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.

Multiple Myeloma (MM) Highly Expresses CGEN-928 and the Anti-CGEN-928 TM21 Antibody Markedly Inhibits the Growth of MM Cells and Enhances the Anti-Myeloma Effects of Dexamethasone, Melphalan and Bortezomib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4067-4067
Author(s):  
Haiming Chen ◽  
Mingjie Li ◽  
Cathy Wang ◽  
Jessica Wang ◽  
Eric Sanchez ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Tumor Cells ◽  
Cell Lines ◽  
Polyclonal Antibody ◽  
Flow Cytometric Analysis ◽  
Therapeutic Options ◽  
Treatment Modalities ◽  
In Cells

Abstract Abstract 4067 Although patients with multiple myeloma (MM) initially respond to current treatment modalities, it remains an incurable disease. Many new therapeutic options have become available during the past several years but nearly all patients develop resistance to currently available therapeutic options. In addition, there is no tumor marker that is uniformly expressed in all MM cells although CD138 is considered to be present on the surface of tumor cells in most cases of MM but generally is only present in a subset of the patients' tumor population and may be absent in the most resistant part of the tumor clone. In order to address these unmet clinical needs, we queried Compugen's MED Platform, an expression database which covers over 40,000 microarray experiments, for genes overexpressed in B cell-derived malignancies including MM and that exhibit low expression levels in normal cells and tissues. One of the most prominent candidates was CGEN-928, which was validated as over-expressed in MM at the mRNA level using an independent panel of both hematological malignancies and normal tissues. In this study, we first investigated whether the previously unidentified membrane antigen CGEN-928 is expressed in cells from human MM cell lines, human MM xenografts and fresh bone marrow (BM) aspirates derived from MM patients using flow cytometric analysis and immunohistochemical staining with the anti-CGEN-928 TM21 polyclonal antibody (Compugen Ltd, Tel Aviv, Israel). Using this antibody, we found that CGEN-928 was highly expressed in cells from the MM1s, U266 and RPMI8226 MM cell lines. Next, we examined CGEN-928 antigen expression in fresh tumor cells from BM aspirates from 17 MM patients and also showed high expression of CGEN-928. Notably, expression of this antigen was not only found on CD138+ MM cells but also on MM tumor cells lacking CD138 expression. We also examined the expression of CGEN-928 using our human MM xenograft models LAGκ-1A (bortezomib-sensitive), LAGκ-1B (bortezomib-resistant) and LAGλ-1 (melphalan-resistant). The bortezomib-sensitive MM tumor LAGκ-1A expresses CD138 whereas the bortezomib-resistant version LAGκ-1B developed from the same patient after the patient developed bortezomib reisistance does not express CD138. Cells from all three tumor types showed high levels of reactivity with the TM21 antibody. Similar to the fresh MM BM samples, CGEN-928 expression was not only found on CD138+ MM cells but also on CD138- tumor cells derived from these human MM xenografts. Because this molecule is highly expressed on MM cells, we hypothesized that the anti-CGEN-928 antibody may show anti-MM effects and enhance the anti-MM effects of other anti-MM drugs. To evaluate this, we examined the effect of the TM21 antibody alone and in combination with dexamethasone, melphalan and bortezomib in vitro using cell proliferation MTT assays. Anti-TM21 polyclonal antibody (100 mg/ml) decreased MM tumor cell proliferation and increased apoptosis in cells from the MM1s, RPMI8226 and U266 cell lines. Next, we determined the effects of combining the anti-CGEN-928 antibody with bortezomib, melphalan or dexamethasone on MM1s cells. Cell proliferation assays demonstrated marked enhanced anti-proliferative effects when CGEN-928 antibody at concentrations of 5, 10, 50 and 100 mg/ml was combined with bortezomib, melphalan or dexamethasone. Further investigations are defining the mechanisms and signal transduction pathways that produce the anti-MM effects of CGEN-928. These preliminary studies suggest that the CGEN-928 antigen is highly expressed in MM and treatment with an anti-CGEN-928 polyclonal antibody produces anti-MM effects alone and in combination with other anti-MM agents; and thus, this antigen may be a target for the treatment of multiple myeloma. Currently, a monoclonal anti-CGEN-928 antibody is in development that will be used by our group to evaluate its anti-MM effects both in vitro and in vivo using our SCID-hu models of human MM. Disclosures: Levy: Compugen Ltd.: Employment. Dassa:Compugen Ltd.: Employment. Cojocaru:Compugen Ltd.: Employment. Berenson:Compugen Ltd.: Research Funding. Levine:Compugen Ltd.: Employment.

Epigenetic Modulation by Vidaza™ induces Apoptosis and Overcomes In Vitro Drug Resistance in Human Multiple Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5001-5001
Author(s):  
Tanyel Kiziltepe ◽  
Laurence Catley ◽  
Teru Hideshima ◽  
Noopur Raje ◽  
Hiroshi Yasui ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Epigenetic Modification ◽  
Biological Effects ◽  
Hematologic Malignancies ◽  
Aberrant Methylation ◽  
Aberrant Expression ◽  
Induced Apoptosis

Abstract Methylation of DNA is an epigenetic modification that plays an important role in the regulation of gene expression in mammalian cells. Although DNA methylation is required for normal cell development and function, aberrant methylation and the resulting aberrant expression of genes, such as tumor suppressor genes and oncogenes, contribute to the development of malignancies. Interestingly, aberrant DNA methylation has recently emerged as one of the most frequent molecular alterations in hematologic malignancies, providing a powerful rationale to use inhibitors of DNA methylation as a novel means of targeting hematologic malignancies. Vidaza™ [Pharmion Corporation] (5-azacytidine), an FDA approved drug for the treatment of myelodysplastic syndromes, is an inhibitor of DNA methylation. Multiple myeloma (MM) is currently an incurable hematological malignancy despite all the conventional and novel therapies, and we here examined the biological effects using Vidaza™ on human MM cells. We demonstrate here that Vidaza™ has significant cytotoxicity in both conventional therapy sensitive (MM1S, RPMI-8226) and resistant (MM1R, RPMI-Dox40, RPMI-LR5) MM cell lines, as well as freshly isolated patient MM tumor cells, with an IC50 of 1.25–4 mM at 72 hours in vitro. Importantly, no cytotoxic effects of Vidaza™ were detected in peripheral blood mononuclear cells (PBMNC) obtained from healthy volunteers at ≤20 mM, suggesting a therapeutic index. Moreover, Vidaza™ overcame 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). Vidaza™ induced apoptosis in MM cells, as determined by flow cytometric analysis using PI and Annexin V staining. Vidaza™-induced apoptosis was associated with PARP, caspase 8 and caspase 9 cleavage. Importantly, pan-caspase inhibitor zVAD-fmk, significantly, but only partially, inhibited apoptosis induced by Vidaza™, suggesting the involvement of both caspase dependent and independent pathways. Taken together, our studies therefore demonstrate that Vidaza™ induces apoptosis and overcomes in vitro drug resistance in MM cells. Ongoing studies are delineating the mechanism of action of Vidaza™ against MM cells to further provide the preclinical rationale for clinical evaluation of Vidaza™, alone or in combination with other agents, to improve patient outcome in MM.

Irinotecan Resistance – In Search of New Markers in CRC.

2021 ◽  
Author(s):  
Jakub Kryczka ◽  
Joanna Boncela
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Expression Profiles ◽  
Resistance Mechanism ◽  
Interaction Network ◽  
In Vitro Study ◽  
Cancer Drug ◽  
New Genes

Abstract Colorectal cancer (CRC) is one of the most prominent causes of cancer death worldwide. Chemotherapeutic regimens consisting of different drugs combinations such as 5-fluorouracil, and oxaliplatin (FOLFOX) or irinotecan (FOLFIRI) have been proven successful in the treatment of CRC. However, chemotherapy often leads to the acquisition of cancer drug resistance followed by metastasis and in the aftermath therapeutic failure. The molecular mechanism responsible for drug resistance is still unclear. The systemic search for new biomarkers of this phenomenon may identify new genes and pathways. To understand the drug resistance mechanism in CRC, the in vitro study based on the molecular analysis of drug-sensitive cells lines vs drug-resistant cells lines has been used. In our study to bridge the gap between in vitro and in vivo study, we compared the expression profiles of cell lines and patient samples from the publicly available database to select the new candidate genes for irinotecan resistance. Using The Gene Expression Omnibus (GEO) database of CRC cell lines (HT29, HTC116, LoVo, and their respective irinotecan-resistant variants) and patient samples (GSE42387, GSE62080, and GSE18105) we compared the changes in the mRNA expression profile of the main genes involved in irinotecan body’s processing, such as transport out of the cells and metabolism. Furthermore, using a protein-protein interaction network of differently expressed genes between FOLFIRI resistant and sensitive CRC patients, we have selected top networking proteins (upregulated: NDUFA2, SDHD, LSM5, DCAF4, and COX10, downregulated: RBM8A, TIMP1, QKI, TGOLN2, and PTGS2). Our analysis provided several potential irinotecan resistance markers, previously not described as such.

The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Potent and Selective Cytotoxicity against CD138 Positive Multiple Myeloma Cells in Vitro and in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1716-1716 ◽  
Author(s):  
Hiroshi Ikeda ◽  
Teru Hideshima ◽  
Robert J. Lutz ◽  
Sonia Vallet ◽  
Samantha Pozzi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Tumor Cells ◽  
Cell Lines ◽  
Xenograft Model ◽  
Growth Delay ◽  
Selective Cytotoxicity ◽  
Bystander Killing

Abstract CD138 is expressed on differentiated plasma cells and is involved in the development and/or proliferation of multiple myeloma (MM), for which it is a primary diagnostic marker. In this study, we report that immunoconjugates comprised of the murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives (nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1) showed cytotoxic activity against CD138-positive MM cells both in vitro and in vivo. These agents demonstrated cytotoxicity against OPM1 and RPMI8226 (CD138-positive MM cell lines) in a dose and time-dependent fashion and were also cytotoxic against primary tumor cells from MM patients. Minimal cytotoxicity was noted in CD138-negative cell lines and no activity was observed against peripheral blood mononuclear cells from healthy volunteers, suggesting that CD138-targeting is important for immunoconjugate-mediated cytotoxicity. Examination of the mechanism of action whereby these immunoconjugates induced cytotoxicity in MM cells demonstrated that treatment triggered G2/M cell cycle arrest, followed by apoptosis associated with cleavage of PARP and caspase-3, -8 and -9. Neither interleukin-6 nor insulin-like growth factor-I could overcome the apoptotic effect of these agents. The level of soluble (s)CD138 in the BM plasma from 15 MM patients was evaluated to determine the potential impact of sCD138 on immunoconjugate function. The sCD138 level in BM plasma was found to be significantly lower than that present in MM cell culture supernatants where potent in vitro cytotoxicity was observed, suggesting that sCD138 levels in MM patient BM plasma would not interfere with immunoconjugate activity. Because adhesion to bone marrow stromal cells (BMSCs) triggers cell adhesion mediated drug resistance to conventional therapies, we next examined the effects of the conjugates on MM cell growth in the context of BMSC. Co-culture of MM cells with BMSCs, which protects against dexamethasoneinduced death, had no impact on the cytotoxicity of the immunoconjugates. The in vivo efficacy of these immunoconjugates was also evaluated in SCID mice bearing established CD138-positive MM xenografts and in a SCID-human bone xenograft model of myeloma. Significant tumor growth delay or regressions were observed at immunoconjugate concentrations that were well tolerated in all models tested. The ability of these agents to mediate bystander killing of proximal CD138-negative cells was also evaluated. While nBT062-SPDB-DM4 was inactive against CD138-negative Namalwa cells cultured alone, significant killing of these CD138-negative cells by nBT062-SPDB-DM4 was observed when mixed with CD138-positive OPM2 cells. This bystander killing may contribute to the eradication of MM tumors by disrupting the tumor microenvironment and/or killing CD138-negative MM tumor cells, such as the putative CD138 negative myeloma stem cells. These studies demonstrate strong evidence of in vitro and in vivo selective cytotoxicity of these immunoconjugates and provide the preclinical framework supporting evaluation of nBT062-based immunoconjugates in clinical trials to improve patient outcome in MM.

A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1841-1841
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Arghya Ray ◽  
Teru Hideshima ◽  
Paul G. Richardson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Advisory Committees ◽  
Acid Analog

Abstract Abstract 1841 Introduction: The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results: We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions: We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures: Hideshima: Acetylon: Consultancy. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

Pralatrexate Has Potent Activity Against Multiple Myeloma In Vitro and In Vivo, and Activity Correlates with Tumor RFC-1 and DHFR Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1831-1831 ◽  
Author(s):  
Michael Mangone ◽  
Luigi Scotto ◽  
Enrica Marchi ◽  
Owen A. O'Connor ◽  
Hearn J. Cho
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Research Funding ◽  
Folate Metabolism ◽  
Nog Mice ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Functional Biomarkers

Abstract Abstract 1831 Multiple myeloma (MM) is the second most common hematologic malignancy. Although there are effective new agents that can induce remission, relapse is inevitable and the disease is currently incurable. Progress in the treatment of this disease demands development of novel therapeutics and identification of functional biomarkers that may be used to distinguish tumors that are susceptible to specific targeted agents, creating a “personalized” therapeutic strategy for individual patients. We investigated these principles with anti-folates, which are not commonly used in MM but have demonstrated activity in this disease. Pralatrexate (PDX, 10-propargyl 10-deazaaminopterin) is a folate analogue that was rationally designed to have high affinity for Reduced Folate Carrier (RFC)-1, an oncofetal protein expressed in many cancers that actively transports folates into cells. PDX induced dose-dependent apoptotic cell death in a subset of human myeloma cell lines (HMCL) and CD138+ MM cells isolated from a clinical specimen. In sensitive cell lines, PDX exhibited 10-fold greater potency compared to the structurally related drug methotrexate (MTX). PDX induced dose-dependent, intrinsic apoptosis in sensitive HMCLs, characterized by cleavage of caspase-3 and -9 and accompanied by the loss of full-length Mcl-1, a Bcl-2 family protein that plays a critical role in drug-induced apoptosis in MM. Furthermore, the activity of PDX is not abrogated by the presence of exogenous interleukin-6 or by co-culture with HS-5 bone marrow stromal cells, both of which exert powerful survival effects on MM cells and can antagonize apoptosis in response to some cytotoxic chemotherapy drugs. Sensitivity to PDX-induced apoptosis correlated with higher relative levels of RFC-1 mRNA in sensitive compared to resistant HMCL. Resistant HMCL also exhibited a dose-dependent up-regulation of dihydrofolate reductase (DHFR) protein, a primary molecular target for anti-folates, in response to PDX exposure, whereas sensitive HMCL did not. These changes in functional folate metabolism biomarkers, high baseline RFC-1 expression and upregulation of DHFR in response to PDX, appeared to be mutually exclusive to sensitive or resistant HMCL, respectively. Importantly, PDX was also effective against sensitive HMCL in vivo in a novel mouse xenograft model. NOD/Shi-scid/IL-2Rγnull (NOG) mice were inoculated with MM.1s HMCL stably transduced to express both GFP and luciferase (GFP-luc). GFP-luc MM.1s cells engrafted into the long bones, pelvis, and vertebral column of NOG mice within 4–7 days after injection of cells, as assessed by in vivo bioluminescent imaging. Treatment with PDX resulted in a significant reduction in tumor burden after two doses. These results demonstrate that PDX has potent anti-myeloma activity in vitro and in vivo, and that RFC-1 expression and DHFR upregulation are robust functional biomarkers that may identify patients who are likely to benefit from PDX therapy. These data support further exploration of PDX therapy in clinical trials for MM and investigation of folate metabolism biomarkers as indices for treatment with this class of drugs. Improved anti-folates such as PDX are a promising class of agents that may be a valuable addition to the arsenal against MM. Disclosures: O'Connor: Celgene: Consultancy, Research Funding; Merck: Research Funding; Novartis: Research Funding; Spectrum: Research Funding.

Anti-Myeloma Activity of a Novel Free Radical Inducer Rrx-001

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4712-4712 ◽  
Author(s):  
Deepika Sharma Das ◽  
Ze Tian ◽  
Arghya Ray ◽  
Durgadevi Ravillah ◽  
Yan Song ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Clinical Trial ◽  
Drug Resistance ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Advisory Committees ◽  
Healthy Donors

Abstract Background and Rationale: Multiple Myeloma (MM) remains incurable despite the advent of novel drugs, highlighting the need for further identification of factors mediating disease progression and resistance. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in MM cells. Studies to date suggest an important role of BM hypoxia (low oxygenation) in MM cell survival, drug resistance, migration, and metastasis. Therapies targeting the MM cell in its BM milieu under hypoxic conditions may therefore achieve responses in patients resistant to various therapies. Recent studies led to the development of a novel aerospace-industry derived Phase 2 molecule RRx-001 with epigenetic and NO-donating properties. RRx-001 generates reactive oxygen and nitrogen species (RONS), which induces oxidative stress in tumor cells. Importantly, RRx-001 is also a potent vascular disrupting agent, which further provides rationale for utilizing RRx-001 as a therapeutic agent since tumor-associated angiogenesis is a characteristic of MM. A Phase I clinical trial has shown RRx-001 to have antitumor activity in heavily pretreated cancer patients and to be safe and well tolerated with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578). Here we examined the anti-MM activity of RRx-001 using in vitro and in vivo models of MM. Materials and methods: MM cell lines, patient MM cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of RRx-001 alone or in combination with other agents. Drug sensitivity, cell viability, apoptosis, and migration assays were performed using WST, MTT, Annexin V staining, and transwell Inserts, respectively. Synergistic/additive anti-MM activity was assessed by isobologram analysisusing “CalcuSyn” software program. Signal transduction pathways were evaluated using immunoblotting. ROS release, nitric oxide generation, and mitochondrial membrane potential was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. DNMT1 activity was measured in protein lysates using EpiQuik DNMT1 assay kit. 5-methyl cytosine levels were analyzed in gDNA samples using methylflash methylated DNA quantification kit from Enzo life sciences; USA. For xenograft mouse model, CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Blood, 2010, 115:834). Statistical significance of data was determined using a Student’st test. RRx-001 was obtained from RadioRx Inc., CA, USA; bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA. Results: Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.001; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies show that RRx-001-triggered apoptosis is associated with 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNA methyltransferase (DNMT1) enzymatic activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. In vivo studies using subcutaneous human MM xenograft models show that RRx-001 is well tolerated and inhibits tumor growth. Finally, combining RRx-001 with bortezomib, SAHA, or pomalidomide induces synergistic anti-MM activity and overcomes drug resistance. Conclusion: Our preclinical studies showing efficacy of RRx-001 in MM disease models provide the framework for clinical trial of RRx-001, either alone or in combination, to improve outcome in relapsed and refractory MM patients. Disclosures Richardson: Oncopeptides AB: 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. Oronsky:RadioRx Inc, : Employment. Scicinski:RadioRx Inc,: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

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

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.

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