SDX-101 Is Cytotoxic and Overcomes Drug Resistance in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3466-3466
Author(s):  
Makoto Hamasaki ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Hiroshi Yasui ◽  
Norihiko Shiraishi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Antineoplastic Agent ◽  
Parp Cleavage ◽  
Family Protein ◽  
Dose Dependent Fashion ◽  
Peripheral Mononuclear Cells ◽  
Induced Apoptosis

Abstract SDX-101 is an oral antineoplastic agent, has been evaluated in a phase I/II study in B-cell malignancies. It induces cytotoxicity at least in part, by significantly inhibiting expression of Mcl-1, an anti-apoptotic Bcl-2 family protein which is highly expressed in CLL cells. Here, we examined the cytotoxicity of SDX-101 against multiple myeloma (MM) cell lines. SDX-101 significantly inhibited growth in MM.1S, U266, RPMI8226 MM cell lines using MTT assays in a time- and dose-dependent fashion, with IC50s of 0.6mM, 1.0mM, and 0.4mM, respectively. In contrast, SDX-101 did not induce cytotoxicity in normal peripheral mononuclear cells (PBMCs) at these concentrations. Importantly, SDX-101 induced cytotoxicity even in dexamethasone (MM.1R)-, doxorubicin (RPMI-Dox40)-, and melphalan (LR5)- resistant MM cell lines. SDX-101 (0.3–1.25mM) triggered apoptosis associated with pro-caspase-3, pro-caspase-8, and PARP cleavage, as confirmed by immunoblotting. Although, interleukin-6 (IL-6) and insulin-like growth factor (IGF)-1 completely abrogates Dex-induced MM cell apoptosis, neither protects against SDX-101-induced apoptosis in MM.1S and RPMI8226 cells. Moreover, dexamethasone, Melphalan, and AS2O3 augment apoptosis induced by SDX-101. Importantly, SDX-101 downregulated both b-catenin and cyclin D1 expression in RPMI8226 cells. Finally, our recent studies demonstrate that the bone marrow (BM) microenvironment promotes MM cell growth, survival, and drug resistance while SDX-101 inhibits viability even if MM cells adherent to BM stromal cells; an environment in which cytotoxic potency is lost with other anti-neoplastic agents. Our data therefore demonstrate that SDX-101 induces apoptosis in MM cells via a mechanism different from conventional MM drugs, and support clinical trials of this agent, alone or in combination with conventional and/or novel agents to improve therapeutic outcome in MM.

Honokiol Overcomes Conventional Drug Resistance in Human Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1488-1488
Author(s):  
Kenji Ishitsuka ◽  
Teru Hideshima ◽  
Makoto Hamasaki ◽  
Raje Noopur ◽  
Kumar Shaji ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Flow Cytometric Analysis ◽  
Caspase 8 ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Human Multiple Myeloma ◽  
Induced Apoptosis

Abstract Honokiol is an active component isolated and purified from Magnolia, a plant used in traditional Chinese medicine. It is an anti-oxidant, and inhibits both xanthine oxidase and angiogenesis. In this study, we first examined the direct toxicity of honokiol against human multiple myeloma (MM) cell lines in vitro. Honokiol significantly inhibited growth of MM cell lines (RPMI8226, U266 and MM.1S) via induction of G1 growth arrest, followed by apoptosis, with IC50 values at 48h of 5 to 10 μg/ml. Moreover, honokiol similarly inhibited growth of doxorubicin (Dox)-resistant (RPMI-Dox40), melphalan resistant (RPMI-LR5), and dexamethasone (Dex)-resistant (MM.1R) cell lines. Furthermore, flow cytometric analysis demonstrated that honokiol (6–10 μg/ml, 48h) induced death of CD38+CD138+ tumor cells isolated from 5 patients with relapsed refractory MM. In contrast, no toxicity was observed in normal peripheral blood mononuclear cells or long term-cultured bone marrow stromal cells (BMSCs) treated with honokiol (≤20 mg/ml). Neither culture of MM cells with BMSCs nor interleukin-6 (IL-6) and insulin like growth factor-1 (IGF-1) protected against honokiol-induced cytotoxicity in MM.1S cells. We next delineated the mechanism of honokiol-triggered cytotoxicity. Honokiol triggered increased expression of Bax and Bad; down regulated Mcl-1 protein expression, followed by caspase-8/9/3 cleavage. Importantly, the pan-caspase inhibitor z-VAD-fmk only partially inhibited honokiol-induced apoptosis in MM.1S cells. Furthermore, honokiol induced apoptosis even in SU-DHL4 cells, which express low level of caspase-8 and -3 and are resistant to both conventional (doxorubicin, melphalan, dexamethason) and novel (bortezomib, revimid) drugs. These results suggest that honokiol may induce apoptosis via both caspase-dependent and -independent pathways. Finally, honokiol inhibited IL-6-induced phosphorylation of ERK1/2, STAT3, and Akt, known to mediate growth, survival, and drug resistance, respectively. Taken together, our results suggest that providing the rationale for clinical evaluation of honokiol to improve patient outcome in MM.

ONC201 Overcomes Chemotherapy Resistance By Upregulation of Bim in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4476-4476 ◽  
Author(s):  
Yong-sheng Tu ◽  
Jin He ◽  
Huan Liu ◽  
Richard Eric Davis ◽  
Robert Z. Orlowski ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Viability ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Wild Type ◽  
Myeloma Cells ◽  
Induced Apoptosis ◽  
Tumor Types

Abstract In multiple myeloma, disease relapse and drug resistance occurs in the majority of myeloma patients after standard treatment despite recent improvements offered by new therapies. Therefore, there is an urgent need for new drugs that can overcome drug resistance and prolong patient survival after failure of standard therapies. ONC201, the founding member of a novel class of anti-tumor agents called impridones, has selective preclinical efficacy against a variety of tumor types. It is currently in phase I and phase II clinical trials for patients with advanced solid tumors and hematological malignancies. Given the pronounced sensitivity of B-cell lymphomas to ONC201, we assessed the efficacy of ONC201 in preclinical models of multiple myeloma. We treated human myeloma cell lines and primary myeloma cells isolated from bone marrow aspirates of myeloma patients with ONC201 for 72 hours. CellTiter-Glo Luminescent and annexin-V binding assays for assessing myeloma cell viability and apoptosis were performed, along with immunoblotting for cleavage of caspases, phosphorylation of signaling kinases, and expression of pro- or anti-apoptotic proteins. ONC201 treatment decreased myeloma cell viability, with IC50 values that were 1 μM to 1.5 μM, even in high risk myeloma cell line RPMI8226. The status of TP53 did not appear to affect the efficacy of ONC201, as MM.1S or NCI-H929 cells with wild-type TP53 and OPM-2 or RPMI8226 with mutated TP53 had a similar sensitivity towards ONC201. These results agree with prior reports in other tumor types that have demonstrated that the efficacy of ONC201 is independent of TP53. Western blot analysis showed increased apoptosis, cleavage of caspase-9, caspase-3, and PARP. We also found that ONC201 induced expression of the pro-apoptotic protein Bim in myeloma cells, which can occur downstream of ERK inactivation. Knockdown of Bim expression in myeloma cells by shRNAs abrogated ONC201-induced apoptosis. Phosphorylation of Bim at Ser69 by Erk1/2 has been shown to promote proteasomal degradation of Bim. In accordance with this mechanism, we observed that ONC201 treatment reduced levels of phosphorylated Erk1/2, an indicator of Erk1/2 kinase activity, and Bim pSer69. In addition, ONC201 induced apoptosis in dexamethasome-, bortezomib-, and carfilzomib-resistant myeloma cell lines with the same efficacy as in wild-type cells. As a rational strategy to increase the efficacy of ONC201 by enhancing its inhibition of proteasome-mediated Bim degradation, we tested combinations of ONC201 with proteasome inhibitors bortezomib or carfilzomib. These combinations were synergistic in reducing cell viability and enhancing Bim expression and PARP cleavage in myeloma cells. Overall, these findings demonstrate that ONC201 inhibits the Erk1/2 signaling pathway and induces Bim expression to induce apoptosis in multiple myeloma regardless of resistance to standard-of-care therapies. Our studies suggest that ONC201 should be evaluated clinically in relapsed/refractory multiple myeloma. Disclosures Allen: Oncoceutics: Employment, Equity Ownership.

Anti-Tumor Activity of a Novel Immunosuppressant FTY720 in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3456-3456
Author(s):  
Hiroshi Yasui ◽  
Teru Hideshima ◽  
Aldo M. Roccaro ◽  
Norihiko Shiraishi ◽  
Makoto Hamasaki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Human Cancer ◽  
Human Peripheral Blood ◽  
Parp Cleavage ◽  
Igf I ◽  
Sphingosine 1 Phosphate

Abstract Sphingosine and its metabolites are bioactive sphingolipids involved in lipid biosynthesis, signal transduction and apoptosis. FTY720, a synthetic sphingosine analogue of myriocine derived from culture filtrates of Isaria sinclairii, has been reported to interact with the sphingosine-1-phosphate specific G protein-linked receptors (S1P1, 3, 4 and 5) (Mandala S et al. Science, 2002) and alter the migration and homing of lymphocytes, thereby inhibiting the immune response (Matloubian M et al. Nature, 2004). Recent studies have also shown that FTY720 induces growth inhibition and/or apoptosis in human cancer cells in vitro as well as in vivo murine model (Azuma H et al. Cancer Research, 2002). To date, however, the biologic sequelae of inhibiting sphingosine-1-phosphate activity on multiple myeloma (MM) cells have not been demonstrated. In the present study, we examined whether FTY720 triggers anti MM activity. FTY720 induced potent cytotoxicity against MM cell lines including MM.1S, U266, RPMI8226, with IC50 at 24 h of 3.0 – 7.0 mM, assessed by trypan-blue exclusion and MTT assays. FTY720 also inhibited growth of doxorubicin (Dox)-resistant RPMI8226-Dox40 and dexamethasone (Dex)-resistant MM.1R cell lines, with IC50 values similar to the parental drug-sensitive cell lines. In contrast, no cytotoxicity of FTY720 was recognized against human peripheral blood mononuclear cells from normal healthy donors. The combination of Dex with FTY720 demonstrated enhanced cytotoxicity compared to either agent alone. Importantly, neither interleukin-6 (IL-6) nor insulin like growth factor-I (IGF-I), which induces MM cell growth and protection against Dex-induced apoptosis, protected against FTY720-induced growth inhibition. The anti-MM mechanisms of action of FTY720 were next studied, and FTY720 induced caspase-dependent apoptosis in MM cell lines: FTY720 triggers caspase−8, −9 and −3 cleavage, followed by PARP cleavage and DNA fragmentation, as confirmed by Western blotting and agarose gel electrophoresis, respectively. Moreover, FTY720 abrogated both IL-6 mediated phosphorylation of Akt-1, STAT3 and p42/44MAPK, and IGF-I mediated Akt-1 phosphorylation. Importantly, paracrine MM cell growth with bone marrow stromal cells was strongly inhibited by FTY720. These results suggest that FTY720 overcomes drug resistance in MM cells and, providing the rationale for its clinical evaluation to improve patient outcome in MM.

JS-K, a GST-Activated Nitric Oxide Generator, Induces Apoptosis and Overcomes In Vitro Drug Resistance in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1593-1593
Author(s):  
Tanyel Kiziltepe ◽  
Kenji Ishitsuka ◽  
Teru Hideshima ◽  
Noopur Raje ◽  
Norihiko Shiraishi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Tumor Cells ◽  
Cell Lines ◽  
Biological Effects ◽  
Treatment Modalities ◽  
Cancer Drug ◽  
Induced Apoptosis

Abstract Multiple myeloma (MM) is currently an incurable hematological malignancy. A major reason for the failure of currently existing therapies is the chemotherapeutic resistance acquired by the MM cells upon treatment. Overexpression of glutathione S-transferases (GST) has been shown as one possible mechanism of anti-cancer drug resistance in a broad spectrum of tumor cells. JS-K (O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) belongs to a class of pro-drugs which are designed to release nitric oxide (NO) on reaction with GST. JS-K can possibly turn GST overexpression to the tumor’s disadvantage by (1) consuming intracellular GSH and preventing drug inactivation; and (2) by exposing tumor cells to high intracellular concentrations of NO. JS-K has potent in vitro and in vivo anti-leukemic activity. The purpose of the present study is to examine the biological effects of JS-K on human MM cells. We demonstrate that JS-K has significant in vitro cytotoxicity on MM cell lines, with an IC50 of 0.3-2 mM at 48 hours. JS-K also induces cytotoxicity on cell lines that are resistant to conventional chemotherapy (i.e., MM1R, RPMI-Dox40, RPMI-LR5, RPMI-MR20). Importantly, no cytotoxic effects of JS-K were detected on peripheral blood mononuclear cells (PBMNC) obtained from healthy volunteers at these doses. Moreover, JS-K could overcome the survival and growth advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells (BMSC). JS-K caused a transient G2/M arrest followed by apoptosis, as determined by flow cytometric analysis using PI, Annexin V and Apo2.7 staining. JS-K-induced apoptosis was associated with caspase 8, 7, 9 and 3 activation. Interestingly, Fas was upregulated by JS-K, suggesting the involvement of death receptor pathway in induction of apoptosis. JS-K also triggered Mcl-1 cleavage and Bcl-2 phosphorylation, suggesting the involvement of mitochondrial pathway. In addition, apoptosis inducing factor (AIF), endonuclease G (EndoG) and cytochrome c were released into the cytosol during apoptosis. Taken together, these findings suggest the involvement of both intrinsic and extrinsic apoptotic pathways in JS-K-induced apoptosis in MM cells. In summary, our studies demonstrate that JS-K induces apoptosis and overcomes in vitro drug resistance in MM cells. Therefore, JS-K is a novel compound which carries significant potential to be included in the repertoire of existing treatment modalities for MM. Ongoing studies are delineating the mechanism of action of JS-K to provide the preclinical rationale for combination therapies to overcome drug resistance and improve patient outcome.

Bone Marrow Stromal Cells Induce Bortezomib Resistance in Multiple Myeloma Cells through Downregulation of miRNA-101-3p Targeting Survivin

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1772-1772 ◽  
Author(s):  
Jahangir Abdi ◽  
Yijun Yang ◽  
Patrick Meyer-Erlach ◽  
Hong Chang
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Molecular Mechanisms ◽  
Marrow Stromal Cells ◽  
Survivin Gene ◽  
Induced Apoptosis

Abstract INTRODUCTION It is not yet fully understood how bone marrow microenvironment components especially bone marrow stromal cells (BMSCs) induce drug resistance in multiple myeloma (MM). This form of drug resistance has been suggested to pave the way for intrinsic (de novo) resistance to therapy in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Hence, deciphering the molecular mechanisms involved in induction of above resistance will help identify potential therapeutic targets in MM combined treatments. Our previous work showed that BMSCs (normal and MM patient-derived) induced resistance to bortezomib (BTZ) compared with MM cells in the absence of stroma. This resistance was associated with modulation of a transcriptome in MM cells, including prominent upregulation of oncogenes c-FOS, BIRC5 (survivin) and CCND1. However; whether these oncogenes mediate BTZ resistance in the context of BMSCs through interaction with miRNAs is not known. METHODS Human myeloma cell lines, 8226, U266 and MM.1s, were co-cultured with MM patient-derived BMSCs or an immortalized normal human line (HS-5) in the presence of 5nM BTZ for 24 h. MM cell monocultures treated with 5nM BTZ were used as controls. Co-cultures were then applied to magnetic cell separation (EasySep, Stem Cell Technologies) to isolate MM cells for downstream analyses (western blotting and qPCR). Total RNA including miRNAs was isolated from MM cell pellets (QIAGEN miRNeasy kit), cDNAs were synthesized (QIAGEN miScript RT II kit) and applied to miScript miRNA PCR Array (SABioscience, MIHS-114ZA). After normalization of all extracted Ct values to 5 different housekeeping genes, fold changes in miRNA expression were analyzed in co-cultures compared to MM cell monocultures using the 2-ΔΔCt algorithm. Moreover, survivin gene was silenced in MM cells using Ambion® Silencer® Select siRNA and Lipofectamine RNAiMAX transfection reagent. Survivin-silenced cells were then seeded on BMSCs and exposed to BTZ. Percent apoptosis of gated CD138+ MM cells was determined using FACS. For our overexpression and 3'UTR reporter experiments, we transiently transfected MM cells with pre-miR-101-3p, scrambled miRNA or pEZX-3'UTR constructs using Endofectin reagent (all from GeneCopoeia). RESULTS BMSCs upregulated survivin gene / protein (a member of inhibitors of apoptosis family) and modulated an array of miRNAs in MM cells compared to MM cells in the absence of stroma. The more noticeably downregulated miRNAs were hsa-miR-101-3p, hsa-miR-29b-3p, hsa-miR-32-5p, hsa-miR-16-5p (4-30 fold) and highly upregulated ones included hsa-miR-221-3p, hsa-miR-409-3p, hsa-miR-193a-5p, hsa-miR-125a-5p (80-330 fold). We focused on miRNA-101-3p as it showed the highest level of downregulation (30 fold) and has been shown to function as an important tumor suppressor in other malignancies. Real time RT-PCR confirmed downregulation of miRNA-101-3p. Moreover, microRNA Data Integration Portal (mirDIP) identified miRNA-101-3p as a putative target for survivin and Luciferase activity assays confirmed binding of miRNA-101-3p to 3'UTR of survivin. In addition, overexpression of miRNA-101-3p downregulated survivin and sensitized MM cells to BTZ-induced apoptosis. Furthermore, silencing of survivin upregulated miRNA-101-3p and increased BTZ-induced apoptosis in MM cell lines both in the absence of BMSCs (Apoptosis range in BTZ-treated conditions: 57.65% ± 4.91 and 28.66% ± 0.78 for si-survivin and scrambled control, respectively, p<0.05) and in the presence of BMSCs (41.23% ± 1.43 and 14.8% ± 0.66, for si-survivin and scrambled control, respectively, p<0.05). CONCLUSION Our results indicate that BMSCs downregulated miRNA-101-3p and upregulated survivin in MM cells compared to MM cells in the absence of stroma. Silencing of survivin or overexpression of miRNA-101-3p sensitized MM cells to BTZ in the presence of BMSCs. These findings suggest that miRNA-101-3p mediates BTZ response of MM cells in the presence of BMSCs by targeting survivin and disclose a role of survivin-miRNA-101-3p axis in regulation of BMSCs-induced BTZ resistance in MM cells, thus provide a rationale to further investigate the anti-myeloma activity of miRNA-101-3p in combination with BTZ as a potential novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals KP772 overcomes multiple drug resistance in malignant lymphoma and leukemia cells in vitro by inducing Bcl-2-independent apoptosis and upregulation of Harakiri

2021 ◽  
Author(s):  
Lisa Kater ◽  
Benjamin Kater ◽  
Michael A. Jakupec ◽  
Bernhard K. Keppler ◽  
Aram Prokop
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Caspase 3 ◽  
Multiple Drug Resistance ◽  
Antineoplastic Agent ◽  
Resistant Cell ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Induced Apoptosis

AbstractDespite high cure rates in pediatric patients with acute leukemia, development of resistance limits the efficacy of antileukemic therapy. Tris(1,10-phenanthroline)tris(thiocyanato-κN)lanthanum(III) (KP772) is an experimental antineoplastic agent to which multidrug-resistant cell models have shown hypersensitivity. Antiproliferative and apoptotic activities of KP772 were tested in leukemia, lymphoma and solid tumor cell lines as well as primary leukemia cells (isolated from the bone marrow of a child with acute myeloid leukemia (AML). The ability to overcome drug resistances was investigated in doxorubicin- and vincristine-resistant cell lines. Real-time PCR was used to gain insight into the mechanism of apoptosis induction. KP772 inhibited proliferation and induced apoptosis in various leukemia and lymphoma cell lines in a concentration-dependent manner (LC50 = 1–2.5 µM). Primary AML cells were also sensitive to KP772, whereas daunorubicin showed no significant effect. KP772 induces apoptosis independently of Bcl-2, Smac, and the CD95 receptor and is also effective in caspase 3-deficient MCF7 cells, indicating that apoptosis is partly triggered independently of caspase 3. mRNA expression profiling revealed an upregulation of the BH3-only Bcl-2 protein Harakiri in the course of KP772-induced apoptosis. Remarkably, KP772 overcame drug resistance to doxorubicin and vincristine in vitro, and the apoptotic effect in resistant cells was even superior to that in non-resistant parental cells. In combination with vincristine, doxorubicin and cytarabine, synergistic effects were observed in BJAB cells. The cytotoxic potency in vitro/ex vivo and the remarkable ability to overcome multidrug resistance propose KP772 as a promising candidate drug for antileukemic therapy, especially of drug-refractory malignancies.Graphic abstract

p53-Independent Anti-Myeloma Activity of Prima-1met

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1826-1826
Author(s):  
Manujendra N Saha ◽  
Hua Jiang ◽  
Chen Mei-Hsi ◽  
Hong Chang
Keyword(s):  
Stress Response ◽  
Cell Lines ◽  
Therapeutic Strategy ◽  
Caspase 3 ◽  
Mononuclear Cells ◽  
Parp Cleavage ◽  
Mutant P53 ◽  
Wild Type ◽  
Cytotoxic Response ◽  
Induced Apoptosis

Abstract Abstract 1826 Background: Although mutation/deletion of p53 is relatively rare (∼10%) in multiple myeloma (MM), this subset of patients are resistant to current therapies with very poor prognosis. Thus, improved therapeutic strategy is urgently required for this high risk group of patients. Prima-1met (p53 reactivation and induction of massive apoptosis), a methylated derivative and more active analog of prima-1, is a small molecule which restores tumor suppressor function to mutant p53 and has shown to induce cytotoxic effects and apoptosis in various human tumor cells harboring mutant p53. However, anti-myeloma activity of prima-1met is unknown. Experimental design: 6 human MM cell lines, primary MM samples from 7 newly diagnosed patients, bone marrow mononuclear cells (BMNCs) and peripheral blood mononuclear cells (PBMNCs) from 3 healthy donors were used for this study. MM.1S and H929 cell lines harbour wild type p53; 8266, U266, and LP1 express mutant p53; and 8226R5 does not express p53 (p53 null). The cells were treated with different dose (2.5-100 μM) of prima-1met for different time periods. The therapeutic effect of prima-1met was studied in these cells using MTT cell viability assay, flow cytometry (FCM), qRT-PCR, and Western blot (WB) analysis. Results: Prima-1met efficiently inhibited the viability of MM cell lines irrespective of p53 status. Similarly, prima-1met induced a dose-dependent cytotoxic response in 5 of the 7 patient samples tested. However, prima-1met only showed limited cytotoxicity toward normal BMNCs or PBMNCs suggesting a preferential killing of MM cells by prima-1met. We next examined whether the declining in the viability of the cells was due to the apoptosis induction by prima-1met. Depending on the cell types, 48 hrs after treatment with 100 μM prima-1met 35–70% Annexin V-positive cells was determined by FCM. The cytotoxic effect of prima-1met was associated with activation of caspase-3, PARP cleavage, up-regulation of Noxa and c-Jun without significant modulation of p53 level. Treatment of cells with prima-1met induced the activation of caspase-8 but not -9 suggesting the association of extrinsic pathway. Furthermore, the pan-caspase inhibitor, Z-VAD-FMK, inhibited the activation of caspase-3 indicating that prima-1met-induced apoptosis in MM cells is caspase-dependent. The apoptotic effect of prima-1met was not significantly affected in H929 cells transfected with p53 siRNA confirming that prima-1met exerts anti-myeloma activity in a p53-independent manner. To further explore the molecular mechanisms associated with prima-1met-induced apoptosis, RNA from DMSO-treated and prima-1met-treated MM cells (MM.1S, U266 and 8266R5) was profiled by Illumina HT-12 microarray and differentially expressed genes were analysed. A significant number of genes associated with stress response such as HASP1B, HSPA6, FOS, ATF3, MYC and EGR-1 were modulated in all 3 cell lines upon prima-1met treatment. Of note, EGR-1 is highly up-regulated (7 to 21-fold) in all 3 cell lines. EGR-1 is a member of the immediate-early gene family and plays a pivotal role in the regulation of cell growth, differentiation and apoptosis. Overexpression of EGR-1 has shown to down-regulate anti-apoptotic protein survivin and induction of apoptosis in myeloma cells. The role of EGR-1 and other relevant stress response genes associated with p53-independent apoptosis induced by prima-1met is currently under investigation. In addition, we found a strong synergistic effect in MM cells harbouring either wild type or mutant p53 when prima-1met was combined with dexamethasone (DXM) or doxorubicin (DOXO). We examined cell cytotoxicity of the combination by using prima-1met and DOXO/DXM at concentrations lower than their maximal cytotoxic concentrations. The combination of 2 μM prima-1met and 0.5 μM DOXO/DXM produced a synergistic response (CI=0.82-0.87) in H929 cells. The combination of 10 μM prima-1met and 2.0 μM DOXO displayed a synergistic cytotoxic response (CI=0.68) in U266 cells. Conclusion: This study is the first to show the potential p53-indpendent anti-myeloma activity of prima-1met. Our results provide the framework for the clinical evaluation of prima-1met either alone or in combination with other chemotherapeutic agents which may offer a novel and more efficient therapeutic strategy for the treatment of MM patients carrying either wild type or mutant p53. Disclosures: No relevant conflicts of interest to declare.

TRAIL/Apo2L ligand selectively induces apoptosis and overcomes drug resistance in multiple myeloma: therapeutic applications

Blood ◽  
2001 ◽  
Vol 98 (3) ◽  
pp. 795-804 ◽  
Author(s):  
Constantine S. Mitsiades ◽  
Steven P. Treon ◽  
Nicholas Mitsiades ◽  
Yoshihito Shima ◽  
Paul Richardson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Mononuclear Cells ◽  
Nuclear Translocation ◽  
Death Receptor 5 ◽  
Therapeutic Applications ◽  
Trail Receptors ◽  
Induced Apoptosis

Abstract Multiple myeloma (MM) remains incurable and novel treatments are urgently needed. Preclinical in vitro and in vivo evaluations were performed to assess the potential therapeutic applications of human recombinant tumor necrosis factor (TNF)–related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) in MM. TRAIL/Apo2L potently induced apoptosis of MM cells from patients and the majority of MM cell lines, including cells sensitive or resistant to dexamethasone (Dex), doxorubicin (Dox), melphalan, and mitoxantrone. TRAIL/Apo2L also overcame the survival effect of interleukin 6 on MM cells and did not affect the survival of peripheral blood and bone marrow mononuclear cells and purified B cells from healthy donors. The status of the TRAIL receptors (assessed by immunoblotting and flow cytometry) could not predict TRAIL sensitivity of MM cells. The anti-MM activity of TRAIL/Apo2L was confirmed in nu/xid/bg mice xenografted with human MM cells; TRAIL (500 μg intraperitoneally daily for 14 days) was well tolerated and significantly suppressed the growth of plasmacytomas. Dox up-regulated the expression of the TRAIL receptor death receptor 5 (DR5) and synergistically enhanced the effect of TRAIL not only against MM cells sensitive to, but also against those resistant to, Dex- or Dox-induced apoptosis. Nuclear factor (NF)-κB inhibitors, such as SN50 (a cell-permeable inhibitor of the nuclear translocation and transcriptional activity of NF-κB) or the proteasome inhibitor PS-341, enhanced the proapoptotic activity of TRAIL/Apo2L against TRAIL-sensitive MM cells, whereas SN50 reversed the TRAIL resistance of ARH-77 and IM-9 MM cells. Importantly, normal B lymphocytes were not sensitized to TRAIL by either Dox, SN50, or PS-341. These preclinical studies suggest that TRAIL/Apo2L can overcome conventional drug resistance and provide the basis for clinical trials of TRAIL-based treatment regimens to improve outcome in patients with MM.

Atiprimod (N-N-diethl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) Inhibits Myeloma in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2401-2401
Author(s):  
Makoto Hamasaki ◽  
Teru Hideshima ◽  
Pierfrancesco Tassone ◽  
Paola Neli ◽  
Kenji Ishitsuka ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Parp Cleavage ◽  
Dose Dependent Fashion ◽  
Orally Bioavailable ◽  
Induced Apoptosis ◽  
Endothelial Growth Factor

Abstract Atiprimod (N-N-diethl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) is an orally-bioavailable cationic amphiphilic compound which significantly inhibits inflammation in rat arthritis and multiple sclerosis models. It acts, at least in part, by significantly inhibiting production of interleukin (IL)-6. Since IL-6 mediates MM cell growth, survival and drug resistance in the bone marrow (BM) microenvironment, we in this study characterized the effect of Atiprimod on human MM cells. We first demonstrated that Atiprimod significantly inhibited growth (p&lt;0.05) in MM.1S, U266, and RPMI8226 MM cell lines in a time- and dose-dependent fashion, with IC50s of 0.5–1.25 mM. It also induced cytotoxicity in dexamethasone (Dex)-, doxorubicin (Dox)-, and melphalan (Mel)-resistant MM cell lines; as well as in patient MM cells, but not in normal PBMCs. Atiprimod triggered MM cell apoptosis via activation of caspase-8 and caspase-3, followed by PARP cleavage. Importantly, neither IL-6 nor insulin-like growth factor (IGF)-1, which completely abrogate Dex-induced apoptosis, protects against Atiprimod-induced apoptosis in MM.1S cells. In combination treatment studies, both conventional (Dex, Dox, Mel) and novel (As2O3) agents augment MM cell apoptosis induced by Atiprimod. Atiprimod inhibits STAT3 and Akt, but not ERK1/2, phosphorylation triggered by IL-6, BAX, Bcl-xl, and Mcl-1 in MM.1S cells. Importantly Atiprimod inhibits both IL-6 and vascular endothelial growth factor (VEGF) secretion in BM stromal cells (BMSCs) triggered by MM cell adherence to BMSCs, as well as associated MM cell growth. Finally, Atiprimod inhibits tumor OPM1 MM cell growth in vivo and prolongs survival in a SCID mouse model. Our data therefore demonstrate that Atiprimod both induces MM cell apoptosis and inhibits cytokine secretion in the BM milieu, providing the framework for ongoing clinical trials of this agent to improve patient outcome in MM.

Inhibition of Notch signaling induces apoptosis of myeloma cells and enhances sensitivity to chemotherapy

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2220-2229 ◽  
Author(s):  
Yulia Nefedova ◽  
Daniel M. Sullivan ◽  
Sophia C. Bolick ◽  
William S. Dalton ◽  
Dmitry I. Gabrilovich
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Notch Signaling ◽  
Mononuclear Cells ◽  
De Novo ◽  
Drug Induced ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells ◽  
Induced Apoptosis

Drug resistance remains a critical problem in the treatment of patients with multiple myeloma. Recent studies have de-termined that Notch signaling plays a major role in bone marrow (BM) stroma-mediated protection of myeloma cells from de novo drug-induced apoptosis. Here, we investigated whether pharmacologic inhibition of Notch signaling could affect the viability of myeloma cells and their sensitivity to chemotherapy. Treatment with a γ-secretase inhibitor (GSI) alone induced apoptosis of myeloma cells via specific inhibition of Notch signaling. At concentrations toxic for myeloma cell lines and primary myeloma cells, GSI did not affect normal BM or peripheral blood mononuclear cells. Treatment with GSI prevented BM stroma-mediated protection of myeloma cells from drug-induced apoptosis. The cytotoxic effect of GSI was mediated via Hes-1 and up-regulation of the proapoptotic protein Noxa. In vivo experiments using xenograft and SCID-hu models of multiple myeloma demonstrated substantial antitumor effect of GSI. In addition, GSI significantly improved the cytotoxicity of the chemotherapeutic drugs doxorubicin and melphalan. Thus, this study demonstrates that inhibition of Notch signaling prevents BM-mediated drug resistance and sensitizes myeloma cells to chemotherapy. This may represent a promising approach for therapeutic intervention in multiple myeloma.

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