RESUMO
The metalloid arsenic is a worldwide environmental toxicant, exposure to which is associated with many adverse outcomes. Arsenic is also an effective therapeutic agent in certain disease settings. Arsenic was recently shown to regulate the activity of the Hedgehog (HH) signal transduction pathway, and this regulation of HH signaling was proposed to be responsible for a subset of arsenic's biologic effects. Surprisingly, these separate reports proposed contradictory activities for arsenic, as either an agonist or antagonist of HH signaling. Here we provide in vitro and in vivo evidence that arsenic acts as a modulator of the activity of the HH effector protein glioma-associated oncogene family zinc finger (GLI), activating or inhibiting GLI activity in a context-dependent manner. This arsenic-induced modulation of HH signaling is observed in cultured cells, patients with colorectal cancer who have received arsenic-based therapy, and a mouse colorectal cancer xenograft model. Our results show that arsenic activates GLI signaling when the intrinsic GLI activity is low but inhibits signaling in the presence of high-level GLI activity. Furthermore, we show that this modulation occurs downstream of primary cilia, evidenced by experiments in suppressor of fused homolog (SUFU) deficient cells. Combining our findings with previous reports, we present an inclusive model in which arsenic plays dual roles in GLI signaling modulation: when GLIs are primarily in their repressor form, arsenic antagonizes their repression capacity, leading to low-level GLI activation, but when GLIs are primarily in their activator form, arsenic attenuates their activity.
Assuntos
Arsênio/farmacologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição Gênica/fisiologia , Animais , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Proteína GLI1 em Dedos de ZincoRESUMO
Tryptophanyl-tRNA synthetase (TrpRS) is an interferon-induced phosphoprotein with autoantigenic and cytokine activities detected in addition to its canonical function in tRNA aminoacylation. The availability of monoclonal antibodies (mAbs) specific for TrpRS is important for development of tools for TrpRS monitoring. A molecular characterization of two mAbs raised in mice, using purified, enzymatically active bovine TrpRS as the inoculating antigen, is presented in this report. These IgG1 antibodies are specific for bovine, human and rabbit but not E. coli TrpRS. Immunoreactivity and specificity of mAbs were verified with purified recombinant hTrpRS expressed in E. coli and TrpRS-derived synthetic peptides. One of the mAbs, 9D7 is able to disaggregate fibrils formed by Ser32-Tyr50 TrpRS-peptide. Epitope mapping revealed that disaggregation ability correlates with binding of 9D7 to this peptide in ELISA and immunocytochemistry. This epitope covers a significant part of N-terminal extension that suggested to be proteolytically deleted in vivo from the full-length TrpRS whereas remaining COOH-fragment possesses a cytokine activity. For epitope mapping of mAb 6C10, the affinity selected phage-displayed peptides were used as a database for prediction of conformational discontinuous epitopes within hTrpRS crystal structure. Using computer algorithm, this epitope is attributed to COOH-terminal residues Asp409-Met425. In immunoblotting, the 6C10 mAb reacts preferably with (i) oligomer than monomer, and (ii) bound than free TrpRS forms. The hTrpRS expression was shown to correlate with growth rates of neuroblastoma and pancreatic cancer cells. Immunohistochemically both mAbs revealed extracellular plaque-like aggregates in hippocampus of Alzheimer's disease brain.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos , Triptofano-tRNA Ligase/imunologia , Doença de Alzheimer/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Bovinos , Linhagem Celular Tumoral , Reações Cruzadas , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Conformação Proteica , Coelhos , Especificidade da Espécie , Triptofano-tRNA Ligase/químicaAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Recidiva Local de Neoplasia/tratamento farmacológico , Idoso , Trióxido de Arsênio , Arsenicais/administração & dosagem , Ensaios Clínicos Fase I como Assunto , Feminino , Fluoruracila/administração & dosagem , Humanos , Leucovorina/administração & dosagem , Masculino , Pessoa de Meia-Idade , Óxidos/administração & dosagem , Resultado do TratamentoRESUMO
In the past decade, the therapeutic potential of arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL) was recognized. This encouraged other investigators to test the efficacy of ATO in the management of other hematological and solid tumor malignancies. Notably, as a single agent, arsenic trioxide did not benefit patients diagnosed with solid tumors. However, when it was combined with other agents, treatment benefit emerged. In this article, we have summarized the outcome of clinical trials that used arsenic trioxide as a single agent as well as in combination settings in patients diagnosed with solid tumors. We have also reviewed possible additional mechanisms by which ATO may be useful as a chemosensitizer in combination therapy. We hope that our review will encourage clinical investigators to rationally combine ATO with additional chemotherapeutic agents in treating patients diagnosed with solid tumors.
Assuntos
Antineoplásicos/uso terapêutico , Arsenicais/uso terapêutico , Neoplasias/tratamento farmacológico , Óxidos/uso terapêutico , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Trióxido de Arsênio , Arsenicais/administração & dosagem , Arsenicais/efeitos adversos , Arsenicais/farmacologia , Encefalopatias/induzido quimicamente , Ensaios Clínicos como Assunto , Terapia Combinada , Aprovação de Drogas , Sinergismo Farmacológico , Proteínas Hedgehog/fisiologia , Doenças Hematológicas/induzido quimicamente , Humanos , Medicina Tradicional Chinesa , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Óxidos/administração & dosagem , Óxidos/efeitos adversos , Óxidos/farmacologia , Radioterapia Adjuvante , Transdução de Sinais/efeitos dos fármacos , Timidilato Sintase/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Resultado do Tratamento , Estados Unidos , United States Food and Drug Administration , Proteína GLI1 em Dedos de ZincoRESUMO
Achyranthes aspera (family Amaranthaceae) is known for its anticancer properties. We have systematically validated the in vitro and in vivo anticancer properties of this plant. However, we do not know its mode of action. Global gene expression analyses may help decipher its mode of action. In the absence of identified active molecules, we believe this is the best approach to discover the mode of action of natural products with known medicinal properties. We exposed human pancreatic cancer cell line MiaPaCa-2 (CRL-1420) to 34 µ g/mL of LE for 24, 48, and 72 hours. Gene expression analyses were performed using whole human genome microarrays (Agilent Technologies, USA). In our analyses, 82 (54/28) genes passed the quality control parameter, set at FDR ≤ 0.01 and FC of ≥±2. LE predominantly affected pathways of immune response, metabolism, development, gene expression regulation, cell adhesion, cystic fibrosis transmembrane conductance regulation (CFTR), and chemotaxis (MetaCore tool (Thomson Reuters, NY)). Disease biomarker enrichment analysis identified LE regulated genes involved in Vasculitis-inflammation of blood vessels. Arthritis and pancreatitis are two of many etiologies for vasculitis. The outcome of disease network analysis supports the medicinal use of A. aspera, viz, to stop bleeding, as a cure for pancreatic cancer, as an antiarthritic medication, and so forth.
RESUMO
Thymidylate synthase (TS) gene contains 28-bp polymorphic sequence and 6-bp insert at the 5'- and 3'-untranslated region, respectively. We investigated the role of these two polymorphic traits on TS mRNA expression in nine different cultured human cancer cell lines in vitro. Three cell lines each were 2R/2R, 2R/3R and 3R/3R genotypes. Six of the nine cell lines tested homozygous for the presence of 6-bp insert (+6/+6) and the rest three lacked this insert (-6/-6). TS expression analyses associated homozygous three repeats (3R/3R) to higher TS expression.
Assuntos
Regiões 5' não Traduzidas/genética , Regulação da Expressão Gênica/genética , Neoplasias/genética , Timidilato Sintase/genética , Sequência de Bases , Linhagem Celular Tumoral , Expressão Gênica , Genótipo , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , RNA Mensageiro , Reação em Cadeia da Polimerase em Tempo Real , Timidilato Sintase/biossínteseRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Achyranthes aspera (Family Amaranthacea) is used for cancer therapy by ayurvedic medical practitioners in India. However, due to the non formal nature of its use, there are no systematic studies validating its medicinal properties. Thus, it's utility as an anti cancer agent remains anecdotal. Earlier, we demonstrated A. aspera to exhibit time and dose-dependent preferential cytotoxicity to cultured human pancreatic cancer cells. In this report we validate in vivo anti tumor properties of A. aspera. MATERIALS AND METHODS: The in vivo anti tumor activity of leaf extract (LE) was tested by intraperitoneal (IP) injections into athymic mice harboring human pancreatic tumor subcutaneous xenograft. Toxicity was monitored by recording changes in behavioral, histological, hematological and body weight parameters. RESULTS: Dosing LE to athymic mice by I.P. injection for 32 days showed no adverse reactions in treated mice. Compared to the control set, IP administration of LE to tumor bearing mice significantly reduced both tumor weight and volume. Gene expression analysis using Real time PCR methods revealed that LE significantly induced caspase-3 mRNA (p<0.001) and suppressed expression of the pro survival kinase Akt-1 (p<0.05). TUNEL assay and immunohistochemistry confirmed apoptosis induction by activation of caspase-3 and inhibiting Akt phosphorylation in treated sets. These results are in agreement with RT PCR data. CONCLUSION: Taken together, these data suggest A. aspera to have potent anti cancer property.
Assuntos
Achyranthes , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Fitoterapia , Extratos Vegetais/uso terapêutico , Animais , Antineoplásicos Fitogênicos/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Injeções Intraperitoneais , Ayurveda , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosforilação , Extratos Vegetais/farmacologia , Folhas de Planta , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES: Pancreatic cancer is one of most deadly because of its aggressive growth and high metastatic ability that correlates with intratumoral hypoxia. Earlier diagnosis and prognosis marker of pancreatic cancer is not yet available. In colorectal cancer, protein biosynthesis enzyme, tryptophanyl-tRNA synthetase (TrpRS), is up-regulated in good-prognosis tumors and down-regulated in metastatic poor-prognosis tumors. Tryptophanyl-tRNA synthetase status in pancreatic cancer is unknown. To correlate metastatic ability with hypoxia and TrpRS as a possible prognostic marker, we examined mRNA and protein expression in 2 human pancreatic cancer cell lines with different metastatic abilities and TrpRS levels using our site-specific monoclonal antibodies directed to conformation-dependent epitopes on pancreatic TrpRS. METHODS: Pancreatic MIAPaCa-2, Panc-1, cervical HeLa, and prostate cancer PC-3 cells were cultivated under normoxia or in hypoxic chamber. Expression of full-length TrpRS, antiangiogenic TrpRS, cyclin B1, hypoxia-inducible factor 1α, and Glut-1 was determined with reverse transcriptase-polymerase chain reaction, immunoblotting, and immunocytochemistry. RESULTS: We demonstrate that hypoxia regulates differentially TrpRS splice forms. Pronounced down-regulation of full-length TrpRS by hypoxia is concomitant with higher metastatic ability. CONCLUSIONS: Tryptophanyl-tRNA synthetase down-regulation by hypoxia may be a factor responsible for low TrpRS in tumors with high metastatic ability. Tryptophanyl-tRNA synthetase recognizability is important for pancreatic cancer prognosis and as a new target for metastasis treatment.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Pancreáticas/enzimologia , Triptofano-tRNA Ligase/metabolismo , Biomarcadores Tumorais/genética , Western Blotting , Hipóxia Celular , Ciclina B1/metabolismo , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Transportador de Glucose Tipo 1/metabolismo , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Invasividade Neoplásica , Oxigênio/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esferoides Celulares , Fatores de Tempo , Triptofano-tRNA Ligase/genéticaRESUMO
AIMS OF THE STUDY: Achyranthes aspera (Family: Amaranthacea) is a medicinal plant used as an anti-cancer agent in ayurveda, a traditional system of medicine practiced in subcontinental India. The aim of the study was to systematically investigate the anti-proliferative properties of Achyranthes aspera leaves extracted in methanol (LE) on human cancer cells in vitro. MATERIALS AND METHODS: We tested time, dose dependent and specific anti-proliferative activity of LE by clonogenic cell survival assay on human cancer and normal epithelial cell lines in vitro. We further investigated its effect on the expression of metastatic and angiogenic genes by real time polymerase chain reaction. On silica gel column, we carried out initial fractionation analysis. RESULTS: LE exhibited time and dose dependent cytotoxicity on several tumor cells. Compared to cancer cells of colon, breast, lung and prostate origin, pancreatic cancer cells were significantly more sensitive to LE. Preliminary mechanistic studies suggested that LE selectively suppressed the transcription of metalloproteases (MMP-1 and -2), inhibitors of MMPs (TIMP-2) and angiogenic factors (VEGF-A and VEGF-B). Fractionation of LE on methanol equilibrated silica gel column resolved into three fractions of which fraction (F 3) was found to be enriched with anti-proliferative activity. CONCLUSION: Methanolic extract of Achyranthes aspera contains potent anti-proliferative compound with specific activity against pancreatic cancer. Further studies are needed to confirm the in vivo anti-tumorigenicity and subsequent chemical characterization of the active molecule(s).
Assuntos
Achyranthes , Antineoplásicos Fitogênicos/uso terapêutico , Inibidores do Crescimento/uso terapêutico , Neoplasias Pancreáticas , Extratos Vegetais/uso terapêutico , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular , Linhagem Celular Tumoral , Inibidores do Crescimento/isolamento & purificação , Células HT29 , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Extratos Vegetais/isolamento & purificação , Folhas de PlantaRESUMO
Resistance to chemotherapy is a major issue in treating malignant diseases. 5-Fluorouracil (5-FU) is the drug of choice in managing colorectal cancer (CRC) patients. However, 5-FU resistance leads to eventual treatment failure. Therefore, delaying or reversing the onset of 5-FU resistance will benefit these terminally ill patient populations. A metabolite of 5-FU irreversibly binds thymidylate synthase (TS) thus inhibiting its activity. Many studies demonstrated that these resistant patients had an increased intratumoral TS level. We used TS-siRNA to reduce TS and resensitize HT29FU CRC cells back to this uracil analogue. We exposed the CRC cell line HT29 to an increasing concentration of 5-FU or 5-fluorouridine (FUR) and established a derivative cell line (HT29FU and HT29FUR). Using real-time polymerase chain reaction (PCR) and Western immunodetection assays, we analyzed the expression of TS and p53 mRNA and protein in control and experimental groups. Cytotoxicity to 5-FU was determined by reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT assay) or trypan blue dye exclusion assay. The HT29FU and HT29FUR cells have a distinct morphology: they are generally asteroid shaped. The half maximal inhibitory concentration (IC(50)) values for the resistant cell line for 5-FU is over 148 microM compared to 5 microM for the sensitive parental cell line. The resistant cell lines expressed more of TS and less of p53. TS-siRNA suppressed TS only. Other pathways were not significantly altered. It also marginally (20%) re-sensitized resistant cells to 5-FU. Restoration of partial sensitivity to 5-FU by TS-siRNA reiterates the primacy of the DNA synthesis pathway in 5-FU mode of action. We speculate that the short half-life of the transiently transfected siRNA may contribute to the marginal restoration of sensitivity. By integrating TS-siRNA expression vector into the genome and regulating its expression, we may be able to reverse 5-FU resistance and make the cells as sensitive as the parental cell line.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/farmacologia , Pirimidinas/farmacologia , Timidilato Sintase/biossíntese , Proteína Supressora de Tumor p53/antagonistas & inibidores , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Indução Enzimática/efeitos dos fármacos , Células HT29 , Humanos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Timidilato Sintase/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: This Phase I study was designed to determine a safe combination dose of 5-fluorouracil (5-FU) and arsenic trioxide (ATO) to treat 5-FU-resistant relapsed/refractory colorectal cancer patients. We studied the effect of ATO in the downregulation of thymidylate synthase (TS) in peripheral blood mononuclear cells and in tumor biopsies. EXPERIMENTAL DESIGN: ATO was administered for 5 consecutive days during the first week and twice during weeks 2 to 3 and once on week 4. 5-FU/leucovorin (LV) was administered on days 8, 15, and 22. A modified accelerated titration design was used. 5-FU was dose escalated first followed by a planned dose increase for ATO. RESULTS: No dose-limiting toxicities were seen in seven patients who received 0.15 mg/kg ATO; grade 3 toxicities were as follows: neutropenia 1, diarrhea 1, and bowel obstruction 1. In patients receiving 0.20 mg/kg ATO, grade 3 toxicities were QTc prolongation 1, fatigue 4, alkaline phosphatase elevation 2, diarrhea 2, and peripheral edema 1. TS gene expression in peripheral blood mononuclear cell decreased in all patients. Eight tumors were biopsied, four showed TS downregulation, three showed upregulations, and one did not change. Estimated median progression-free survival and overall survival were 3.1 and 13.9 months, respectively. In patients who showed TS increase or no change versus TS reduction, estimated median progression-free survival was 2.6 versus 7.9 months (P = 0.188) and overall survival was 8.6 versus 11.7 months (P = 0.44), respectively. CONCLUSIONS: Thus, we determined 0.20 mg/kg ATO, 2,600 mg/m(2) 5-FU, and 500 mg/m(2) leucovorin (LV) to be the recommended phase II dose.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Arsenicais/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/administração & dosagem , Leucovorina/administração & dosagem , Óxidos/administração & dosagem , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Trióxido de Arsênio , Arsenicais/efeitos adversos , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Óxidos/efeitos adversos , Timidilato Sintase/metabolismoAssuntos
Fracionamento Celular/métodos , DNA/isolamento & purificação , Preservação de Sangue , Soluções Tampão , Fracionamento Celular/economia , Fracionamento Celular/instrumentação , Centrifugação , Criopreservação , DNA/sangue , Eletroforese em Gel de Ágar , Humanos , Peso Molecular , RibonucleasesRESUMO
Despite the introduction of several novel anticancer agents almost 50% of colorectal cancer (CRC) patients die for cancer suggesting the necessity of new therapeutical approaches. In this study we demonstrated that the HDAC inhibitor vorinostat exerted potent antiproliferative effect in a panel of mut- and wt-p53 human CRC cell lines. Moreover, in combination with 5-fluorouracil modulated by folinic acid (5FU-FA) or with Raltitrexed (RTX), both commonly used in the treatment of this disease, it showed a clear schedule-dependent synergistic antiproliferative interaction as demonstrated by calculating combination indexes. Only simultaneous, or 24 h pretreatment with vorinostat followed by either agent, produced synergistic effect paralleled by evident cell cycle perturbations with major S-phase arrest. Moreover, we provided for the first time evidences that vorinostat can overcome resistance to both 5FU and RTX. Downmodulation of Thymidilate synthase (TS) protein induced by vorinostat within 24 h, represented a key factor in enhancing the effects of both drugs in sensitive as well as resistant tumor cells. Furthermore, p53, whose wild-type expression is critical for sensitivity to 5FU and RTX, was upregulated by vorinostat in wt- and downregulated in mut-p53 cells, suggesting an additional mechanism of the antiproliferative synergistic interactions observed. Overall these data add new insights in the mechanism of vorinostat antitumor effect and suggested that the association of vorinostat plus 5FU-FA and/or RTX should be clinically explored.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Timidilato Sintase/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Leucovorina/administração & dosagem , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , Tiofenos/administração & dosagem , Tiofenos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , VorinostatRESUMO
Arsenic trioxide (As2O3) has demonstrated effectiveness in treating acute promyelocytic leukemia (APL). Therefore the FDA has approved it to treat APL. In patients with refractory metastatic colorectal carcinoma (CRC), we assessed the efficacy and toxicity of As2O3/AA (ascorbic acid) as the outcome of this trial. Five patients with refractory metastatic CRC who failed all previous standard chemotherapy were enrolled in this study. They were treated with 0.25 mg/kg body weight/day As2O3 and 1000 mg/day of ascorbic acid for 5 days a week for 5 weeks. Each treatment cycle extended for 7 weeks with 5 weeks of treatment and 2 weeks of rest. All the patients developed moderate to severe toxic side effects to arsenic trioxide/AA therapy and therefore the study was discontinued. No CR (complete remission) or PR (partial remission) was observed. CT scans demonstrated stable or progressive disease. Three of the five patients died within 2 to 5 months after cessation of the therapy. None of the deaths could be related to this clinical trial. Two years of follow-up study showed that two patients were alive with stable disease. Under the current treatment regimen all patients developed moderate to severe side effects with no clinically measurable activity. As an alternate, efforts may be made to reduce the dose and arsenic trioxide may be combined with other standard regimen in reversing the chemo resistance.