Evaluation of Anti-Neoplastic Effects of a New Hemostatic Agent Ankaferd Blood Stopper on Myeloma Cell Line and Plasmocytoma Development in Balb/c Mice: Results of the First in Vitro and in Vivo Study

Abstract Ankaferd Blood Stopper (ABS), a unique traditional herbal mixture, has been used topically to stop bleeding for centuries in Anatolia. As well as ABS has been used as a blood-stopping agent, it may also have a considerable therapeutic benefit, because of its anti-infective, anti-neoplastic, and wound healing properties. The aim of this study is to investigate the anti-neoplastic effects of the ABS on myeloma cell line, in vitro and on the plasmocytoma development in Balb/c mice by intraperitoneal injection of pristane, in vivo. We therefore sought to evaluate the efficacy of ABS on MM cells and to study the modulation of cell-death pathways. The cytotoxicity of ABS against the MM cell lines (RPMI-8226, and ARH-77) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-dye reduction assay. Responses to ABS by RPMI-8226 and ARH-77 cell lines were dose dependent but not time dependent. The IC50 values for RPMI 8226 and ARH 77 myeloma cell lines in 24h were 12,84 μL/mL and 13,86 μL/mL, respectively. Various cell-death characteristics such as caspase-3, Bcl-2, Bax were studied in response to ABS, but we couldn’t demonstrate specific features of apoptotic cell death, in vitro. We have also investigated the effect of the ABS on the pristane (2.6.10.14-tetramethylpentadecane)-induced plasmacytoma (PCT) development on six-week-old BALB/c mice. Three groups of mice were treated with intraperitoneal ABS (1 mg/kg, 0.5mg/kg, and 0,1mg/kg) per-week for eight weeks after pristane-induced PCT development. The study was stopped at twelfth week, the remaining mice were autopsied, and peritoneal tissues were examined histologically for PCTs. A database of different groups’ mice was analyzed using Kaplan-Meier and Cox regression statistics based on variables. Kaplan-Meier analysis revealed a difference of the survival of pristane-induced alone between the groups of pristane-induced plus ABS 1 mg/kg, 0.5mg/kg, and 0.1mg/kg. (Log-rank, p=0.016; p<0.001 and p<0.001; respectively). The present results indicate that direct anti-tumor effect of ABS on pristane-induced PCT and significantly increased survival. This hypothesis needs to now be further investigated in clinical trials. Disclosures No relevant conflicts of interest to declare.

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Combination of Akt/PKB Inhibition (Perifosine) and Farnesyl Transferase Inhibition (Tipifarnib) Results in Increased Cell Death in Myeloma Cells Lines.

Blood ◽

10.1182/blood.v106.11.1568.1568 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1568-1568 ◽

Cited By ~ 1

Author(s):

Rajni Sinha ◽

Ebenezer David ◽

Emily Zeilter ◽

Claire Torre ◽

Jonathan L. Kaufman ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Cell Death ◽

Combination Therapy ◽

Cell Line ◽

Cell Lines ◽

Single Agent ◽

Myeloma Cell ◽

Myeloma Cell Line

Abstract Introduction Multiple myeloma is a clonal plasma cell malignancy characterized by proliferation and accumulation of plasma cells in the bone marrow. Most patients are incurable with the current treatment modalities. Clearly novel agents are needed to improve the outcome for patients with myeloma. We have previously shown that the combination of bortezomib and tipifarnib results in synergistic myeloma cell death. This increase in apoptosis is associated with down regulation of phosphorylated AKT, a potent anti-apoptotic signaling molecule. Therefore, agents that target AKT represent ideal compounds for further study in myeloma. Perifosine is a novel, oral bioavailable alkylphospholipid. Perifosine has displayed apoptotic and antipropliferative activity in vitro and in vivo in several human cancer models including leukemia. Perifosine exerts its actions by interfering with key intracellular pathways including AKT, MAPK, JNK, p21waf1. Our hypothesis is that targeting AKT via multiple upstream pathways will result in increased myeloma cell apoptosis. Therefore, we assessed the effects of single agent perifosine with and without tipifarnib on multiple myeloma cell lines. Method The myeloma cell line RPMI8226 was used. Cell viability and proliferation were assessed using MTT assays. Cells were incubated with increasing concentrations of both agents alone and in combination. Cell proliferation was assayed at 24, 48 and 72 hours. Western blots were then carried out to evaluate the effects of the intracellular protein PDK1, one of the critical signaling molecules that phosphorylates and activates AKT. Results As we and others have previously shown, tipifarnib at concentrations that can be achieved clinically is associated with minimal cytotoxicity. At 5 μM, tipifarnib decrease proliferation by only 20%. In contrast, there is a potent dose response effect of single agent perifosine (Fig. 1). These results were apparent as early as 24 hours. When tipifarnib at 5 μM is used in combination with a subtherapeutic dose of perifosine (2 μM), there is a marked decrease in cell proliferation (Fig. 2). In addition, combination therapy resulted in a reduction in the phosphorylated form of PDK1, a critical finding that was not seen with either drug alone. Conclusion Combination therapy with tipifarnib and perifosine results in less cell proliferation compared to either agent used alone in the RPMI8226 myeloma cell line. The dosages employed in these in-vitro studies are lower than those used in previously published data and are clinically achievable. Studies targeting other cell lines including MM.1R, MM.1S, and U266 are in progress. Analysis of AKT, Caspase 3, 8 and 9 are being explored to help delineate the mechanism of this novel combination. The goal is to develop further effective treatment options for patients with myeloma. Figure 1 Figure 1. Figure 2 Figure 2.

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Simvastatin Induces Death of Multiple Myeloma Cell Lines

Journal of Investigative Medicine ◽

10.1136/jim-52-05-34 ◽

2004 ◽

Vol 52 (5) ◽

pp. 335-344 ◽

Cited By ~ 11

Author(s):

Naomi Gronich ◽

Liat Drucker ◽

Hava Shapiro ◽

Judith Radnay ◽

Shai Yarkoni ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Death ◽

Cell Lines ◽

Myeloma Cell ◽

Cytoplasmic Calcium ◽

Multiple Myeloma Cell ◽

Cycle Arrest ◽

Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

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The Bcl-2—IP3 Receptor Interaction As a Novel Target for Therapeutics in Myeloma

Blood ◽

10.1182/blood.v120.21.4011.4011 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4011-4011

Author(s):

Ming-Jin Chang

Keyword(s):

Cell Line ◽

Cell Lines ◽

Cytosolic Calcium ◽

Myeloma Cell ◽

Receptor Interaction ◽

Ip3 Receptor ◽

Myeloma Cell Line ◽

Chemotherapy Drugs ◽

Calcium Elevation

Abstract Abstract 4011 The anti-apoptotic Bcl-2 (B-cell lymphoma 2) protein was first identified in follicular lymphoma, where the Bcl-2 gene was dysregulated as a result of chromosomal translocation. The anti-apoptotic activity of Bcl-2 can be attributed to two mechanisms conferred by structurally distinct domains. First, the BH domains 1–3 of Bcl-2 dimerize with pro-apoptotic members of the Bcl-2 family, such as Bax and Bak, and maintain mitochondrial integrity. Small molecules targeting these domains on Bcl-2 (e.g. ABT-737 and ABT-263/Navitoclax®) are under clinical evaluation. A less explored activity of Bcl-2 is that its BH4 domain functionally interacts with the IP3 receptor located on the endoplasmic reticulum (ER) membrane, the major organelle involved in regulating calcium homeostasis. Through this interaction, Bcl-2 inhibits IP3 receptor channel opening and prevents excessive calcium release from ER that can be harmful to cells. Our strategy is to disrupt the Bcl-2—IP3 receptor interaction to induce cell death in lymphoid malignant cells. Previously, our group has shown that an IP3 receptor derived peptide, or IDP, blocks the Bcl-2—IP3 receptor interaction and thereby induces elevation of cytosolic calcium in lymphoma cell lines. A modified version of the peptide—IDP-DD/AA, with an asparaginase cleavage site removed, is more potent in inducing calcium elevation as well as apoptosis in primary chronic lymphocytic leukemia (CLL) cells. Concentrations of the IDP-DD/AA peptide that killed CLL cells did not affect the viability of normal lymphocytes, suggesting a therapeutic window. We further exploited the cytotoxic activities in a panel of leukemia/lymphoma/myeloma cell lines and found that over 60% of myeloma cell lines tested were sensitive to the IDP-DD/AA peptide but not to the control peptide. Furthermore, in the sensitive myeloma cell lines treated with the IDP-DD/AA peptide, high amplitude cytosolic calcium elevation preceded caspase-3 activation and apoptotic morphology, suggesting the involvement of a calcium-mediated intrinsic apoptotic pathway. The in vivo anti-tumor activity of the IDP peptide was assessed in a myeloma xenograft model. Subcutaneous tumors were established in nude mice using the human myeloma cell line, NCI-H929. Randomly grouped mice treated by in-tumor injection of the IDP-DD/AA peptide showed prominent inhibition of tumor growth compared to saline-injected control. One of three mice treated with the IDP-DD/AA peptide showed no visible sign of tumor at the end of treatment. The data proffer promise of therapeutic use of the IDP peptide in myeloma disease. The effect of combinatory treatment of the IDP-DD/AA peptide and current anti-myeloma drugs (Bortezomib/Valcade®, doxorubicin/Adriamycin®, melphalan) were evaluated. Co-treatment of the myeloma cell line NCI-H929 cells with the IDP-DD/AA peptide and each one of the three anti-myeloma drugs induced synergistic cytotoxicity with combinatory indices (CI) less than 1. The data suggest that the IDP-DD/AA peptide augments cell killing activity of current chemotherapy drugs. In conclusion, the IDP-DD/AA peptide represents a novel anti-cancer agent that targets the anti-apoptotic Bcl-2 protein. The peptide exhibits both in vitro and in vivo anti-myeloma activity. Moreover, the IDP-DD/AA peptide can function as a single agent (in myeloma or CLL) as well as in combination with current anti-myeloma chemotherapy drugs. Disclosures: No relevant conflicts of interest to declare.

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Sphingolipids as a novel target for the treatment of multiple myeloma.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e19534 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e19534-e19534

Author(s):

Yubin Kang ◽

Jagadish Kummetha Venketa

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Line ◽

Cell Lines ◽

Apoptotic Cell ◽

Myeloma Cell ◽

Sphingolipid Metabolism ◽

Myeloma Cell Line ◽

Myeloma Cells ◽

Novel Target

e19534 Background: Multiple myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ~10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding the disease’s molecular pathways and for identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in tumor cell proliferation and in tumor sensitivity to anticancer drugs. We hypothesize that altered sphingolipid metabolism plays an important role in the pathogenesis of MM, thus providing a novel target in the treatment of MM. Methods: We first assayed sphingolipid metabolism including sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+myeloma cells, and in publically available dataset. We then tested the efficacy of the selective SK2 inhibitor (ABC294640) and the SK2 shRNA in killing myeloma cells in vitro. Results: 1) Compared to immortalized B cells, the levels of pro-apoptotic ceramides were decreased whereas the proliferative sphingosine 1-phosphate (S1P) was increased in myeloma cell lines. 2) The expression of several key sphingolipid-metabolizing genes including sphingosine kinase (SK) 1 and 2 was altered in freshly isolated human primary bone marrow myeloma cells and in publically available microarray dataset. 3) The selective SK2 inhibitor (ABC294640) induces apoptotic cell death and inhibits myeloma cell growth with an IC50of ~20 μM in 9 myeloma cell lines. 4) Interestingly, OPM-1 myeloma cell line was extremely sensitive to ABC294640 with an IC50of <5 µM whereas U266 myeloma cell line was resistant to ABC294640. SK2 shRNA induced apoptotic cell death in OPM-1, but not in U266 cells. We are currently investigating the molecular mechanisms underlying the resistance of U266 myeloma cells to ABC294640. Conclusions: Our data demonstrated that sphingolipid metabolism provides an attractive target in the treatment of refractory/relapased multiple myeloma.

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Non-genotoxic hepatocarcinogenesis in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis

Journal of Cell Science ◽

10.1242/jcs.104.2.307 ◽

1993 ◽

Vol 104 (2) ◽

pp. 307-315 ◽

Cited By ~ 1

Author(s):

A.C. Bayly ◽

N.J. French ◽

C. Dive ◽

R.A. Roberts

Keyword(s):

Cell Death ◽

Cell Line ◽

Cell Lines ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Peroxisome Proliferator ◽

Peroxisome Proliferators ◽

Hepatoma Cell Lines

A range of hepatoma cell lines (RH1, HTC, FaO, 7800C1 and MH1C1), has been studied with the aim of establishing an in vitro model to investigate the molecular mechanisms of hepatocarcinogenicity induced by the peroxisome proliferator class of non-genotoxic carcinogens. In view of speculation that peroxisome proliferators suppress hepatocyte apoptosis in vivo, we have placed particular emphasis on evaluating whether hepatoma cell lines retain the ability to undergo apoptotic cell death. Expression of the liver-specific differentiation marker albumin and the peroxisome proliferator-activated receptor (PPAR) was highest in the Reuber hepatoma cell line, FaO. This cell line also demonstrated the most marked response to the peroxisome proliferator nafenopin with a 2.2-fold induction of the microsomal enzyme cytochrome p450IVA1. This response was found to display intercellular heterogeneity by immunocytochemistry. Thus, the FaO cell line maintained characteristics of hepatocytes, both in vivo and in vitro, in terms of expression of constitutive and inducible markers. However, none of the cell lines tested mirrored the hyperplastic response of hepatocytes to nafenopin, since no increase in cell growth kinetics was observed on addition of nafenopin to the growth medium. The mode of cell death in confluent FaO cultures was characterised as apoptosis, by fluorescence microscopy and agarose gel electrophoresis of extracted DNA. Cells detaching from confluent FaO cultures exhibited chromatin condensation and DNA fragmentation patterns characteristic of cels undergoing apoptotic death.Interestingly, no apoptosis was seen in monolayer cells, suggesting that apoptosis in vitro is associated with cell shrinkage and detachment similar to that documented for the liver in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

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Zoledronate Can Overcome Drug Resistance in a Dexamethasone Resistant Myeloma Cell Line.

Blood ◽

10.1182/blood.v104.11.4894.4894 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4894-4894

Author(s):

Masayuki Koizumi ◽

Chiaki Nakaseko ◽

Chikako Ohwada ◽

Ryuko Cho ◽

Miki Nishimura ◽

...

Keyword(s):

Dna Synthesis ◽

Cell Line ◽

Cell Lines ◽

Antitumor Effect ◽

Annexin V ◽

Myeloma Cell ◽

Myeloma Cell Line ◽

Human Myeloma Cell Line ◽

Induced Apoptosis

Abstract <Introduction> Multiple myeloma (MM) is almost invariably fatal despite all available chemotherapeutic and supportive treatment. Initial treatment of MM with dexamethasone (Dex), a key drug for MM chemotherapy, effectively induces myeloma cell death. However, prolonged drug exposure results in the development of Dex-resistance and clinical failures. Therefore, one of the most important issues in myeloma therapy is to overcome resistance to Dex at the stage of clinical refractoriness. Several recent in-vitro studies have demonstrated an antitumor effect of nitrogen-containing amino-bisphosphonates (N-BPs) in some tumor cell lines, including myeloma cell lines. However, few data are available concerning the effects of N-BPs on Dex resistant myeloma cell lines. In this study, we have established a Dex-resistant human myeloma cell line and investigated the antitumor effect of the third generation bisphosphonate Zoledronate (ZOL) on the Dex-resistant subline. <Methods> 1) Development of de novo resistance through continuous exposure to Dex. A Dex-resistant human myeloma cell line (Dex-R) was selected from the Dex-sensitive myeloma cell line RPMI8226 by continuously exposing cells to gradually increasing doses of Dex. Resistance was validated by flow cytometry. 2) Apoptosis assay. Dual-color FACS with propidium iodide (PI) and Annexin V was used to detect Dex-induced apoptosis in RPMI8226 and Dex-R. 3) Surface markers. Phenotypes of sensitive and resistant cells were compared by FACS analysis. 4) Cell Proliferation and DNA synthesis assay. RPMI8226 and Dex-R cells were cultured in 96 wells plates for 72 hours in the absence or presence of Dex (1 μM) or increasing concentrations of ZOL (2.5 to 50 μM). Cell growth was assessed by WST-8 assay kit. DNA synthesis was measured according to thymidine uptake. 5) Actin staining. RPMI8226 and Dex-R cells were cultured for 48 hours in the absence or presence of Dex (2 μM) or ZOL (40 μM). The distribution of f-actin stained by fluorescent phalloidin was investigated by confocal fluorescence microscopy. <Results> The developed subline Dex-R showed reduced apoptotic and antiproliferative responses to Dex treatment. Flow cytometry on 24 hours Dex exposure revealed significantly low percentage of apoptotic (Annexin V positive and PI negative) population in Dex-R cells compared with RPMI8226 cells (6.7% vs. 29.9%). Our data demonstrate that Dex-R cells showed increased CD38, CD44, CD49d (VLA-4) expression, and decreased CD45, CD95 (Fas), CD138 (Syndecan-I) expression as compared to native RPMI8226 cells. ZOL induced apoptosis and inhibited DNA synthesis and cell proliferation in both RPMI8226 and Dex-R cell lines in a dose-dependant manner. Interestingly, some modifications of the cellular morphology were observed in ZOL-treated cells. Rhodamine-phalloidin staining of the f-actin cytoskeleton showed disruption of the cytoskeleton in these deformed cells. This fibroblast-like cellular morphology was common to Dex-R and RPMI8226 cells treated with ZOL, but was not detected under Dex treatment. This result might reflect different mechanism of antitumor effect between Dex and ZOL.. <Conclusion> Our results suggest that ZOL can induce myeloma cell death in vitro in a different mechanistic way than Dex. ZOL might be effective as an antitumor drug for Dex resistant myeloma.

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Interferon γ Enhances the Anti-Myeloma Activity of the Fully Human Anti-HLA-DR Monoclonal Antibody 1D09C3.

Blood ◽

10.1182/blood.v108.11.656.656 ◽

2006 ◽

Vol 108 (11) ◽

pp. 656-656

Author(s):

Carmelo Carlo-Stella ◽

Anna Guidetti ◽

Massimo Di Nicola ◽

Cristiana Lavazza ◽

Loredana Cleris ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Median Survival ◽

Plasma Cells ◽

Interferon Γ ◽

Hla Dr ◽

Ifn Γ ◽

Rpmi 8226

Abstract The fully human anti-HLA-DR antibody 1D09C3 exerts a potent anti-lymphoma activity both in vitro and in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. 1D09C3 is currently tested in phase I clinical trials in patients with relapsed/refractory B-cell malignancies. To investigate whether 1D09C3 might represent a treatment modality to target malignant plasma cells, we re-evaluated HLA-DR expression on CD138+ plasma cells. Additionally, we investigated the capacity of interferon γ (IFN-γ ) to upregulate HLA-DR expression on myeloma cell lines, and tested in vitro and in vivo the anti-myeloma activity of 1D09C3 alone or in combination with IFN-γ . Bone marrow CD138+ cells were enriched using an immunomagnetic method from 60 multiple myeloma (MM) patients. Three-color flow cytometry revealed a highly heterogeneous HLA-DR expression on plasma cells. CD138+HLA-DR+ cells were detected in 31/60 patients (52%), with 15/60 patients (25%) having ≥ 20% CD138+HLA-DR+ cells (median, 50%; mean, 54%; range, 23 - 100), and 3 patients (5%) displaying 100% CD138+HLA-DR+ cells. Two thirds of HLA-DR+ patients expressed CD45 on CD138+ cells, suggesting that 1D09C3 might target self-renewing plasma cells. HLA-DR expression was not associated with distinct cytogenetic abnormalities. Since primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines (n = 6) with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin-V/propidium iodide double staining showed that 1D09C3-induced cell death strongly correlated with constitutive HLA-DR expression. Interestingly, induction of HLA-DR by IFN-γ restored the sensitivity of HLA-DR dim cell lines (i.e., RPMI-8226, KMS-11) to the cytotoxic activity of 1D09C3. As compared to controls, exposure of RPMI-8226 and KMS-11 cell lines to IFN-γ and 1D09C3 significantly increased cell death to 45% (P ≤ 0.0001) and 40% (P ≤ 0.0001), respectively. The in vivo activity of 1D09C3 was analyzed by xenografting NOD/SCID mice with the KMS-11 cell line. A significant increase of median survival over controls was detected in mice treated with 1D09C3 alone at either 3 mg/mouse (92 vs 48 days, P ≤ 0.0001) or 6 mg/mouse (89 vs 48 days, P ≤ 0.0001). The combined treatment with IFN-γ plus 1D09C3 (3 mg/mouse) resulted in a significant increase of median survival as compared to controls (147 vs 48 days, P ≤ 0.0001) or mice receiving 1D09C3 alone (147 vs 92 days, P ≤ 0.03). The better therapeutic activity of the combined IFN-γ /1D09C3 treatment over 1D09C3 alone was further demonstrated by a 2-fold increase of mice being disease-free at 150 days after xenograft (47% vs 25%). No mice experienced any apparent treatment-related toxicity. In conclusion, our data demonstrate that: one third of patients with MM express significant levels of HLA-DR on CD138+ cells; IFN-γ-induced upregulation of HLA-DR results in a potent enhancement of the in vivo anti-myeloma activity of 1D09C3.

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Thymoglobulin (Polyclonal Rabbit Anti Thymocyte Globulin) Has In Vivo Activity in a Mouse Model of Myeloma.

Blood ◽

10.1182/blood.v108.11.3487.3487 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3487-3487

Author(s):

Michael Timm ◽

Linda Wellik ◽

Teresa Kimlinger ◽

Jessica Haug ◽

Michael Kline ◽

...

Keyword(s):

Flow Cytometry ◽

Tumor Growth ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Myeloma Cell ◽

Solid Organ ◽

Polyclonal Rabbit

Abstract Background: Multiple myeloma remains incurable with current approaches and newer therapies are needed to improve the outcome of these patients. While monoclonal antibody based therapies have been successful in some of the hematological malignancies, such approaches have had limited efficacy in the setting of myeloma. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) (Thymo) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and solid organ transplants. Given the polyclonal nature of this product, with antibodies against different B cell antigens, we evaluated the in vitro and in vivo activity of Thymo in myeloma. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity following drug treatment was measured using the MTT viability assay. Apoptosis was measured by flow cytometry using Annexin V/PI in cell lines and Apo 2.7 in primary patient plasma cells. Shifts in expression of a variety of different B cell and plasma cell antigens were examined on several different myeloma cell lines following Thymo treatment in order to identify the potential antigenic targets. In vivo activity of thymo was evaluated in a SCID plasmacytoma model injected with RPMI myeloma cell lines. Results: rATG was cytotoxic in vitro to several MM cell lines (RPMI 8226, U266, OPM1, OPM2) including the IL-6 dependent cell line Kas6/1 with LC50 of around 1 mg/mL. Additionally, thymo was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). Thymo induced apoptosis in MM cell lines and in patient derived primary myeloma cells. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan. Using competitive flow cytometry, we identified CD138, CD38, Cd45, CD126, CD49d (VLA4), as well as CD20 as antigens likely to be targeted by Thymo. Tumor bearing mice injected with Thymo at two different doses (5 mg/kg and 10 mg/kg for five days) had significantly delayed tumor growth compared to non-injected mice, and this translated into a better survival for these mice. Mice receiving 10 mg/kg dose had a slower tumor growth compared to 5 mg/kg dose (Figure). Conclusions: Thymoglobulin has promising in vitro and in vivo activity in the setting of myeloma. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents. Based on these results, we are in the process of initiating a clinical trial combining Thymo with Melphalan. Figure Figure

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Pyrroline-5-Carboxylate Reductase 1: A Novel Target for Sensitizing Myeloma to Cytotoxic Agents By Inhibition of PRAS40-Mediated Protein Synthesis

Blood ◽

10.1182/blood-2021-149156 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1574-1574

Author(s):

Inge Oudaert ◽

Hatice Satilmis ◽

Philip Vlummens ◽

Anke Maes ◽

Elke De Bruyne ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Protein Synthesis ◽

Apoptotic Cell ◽

Myeloma Cell ◽

Tumor Burden ◽

Rpmi 8226 ◽

Carboxylate Reductase

Abstract Introduction Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both cancer cell survival and drug resistance. In this study, we aimed to identify which metabolic changes and downstream pathways are involved in myeloma cell growth and persistence. Methods Correlation of pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) with overall survival was investigated in the gene-expression data of MM patients (MMRF CoMMpass trial). To perform a tracer study, RPMI-8226 cells were supplemented with 13C-glutamine for 48h in both normoxia and hypoxia (<1% O 2, by chamber). For further in vitro investigation, 2 human MM cell lines (OPM-2 and RPMI-8226) were used. Proline concentrations in cell lysates were measured by ELISA-based proline assay kit. We used siRNA to establish a knockdown of PYCR1 and/or PYCR2. Levels of apoptosis were measured using AnnexinV and 7-AAD positivity on flow cytometry. Differential protein expression was evaluated with western blot. Proliferation was measured by assessing BrdU incorporation through flow cytometry. Pargyline was used as a PYCR1 inhibitor. All in vitro experiments were performed in hypoxic conditions. For the in vivo murine experiment, C57BL/KalwRij mice were inoculated with 1 million of eGFP+ 5TGM1 cells, and treated with vehicle, bortezomib (0.6 mpk, 2x/week, starting day 14), pargyline (100 mpk, 5x/week, starting day 1) or combination of both. Tumor burden was measured by flow cytometry when vehicle mice reached end-stage. Results Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are 2 mitochondrial enzymes that facilitate the last step in the enzymatic conversion of glutamine to proline. High expression of both enzymes correlated with a lower overall survival in the CoMMpass trial. Moreover, MM cells from relapse/refractory patients expressed significant higher levels of PYCR1. We performed a tracer study with RPMI-8226 cells, revealing an increased conversion of 13C-glutamine to proline in hypoxia compared to normoxia. We confirmed these results by increased proline production after 48h of hypoxic culture. SiRNA-mediated knockdown of PYCR1 or both PYCR1/2 combined with bortezomib increased apoptotic cell death in OPM-2 and RPMI-8226, which we confirmed by detecting upregulation of cleaved PARP and cleaved CASPASE 3 levels. In contrast, PYCR2 knockdown combined with bortezomib did not significantly alter apoptosis. Further investigation revealed that PYCR1 knockdown reduced proliferation, and led to a decrease in p-AKT, p-p42/44 MAPK and c-MYC levels. Mechanistically, we found that PYCR1 silencing affected protein synthesis, as shown by a downregulation of p-PRAS40, p-MTOR, p-p70, p-S6, p-4EBP1 and p-EIF4e levels. Next, we evaluated whether the clinically relevant anti-hypertensive agent and PYCR1 inhibitor, pargyline, was capable of inducing myeloma cell death. In vitro, pargyline reduced proline production, MM viability and increased apoptotic cell death. Pargyline was also capable of reducing viability in CD138+ cells of primary patient samples . Finally, in vivo combination of pargyline with bortezomib significantly reduced tumor burden in the 5TGM1 model. On protein level, we also observed a significant decrease in p-4EBP1 and p-EIF4e in the freshly isolated 5TGM1 cells for the combination therapy. Conclusion Hypoxia increased glutamine-to-proline conversion in myeloma cells by stimulating PYCR activity. Knockdown of PYCR1 and PYCR1/2 increased bortezomib efficacy and inhibited proliferation. Mechanistically, PYCR1 interference reduced PRAS40-mediated protein synthesis. Pargyline, a PYCR1 inhibitor, also reduced MM viability and increased apoptosis. In vivo, pargyline combined with bortezomib significantly reduced tumor burden in the 5TGM1 model compared to both single agents. In conclusion, this study identifies PYCR1 as a novel target in MM therapy. Disclosures De Veirman: Active Biotech AB: Research Funding. OffLabel Disclosure: Pargyline is a antihypertensive agent and irreversible MAO B inhibitor that also inhibits PYCR1. Pargyline is not approved by the FDA as a PYCR1 inhibitor.

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