TRAILR Triggering by Human Antibodies Induces Apoptosis through an Early Cleavage of Mcl-1 and Caspase 3 in Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3397-3397
Author(s):  
Catherine Pellat-Deceunynck ◽  
Patricia Gomez-Bougie ◽  
Sylvanne Daniels ◽  
Alexandrine Geffroy-Luseau ◽  
Regis Bataille ◽  
...  
Keyword(s):  
Cell Lines ◽  
Caspase 3 ◽  
Chromosomal Translocation ◽  
Myeloma Cell ◽  
Caspase 8 ◽  
Early Cleavage ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Human Antibodies

Abstract We investigated TRAILR expression and sensitivity of myeloma cells in vitro. This study was done using a panel of 20 myeloma cell lines that are representative of primary myeloma cells (14q chromosomal translocation, IL-6 dependency, phenotype, oncogenes mutation). TRAILR were stimulated with agonistic human antibodies directed against either TRAIL-R1/DR4 (HGS-ETR1, mapatumumab) or TRAIL-R2/DR5 (HGS-ETR2), provided by Human Genome Sciences, Rockville, MD. This approach allowed us to analyze the contribution of each receptor separately. We show that a wide majority of cell lines, 16 of 20 were killed upon either TRAIL-R1 or R2 stimulation in the presence or absence of IL-6. However, 4 cell lines were resistant to HGS-ETR1 and 6 to HGS-ETR2 and 3 to both. Activation of both caspase 8 and Bid has been extensively described as being associated with TRAIL response. Indeed, we observed an activation of both caspase 8 and Bid. Cleaved molecules were detected 6 to 18h after antibody addition but after detection of cellular apoptosis. However, we show that Mcl-1L, a key molecule for myeloma survival, was downregulated and cleaved as soon as 3h after Ab addition. The cleaved form of Mcl-1 has been shown to behave like a proapoptotic molecule. Since caspase 3 has been reported to cleave Mcl-1, we looked at caspase 3 activation. Indeed, we observed that caspase 3 cleavage occured early and concomitantly to the one of Mcl-1. Our data show that in a wide majority of myeloma cell lines (80%) TRAILR triggering induces massive apoptosis that was not prevented by IL-6, the major myeloma cell growth and survival factor. Moreover, apoptosis induced upon TRAILR triggering was fully correlated to an early cleavage of both caspase 3 and Mcl-1 and to a delayed one of both caspase 8 and Bid.

Baicalein, a component of Scutellaria radix from Huang-Lian-Jie-Du-Tang (HLJDT), leads to suppression of proliferation and induction of apoptosis in human myeloma cells

Blood ◽  
2005 ◽  
Vol 105 (8) ◽  
pp. 3312-3318 ◽  
Author(s):  
Zi Ma ◽  
Ken-ichiro Otsuyama ◽  
Shangqin Liu ◽  
Saeid Abroun ◽  
Hideaki Ishikawa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Caspase 3 ◽  
Herbal Medicines ◽  
Myeloma Cell ◽  
Suppressive Effect ◽  
Caspase 9 ◽  
Myeloma Cells ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

Abstract In the search for a more effective adjuvant therapy to treat multiple myeloma (MM), we investigated the effects of the traditional Chinese herbal medicines Huang-Lian-Jie-Du-Tang (HLJDT), Gui-Zhi-Fu-Ling-Wan (GZFLW), and Huang-Lian-Tang (HLT) on the proliferation and apoptosis of myeloma cells. HLJDT inhibited the proliferation of myeloma cell lines and the survival of primary myeloma cells, especially MPC-1- immature myeloma cells, and induced apoptosis in myeloma cell lines via a mitochondria-mediated pathway by reducing mitochondrial membrane potential and activating caspase-9 and caspase-3. Further experiments confirmed that Scutellaria radix was responsible for the suppressive effect of HLJDT on myeloma cell proliferation, and the baicalein in Scutellaria radix showed strong growth inhibition and induction of apoptosis in comparison with baicalin or wogonin. Baicalein as well as baicalin suppressed the survival in vitro of MPC-1- immature myeloma cells rather than MPC-1+ myeloma cells from myeloma patients. Baicalein inhibited the phosphorylation of IkB-α, which was followed by decreased expression of the IL-6 and XIAP genes and activation of caspase-9 and caspase-3. Therefore, HLJDT and Scutellaria radix have an antiproliferative effect on myeloma cells, especially MPC-1- immature myeloma cells, and baicalein may be responsible for the suppressive effect of Scutellaria radix by blocking IkB-α degradation. (Blood. 2005;105:3312-3318)

TP53 Positively Regulates Cell Death through TRAILR2 (DR5) but Negatively through TRAILR1 (DR4) In Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 136-136
Author(s):  
Sylvanie Surget ◽  
Patricia Gomez-Bougie ◽  
David Chiron ◽  
Robin Humphreys ◽  
Philippe Moreau ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Caspase 8 ◽  
Myeloma Cells ◽  
Trail Receptors ◽  
Tp53 Status

Abstract Abstract 136 Mapatumumab and lexatumumab are human antibodies that bind and activate death receptors TRAILR1/TNFSF10A/DR4 and TRAILR2/TNFSF10B/DR5, respectively. Treatment of primary myeloma cells and myeloma cell lines with these mAbs induced cell death. Mapatumumab induced cell death more effectively than lexatumumab in a panel of 30 human myeloma cell lines (HMCLs). Interestingly, sensitivity to mapatumumab and lexatumumab was mutually exclusive and related to TP53 status (p=0.006). Indeed, wildtype TP53 HMCLs (n=9) were sensitive to lexatumumab (mean of death 40%) but resistant to mapatumumab (mean of death 7%). In contrast, abnormal (n=21) TP53 HMCLs were resistant to lexatumumab (mean of death 7%) but sensitive to mapatumumab (mean of death 44%). Of note, killing by lexatumumab was correlated to TRAILR2 expression while no correlation was found for TRAILR1 expression and lapatumumab killing. Transcriptomic analysis of 30 HMCLs revealed that HMCLs with abnormal TP53 status underexpressed 4 well-known p53 target genes, MDM2, CDKN1A, Bax and TRAILR2 (p<0.01). To activate p53 pathway in myeloma cells, we used melphalan at low doses. Melphalan treatment of wildtype HMCLs, but not of TP53 abnormal HMCLs, increased TRAILR2 expression and cell death mediated by lexatumumab. In contrast, melphalan did not alter TRAILR1 expression or mapatumumab-induced killing, suggesting that TRAILR2 but not TRAILR1 is a p53 target in myeloma cells. Silencing TP53 significantly increased mapatumumab apoptosis (>50% p<0.01). In good agreement with Bax underexpression in TP53 abnormal HMCLs, extensive silencing of key molecules of both extrinsic (caspase 8, caspase 3) and intrinsic pathways of apoptosis (caspase 9, Bid, Bim, Bax) showed that mapatumumab killing was dependent on the extrinsic pathway of apoptosis only: only silencing of caspase 8 or caspase 3 inhibited mapatumumab killing. Altogether, these data show that killing through TRAIL receptors is differentially regulated by p53 in myeloma cells, positively for TRAILR2 but negatively for TRAILR1. Interestingly, myeloma cells with an abnormal p53 that are more resistant to all drugs are more sensitive than wt ones to killing through TRAILR1 making this pathway very attractive for p53 deficient myeloma cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ablation of CD28-86 Signaling Results in Induction of Both Caspase-Dependent and Caspase-Independent Cell Death in Myeloma Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4726-4726
Author(s):  
Catherine M Gavile ◽  
Ajay K. Nooka ◽  
Sagar Lonial ◽  
Kelvin P Lee ◽  
Lawrence H. Boise
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Family Members ◽  
Myeloma Cell ◽  
Chemotherapeutic Agents ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Survival Mechanisms ◽  
Survival Signals

Abstract Multiple myeloma is a disease of long-lived plasma cells (LLPCs), and is the 2nd most common hematologic malignancy. In recent years, pharmacologic advances have led to an increase in median and overall survival rates. However, the disease remains incurable for the majority of patients, and research on the underlying survival mechanisms of myeloma cells is relevant for discovering new therapeutic approaches that could eventually lead to a cure. Myeloma cells retain most of the physiological characteristics of their normal counterpart – the LLPC. They secrete antibodies, express CD138, and home and reside in the bone marrow, where they are heavily reliant on growth and survival signals from the stromal microenvironment. Recent reports have shown that the CD28-86 costimulation pathway is important for the generation and survival of LLPCs. Consistent with a pro-survival function, previous studies have demonstrated that CD28 and CD86 high expression are poor prognostic indicators for myeloma patients. Additionally we have shown that CD28 signaling mediates resistance to different chemotherapeutic agents. To better understand the role of CD28 and CD86 in myeloma we have been studying the effects of loss of expression or blockade in myeloma cell lines and patient samples. We have shown that myeloma cells also require CD28-86 signaling for their survival, as knockdown of either CD28 or CD86 via shRNAs, or blockade with CTLA4Ig (Abatacept), led to cell death in 5 myeloma cell lines and 1 patient sample. We have also shown that CD28-86 signaling regulates expression of integrins (β7, β1) that play important roles in cell-cell or cell-matrix interactions that facilitate cell growth and survival. Taken together, our previous work indicates that the CD28-86 signaling pathway plays an important role in maintaining myeloma cell viability. Interestingly, our data indicate that CD86 relays a survival signal that is different from its function as a CD28 ligand. Overexpression of an shRNA-resistant CD86 (CD86FLm) protected against CD86 silencing, while overexpression of CD86TLm (where the intracellular domain of CD86 has been deleted) does not, indicating that the cytoplasmic tail of CD86 plays a role in myeloma cell survival. In order to determine the survival mechanisms mediated by this signaling pair, we investigated different pathways known to protect myeloma cells from pro-apoptotic signals.We first demonstrated that exogenous IL-6, a myeloma growth and survival factor, cannot protect against cell death from CD28 or CD86 silencing, suggesting that the CD28-CD86 pathway is distinct from IL-6 signaling and provides survival signals that are complementary to IL6 receptor signaling. In contrast, overexpression of pro-survival Bcl-2 family members protects against cell death induced by silencing of CD28 and CD86. However, when we performed expression analyses (RNA-seq, pRT-PCR and Western blot), no consistent significant changes were observed in any of the Bcl-2 family members following CD28 or CD86 knockdown. Since Bcl-2 proteins can inhibit both apoptotic and non-apoptotic forms of cell death (e.g. autophagy), we determined if the cell death was caspase dependent. Caspase-3 is activated by CD28 or CD86 silencing or CTLA4Ig treatment. However pan-caspase inhibitors Boc-D-FMK or QVD-Oph can only partially protect against this cell death despite demonstrating a complete blockade of caspase-3 cleavage. Overall, our data show that cell death induced upon ablation of CD28-86 signaling is pleiotropic, as it appears to be both caspase-dependent and caspase-independent. We will present data on the mechanism of non-apoptotic death (autophagy or necrosis). Preliminary data indicates that autophagy is activated by CD28/CD86 silencing. Our data suggest that blocking the CD28-86 pathway may be a viable therapeutic addition to current regimens since it induces myeloma cell death through multiple mechanisms and therefore may not be susceptible to drug resistance that is associated with relapsed/refractory disease. Disclosures No relevant conflicts of interest to declare.

Plasma Levels of SDF-1 and Expression of SDF-1 Receptor on Multiply Myeloma Cells of Human Multiply Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4874-4874
Author(s):  
Caixia Li ◽  
De Pei Wu ◽  
Junjie Cao ◽  
Xiaojin Wu ◽  
Xiao Ma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Levels ◽  
Myeloma Cell ◽  
Migration Ability ◽  
Myeloma Cells ◽  
And Migration ◽  
Cxcr4 Axis ◽  
Healthy Persons

Abstract Multiple myeloma(MM) is a monoclonal expansion of malignant cells with a plasmablast-plasma cell morphology that is almost exclusively localized to the bone marrow, except at the final stages of disease, when they proliferate in the extramedullary area. The mechanisms of the selective homing of MM cells to the bone marrow compartment are poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 contribute to stem cell homing and play a role in trafficking of leukemic cells. In this study we have investigated expression and biological behavior of SDF-1/CXCR4 in MM-derived cell lines and primary MM cells. FACS and RT-PCR analysis was used to study the expression of CXCR4 and ICAM-1(CD54) on the surface of MM cells from 4 IL-6 dependant cell lines (XG1,XG2,XG6 and XG7) and 25 freshly isolated tumor samples from patients with diagnosed MM. Mononuclear cells were purified by positive selection of magnetical and FACS sorting. Chemotaxis assay through transwell bore polycaronate and ELISA assay were employed to monitor the SDF-1, IL-6, and sICAM-1 levels. We found that[circ1]Fresh MM cells and MM cell lines expressed various levels of functional CXCR4 ranging from 23.1% to 77.7%,which was correlated with the in vitro migration ability of MM cells[(23.2±1.08)%, P<0.01]; [circ2]SDF-1 levels in the bone marrow(BM) of MM patients were significantly higher than the those of healthy persons (3489.23±651.63)pg/ml, (2818.57±597.79)pg/ml, P<0.05; but plasma levels of SDF-1 in peripheral blood of MM patients were lower than those of healthy persons[(1973±133)pg/ml, (2334.857±574.92), P=0.062]; [circ3]Plasma levels of PCL(4097.14±680.71) were significantly higher than those of healthy persons, P<0.01. The results firstly demonstrated abnormal expression of SDF-1 and its receptor CXCR4 on Human MM cells, which is closely correlated with the migration of MM cells. Furthermore, we discovered that SDF-1 could up-regulate the expression of ICAM-1 on MM cells; the plasma level of soluble ICAM-1 was correlated with the expression of CXCR4 on MM cells. These findings suggested that SDF-1/CXCR4 axis play a key role on the trafficking of MM cells via mediating the effect of adhesion molecules. Moreover, we observed higher plasma levels of IL-6 in PB of 60% MM patients compared with those of healthy individuals. Finally, the levels of IL-6 were closely correlated with SDF-1 levels (γ=0.8, P<0.01), These data indicated that in the IL-6-dependent myeloma cell lines or fresh myeloma samples and myeloma cell growth triggered by SDF-1 maybe due to up-regulation of autocrine and paracrine IL-6 by myeloma cells and stromal cells in BM. The results suggested that the expression of CXCR4 have an essential role in the proliferation and migration of myeloma cells in patients with multiple myeloma.In conclusion, MM cells expressed various levels of functional CXCR4, which were correlated with the migration ability of MM cells in vitro; SDF-1/CXCR4 axis plays a key role in the trafficking of MM cells via mediating the effect of adhesion molecules; The plasma levels of IL-6 closely correlated with SDF-1 plasma levels, myeloma cell growth triggered by SDF-1 may be due to up-regulation of autocrine and paracrine IL-6 by myeloma cells and stromal cells in BM. All these suggested that the expression of CXCR4 play an essential role in the proliferation and migration of myeloma cells in patients with multiple myeloma.

Inhibition of p38α MAPK Reduces Tumor Burden, Prevents the Development of Myeloma Bone Disease, and Increases Survival in the 5T2 and 5T33 Murine Models of Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3436-3436 ◽  
Author(s):  
Karin Vanderkerken ◽  
Satya Medicherla ◽  
Les Coulton ◽  
Benjamin Van Camp ◽  
Andy Protter ◽  
...  
Keyword(s):  
Bone Disease ◽  
Critical Role ◽  
Disease Free Survival ◽  
Myeloma Cell ◽  
Bone Lesions ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Myeloma Bone Disease ◽  
Osteolytic Bone Disease

Abstract The bone microenvironment plays a critical role in supporting the growth and survival of myeloma cells and the development of osteolytic bone disease. Signalling through p38 α MAPK mediates synthesis of myeloma cell survival factors by stromal cells; whereas, inhibiting p38 α MAPK reduces myeloma cell proliferation and inhibits osteoclast formation in vitro. However, it is unclear whether p38 α MAPK inhibition will prevent the growth and survival of myeloma cells and the bone disease in vivo. The aim of this study was to determine whether SCIO-469, a selective p38 α MAPK inhibitor, would inhibit myeloma growth and prevent the development of bone disease in the 5TMM syngeneic models of myeloma. Treatment of 5TMM cells, in vitro, with SCIO-469 resulted in a clear inhibition of p38 phosphorylation, as assessed by Western blotting and an inhibition up to 35% of stromal cell induced 5T33MM proliferation. Injection of 5T2MM murine myeloma cells into C57Bl/KaLwRij mice resulted in the growth of myeloma in bone and the development of bone disease characterized by increased osteoclast surface (p<0.05), a reduction in cancellous bone (p<0.01) and the presence of osteolytic bone lesions on x-ray (p<0.01). Treatment of 5T2MM-bearing mice with SCIO-469 (150mg/kg in the diet, therapeutical treatment from paraprotein detection) resulted in a 42% decrease in serum paraprotein and prevented development of osteolytic lesions (p<0.01). Injection of 5T33MM cells into C57Bl/KaLwRij mice also resulted in the development of myeloma but not associated bone disease. Treatment of 5T33MM-bearing mice from the time of tumor cell injection with SCIO-469 resulted in a decrease in serum paraprotein (8.8+/−1.4g/dl to 0.04+/− 0.03g/dl, p<0.001) and a reduction in the proportion of tumor cells in the bone marrow (67 +/− 8.1% to 1.09 +/− 0.58%, p<0.001). Kaplan-Meier analysis demonstrated an increase in disease-free survival (veh=27.5 days vs 96 days, p<0.001) after treatment of the mice with SCIO-469. These data demonstrate that targeting p38 α MAPK with SCIO-469 is associated with an anti-myeloma effect, which indirectly prevents the development of myeloma bone disease.

The Expression of CD56 Is Frequently Accompanied with the Expression of Neuronal Cell Markers and Its Down-Regulation Is Induced by IL-6 in Human Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3534-3534
Author(s):  
Mohd S. Iqbal ◽  
Ken-ichiro Otsuyama ◽  
Karim Shamsasenjan ◽  
Saeid Abroun ◽  
Jakia Amin ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Cell Lines ◽  
Cell Lineage ◽  
Neuronal Cell ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Cell Markers ◽  
Phenotypic Changes ◽  
Human Myeloma Cells

Abstract Human myeloma cells have the marked phenotypic heterogeneity of surface marker expressions, possibly because of loss of PAX-5 expression. Especially, ectopic expression of CD56, one of non-B cell lineage markers, is frequently detected on primary myeloma cells from more than 80% patients with overt myeloma. However, only 2 (NOP2 and AMO1) out of 10 myeloma cell lines were CD56(+). In primary myeloma cells as well as CD56(−) myeloma cell lines, the treatment with forskolin could induce the expression of CD56 in the in vitro culture. In most CD56(+) primary myeloma cells as well as myeloma cell lines, the expressions of neuronal cell markers such as neuron specific enolase (NSE), nestin, β-tubulin III or chromogranin A were found coincidentally. By gene expression profiling, CD56(+) myeloma cell lines showed the marked expressions of transcription factors involved in neuronal cell lineage. On the other hand, addition of IL-6 down-regulated the expression of CD56 in CD56(+) myeloma cell lines in the in vitro culture. In 13 out of 60 patients with overt myeloma, these myeloma cells showed CD56(−) and their values of plasma CRP were significantly increased and MPC-1(−)CD45(+) immature myeloma cells were also increased compared to those in CD56(+) myeloma cases. Therefore, these results indicate that the expression of CD56 is possibly due to phenotypic changes into neuronal cell lineage, and IL-6 can block these phenotypic changes, keeping PAX-5(−) myeloma cells being uncommitted cells to any lineage.

SDF-1 Is Responsible for the Constitutively High NF-kB Activity in Human Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4737-4737
Author(s):  
Abul Islam ◽  
Ken-ichiro Otsuyama ◽  
Jakia Amin ◽  
Saeid Abroun ◽  
Karim Shamsasenjan ◽  
...  
Keyword(s):  
Cell Lines ◽  
Target Genes ◽  
Plasma Cells ◽  
Plasma Concentrations ◽  
Myeloma Cell ◽  
Binding Activity ◽  
Myeloma Cells ◽  
Expression Levels ◽  
Human Myeloma Cells

Abstract The chemokine, stromal cell-derived factor 1 (SDF-1; CXCL12) and its receptor, CXCR4 are considered to be essentially required for plasma cell homing to the bone marrow (BM). It is well known that plasma cells in the BM (long-lived plasma cells) survive for a long time and have the constitutively high NF-kB activity. Since human myeloma cells are considered to be derived from these committed long-lived plasma cells, we investigated the role of SDF-1 on the survival of primary myeloma cells from myeloma patients and the possible relationship with NF-kB activity. First, we confirmed that all primary myeloma cells expressed CXCR4 but not CCR9 or CCR10 receptors on their surface and the levels of CXCR4 expression apparently correlated with maturity of BM plasma cells; mature myeloma cells (MPC-1+) as well as polyclonal plasma cells expressed higher levels of CXCR4 than those on immature myeloma cells (MPC-1-). The production of SDF-1 was found strongly in BM stromal cells but not in primary myeloma cells as well as myeloma cell lines. On the other hand, high DNA binding activity of NF-kB was constitutively detected in primary myeloma cells as well as myeloma cell lines, and these NF-kB activities significantly correlated with the expression levels of CD54 on their surface, for CD54 gene is one of the strict NF-kB target genes. Based on the expression levels of CD54 protein, interestingly, primary myeloma cells showed weaker NF-kB activities than those in monoclonal plasma cells from MGUS and polyclonal plasma cells from polyclonal gammopathy. Plasma concentrations of SDF-1 were also significantly correlated to the expression levels of CD54 on primary myeloma cells significantly (P<0.01). Furthermore, it was confirmed that addition of SDF-1 significantly increased the expression levels of CD54 in the in vitro culture of primary myeloma cells. Therefore, these results indicate that SDF-1 is responsible for high expression levels of CD54 and possibly the constitutively high NF-kB activity in primary myeloma cells.

Nocodazole Induces Myeloma Cell Death by Disrupting the Microtubular Network, Resulting in G2/M Arrest

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3673-3673
Author(s):  
Rentian Feng ◽  
Jorge A Rios ◽  
Markus Mapara ◽  
Suzanne Lentzsch
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Caspase 8 ◽  
Inhibition Rate ◽  
Growth And Survival ◽  
Microtubular Network

Abstract Patients with relapsed multiple myeloma (MM) previously treated with bortezomib and lenalidomide often fail to respond to further therapies. To identify potential new treatment approaches for MM, we used Luminex technology to screen a library of 1,120 compounds provided by the Multiple Myeloma Research Foundation. By multiplex cytokine array, we identified benzimidazoles including the anthelmintics mebendazole, fenbendazole, albendazole, nocodazole and pyrvinium pamoate, as inhibiting the production of cytokines essential for MM cell growth and survival, such as IL-6 (inhibition rate 40–70%), MIP-1α (inhibition rate 65–75%), VEGF (inhibition rate 75%), and soluble IL-6R (inhibition rate 40–52%). Consequently, these anthelmintics demonstrated dose-dependent inhibition of myeloma cell (RPMI-8226, H929, U266 and MM1S) proliferation. The lead compound, nocodazole, caused nuclear fragmentation and caspase-8 activation in MM cell lines and primary CD138+ cells in dose- and time-dependent fashion (IC50: 30–60 nM). Importantly, growth and survival signals provided by bone marrow stromal cells in bone marrow co-cultures failed to protect MM cells from nocodazole-induced cell death. In the apoptotic cells, caspase-8 was more activated than caspase-9, suggesting that mitochondrial signaling is not a major apoptotic pathway. Cell cycle analysis indicated that G2/M cell cycle arrest reached a peak at 17 hr. Sub-G1 proportion was strongly increased after treatment for 24 hr in all tested cell lines. Electron microscope (EM) and nuclear staining studies consistently showed the accumulation of metaphase cells, and morphologic elongation at 7 hr, at which time G2/M arrest was obvious. Most of the elongated cells had only one nucleus, suggesting that they failed to progress to mitosis due to overall microtubular network disarray. We conclude that nocodazole exposure induced microtubular network disarray with cell elongation, and G2/M arrest with a late stage mitotic block resulting in cell death. Benzimidazoles including nocodazole, traditionally used as antihelmintic drugs, have shown antitumor activity against hepatocellular, lung and adrenocortical carcinoma, and melanoma. In our study, we identified the anthelmintic compound nocodazole as a new anti-myeloma agent. Nocodazole warrants further investigation for its anti-MM effects in vitro and in vivo.

Arsenic Trioxide-Mediated Growth Inhibition of Myeloma Cells Is Associated with An Extrinsic Signaling Pathway Involved in Caspase 3 and Caspase 8.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4899-4899
Author(s):  
Jumei Shi ◽  
Yi Wu ◽  
Siqing Wang ◽  
Xiuqin Meng ◽  
Rong Wei ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Arsenic Trioxide ◽  
Signaling Pathway ◽  
Molecular Mechanism ◽  
Caspase 3 ◽  
Myeloma Cell ◽  
Caspase 8 ◽  
Myeloma Cells

Abstract Abstract 4899 Arsenic trioxide (ATO) is a well-known inhibitor of cell proliferation in certain forms of malignancy and has been successfully used in the treatment of acute promyelocytic leukemia. Preclinical and clinical studies showed that ATO has anti-myeloma effects both as a single agent and in the combination therapy; however, the underlying molecular mechanism remains elusive. This study was performed to evaluate the molecular mechanism underlying its anti-myeloma activities. Cells from OPM2, U266, RPMI8226 myeloma cell lines and patients diagnosed with myeloma (n=6) were cultured with various concentrations of ATO for 4 days. Cell growth and viability were assayed by trypan blue dye exclusion. Cell cycle and apoptosis were analyzed by flow cytometry using CellQuest software and Vybrant Apoptosis Assay Kit. Alterations of the signaling pathways induced by ATO were tested by real-time PCR and western blot. ATO induced potent inhibition of myeloma cell growth compared with untreated control cells. Further investigation showed that ATO down-regulated c-Myc and phosphorylated (p)-Rb, while it up-regulated p53, p21Clip1, and p27Kip1 proteins, resulting in G2/M cell cycle arrest and cell growth inhibition. ATO treatment increased mRNA levels of interferon regulatory factor-1 (IRF-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as well as protein levels of caspase 8 and cleaved caspase 3, indicating involvement of the extrinsic apoptotic pathway. No significant change was detected in the expression levels of Bax, Bcl-xL caspase 9 and Bcl-2, indicating that the intrinsic signaling pathway was not involved. A pan-caspase inhibitor abrogated ATO-induced apoptosis of myeloma cells. Our data suggest that ATO induces apoptosis in MM cells most likely through an extracellular signaling pathway. Disclosures No relevant conflicts of interest to declare.

Inhibition of HIF1A Signaling by a Novel Class of Sulfonanilides for Targeted Treatment of Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2856-2856 ◽  
Author(s):  
Dirk Hose ◽  
Anja Seckinger ◽  
Hartmut Goldschmidt ◽  
Tobias Meiβner ◽  
Blanka Rebacz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Chromosomal Aberrations ◽  
Myeloma Cell ◽  
Proliferation Index ◽  
The Novel ◽  
Myeloma Cells ◽  
Signaling Inhibitors ◽  
Exposure Levels

Abstract Abstract 2856 Poster Board II-832 BACKGROUND. Molecular profiling of multiple myeloma allows the identification of novel targets, including HIF1A, and evaluation of their expression within large cohorts of patients. We report here the expression of HIF1A in myeloma and for the first time the preclinical testing of 4 members of a novel class of sulfonanilide HIF1A signaling inhibitors. PATIENTS AND METHODS. Expression of HIF1A was assessed using Affymetrix DNA-microarrays in 329 samples of CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3). Proliferation of primary myeloma cells (n=67) was determined by propidium iodine staining. The effect of the novel HIF1A signaling inhibitors ELR510490, ELR510454, ELR510444 and ELR105813 on the proliferation of 12 human myeloma cell lines and the first three on the survival of 5 primary myeloma cell-samples cultured within their microenvironment was tested, and their ability to inhibit HIF1A signaling was examined using a cell-based reporter assay. Studies were also conducted to determine in vitro stability (in plasma and microsomes), as well as single-dose PK (SDPK) parameters and maximum tolerated dose (MTD) levels after dosing in mice. RESULTS. We found (i) HIF1A to be expressed by 95.4% of CD138-purified primary myeloma cell samples from previously untreated patients. (ii) HIF1A expression shows a weak but significant correlation (r=0.3, p<0.001) with a gene expression based proliferation index. (iii) Of the chromosomal aberrations tested, myeloma cells of patients with presence of a translocation t(4,14) show a significantly higher expression of HIF1A (p<0.001) vs. patients without. Myeloma cells of hyperdiploid patients show a significantly lower expression of HIF1A (p=0.02) vs. non hyperdiploid patients. (iii) HIF1A expression does not show a correlation with event-free or overall survival. (iv) The sulfonanilides ELR510490, ELR510444, ELR510454 and ELR105813 completely inhibit proliferation of all tested myeloma cell lines at nM concentrations. (v) The compounds tested, i.e. ELR510490, ELR510444, ELR510454, are active on all primary myeloma cell-samples tested. (vi) The compounds show a pronounced effect on the HIF1A signaling pathway at EC50s of 1-25nM. (vii) Pre-clinical pharmacology data for the compounds ELR510444 and ELR510490 in mice indicate favorable absorption, distribution, metabolism, and excretion (ADME) profiles as well as exposure levels upon dosing at well-tolerated levels that are significantly above the in vitro EC50 in all the cell lines tested. CONCLUSION. HIF1A is expressed in almost all primary myeloma cells. The novel HIF1A signaling inhibitors tested are very active on myeloma cell lines as well as primary myeloma cells and show favorable in vivo profiles with exposure levels in mice significantly higher than the concentrations required for the inhibition of cell proliferation or apoptosis induction in vitro. This class of compounds thus represents a promising weapon in the therapeutic arsenal against multiple myeloma. Disclosures: Rebacz: ELARA Pharmaceuticals: Employment. Lewis:ELARA Pharmaceuticals: Employment. Schultes:ELARA Pharmaceuticals: Employment.

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