Advanced Glycation End Products (AGEs)-Mediated Cell Growth of Human Acute Myelogenous Leukemia Via Mitogen-Activated Protein (MAP) Kinase Pathways.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2762-2762
Author(s):  
Ju Young Kim ◽  
Hyun Ki Park ◽  
Jin Sun Yoon ◽  
Eun Shil Kim ◽  
Kwang Sung Ahn ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Myelogenous Leukemia ◽  
Dependent Manner ◽  
Glycation End Products ◽  
Mitogen Activated Protein ◽  
Acute Myelogenous ◽  
Dose Dependent

Abstract Advanced glycation end products (AGEs) are products of non-enzymatic glycation/oxidation of proteins/lipids that accumulate slowly during natural aging and at a much accelerated rate in a variety of disorders such as diabetes, renal failure, and Alzheimer’s disease. AGE modifications do not only change the physicochemical properties of the afflicted molecules, but also induce cellular signaling, activation of transcription factors and subsequent gene expression in vitro and in vivo. Most of the biologic activities associated with AGEs have been transduced by receptor for AGE (RAGE). Recently, AGEs are known to be in association with diverse cancers in terms of cellular proliferation and metastasis. However, little is known about the role of AGEs in acute myelogenous leukemia (AML). Here we examined the effects of the AGEs-RAGE interaction on the cell proliferation and intracellular signaling of AGEs in human leukemia cell lines. Expression of RAGE was observed in 8 AML cell lines examined, and up-regulated by treatment of AGE. AGE induced the proliferation of AML cell lines, HL60 and HEL, in a dose-dependent manner. Treatment with 5 μM of antisense S-ODN for RAGE did effectively inhibit cell growth of HEL cells. Exposure of HL60 and HEL with AGE induced a significant increase in the numbers of cells in S phase of cell cycle in a dose-dependent manner. AGE enhanced the expression of cell cycle regulatory proteins such as cyclin-dependent kinase (CDK) 2/4/6, cyclin D1/E/B in a dose- and a time-dependent manner. In addition, the protein levels of the cyclin-dependent kinase inhibitor (CDKI), p21 and p27, were decreased by 24 hr exposure of AGE from 10 to 200 μg/ml in HEL. Furthermore, treatment of HEL with 200 μg/ml of AGE triggered activation of mitogen-activated protein (MAP) kinases, Erk, Akt, and p38, pathways and in nuclear translocation of transcription factors NF-kB. These results indicated that AGE induced the cell growth of human AML cells, HL60 and HEL, via augmentation of cell cycle and activation of MAPK kinase pathways. Up-regulation of RAGE by exposure of AGE suggested that cellular proliferation of AML cells might be mediated in autocrine fashion.

Cyclin-Dependent Kinase 4/6 Inhibitor Abemaciclib Exerts Dose-Dependent Cytostatic and Cytocidal Effects on Multiple Myeloma Cells Via Autophagy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4478-4478 ◽  
Author(s):  
Noriyoshi Iriyama ◽  
Hirotsugu Hino ◽  
Shota Moriya ◽  
Masaki Hiramoto ◽  
Yoshihiro Hatta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Annexin V ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Dose Dependent

Abstract Background:Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of abnormal plasma cells in the bone marrow. D-type cyclins (CCNDs), an important family of cell cycle regulators, are thought to be implicated in multiple myeloma (MM) development because CCNDs are commonly expressed in myeloma cells. CCND is known to positively regulate the cell cycle from G1 to S-phase initiation by binding to cyclin-dependent kinase (CDK) 4/6, resulting in potentiation of myeloma cell growth. These findings suggest a possible role for CDK4/6-targeting therapy in MM, yet the details remain incompletely understood. In this regard, we investigated the biological activity of abemaciclib, a potent, highly selective CDK4/6 inhibitor, in myeloma cell lines, to elucidate the mechanisms underlying the involvement of the CCND-CDK4/6 complex in cell cycle regulation and survival. Methods:The effects of abemaciclib on myeloma cells were investigated using three myeloma cell lines, KMS12-PE (CCND1-positive and CCND2-negative), RPMI8226 (CCND1-negative and CCND2-positive), and IM-9 (both CCND1- and CCND2-positive). Cell growth was assessed by trypan blue exclusion assay. Cell cycle analysis was performed using propidium iodide (PI) and apoptosis was measured using annexin V/PI staining via flow cytometry. Cell cycle regulated proteins, including p21 and p27, and phosphorylated proteins, including STAT1, STAT3, ERK, JNK, p38, and AKT, were evaluated using a phospho-flow method. Autophagy was assessed using CYTO-ID via flow cytometry. PARP cleavage was investigated via western blotting. Clarithromycin, an antibiotic agent belonging to the macrolide class, was used as an autophagy inhibitor. Results:Abemaciclib inhibited myeloma cell growth in a dose-dependent manner in all the cell lines evaluated, with significant differences seen at a concentration of 320 nM. Annexin V/PI staining revealed that 1 μM abemaciclib showed little or no effect on apoptosis, but 3.2 μM abemaciclib induced apparent myeloma cell apoptosis, with an increase in both the early and late apoptotic fractions. Therefore, 1 and 3.2 μM of abemaciclib were used in subsequent experiments for the assessment of cell growth and apoptosis, respectively. Cell cycle analyses revealed that 1 μM abemaciclib increased the fraction of cells in G0/G1 phase and decreased the fraction in S-G2/M phase. Furthermore, this effect was associated with the upregulation of p21 and p27 in the evaluated myeloma cells. PARP cleavage was observed in KMS12-PE cells treated with 3.2 μM abemaciclib, but not 1 μM, suggesting a close connection between the degree of PARP cleavage and apoptosis in myeloma cells. Importantly, abemaciclib induced autophagy in a dose-dependent manner. However, no apparent inhibitory effect on the autophagy-related phosphorylated proteins STAT1 (Y701), STAT3 (Y705), ERK (T202/Y204), JNK (T183/Y185), p38 (T180/Y182), or AKT (Y315) was observed in myeloma cells treated with 3.2 μM abemaciclib. To investigate the role of abemaciclib-induced autophagy on myeloma cell apoptosis, we further assessed the apoptotic effect of 3.2 μM abemaciclib or 50 μg/mL clarithromycin, alone or in combination. Clarithromycin did not induce apoptosis of myeloma cells. Importantly, clarithromycin treatment in combination with abemaciclib attenuated the apoptotic effect of abemaciclib. Discussion & Conclusions: Although the underlying mechanisms conferring the level of CCND expression are known to differ greatly (e.g., CCND translocation, hyperdiploidy, or activation of upstream pathways of CCND transcription), the results of the current study indicate that the CCND-CDK4/6 complex is closely involved in myeloma cell growth and survival regardless of the CCND family member present. In addition, we demonstrate that abemaciclib exerts multiple effects, such as myeloma cell apoptosis, via the PARP pathway or autophagy, as well as cell cycle regulation. Because abemaciclib in combination with clarithromycin inhibits myeloma cell apoptosis, the autophagy induced by abemaciclib is considered to have a critical role in the induction of apoptosis, so-called "autophagic cell death." These results provide novel insights into a possible therapeutic approach using abemaciclib to target CDK4/6 in patients with MM, and offer new possibilities for combination therapy with CDK4/6 inhibitors and autophagy regulators. Disclosures Iriyama: Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau. Hatta:Novartis Pharma: Honoraria.

Leptin-Induce Proliferation Mediated by JAK/STAT and MAPK Activation in Acute Myelogenous Leukemia Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4273-4273
Author(s):  
Hyun Ki Park ◽  
Ju Young Kim ◽  
Jin Sun Yoon ◽  
Eun Shil Kim ◽  
Kwang Sung Ahn ◽  
...  
Keyword(s):  
Cell Lines ◽  
Acute Myelogenous Leukemia ◽  
Cellular Proliferation ◽  
Short Form ◽  
Janus Kinase ◽  
Myelogenous Leukemia ◽  
Dependent Manner ◽  
Mapk Activation ◽  
Stat3 Phosphorylation ◽  
Acute Myelogenous

Abstract Leptin, secreted as a product of the ob gene that is mainly produced by adipose tissue, has been involved in the regulation of energy metabolism. Recently, leptin has been suggested to stimulate normal cells as well as various cancer cells including acute myelogenous leukemia (AML). However, the molecular mechanism of leptin as a proliferative effector is not poorly understood in AML. In the present study, we show that leptin-induced cellular proliferation is mediated by JAK/STAT and MAPK activation in AML. Expression of the long form (Ob-Rb) and the short form (Ob-Ra) of leptin receptor (Ob-R) was observed in 10 AML cell lines examined, and up-regulated by treatment of leptin. Leptin induced the proliferation of HEL, which was shown to express the highest Ob-R among AML cell lines, in a dose-dependent manner. Treatment with 100ng/ml of leptin enhanced the expression of Janus kinase 2 (JAK2) phosphorylation, signal transducer and activator of transcription (STAT)-3 phosphorylation and mitogen-activated protein kinases (MAPKs) in HEL. Blocking of STAT3 phosphorylation with a specific inhibitor, AG490 (50 μM), significantly reduced leptin-induced ERK1/2 phosphorylation and cellular proliferation of HEL, whereas blocking of ERK1/2 activation by a specific ERK1/2 kinase inhibitor, PD98059 (25 μM), did not affect the STAT3 phosphorylation and leptin-induced proliferation in HEL. Furthermore, knockdown of Ob-R expression with small interfering RNA (siRNA) reduced leptin-induced proliferation of HEL, and also significantly attenuated leptin-induced STAT3 and ERK1/2 activation. This results provides that leptin promotes AML cell growth by activating JAK/STAT3 and MAPK, although not directly dependent on ERK. Blocking as direct receptor level could be a rational therapeutic strategy of AML.

scholarly journals Effect of interferon on colony formation in culture by blast cell progenitors in acute myeloblastic leukemia

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 549-552 ◽  
Author(s):  
R Taetle ◽  
RN Buick ◽  
EA McCulloch
Keyword(s):  
Colony Formation ◽  
Antitumor Agents ◽  
Myelogenous Leukemia ◽  
Dependent Manner ◽  
Self Renewal ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Cellular Events ◽  
Acute Myelogenous ◽  
Dose Dependent

Abstract The effect of purified human fibroblast interferon on primary and secondary colony formation by blast progenitors from the peripheral blood of patients with acute myelogenous leukemia was examined. Interferon inhibited blast progenitors and normal granulocyte/macrophage progenitors (CFU-C) in a dose-dependent manner. The magnitude of this effect on blast progenitors and CFU was similar. Interferon also inhibited secondary plating of blast progenitors (self- renewal). This effect was in marked contrast to the effect of adriamycin, which reduced primary plating efficiency of blast progenitors but did not affect self-renewal. Inhibition of blast progenitor proliferation by interferon was markedly reduced when interferon was added after 24 hr of culture and was absent when added after 72 hr. Inhibition of self-renewal was observed even when interferon was added at 72 hr. We conclude that interferon inhibits both primary proliferation and self-renewal of blast progenitors and that this effect is not due to reduction in the number of primary colonies. These experiments provide an example of how cell culture techniques may be used to test antitumor agents for effects on important cellular events other than general cytotoxicity.

scholarly journals Autocrine stimulation of interleukin 1 beta in acute myelogenous leukemia cells.

1987 ◽  
Vol 166 (5) ◽  
pp. 1597-1602 ◽  
Author(s):  
K Sakai ◽  
T Hattori ◽  
M Matsuoka ◽  
N Asou ◽  
S Yamamoto ◽  
...  
Keyword(s):  
Acute Myelogenous Leukemia ◽  
Interleukin 1 ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Acute Myelogenous ◽  
Autocrine Stimulation ◽  
Basal Proliferation ◽  
Culture Supernatants ◽  
Dose Dependent

A significant increase in CD25 antigen-positive cells by IL-1 was observed in cells of a patient with M7 acute myelogenous leukemia. Basal proliferation and expression of CD25 antigen by the M7 leukemic cells were inhibited by addition of anti-IL-1 beta antibody in a dose-dependent manner, but not by rabbit anti-IL-1 alpha antibody. Culture supernatants of these leukemic cells contained IL-1 activity, which was specifically inhibited by addition of anti-IL-1 beta antibody, and Northern blot analysis detected intracellular IL-1 beta mRNA. These results indicated that autocrine secretion of IL-1 beta was involved in proliferation of some myelogenous leukemic cells.

Janus Kinase (JAK)-2 Does Not Play a Role in Bcr-Abl Signaling in Philadelphia Chromosome (Ph)-Positive (p190) Acute Lymphoblastic Leukemia (ALL) Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4241-4241
Author(s):  
Stefan H. Faderl ◽  
Quin Van ◽  
Patricia E. Koch ◽  
David M. Harris ◽  
Inbal Hallevi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Janus Kinase ◽  
Myelogenous Leukemia ◽  
Dependent Manner ◽  
Western Immunoblotting ◽  
Gm Csf ◽  
Dose Dependent

Abstract Novel immunochemotherapy regimens combined with imatinib mesylate (IA) have significantly improved treatment outcome of Ph+ ALL. Nevertheless, most adult patients with Ph+ ALL relapse and succumb to their disease. Recent reports suggested that Jak-2 is engaged in the signaling of Bcr-Abl in chronic myelogenous leukemia (CML) cells. Because Jak-2 inhibitory agents are currently investigated in clinical trials, we sought to explore the role of Jak-2 in the signaling of Bcr-Abl in Ph+ ALL assuming that inhibition of Jak-2 might be beneficial in the treatment of Ph+ ALL. To do this, we used our Ph+ (p190) ALL cell lines Z-119 and Z-181 (Estrov et al. J Cell Physiol166: 618, 1996). We chose these cells because in both lines Jak-2 can be activated. Both Z-119 and Z-181 cells express granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors and GM-CSF activates Jak-2 and stimulates the proliferation of both cell lines. Using a clonogenic assay, we found that IA inhibited the proliferation of these cells at concentrations ranging from 50 to 500 nM. Because Bcr-Abl was found to activate the signal transducer and activator of transcription (STAT)-5 in CML cells, we used Western immunoblotting and found that IA inhibited the phosphorylation (p) of STAT5 in a dose-dependent manner in Ph+ ALL cells. To test whether JAk-2 plays a role in Bcr-Abl (p190) signaling we incubated Z-181 cells for 4 hours with or without 50, 100, 250, and 500 nM IA, extracted cellular protein and immunoprecipitated total STAT5 protein. Then, using Western immunoblotting we detected the Bcr-Abl p190 protein in all STAT5 immunoprecipitates and by using specific pSTAT5 antibodies, we demonstrated that IA induced a dose-dependent reduction in the levels of pSTAT5, but not of p190 protein, suggesting that the p190 Bcr-Abl kinase binds to and activates STAT5. Remarkably, neither Jak-2 nor pJak-2 was detected in either immunoprecipitate. To further delineate the role of Jak-2 in Bcr-Abl signaling we extracted protein from Z-181 cells and immunoprecipitated Jak-2. Neither Bcr-Abl nor STAT5 was detected in these immunoprecipitates, confirming that Jak-2 does not bind Bcr-Abl p190 protein and does not participate in the activation of STAT5. Taken together, our data suggest that Bcr-Abl (p190) binds and phosphorylates STAT5 whereas, Jak-2 is not engaged in Bcr-Abl (p190) signaling in Ph+ ALL cells.

scholarly journals Decorin induced by progesterone plays a crucial role in suppressing endometriosis

2014 ◽  
Vol 223 (2) ◽  
pp. 203-216 ◽  
Author(s):  
Yoshihiro Joshua Ono ◽  
Yoshito Terai ◽  
Akiko Tanabe ◽  
Atsushi Hayashi ◽  
Masami Hayashi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mrna Expression ◽  
Cell Lines ◽  
Stromal Cells ◽  
Crucial Role ◽  
Dependent Manner ◽  
Endometrial Stromal Cells ◽  
Endometrial Cells ◽  
Cycle Arrest ◽  
Dose Dependent

Dienogest, a synthetic progestin, has been shown to be effective against endometriosis, although it is still unclear as to how it affects the ectopic endometrial cells. Decorin has been shown to be a powerful endogenous tumor repressor acting in a paracrine fashion to limit tumor growth. Our objectives were to examine the direct effects of progesterone and dienogest on the in vitro proliferation of the human ectopic endometrial epithelial and stromal cell lines, and evaluate as to how decorin contributes to this effect. We also examined DCN mRNA expression in 50 endometriosis patients. The growth of both cell lines was inhibited in a dose-dependent manner by both decorin and dienogest. Using a chromatin immunoprecipitation assay, it was noted that progesterone and dienogest directly induced the binding of the decorin promoter in the EMOsis cc/TERT cells (immortalized human ovarian epithelial cells) and CRL-4003 cells (immortalized human endometrial stromal cells). Progesterone and dienogest also led to significant induced cell cycle arrest via decorin by promoting production of p21 in both cell lines in a dose-dependent manner. Decorin also suppressed the expression of MET in both cell lines. We confirmed that DCN mRNA expression in patients treated with dienogest was higher than that in the control group. In conclusion, decorin induced by dienogest appears to play a crucial role in suppressing endometriosis by exerting anti-proliferative effects and inducing cell cycle arrest via the production of p21 human ectopic endometrial cells and eutopic endometrial stromal cells.

Inhibition of Human Plasmacytoma Cell Growth by a Novel JAK Kinase Inhibitor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 644-644
Author(s):  
Renate Burger ◽  
Steven Legouill ◽  
Yu-Tzu Tai ◽  
Reshma Shringarpure ◽  
Klaus Podar ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Cell Lines ◽  
Hormone Receptors ◽  
Critical Role ◽  
Myeloma Cell ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Jak Kinase ◽  
Dose Dependent

Abstract Novel strategies in cancer therapy aim at inhibiting distinct signal transduction pathways that are aberrantly activated in malignant cells. Protein tyrosine kinases of the JAK family are associated with a number of cytokine and cytokine-like hormone receptors and regulate important cellular functions such as proliferation, survival, and differentiation. Constitutive or enhanced JAK activation has been implicated in neoplastic transformation and abnormal cell proliferation in various hematological malignancies. In multiple myeloma (MM), JAK kinases play a critical role because of their association with cytokine receptors of the IL-6/gp130 family. A novel small-molecule inhibitor was developed that shows a 100 to 1,000-fold selectivity for JAK1, JAK2, JAK3, and TYK2 relative to other kinases including Abl, Aurora, c-Raf, FGFR3, GSK3b, IGF-1R, Lck, PDGFRa, PKBb, and Zap-70. Growth of MM cell lines and primary patient cells was inhibited by this compound in a dose-dependent manner. The IL-6 dependent cell line INA-6 and derived sublines were sensitive to the drug, with IC50’s of less than 1 mM, in [3H]-thymidine uptake and a colorimetric, tetrazolium compound (MTS) based assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI). Importantly, INA-6 and patient tumor cell growth was also inhibited in the presence of bone marrow stromal cells, which by themselves remained largely unaffected. Growth suppression of INA-6 correlated with a significant and dose-dependent increase in the percentage of apoptotic cells, as evaluated by Apo2.7 staining after 48 hours of drug treatment. In addition, the compound blocked IL-6 induced phosphorylation of STAT3, a direct downstream target of JAK kinases and important transcription factor triggering anti-apoptotic pathways. In other myeloma cell lines, the drug overcame the protective effect of gp130 cytokines on dexamethasone induced apoptosis. In MM1.S cells, it completely blocked IL-6 induced phosphorylation of SHP-2 and AKT, both known to mediate the protective effects of IL-6. In contrast, AKT phosphorylation induced by IGF-1 remained unchanged, demonstrating selectivity of the compound. These studies show that disruption of JAK kinase activity and downstream signaling pathways inhibits myeloma cell growth and survival as well as circumvents drug resistance, thereby providing the conceptual basis for the use of JAK kinase inhibitors as a novel therapeutic approach in MM.

Raf Inhibitor BAY 43-9006 Induces Bim Dephosphorylation and Activates the Intracellular Apoptotic Pathway in AML.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3367-3367 ◽  
Author(s):  
Weiguo Zhang ◽  
Marina Konopleva ◽  
Teresa McQueen ◽  
Jorje Cortes ◽  
James McCubrey ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Leukemia Cell ◽  
Mapk Signaling ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Apoptotic Pathway ◽  
Apoptosis Protein ◽  
Dose Dependent

Abstract We have previously demonstrated constitutive activation of MAPK signaling in 70% of primary AML samples (Millela et al, JCI108:851–859, 2001), suggesting that upstream kinases (Raf and MEK) may play a role in the leukemic transformation of myeloid cells. BAY 43-9006 is a small molecule Raf kinase inhibitor that has demonstrated potent anti-tumor activity against solid human tumors in xenograft models. In this study, we tested the hypothesis that BAY 43-9006 inhibits leukemia cell growth and/or induces apoptosis by suppressing the activity of the MAPK pathway. In the in vitro kinase assay, BAY 43-9006 inhibited both Raf-1 and B-Raf-mediated MEK1 phosphorylation in a dose-dependent manner, with Raf-1 kinase being more sensitive to the inhibitory effects of BAY 43-9006 (IC50Raf-1, 1.37 μM vs. IC50B-Raf, 4.64 μM). BAY 43-9006 suppressed MEK1/2 and ERK phosphorylation in the AML cell lines OCI-AML3, HL-60, U937 and KG-1 in a dose-dependent manner after 24 hr treatment. Unexpectedly, BAY 43-9006 also inhibited AKT phosphorylation on Ser473 (after 4.5 hrs). BAY 43-9006 inhibited growth of AML cells in a dose- and time-dependent manner. The 50% inhibitory concentration (IC50) of BAY 43-9006 was 0.39, 1.14, 2.86 and 2.80 μM, respectively in OCI-AML3, HL-60, U937 and KG-1 cells after 72 hrs. This growth-inhibitory effect was mediated by a dose-dependent induction of cell cycle arrest in G1 mediated by the down-regulation of the cell cycle-related proteins cyclin E, cdk2 and cdc2, followed by induction of apoptosis after 72 hrs. In primary AML patient samples, BAY 43-9006 not only inhibited cell growth and induced apoptosis after 48–72 hrs in vitro, but also preferentially inhibited colony formation of AML progenitor cells compared to normal bone marrow cells [IC50: 2.33 μM vs. 9.34μM (CFU-GM), 5.69 μM (Erythroid) and 3.75 μM (Mixed), respectively]. Time-course analyses demonstrated that BAY 43-9006 suppressed phosphorylation of the pro-apoptotic protein Bim (at 4.5 hrs), caused loss of the mitochondrial membrane potential and cytochrome c release (at 6 hrs) followed by cleavage of caspases-3 and -9 but not of caspase-8, suggesting primary involvement of the intrinsic mitochondrial pathway. Furthermore, the pro-apoptotic proteins Bim and Bax were up-regulated after 48 hrs of BAY 43-9006 treatment, and the level of the inhibitor-of-apoptosis protein Survivin was down-regulated after 48 hrs. In summary, our data demonstrates that BAY 43-9006 inhibits Raf-MEK-ERK signaling and induces apoptosis in AML via Bim de-phosphorylation and activation of the intrinsic apoptotic pathway. The potential of BAY 43-9006 in the therapy of AML patients will be tested in a Phase I clinical trial.

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.

Biologic Sequelae of CDC2 Inhibition in MM.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4797-4797
Author(s):  
Mariateresa Fulciniti ◽  
Pierfrancesco Tassone ◽  
Teru Hideshima ◽  
Kenneth C. Anderson ◽  
Nikhil C. Munshi
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Cell Cycle Checkpoint ◽  
Time Dependent ◽  
Thymidine Uptake ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Apoptotic Pathway ◽  
M Phase

Abstract Multiple Myeloma (MM) is a malignant proliferation of plasma cells characterized by disruption of cell cycle checkpoint controls which maintain G2M transition and/or mitosis. CDC2 is the cyclin-dependent kinase that normally drives cells into mitosis and is universally expressed in MM. To examine the biologic role of CDC2 in MM, we evaluated cellular and molecular effects of Terameprocol (M4N, tetra-O-methyl nordihydroguaiaretic acid) that has been shown to inhibit cell cycle progression at the G2/M phase by inhibiting the transcription of sp-1 dependent expression of CDC2. We observed that Terameprocol downregulated the expression of cdc2 in a time-dependent manner, with a maximal effect at 24h. This was associated with induction of G2/M growth arrest in a panel of MM cell lines (INA6, OPM1, OPM2, MM1S, RPMI-8226, U266), as determined by PI staining. Interestingly, Terameprocol treatment led to increase in p21waf1 protein levels. Importantly, we observed inhibition of DNA synthesis by Terameprocol in a dose- and time-dependent manner, with IC50 range from 1–20 uM for a 24 hours period of treatment, as assessed by 3H-thymidine uptake. Longer exposure of MM cells to Terameprocol resulted in cytoxicity, as assessed by MTT assay, via induction of apoptosis, evidenced by Annexin V+ /PI staining, in all the MM cell lines tested. Terameprocol -induced apoptosis is predominantly associated with caspase-9 and caspase-3, but not caspase-8 activation, suggesting that Terameprocol triggers intrinsic apoptotic pathway in MM cells. Our results show that genes that control entry and progression of G2/M phase, especially cdc2, may be an attractive target for MM therapy and Terameprocol represents a prototypic agent for the control of unregulated cellular proliferation in MM.

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