Sunday, December 5, 2004, 06:00 PM
[2457] Comparisson of Molecular Mechanisms Altered by Treatment with Different Drugs (Doxorubicin, Melphalan, Bortezomib, Aplidin and Arsenic Trioxide) in Multiple Myeloma Patients Using Oligonucleotide Microarrays.
Session Type: Poster Session 670-II
Patricia Maiso, Norma C. Gutierrez, Ricardo Lopez-Perez, Gema Mateo, Isabel M. Isidro, Encarna Ferminan, Xonia Carvajal-Vergara, Juan C. Montero, Jesus M. Hernandez, Atanasio Pandiella, Jesus F. San Miguel. Hematology, University Hospital, Salamanca, Spain; Hematology, Cancer Research Center, Salamanca, Spain
The development of novel targeted therapies in Multiple Myeloma (MM) has opened promising expectations for the treatment of this incurable hematological malignancy. However, the molecular mechanisms of both novel biologically based therapies and conventional treatments are still unclear. The purpose of the present study was to evaluate the changes in the gene expression profile of the human multiple myeloma cell line (MM.1S) following exposure to Doxorubicin, Melphalan, Bortezomib, Aplidin and Arsenic Trioxide. We have focused on the analysis of the early steps of activation of molecular mechanisms that lead to MM cell death. For this purpose we investigated with a time-course the onset of the apoptosis for every drug, according to the flow cytometric analysis with Annexin V- FITC Apoptosis Detection Reagent Kit. Based on these results the optimal concentration and time of exposure for each of the drugs were as follows Melphalan 50 µM, 9hours; Doxorubicin 1 µM, 17hours; Bortezomib 10 nM, 6 hours; Aplidin 50 nM, 4 hours and Arsenic Trioxide 5 mM, 3 hours. Affymetrix HG-U133A array containing around 15,000 full-length genes was used for mRNA expression profiling. All the experiments were performed in duplicate. The DNA- Chip Analyzer (DChip) was used to normalize and compare samples. Genes with expression changes greater than twofold in either direction were considered significant. A total of 269, 74, 74, 808 and 525 genes showed a significantly altered expression pattern, in response to Melphalan, Doxorubicin, Bortezomib, Aplidin and Arsenic Trioxide, respectively. Our results demonstrate that treatment with Melphalan inhibits DNA replication and transcription (underexpression of POLA, RFC1) as well as proliferation and survival (underexpression of IGF-1); it blocks the cell cycle (overexpression of CDKN1A and PA26) and induces apoptosis (overexpression of GADD45B). Doxorubicin deregulates many genes involved in cell cycle arrest (high expression of CDKN1A, PA26, GADD45A and low expression of CCND2 y CDC20) as well as up-regulation of several members of the TNF family (CD95, TRAILR2 and CD27). Bortezomib increases the expression of many “heat shock proteins” (HSP 110, HSP 70B, HSP 70B´) and decreases the level expression of IGF-1. Aplidin triggers early induction of many genes involved in apoptosis (overexpression of MAP4K3 and EGR2) and down-regulation of genes that play and important role in G2/M phase transition (NEK2, CENPF, BUB1). Arsenic Trioxide induces underexpression of essential genes for G1/S phase transition and cell cycle progression (CDC7L1, CDC25A) as well as underexpression of genes that mediate spindle formation and cromosome segregation (STK6, PRC1, HCAP-G, ZWINT, KIF4A, BUB1). Moreover, Arsenic Trioxide alike Bortezomib treatment up-regulates several HSP (HSP 40, HSP70B, HSP27). TNFSF9 which inhibits proliferation was the only gene up-regulated by all the drugs. Microarray technology demonstrates that treatment with novel targeted therapies (Bortezomib, Aplidin and Arsenic Trioxide) induces deregulation of molecular mechanisms which are not involved in anti-myeloma activity of conventional treatments (Melphalan, Doxorubicin). In addition it is an efficient tool to understand the differences in mechanisms of action of novel drugs.
Abstract #2457 appears in Blood, Volume 104, issue 11, November 16, 2004
Keywords: Microarray analysis|Multiple myeloma|Drug sensitivity
Poster Session: New Targets and Immune Based Therapy (6:00 PM-7:30 PM)
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Sunday, December 5, 2004, 06:00 PM
[2467] Arsenic Trioxide Shows Synergistic Anti-Myeloma Effects When Combined with Bortezomib and Melphalan In Vitro and Helps Overcome Resistance of Multiple Myeloma Cells to These Treatments In Vivo.
Session Type: Poster Session 680-II
Richard A. Campbell, Haiming Chen, Daocheng Zhu, Janice C. Santos, Benjamin Bonavida, Shen Pang, Jonathan Said, James R. Berenson. Hematology/Oncology, Institute for Myeloma & Bone Cancer Research, West Hollywood, CA, USA; Medicine - MIMG, UCLA School of Medicine, Los Angeles, CA, USA; Dental & Oral Biology, UCLA School of Dentistry, Los Angeles, CA, USA; Medicine - Pathology, UCLA School of Medicine, Los Angeles, CA, USA
Arsenic trioxide (ATO) induces apoptosis of plasma cells through a number of mechanisms including inhibiting DNA binding by NF-?B. These results suggest that this agent may be synergistic when combined with other active anti-myeloma drugs. To evaluate this we examined the effect of ATO alone and in combination with anti-myeloma treatments evaluated in vitro with MTT assays and using our severe combined immunodeficient (SCID)-hu murine myeloma models. First, we determined the effects of combining ATO with bortezomib or melphalan on the myeloma cell lines RPMI8226 and U266. Cell proliferation assays demonstrated marked synergistic anti-proliferative effects of ATO at concentrations ranging from 5x10-5M – 5x10-9M and melphalan concentrations ranging from 3x10-5M – 3x10-9M. Similar effects were observed when these cell lines were treated with bortezomib and varying concentrations of ATO (5x10-5 M – 5x10-10 M). We also investigated the potential of ATO to increase the efficacy of anti-myeloma therapies in our SCID-hu murine model LAG?-1 (Yang H et al. Blood 2002). Each SCID mouse was implanted with a 0.5 cm3 LAG?-1 tumor fragment into the left hind limb muscle. Mice were treated with ATO alone at 6.0 mg/kg, 1.25 mg/kg, 0.25 mg/kg, and 0.05 mg/kg intraperitoneally (IP) daily x5/week starting 19 days post-implantation. Mice receiving the highest dose of ATO (6.0 mg/kg) showed marked inhibition of tumor growth and reduction of paraprotein levels while there was no effect observed in all other treatment groups. Next, 27 days following implantation of our LAG?-1 intramuscular (IM) tumor, LAG?-1 mice were treated with ATO (1.25 mg/kg) IP, bortezomib (0.25 mg/kg), or the combination of both drugs at these doses in the schedules outlined above. ATO or bortezomib treatment alone had no anti-myeloma effects at these low doses consistent with our previous results whereas there was a marked decrease in both tumor volume (57%) and paraprotein levels (53%) in mice receiving the combined therapy. The combination of melphalan and ATO was also evaluated in this model. LAG?-1 bearing mice received therapy with melphalan IP x1/weekly at 12.0 mg/kg, 6.0 mg/kg, 0.6 mg/kg, and 0.06 mg/kg starting 22 days post-implantation and showed no anti-myeloma effects. Twenty-eight days following implantation of LAG?-1 tumor, mice received ATO (1.25 mg/kg) or melphalan (0.6 mg/kg) alone at doses without anti-myeloma effects, or the combination of these agents at these doses. The animals treated with these drugs alone showed a similar growth and increase in paraprotein levels to control mice whereas the combination of ATO and melphalan at these low doses markedly suppressed the growth of the tumor by >50% and significantly reduced serum paraprotein levels. These in vitro and in vivo studies suggest that the addition of ATO to other anti-myeloma agents is likely to result in improved outcomes for patients with drug resistant myeloma. Based on these results, these combinations are now in clinical trials with promising early results for patients with drug resistant myeloma.
Abstract #2467 appears in Blood, Volume 104, issue 11, November 16, 2004
Keywords: Multiple myeloma|SCID mice|Arsenic trioxide
Poster Session: New Targets and Immune Based Therapy (6:00 PM-7:30 PM)
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Sunday, December 5, 2004, 06:00 PM
[2470] Ascorbic Acid Overcomes Drug Resistance in Myeloma and Significantly Increases the Anti-Myeloma Effects of Both Arsenic Trioxide and Melphalan In Vitro and In Vivo.
Session Type: Poster Session 683-II
Richard A. Campbell, Haiming Chen, Daocheng Zhu, Janice C. Santos, Benjamin Bonavida, Shen Pang, Jonathan Said, James R. Berenson. Hematology/Oncology, Institute for Myeloma & Bone Cancer Research, West Hollywood, CA, USA; Medicine - MIMG, UCLA School of Medicine, Los Angeles, CA, USA; Dental & Oral Biology, UCLA School of Dentistry, Los Angeles, CA, USA; Medicine - Pathology, UCLA School of Medicine, Los Angeles, CA, USA
Glutathione levels have previously been shown to be associated with the development of resistance to a variety of anti-myeloma therapies. Ascorbic acid (AA) depletes intracellular glutathione levels which, in turn, should increase the sensitivity of tumor cells to anti-myeloma agents such as arsenic trioxide (ATO) and melphalan. To determine the synergistic effects of combining AA,with ATO and/or melphalan, we evaluated the effects of these combinations with MTT assays on myeloma cell lines in vitro and using our severe combined immunodeficient (SCID)-hu murine myeloma models. We determined the synergistic effects of combining AA with ATO and/or melphalan on the myeloma cell lines RPMI8226, 8226/dox, U266, and U266/dox in vitro. MTT assays demonstrated marked synergistic anti-proliferative effects of AA at 10 mM when added to these cell lines in the presence of ATO concentrations ranging from 5x10-5 M – 5x10-9 M, and melphalan concentrations ranging from 3x10-5 M – 3x10-9 M. In order to provide further evidence for the clinical relevance of these synergistic effects of AA, we investigated the potential of AA to increase the efficacy of current anti-myeloma therapies in our SCID-hu murine model of human myeloma LAG?-1 (Yang H et al. Blood 2002). Each SCID mouse was implanted with a 0.5 cm3 LAG?-1 tumor fragment into the left hind limb muscle. Twenty-eight days following implantation, mice then received treatment intraperitoneally (IP) with either AA (300 mg/kg) daily x5/week, ATO (1.25 mg/kg) daily x5/week, or melphalan (3.0 mg/kg) x1/week, or the combination of these agents. AA, ATO, and melphalan alone have no anti-myeloma effects at these doses, whereas AA+melphalan results in significantly decreased tumor burden and paraprotein levels. The most profound anti-myeloma effects were observed in animals treated with all three drugs together. These data show not only the additional synergistic anti-myeloma effects of AA on both ATO and melphalan in vitro but for the first time suggest that these effects are also present in vivo. This provides the rationale for combining AA with these agents in myeloma patients with resistant disease. In support of this, early results of clinical trials using the combination of AA, ATO and low doses of oral melphalan are promising.
Abstract #2470 appears in Blood, Volume 104, issue 11, November 16, 2004
Keywords: Multiple myeloma|Chemotherapy|SCID mice
Poster Session: New Targets and Immune Based Therapy (6:00 PM-7:30 PM)
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Sunday, December 5, 2004, 06:00 PM
[2475] A Green Tea Component, Catechin, Induces Apoptosis of Human Myeloma Cells and Markedly Enhances Arsenic Trioxide-Induced Cytotoxicity Via Production of Reactive Oxygen Species (ROS).
Session Type: Poster Session 688-II
Tomonori Nakazato, Keisuke Ito, Yoshitaka Miyakawa, Yasuo Ikeda, Masahiro Kizaki. Department of Internal Medicine, Division of Hematology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan
Multiple myeloma is a plasma cell malignancy that remains incurable despite the use of conventional and high dose chemotherapy with hematopoietic stem cell transplantation, and so novel therapeutic approaches are urgently required in the clinical settings. Recent understanding of the biology of myeloma has led to the development of biological treatments such as thalidomide and bortezomib, which target the myeloma cell and the bone marrow microenvironment. These agents have shown remarkable activity against refractory multiple myeloma in the early clinical trials, but prolonged drug exposures may result in the development of de novo drug resistance in some cases. Therefore, identification and validation of more novel targeted therapies to overcome drug resistance and improve patient outcome of multiple myeloma will be needed. Recently, green tea attracted much attention due to its beneficial health effects; the polyphenolic compound, (-)-epigallocatechin-3-gallate (EGCG), has potent chemopreventive effects against various tumors. EGCG has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of EGCG as a novel therapeutic agent for the patients with multiple myeloma. EGCG rapidly induced apoptotic cell death in various myeloma cell lines (U266, IM9, RPMI 8226, and HS-Sultan) and fresh myeloma cells from patients in a dose (0-100 µM)- and time (0-72 h)-dependent manner. IM9 cells were most sensitive to EGCG with an IC50 of 17 µM, and cell growth was suppressed as early as 6 h and the typical morphological appearance of apoptosis was observed. Treatment with EGCG (20 µM) increased the population of cells in the G0/G1 phase with the reduction of S phase followed by the appearance of a sub-G1 DNA contents, indicating that EGCG led to cell cycle arrest at G1 phase followed by apoptosis. EGCG-induced apoptosis in myeloma cells was in association with the loss of mitochondrial transmembrane potentials (??m), the release of cytochrome c, Smac/DIABLO and AIF from mitochondria into the cytosol, and the activation of caspase-3 and -9. Treatment with 20 µM EGCG for 1 h in IM9 cells as well as fresh myeloma cells from patients showed rapid elevation of intracellular reactive oxygen species (ROS) production. Antioxidant, catalase and Mn-SOD completely blocked ROS generation, the loss of ??m, caspase-3 activation and consequently inhibited EGCG-induced apoptosis in IM9 cells, suggesting that ROS plays a key role in EGCG-induced apoptosis in myeloma cells. Recently, arsenic trioxide (AS) was reported to inhibit the proliferation of myeloma cells by induction of apoptosis via intracellular production of ROS. Therefore, we further tested the possibility of using an ROS-producing agent, EGCG, to enhance the activity of AS. The combination with AS and EGCG significantly enhanced induction of apoptosis compared to AS or EGCG alone via decreased intracellular GSH levels and increased production of ROS in all myeloma cells, suggesting that EGCG potentiated AS-induced cytotoxicity. In conclusion, EGCG has potential as a novel therapeutic agent for patients with multiple myeloma via induction of apoptosis mediated by modulation of the molecules of the redox system. Furthermore, the combination of EGCG and ROS-producing agents such as AS may provide a new strategy to enhance therapeutic activity for the patients with multiple myeloma.
Abstract #2475 appears in Blood, Volume 104, issue 11, November 16, 2004
Keywords: Experimental therapeutics|Drug resistance|Caspase
Poster Session: New Targets and Immune Based Therapy (6:00 PM-7:30 PM) |
