Myeloma Cell Proliferation Is Inhibitied by the Activation of Adenosine Monophosphate Activated Protein Kinase (AMPK).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5045-5045
Author(s):  
Philipp Baumann ◽  
Sonja Mandl-Weber ◽  
Bertold Emmerich ◽  
Christian Straka ◽  
Daniel Franke ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Adenosine Kinase ◽  
Intracellular Signalling ◽  
Phase Cell ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell

Abstract In multiple myeloma (MM), a network of cytokines in the bone marrow microenvironment promotes myeloma cell proliferation. Consequent inhibition of intracellular signalling in the myeloma cells seems to be a promising strategy to encounter disease progression. The multiple myeloma cell lines U266, OPM-2, RPMI-8226 and NCI-H929 were incubated with the AMPK activators AICAr and D942. Basal and cytokine stimulated proliferation rates of myeloma cells were measured by the WST-1 assay. Alterations of the cell cycle were determined by flow cytometry after staining with propidium iodide. Intracellular signalling was shown by western blotting. The AMPK activators 5-aminoimidazole-4-carboxamide (AICAr) and D942 induced inhibition of proliferation in multiple myeloma cell lines. AICAr also induced a S-phase cell cycle arrest in all four tested cell lines and led to phosphorylation and herewith activation of AMPK. Furthermore, the inhibition of a nucleoside transporter by nitrobenzyl-thio-9-β-D-ribofuranosylpurine (NBTI), inhibition of the adenosine kinase by iodotubericidine and inhibition of AMPK by AMPKI Compound C reversed AICAr effects, indicating that the cellular effects of AICAr were mediated by AMPK. Activation of AMPK inhibited basal extracellular-signal regulated kinase (ERK), mTOR and P70S6 kinase (P70S6K) signalling and blocked cytokine induced increase of proliferation, which again was due to inhibition of ERK and P70S6K signalling. Troglitazone, a representative of a group of anti-diabetic drugs, similarly inhibited myeloma cell proliferation, activated AMPK and decreased ERK and P70S6K signalling. We demonstrate for the first time that myeloma cell proliferation is controlled by AMPK activity. Consequently, targeting this pathway by inhibitors like glitazones provides a novel strategy in myeloma therapy.

Simvastatin Induces Death of Multiple Myeloma Cell Lines

2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytoplasmic Calcium ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

Molecular Characterization of Human Multiple Myeloma Cell Lines by Genome-Wide Profiling Identifies Kinase Pathway Alterations.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1694-1694
Author(s):  
Carolina Elosua ◽  
Purificacion Catalina ◽  
Brian A Walker ◽  
Nicholas J Dickens ◽  
Athanasia Avramidou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Myeloma Cell ◽  
Molecular Pathogenesis ◽  
Gene Localization ◽  
Cycle Progression ◽  
Multiple Myeloma Cell ◽  
Human Multiple Myeloma

Abstract Multiple Myeloma (MM) is a malignancy depicted by clonal expansion of plasma cells in the bone marrow. There are two broad genetic subtypes of multiple myeloma as defined as hyperdiploid multiple myeloma (H-MM), characterized by trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21, and nonhyperdiploid multiple myeloma (NH-MM) associated with primary translocations involving the immunoglobulin heavy chain (IgH). These two subtypes of multiple myeloma have two different molecular pathogenesis given that characteristic changes of each have been already observed. In order to contribute to the understanding of this malignancy and to unveil the different molecular pathogenesis, our interest is focused on Human Multiple Myeloma Cell lines (HMCLs), as a model, and a broad but specific group of enzymatic proteins: the Kinases. Kinase hyperactivity or lack of it often results in disregulation of cellular pathways involved in proliferation and survival. In our study, we describe the patterns of genetic lesions and molecular pathogenesis of 11 HMCLs with Single Nucleotide Polymorphism (SNP)-based mapping arrays from Affymetrix Human Mapping 500K array set. This technique allows the examination and identification of copy number changes, bi-allelic deletions and the identification of loss of heterozygosity (LOH) due to loss and uniparental disomy, as well as gene localization and identification. The 11 HMCLs utilized are characterized for their structural alterations and not by hyperdiploidy. In addition, so as to fulfill the selection criteria, a minimum of 3 cell lines must present the alterations cited below. The most frequently identified alterations were located as follows: Previously described gains were observed in 1q, 7q, 8, 11q, 18, 19, and 20q; but also found at 4q. The bi-allelic deletions were ascertained on 3p. Similarly, we identified the regions of hemizygotic deletions on 1, 2q, 6q, 8q, 9p, 11q, 12, 13q, 14q, 17p, and 20p. In addition, described regions of homozygotic deletions were detected on 1p, 6q, 8p, 13q, 16q, and 22q, and furthermore located on 2q, 3, 4q, 9, 10q, 12p, and 20p. Finally, the uniparental disomies (UPDs) obtained were traced on 1q, 4q, 8q, 10q, and 22q. These identified alterations are affecting a series of enzymatic genes belonging to targeted pathways. Within the chromosomes 1, 10, 11, 14, and 16 we have localized kinases that are part of the PI3K/AKT pathway, which affect to a number of intracellular and extracellular myeloma growth cytokines. In the chromosomes 1, 6, 12, and 19 we identified a series of Cyclin-Dependent Kinases that are critical regulators of cell cycle progression and RNA transcription, since they regulate and control the cyclins, cell cycle regulatory proteins, which can provoke dysregulation and abnormally accelerated cell cycle progression. And finally on chromosomes 1, 2, 14, 21, and 22 we observed certain Aurora and related kinases, as another family of the cell cycle regulators and often aberrantly activated in human tumor cells, they facilitate transit from G2 through cytokinesis. These mutated kinases may be potential targets for therapeutics. Our data demonstrates the genomic complexity of multiple myeloma enhancing our understanding of the molecular pathogenesis of the disease and the importance of the HMCLs as a model.

Antitumor Effects and Mechanisms of Curcumin On Human Multiple Myeloma Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4896-4896
Author(s):  
Qingxian Bai ◽  
Qifa Liu
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Synergistic Effect ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Dependent Manner ◽  
Antitumor Effects ◽  
Low Concentration ◽  
Multiple Myeloma Cell ◽  
Human Multiple Myeloma

Abstract Abstract 4896 BackgroundF Multiple myeloma(MM) is a malignant plasma disease, which is characterized as high relapse rate and high resistance to chemotherapy. Curcumin is a polyphenol derived from the rhizome of Curcuma spp. It possesses diverse pharmacologic actions, such as antitumor, anti-inflammatory,anti- oxidation properties .Curcumin has the property of inhibit multiple tumor cell lines, in which included multiple myeloma cell. The real mechanism is not completely clear yet. We explored the mechanisms of curcumin on human multiple myeloma cell lines (RPMI8226 and H929), and investigated whether the combination of curcumin and adriamycin(Adr) has a synergistic effect. MethodsF The effect of curcumin on proliferation of RPMI8226 and H929 was observed with MTT assay. The synergetic effect of curcumin and Adr was analyzed by median-effect principle. Cell cycle distribution and apoptosis were studied with flow cytometry. Expression of surviving, bcl-2, bax mRNA was detected by RT-PCR. ResultsF Curcumin could inhibit the proliferation of RPMI8226 and H929 cells in a time- and dose-dependent manner. The IC50 values for RPMI8226 and H929 cell line were 12.15 μmol/L,17.24μmol/L respectively. The combination of curcumin and Adr showed synergistic effect even at low concentration of Adr. Apoptotic ratio of treated cells was significantly higher than untreated controls (36.9% vs 10.6%, p<0.05). Cells treated with curcumin showed cell cycle arrest at G2/M phase. Curcumin upregulated expression of survivin, bcl-2, while bax mRNA was significantly downregulated. ConclusionF Curcumin could suppress the proliferation of multiple myeloma cells and induce apoptosis. Adr combining with curcumin can show synergistic effect at low concentration of Adr. The mechanism of curcumin's antitumous effect might be related to down-regulation of surviving, bcl-2 mRNA and up-regulation of bax mRNA. Disclosures No relevant conflicts of interest to declare.

Aptamer TY04 Inhibits Multiple Myeloma Cell Growth Via Cell Cycle Arrest

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5390-5390
Author(s):  
Jing Liu ◽  
Hong-Juan Dai ◽  
Bian-Ying Ma ◽  
Jian-Hui Song ◽  
Hui-yong Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Growth ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
M Phase ◽  
Target Molecules

Abstract Multiple myeloma (MM), also known as plasma cell myeloma, is characterized by accumulation of clonal plasma cells in the bone marrow and overproduction of monoclonal immunoglobulin (Ig) in the blood or urine. MM accounts for approximately 10% of all hematologic malignancies. Despite recent advances in the understanding and treatment of this disease, MM remains an incurable disease in the vast majority. With conventional chemotherapy, the 5-year median survival rate for MM patients is approximately 25%. Aptamers are single-stranded RNA or DNA sequences that bind to target molecules with high affinity and specificity. Compared with antibodies, aptamers have unique advantages including easy chemical synthesis and modification, low toxicity, lack of immunogenicity, and rapid tissue penetration, Based on these advantages, aptamers show great potential for therapeutic application. The aptamer TY04 is a single-stranded DNA (ssDNA) generated by a method named cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX), We found TY04 strongly inhibited the growth of multiple myeloma cell lines including MM1.S, NCI-H929, KM3 and OPM2,The concentration of TY04 to inhibit 50% cell growth (IC50) on MM1.S was 3.89 μM. In contrast, TY04 had no effect on the growth of non-tumor cell lines — immortal B lymphoblastoid cell lines. Next, we used MM1.S cell line as the model to study the mechanism of TY04 anti- multiple myeloma. Flow cytometry analysis showed that TY04 with the sequence specifically bind to MM1.S cells when compared with unselected ssDNA library control. To investigate whether the target molecules of TY04 are membrane proteins on cell surface, MM1.S cells were treated with trypsin and proteinase k for 2 or 10 minutes before incubation with TY04. The result revealed that TY04 lost partly recognition ability on treated cells, indicating that the target molecules were most likely membrane proteins. Furthermore, we evaluated the cell cycle distribution of MM1.S after TY04 treatment. We found that TY04 significantly caused cell-cycle arrest in G2/M phase. The percentage of G2/M phase cells increased from 30.1±1.56 to 53.2±6.36. To identify the underlying molecular mechanism, G2/M-related proteins were assayed by flow cytometry. Following TY04 treatment, a concomitant inhibition of ERK1/2, cyclin B, CDK1 and γ-tubulin expression occurred. Meanwhile, human cell cycle PCR array was used to analyze the expression of 84 genes key to cell cycle regulation in TY04-treated MM1.S cells. Our results indicated that aptamer TY04 decreased the genes expression of CCNB1, CCNB2, BIRC5, BRCA1 and CCNH, which were involved in the progress of G2/M phase. All these results are significant in that they provide a framework for further exploring the use of TY04 as a novel anti-multiple myeloma agent. Disclosures: No relevant conflicts of interest to declare.

Metabolomic, Proteomic and Genomic Profiling Identifies Biomarakers of Sensitivity to Glutaminase Inhibitor CB-839 in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1802-1802 ◽  
Author(s):  
Andrew L MacKinnon ◽  
Mark Bennett ◽  
Matt Gross ◽  
Julie Janes ◽  
Weiqin Li ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Tca Cycle ◽  
Myeloma Cell ◽  
Akt Inhibitor ◽  
Employment Equity ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell ◽  
Equity Ownership ◽  
Resistant Cells

Abstract Introduction Glutaminase is a mitochondrial enzyme that converts glutamine to glutamate to support several metabolic processes including amino acid and nucleotide synthesis, maintenance of cellular redox homeostasis, and the replacement of TCA cycle intermediates. Selective glutaminase inhibitors BPTES and CB-839 have anti-proliferative activity in several pre-clinical cancer models including breast, pancreatic, lung, renal, brain, leukemia, and lymphoma. Across a panel of twenty-nine multiple myeloma cell lines, we found that glutaminase inhibition with CB-839 caused tumor cell death or growth inhibition in only a subset of cell lines. To identify biomarkers that predict sensitivity to CB-839 in multiple myeloma cells, we profiled cellular metabolites, mRNA transcripts, and signaling pathways in eight multiple myeloma cell (four CB-839-sensitive and four CB-839-resistant). Results Proteomic analysis showed that CB-839 treatment suppressed the activity of the amino-acid sensing kinase mTORC1 in CB-839-sensitive cells, leading to down regulation of protein synthesis and expression of metabolic genes. Analysis of steady-state levels of intra-cellular metabolites revealed that CB-839-sensitive cells had more profound decreases in nucleotide levels and less pronounced increases in essential amino acids upon CB-839 treatment compared to CB-839-resistant cells. This suggests that the metabolic response to glutaminase inhibition is fundamentally different in sensitive versus resistant multiple myeloma cell lines. Consistent with the in vitro data, in a xenograft model with the CB-839-sensitive cell line RPMI8226, CB-839 treatment produced a 71% reduction in tumor growth that was associated with reduced levels of intratumoral nucleotides and no changes in the levels of essential amino acids. We next explored protein biomarkers that predict resistance to CB-839 and found that pyruvate carboxylase (PC) expression strongly correlated with resistance. siRNA-mediated knockdown of PC reduced TCA cycle activity and sensitized cells to CB-839 treatment, suggesting that PC can rescue cells from glutaminase inhibition by supporting anapleurotic utilization of glucose. This hypothesis was further substantiated by the observation that treatment of CB-839-resistant cells with the AKT inhibitor MK2206 led to a decrease in glucose utilization, and when combined with CB-839, produced a significant decrease in TCA cycle activity and a profound synergistic anti-proliferative response. Conclusion Multiple myeloma cells show varying anti-proliferative responses to glutaminase inhibition by CB-839. CB-839 treatment inhibits mTORC1 pathway signaling and causes decreases in nucleotides in sensitive multiple myeloma cells. Multiple myeloma cells that are resistant to glutaminase inhibition have high expression of PC, which may allow these cells to utilize glucose instead of glutamine to resupply TCA cycle intermediates. Knockdown of PC or treatment with an AKT inhibitor causes cells to utilize less glucose and sensitizes resistant cells to glutaminase inhibition with CB-839. CB-839 is currently being evaluated in Phase 1 clinical trials for the treatment of various solid and hematological cancers including multiple myeloma. We are exploring the utility of PC and mTORC1 pathway signaling biomarkers to identify multiple myeloma patients that may respond to CB-839 treatment. Disclosures MacKinnon: Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Gross:Calithera Biosciences: Employment, Equity Ownership. Janes:Calithera Biosciences: Employment, Equity Ownership. Li:Calithera Biosciences: Employment, Equity Ownership. Rodriquez:Calithera Biosciences: Employment, Equity Ownership. Wang:Calithera Biosciences: Employment, Equity Ownership. Zhang:Calithera Biosciences: Employment, Equity Ownership. Parlati:Calithera Biosciences: Employment, Equity Ownership.

SDX-308, a nonsteroidal anti-inflammatory agent, inhibits NF-κB activity, resulting in strong inhibition of osteoclast formation/activity and multiple myeloma cell growth

Blood ◽  
2006 ◽  
Vol 109 (5) ◽  
pp. 2130-2138 ◽  
Author(s):  
Rentian Feng ◽  
Gülsüm Anderson ◽  
Guozhi Xiao ◽  
Gary Elliott ◽  
Lorenzo Leoni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Bone Resorption ◽  
Bone Destruction ◽  
Structural Analog ◽  
Myeloma Cell ◽  
Osteoclast Formation ◽  
Myeloma Cells ◽  
Osteoclast Activity ◽  
Multiple Myeloma Cell

Abstract Multiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities, additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac, known as SDX-308, and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101, a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation, and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308, as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-κB activation and osteoclast formation in an osteoclast cellular model, RAW 264.7. SDX-308 effectively suppressed TNF-α–induced IKK-γ and IκB-α phosphorylation and degradation and subsequent NF-κB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity, potentially by controlling NF-κB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study.

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.

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.

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.

Triggering of Toll-Like Receptor-4 In Human Multiple Myeloma Cells Promotes Proliferation and Alters Cell Responses to Immune and Chemotherapy Drug Attack

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1905-1905
Author(s):  
Zhen Cai ◽  
Hanying Bao ◽  
Peilin Lu ◽  
Lijuan Wang ◽  
Donghua He ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Response To Chemotherapy ◽  
Tlr4 Ligand ◽  
Induced Apoptosis

Abstract Abstract 1905 Multiple myeloma (MM) is a fatal plasma cell malignancy mainly localized in the bone marrow. The clonal expansion of tumor cells is associated with the disappearance of normal plasma cells and with a marked depression in the production of normal immunoglobulin (Ig). This makes MM patients highly vulnerable to bacterial, fungal and viral infections and recurrent infections remain to be a major cause of death in MM patients. It has been shown that most primary myeloma cells and cell lines express multiple Toll-like receptors (TLRs). Among them, TLR4 is most frequently expressed. To investigate TLR-initiated responses in MM cells including proliferation, anti-apoptosis and immune escape, we first screened four commonly used human myeloma cell line (HMCL) for the expression of major TLRs by RT-PCR. Surprisingly, all the HMCL expressed multiple TLRs. We also examined primary myeloma cells from 4 patients with MM and our results showed that TLR4 was expressed by all the tumor cells. We incubated myeloma cells with LPS, the natural ligand for TLR4, and found that cell proliferation increased significantly. Targeting TLRs on malignant B cells can induce resistance to chemotherapeutic agents but can also be exploited for combined therapeutic approaches. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of HMCL cells. Myeloma cells were pretreated for four hours with LPS before being induced apoptosis by adriamycin. Results showed that LPS pretreatment partially protected the cells from adriamycin-induced apoptosis. The TLR signaling pathway activates several signaling elements, including NF-kB and ERK/JNK/p38 MAPKs, which regulate many immunologically relevant proteins. Time-dependent MAPK phosphorylation was measured to assess the activation of these kinases upon treatment with LPS in cell lines. ERK1/2, p38, and JNK phosphorylation and NF-kB were significantly up-regulated following LPS treatment. Moreover, our findings demonstrated that LPS-induced cell proliferation was dependent on JNK, ERK and p38 signaling. IL-18, a recently described member of the IL-1 cytokine superfamily, is now recognized as an important regulator of innate and acquired immune responses. In this study, we found that LPS induced IL-18 secretion and activated MAPK and NF-kB signaling simultaneously. Therefore, our results suggest that activation of the MAPK signaling and secretion of IL-18 are interconnected. Tumors evade immune surveillance by multiple mechanisms, including the production of factors such as TGF-β and VEGF, which inhibit and impair tumor-specific T cell immunity. Our study also showed that T cell proliferation induced by allostimulatory cells decreased when the HMCL were pre-treated with LPS. Moreover, immunoregulatory molecules on HMCL, such as B7-H1, B7-H2 and CD40, were upregulated after treatment with LPS, suggesting that TLR4 ligand LPS facilitates tumor cell evasion of the immune system. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks. Disclosures: No relevant conflicts of interest to declare.

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