Multiple Myeloma Cell U266 Research Articles

Abstract 5548: Novel small-molecule delta opioid receptor ligands modeled from naltrindole inhibit the proliferation of human U266 multiple myeloma cells.

Abstract Multiple myeloma is an invasive plasma cell neoplasm that proliferates in the bone marrow, and is responsible for 10% of all hematological malignancies. Naltrindole, synthesized by Portoghese and colleagues (1988) is a synthetic alkaloid derived from naltrexone, and has the pharmacological profile of a selective delta opioid receptor antagonist. Naltrindole also acts as a potent immunosuppressant in the allogeneic mixed lymphocyte reaction and in inhibiting renal graft rejection, and Gaveriaux-Ruff and colleagues (2001) demonstrated that a non-opioid receptor target was involved in the immunosuppressant activity of naltrindole. In a recent report (Mundra et al., J. Pharmacol. Exp. Ther. 342: 273-287, 2012), naltrindole inhibited human multiple myeloma cell proliferation in vitro and in a murine xenograft model in vivo, by interaction with a non-opioid receptor target. In this study, it was discovered that four novel small-molecule delta opioid receptor ligands (DOPs) derived from naltrindole by rational design (see Peng et al., Bioorgan. & Med. Chem. 17: 6442-6450, 2009) competed for specific 3H-naltrindole binding to human U266 multiple myeloma cells. The apparent dissociation constant, KD, of naltrindole binding to intact multiple myeloma cells was 20 μM, and the Bmax was 1.8 nmol/mg protein. Whole cells were incubated with 10 nM 3H-naltrindole in the absence and presence of 500 μM unlabeled naltrindole (to define specific binding), and the ability of the DOP ligands to displace specific naltrindole binding was assayed at room temperature following a 30 min incubation of each DOP compound at 50 μM. DOP 108 inhibited specific naltrindole binding down to 22 ± 6 % of control (78% inhibition of binding); DOP 121 reduced binding to 36 ± 10 % control; DOP 126 inhibited binding to 12 ± 5 % control, and DOP 402 inhibited binding to 16 ± 7 % control. More detailed competition curves will be performed, but it appears that the affinity of the DOP ligands for the naltrindole binding site are similar or higher than naltrindole itself. When U266 multiple myeloma cells were incubated at 37°C for 72 h in the presence of naltrindole or the DOP ligands, each at 50 μM, the number of viable myeloma cells was inhibited to 42 ± 7, 49 ±11, 31 ± 8, 29 ± 8, and 28 ± 9 % of the number of control untreated cells by naltrindole, DOP 108, DOP 121, DOP 126, and DOP 402, respectively, demonstrating that the efficacy of the DOP compounds to inhibit multiple myeloma cell growth was similar or better than naltrindole. This study provides a rationale basis for further evaluation of the DOP compounds as anti-multiple myeloma therapeutic agents. Citation Format: Richard D. Howells, Jyoti Joshi Mundra, Alessandro Howells, Giovanni Howells, Youyi Peng, William Welsh. Novel small-molecule delta opioid receptor ligands modeled from naltrindole inhibit the proliferation of human U266 multiple myeloma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5548. doi:10.1158/1538-7445.AM2013-5548

Scytonemin inhibits cell proliferation and arrests cell cycle through downregulating Plk1 activity in multiple myeloma cells

Multiple myeloma is the second most common hematologic malignancy. During the pursuit for novel and more selective anticancer drugs, different approaches have pointed to polo-like kinase 1 (Plk1) as a promising target. So we used a novel agent, scytonemin, to inhibit the activity of Plk1 to investigate the effect of Plk1 in multiple myeloma cells. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to examine the effect of scytonemin on the cell proliferation of three multiple myeloma cell lines with different concentration and different time. Flow cytometry was used to examine the effect of scytonemin on the cell cycle of multiple myeloma U266 cells with different concentration and different time. Moreover, the expression of Plk1 was analyzed by Western blot and real-time PCR in myeloma U266 cells with the treatment of scytonemin. Statistical analysis was used to analyze the effect of scytonemin on the cell proliferation and cell cycle with different concentration and different time and the association between Plk1 expression and activity with the treatment of scytonemin. Scytonemin was able to inhibit the proliferation of three myeloma cells in a dose-dependent manner, while U266 was the most sensitive one to scytonemin. Treatment with 3 and 4 μM scytonemin gradually increased the percentage of cells in the G2-M phase in U266 cells upon 48- and 72-h treatment. Scytonemin (at 3 and 4 μM concentration) inhibited multiple myeloma cell growth associated with downregulation of the activity of Plk1 but no effect on the expression of Plk1. Scytonemin, representing a novel Plk1 inhibitor, induced the inhibition of cell growth and cell cycle arrest in multiple myeloma cells by specifically decreasing Plk1 activity. Taken together, scytonemin is a promising novel agent for the treatment of multiple myeloma.

Pigment epithelium-derived factor (PEDF) inhibits survival and proliferation of VEGF-exposed multiple myeloma cells through its anti-oxidative properties

Vascular endothelial growth factor (VEGF) has been reported not only to induce angiogenesis within the bone marrow, but also directly stimulate the proliferation and survival of multiple myeloma cells, thus being involved in the development and progression of this second most common hematological malignancy. We, along with others, have found that pigment epithelium-derived factor (PEDF) has anti-angiogenic and anti-vasopermeability properties both in cell culture and animal models by counteracting the biological actions of VEGF. However, effects of PEDF on VEGF-exposed myeloma cells remain unknown. In this study, we examined whether and how PEDF could inhibit the VEGF-induced proliferation and survival of myeloma cells. PEDF, a glutathione peroxidase mimetic, ebselen, or an inhibitor of NADPH oxidase, diphenylene iodonium significantly inhibited the VEGF-induced reactive oxygen species (ROS) generation, increase in anti-apoptotic and growth-promoting factor, myeloid cell leukemia 1 (Mcl-1) expression, and proliferation in U266 myeloma cells. VEGF blocked apoptosis of multiple myeloma cells isolated from patients, which was prevented by PEDF. PEDF also reduced p22phox levels in VEGF-exposed U266 cells. Furthermore, overexpression of dominant-negative human Rac-1 mutant mimicked the effects of PEDF on ROS generation and Mcl-1 expression in U266 cells. Our present study suggests that PEDF could block the VEGF-induced proliferation and survival of multiple myeloma U266 cells through its anti-oxidative properties via suppression of p22phox, one of the membrane components of NADPH oxidase. Suppression of VEGF signaling by PEDF may be a novel therapeutic target for multiple myeloma.

TNFαMediated IL-6 Secretion Is Regulated by JAK/STAT Pathway but Not by MEK Phosphorylation and AKT Phosphorylation in U266 Multiple Myeloma Cells

IL-6 and TNFα were significantly increased in the bone marrow aspirate samples of patients with active multiple myeloma (MM) compared to those of normal controls. Furthermore, MM patients with advanced aggressive disease had significantly higher levels of IL-6 and TNFα than those with MM in plateau phase. TNFα increased interleukin-6 (IL-6) production from MM cells. However, the detailed mechanisms involved in signaling pathways by which TNFα promotes IL-6 secretion from MM cells are largely unknown. In our study, we found that TNFα treatments induce MEK and AKT phosphorylation. TNFα-stimulated IL-6 production was abolished by inhibition of JAK2 and IKKβ or by small interfering RNA (siRNA) targeting TNF receptors (TNFR) but not by MEK, p38, and PI3K inhibitors. Also, TNFα increased phosphorylation of STAT3 (ser727) including c-Myc and cyclin D1. Three different types of JAK inhibitors decreased the activation of the previously mentioned pathways. In conclusion, blockage of JAK/STAT-mediated NF-κB activation was highly effective in controlling the growth of MM cells and, consequently, an inhibitor of TNFα-mediated IL-6 secretion would be a potential new therapeutic agent for patients with multiple myeloma.

Design and biological characterization of hybrid compounds of curcumin and thalidomide for multiple myeloma

In our efforts to develop effective treatment agents for human multiple myeloma (MM), a series of hybrid molecules based on the structures of thalidomide (1) and curcumin (2) were designed, synthesized, and biologically characterized in human multiple myeloma MM1S, RPMI8226, U266 cells, and human lung cancer A549 cells. The biological results showed that two hybrid compounds, 5 and 7, exhibited significantly improved lethal effects towards all three human MM cell models compared to 1 or 2 alone, as well as the combination of 1 and 2. Furthermore, mechanistic studies in U266 cells demonstrated that 5 and 7 can induce the production of reactive oxygen species (ROS) and cause G1/S arrest, thus leading to apoptosis and cell death. Additionally, they exhibited inhibitory effects on NFκB activation in A549 cells. Collectively, the results obtained from these hybrid compounds strongly encourage their further optimization as new leads to develop effective treatment agents for human MM.

Anticancer activity in human multiple myeloma U266 cells: synergy between cryptotanshinone and arsenic trioxide

Arsenic trioxide (As2O3) has been recently established as one of the most effective drugs for the treatment of patients with acute promyelocytic leukemia. However, it has exhibited to be less efficient for the non-promyelocytic leukaemia or other types of malignant tumors. The purpose of the present study was to explore new therapeutic strategies based on As2O3 for human multiple myeloma. Here, we first report cryptotanshinone (CPT) and As2O3 synergy for enhanced cytotoxicity in human multiple myeloma U266 cells. In particular, the apoptosis related proteins (e.g., cleaved poly (ADP-ribose) polymerase (PARP), caspase-3 and -9) were significantly increased by the combination treatment (iAs(III) + CPT), whereas, the expression of survival proteins such as Bcl-2 and survivin was suppressed, suggesting that the induction of apoptosis through mitochondrial-mediated apoptotic pathway. In addition, there were no appreciable effects observed in cells after exposure to either As2O3 or CPT alone. In order to better understand the molecular mechanism, we further determined the phosphorylation of STAT3, JNK, ERK and p38. Interestingly, phosphorylation of JNK and p38 were remarkably induced by combination treatment, and no significant inhibition of STAT3 or ERK was observed. In addition, induction of apoptosis in human multiple myeloma cells was partially abrogated only by pretreatment with JNK inhibitor and not by p38 inhibitor, suggesting that JNK pathway may play an important role in induction of apoptosis by the combination of iAs(III) and CPT. Further studies are needed to evaluate this synergistic anticancer effect in vivo. In the near future, this new approach might be used clinically for multiple myeloma (MM) treatment.

Paclitaxel-Fe3O4 nanoparticles inhibit growth of CD138–  CD34– tumor stem-like cells in multiple myeloma-bearing mice

BackgroundThere is growing evidence that CD138− CD34− cells may actually be tumor stem cells responsible for initiation and relapse of multiple myeloma. However, effective drugs targeted at CD138− CD34− tumor stem cells are yet to be developed. The purpose of this study was to investigate the inhibitory effect of paclitaxel-loaded Fe3O4 nanoparticles (PTX-NPs) on CD138− CD34− tumor stem cells in multiple myeloma-bearing mice.MethodsCD138− CD34− cells were isolated from a human U266 multiple myeloma cell line using an immune magnetic bead sorting method and then subcutaneously injected into mice with nonobese diabetic/severe combined immunodeficiency to develop a multiple myeloma-bearing mouse model. The mice were treated with Fe3O4 nanoparticles 2 mg/kg, paclitaxel 4.8 mg/kg, and PTX-NPs 0.64 mg/kg for 2 weeks. Tumor growth, pathological changes, serum and urinary interleukin-6 levels, and molecular expression of caspase-3, caspase-8, and caspase-9 were evaluated.ResultsCD138− CD34− cells were found to have tumor stem cell characteristics. All the mice developed tumors in 40 days after injection of 1 × 106 CD138− CD34− tumor stem cells. Tumor growth in mice treated with PTX-NPs was significantly inhibited compared with the controls (P < 0.005), and the groups that received nanoparticles alone (P < 0.005) or paclitaxel alone (P < 0.05). In addition, the PTX-NPs markedly inhibited interleukin-6 secretion, increased caspase-8, caspase-9, and caspase-3 expression, and induced apoptosis of tumor cells in the treated mice.ConclusionPTX-NPs proved to be a potent anticancer treatment strategy that may contribute to targeted therapy for multiple myeloma tumor stem cells in future clinical trials.

Set9, NF-κB, and microRNA-21 mediate berberine-induced apoptosis of human multiple myeloma cells

To investigate the mechanisms by which berberine suppressed the proliferation of human multiple myeloma cells. Human U266 multiple myeloma cell line was tested. Cell proliferation, apoptosis, ultramicrostructure and secretion function were examined using Cell Counting Kit-8 (CCK8), flow cytometry (FCM), electron and fluorescence microscopy, as well as ELISA assay. The microRNAs (miRs) and transcription factors in U266 cells were detected using arrays and verified by qRT-PCR. EMSA and luciferase assays were used to verify the p65-dependent transactivation of miR-21 gene. Treatment of U266 cells with berberine (40-160 μmol/L) suppressed cell proliferation and IL-6 secretion in dose- and time-dependent manners. Meanwhile, berberine dose-dependently induced ROS generation, G(2)/M phase arrest and apoptosis in U266 cells, and decreased the levels of miR-21 and Bcl-2. Overexpression of miR-21 counteracted berberine-induced suppression of cell proliferation and IL-6 secretion. In U266 cells treated with berberine (80 μmol/L), the activity of NF-κB was decreased by approximately 50%, followed by significant reduction of miR-21 level. berberine (80-160 μmol/L) increased the level of Set9 (lysine methyltransferase) by more than 2-fold, caused methylation of the RelA subunit, which inhibited NF-κB nuclear translocation and miR-21 transcription. In U266 cells treated with berberine (80 μmol/L), knockdown of Set9 with siRNAs significantly increased NF-κB protein level accompanying with a partial recovery of proliferation. In U266 cells, berberine suppresses NF-κB nuclear translocation via Set9-mediated lysine methylation, leads to decrease in the levels miR21 and Bcl-2, which induces ROS generation and apoptosis.

Brucine induces the apoptosis of U266 multiple myeloma cells by phosphorylation of c-Jun

The aim of this study was to investigate the mechanism of the apoptotic effect of brucine on human multiple myeloma (MM) cells. U266 cells (5x104) were plated in the presence or absence of brucine (0, 0.05, 0.1, 0.2 and 0.4 mg/ml) in 96‑well culture plates for 24‑72 h. The anti-proliferative response to brucine was assessed by MTT assay. Analysis of the cell cycle of U266 cells treated with or without brucine was performed using flow cytometry. The expression change of c-Jun following treatment with brucine or brucine plus the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 was detected using RT-PCR. Brucine appeared to have an effect on apoptosis in a dose- and time‑dependent manner. Cell cycle analysis using flow cytometry revealed the accumulation of cells at the sub-G0/G1 phase. The apoptotic rates were 4.137, 10.55, 12.31, 27.67 and 29.67% (0, 0.05, 0.1, 0.2, 0.4 mg/ml brucine, respectively; P<0.01). The gray scale values were 0.7961 ± 0.007 and 0.4683 ± 0.003 (mRNA expression of c-Jun of U266 cells with or without SP600125, respectively). Concentrations of ≤ 0.4 mg/ml brucine induced apoptosis in U266 cells. Thus, brucine‑induced apoptosis in U266 cells occurs via the JNK signaling pathway and phosphorylation of c-Jun.

A 3-Dimensional Co-Culture Model to Investigate Adhesion-Mediated Drug Resistance in Multiple Myeloma

AbstractAbstract 1826 Background:In multiple myeloma (MM), evidence suggests that the adhesion of malignant plasma cells to bone marrow microenvironment elements is central to MM growth and drug resistance, otherwise referred to as adhesion–mediated drug resistance. As other investigators have shown, the MM-stroma interaction is in part mediated by adhesion molecules like N-cadherin. Goal:To develop a 3-dimensional MM in vitro co-culture model where MM cells interact with stromal elements mimicking the bone marrow microenvironment and use the model to investigate N-cadherin mediated drug resistance. Methods:We co-cultured U266 cells, a MM cell line, with mesenchymal stromal cells (MSCs) isolated from Wharton's jelly (WJ) in a synthetic copolymer (polyglycolic acid/ poly L-lactic acid 90/10 copolymer) (3-dimensional culture conditions). For controls, we cultured U266 cells over a monolayer of WJMCS (2-dimensional conditions) or U266 cells in suspension. Cell proliferation was measured by Ki-67 immunohistochemistry (IHC) stain and lambda light chain expression was measure by enzyme-linked immunosorbent assay (ELISA) and IHC stains. Bortezomib in 1, 5, and 10 nM concentrations was used to treat U266 cells for 24 hours under the three culture conditions. Cytotoxicity was measured by modified Alamar Blue assay. Results:U266 cells in our 3-D model adhered to stromal cells and formed clusters close to matrix material. U266 cells proliferated as evidenced by positive Ki-67 stain, and expressed lambda light chain by IHC and ELISA. Lambda light expression measured by ELISA peaked at 1 week of co-culture in the 3-D model. Though N-cadherin was expressed in both stromal cells and U266 MM cells in 3-D, it was expressed only in stromal cells in 2-D culture conditions. Finally, pre-incubating U266 cells with N-cadherin blocking antibody resulted in less population of the 3-D model by U266 MM cells. Treatment of U266 with bortezomib resulted in less cell cytotoxicity in 3-D and 2-D co-culture conditions compared to U266 cells treated in suspension. Conclusions:Using a 3-D in vitro co-culture model, we demonstrated that only 3-D co-culture conditions resulted in N-cadherin expression in U266 MM cell line. We also demonstrated that U266 population of the 3-D model was successfully blocked using an N-cadherin blocking antibody. We propose N-cadherin mediated adhesion as a mechanism to explain reduced cytotoxicity to bortezomib in 3-D. We propose this model to be used to investigate adhesion-mediated drug resistance. Disclosures:No relevant conflicts of interest to declare.

Targeting Aminopeptidases by Tosedostat (TST) (CHR2797), Alone and with LBH589, Induces Significant Cytotoxicity Against Human Multiple Myeloma (MM) Cells

AbstractAbstract 1847Aminopeptidases (AP) are necessary for the growth and development of malignant cells and have a selectively important role in the maintenance of intracellular amino acid (AA) levels in neoplastic cells. CHR2797 is a novel, low nanomolar inhibitor of the M1 family of AP, a group of metalloenzymes containing a central Zn2+ ion. CHR2797 has antiproliferative and apoptotic effects against MM in vitro by inducing the AA deprivation response (AADR). TST, an oral, chronically administered agent with a good safety profile has demonstrated activity in patients with relapsed/refractory AML and is currently under study as part of combination therapy for untreated elderly patients with AML. At the epigenetic regulatory level, Zn-dependent histone deacetylase (HDAC) cause the deacetylation of histone and non-histone cellular proteins which are critical for gene expression, inducing apoptosis and cell cycle arrest in cancer cells. LBH589 (Panobinostat) is an established pan-HDAC inhibitor with potent in vitro anti-cancer activity in many hematological malignancies. The clinical efficacy of Panobinostat is currently being studied in several Phase II/III clinical trials with particular promise seen in the treatment of MM. Here we examined the potential therapeutic effect of CHR2797, alone and with LBH589, against MM cells. Using MTS and CTG assays, CHR2797, at clinically achievable concentrations, decreased survival and proliferation in MM1S and IL-6-dependent ANBL6 cells, in the presence or absence of bone marrow stromal cells following 72 hours incubation. CHR2797 induces apoptosis in MM cells via activation of Caspase 3/7 and 9 but not Caspase 8. Significantly, CHR2797 (10 μM) induced apoptosis in patient MM cells, as seen by % of annexin V and PI from 22 + 1.5% to 39 + 2.3% after 48h incubation. Combined treatment with CHR2797 and LBH589 in MM cells (MM1S, ANBL6, and INA6) further reduced cell viability following 72 hour incubation when compared with CHR2797 treatment alone, as determined by CTG viability luminescent assay. Both drugs together also augmented growth inhibitory effects when compared with single agent alone, after 72 hours incubation followed by MTS assay. Importantly, the combination of both drugs increased caspase 3/7- & 9-mediated apoptosis than CHR2797 alone in these MM cells following 24h-treatment. Cell cycle analysis (CHR2797 at 1μM; LBH589 at 1 nM) showed an increased growth arrest in G0/G1 cells in MM1R cells treated with both drugs versus CHR2797 alone after 24 hours: 68.5±3.3% versus 36±2.5%. Furthermore, CHR2797 inhibited anti-apoptotic protein Mcl-1 in MM1R and U266 MM cells by immunoblottings. Combined treatment with CHR2797 and LBH589 further blocked Mcl-1 when compared with either treatment alone after 24 hours incubation. Together, these results show that the combination of CHR2797 and LBH589 enhanced anti-myeloma effects when compared with either drug alone. This combination, which also has the potential of being without overlapping clinical toxicities, provides a promising novel approach to anti-myeloma therapy. Disclosures:Singer:Cell Therapeutics, Inc: Employment, Equity Ownership. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees.

Characterization of TFDP1 As Novel Regulatory Gene in Multiple Myeloma

AbstractAbstract 569Alteration in expression and function of transcription factors (TF) has been frequently associated with neoplastic transformation and hence represent important target for cancer therapy. We evaluated expression profile of 172 newly-diagnosed uniformly-treated multiple myeloma (MM) patients. Our oncogenomic analysis of this large annotated patient samples identified TFDP1 as one of the most important gene affecting both overall and event-free survival in MM: elevated DP1 expression was a marker predictive of adverse clinical outcome. Importantly, association of DP1 protein partners, E2F1 and RB, was not observed with poor clinical outcome. This discordance between E2F1 and DP1 levels provides the significance to further evaluate role of DP1 in MM. We have observed that expression of DP1 is pronounced and mostly nuclear in all MM cell lines and primary cells and it is further markedly upregulated upon MM-bone marrow stromal cell (BMSC) interaction. Moreover, RNAi-mediated suppression of DP1 expression inhibited MM cell growth independently of pRB and p53 status, inducing cell cycle arrest and further apoptosis, revealing that E2F/DP activity is indeed essential for MM cell proliferation and survival. Interestingly, interaction with BMSC was able to partly overcome the inhibitory effect of DP1 silencing on MM cell growth. We next evaluted differential DP1 and E2F1 DNA-binding regions and targets in MM1S and U266 MM cells using chromatin immunoprecipitation with E2F1 and DP1 specific antibodies followed by genome wide sequencing (ChIP-Seq) along with gene expression profiling (GEP) after DP1 and E2F1 silencing in same MM cell lines. E2F1 and DP1 direct targets in myeloma have being identified by integrating differential GEP information with the bound target genes defined by ChIP-seq, providing insight into DP1 and E2F1 target genes and function, and identifying exclusive DP1 binding genes in MM with potential functional consequences. We have identified 1392 common genes, 169 E2F1 genes and 221 of distinct targets of DP1 in MM. We have used same approach to identify overlapping and distinct miR targets of DP1 and E2F1.This analysis is providing insight into potential DP1 targets that may predict poor clinical outcome. As interactions between DP1 with E2Fs is central to the regulation of its activity, disruption of this interaction represents a potential therapeutic target. We have investigated a polypeptide corresponding to residues 163 to 199 of DP1 which disrupt DP1-E2F interactions and observed inhibition of MM cell proliferation as evaluated by [3H]-Thymidine uptake after treatment with the peptide in a dose and time dependent manner. In conclusion, we have validated DP1 as an important functional gene in MM, and identified, using ChIP-seq, DP1 - E2F1 regulated targets with potential functional implications. Disruption of this interaction provides a novel therapeutic approach for clinical application of DP1 targeted therapy in MM. Disclosures:Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

Celastrol Overcomes Chemoresistance in Vitro and Potentiates the Effect of Bortezomib Against Human Multiple Myeloma in Nude Mice Mode

AbstractAbstract 5017 Introduction:Multiple myeloma (MM) is a B cell malignancy characterized by clonal proliferation of B cell in the bone marrow with low proliferative index. Despite the advent of novel therapeutics in addition to conventional chemotherapeutics, MM remains incurable because of the development of chemoresistance. Persistent activation of NF-κB/STAT3 signaling pathways and deregulation of apoptosis is considered to play an important role in the development of chemoresistance. The use of anticancer drugs derived from natural sources may be able to overcome resistance without some of the debilitating side effects of conventional chemotherapy. Celastrol is one such compound that has gained substantial attention recently for its anti-inflammatory and anticancer activities and is derived from the Chinese medicinal plant ‘Tripterygium wilfordii.We have demonstrated that celastrol overcomes the chemoresistance and induce apoptosis in MM cells by inhibiting NF-κB and STAT 3 pathways cell lines sensitive and resistant to various chemotherapeutic agents and Bortezomib. Our experimental findings have indicated that celastrol in combination with bortezomib/thalidomide can inhibit proliferation, induce apoptosis and overcome chemoresistance in MM cells in synergistic manner. We also observed that celastrol inhibited the activation of NF-κB and STAT3 and downregulated the expression of various genes involved in MM proliferation, survival and angiogenesis. Materials and Methods:Male athymic balb/c nude mice were implanted with 2×106 cells with either Human MM U266 cell lines subcutaneously. When tumors have reached more than 0. 3 cm in diameter, the mice were randomized into four groups. Group I (control) received corn oil 100 ul i. p. for five days a week, group II received 0. 25 mg/kg celastrol in 100ul corn oil for five days a week, group III received 0. 25 mg/kg bortezomib in 100 ul corn oil i. p. weekly and group IV received 0. 25mg/kg celastrol in 100 ul corn oil i. p. 5 days a week and 0. 25 mg/kg bortezomib in 100 ul corn oil i. p. weekly for 3 consecutive weeks. The tumor volume and body weight of the mice were monitored twice a week for the duration of the experiment. On completion of the treatment period, mice were euthanized by i. p. phentobarbital (40 mg/kg b. w) followed by cervical dislocation and then tumors were dissected and diameters measured. The tumor volume was calculated using the formula [L × W2]/2, where W and L are the width (short diameter) and the length (long diameter) of the tumor and the tumors were subjected to histological examination. Results:In the MM xenograft mice model, we observed that celastrol potentiated the antitumor effects of bortezomib and this correlated with significant suppression of NF-κB, STAT3, COX-2 and VEGF which was demonstrated by IHC. Overall, our data indicates that celastrol could be a potential therapeutic agent for the treatment of MM, especially in combination with the novel anti-myeloma agents. Disclosures:No relevant conflicts of interest to declare.

Bortezomib Could Reduce Cell Viability by Activation of Heme Oxygenase 1 in Multiple Myeloma Cells

AbstractAbstract 5005Bortezomib (PS-341, Velcade®) specifically targeting the ubiquitin-proteosome pathway, is the first defined therapeutic proteosoma inhibitor approved by FDA for treatment of Multiple Myeloma (MM). We explored the hypothesis that the activity of Bortezomib in MM cells may involve the heme oxygenase 1 (HMOX1), a enzyme with pleiotropic effects, catalysing the degradation of heme to carbon monoxide, biliverdin and ferrous iron.After 24h of treatment with Bortezomib at 10nM, the U266 and SKM-M1 MM cell lines showed a decrease of cell viability around 50% and an increase of HMOX1 protein and mRNA expression of 40 fold (p<0. 001). Using a colorimetric assay we observed that Bortezomib increased also the HMOX1enzymatic activity (p<0. 03). To elucidate the significance of the HMOX1 increase after bortezomib treatment, we treated myeloma cells with Bortezomib plus an inducer (Hemin 10uM) or an inhibitor (Tin, Sn-Mesoprotoporphyrin, 10uM) of the enzyme activity. Treatment with Hemin and Tin alone not changed the cell viability. Cell viability was reduced (p<0. 003) by addition of Hemin and was increased (p<0. 002) by Tin addiction with respect to Bortezomib alone. Conclusion:Bortezomib increases HMOX1expression and its enzymatic activity and this increase could be an important mechanism of Bortezomib induced cytotoxicity in MM cells. Modulation of HMOX1 activity has effect on Bortezomib induced cell death. Disclosures:Off Label Use: Eltrombopag is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune (idiopathic) thrombocytopenic purpura (ITP).

Blockade of the Hedgehog Signaling Pathway by the Novel Agent NVP-LDE225 Induces Differentiation, Prevents De-Differentiation and Inhibits Proliferation of Multiple Myeloma Stem Cells in Vitro

AbstractAbstract 3950Chemotherapy-resistant cancer stem cells are a major cause of relapse. It is considered that the small population of CD38++ cells which also exhibit “side population” (SP) characteristics after Hoescht 33342 staining contain chemotherapy resistant multiple myeloma (MM) stem cells. The hedgehog (Hh) signalling pathway is a key regulator of stem cell proliferation and differentiation. A range of Hh inhibitors are currently under development as therapeutics. Our aim was to determine the effect that NVP-LDE225, a novel Hh pathway inhibitor has on MM stem cells.NVP-LDE225 was kindly supplied by Novartis. Side population (SP) cells, considered to contain the MM stem cells, were detected after Hoescht 33342 staining using a BD ARIA II flow cytometer, Proliferation was monitored with CFSE tracking and viability with propidium iodide. MM cell lines RPMI8226, KMS-11, OPM2, U266 and primary MM cells were included. Expression of Hh pathway proteins PTCH1, Smo and Gli1 was determined by flow cytometry. Bone marrow samples were obtained from patients with myeloma after informed consent.CD38++ SP cells were identified in all 4 MM cell lines tested and 90% (26/29) of MM BM samples with the percentage of SP cells ranging from 0 to 9.5% of CD38++ cells. There was no correlation between %SP and %CD38++ and no significant difference in %SP during therapy. PTCH1 expression was higher on SP cells of KMS-11, OPM2 and U266 than non-SP cells (80.5% vs 51.7%) suggesting that the canonical Hh pathway was upregulated in CD38++ SP cells. PTCH1 expression varied from 0.1 – 87% on patient’s CD38++ cells. In vitro dose response curves demonstrated increased killing of plasma cell lines above 1μM NVP-LDE225. The IC50 was 4.7uM for KMS-11 SP cells and 3.4uM for KMS-11 non-SP cells. During culture of flow-sorted CD38++ SP+ and non-SP cells (RPMI 8226 and KMS11), fluctuations in %SP suggest that SP cells differentiated to non-SP cells but also that the SP phenotype is unstable. This is supportive of the concept that the stem cell compartment is in a state of flux. The presence of NVP-LED225 caused a 90% inhibition of non-SP returning to SP and induced an additional 79% differentiation of SP to non-SP KMS11 cells on day 2. Proliferation studies using CFSE tracking of sorted SP cells demonstrated a 34% reduction in proliferation after exposure to NVP-LDE225. Studies with 9 primary human bone marrow samples from patients with MM confirmed the data from cell lines demonstrating that differentiation was significantly induced (t=2.78; p<0.0275) and de-differentiation was blocked.The studies confirm there is flux, to and from the SP and presumably MM stem cell compartment, that the canonical Hh pathway is upregulated in MM SP cells, and that the Hh pathway inhibitor NVP-LDE225 can induce SP differentiation, block de-differentiation, inhibit SP proliferation and at high concentrations is cytotoxic to all cells including MM stem cells. NVP-LDE25 could synergise with other maintenance therapy to reduce drug resistance by inducing the differentiation of chemo-resistant MM stem cells. Disclosures:No relevant conflicts of interest to declare.

Targeting Cereblon with the High Affinity Immunomodulatory Compound CC-220: A Novel Therapeutic Agent for B Cell Dyscrasias

AbstractAbstract 1055 Background:Cereblon (CRBN) is a component of the E3 ubiquitin ligase complex including CUL4A, DDB1, and ROC-1, and was found to be the molecular binding target of thalidomide (Thalomid®), lenalidomide (Revlimid®), and pomalidomide. CC-220 is a novel immunomodulatory compound developed with increased potency and is currently in development for the treatment of immune conditions. The effect of CC-220 on CRBN binding, ubiquitination, and cell proliferation was profiled. Methods:Binding studies to CRBN were conducted using thalidomide analog-conjugated beads in a competitive assay. Endogenous CRBN from human U266 multiple myeloma (MM) cells was measured by incubating cell extracts with varying concentrations of either CC-220 or pomalidomide as a positive control. Affinity beads coupled to a thalidomide acid analog were incubated with the U266 extracts and, after extensive washing of the beads, the bound proteins were eluted. CRBN binding to the thalidomide-coupled affinity beads was determined by quantitative CRBN immunoblot determination. CRBN ubiquitination was measured in HEK293T cells, which were transfected with an amino-terminal His-biotin-tagged CRBN construct, then preincubated with compounds for one hour followed by treatment with the MG132 proteasome inhibitor (to arrest degradation of ubiquitinated proteins). Cells were lysed and processed to measure CRBN ubiquitination by SDS-PAGE and immunoblot analysis using an anti-ubiquitin antibody. Cell proliferation studies were conducted in lenalidomide-sensitive and -refractory multiple myeloma cells. Lenalidomide-resistant or -sensitive H929 MM cell lines were treated with CC-220 for 5 days, and then cell proliferation and viability were assessed by 7-aminoactinomycin D (7-AAD) staining. T-cell costimulation was measured in purified primary human T cells stimulated using immobilized anti-CD3 antibody in cell culture for 2 days, and cytokine secretion was measured by ELISA. Immunoglobulin M and G (IgG and IgM) production was measured from normal donor peripheral blood mononuclear cells by culturing in the presence of the B cell differentiation factors recombinant human IL-2 (20 U/mL), IL-10 (50 ng/mL), IL-15 (10 ng/mL), His-tagged CD40 Ligand (50 ng/mL), polyHistidine mouse IgG1 antibody (5 μg/mL), and ODN 2006-Human TLR9 ligand (10 μg/mL) for 4 days, followed by IL-2, IL-10, IL-15, and IL-6 (50 ng/mL) for an additional 3 days. IgM and IgG were measured by ELISA. Results:In the competitive CRBN binding studies, preincubation with pomalidomide at a concentration of 3 μM resulted in approximately 50% less CRBN bound to the affinity beads, while CC-220 at a concentration of 0.1 μM resulted in similar CRBN binding. CRBN ubiquitination studies in the transfected HEK293T cells resulted in the following potencies: CC-220 IC50 = 0.19 μM; lenalidomide IC50 = 12.9 μM; and pomalidomide IC50 = 21.6 μM. The IC50 value for inhibition of proliferation by CC-220 shifted from 0.01 μM in the parental H929 cell line and 0.04 μM in the DMSO-treated subclone to 0.51–1.58 μM in the lenalidomide-resistant subclones. A 50% decrease in cell cycle (S-phase) was evident after 24 hours of treatment of H929 cells with CC-220. At 48 hours, CC-220 decreased expression of survivin and retinoblastoma protein (pRB) and increased expression of the cyclin-dependent kinase inhibitor p27. CC-220 costimulated IL-2 production by T cells with an EC50 of approximately 0.29 nM, compared with 10 nM for pomalidomide. CC-220 inhibited IgM and IgG production with an IC50 of 0.35 and 2.1 nM, respectively, compared to 17 nM and 63 nM for pomalidomide. Conclusions:The results indicate that CC-220 binds to CRBN with approximately 30-fold higher affinity than pomalidomide, and inhibits CRBN ubiquitination with approximately 110-fold greater potency than pomalidomide in this system. CC-220 is approximately 34-fold more potent than pomalidomide for costimulating IL-2 production by T cells, and is 30- to 48-fold more potent than pomalidomide for inhibiting immunoglobulin production. In summary, CC-220 is a novel high affinity CRBN ligand with cellular potencies 1 or 2 orders of magnitude greater than that of pomalidomide, and is currently in development for the treatment of immune conditions, including those involving B cell dyscrasias. Disclosures:Schafer:Celgene: Employment, Equity Ownership. Rychak:Celgene: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Parton:Celgene Corp: Employment, Equity Ownership. Capone:Celgene Corp: Employment, Equity Ownership. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership.

The Rheumatoid Arthritis Drug Auranofin Has Significant in Vitro Activity in MCL and DLCL and Is Synergistic with a Glutathione Depleting Agent

Abstract 1658There is great interest in repurposing drugs currently available in the pharmacopeia to other indications like cancer as such an approach would significantly shorten the time needed and cost encumbered to provide new and effective cancer therapeutics. One such drug is the gold compound Auranofin (AF), which is FDA approved as a treatment for rheumatoid arthritis (RA). We demonstrate that AF induces MCL cell death with an LD50 of 1000 nM, 500 nM and 90 nM for Granta and Jeko cell lines, and cells derived from a patient biopsy, respectively. The increased sensitivity of primary MCL patient specimens to AF is confirmed on 4 additional patient samples tested. AF similarly induced DLBCL cell death with an LD50 of 500 nM, 500 nM and 1000 nM for OCI Ly-10, SUDHL-6 and −4 cell lines, respectively. Exposure of Granta cells to an AF concentration that induced cell death resulted in the generation of reactive oxygen species (ROS). Pre-treatment of these cells with N-acetyl-cysteine (NAC) or glutathione (GSH) abrogated both ROS generation and the induction of cell death supporting the notion that AF induces NHL cell death through a redox dependent pathway. Although AF does increase mitochondrial membrane permeability (although not through the classical permeability transition pore), the major mechanism through which AF induces NHL cell death is activation of the extrinsic apoptotic pathway. In this regard, AF induces the activation of caspases 7 and 8 and results in PARP cleavage, all of which are blocked by NAC. Despite the fact that AF is a known inhibitor of thioredoxin reductase (TR), its cytotoxic effect is independent of TR inhibition, as we observe no difference in the response to AF in U266 multiple myeloma cells transfected with an expression vector which results in the over-expression of TR. Given the redox dependence of AF-induced cytotoxicity we hypothesized that inhibition of the glutathione system with buthionine sulphoximine (BSO) would further augment AF induced cell death. To test this hypothesis, Granta cells were exposed to both AF and BSO. Significant synergistic interactions of these drugs were seen when tested in a Laska analysis of synergy. For example, whereas the LD50 for AF alone in Granta cells was 1000 nM, the LD50 for AF in combination with 5 μM BSO was 200nm; for Ly-10 cells, the LD50 for AF±BSO was 400 nm vs. 190 nM, respectively, and for SUDHL-6, 411 nM vs. 185 nM, respectively. Similar results were seen in MCL cells flow cytometrically sorted from patient biopsy specimens. As observed in studies using AF alone, the cytotoxic effects of the combination were blocked with both NAC and GSH. Similar to results with AF alone, the synergistic effects of AF and BSO on NHL cytotoxicity were independent of their effects on TR. Exposure of Granta cells to AF resulted in NF-κB inhibition. NF-κB was further inhibited with concomitant exposure to BSO over-expression of relA in Granta cells, however, had no effect on AF and BSO induced cell death, suggesting the synergistic effects of AF and BSO on NHL cell death may be NF-κB-independent. Finally, we demonstrate that AF and BSO modify free thiols on the plasma membrane. As we have recently shown that the redox agent parthenolide induces NHL death in part by modifying surface protein thiols, AF and BSO may induce NHL cell death through a similar mechanism. In summary, AF induces both MCL and DLCL cell death through a redox-dependent pathway that involves the extrinsic apoptotic pathway. Profound synergy is seen with concomitant depletion of glutathione by BSO. The data presented above, along with the fact that AF is a drug having a favorable safety profile in patients, and is an FDA-approved drug for the treatment of RA makes it an attractive candidate for further study as a single agent and in rational combination with other redox active drugs. Disclosures:No relevant conflicts of interest to declare.

Changes in protein profiles of multiple myeloma cells in response to bortezomib

The objective of this study was to determine the changes in protein profiles of U-266 multiple myeloma cells in response to bortezomib. Bortezomib inhibited cell proliferation and increased the loss of mitochondrial membrane potential and caspase-3 activity in a dose-dependent manner. DECODON Delta2D Version 4.3 software demonstrated 37 differentially expressed protein spots: five proteins were newly formed, 10 proteins were lost, 12 proteins were up-regulated and 10 proteins were down-regulated in bortezomib-treated cells as compared to untreated cells. Some of the identified proteins after mass spectrometric analysis were as follows: apoptosis regulatory protein Siva (newly formed), caspase recruitment domain-containing protein 14 (lost), Ras-related protein Rab-25 (up-regulated), nuclear factor κB (NF-κB) p105 subunit (down-regulated). In summary, differentially expressed proteins of MM U-266 cells in response to bortezomib were analyzed and identified. The data obtained from this study may indicate the use of bortezomib for the treatment of various diseases.

Brazilin Induces Apoptosis and G2/M Arrest via Inactivation of Histone Deacetylase in Multiple Myeloma U266 Cells

Although brazilin [7,11b-dihydrobenz(b)indeno[1,2-d]pyran-3,6a,9,10(6H)-tetrol] isolated from Caesalpinia sappan was known to have various biological activities, including anti-inflammation, antibacteria, and antiplatelet aggregation, there is no report yet on its anticancer activity. In the present study, the anticancer mechanism of brazilin was elucidated in human multiple myeloma U266 cells. We found that brazilin significantly inhibited the activity of histone deacetylases (HDACs), transcription factors involved in the regulation of apoptosis and cell cycle arrest in U266 cells. Consistently, brazilin enhanced acetylation of histone H3 at Lys 23, indicating activation of histone acetyltransferase (HAT), and also suppressed the expressions of HDAC1 and HDAC2 at both protein and mRNA levels. Additionally, brazilin significantly increased the number of sub-G1 cell population and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells undergoing apoptosis and also activated caspase-3 and regulated the expression of Bcl-2 family proteins, including Bax, Bcl-x(L), and Bcl-2 in U266 cells, indicating that brazilin induces apoptosis through the mitochondria-dependent pathway. Interestingly, cell cycle analysis revealed that brazilin induced G2/M phase arrest along with apoptosis induction. Consistently, brazilin attenuated the expression of cyclin-dependent kinases (CDKs), such as cyclin D1, cyclin B1, and cyclin E, and also activated p21 and p27 in U266 cells. Furthermore, HAT inhibitor anacardic acid reversed activation of acetyl-histone H3 and cleavage of PARP induced by brazilin, while pan-caspase inhibitor Z-VAD-FMK001 did not affect the expression of HDAC induced by brazilin that brazilin mediates apoptosis via inactivation of HDAC in U266 cells. Notably, brazilin significantly potentiated the cytotoxic effect of standard chemotherapeutic agents, such as bortezomib or doxorubicin, in U266 cells. When our findings are taken together, they suggest that brazilin has potential as a chemotherapeutic agent alone or in combination with an anticancer agent for multiple myeloma treatment.

The hypoxia-mimetic agent cobalt chloride induces cell cycle arrest and alters gene expression in U266 multiple myeloma cells

Hypoxia is a common feature of tumors that occurs across a wide variety of malignancies. Multiple myeloma is an incurable malignant disorder of plasma cells in the bone marrow. Although bone marrow hypoxia is crucial for normal hematopoiesis, the effect of hypoxia on multiple myeloma is poorly understood. In this study, we demonstrated that cobalt chloride (CoCl2)-mediated hypoxia decreased cell viability and altered gene expression in U266 human multiple myeloma cells. CoCl2 induced the loss of cell viability in a concentration-dependent manner. In addition, FACS analysis revealed that the loss of cell viability was related to apoptosis. Using microarray analysis, we identified mRNA expression profile changes in response to CoCl2 treatment in U266 cells. Four hundred and fifty-two mRNAs exhibited >2-fold changes in expression in CoCl2-treated U266 cells compared to their expression in control cells. A follow-up bioinformatics study revealed that a great number of genes with altered expression were involved in apoptosis, cell cycle, transcription and development. In conclusion, these results provide novel evidence that CoCl2-mediated hypoxia affects the expression profiles of genes that are functionally related to apoptosis and angiogenesis in U266 multiple myeloma cells.