scholarly journals Seliciclib (CYC202 or R-roscovitine), a small-molecule cyclin-dependent kinase inhibitor, mediates activity via down-regulation of Mcl-1 in multiple myeloma

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 1042-1047 ◽  
Author(s):  
Noopur Raje ◽  
Shaji Kumar ◽  
Teru Hideshima ◽  
Aldo Roccaro ◽  
Kenji Ishitsuka ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Kinase Inhibitor ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Down Regulation ◽  
Induce Cell Cycle Arrest ◽  
Caspase Cleavage ◽  
Stat3 Phosphorylation

AbstractCyclin-dependent kinase (CDK) inhibitors have the potential to induce cell-cycle arrest and apoptosis in cancer cells. Seliciclib (CYC202 or R-roscovitine) is a potent CDK inhibitor currently undergoing phase-2 clinical testing in lung and B-cell malignancies. Here we studied the in vitro cytotoxic activity of seliciclib against multiple myeloma (MM) cells. Our data demonstrate that seliciclib has potent cytotoxicity against MM cells that are both sensitive and resistant to conventional therapy as well as primary MM cells from patients. Cell-cycle and Western blot analysis confirmed apoptosis. Importantly, seliciclib triggered a rapid down-regulation of Mcl-1 transcription and protein expression independent of caspase cleavage. Adherence of MM cells to bone marrow stromal cells (BMSCs) induced increased Mcl-1 expression associated with signal transducer and activator of transcription 3 (STAT3) phosphorylation, which was inhibited in a time- and dose-dependent manner by seliciclib. Furthermore, seliciclib inhibited interleukin 6 (IL-6) transcription and secretion triggered by tumor cell binding to BMSCs. Up-regulation of Mcl-1 expression in cocultures was only partially blocked by neutralizing antibody to IL-6, suggesting alternative mechanisms of Mcl-1 modulation by seliciclib. Finally, combination studies of seliciclib with doxorubicin and bortezomib show in vitro synergism, providing the rationale for testing these drug combinations to improve patient outcome in MM.

345 GENE EXPRESSION IN OOCYTES AFTER MEIOTIC INHIBITION WITH THE CYCLIN-DEPENDENT KINASE INHIBITOR BUTYROLACTONE I

2010 ◽  
Vol 22 (1) ◽  
pp. 329
Author(s):  
C. L. V. Leal ◽  
S. Mamo ◽  
T. Fair ◽  
P. Lonergan
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Relative Abundance ◽  
Kinase Inhibitor ◽  
Expression Profiles ◽  
Cyclin Dependent Kinase ◽  
Oocyte Competence ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Bovine Oocytes

Once removed from the follicle, mammalian oocytes resume meiosis spontaneously and progress through breakdown of the germinal vesicle to the matured state at metaphase II. The ability to reversibly inhibit such meiotic resumption has been reported and is a potentially useful method for studying developmental competence acquisition in oocytes as well as in some cases allowing flexibility in an IVF system where oocytes are collected from distant locations or on different days. The aim of the present study was to determine the effect of temporary inhibition of meiotic resumption using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes. Immature bovine oocytes were recovered from the ovaries of slaughtered heifers at a commercial abattoir and assigned to 1 of 4 groups: (1) Control: immature oocytes were collected either immediately or (2) after IVM for 24 h in TCM-199 containing 10 ng mL-1 EGF and 10% (v/v) FCS, (3) Inhibited oocytes collected either 24 h after incubation in the presence of 100 μM BLI in TCM-199 with 3 mg mL-1 BSA or (4) after meiotic inhibition for 24 h followed by in vitro maturation. All cultures were carried out at 38.5°C under 5% CO2 in air and maximum humidity. For mRNA relative abundance analysis, cumulus cells were removed and pools of 10 denuded oocytes were snap frozen in liquid nitrogen and stored at -80°C until use. A total of 42 transcripts, previously reported to be related to cell cycle regulation and/or oocyte competence were evaluated by quantitative real time PCR. Differences in relative abundance were analyzed by ANOVA and Student’s t-test. The majority of transcripts were downregulated (P < 0.05) after IVM in control oocytes (23 out of 42) and the same pattern was observed in inhibited oocytes that were allowed to mature. Twelve transcripts remained stable (P > 0.05) after IVM in control oocytes; of these, only two (PTTG1 and INHBA) did not show the same pattern in inhibited and matured oocytes. Few genes (7) were upregulated after IVM in control oocytes (P < 0.05) and of these, three (PLAT1, RBP1, and INHBB) were not upregulated in inhibited oocytes after IVM. Inhibited oocytes showed similar levels of expression (P > 0.05) as immature control oocytes, except for two genes (LUM and INHBB), which were increased in these oocytes (P < 0.05). The expression profiles of cell cycle genes were mostly unaffected by the BLI treatment. The few genes affected were previously reported as competence-related and could be useful markers of oocyte competence following pretreatment. In conclusion, the changes occurring in transcript abundance during oocyte maturation in vitro were to a large extent mirrored following inhibition of meiotic resumption prior to IVM and subsequent release from inhibition and maturation. CLV Leal was supported by CNPq, Brazil (PDE 201487/2007-1); Supported by Science Foundation Ireland (07/SRC/B1156).

Capsaicin induces G1/S cell cycle arrest leading to apoptosis of multiple myeloma cells through suppression of STAT3 activation and STAT3-regulated gene products

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20039-20039
Author(s):  
M. Bhutani ◽  
A. K. Pathak ◽  
G. Sethi ◽  
B. B. Aggarwal
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Hot Pepper ◽  
Dependent Manner ◽  
Antiapoptotic Proteins ◽  
Cycle Arrest ◽  
Stat3 Phosphorylation ◽  
Synergistic Cytotoxicity

20039 Background: Agents that can block activated STAT3, a central player for proliferation, cell survival and chemoresistance, have a potential as therapeutic agents for the treatment of Multiple Myeloma (MM). Capsaicin, a spicy component of hot pepper, is a homovanillic acid derivative that preferentially induces certain cancer cells to undergo apoptosis. We have previously shown that Capsaicin blocked activation of NF-kappa B in human myeloid ML-1a cells. In this study we evaluated the effect of Capsaicin on STAT3 in MM cells. Methods: We used U266, a well-characterized MM cell line, which constitutively expresses activated STAT3. MM cells treated with Capsaicin were subjected to western blot analysis with specific antibodies to STAT3, tyrosyl phosphorylated STAT3 and STAT5. The effect of Capsaicin on nuclear-cytoplasmic compartment of STAT3 was studied by immunocytochemistry. The antiproliferative effect of Capsaicin was determined by the MTT assay and the effect on the cell cycle was determined by flowcytometry. Apoptosis of cells was measured using the Live and Dead assay. To determine the downstream targets like antiapoptotic proteins (Bcl-xL, Bcl-2, and Survivin), and cell cycle-regulators (cyclin D1) immunoblot analysis of Capsaicin treated cells was performed. Results: Capsaicin suppressed the constitutive activation of STAT3 in human MM cells in a dose- and time-dependent manner, prior to cell death. Capsaicin’s effect on STAT3 was specific as STAT5 was unaffected. Capsaicin depleted nuclear pool of STAT3 in U266 cells. Abrogation of constitutive STAT3 phosphorylation in MM cells induced G1 cell cycle arrest. The antiapoptotic proteins BCl-xl, suvivin, cyclin D1, and Bcl-2, which are encoded in target genes of STAT3, were down regulated by Capsaicin, followed by induction of apoptosis through activation of caspase-3. We further demonstrated that low dose combined Capsaicin and thalidomide/ bortezomib treatment triggered synergistic cytotoxicity. Conclusions: These findings suggest that the antitumor activity of Capsaicin is at least partially due to inhibition of STAT3 pathway and provide a basis for potential application of Capsaicin for treatment of relapsed and refractory MM. No significant financial relationships to disclose.

Unscheduled re-entry into the cell cycle induced by NGF precedes cell death in nascent retinal neurones

2000 ◽  
Vol 113 (7) ◽  
pp. 1139-1148 ◽  
Author(s):  
J.M. Frade
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Cycle Progression ◽  
Kinase Inhibitor ◽  
Ganglion Cells ◽  
Neurotrophin Receptor ◽  
Dependent Manner ◽  
Apoptotic Effect ◽  
Induced Apoptosis

During their early postmitotic life, a proportion of the nascent retinal ganglion cells (RGCs) are induced to die as a result of the interaction of nerve growth factor (NGF) with the neurotrophin receptor p75. To analyse the mechanisms by which NGF promotes apoptosis, an in vitro culture system consisting of dissociated E5 retinal cells was established. In this system, NGF-induced apoptosis was only observed in the presence of insulin and neurotrophin-3, conditions that favour the birth of RGCs and other neurones expressing the glycoprotein G4. The pro-apoptotic effect of NGF on the G4-positive neurones was evident after 10 hours in vitro and was preceded by a significant upregulation of cyclin B2, but not cyclin D1, and the presence of mitotic nuclei in these cells. Brain-derived neurotrophic factor prevented both the increase of cyclin B2 expression in the G4-positive neurones and the NGF-induced cell death. Finally, pharmacologically blocking cell-cycle progression using the cyclin-dependent kinase inhibitor roscovitine prevented NGF-induced cell death in a dose-dependent manner. These results strongly suggest that the apoptotic signalling initiated by NGF requires a driving stimulus manifested by the neuronal birth and is preceded by the unscheduled re-entry of postmitotic neurones into the cell cycle.

Primitive Hematopoietic Cells Resist HIV-1 Infection Via P21Waf1/Cip1/Sdi1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 490-490
Author(s):  
Jie Lin Zhang ◽  
Clyde S. Crumpacker ◽  
David T. Scadden
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Hematopoietic Cells ◽  
Surrogate Marker ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Hematopoietic Stem ◽  
Hiv 1

Abstract Hematopoietic stem cells are resistant to HIV-1 infection. We have identified a novel mechanism by which the cyclin-dependent kinase inhibitor, p21Waf1/Cip1/Sdi1 (p21), known for its regulation of stem cell pool size (1,2), restricts HIV-1 infection of primitive hematopoietic cells in a non-cell cycle dependent manner. Knocking down p21 by siRNA increased HIV-1 infection and induction of p21 expression by phorbol ester (TPA) blocked HIV-1 replication. P21 did not affect the overall levels of cDNA synthesis, but significantly blocked viral integration and resulted in marked increase in 2-LTR circles, a surrogate marker of abortive integration. Consistent with these observations, p21 coimmunoprecipitated with viral integrase and both were detected in the preintegration complex (PIC). Furthermore, silencing p27Kip1 and p18INK4C, cyclin dependent kinase inhibitors related to p21 that affect cell cycle, revealed no impact on viral DNA integration. A closely related dual-tropic lentivirus with a distinct integrase, SIVmac-251 and the other cell-intrinsic inhibitors of HIV-1, Trim5a, PML, Murr1, and IFN-a were unaffected by p21. These results indicate a new function for p21, participating in prevention of HIV integration into the cellular genome. Therefore p21 is an endogenous cellular component in stem cells that provides a unique molecular barrier to HIV-1 infection and may explain the basis for these cells being an uninfected ‘sanctuary’ in HIV disease.

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.

The Dual PIM/PI3-K Inhibitor Ibl-202 Overcomes Microenvironmental Mediated Resistance in Multiple Myeloma and Prevents PIM1 Induced CXCR4 Upregulation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5350-5350
Author(s):  
Mairead Reidy ◽  
Marianne VanDijk ◽  
Michael O'Neill ◽  
Michael O'Dwyer
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Surface Expression ◽  
Down Regulation ◽  
Pim Kinases ◽  
Ic50 Values ◽  
And Migration

Abstract Background: The interaction of multiple myeloma (MM) cells with bone marrow (BM) cells along with factors in the BM milieu such as chemokines and cytokines play a crucial role in both progression of MM and drug resistance. Activation of the PI3-K/Akt survival pathway is a characteristic of both human MM cell lines and patient samples. This activation can be linked to BM microenvironmental signalling and use of proteasome inhibitors in treatment, suggesting this as a crucial point of therapeutic intervention to abrogate growth and survival signals in MM. However, the efficacy of such therapeutics has been modest and is likely to be compromised by the stimulation of compensatory signalling pathways, such as the PIM kinases, which like the PI3-K/Akt pathway are also induced by BM microenvironmental influences and share similar downstream targets. These proto-oncogenic kinases are constitutively active and play an important role in proliferation and survival in MM. The influence of these kinases on homing and migration has been observed in other malignancies, this has yet to be reported in MM. Here we report the effects of a dual inhibitor of PIM/PI3-K, IBL-202, and provide novel insights into effects on cell survival, signaling and migration. Methods: We investigated the effect of IBL-202 against a panel of MM cell lines (MM.IS, NCI-H929s, KMS11 and RPMI-8226) and primary MM patient samples. The in vitro efficacy of IBL-202 was compared to that of single pan-PIM inhibitors pPIMi and AZD1208 and also the pan-PI3-K inhibitor GDC-0941. Apoptosis was measured with AnnexinV staining and cell cycle analysed with Edu/DAPI staining. To mimic BM microenvironmental conditions MM cells were cultured under hypoxic conditions (1% O2) and in co-culture with the human stromal cell line HS5. Surface expression of CXCR4 was assessed in MM cell lines by flow cytometry. PIM kinases, pCXCR4 and downstream targets of PIM/PI3-K were examined by western blot. Transwell migration assays were carried out in the presence of 50ng SDF-1α for 4h @ 37o C. Results: Simultaneous inhibition of PIM and PI3-K using IBL-202 in vitro was significantly more potent at inducing apoptosis than GDC-0941, pPIMi or AZD1208 in all MM cell lines tested. IC50 values were under 1μM for IBL-202 at 48h whilst in comparison the pan PIM inhibitors pPIMi and AZD1208 scored IC50 values between 5 and 10μM. The IC50 for GDC-0941 was on average 5μM (Figure 1). At the molecular level there was a notable decrease in phosphorylation of known PIM/PI3-K targets Akt (Ser473), Bad (Ser112) and the translational targets S6 (Ser235/236) and 4EBP1 (Thr37/46). The levels of total proteins were unchanged. Treatment with increasing doses of IBL-202 led to a marked reduction in cells in S phase of the cell cycle. These changes were paralled by down regulation of the cell cycle promoting proteins cyclin D1 and c-myc. IBL-202 was also effective in inducing apoptosis in primary MM patient samples (n=4) after just 24h as assessed by Annexin-V staining (Figure 2). To explore the role of the BM microenvironment we co-cultured MM cell lines with HS5s. This led to strong induction of PIM2 in MM cells. While MM cells in this setting were protected from Bortezomib-induced cell death, the apoptotic effect of IBL-202 was enhanced. In a further effort to mimic the tumour microenvironment we cultured MM cell lines in hypoxia. This may be of particular relevance as Pim-1 has been reported to be a pivotal regulator involved in hypoxia-induced chemoresistance. MM cells were further sensitised to IBL-202 in hypoxia. In addition, hypoxia increased the surface expression of CXCR4, a chemokine receptor critical for homing of MM cells to the bone marrow, with a concomitant increase in PIM1. Treatment of MM cell lines with IBL-202 reduced the level of PIM1 and CXCR4 Ser339 phosphorylation, along with down regulation of CXCR4 surface expression resulting in reduced migration of MM cells along an SDF-1 gradient. Conclusion: Together these data provide direct evidence of the potency of IBL-202 in MM in conditions that mimic the BM microenvironment. Moreover, they indicate a potential role for PIM kinases in facilitating dissemination and invasiveness of MM by CXCR4 and provide an added rationale for targeting PIM kinases in MM. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures O'Neill: Inflection Biosciences: Employment.

scholarly journals A Conserved Gammaherpesvirus Cyclin Specifically Bypasses Host p18INK4cTo Promote Reactivation from Latency

2015 ◽  
Vol 89 (21) ◽  
pp. 10821-10831 ◽  
Author(s):  
Lisa M. Williams ◽  
Brian F. Niemeyer ◽  
David S. Franklin ◽  
Eric T. Clambey ◽  
Linda F. van Dyk
Keyword(s):  
Common Property ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Genetic Ablation ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Viral Cyclin

ABSTRACTGammaherpesviruses (GHVs) carry homologs of cellular genes, including those encoding a viral cyclin that promotes reactivation from latent infection. The viral cyclin has reduced sensitivity to host cyclin-dependent kinase inhibitorsin vitro; however, thein vivosignificance of this is unclear. Here, we tested the genetic requirement for the viral cyclin in mice that lack the host inhibitors p27Kip1and p18INK4c, two cyclin-dependent kinase inhibitors known to be important in regulating B cell proliferation and differentiation. While the viral cyclin was essential for reactivation in wild-type mice, strikingly, it was dispensable for reactivation in mice lacking p27Kip1and p18INK4c. Further analysis revealed that genetic ablation of only p18INK4calleviated the requirement for the viral cyclin for reactivation from latency. p18INK4cregulated reactivation in a dose-dependent manner so that the viral cyclin was dispensable in p18INK4cheterozygous mice. Finally, treatment of wild-type cells with the cytokine BAFF, a known attenuator of p18INK4cfunction in B lymphocytes, was also able to bypass the requirement for the viral cyclin in reactivation. These data show that the gammaherpesvirus viral cyclin functions specifically to bypass the cyclin-dependent kinase inhibitor p18INK4c, revealing an unanticipated specificity between a GHV cyclin and a single cyclin-dependent kinase inhibitor.IMPORTANCEThe gammaherpesviruses (GHVs) cause lifelong infection and can cause chronic inflammatory diseases and cancer, especially in immunosuppressed individuals. Many GHVs encode a conserved viral cyclin that is required for infection and disease. While a common property of the viral cyclins is that they resist inhibition by normal cellular mechanisms, it remains unclear how important it is that the GHVs resist this inhibition. We used a mouse GHV that either contained or lacked a viral cyclin to test whether the viral cyclin lost importance when these inhibitory pathways were removed. These studies revealed that the viral cyclin was required for optimal function in normal mice but that it was no longer required following removal or reduced function of a single cellular inhibitor. These data define a very specific role for the viral cyclin in bypassing one cellular inhibitor and point to new methods to intervene with viral cyclins.

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.

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