YM155, a Survivin Suppressant, Induces Cell Death Via Suppression Of c-Myc Expression In Multiple Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1667-1667
Author(s):  
Shigeki Ito ◽  
Maki Asahi ◽  
Ryousei Sasaki ◽  
Tatsuo Oyake ◽  
Hideto Tamura ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Conflicts Of Interest ◽  
Mrna Level ◽  
Inhibitor Of Apoptosis ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Inhibitor Of Apoptosis Protein ◽  
Apoptosis Protein

Abstract Survivin is a member of the inhibitor of apoptosis protein (IAP) family with its dual roles in mitosis and apoptosis, and emerges as an attractive target for cancer therapy. Recent reports have demonstrated that survivin overexpression is associated with drug resistance and poor outcome in hematological malignancy including multiple myeloma (MM). YM155, a novel molecular targeted agent, suppresses survivin through the inhibition of transcription. However, the effect of this agent on MM cells has not been elucidated. In this study, we investigated the effect of YM155 on proliferation and survival of five human MM cell lines. YM155 inhibited the proliferation of these cells in a dose- and time-dependent manner (IC50 = 10 nM in 3 and 100 nM in 2 cell lines, respectively). Annexin V assay showed that YM155 induced apoptosis in these cells. To better understand these effects of YM155 on MM cells, we evaluated the intracellular signaling and apoptosis-associated protein status. Immunoblot analyses showed that YM155 reduced not only survivin but also myeloid cell leukemia sequence 1 (Mcl-1) and X-linked inhibitor of apoptosis protein (XIAP) expression. We also observed the activation of caspase-3 and poly(ADP-ribose) polymerase in YM155-treated cells, indicating that YM155 induces caspase-dependent apoptosis. YM155 did not affect phosphorylation status of Erk1/2 and STAT3. Interestingly, we found that YM155 suppressed c-Myc and interferon regulatory factor 4 (IRF4) expression, both of which are recognized as an important oncogene in the pathogenesis of MM. In addition, c-Myc and IRF4 protein levels were reduced at 6 and 12 hours after treatment with YM155, respectively. As IRF4 and c-Myc form a positive feedback loop in myeloma cells, this observation indicates that c-Myc inhibition by YM155 treatment might lead to subsequent inhibition of IRF4 expression, and thus raises the possibility of YM155 target for c-Myc rather than IRF4. We next examined the mechanism of downregulation of c-Myc in RPMI8226 cells. Real-time quantitative RT-PCR assay showed that YM155 treatment reduced c-Myc mRNA level. On the other hand, proteasome inhibitor did not prevent the suppression of c-Myc expression by YM155 treatment. These Results suggest that YM155 transcriptionally at least in part represses c-Myc in RPMI8226 cells. In conclusion, YM155 suppresses cell proliferation and survival in MM cells in part via not only inhibiting anti-apoptotic proteins such as survivin, Mcl-1 and XIAP but also repressing c-Myc oncogene. Further study is needed to clarify the molecular mechanism of downregulation of c-Myc induced by YM155. Our Results may provide a platform for clinical trials of YM155 in MM. Disclosures: No relevant conflicts of interest to declare.

Targeting Syk (spleen tyrosine kinase) Inhibits the Proliferation, Migration and Viability of Multiple Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4071-4071
Author(s):  
Ruth-Miriam Körber ◽  
Stefanie AE Held ◽  
Solveig Daecke ◽  
Marie von Lilienfeld-Toal ◽  
Anita Bringmann ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Map Kinase ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Conflicts Of Interest ◽  
B Cell Lymphoma ◽  
High Dose ◽  
Dependent Manner

Abstract Abstract 4071 Introduction: Multiple myeloma (MM) is a malignant plasma cell disorder characterized by clonal expansion of single plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of active drugs like bortezomib or lenalidomide that have been associated with improved survival MM is still incurable. Thus, the identification of novel molecular targets and additional treatment options are warranted. In B-cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma, the inhibition of the tyrosine kinase Syk gave promising preclinical and first clinical results.In our study, we analyzed the potential of Syk as a target in MM. Methods: The MM cell lines AMO-1, U266, MMS-1 and RPMI8226 and primary MM cells were treated with the Syk-inhibitors BAY61-3606 or piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Results: Incubation of MM cell lines with Syk-inhibitors resulted in a reduced proliferation and and SDF-1 induced migration, that was accompanied by a concentration dependent inhibition of the MAP kinase signaling characterized by reduced phosphorylation of ERK an p38 molecules. Furthermore, the nuclear localized expression of the NF-kB members RelA and RelB was inhibited in treated cells. In addition, Syk inhibition induced apoptosis in MM cell lines and primary MM cells in a dose-dependent manner as demonstrated by flow cytometry, caspase-3 activity and PARP-1 cleavage. While there was no effect on the expression of xIAP, survivin or MCL-1, Syk inhibition reduced the expression of pro-apoptotic Bcl-2 and Bcl-xl molecules and increased the release of cytochrome c, indicating that the apoptotic cell death is mediated via the internal mitochondrial pathway. Combination of piceatannol with lenalidomide and orally bioavailable dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235but not with bortezomib or dexamethasone enhanced the cytotoxic effects of the compound. In line with the results from previous experiments the addition of MAPK inhibitors PD98059, SP600125, U0126, SB202190 and SB203580 to piceatannol further increased the efficacy of Syk inhibition. Conclusions: Our results show that Syk inhibition might represent a promissing new treatment option in MM with an increased efficacy when combined with lenalidomide or MAP kinase inhibitors. Disclosures: No relevant conflicts of interest to declare.

X-linked inhibitor of apoptosis protein (XIAP) inhibition in systemic sclerosis (SSc)

2021 ◽  
pp. annrheumdis-2020-219822
Author(s):  
Christina Bergmann ◽  
Ludwig Hallenberger ◽  
Sara Chenguiti Fakhouri ◽  
Benita Merlevede ◽  
Amelie Brandt ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Transforming Growth Factor Beta ◽  
Topoisomerase I ◽  
Transforming Growth Factor ◽  
Inhibitor Of Apoptosis ◽  
Dependent Manner ◽  
Cultured Fibroblasts ◽  
Multifunctional Protein ◽  
Inhibitor Of Apoptosis Protein ◽  
Apoptosis Protein

ObjectiveX-linked inhibitor of apoptosis protein (XIAP) is a multifunctional protein with important functions in apoptosis, cellular differentiation and cytoskeletal organisation and is emerging as potential target for the treatment of various cancers. The aim of the current study was to investigate the role of XIAP in the pathogenesis of systemic sclerosis (SSc).MethodsThe expression of XIAP in human skin samples of patients with SSc and chronic graft versus host disease (cGvHD) and healthy individuals was analysed by quantitative PCR, immunofluorescence (IF) and western blot. XIAP was inactivated by siRNA-mediated knockdown and pharmacological inhibition. The effects of XIAP inactivation were analysed in cultured fibroblasts and in the fibrosis models bleomycin-induced and topoisomerase-I-(topoI)-induced fibrosis and in Wnt10b-transgenic mice.ResultsThe expression of XIAP, but not of other inhibitor of apoptosis protein family members, was increased in fibroblasts in SSc and sclerodermatous cGvHD. Transforming growth factor beta (TGF-β) induced the expression of XIAP in a SMAD3-dependent manner. Inactivation of XIAP reduced WNT-induced fibroblast activation and collagen release. Inhibition of XIAP also ameliorated fibrosis induced by bleomycin, topoI and overexpression of Wnt10b in well-tolerated doses. The profibrotic effects of XIAP were mediated via WNT/β-catenin signalling. Inactivation of XIAP reduces binding of β-catenin to TCF to in a TLE-dependent manner to block WNT/β-catenin-dependent transcription.ConclusionsOur data characterise XIAP as a novel link between two core pathways of fibrosis. XIAP is overexpressed in SSc and cGvHD in a TGF-β/SMAD3-dependent manner and in turn amplifies the profibrotic effects of WNT/β-catenin signalling on fibroblasts via transducin-like enhancer of split 3. Targeted inactivation of XIAP inhibits the aberrant activation of fibroblasts in murine models of SSc.

HIF-1 Supports the Survival of Multiple Myeloma Cells through the Induction of Survivin Gene.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 110-110 ◽  
Author(s):  
Keita Kirito ◽  
Hu Yongzhen ◽  
Kozue Yoshida ◽  
Toru Mitsumori ◽  
Kei Nakajima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cancer Cells ◽  
Cell Lines ◽  
Dependent Manner ◽  
Survivin Mrna ◽  
Myeloma Cells ◽  
Survivin Gene ◽  
Growth And Survival ◽  
Induced Apoptosis

Abstract In spite of the recent development of therapeutic strategies, multiple myeloma (MM) still remains incurable. Several cytokines and chemokines contribute to progression of the disease and acquisition of resistance to chemotherapy. These humoral factors support the growth and survival of myeloma cells through the regulation of transcription factors including NF-κB, Stat3 and FOXO3a. Hypoxia inducible factor-1 (HIF-1) is an important transcription factor that is activated under low oxygen tension and controls dozens of genes involved in angiogenesis, energy production and resistance to apoptosis. Interestingly, HIF-1 is frequently activated in cancer cells even under normoxic condition and it is well established that HIF-1 expression and activation correlates with tumor progression and resistance to cancer treatments. In this study, we investigated whether HIF-1 is involved in the biology of multiple myeloma. To this end, we used three MM cell lines U266, RPMI8226 and KMM-1. After informed consent, we also prepared primary MM cells from bone marrow samples of patients (n=5) using anti-CD138 magnetic beads. Initially, we treated MM cells with insulin-like growth factor-1 (IGF-1) and IL-6, both of which are major growth and survival factors for myeloma cells. Treatment with IGF-1 and, to be a lesser degree, IL-6 clearly enhanced expression of HIF-1α, a subunit of HIF-1, in all three cell lines. Similar results were obtained from isolated primary MM cells. Based on several lines of evidence that survivin, a member of inhibitor of apoptosis (IAP) family protein, is transcriptionally regulated by HIF-1 in breast cancer cells, and that this anti-apoptotic factor is important for growth of MM cells, we examined whether HIF-1 supports the survival of MM cells through the induction of survivin. Quantitative RT-PCR assay revealed that IGF-1 increased survivin mRNA both in MM cell lines and in primary MM cells. In addition, IGF-1 activated survivin gene promoter containing a HIF-1-binding site. To confirm that IGF-1-induced activation of survivin gene is mediated by HIF-1, we treated MM cell lines with echinomycin, an inhibitor of DNA-binding activity of HIF-1. As expected, echinomycin inhibited IGF-1-induced survivin gene expression in a dose-dependent manner. The inhibitor also induced apoptosis of MM cells, and IGF-1 could not rescue the MM cells from echinomycin-induced apoptosis. Furthermore, echinomycin enhanced melphalan-induced apoptosis of MM cells. To further examine the involvement of HIF-1 in IGF-1-induced survivin gene expression, we generated three independent HIF-1α knockdown KMM-1 clones using siRNA system. Survivin mRNA was not detected in the HIF-1α knockdown cells, and these clones easily underwent apoptosis even in the presence of IGF-1, compared to the parental cells. Taken together, HIF-1 plays a pivotal role in survival of MM cells through the induction of survivin gene. In conclusion, HIF-1 might be an attractive therapeutic target for MM.

Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1915-1915
Author(s):  
Unn-Merete Fagerli ◽  
Thorsten Stühmer ◽  
Toril Holien ◽  
Randi Utne Holt ◽  
Ove Bruland ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factors ◽  
Cell Lines ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Transcriptional Response ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Membrane Lipid Biosynthesis As a Potential Therapeutic Target in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1836-1836
Author(s):  
Carolyne Bardeleben ◽  
Alan Lichtenstein
Keyword(s):  
Multiple Myeloma ◽  
Er Stress ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Membrane Lipid ◽  
Dependent Manner ◽  
Kennedy Pathway ◽  
Phosphatidylcholine Synthesis

Abstract Abstract 1836 Phosphatidylcholine (PC) is the most prominent phospholipid in mammalian endoplasmic reticulum (ER) membranes. The rate-limiting step in PC synthesis through the Kennedy pathway is the conversion of phosphocholine + cytidine triphosphate (CTP) to cytidine diphosphocholine, (CDP)-choline, via the enzyme CTP:phosphocholine cytidylyltransferase (CCT) (see figure). Multiple myeloma (MM) cells may be particularly dependent on this biosynthetic reaction because of their high consistent level of ER stress and requirement to continuously replenish ER membranes. Indeed, CCT-null mice have a defect in differentiation of B lymphocytes to plasma cells and deficiencies in Ig synthesis. To test whether this pathway remains critical in survival of malignant MM cells, we exposed MM cell lines to an inhibitor shown to inhibit CCT activity, HexPC. HexPC induced apoptosis in all MM cell lines in a concentration- and time-dependent manner. The addition of lysophosphatidylcholine (LPC), presumably converted to PC independently of the Kennedy pathway, completely rescued MM cell apoptosis. In contrast, similar concentrations of LPC in the same cell lines could not rescue apoptosis induced by bortezomib. An additional intervention to inhibit phosphatidylcholine synthesis, namely inducing pyrimidine starvation, also resulted in MM cell apoptosis and down-regulation of CDP-choline levels. Apoptosis of MM cells induced by HexPC was associated with induction of ER stress as shown by enhanced phosphorylation of IRE1 and eIF-2alpha. This ER stress was also prevented when LPC was added to HexPC although LPC could not prevent similar ER stress induced by bortezomib. These results underscore the importance of this phosphatidylcholine synthesis pathway in MM cells and provide new targets for future therapy. Disclosures: No relevant conflicts of interest to declare.

Inhibition Of PI3K Classia Kinases Using GDC0941 Overcomes Protection Of Multiple Myeloma Cells In The Bone Marrow Microenvironment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3169-3169
Author(s):  
Hugh Kikuchi ◽  
Amofa Eunice ◽  
Maeve McEnery ◽  
Farzin Farzaneh ◽  
Stephen A Schey ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Bone Resorption ◽  
Cell Lines ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Pi3k Pathway ◽  
Dependent Manner ◽  
Dose Dependent

Abstract Despite of newly developed and more efficacious therapies, multiple myeloma (MM) remains incurable as most patient will eventually relapse and become refractory. The bone marrow (BM) microenvironment provides niches that are advantageous for drug resistance. Effective therapies against MM should ideally target the various protective BM niches that promote MM cell survival and relapse. In addition to stromal mesenchymal/myofibroblastic cells, osteoclasts play a key supportive role in MM cell viability. Additionally, 80% of patients develop osteolytic lesions, which is a major cause of morbidity. Increased osteoclast activity is characteristic in these patients and targeting osteoclast function is desirable to improve therapies against MM. Osteoclasts need to form an F-actin containing ring along the cell margin that defines a resorbing compartment where protons and degradative enzymes are secreted for dissolution of bone mineral. Remodelling of F-actin and vesicle secretion are regulated by the class IA PI3K pathway during osteoclastic bone resorption. Additionally, it has recently been shown that inhibition of the class IA PI3K pathway in MM cells with GDC0941 induces apoptosis-mediated killing. We hypothesised that GDC0941 could be used as a therapeutic agent to overcome MM-induced osteoclast activation. GDC0941 inhibited maturation of osteoclasts derived from BM aspirates from MM patients in a dose dependent manner. This correlated with decreased bone resorption of osteoclasts cultured on dentine discs. Exposure of mature osteoclasts to GC0941 resulted in abnormal organisation of larger F-actin rings, suggesting a negative effect on the dynamics of the actin cytoskeleton required for bone resorption. We also found that GDC-0941 can prevent protection of the MM cell lines MM1.S and MM1.R by osteoclasts against killing. GDC-0941 alone blocked MM cell proliferation independently of the presence of BM stromal cells and synergised with other therapeutic agents including Lenalidomide, Pomalidomide, Bortezomid and Dexamethasone. We also found that in the presence of MM cells, Dexamethasone (a drug commonly used alone or in combination with new drugs against MM) induced the proliferation of BM stromal cells and adhesion of MM cells on this protective stroma in a dose dependent manner. Dexamethasone is highly effective at MM cell killing when cells are cultured alone. However, we found that at low doses (below 1 uM) and in the presence of BM stromal cells, Dexamethasone could induce MM cell proliferation. GDC0941 enhanced Dexamethasone killing even in the presence of BM stromal cells by blocking Dexamethasone-induced stromal cell proliferation and adhesion of MM cells on the stroma. Targeting individual the PI3K Class IA isoforms alpha, beta, delta or gamma proved to be a less efficient strategy to enhance Dexamethasone killing. Previous work has shown that efficacy of targeting individual PI3K Class I A isoforms would be low for activation of caspases in MM cells as it would be dependent on relative amounts of isoforms expressed by the MM patient. GDC-0941 also inhibited the proliferation of MM1.R and RPMI8266 MM cell lines, which are less sensitive to treatment to Dexamethasone. Co-culture of MM cells with BM stromal cells induced the secretion of IL-10, IL-6, IL-8, MCP-1 and MIP1-alpha. The dose-dependant increased proliferation of Dexamethasone-treated MM cells in the presence of the BM stroma correlated with the pattern of secretion of IL-10 (a cytokine that can induce B-cell proliferation) and this was blocked by the combination of Dexamethasone with GDC0941. GDC-0941 alone or in combination with Dexamethasone was more efficacious at inducing MM cell apoptosis in the presence of the BM stroma cells vs treatment of MM cells alone. These are very encouraging results as they suggest that GDC-0941 in combination with Dexamethasone would be potentially highly efficacious for targeting MM cells in the BM microenvironment. We are currently performing in vivo data using C57BL/KaLwRij mice injected with 5T33-eGFP MM cells that will be discussed at the meeting. We propose that MM patients with active bony disease may benefit from treatment with GDC0941 alone or in combination with currently used therapeutic drugs against MM. Disclosures: No relevant conflicts of interest to declare.

Translocation of the inhibitor of apoptosis protein c-IAP1 from the nucleus to the Golgi in hematopoietic cells undergoing differentiation: a nuclear export signal-mediated event

Blood ◽  
2004 ◽  
Vol 104 (7) ◽  
pp. 2035-2043 ◽  
Author(s):  
Stéphanie Plenchette ◽  
Séverine Cathelin ◽  
Cédric Rébé ◽  
Sophie Launay ◽  
Sylvain Ladoire ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
B Cell Lymphoma ◽  
Site Directed Mutagenesis ◽  
Inhibitor Of Apoptosis ◽  
Monocytic Cell ◽  
Inhibitor Of Apoptosis Protein ◽  
Apoptosis Protein ◽  
Export Signal

Abstract The caspase inhibitor and RING finger-containing protein cellular inhibitor of apoptosis protein 1 (c-IAP1) has been shown to be involved in both apoptosis inhibition and signaling by members of the tumor necrosis factor (TNF) receptor family. The protein is regulated transcriptionally (eg, is a target for nuclear factor-κB [NF-κB]) and can be inhibited by mitochondrial proteins released in the cytoplasm upon apoptotic stimuli. The present study indicates that an additional level of regulation of c-IAP1 may be cell compartmentalization. The protein is present in the nucleus of undifferentiated U937 and THP1 monocytic cell lines. When these cells undergo differentiation under phorbol ester exposure, c-IAP1 translocates to the cytoplasmic side of the Golgi apparatus. This redistribution involves a nuclear export signal (NES)-mediated, leptomycin B-sensitive mechanism. Using site-directed mutagenesis, we localized the functional NES motif in the caspase recruitment domain (CARD) of c-IAP1. A nucleocytoplasmic redistribution of the protein was also observed in human monocytes as well as in tumor cells from epithelial origin when undergoing differentiation. c-IAP1 does not translocate from the nucleus of cells whose differentiation is blocked (ie, in cell lines and monocytes from transgenic mice overexpressing B-cell lymphoma 2 [Bcl-2] and in monocytes from patients with chronic myelomonocytic leukemia). Altogether, these observations associate c-IAP1 cellular location with cell differentiation, which opens new perspectives on the functions of the protein. (Blood. 2004;104:2035-2043)

Valproic Acid Inihibts Histone Deacetylases and Proliferation and Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5165-5165
Author(s):  
Martin Kaiser ◽  
Ulrike Heider ◽  
Ivana Zavrski ◽  
Jan Sterz ◽  
Kurt Possinger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Valproic Acid ◽  
Cell Lines ◽  
Myeloma Cell ◽  
G1 Phase ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Cycle Arrest ◽  
Dose Dependent

Abstract Multiple myeloma remains an incurable disease in the majority of the patients and novel treatment strategies are urgently needed. A new class of drugs, the histone deacetylase (HDAC) inhibitors take influence in epigenetic modifications and have antiproliferative effects in some malignancies. Valproic acid (VPA) is an anticonvulsant drug and was recently shown to inhibit HDACs and suppress tumor growth. The drug is currently being evaluated in clinical studies in acute myeloid leukemia. Its effects on myeloma cells are unknown. The aim of this study was to evaluate the effects of VPA on proliferation, apoptosis and HDAC inhibition in multiple myeloma cell lines as well as in sorted human bone marrow multiple myeloma cells. Myeloma cell lines, OPM-2, NCI-H929, LP-1, and freshly isolated multiple myeloma cells from bone marrow aspirates were exposed to different concentrations of VPA for 4 to 72 hours. Cell proliferation, cell cycle distribution and apoptosis were assayed in reaction to the treatment. Proliferation decreased noticeably and apoptosis was induced in a dose-dependent manner in multiple myeloma cell lines as well as in freshly sorted primary myeloma cells. After 48 hours of incubation with VPA at 1 mM, approximately 46%, 52% and 25% of OPM-2, NCI-H929 and LP-1 cell lines had undergone specific apoptosis, respectively. Freshly sorted primary bone marrow myeloma cells from patients showed also specific apoptosis. In cell cycle analysis by flow cytometry, the population of cells in the G0/G1 phase increased, whereas cells in the S phase decreased in a time and dose dependent manner. Incubation of the cell line OPM-2, for example, with 1 mM VPA for 48 hours decreased the proportion of cells in the S phase from 39 % to 6 % of the total cell count and increased cells in the G0/G1 phase from 49 % to 85 %. Acetylation of histones and expression of cyclin D1 and the cell cycle regulators p21 and p27 were studied by western blot. Histone acetylation and p21 concentrations increased after VPA treatment whereas levels of p27 remained constant. A decrease in cyclin D1 concentrations was observed. Subapoptotic doses of VPA significantly decreased the production of VEGF in OPM-2 cell line. These data show that treatment with valproic acid effectively inhibits histone deacetylase activity, leading to the accumulation of acetylated histones in multiple myeloma cells. Parallel upregulation of cell cycle inhibitors like p21WAF1 was observed, together with a reduction of cyclin D1 levels. Myeloma cell proliferation was inhibited in a time and dose dependent manner and cell cycle arrest in the G0/G1 phase was induced by VPA treatment. VPA potently induced apoptosis in all human myeloma cell lines as well as in sorted primary multiple myeloma cells in a dose and time dependent manner. These results show for the first time that VPA acts as an HDAC inhibitor in multiple myeloma cells, induces G1 cell cycle arrest, potently inhibits tumor growth and markedly induces apoptosis. In addition to its direct antitumor effect, valproic acid may exert an antiangiogenic effect by reducing VEGF production in myeloma cells. These data provide the framework for clinical studies with valproic acid in multiple myeloma.

Novel Association Between Thyroid Hormones and Multiple Myeloma Cell Proliferation: a MAPK Dependent Activity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2836-2836
Author(s):  
Osnat Ashur-Fabian ◽  
Keren Cohen ◽  
Aleck Hercbergs ◽  
Martin Ellis
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Thyroid Hormones ◽  
Cancer Cells ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cell Surface Receptor ◽  
Dependent Manner ◽  
Santa Cruz ◽  
Myeloma Cells

Abstract Abstract 2836 Poster Board II-812 Background: Multiple myeloma (MM) is a plasma cell neoplasia accounting for more than 10% of hematological malignancies. Since the disease was first described in England around 1850, MM has been very resistant to treatment with common relapses. It has a poor prognosis with a median survival of 3–5 years, despite all treatment approaches. In recent years, evidence has been provided that thyroid hormones (T3 and T4) may play a permissive role in various cancer cells including breast, brain, prostate and lung, enhancing tumor cell proliferation. Deprivation of these hormones decreases cancer cell proliferation and enhances cell death and response rates to chemotherapy and radiation therapy. It was recently discovered that T3 and T4 exert their proliferating actions through binding to aVb3 integrin, a common cell surface receptor, leading to mitogen-activated protein kinase (MAPK) activation and downstream intra cellular and nuclear events. Interestingly, aVb3 expression is increased during tumor progression and a spectrum of cancer cells, including MM, interact with this central integrin for their invasion, spreading and proliferation. In the current study, we hypothesized that that MM cells, similar to other cancer cells, are thyroid hormones sensitive and aimed to further investigate and characterize their effects on cell survival, proliferation and MAPK signaling. In addition, the additive/ supra additive effects of hypothyroid induction in MM cells on bortezomib's activity were evaluated. Methods: Cell lines: MM cell lines, RPMI 8226, U266, ARP1, ARK and CAG are cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS/antibiotics. Reagents and chemicals: Bortezomib (Velcade) is obtained from the hospital pharmacy. T3, T4, tetrac RGD and RGE peptides (Sigma-Aldrich). PE conjugatedb3 monoclonal antibodies (LM609) and mouse IgG are from Chemicon International. phosphorylated MAPK ERK1/2, p38, JunK antibodies are from Cell Signaling (Danvers, MA). Alpha tubulin and PCNA antibodies are from Santa Cruz Biothecnology (Santa Cruz, CA, USA) WST-1 cell proliferation assay: WST-1 (10% final concentration) is incubated at 37°C for 2 h and read using microELISA reader at 440nm. Flow cytometry : Cell cycle: Cells are harvested, fixed and stained with DNA propidium iodide (PI) (50 μg/ml) /RNAse A (10mg/ml) and analyzed for DNA content by FACS. Analysis of apoptosis/necrosis: Cells (105) are incubated with 10 μl Annexin V (FITC conjugated)/5 μl PI and analyzed by FACS (Annexin+/PI-, early apoptosis; Annexin+/PI+, late apoptosis/necrosis). aVb3 in MM cells: Cells are harvested in RPMI 1640 and directly labeled with PE-aVb3 mAbs (10 mg/ml) and analyzed by FACS. Isotype-matched antibody, serves as negative control.Western blotting: Whole cell lysates were separated on 5-8% polyacrylamid gels and analyzed by western blot using antibodies for phosphorylated MAPK ERK1/2, p38, JunK and PCNA.Statistical analyses: Results were analyzed using unpaired students t test. Results: The sensitivity of myeloma cells to thyroid hormones was explored by addition of increasing concentrations of T3 and T4 to several myeloma cell lines. Results demonstrate that T3 and T4 significantly induced proliferation and cell number in these cells in accordance with PCNA protein elevation. This proliferating action was MAPK related, with phosphor ERK1/2, p38 and JunK elevated in a dose dependent manner. Mimicking hypothyroidism in the cells by using condition medium or T4 analog that block thyroid hormones binding to the integrin, tetrac, inhibited proliferation, increased apoptosis/necrosis and produced G2M arrest. Moreover, supra additive/additive “drug sparing” effects of tetrac-botezomib were observed with significant reduction in survival and increase in apoptosis. Discussion: We present here, for the first time in myeloma, indication that myeloma cells, similar tp reports from other cancer types, are thyroid hormones sensitive and that hypothyroidism induction inhibits cell proliferation and sensitizes response to bortezomib. Conclusions: As most MM patients still relapse, new drugs combinations are needed to overcome resistance. Our novel chemosensitizing approach may potentially demonstrate the importance of thyroid hormones status in this disease and may suggest a protective effect of sub clinical hypothyroidism in MM as a useful and unique adjunct for MM therapy. Disclosures: No relevant conflicts of interest to declare.

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