OSU-HDAC42, a Novel Histone Deacetylase Inhibitor, Induces Apoptosis in a Caspase-Dependent Manner and Induces p21WAF1/CIP1 and p16 Expression in Multiple Myeloma Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5078-5078
Author(s):  
Valerie L. White ◽  
Shuhong Zhang ◽  
David Lucas ◽  
Ching-Shih Chen ◽  
Sherif S. Farag
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitor ◽  
Myeloma Cell ◽  
Dependent Manner ◽  
In Vitro Activity ◽  
Myeloma Cells ◽  
Deacetylase Inhibitor

Abstract Multiple myeloma (MM) is a neoplastic disorder characterized by accumulation of slowly-proliferating clonal plasma cells. OSU-HDAC42 [a.k.a. (S)-HDAC-42] is a novel histone deacetylase inhibitor that induces apoptosis in various types of cancer cells and is being developed as an anti-cancer therapy in the NCI Rapid Access to Intervention Therapy (RAID) program. In this study, we tested the in vitro activity of OSU-HDAC42 against human MM cells. OSU-HDAC42 induced myeloma cell death, with an LC50 of less than 1.6μM after 48 hours in the four cell lines tested - U266, IM-9, RPMI 8226 and ARH-77 using the MTT assay. OSU-HDAC42 induced cleavage of caspases 3, 8 and 9, as well as polyADP-ribose polymerase (PARP). Addition of the pan-caspase inhibitor Q-VD-OPH before exposure to the drug prevented apoptosis at 48 hours, as determined by Annexin V/propidium iodide staining. These results indicate that OSU-HDAC42 induced apoptosis by a mainly caspase-dependent manner. Bax expression was up-regulated at 24 and 48 hours, while Bcl-2 remains relatively constant. Mcl-1 showed increasing cleavage at increasing doses of OSU-HDAC42. These findings support a mitochondrial pathway of apoptosis. Cell cycle suppressor proteins p21WAF1/CIP1 and p16 were also significantly induced after treatment with the drug, suggesting that OSU-HDAC42 may also acts on pathways to halt cell cycle progression. In addition, the gp130 (signal-transducing) subunit of the IL-6 receptor was down-regulated by OSU-HDAC42 exposure. The tyrosine-phosphorylated form of STAT3, which is phosphorylated by dimerized gp130, was also dramatically reduced following incubation with OSU-HDAC42, supporting the finding that gp130 expression is diminished. As IL-6 is an important growth and survival factor for MM cells, down-regulation of gp130 may be an important mechanism for the activity of OSU-HDAC42 against MM cells. TRAIL, FasL, XIAP, and p53 expression were not affected by OSU-HDAC42. While other HDAC inhibitors have been shown to activate the death receptor pathway or down-regulate XIAP, this was not observed with OSU-HDAC42 in myeloma cells. In conclusion, OSU-HDAC42 has in vitro activity against myeloma cells and acts via activation of caspases, inducing the cell cycle suppressors p21WAF1/CIP1 and p16, as well as interfering with the IL-6 signal transduction pathway.

Phenyhexyl Isothiocyanate, a Novel Inhibitor of Histone Deacetylation and Methylation, Induce Growth Arrest and Apoptosis of Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5033-5033
Author(s):  
Quanyi Lu ◽  
Jane Feng ◽  
Karen Seiter ◽  
Tauseef Ahmed ◽  
Dicky J.W. Chiao ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Hdac Inhibitors ◽  
Hematologic Malignancies ◽  
Myeloma Cell ◽  
Histone Deacetylation ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Human Myeloma Cell Line ◽  
Endothelial Growth Factor

Abstract Histone deacetylase (HDAC) inhibitors are emerging as a promising family of new agents in the therapy of hematologic malignancies. We have shown that phenyhexyl isothiocyanate(PHI) is a novel histone deacetylase inhibitor and can also modulate histone methylation in leukemia cells. In this study we investigated the effect of PHI on human myeloma cell line RPMI8226 in vitro. We have observed that cell proliferation was inhibited by PHI in a dose and time dependent manner. Cell cycle analysis indicated an arrest in G0/G1 phase, and significant apoptosis was detected in PHI treated RPMI8226 cells. The accumulation of P21 and reduced Levels of PCNA were detected. We also examined the effect of PHI on vascular endothelial growth factor (VEGF) production by the myeloma cells. Treatment with 20μM PHI for 24hrs led to a decrease of VEGF concentration by 50% of that secreted by the control cells. VEGF production further decreased to 25% when the cells were exposed to PHI for 48hrs. These data suggest that myeloma cells are sensitive to the novel HDAC inhibitor, and PHI may become a novel agent in multiple myeloma therapy. PHI Inhibit VEGF production by Myeloma Cells PHI Inhibit VEGF production by Myeloma Cells

NVP-LAQ824 is a potent novel histone deacetylase inhibitor with significant activity against multiple myeloma

Blood ◽  
2003 ◽  
Vol 102 (7) ◽  
pp. 2615-2622 ◽  
Author(s):  
Laurence Catley ◽  
Ellen Weisberg ◽  
Yu-Tzu Tai ◽  
Peter Atadja ◽  
Stacy Remiszewski ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Histone Deacetylase ◽  
Hdac Inhibitors ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Significant Activity ◽  
Dependent Manner

Abstract Histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in hematologic malignancies. Here we show that NVP-LAQ824, a novel hydroxamic acid derivative, induces apoptosis at physiologically achievable concentrations (median inhibitory concentration [IC50] of 100 nM at 24 hours) in multiple myeloma (MM) cell lines resistant to conventional therapies. MM.1S myeloma cell proliferation was also inhibited when cocultured with bone marrow stromal cells, demonstrating ability to overcome the stimulatory effects of the bone marrow microenvironment. Importantly, NVP-LAQ824 also inhibited patient MM cell growth in a dose- and time-dependent manner. NVP-LAQ824-induced apoptotic signaling includes up-regulation of p21, caspase cascade activation, and poly (adenosine diphosphate [ADP]) ribose (PARP) cleavage. Apoptosis was confirmed with cell cycle analysis and annexin-propidium iodide staining. Interestingly, treatment of MM cells with NVPLAQ824 also led to proteasome inhibition, as determined by reduced proteasome chymotrypsin-like activity and increased levels of cellular polyubiquitin conjugates. Finally, a study using NVP-LAQ824 in a preclinical murine myeloma model provides in vivo relevance to our in vitro studies. Taken together, these findings provide the framework for NVP-LAQ824 as a novel therapeutic in MM. (Blood. 2003;102:2615-2622)

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.

NSC 338258 Represents a Novel Anti-Neoplastic Agent for the Treatment of Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3469-3469
Author(s):  
Erming Tian ◽  
Terry Landowski ◽  
Owen Stephens ◽  
Shmuel Yaccoby ◽  
Bart Barlogie ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Myeloma Cell ◽  
Current Data ◽  
Apoptotic Pathway ◽  
Synthetic Compound ◽  
Myeloma Cells

Abstract We have shown that high-risk multiple myeloma (MM) can be linked to amplification and overexpression of the cell cycle regulator CKS1B. With this in mind we correlated high expression of this gene in the NCI60 cell line panel with IC50 of over 10,000 anticancer compounds. NSC 338258 (EPED3) was identified in this search. EPED3 is a highly stable, hydrophilic derivative of ellipticine. In vitro, this synthetic compound exhibits drastic cytotoxic activity on myeloma cells, which is unique among most ellipticine derivatives. This efficacy of EPED3 was examined in myeloma cells co-cultured with bone marrow stromal cells. Despite the protective influence of stromal cells, micromolar concentrations of EPED3 were highly effective at killing myeloma cells; however, this lethal activity was exclusive of stromal cells. In co-culture, EPED3-induced cell cycle arrest and massive apoptotic progression appears to be a consequence of its instant impact on cytoplasmic organelles, particularly mitochondria. Disruption of mitochondrial and endoplasmic distribution of cytochrome c initiated the intracellular proteolytic cascade through the intrinsic apoptotic pathway. Effects of EPED3 treatment were further evaluated in myeloma cell lines with selective tolerances to doxorubicin, dexamethasone, and Velcade; EPED3 overcame these acquired drug resistances. In addition, the potency of EPED3 was tested on mononuclear cells isolated from peripheral blood of healthy donors. Under mitogenic stimulation, EPED3 had no significant growth inhibition effects within a range of concentrations killing myeloma cells. Collectively, our current data suggest that EPED3 is an extraordinary agent that, in vitro, targets mitochondrial function to rapidly deplete chemical energy and to initiate apoptosis in myeloma cells at low concentration, while leaving healthy stromal and mononuclear cells unharmed.

Pegylated Recombinant Human Arginase, Rharg-Peg5,000MW, Targets Cell Cycle Machinery in Multiple Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4095-4095
Author(s):  
Delong Liu ◽  
Xianghua Lin ◽  
Quanyi Lu ◽  
Thomas Leung ◽  
Paul N.M. Cheng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Control Cell ◽  
Antitumor Agent ◽  
Myeloma Cell ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell ◽  
G2m Phase

Abstract Arginase has been shown to inhibit growth of human hepatocellular carcinoma by depletion of arginine. We have studied the effects of the pegylated human recombinant arginase (BCT-100, rhArg-peg5,000MW) on RPMI8226 cells, a multiple myeloma cell line. This study showed that three day exposure of the myeloma cells to pegylated rhArg at a concentration of 0.08 IU/ml and 0.48 IU/ml resulted in growth suppression of 10% and 70% respectively, as compared to untreated control. Cell cycle analysis revealed significant decreases in the proportion of cells in both S- and G2M-phase and a concomitant increase of cells in G1-phase in a time- and concentration- dependent manner. We further studied the mechanisms of cell cycle arrest induced by the pegylated rhArg. The pegylated rhArg inhibited both cyclin-dependent kinases CDK2 and CDK4, enhanced the expression of the CDK inhibitor p21, and reduced the expression of cyclinD1, D2, and E. The level of phosphorylated Rb protein was also found to be significantly decreased. The regulators of cell cycle have thus been revealed as targets of pegylated rhArg for myeloma growth arrest. The pegylated rhArg may serve as a novel antitumor agent for multiple myeloma.

Hypoxia Activated Prodrug TH-302 for the Treatment of Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3838-3838
Author(s):  
Jinsong Hu ◽  
Damian R Handisides ◽  
Els Van Valckenborgh ◽  
Hendrik De Raeve ◽  
Eline Menu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Solid Tumors ◽  
Cycle Phase ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Hypoxic Conditions

Abstract Abstract 3838 Poster Board III-774 Hypoxia is known to be linked to increased metastatic potential and a treatment-resistant phenotype leading to rapid progression and poor prognosis in solid tumors. We confirmed previous data[1] on hypoxia in human multiple myeloma (MM) in the 5T33MM syngeneic murine model of MM by using H&E staining and Hypoxyprobe (Pimonidazole) staining on consecutive serial sections from both naive mice and 5T33MMvv diseased mice. We observed a physiological hypoxic situation in MM diseased bone marrow. Given the contribution of hypoxia to tumor progression and drug resistance, a number of hypoxia-targeted therapeutics are under development. TH-302 is a new hypoxia-activated prodrug (HAP) that is currently being evaluated in the clinical trials as monotherapy and in combination with standard chemotherapy regimens for the treatment of solid tumors. The aims of the current study are (1) to demonstrate the effects of TH302 on MM cells in hypoxic conditions, focusing on apoptosis and cell cycle and associated signaling pathways and (2) to evaluate potential therapeutic effects when used in an experimental mouse MM model. We evaluated the effects of TH-302 in vitro on the murine 5T33MMvt cell line and the human LP-1, MMS-1, RPMI-8226, Karpas MM cell lines. Flow cytometry analysis revealed that TH-302 (0.5-50μM) can induce significant Go/G1 cell cycle phase arrest and apoptosis in hypoxic conditions (both 1% and 0% O2) in a concentration dependent manner, in contrast to normoxic conditions (20% O2) (p<0.001). Western blot confirmed that treatment with TH-302 in hypoxic conditions down-regulates cyclin D1/2/3, CDK4/6 and pRb expressions, but CDK2 expression was not disturbed. Furthermore, treatment with TH-302 in hypoxic conditions down-regulates the anti-apoptotic proteins BCL-2 and BCL-xL, as well as up-regulates the expression of three proapoptotic proteins: cleaved caspase-3, 9 and PARP. The expression pattern of Bax was however not influenced. The expression of p21 and p27 decreased in hypoxic condition after treatment with TH-302. Further studies conducted in the 5T33MMvv mouse model demonstrated that animals treated prophylactically with TH-302 (12.5 mg/kg, 25 mg/kg and 50 mg/kg, i.p.) for 3 weeks from day 1 after tumor inoculation showed decreased serum paraprotein (12.5 mg/kg, 32% decrease, p<0.05; 25 mg/kg, 77% decrease, p<0.001; 50 mg/kg, 54% decrease, p<0.001), compared to vehicle-treated 5T33MMvv mice (n=10). The frequency of apoptotic multiple myeloma cells in bone marrow sections was also significantly increased (12.5 mg/kg, 2.5 fold, p<0.05; 25mg/kg, 2.1 fold, p<0.05; 50mg/kg, 3.1 fold, p<0.01). Treatment with TH-302 resulted in no adverse events, any observable detriment to the mice or weight loss (p>0.05). In conclusion, these results show that hypoxia-activated treatment with TH-302 activates apoptosis and induces cell cycle arrest in MM cells, under hypoxic conditions, both in vitro and in vivo and therefore represents a promising therapeutic approach for multiple myeloma. Reference [1] Simona Colla, Paola Storti, Gaetano Donofrio, et al. Hypoxia and Hypoxia Inducible Factor (HIF)-1α in Multiple Myeloma: Effect on the Pro-Angiogenic Signature of Myeloma Cells and the Bone Marrow Microenvironment, 50th ASH annual meeting, http://ash.confex.com/ash/2008/webprogram/Paper13156.html Disclosures: Handisides: Treshold Pharmaceuticals: Employment. Liu:Treshold Pharmaceuticals: Employment. Sun:Treshold Pharmaceuticals: Employment. Hart:Treshold Pharmaceuticals: Employment. Vanderkerken:Treshold Pharmaceuticals: Research Funding.

Significant in-Vitro Activity of a Novel JAK2 Inhibitor in Multiple Myeloma Associated with Preferential Cytotoxicity for CD45+ Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2848-2848
Author(s):  
Vijay Ramakrishnan ◽  
Jessica Haug ◽  
Teresa Kimlinger ◽  
Timothy Halling ◽  
Linda Wellik ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Research Funding ◽  
Myeloma Cell ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Myeloma Cells ◽  
Stat Pathway

Abstract Abstract 2848 Poster Board II-824 Background: Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. IL-6 is a critical cytokine involved in myeloma cell proliferation and survival and exerts its activity primarily through the JAK/STAT pathway. In addition to IL6, other cytokines are also believed to cross talk with the JAK/STAT pathway, making it a crucial interface for survival signals. It has been implicated in myeloma cell interaction with the microenvironment and resistance to apoptotic stimuli from different drugs, and represents a potential therapeutic target. We examined the pre-clinical activity of a novel JAK2 tyrosine kinase inhibitor TG101209. Methods: TG101209 (N-tert-butyl-3-(5-methyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-ylamino)-benzenesulfonamide) was synthesized by TargeGen Inc. (San Diego, CA, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and using Apo2.7 in primary patient cells. CD45 expression was estimated using flow cytometry and cells were gated by their CD45 expression to assess differential effects of the drug. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: TG101209 resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 48 hours, with minimal increase seen up to 96 hours of incubation. At 48 hours of incubation, the median inhibitory concentration was between 2 and 4uM with similar IC50 seen for myeloma cell lines sensitive or resistant to conventional therapies. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Increasing doses of IL6 was not able to rescue the cells from the drug. Dose dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Addition of the pan-caspase inhibitor zvad-fmk did not prevent drug-induced apoptosis confirming non-caspase mediated mechanisms of cell death as well. Primary myeloma cells from several patients were treated with increasing doses of the drug and IC50 similar to cell lines were seen in 8/10 patient samples tested. Interestingly, evaluation of U266 cell lines, which have a mix of CD45+ and negative cells as well as primary patient cells demonstrated more profound cytotoxicity and anti-proliferative activity of the drug on the CD45+ population relative to the CD45- cells. Immunoblotting studies demonstrated significant down regulation of IL-6 induced pSTAT3 with minor effects on the pERK and pAkt. The effect on pSAT3 was sustained compared to that on pERK and pAkt. This was accompanied by significant down regulation of Bcl-xL. Studies in a mouse model of myeloma are planned. Conclusion: These studies demonstrate significant in-vitro activity of JAK2 inhibition in multiple myeloma. In particular, the preferential targeting of CD45 cells, considered to reflect the proliferative compartment in myeloma holds out the promise for more sustained impact on the disease from a therapeutic standpoint. This is likely explained by the increased sensitivity of the CD45 cells to cytokines as a result of higher expression of different cytokine receptors as has been previously shown. This leads to increased activity of and dependence of the cells on the JAK-STAT pathway and likely explains the increased effect of the pathway inhibition. These studies form the framework for clinical evaluation of the drug in the setting of myeloma. Disclosures: Kumar: CELGENE: Research Funding; MILLENNIUM: Research Funding; BAYER: Research Funding; GENZYME: Research Funding; NOVARTIS: Research Funding.

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