Low Concentration DMSO Stimulates Cell Growth and In vitro Transformation of Human Multiple Myeloma Cells

2015 ◽  
Vol 5 (1) ◽  
pp. 65-74 ◽  
Author(s):  
Jianguo Wen ◽  
Yijiu Tong ◽  
Youli Zu
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Myeloma Cells ◽  
Low Concentration ◽  
In Vitro Transformation ◽  
Human Multiple Myeloma

scholarly journals Pterostilbene Inhibits Human Multiple Myeloma Cells via ERK1/2 and JNK Pathway In Vitro and In Vivo

2016 ◽  
Vol 17 (11) ◽  
pp. 1927 ◽  
Author(s):  
Bingqian Xie ◽  
Zhijian Xu ◽  
Liangning Hu ◽  
Gege Chen ◽  
Rong Wei ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Jnk Pathway ◽  
Myeloma Cells ◽  
Human Multiple Myeloma

scholarly journals Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 11-13 ◽  
Author(s):  
XG Zhang ◽  
B Klein ◽  
R Bataille
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
S Phase ◽  
Myeloma Cells ◽  
Severity Of The Disease

Abstract It has recently been demonstrated that interleukin-6 (IL-6) is a potent myeloma-cell growth factor in the majority of patients with multiple myeloma (MM). Using an anti-bromodeoxyuridine monoclonal antibody (MoAb) to specifically count myeloma cells in the S-phase (ie, labeling index, LI), we demonstrate that the IL-6 responsiveness of myeloma cells in vitro is directly correlated with their LI in vivo. Myeloma cells from all 13 patients with high LIs in vivo (greater than or equal to 1%) responded in vitro to IL-6, the strongest response occurring in cells from five patients with plasma-cell leukemia. In contrast, the cells of only two of eight patients with low myeloma-cell LIs in vivo (less than 1%) responded to IL-6 in vitro. After seven days of culturing with 1,000 U/mL recombinant IL-6 (rIL-6), the median LI value in the first group of patients (in vivo LI greater than or equal to 1%) was 11%, ie 11 times higher (P less than .01) than the median LI value (1%) in the second group of patients (in vivo LI less than 1%). Thus, the in vitro IL-6 responsiveness of myeloma cells is directly related to their in vivo proliferative status, and hence to the severity of the disease.

Kinome-Wide RNAi Studies in Human Multiple Myeloma Identify a Lymphoid Restricted Kinase GRK6 as a Selectively Vulnerable Target That Regulates STAT3/MCL1.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 601-601
Author(s):  
Rodger E. Tiedemann ◽  
Yuan Xiao Zhu ◽  
Jessica Schmidt ◽  
Hongwei Yin ◽  
Quick Que ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Epithelial Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Lymphoid Tissues ◽  
Myeloma Cells ◽  
Stat3 Phosphorylation ◽  
Human Multiple Myeloma

Abstract Abstract 601 A paucity of validated kinase targets in human multiple myeloma (MM) has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in MM tumor lines bearing common t(4;14), t(14;16) and t(11;14) translocations to identify critically vulnerable kinases in MM tumor cells without regard to preconceived mechanistic notions. Primary screening was performed in duplicate using an 1800-oligo siRNA library in a single-siRNA-per-well format. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. After 96 hours, viability was measured by ATP-dependent luminescence. Fifteen kinases were consistently vulnerable in MM cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. While several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly the G-protein coupled receptor kinase, GRK6, appeared selectively vulnerable in MM. GRK6 inhibition is selectively lethal to KMS11, OPM1, H929, KMS18 and OCI-MY5 myeloma cells and has minimal effect on 293, MCF7, SF767, A375 or A549 epithelial cells. Persistent expression of FLAG-GRK6 via cDNA rescued KMS11 cells from the lethal effect of a 3'UTR-targeted GRK6 siRNA, but not from control siRNA, validating identification of GRK6 as an essential myeloma survival kinase. Furthermore, concordant results were obtained using four different exon-based GRK6 siRNA, all of which induced GRK6 silencing and similar inhibition of KMS11 proliferation and viability. Significantly, GRK6 is ubiquitously expressed in lymphoid tissues and myeloma, but by comparison appears absent or only weakly expressed in most primary human somatic tissues. From co-immunoprecipitation experiments we demonstrate that GRK6 is highly expressed in myeloma cells via direct association with the HSP90 chaperone. Inhibition of HSP90 with geldanamycin blocks GRK6 protein expression. Importantly, direct GRK6 silencing causes rapid and selective suppression of STAT3 phosphorylation that is associated with sustained reductions in total MCL1 protein levels and MCL1 phosphorylation (within 24 hours), providing a potent mechanism for the cytotoxicity of GRK6 inhibition in MM tumor cells. GF109203X is an inhibitor of both protein kinase C and of GRK6 that causes near total inhibition of these kinases in vitro at distinct concentrations of 0.1μM and 1-10μM respectively. Notably, GF109203X was substantially cytotoxic to 10/14 myeloma tumor lines at concentrations most consistent with GRK6 inhibition (5-20μM), and was selectively more cytotoxic to myeloma tumor cells than to non-myeloma cell lines (P=0.01), highlighting the potential of GRK6 as a pharmaceutical target for selective therapeutic intervention in myeloma. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. Disclosures: Perkins: MMRC: Employment. Reeder:Celgene: Research Funding; Millennium: Research Funding. Fonseca:Otsuka: Consultancy; BMS: Consultancy; Amgen: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

Sphingolipids As a Novel Target For The Treatment Of Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3163-3163 ◽  
Author(s):  
Jagadish Kummetha Venkata ◽  
Robert K Stuart ◽  
Luciano J Costa ◽  
Ningfei An ◽  
Houjian Cai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloma Cell ◽  
Sphingolipid Metabolism ◽  
Myeloma Cells ◽  
S1p Receptors

Abstract Introduction Multiple Myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ∼10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding of the disease’s molecular pathways and identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in cancer biology. In particular, sphingosine kinases (SK1 and SK2) provide a potential site for manipulation of the ceramide / sphingosine 1-phosphate (S1P) rheostat that regulates the balance between tumor cell proliferation and apoptosis, as well as tumor sensitivity to drugs. Currently, very little is known about sphingolipid metabolism in MM. We herein for the first time provide a detailed analysis of sphingolipid metabolism in MM and demonstrate the potential of targeting SK2 for the treatment of MM. Methods We first quantified sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+ myeloma cells, and in a publically available gene expression dataset from MM patients. We then tested the anti-myeloma activity of SK2-specific shRNA and determined the efficacy of a selective SK2 inhibitor (ABC294640) in killing myeloma cell lines and primary human myeloma cells in vitro. The mechanistic pathway of apoptosis was analyzed by immunoblotting and flowcytometry. MM cell lines stably expressing luciferase and eGFP were generated for xenograft experiments and for in vitro co-cultures with stromal cells. Results From the publically available GSE6477 microarray data set, we found that one third of the genes involved in sphingolipid metabolism were significantly different in CD138+ MM cells from newly diagnosed MM patients compared to normal individuals, including SK2 and S1P receptors. In 5 MM cell lines compared to immortalized B cells (IBC), 19 key sphingolipid metabolites were measured, and we found that ceramides were significantly reduced whereas S1P was significantly increased. mRNA analyses of 11 sphingolipid metabolizing genes including S1P receptors in 7 MMs showed that SK1, SK2, and alkaline ceramidases were significantly increased compared to IBC. Furthermore, we isolated CD138+ myeloma cells from 21 MM patients and found that 13 of the patients had higher SK2 expression in CD138+ MM cells compared to CD138-cells. These data demonstrated abnormal sphingolipid metabolism and dys-regulated SK2 in myeloma cells. We generated SK2-specific shRNA and found that SK2 shRNA down-regulated SK2 mRNA, inhibited proliferation, and induced death in myeloma cells, suggesting that SK2 is important in myeloma cell survival. We then tested the efficacy of ABC294640 (the most-advanced, non-lipid SK2 inhibitor) in 6 MM cell lines. ABC294640 inhibited myeloma cell growth with an IC50s of ∼30 μM, including steroid-resistant and doxorubicin-resistant myeloma cells. ABC294640 inhibited MM cell growth as early as 6 hours after exposure and induced apoptotic cell death as demonstrated by Annexin V staining, PARP cleavage and caspase 9 activation. ABC294640 inhibited primary human CD138+MM cells with the same efficacy as with MM cell lines, demonstrating the potential of ABC294640 for the treatment of MM. Additionally, we found that blocking S1P receptors with FTY720 (a S1PR agonist with receptor degradation) induced apoptosis in MM cells. We performed extensive mechanistic and signaling pathway analyses and found that ABC294640 inhibited Mcl-1 and C-Myc expression, but had no effects on Bcl2. Furthermore, ABC294640 induced cell death by directing Mcl-1 to proteosomal degradation. MM is dependent on the bone marrow niche microenvironment for survival and progression. We found that ABC294640 was effective in inducing apoptosis in MM cells even in the presence of stromal cells. Finally, we are currently testing the in vivo effect of ABC294640 alone and in combination with bortezomib, thalidomide and dexamethasone in MM xenograft model transplanted with MM cells stably expressing luciferase. Our early preliminary results were encouraging. Conclusion Our data demonstrate that sphingolipid metabolism is abnormal and provides an attractive target in the treatment of refractory/relapsed MM. Disclosures: Costa: Otsuka: Research Funding.

scholarly journals Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 11-13 ◽  
Author(s):  
XG Zhang ◽  
B Klein ◽  
R Bataille
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
S Phase ◽  
Myeloma Cells ◽  
Severity Of The Disease

It has recently been demonstrated that interleukin-6 (IL-6) is a potent myeloma-cell growth factor in the majority of patients with multiple myeloma (MM). Using an anti-bromodeoxyuridine monoclonal antibody (MoAb) to specifically count myeloma cells in the S-phase (ie, labeling index, LI), we demonstrate that the IL-6 responsiveness of myeloma cells in vitro is directly correlated with their LI in vivo. Myeloma cells from all 13 patients with high LIs in vivo (greater than or equal to 1%) responded in vitro to IL-6, the strongest response occurring in cells from five patients with plasma-cell leukemia. In contrast, the cells of only two of eight patients with low myeloma-cell LIs in vivo (less than 1%) responded to IL-6 in vitro. After seven days of culturing with 1,000 U/mL recombinant IL-6 (rIL-6), the median LI value in the first group of patients (in vivo LI greater than or equal to 1%) was 11%, ie 11 times higher (P less than .01) than the median LI value (1%) in the second group of patients (in vivo LI less than 1%). Thus, the in vitro IL-6 responsiveness of myeloma cells is directly related to their in vivo proliferative status, and hence to the severity of the disease.

Gadolinium Containing Contrast Agent Promotes Multiple Myeloma Cell Growth: Implication for Clinical Use of MRI in Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1809-1809
Author(s):  
Mariateresa Fulciniti ◽  
Swaminathan Sundararaman ◽  
Puru Nanjappa ◽  
Samir B Amin ◽  
Prajwal Chevireddy ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Contrast Agent ◽  
Conflicts Of Interest ◽  
Bone Lesions ◽  
Myeloma Cells ◽  
Growth Promoting

Abstract Abstract 1809 Poster Board I-835 Bone marrow infiltration by myeloma cells and osteolytic bone lesions are the major features of Multiple Myeloma. Magnetic Resonance Imaging (MRI) has been used in MM not only to image bone marrow (BM) and to identify lytic bone disease but to also evaluate therapeutic response and prognosis. Gadolinium (Gd)-based contrast agents are frequently used to enhance MRI resolution. We evaluated effect of the most common Gd-containing agent, Omniscan, on myeloma cells. We observed that Omniscan induced both time and dose dependent MM cell growth in vitro (8-20 fold increase relative to control). Importantly, the presence of BMSC enhanced the effect of Omniscan on growth of both MM cell lines and primary MM cells. However, Omniscan was not able to overcome cytotoxic effects of conventional and novel agents in MM. This growth promoting effects were not observed on normal BM stromal cells. Evaluating the molecular mechanism of action of Omniscan on MM cells, we observed time dependent ERK1/2 phosphorylation as well as reversal of growth promoting effects of Omniscan by specific inhibition of ERK signaling; however, Omniscan had no effect on STAT3 and AKT signaling pathways. Next, we investigated in vivo effect of Omniscan in a murine xenograft model of MM. Following detection of tumor, mice were treated with either iv Omniscan or PBS. Treatment with Omniscan significantly induced MM tumor growth compared to control mice (1042 ±243 mm3 vs 502 ±137 mm3 respectively; p=0.0001). Finally in autopsies in 8 MM patients with repeated exposure to Omniscan, we quantified gadolinium in various tissues using Inductively-coupled mass spectrometry. We observed massive quantities of gadolinium accumulation in tissues of these MM patients regardless of their renal function. These results, confirming both in vitro and in vivo growth promoting effects of Gd-containing contrast agent on MM, suggest the need for further analysis of the mechanism of its action on myeloma cells and careful analysis of its clinical impact in MM patients undergoing MRI evaluation. Disclosures No relevant conflicts of interest to declare.

Trolox Improves the Sensitivity of Multiple Myeloma to Arsenic Trioxide in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4920-4920
Author(s):  
Michael Sebag ◽  
Xian-Fang Huang ◽  
Nicolas Garnier ◽  
Wilson H. Miller ◽  
Koren Mann
Keyword(s):  
Oxidative Stress ◽  
Multiple Myeloma ◽  
Cell Growth ◽  
Mouse Model ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Malignant Cells ◽  
Myeloma Cells

Abstract Abstract 4920 Arsenic trioxide (ATO) induces apoptosis and promotes differentiation of acute promyelocytic leukemia (APL) cells, but has less activity in other types of cancers. One factor that may impede ATO success outside of APL is its toxicity profile, which limits in vivo concentrations and therefore, therapeutic benefit. We have reported that trolox, an analogue of alpha tocopherol, can augment ATO sensitivity in a variety of malignant cells, while protecting non-malignant cells from ATO toxicity. In this current study, we have focused on Multiple Myeloma (MM), a plasma cell malignancy that often shows resistance to apoptosis, drug inhibition and remains incurable despite tremendous recent advances. Although ATO has activity against MM cells in vitro, clinical trials of ATO, given as a solo agent, in MM have shown limited promise. To see if the addition of trolox could augment ATO toxicity, a panel of human myeloma cell lines (HMCLs, n=9) representing the genetic diversity seen in this disease, were treated with increasing concentration of ATO with and without 100uM trolox. Cell growth was assessed by MTT viability assays and virtually all cell lines were sensitive to varying doses of ATO. Four cell lines (U266, KMS11, MM1R, MM1S) showed profound inhibition of cell growth with very low concentrations of ATO (<1uM). Trolox (100uM) alone had no effect on cell growth, but in concert with ATO further decreased cell growth by up to 50% as compared to the same dose of ATO alone in virtually all cell lines. To further elucidate the mechanism of growth inhibition, annexin V assays were performed by flow cytometry to measure apoptosis. In all cell lines (n=9), a clear increase in the apoptotic fraction was noted when trolox was added to varying doses of arsenic. To test whether oxidative stress plays a role in ATO-mediated apoptosis of myeloma cells, we looked at the induction of a stress response protein (HO-1), a marker of oxidative stress induced by ATO. Western blot analysis revealed that in all myeloma cells tested, HO-1 was dramatically and quickly induced by ATO and further induced by the addition of trolox, indicating a pro-oxidant activity of trolox in the malignant cells. While the mechanism of trolox enhancement of ATO function remains largely unknown, intracellular concentrations of ATO in MM cells, as measured by inductively coupled plasma mass spectrometry, suggest that trolox does not work by augmenting ATO import or intracellular accumulation. To test the efficacy of ATO with trolox in vivo, we used a novel transgenic mouse model of MM that has been shown to faithfully mimic the human disease and its response to treatment (Chesi et al, Cancer Cell 2008 Feb;13(2):167-80). We first treated MM afflicted mice with a low dose of ATO (5.0mg/kg) and Trolox (50mg/kg) to assess for toxicity and tolerability. This dose was well tolerated in all mice when given for 10 days with no obvious toxic effects. Serum protein electrophoresis performed at the end of the 10 day treatment period revealed that even at this low starting dose, one of three mice showed a 30% reduction in its paraprotein peak, while the others remained stable. Further studies with higher ATO concentrations in the same mouse model are underway. In conclusion, these data support the role of ATO plus Trolox, as a promising anti-myeloma therapy. Disclosures No relevant conflicts of interest to declare.

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