Baicalein Inhibites Multiple Myeloma Cells Proliferation and Migration by Interfering Wnt Signaling Pathway

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5019-5019
Author(s):  
Chaoping Xu ◽  
Shangqin Liu ◽  
Jianying Huang ◽  
Yicheng Zhang ◽  
Li He ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cyclin D1 ◽  
Mrna Expression ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
And Migration ◽  
Proliferation And Migration

Abstract Abstract 5019 Background: The Wnt/β-catenin signaling pathway has been implicated in a variety of tumor cell survival and proliferation, including myeloma cells. It can facilitate myeloma cell proliferation by upregulating the expression of many related genes, such as c-myc, cyclin D1, MMP and so on. We have previously demonstrated that Baicalein, a important component of Scutellaria radix from Huang-Lian-Jie-Du-Tang(HLJDT), can inhibit cell proliferation and induce cell death in myeloma cell through suppressing NF-κB activation (Ma Z, et al., Blood, 2005, 105(8): 3312–3318). In clinic, we found that HLJDT had a very good effective in multiple myeloma(MM) patients treatment (data not show). But, the precise molecular mechanism of HLJDT exerts its anti-tumor effects remains poorly understood. Here we further evaluated alternative mechanisms which is responsible for inhibition myeloma cell proliferation and migration by baicalein. Methods: Myeloma cell lines, RPMI8226 and U266 cells, were used for the analysis. MTT assay was used to assess cell viability in RPMI8226 and U266 cells which were exposed to baicalein with different concentrations and time period. Immunofluorescence assay and western blot analysis were used to assess β-catenin protein expression in MM cell lines. Transwell chamber migration assay was used to assess migration ability of myeloma cells treated with different concentrations of baicalein. RT-PCR analysis was used to assess β-catenin, c-myc, cyclin D1 and integrin β7 mRNA expression. Results: Baicalein can inhibit the MM cell lines proliferation in a dose- and time-dependent manner as measured by MTT assay. Immunofluorescence images and western bloting revealed that treatment of baicalein decreased the β-catenin protein expression level in myeloma cells. The mRNA expression level of β-catenin, c-myc and cyclin D1 was also decreased by baicalein treatment. In addition, we found that baicalein has an ability to inhibit the migration of MM cell llines, RPMI8226 and U266 cell, by decreasing the integrin β7 mRNA expression in a dose-dependent manner. Conclusion: Baicalein can inhibit myeloma cell proliferation is related to downregulation of β-catenin, c-myc and cyclin D1 genes expression, and the inhibition of myeloma ia associated with suppression of integrin β7 gene expression. Thus, HLJDT may have a potential of clinical applications in MM patient treatment. Disclosures: No relevant conflicts of interest to declare.

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.

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.

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.

Upregulation of MMP-2 & 9 by Co-Culture of Human Myeloma Cell Lines and Endothelial Cells May Be Responsible for Protection Against Cytotoxic Drugs.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5080-5080
Author(s):  
Shankaranarayana Paneesha ◽  
Raghu Adya ◽  
Hemali Khanji ◽  
Ed Leung ◽  
C. Vijayasekar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Human Myeloma Cell

Abstract Multiple myeloma is a clonal lymphoproliferative disorder characterised by the proliferation of plasma cells in the bone marrow. Inspite of good initial response, it is associated with universal relapse. We hypothesise this is due to sanctuary provided to myeloma cells by the endothelium. Matrix metalloproteinases (MMPs) are shown play a role in cell growth, invasion, angiogenesis, metastasis and bone degradation. We show here the protection offered by endothelial cells to human myeloma cell lines in in-vitro co-culture with upregulation of MMP-2 & 9 and the role of GM6001 MMP inhibitor (Ilomastat) in overcoming this protection. Human myeloma cell lines (H929, RPMI 8226, U266 & JJN3) with or without endothelial cells (human umbilical vein endothelial cells and EaHy 926 cell line) in-vitro co-culture were treated with melphalan, dexamethasone, arsenic trioxide and Ilomastat. Cytotoxicity/proliferation were assessed by the alamarBlue™ assay (Serotec) and validated by Annexin V-FITC apoptosis detection Kit (Calbiochem) and BrDU proliferation assay (BD Pharmingen™). Gelatin Zymography was used to demonstrate activity of MMP-2 & 9 in the supernatant. MMP-2 and 9 mRNA expression was quantified by Real Time Quantitative PCR (ROCHE). Co-culture of human myeloma cell lines with endothelial cells lead to increase in the proliferation of myeloma cell lines and also protected them from the cytotoxicity of chemotherapeutic agents. MMP-2 & 9 activity was upregulated by the co-culture. MMP-2 mRNA expression in human myeloma cell lines increased following 4 hr co-culture. Treatments with Ilomastat lead to the suppression of proliferation in co-culture in a dose dependent manner, associated with a reduction of MMP-2 and 9 activity. Our study shows endothelial cells offer protection to human myeloma cell lines in the presence of cytotoxic agents. This may result in the sanctuary of myeloma cells in bone marrow leading to ultimate relapse of disease. Our study also demonstrates the upregulation of MMP-2 and 9 by co-culture and increased cytotoxicity achieved by the inhibition of MMPs. Further studies are needed to determine the exact role of MMPs in myeloma biology as MMP inhibition may be an interesting therapeutic target and help in averting relapse in multiple myeloma.

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.

A Critical Role for PIM2 Kinase in Multiple Myeloma Through NF-κB Activation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1839-1839
Author(s):  
Veerendra Munugalavadla ◽  
Leanne Berry ◽  
Jae Chang ◽  
Geoffrey Del Rosario ◽  
Jake Drummond ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Combination Treatment ◽  
Single Agent ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Pim Kinases ◽  
Pi3k Inhibition

Abstract Abstract 1839 The PIM kinases are a family of 3 growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. Although PIM-1, -2 have been noted as highly expressed in multiple myeloma (MM) (Claudio JO et al., 2002), there are few data to support potential therapeutic utility of PIM inhibition in this indication. Here we show that the myeloma cell lines express all PIM protein isoforms to varying extents, and we describe the properties of a novel pan-PIM inhibitor GNE-652 with picomolar biochemical potency, an excellent selectivity profile, and favorable ADME properties. Myeloma cell lines and patient samples exhibit a striking prevalence of response to GNE-652 (23 of 25 lines with IC50 < 1 micromolar, median < 0.1 micromolar) and synergy in combination with the PI3K inhibitor GDC-0941 (mean combination index values ∼0.2 (n=25)). MM cells respond to this combination with cell cycle arrest and marked apoptosis in vitro. Conversely, a PIM-1, -3 selective inhibitor, GNE-568, failed to suppress MM cell growth and also failed to provide synergy in combination with PI3K inhibition, suggesting PIM-2 is a critical driver of MM cell growth & survival. Additional results suggest that PIM signaling converges on both TORC1 and AKT to generate differential synergies with PI3K/AKT/mTOR pathway inhibitors. PIM has been shown to potentially inactivate PRAS40, a negative regulator of TORC1 (Zhang et al., 2009). We demonstrate that PIM or PI3K inhibition caused a loss of phosphorylation on PRAS40 and resulted in a physical association of PRAS40 and TORC1 and a decrease in phosphorylated p70S6K and S6RP. These reductions were apparent in 7 of 7 cell lines assayed and enhanced by the combination of PI3K and PIM inhibition. Consistent with prior reports (Hammerman et al., 2005), we show that a second node of convergence between PIM and TORC1 is 4E-BP1. Both GDC-0941 and GNE-652 treatments reduced phosphorylation of 4E-BP1 in all the myeloma cell lines tested. Since dephosphorylated 4E-BP1 competes with eIF4G for the mRNA cap binding factor eIF4E, we assayed immunoprecipitates of eIF4E for the presence of eIF4G and 4E-BP1 and observed increased BP1 and decreased 4G. The combination treatment significantly enhanced the loss of 4G relative to either single agent, and importantly, even at 5 × IC50 concentrations for single agents, combination drug treatment achieved greater extent of effect than single agent treatment. It has been hypothesized that a subset of mRNAs are particularly sensitive to inhibition of cap-dependent translation, including a number of oncogenes such as cyclin D1. We noted across 7 different myeloma cell lines, strong decreases in levels of cyclin D1, and D3 that were further decreased by combination treatment of PIM and PI3K inhibition. In summary, we have identified several points at which PIM and PI3K/AKT/mTOR converge to provide synergy in multiple myeloma cell lines. As PIM isoforms are highly expressed in MM cells, we hypothesized that this could be due to proteosomal-mediated stability, and interestingly, MG132 and velcade each stabilized all PIM isoforms. It is commonly known that the JAK/STAT pathway regulates PIM transcription, but we show JAK inhibitors failed to abolish the expression of PIM in myeloma cells, suggesting a role for additional regulators. Recent genome sequencing studies from human myeloma samples (Chapman MA et al., 2011) confirmed the prevalence of NF-kB pathway activation, consistent with prior observations made in MM cell lines (Demchenko YN et al., 2010). The relationship of PIM and NF-kB is controversial in the literature (Hammerman PS et al., 2004 & Zhu N et al., 2002), with some groups placing PIM upstream of NF-kB and others the converse. Using an IκBα inhibitor, BMS-345541, we have examined the role for NF-kB in the regulation of PIM kinases. Here, we show that the BMS-345541 could preferentially suppress PIM2 expression in a dose dependent manner while PIM 1, 3 levels are modestly affected, suggesting that the high levels of PIM2 expression observed are partly driven by deregulation of the NF-kB pathway in MM. In conclusion, we provide pharmacological and biochemical evidence to suggest that PIM2 differentially regulate growth and survival of myeloma cells. Our results provide the rationale for further preclinical development of PIM inhibitors and the basis for a possible clinical development plan in multiple myeloma. Disclosures: Munugalavadla: Genentech: Employment. Berry:Genentech: Employment. Chang:Genentech: Employment. Rosario:Genentech: Employment. Drummond:Genentech: Employment. Du:Genentech: Employment. Fitzgerald:Genentech: Employment. Friedman:Genentech: Employment. Gould:Genentech: Employment. Maecker:Genentech: Employment. Moffat:Genentech: Employment. Slaga:Genentech: Employment. Xiaojing:Genentech: Employment. West:Genentech: Employment. Yu:Genentech: Employment. Ebens:Genentech: Employment.

scholarly journals Knockdown of lncRNA BDNF-AS inhibited the progression of multiple myeloma by targeting the miR-125a/b-5p-BCL2 axis

2022 ◽  
Vol 19 (1) ◽  
Author(s):  
Min Chu ◽  
Yingchao Fan ◽  
Liting Wu ◽  
Xiaoyan Ma ◽  
Jinfeng Sao ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Expression Levels ◽  
And Migration ◽  
Related Proteins ◽  
Proliferation And Migration

Abstract Purpose This study aimed to explore the role of long non-coding RNA (lncRNA) BDNF-AS in the progression of multiple myeloma (MM). Methods The expression of BDNF-AS, miR-125a-5p, and miR-125b-5p in MM serum and cell lines were detected by quantitative reverse transcriptase PCR (qRT-PCR). The binding relationships between miR-125a/b-5p and BDNF-AS or Bcl-2 were predicted by Starbase and verified by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2′-deoxyuridine (EdU) staining assay. Cell migration was evaluated by wound healing assay. The expression levels of apoptosis-related proteins were evaluated by Western blot analysis. The role of BDNF-AS was also investigated in a xenograft tumor model in vivo. Results BDNF-AS was significantly upregulated, while miR-125a-5p and miR-125b-5p were downregulated in MM serum and corresponding cancer cell lines. Knockdown of BDNF-AS effectively inhibited the proliferation and migration of MM.1S and U266 cells, and co-transfection of miR-125a-5p or miR-125b-5p inhibitor and sh-BDNF-AS enhanced cell proliferation and migration compared with that in sh-BDNF-AS group. Knockdown of miR-125a-5p or miR-125b-5p significantly enhanced the proliferation and migration of MM.1S and U266 cells, and co-transfection of sh-Bcl-2 and miR-125a/b-5p inhibitor inhibited cell proliferation compared with that in miR-125a/b-5p inhibitor group. Moreover, knockdown of BDNF-AS increased the expression levels of apoptosis-related proteins (cleaved caspase 3 and cleaved PARP), while knockdown of miR-125a-5p or miR-125b-5p reduced the expression levels of these apoptosis-related proteins compared with knockdown of BDNF-AS. Furthermore, knockdown of BDNF-AS effectively suppressed MM tumor growth in vivo. Conclusion Our findings revealed that knockdown of BDNF-AS inhibited the progression of MM by targeting the miR-125a/b-5p-Bcl-2 axis, indicating that BDNF-AS might serve as a novel drug target for MM.

1′-Acetoxychavicol Acetate (ACA) Is a Novel NF-κB Inhibitor with Significant Activity Against Multiple Myeloma in Vitro and in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 765-765 ◽  
Author(s):  
Keisuke Ito ◽  
Tomonori Nakazato ◽  
Yoshitaka Miyakawa ◽  
Ming Ji Xian ◽  
Taketo Yamada ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Serine Phosphorylation ◽  
Significant Activity ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract 1′-acetoxychavicol acetate (ACA) is a component of traditional Asian condiment, obtained from rhizomes of the commonly used ethno-medicinal plant Languas galanga (Zingiberacetate). Recent extensive studies revealed that ACA has potent chemopreventive effects against various tumors. More recently, we have reported that ACA induces apoptosis of myeloid leukemic cells via mitochondrial- and Fas-mediated dual pathway. The transcription factor NF-κB confers significant survival potential in myeloma cells; therefore, it has emerged as a therapeutic target for the treatment of multiple myeloma. Multiple myeloma is an incurable hematological disorders, which has been fatal outcome despite of high dose chemotherapy with stem cell transplantation; therefore, a novel biologically based therapeutic approach is desired. In this study, we investigated the effects of ACA on myeloma cells in vitro and in vivo, and further examined the molecular mechanisms of ACA-induced apoptosis in myeloma cells. ACA dramatically inhibited cellular growth of various human myeloma cell lines (RPMI8226, U266, IM9, and HS-Sultan) as well as freshly isolated myeloma cells from patients, but not normal bone marrow cells, in a dose (0-20 μM)- and time (0-24 h)-dependent manner. Cultivation with 10 μM ACA rapidly increased the population of cells in the G0/G1 phase with a reduction of cells in the S phase, and a strong induction of apoptosis was shown by the appearance of a hypodiploid DNA peak with sub-G1 DNA content 3 h after treatment. Treatment with ACA induced both caspase-3, -9, and caspase-8 activities, suggesting that ACA-induced apoptosis in myeloma cells mediates both mitochondrial- and Fas-dependent pathways. Furthermore, we investigated the effects of ACA on NF-κB activity in myeloma cells, and were able to demonstrate that ACA significantly inhibited serine phosphorylation and degradation of IκBα in a time-dependent manner. ACA rapidly decreased the nuclear expression of NF-κB, but increased the accumulation of cytosol NF-κB in RPMI8226 cells, indicating that ACA inhibits translocation of NF-κB from the cytosol to the nucleus. In addition, we also confirmed the inhibitory effects of ACA on NF-κB activation by ELISA in myeloma cell lines and fresh samples. ACA had a synergistic proapoptotic effect with another NF-κB inhibitor, MG-132 and TLCK. In contrast, NF-κB activator, PMA, dramatically abrogated ACA-induced apoptosis in myeloma cells. These in vitro studies prompted us to examine whether the effects of ACA are equally valid in vivo. To evaluate the effects of ACA in vivo, RPMI8226-transplanted NOD/SCID mice were treated with ACA. Tumor weight decreased in the mice that were injected ACA (mean weight: 0.04±0.06 g in the ACA-treated group vs. 0.63±0.29 g in the control group; p<0.01). During the treatment, ACA-treated mice appeared healthy, and pathological analysis at autopsy revealed no ACA-induced tissue changes in any of the organ, indicating that ACA might be developed as a new potent anti-cancer agent for the management of multiple myeloma. In conclusion, ACA has an inhibitory activity of NF-κB, and induces apoptosis of myeloma cells in vitro and in vivo. Therefore, ACA provides the new biologically based therapy for the treatment of multiple myeloma patients as a novel NF-κB inhibitor.

Brain-Derived Neurotrophic Factor Induces Proliferation, Migration and VEGF Secretion in Human Multiple Myeloma Cells Via Activation of MEK-ERK and PI3K/Akt Signaling.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1697-1697
Author(s):  
Yu Hu ◽  
Chun-yan Sun ◽  
Jing Huang ◽  
Lu Zhang ◽  
Zhang-bo Chu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Myeloma Cell ◽  
Akt Signaling ◽  
Vegf Expression ◽  
Myeloma Cells ◽  
Cell Proliferation And Migration ◽  
Erk Phosphorylation ◽  
And Migration ◽  
Rpmi 8226 ◽  
Proliferation And Migration

Abstract Multiple myeloma (MM) is an incurable form of cancer characterized by accumulation of malignant plasma cells in the bone marrow. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily that is overexpressed in patients with MM. Recent data indicate that in addition to its neuropoietic actions, BDNF is crucially involved in the angiogenesis and may be developed as a new angiogenic factor. Our previous study has defined a functional role of BDNF activation in MM cell proliferation and migration. In this study, we investigated the ability of BDNF activation to regulate VEGF levels in MM cells and the involvement of the different signaling cascades in the proliferation, migration and the stimulation of VEGF production. Myeloma cell lines, RPMI 8226, U266, KM3, and freshly isolated multiple myeloma cells from bone marrow aspirates of patients were exposed to different concentrations of BDNF for 48 hours. Using ELISA analysis, we here demonstrated the increased expression and secretion of VEGF by myeloma cell lines and sorted primary myeloma cells stimulated with BDNF. In the presence of 100 ng/ml BDNF, median VEGF concentrations in the conditioned media increased by 46% over controls in RPMI 8226(P &lt;0.05), by 21% in U266 (P &gt;0.05), by 69% in KM3 (P &lt;0.01), and by 81% in patient MM cells (P &lt;0.01). The results of RT-PCR assays are consistent with these data. To characterize downstream signaling molecules, we first assessed the activation states of ERK1/2 and Akt by western blot. In time-course experiments, specific phosphorylation of ERK1/2 was observed in RPMI 8226, U266 and KM3. ERK phosphorylation was inhibited by pretreatment with MEK-1 inhibitor PD98059. However, treatment with PI3K inhibitor LY249002 or wortmannin had no effect on ERK phosphorylation. BDNF also induced phosphorylation of Akt signaling pathway. Notably, this induction of phosphorylation was inhibited by pretreatment with the LY294002 but not by PD98059. These results show that activation of ERK and Akt pathways occur separately from each other without any cross talk in MM. To further determine the specific signaling pathways involved in the BDNF-induced tumor cell proliferation and migration, a pharmacological approach was taken. Proliferation induced by BDNF was blocked by 50 μM PD98059 significantly as determined by [3H]thymidine incorporation assays. However, the PI3K inhibitor (LY294002 and wortmannin) had little effect on BDNF-induced MM proliferation. In contrast, using transwell migration assay, we demonstrate that migration of RPMI 8226 cells induced by BDNF was inhibited by means of 50 μM LY294002 but not by PD98059. Furthermore, treatment with PI3K inhibitors LY294002 inhibited the secretion of VEGF, whereas the MEK-1 inhibitor PD98059 had no significant effect on BDNF-induced VEGF expression and secretion. Taken together, these data indicate that BDNF-induced proliferation requires activation of MEK, whereas BDNF-controlled migration and VEGF expression require activation of PI3K. These provide the framework for novel therapeutic strategies targeting BDNF signaling to inhibit proliferation and progressive disease in MM.

Potential Use of Sphingosine Kinase-2 Selective Inhibitors for the Treatment of Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5105-5105
Author(s):  
Ningfei An ◽  
Yeong-Bin Im ◽  
Peng Gao ◽  
Luciano J Costa ◽  
Robert K Stuart ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Therapeutic Potential ◽  
Myeloma Cell ◽  
Cancer Center ◽  
Selective Inhibitors ◽  
Myeloma Cells

Abstract Abstract 5105 Multiple myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ∼10,600 deaths annually. Despite the widespread use of several highly active chemotherapy agent (thalidomide, lenalidomide and bortezomib) and the incorporation of autologous hematopoietic stem cell transplantation, MM remains an incurable disease, suggesting the need for a better understanding of the disease's molecular pathways and for the development of novel agents. Sphingolipid metabolism is being increasingly recognized as a key pathway in cancer biology. Among a series of sphingolipid-metabolizing enzymes, sphingosine kinases (SK-1 and -2) are considered to be key regulators of cancer cell proliferation. SK phosphorylates sphingosine to sphingosine-1-phosphate (S1P) and affects the delicate balance between apoptotic ceramide/sphingosine and proliferative S1P. SKs are highly expressed in several solid tumors including pancreatic cancer and ovarian cancer. Currently, however, very little is known about how the SK is expressed in MM, and how sphingolipids respond to drugs targeting SK. We hypothesize that SKs play important roles in the pathogenesis of MM, thus providing a novel target for the treatment of MM. Apogee Biotechnology Corporation has generated new, orally bioavailable small molecule SK inhibitors that have in vitro and in vivo activity in a variety of models of cancer and inflammatory diseases. Several advantages of these SK inhibitors over other anti-MM drugs include: the role of the target in multiple key pathways driving MM; the low toxicity observed in GLP toxicology testing; the oral bioavailability that will simplify administration; and the ability to be combined with the standard drugs for MM (i.e, bortezomib). ABC294640, a SK2-selective inhibitor has recently entered single-agent clinical testing in advanced pancreatic cancer at our institution. In the current study, we investigated the expression patterns of SK2 in myeloma cells and the therapeutic potential of ABC294640 in the treatment of MM. We found that SK2 is highly expressed in several myeloma cell lines and in primary human CD138+ myeloma cells. Compared to the A498 human kidney adenocarcinoma cell line, the expression levels of SK2 mRNA were 1.4–12 fold higher in myeloma cell lines. The expression level of SK2 mRNA was > 62 fold higher in primary human CD138+ myeloma cells, compared to CD138- cells (n = 4). When myeloma cells were treated with ABC294640, cell proliferation was effectively inhibited with IC50 of ∼20 μM, including steroid resistant MM1.R myeloma cells. The degree of cell growth inhibition by ABC294640 correlated well with the expression level of SK2 mRNA in the myeloma cells. We also found that ABC294640 induces PARP cleavage and caspase 3 and 9 activation, indicating that the SK2 inhibitor induces apoptotic cell death. We are currently testing the effects of ABC294640 alone and in combination with dexamethasone or proteasome inhibitors in vivo in the XBP-1s transgenic MM model. Preclinical toxicities of the drug combinations will also be determined in the mouse models. Our studies provide the first evidence of SK2 in the pathogenesis of MM, and suggest excellent therapeutic potential of SK2-selective inhibitors for the treatment of MM. This work is supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, and ASCO Conquer Cancer Foundation Career Development Award Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document