ATO Treatment Research Articles

TRAIL as the ATO-Target Gene Uniquely Activated In the Hyperdiploid Subtype of Myeloma with Prognostic Relevance, Resulting In Better Prognosis

AbstractAbstract 1904Arsenic trioxide (ATO) is a well-known inhibitor of cell proliferation in certain forms of malignancy and has been successfully used in the treatment of acute promyelocytic leukemia. Preclinical and clinical studies showed that ATO has anti-myeloma effects both as a single agent and in the combination therapy; however, the underlying mechanism remains elusive. In this study, the molecular mechanisms of ATO-induced myeloma apoptosis were explored on four myeloma cell lines OPM2, U266, RPMI8226, and KMS28PE of wild type or mutant p53 status and six primary myeloma cells. Gene expression profiling (GEP) of CD138+ bone marrow plasma cells from 22 healthy individuals (NPC), 44 patients with monoclonal gammopathy of undetermined significance (MGUS), and 351 newly diagnosed MM patients were published previously (Zhan et al. Blood. 2006;108:2020-8. Shaughnessy et al. Blood. 2007;109:2276-84.); and GEP from 9 myeloma cell lines were used in this study from the unpublished data of the University of Utah. Cell growth and viability were assayed by trypan blue dye exclusion. Cell cycle and apoptosis were analyzed by flow cytometry using CellQuest software and Vybrant Apoptosis Assay Kit. Alterations of the signaling pathways induced by ATO were tested by real-time PCR and western blot. GEP was performed by using the Affymetrix U133Plus2.0 microarray. ATO induced potent inhibition of myeloma cell growth and myeloma cell apoptosis, compared with controls. Further investigation showed that ATO down-regulated c-Myc and phosphorylated (p)-Rb while up-regulating p53, p21Cip1, and p27Kip1 proteins, resulting in G0/G1 or G2/M cell cycle arrest. ATO treatment increased mRNA levels of interferon regulatory factor-1 and TRAIL, as well as protein levels of caspase 8 and cleaved caspase 3, indicating involvement of the extrinsic apoptotic pathway in the mutated p53 myeloma cells. ATO also activated caspases 3 and 9, indicating involvement of the intrinsic apoptotic pathway in the wild type p53 myeloma cells. The usage of ATO and TRAIL agonist together has a synergistic effect, indicated by a combination index of less than 1. More importantly, these molecular changes induced by ATO-treated myeloma cells are very similar to the baseline expression pattern of hyperdiploid myeloma, which has a relative good prognosis with high expression of TRAIL and interferon related genes. Together, our data suggest that ATO induces apoptosis in MM through either extrinsic or intrinsic signaling pathway depending on the p53 genetic background. These observations may be employed as prognostic tools and lead to novel therapies in primary myelomas. AcknowledgmentsThis work was supported by grants from National Natural Science Foundation of China (30973450 to JS), start-up funds from Shanghai Tenth People's Hospital (JS), institutional start-up funds from the University of Utah School of Medicine and the Huntsman Cancer Institute (FZ), the National Institutes of Health grant RO1 (CA115399 to GT, FZ) and Senior Award from the Multiple Myeloma Research Foundation (FZ). Disclosures:Zhan:University of Utah: Employment, patent Submission.

MicroRNA-dependent regulation of PTEN after arsenic trioxide treatment in bladder cancer cell line T24

Arsenic trioxide has shown remarkable biological activity against bladder cancer in some clinical studies. However, the mechanism of its action is unknown. Our aim was to find the relationship between miRNAs and arsenic trioxide treatment by using T24 human bladder carcinoma cells. By performing microRNA microarray and quantitative real-time PCR after ATO treatment, we found that expression levels of several miRNAs, in particular, miRNA-19a, were significantly decreased in T24 cell line. Furthermore, cell proliferation assay, flow cytometry analysis, prediction of miRNA targets, Western blot analysis, and luciferase reporter assay were performed to determine the role of mir-19a in affecting the biological behaviors of T24 cells. Several miRNAs were up-regulated or down-regulated in T24 cells treated with arsenic trioxide compared to their controls. If only changes above two folds were considered, two miRNAs were identified, miRNA-19a was down-regulated, while miRNA-222* was up-regulated. Among them, knockdown of miRNA-19a by anti-miRNA-19a transfection showed a positive therapeutic effect in bladder cancer cells by inhibiting cell growth and inducing cell apoptosis targeting PTEN through the PTEN/Akt pathway. Besides this, a synergy effect was detected between knockdown of miRNA-19a and arsenic trioxide. Arsenic trioxide altered miRNA expression profile in T24 cells. It seems miRNA-19a plays a critical role in the mechanism of arsenic trioxide treatment in bladder cancer. The synergy effect between miRNA-19a and arsenic trioxide that advocates targeting the mir-19a may represent a potential approach to enhance the efficacy and safety of ATO to treat bladder cancer by a decrease in dose.

Effect of arsenic trioxide (Trisenox) on actin organization in K-562 erythroleukemia cells.

Actin is one of the cytoskeletal proteins that take part in many cellular processes. The aim of this study was to show the influence of Trisenox (arsenic trioxide), on the cytoplasmic and nuclear F-actin organization. Arsenic trioxide is the proapoptotic factor. Together with increasing doses, it caused the increase in the number of cells undergoing apoptosis. Under arsenic trioxide treatment, cytoplasmic and nuclear F-actin (polymerized form of G-actin) was found reorganized. It was transformed into granulated structures. In cytometer studies fluorescence intensity of cytoplasmic F-actin after ATO treatment decreasing urgently in comparison to control. The obtained results may suggest the involvement of F-actin in apoptosis, especially in chromatin reorganization.

Synergistic Anti-Cancer Effects of Arsenic Trioxide and Paclitaxel On Human Malignant Lymphocytes.

Abstract 4809Arsenic trioxide (ATO), an effective drug in the treatment of acute promyelocytic leukemia (APL), and its combination with other chemotherapeutic drugs have been shown to present some activities in other malignant cells besides APL. Increasing reports showed that ATO produces mitotic arrest possibly through targeting to tubulins before apoptosis induction in human tumor cells. Aberrations in the control of cell cycle progression occur in the majority of human malignancies; hence, tubulins are promising targets for anticancer therapy. Here, we define the cellular effects with ATO, alone or in association with paclitaxel, the first identified microtubules stabilizing agent, with respect to inhibition of cell proliferation and cell cycle progression and induction of apoptosis in malignant lymphocytic cell lines Jurkat, Raji, and NAMALWA as well as fresh acute lymphocytic leukemic (ALL) cells. Our results showed that the co-treatment of ATO and PTX at their lower concentrations could significantly induce mitotic arrest followed by growth inhibition and apoptosis in these cell lines. The combined treatment of ATO with PTX also synergistically decreased the cell viability in primary ALL cells. In vivo and in vitro experiments showed that the combination treatment did not increase the microtubules polymerization compared to ATO or PTX treatment alone. Instead, this synergistic action was attributed to the increase of phosphorylation of cyclin-dependent kinase 1 (Cdk1) on Thr161 and the promotion of the dysregulated activation of Cdk1. Furthermore, we found that the ATO/PTX combination significantly enhanced the activation of spindle checkpoint by inducing the formation of the inhibitory checkpoint complex BubR1/Cdc20. These results provide a rational basis for clinical trials of the combined regimen of ATO and PTX in the treatment of lymphocytic neoplasm. Disclosures:No relevant conflicts of interest to declare.

Arsenic Trioxide-Mediated Growth Inhibition of Myeloma Cells Is Associated with An Extrinsic Signaling Pathway Involved in Caspase 3 and Caspase 8.

Abstract 4899Arsenic trioxide (ATO) is a well-known inhibitor of cell proliferation in certain forms of malignancy and has been successfully used in the treatment of acute promyelocytic leukemia. Preclinical and clinical studies showed that ATO has anti-myeloma effects both as a single agent and in the combination therapy; however, the underlying molecular mechanism remains elusive. This study was performed to evaluate the molecular mechanism underlying its anti-myeloma activities. Cells from OPM2, U266, RPMI8226 myeloma cell lines and patients diagnosed with myeloma (n=6) were cultured with various concentrations of ATO for 4 days. Cell growth and viability were assayed by trypan blue dye exclusion. Cell cycle and apoptosis were analyzed by flow cytometry using CellQuest software and Vybrant Apoptosis Assay Kit. Alterations of the signaling pathways induced by ATO were tested by real-time PCR and western blot. ATO induced potent inhibition of myeloma cell growth compared with untreated control cells. Further investigation showed that ATO down-regulated c-Myc and phosphorylated (p)-Rb, while it up-regulated p53, p21Clip1, and p27Kip1 proteins, resulting in G2/M cell cycle arrest and cell growth inhibition. ATO treatment increased mRNA levels of interferon regulatory factor-1 (IRF-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as well as protein levels of caspase 8 and cleaved caspase 3, indicating involvement of the extrinsic apoptotic pathway. No significant change was detected in the expression levels of Bax, Bcl-xL caspase 9 and Bcl-2, indicating that the intrinsic signaling pathway was not involved. A pan-caspase inhibitor abrogated ATO-induced apoptosis of myeloma cells. Our data suggest that ATO induces apoptosis in MM cells most likely through an extracellular signaling pathway. DisclosuresNo relevant conflicts of interest to declare.

Effects of atorvastatin and losartan on monocrotaline-induced pulmonary artery remodeling in rats

Structural remodeling of pulmonary artery plays an important role in the maintaining of sustained pulmonary arterial hypertension (PAH). The anti-remodeling effects of statins have been reported in systemic hypertension. In this study, we investigated the effects of Ato and losartan (Los) on monocrotaline-induced pulmonary artery remodeling in rats. Forty SD rats were assigned into: normal control (Ctr), PAH, Los, Ato treated groups. The mean pulmonary arterial pressure was reduced by Los and Ato treatment (mmHg: PAH, 36.1 4.2; Los, 29.8 4.8; Ato, 27.6 4.1; P<0.05).

Use of a Fluorescently Labeled Poly-Caspase Inhibitor for in Vivo Detection of Apoptosis Related to Vascular-Targeting Agent Arsenic Trioxide for Cancer Therapy

Arsenic trioxide (ATO, Trisenox) is a potent anti-vascular agent and significantly enhances hyperthermia and radiation response. To understand the mechanism of the anti-tumor effect in vivo we imaged the binding of a fluorescently-labeled poly-caspase inhibitor (FLIVO) in real time before and 3 h or 24 h after injection of 8 mg/kg ATO. FSaII tumors were grown in dorsal skin-fold window chambers or on the rear limb and we observed substantial poly-caspase binding associated with vascular damage induced by ATO treatment at 3 and 24 h after ATO injection. Flow cytometric analysis of cells dissociated from the imaged tumor confirmed cellular uptake and binding of the FLIVO probe. Apoptosis appears to be a major mode of cell death induced by ATO in the tumor and the use of fluorescently tagged caspase inhibitors to assess cell death in live animals appears feasible to monitor and/or confirm anti-tumor effects of therapy.

ATORVASTATIN PREVENTED AND PARTIALLY REVERSED ADRENOCORTICOTROPIC HORMONE-INDUCED HYPERTENSION IN THE RAT

1. Adrenocorticotropic hormone (ACTH)-induced hypertension is associated with nitric oxide (NO) deficiency and increased oxidative stress. Atorvastatin (Ato), an HMG-Co-enzyme-A reductase inhibitor has been reported to enhance availability of NO. The aim of the study was to assess whether pretreatment with Ato would prevent the development of ACTH-induced hypertension and whether established ACTH-induced hypertension could be reversed with subsequent administration of Ato in rats. 2. Male Sprague-Dawley rats (n = 60) were treated with Ato (30 mg/kg per day in drinking water) or tap water for 15 days. ACTH (0.2 mg/kg per day s.c) or saline was started 4 days after Ato treatment or non-treated rats and continued for 11-13 days (prevention study). In the reversal study, Ato was given on day 8 of ACTH/Saline treatment for 5 days. Systolic blood pressure (SBP) was measured on alternate days using the tail cuff method. 3. Adrenocorticotropic hormone treatment increased SBP (110 +/- 2-136 +/- 2 mmHg, P < 0.001) and aortic superoxide production (P < 0.001). Ato alone did not alter SBP, but Ato pretreatment prevented ACTH-induced hypertension compared with that in rats treated with ACTH alone (118 +/- 2 and 136 +/- 2 mmHg, respectively, P cent < 0.01). Ato partially reversed ACTH-induced hypertension (124 +/- 3 and 136 +/- 2 mmHg, respectively, P cent < 0.05). Plasma nitrate/nitrite (NOx) was decreased in ACTH-treated rats compared with saline treated rats (6.6 +/- 0.4 saline and 4.5 +/- 0.5 micromol/L ACTH, P < 0.001). Atorvastatin affected neither plasma NOx nor aortic superoxide production. 4. Atorvastatin prevented and partially reversed ACTH-induced hypertension in the rat.

Glutathione and Glutathione Utilizing Enzymes Are Key Determinants in the Sensitivity of Myeloma Cells to Arsenic Trioxide as Well as Its Mechanism of Action.

Arsenic trioxide (ATO, Trisenox) is currently being tested in clinical trials as a single agent or in combination with other agents that have activity in multiple myeloma (MM). We and others have demonstrated that glutathione (GSH) levels can influence the ability of ATO to induce cell death in MM cell lines/patient samples and based on these data have initiated a trial to test the safety and efficacy of the combination of ATO and ascorbic acid for the treatment of refractory/relapsed MM. Therefore we performed expression profiling on 4 MM cell lines treated with ATO over a 48 timecourse. Affymetrix Hu133 2.0 plus arrays were hybridized and up to 7035 out of 54,675 probe sets displayed a change and up to 1546 probes sets displayed 2 fold or greater changes compared to untreated cells at 6 hrs. By only looking at genes that increased in all four cell lines we restricted our search to less than 365 probes sets at any given time point. Interestingly the cells appear to have initiated several pathways that are consistent with an attempt to enhance GSH synthesis. Upregulation of transporters of cysteine (xCT) and glycine (Glyt1) as well as enzymes that convert methionine to cysteine (cystathionase) and serine to glycine (serine hydroxymethyl transferase-1) was observed. Moreover the rate-limiting step of the glutathione salvage pathway gamma-glutamyltransferase is also upregulated. Together this suggests an increase in the building blocks for GSH that can be used for de novo synthesis. The rate limiting step for this reaction is performed by gamma-glutamate cysteine ligase which both the catalytic and modifier subunits are upregulated. GSH can also be regenerated from GSSG by glutathione reductase (GR) in an NADPH-dependent fashion. Both GR and the NADPH generating malic enzyme are also upregulated following treatment with ATO. While consistent with our previous findings the data do not provide much insight as to how the GSH is utilized. The only GSH utilizing enzyme that was observed to be upregulated were the cytosolic and mitochondrial forms of glutaredoxin. Glutathione peroxidase (GPx) activity is not altered by ATO treatment. However GPx baseline expression and activity do correlate with sensitivity of MM cell lines to ATO. We also determined GSTP1 activity in the cells and found that it was expressed in 4/5 MM cell lines tested. In contrast to GPx, GSTP1 baseline expression did not correlate with sensitivity to ATO. However this pattern of expression correlated with our previous findings regarding these cells demonstrating differences in caspase dependence of ATO-induced cell death. The one line that did not express GSTP1, RPMI 8226, also utilizes caspase-independent mechanisms of cell death. Transfection of the GSTP1A allele into these cells could render cells more resistant to ATO-induced apoptosis at concentrations of ATO that are not likely to be achieved in patients. Interestingly transfection of the GSTP1B allele could not render cells more resistant, however like GSTP1A it resulted in inhibition of the caspase-independent pathway. Taken together these data confirm that GSH is an important modulator of ATO therapy and that GPx expression may determine the sensitivity of cells to ATO while GSTP1 can affect the mechanism of action by which ATO-induces apoptosis.

Glutathione-Depletion Potently Enhances the Cytotoxic Activity of Arsenic Trioxide in Imatinib Sensitive and Resistant Bcr-Abl Positive Cell Lines.

Arsenic trioxide (ATO, Trisenox™) is currently being investigated as a potential drug to optimize imatinib based treatment of chronic myelogenous leukemia (CML). ATO has antileukemic activity against imatinib sensitive and resistant CML cell lines and primary cells derived from patients with CML. However, preliminary clinical data reveal only moderate activity of ATO in CML (imatinib sensitive or resistant). ATO-activity in acute myelogenous leukemia or multiple myeloma cells is inversely related to the intracellular Glutathione- (GSH-) content with cells bearing low GSH-levels being the most sensitive. To investigate whether this relationship plays a role in CML cell lines sensitive or resistant to the Bcr-Abl inhibitor imatinib, we determined the antiproliferative activity of ATO with regard to the cellular GSH-levels. Furthermore, we examined the ability of GSH-depletion as a therapeutic tool to sensitize CML cells to ATO. MTS-proliferation assays were performed to determine the concentration of ATO needed to induce 50% cellular growth inhibition (IC50). Cell lines used were the imatinib sensitive CML blast crisis lines AR230-s, KCL22-s, LAMA-s as well as their imatinib resistant derivatives AR230-r1, KCL22-r1, LAMA-r1. Known ATO-sensitive AML cell lines (NB4, HL60) with low GSH-content were also included in this study. Intracellular GSH-levels were measured biochemically using a commercially available kit. Protein content was analyzed using the Bradford method. ATO toxicity was analyzed using trypan blue exclusion and flow cytometric analysis of Annexin/PI-stained cells. MTS-proliferation assays indicate a cell type dependent activity of ATO with IC50-values ranging from 0.37±0.03 μM (NB4) up to 6.9±1.43 μM (AR230-r1). The most sensitive NB4 cells express low GSH-levels (8.74±2.9 nmol/mg), whereas highly resistant AR230-r1 cells reveal a threefold increased GSH-content (26.7±8nmol/mg). Imatinib resistance in AR230-r1 and LAMA-r1 cells is not associated with significant GSH-content modulation when compared to the imatinib naïve counterparts (23.6±5.4nmol/mg [AR230-s] vs 26.7±8nmol/mg [AR230-r1]; 8.3±1.8nmol/mg [LAMA-s] vs 6.9±0.2 [LAMA-r1]). Treatment of AR230-s cells with 100 μM of the GSH-depleting agent L-Buthionine-Sulfoximine (BSO) for 12 h leads to significant downregulation of cellular GSH (23.6±5.4 nmol/mg [control] vs 4,25±0,53 nmol/mg [100 μM BSO]). Treatment with BSO alone does not affect cellular viability nor induces apoptosis. Subsequent cotreatment of AR230-s cells with ATO (1 μM) and BSO (100 μM) for 24 h reduces viability to 31.6 % compared to untreated cells. In contrast, treatment with 1 μM ATO alone does not affect viability. Flow cytometric analysis of apoptosis reflects viability data with a 5.5 fold increase of apoptotic cells in the combined treated fraction. Similar data were generated using the KCL22-s cell line. Experiments using primary patient cells are currently in progress. Our data indicate that high intracellular GSH-content confers relative resistance to ATO in Bcr-Abl positive cell lines regardless whether they are imatinib sensitive or resistant. A promising tool to increase the antileukemic activity of ATO is the application of GSH-depleting agents. Therefore, GSH-dependent response to ATO treatment needs further investigation in individual CML patients.