-ligand interleukin-1–converting enzyme (FLICE)–inhibitory protein (c-FLIP), an endogenous antagonist of caspase-8. However, reductions of FLIP achieved by FLIP antisense oligonucleotides were insufficient for triggering apoptosis, indicating that CDDO has other targets in CLL B cells besides FLIP. These data suggest that the synthetic triterpenoid CDDO should be further explored as a possible therapeutic agent for treatment of chemo-resistant CLL.

An Investigational Novel Orally Bioavailable Proteasome Inhibitor MLN9708/MLN2238 Triggers Cytotoxicity In Multiple Myeloma Cells Via p21- and Caspase-8-Dependent Signaling Pathway

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2992-2992
Author(s):  
Dharminder Chauhan ◽  
Ze Tian ◽  
Teru Hideshima ◽  
Nikhil C. Munshi ◽  
Paul G Richardson ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Boronic Acid ◽  
Proteasome Inhibitor ◽  
Annexin V ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Advisory Committees ◽  
Orally Bioavailable ◽  
Induced Apoptosis ◽  
Rpmi 8226

Abstract Abstract 2992 Background and Rationale: Therapeutic targeting of Ubiquitin-Proteasome Signaling (UPS) is exemplified by the FDA approval of dipeptidyl boronic acid bortezomib first-in-class proteasome inhibitor for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM). As with other agents, however, dose-limiting toxicities and the development of resistance limit its long-term utility. MLN9708 (Millennium Pharmaceuticals, Inc., Cambridge, MA) is a selective, orally bioavailable proteasome inhibitor currently in phase I clinical development. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form. Similar to bortezomib, MLN2238 is a boronic acid analog; however, it is distinct from bortezomib since preclinical data demonstrates it has a shorter proteasome dissociation half-life, as well as improved pharmacokinetics, pharmacodynamics, and antitumor activity in xenograft models versus bortezomib. In the present study, we examined the anti-tumor activity of MLN2238 in MM cells using in vitro model systems. Methods and Model: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12BM, OPM2, H929, LP1, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability, proliferation, and apoptosis assays were performed using Trypan blue, MTT, CellTiter-Glo, thymidine incorporation, and Annexin V staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Statistical significance of data was determined using a Student t test. Results: We first confirmed the functional specificity of MLN2238 using different experimental strategies: 1) Examination of the proteasome activity using human erythrocyte 20S proteasomes and fluorogenic substrates showed that MLN2238, like bortezomib, primarily inhibits chymotrypsin-like proteasome activity (EC50 = 9 ± 2.3 nM versus bortezomib: 5.5 ± 1.5 nM); 2) Treatment of MM.1S MM cells with MLN2238 induces a time- and dose-dependent accumulation of ubiquitinated proteins; and 3) Washout experiments showed that MLN2238 is a reversible proteasome inhibitor (P < 0.001; n=3). We next examined the effects of MLN2238 in MM cells. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, U266, OPM2, H929, LP1, KMS-12BM, and INA-6) and primary patient cells for 48h significantly decreased their viability (IC50 range 12.5 – 25 nM) (P < 0.001; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for MLN2238. MLN2238-triggered apoptosis was confirmed in MM.1S and H929 cells, evidenced by a marked increase in Annexin V+ and PI- cell population (P < 0.001, n=3). Importantly, MLN2238 induced apoptosis in MM cells even in the presence of MM bone marrow stromal cells. Mechanistic studies showed that MLN2238-triggered apoptosis in MM cells is associated with 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) activation of p53, Noxa, PUMA, Bid, E2F, and pAKT; 3) downregulation of MM cell growth and survival proteins: NF-kB, Bcl2, phospho-Rb protein, and cyclin-A; 4) inhibition of migration of MM cells and angiogenesis; and 5) increased expression of CDK inhibitor p21. Importantly, blockade of p21 using both siRNA strategy and p21- knockout HCT116 cells showed significant abrogation of MLN2238-induced cell death (P value < 0.001; n=3). We next examined the requirement for caspase-8 versus caspase-9 during MLN2238-induced apoptosis. Incubation of MM.1S cells with pan-caspase inhibitor (Z-VAD-FMK) markedly abrogates MLN2238-induced apoptosis. Inhibition of caspase-8 (IETD-FMK) led to a significant decrease in MLN2238-triggered cell death, whereas inhibition of caspase-9 (LEHD-FMK) only moderately blocked MLN2238-triggered decreased viability in MM.1S cells (P < 0.001, n=3). These data suggest that MLN2238-induced apoptosis in MM cells is predominantly mediated by caspase-8 extrinsic apoptotic pathway. Finally, combination of MLN2238 with lenalidomide or dexamethasone triggered synergistic anti-MM activity. Conclusions: Our preclinical study supports further clinical evaluation of MLN9708, either alone or in combination, as a potential MM therapy. Disclosures: Munshi: Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

MEK1 Blockade Sensitizes Multiple Myeloma Tumor Cells to Arsenic Trioxide (ATO)-Induced Apoptosis: A Functional and Molecular Study.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1372-1372
Author(s):  
Paolo Lunghi ◽  
Nicola Giuliani ◽  
Laura Mazzera ◽  
Francesca Morandi ◽  
Vittorio Rizzoli ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Combination Index ◽  
Combined Treatment ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Myeloma Cells ◽  
Induction Of Apoptosis ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
Rpmi 8226

Abstract Preliminary data from clinical studies indicate that ATO has clinical activity as a single agent in human multiple myeloma (MM), and combination therapies are being investigated. We recently reported that PD184352 (PD) ( Pfizer), a highly selective inhibitor of MEK1, strikingly enhances ATO-mediated apoptosis in NB4, K562 cell lines and in Acute Myelogenous Leukemia via multiple intrinsic apoptotic pathways activation. The aim of this study was to investigate whether the combined treatment with PD and ATO has cytotoxic effects on MM cells. We first analyzed the pharmacologic interactions between PD (2 μM) and ATO (1–2 μM) using a fixed-ratio experimental design on 7 human myeloma cell lines (HMCL) with varying p53 status (RPMI 8226, U266, XG-1, XG-6, JJN3, HS-SULTAN, NCI-H929) and found that the combined treatment resulted in the synergistic (Combination Index &lt;1.0) induction of apoptosis in NCI-H929, XG-1, XG-6, RPMI 8226, SULTAN and JJN3 HMCL. Conversely, the combination of PD plus ATO had a slight antagonistic effect on apoptosis induction in U266 HMCL (Combination Index &gt;1). Moreover, PD plus ATO-induced cytotoxicity on HMCL was maintained also in presence of IL-6 (20 ng/ml) or in a co-culture system with bone marrow stromal cells. Similarly to HMCL we found that the treatment with PD significantly enhanced the apoptosis of fresh purified MM cells induced by ATO (P&lt; .01) in 8 out of 11 patients of MM analyzed. Conversely, PD treatment partially attenuated (n=2) or did not affect (n=1) the ATO cytotoxicity in normal bone marrow B cells. To investigate the molecular mechanisms by which PD plus ATO induced MM cell apoptosis first we compared the effect on caspase activation in myeloma cells expressing wild type (wt) p53 or mutated p53. Using caspase blocking peptides, specific siRNA against caspase-8 or caspase-9 and Western immunoblotting we demonstrated the involvement of primarily caspase-8 and -3 in PD plus ATO-induced apoptosis in myeloma cells with mutated p53 and primarily caspase-9 and -3 in dual treated cells expressing wt p53. In addition, PD plus ATO induced a p53-dependent up regulation of Puma, Bax and Bak in HMCL with wt p53, and p53-specific siRNA significantly (P&lt; .01) reduced the induction of apoptosis in dual treated cells. The caspase-8-mediated proteolytic activation of BID, a key protein involved in the cross-talk between the intrinsic and extrinsic apoptotic pathways, closely correlated with the caspase-9 activation and loss of mitochondrial membrane potential observed in dual treated HMCL with mutated p53. Finally, in the responsive HMCL both with wt or mutated p53, the combined treatment increased the level of the pro-apoptotic Bim (PD-mediated) and decreased its neutralizing anti-apoptotic protein Mcl-1 (ATO-mediated) causing an imbalance between these proteins that positively influenced the pro-apoptotic efficacy of the combination. In conclusion, our data indicate that the disruption of MEK pathway potentiates the apoptotic effect of ATO in MM cells through the activation of both extrinsic and intrinsic (caspase-8-BID-mediated) pathways in HMCL with mutated p53 or through the primarily activation of intrinsic pathway in HMCL with wt p53, with the positive contribution of Bim pathway. These findings suggest that a strategy combining ATO with disruption of MEK pathway could represent an effective therapeutic strategy for the treatment of MM.

Lamotrigine Attenuates Proteasome Inhibition-Induced Apoptosis by Suppressing the Activation of the Mitochondrial Pathway and the Caspase-8- and Bid-Dependent Pathways

2016 ◽  
Vol 41 (10) ◽  
pp. 2503-2516 ◽  
Author(s):  
Yoon Jeong Nam ◽  
Arum Kim ◽  
Min Sung Lee ◽  
Yong Kyoo Shin ◽  
Dong Suep Sohn ◽  
...  
Keyword(s):  
Proteasome Inhibition ◽  
Mitochondrial Pathway ◽  
Caspase 8 ◽  
Induced Apoptosis

Intracellular regulation of tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis in human multiple myeloma cells

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2162-2171 ◽  
Author(s):  
Nicholas Mitsiades ◽  
Constantine S. Mitsiades ◽  
Vassiliki Poulaki ◽  
Kenneth C. Anderson ◽  
Steven P. Treon
Keyword(s):  
Multiple Myeloma ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Protein Synthesis Inhibitor ◽  
Caspase 8 ◽  
Data Set ◽  
Apoptosis Protein ◽  
Induced Apoptosis ◽  
Necrosis Factor ◽  
Resistant Cells

Abstract Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL, Apo2 ligand) effectively kills multiple myeloma (MM) cells in vitro irrespective of refractoriness to dexamethasone and chemotherapy. Because clinical trials with this anticancer agent are expected shortly, we investigated the signaling pathway of TRAIL-induced apoptosis in MM. We detected rapid cleavage of caspases-8, -9, -3, and -6, as well as the caspase substrates poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor-45 (DFF45), but not caspase-10, upon TRAIL treatment in sensitive MM cells, pointing to caspase-8 as the apical caspase of TRAIL signaling in MM cells. These phenomena were not observed or were significantly delayed in TRAIL-resistant MM cells, suggesting that resistance may arise from inhibition at the level of caspase-8 activation. Higher levels of expression for various apoptosis inhibitors, including FLICE-inhibitory protein (FLIP), and lower procaspase-8 levels were present in TRAIL-resistant cells and sensitivity was restored by the protein synthesis inhibitor cycloheximide (CHX) and the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), which both lowered FLIP and cellular inhibitor of apoptosis protein-2 (cIAP-2) protein levels. Forced expression of procaspase-8 or FLIP antisense oligonucleotides also sensitized TRAIL-resistant cells to TRAIL. Moreover, the cell permeable nuclear factor (NF)–κB inhibitor SN50, which sensitizes TRAIL-resistant cells to TRAIL, also inhibited cIAP2 protein expression. Finally, CHX, BIM, and SN50 facilitated the cleavage and activation of procaspase-8 in TRAIL-resistant cells, confirming that inhibition of TRAIL-induced apoptosis occurs at this level and that these agents sensitize MM cells by relieving this block. Our data set a framework for the clinical use of approaches that sensitize MM cells to TRAIL by agents that inhibit FLIP and cIAP-2 expression or augment caspase-8 activity.

scholarly journals Mitochondrial Cell Death Suppressors Carried by Human and Murine Cytomegalovirus Confer Resistance to Proteasome Inhibitor-Induced Apoptosis

2005 ◽  
Vol 79 (19) ◽  
pp. 12205-12217 ◽  
Author(s):  
A. Louise McCormick ◽  
Christopher D. Meiering ◽  
Geoffrey B. Smith ◽  
Edward S. Mocarski
Keyword(s):  
Cell Death ◽  
Viral Genome ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibition ◽  
Murine Cytomegalovirus ◽  
Viral Inhibitor ◽  
Infected Cells ◽  
Induced Apoptosis ◽  
Functional Copy ◽  
Inactivating Mutations

ABSTRACT Human cytomegalovirus carries a mitochondria-localized inhibitor of apoptosis (vMIA) that is conserved in primate cytomegaloviruses. We find that inactivating mutations within UL37x1, which encodes vMIA, do not substantially affect replication in TownevarATCC (Towne-BAC), a virus that carries a functional copy of the betaherpesvirus-conserved viral inhibitor of caspase 8 activation, the UL36 gene product. In Towne-BAC infection, vMIA reduces susceptibility of infected cells to intrinsic death induced by proteasome inhibition. vMIA is sufficient to confer resistance to proteasome inhibition when expressed independent of viral infection. Murine cytomegalovirus m38.5, whose position in the viral genome is analogous to UL37x1, exhibits mitochondrial association and functions in much the same manner as vMIA in inhibiting intrinsic cell death. This work suggests a common role for vMIA in rodent and primate cytomegaloviruses, modulating the threshold of virus-infected cells to intrinsic cell death.

Novel targets for endoplasmic reticulum stress-induced apoptosis in B-CLL

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2713-2723 ◽  
Author(s):  
Emanuela Rosati ◽  
Rita Sabatini ◽  
Giuliana Rampino ◽  
Filomena De Falco ◽  
Mauro Di Ianni ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Apoptosis ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Minor Role ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Induced Apoptosis

Abstract A better understanding of apoptotic signaling in B-chronic lymphocytic leukemia (B-CLL) cells may help to define new therapeutic strategies. This study investigated endoplasmic reticulum (ER) stress signaling in spontaneous apoptosis of B-CLL cells and whether manipulating ER stress increases their apoptosis. Results show that a novel ER stress-triggered caspase cascade, initiated by caspase-4 and involving caspase-8 and -3, plays an important role in spontaneous B-CLL cell apoptosis. ER stress-induced apoptosis in B-CLL cells also involves CHOP/GADD153 up-regulation, increased JNK1/2 phosphorylation, and caspase-8–mediated cleavage of Bap31 to Bap20, known to propagate apoptotic signals from ER to mitochondria. In ex vivo B-CLL cells, some apoptotic events associated with mitochondrial pathway also occur, including mitochondrial cytochrome c release and caspase-9 processing. However, pharmacologic inhibition studies show that caspase-9 plays a minor role in B-CLL cell apoptosis. ER stress also triggers survival signals in B-CLL cells by increasing BiP/GRP78 expression. Manipulating ER signaling by siRNA down-regulation of BiP/GRP78 or treating B-CLL cells with 2 well-known ER stress-inducers, tunicamycin and thapsigargin, increases their apoptosis. Overall, our findings show that ER triggers an essential pathway for B-CLL cell apoptosis and suggest that genetic and pharmacologic manipulation of ER signaling could represent an important therapeutic strategy.

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