Against Acute Promyelocytic Leukemia Research Articles

Arsenic trioxide versus Realgar-Indigo naturalis formula in non-high-risk acute promyelocytic leukemia: a multicenter, randomized trial.

Realgar-Indigo Naturalis Formula (RIF) is an oral form of arsenic that is effective against acute promyelocytic leukemia (APL). This multicenter, randomized, controlled trial compared the efficacy of all-trans retinoic acid (ATRA) plus RIF with ATRA plus arsenic trioxide (ATO) in a simplified regimen for non-high-risk APL. Following induction therapy with ATRA and ATO, participants were randomly assigned to receive either ATRA plus ATO or ATRA plus RIF both in a 2-week on 2-week off schedule for consolidation therapy. Once achieving molecular complete remission, the regimen was administered for a total of 6 cycles. All of 108 eligible patients achieved hematological complete remission after induction therapy. The median follow-up time was 29 months. The primary endpoint of two-year disease-free survival was 97% in the ATRA-RIF arm and 98% in the ATRA-ATO arm, respectively. (The ATRA-RIF arm was found to be non-inferior to the ATRA-ATO arm, (P < .01), with a percentage difference of -1% (95%CI, -4.8 to 6.9). No deaths have been observed. Most adverse events were moderate. This study confirms the noninferiority of RIF to ATO for non-high-risk APL, while also offering a more favorable regimen schedule for post-remission therapy. (ClinicalTrials gov. Identifier: as NCT02899169).

Additive antitumor effect of arsenic trioxide with exposure to ionizing radiation to human acute promyelocytic leukemia HL‑60 cells.

Arsenic trioxide (ATO) is expected to be a chemical drug with antitumor activity against acute promyelocytic leukemia (APL), a type of acute myeloid leukemia. In Japan, its antitumor effects were confirmed in clinical trials for APL, and it has been approved in various countries around the world. However, there have been no reports on ATO's antitumor effects on radioresistant leukemia cells, which can be developed during radiotherapy and in combination with therapeutic radiation beams. The present study sought to clarify the antitumor effect of ATO on APL cells with radiation resistance and determine its efficacy when combined with ionizing radiation (IR). The radiation‑resistant HL60 (Res‑HL60) cell line was generated by subjecting the native cells to 4‑Gy irradiation every week for 4 weeks. The half‑maximal inhibitory concentration (IC50) for cell proliferation by ATO on native cell was 0.87 µM (R2=0.67), while the IC50 for cell proliferation by ATO on Res‑HL60 was 2.24 µM (R2=0.91). IR exposure increased the sub‑G1 and G2/M phase ratios in both cell lines. The addition of ATO resulted in a higher population of G2/M after 24 h rather than 48 h. When the rate of change in the sub‑G1 phase was examined in greater detail, the sub‑G1 phase in both control cells without ATO significantly increased by exposure to IR at 24 h, but only under the condition of 2 Gy irradiation, it had continued to increase at 48 h. Res‑HL60 supplemented with ATO showed a higher rate of sub‑G1 change at 24 h; however, 2 Gy irradiation resulted in a decrease compared with the control. There was a significant increase in the ratio of the G2/M phase in cells after incubation with ATO for 24 h, and exposure to 2 Gy irradiation caused an even greater increase. To determine whether the inhibition of cell proliferation and cell cycle disruptions is related to reactive oxygen species (ROS) activity, intracellular ROS levels were measured with a flow cytometric assay. Although the ROS levels of Res‑HL60 were higher than those of native cells in the absence of irradiation, they did not change after 0.5 or 2 Gy irradiation. Furthermore, adding ATO to Res‑HL60 reduced intracellular ROS levels. These findings provide important information that radioresistant leukemia cells respond differently to the antitumor effect of ATO and the combined effect of IR.

Application of Ethyl Cellulose and Ethyl Cellulose + Polyethylene Glycol for the Development of Polymer-Based Formulations using Spray-Drying Technology for Retinoic Acid Encapsulation.

Ethyl cellulose (EC)-based microparticles, with and without the incorporation of polyethylene glycol (PEG) as a second encapsulating agent, were prepared using the spray-drying process for the encapsulation of retinoic acid (RA). The production of a suitable controlled delivery system for this retinoid will promote its antitumor efficiency against acute promyelocytic leukemia (APL) due to the possibility of increasing the bioavailability of RA. Product yield ranged from 12 to 28% in all the microparticle formulations, including unloaded microparticles and RA-loaded microparticles. Microparticles with a mean diameter between 0.090 ± 0.002 and 0.54 ± 0.02 µm (number size distribution) and with an irregular form and rough surface were obtained. Furthermore, regarding RA-loaded microparticles, both polymer-based formulations exhibited an encapsulation efficiency of around 100%. A rapid and complete RA release was reached in 40 min from EC− and EC + PEG-based microparticles.

Ascorbic Acid Sensitizes Colorectal Carcinoma to the Cytotoxicity of Arsenic Trioxide via Promoting Reactive Oxygen Species-Dependent Apoptosis and Pyroptosis

Arsenic trioxide (ATO) is an effective therapeutic agent against acute promyelocytic leukemia (APL); however, its anti-tumor effect on solid tumors such as colorectal cancer (CRC) is still in debate. Ascorbic acid (AA) also produces a selective cytotoxic activity against tumor cells. Here, we exploit the potential benefit of ATO/AA combination in generating cytotoxicity to CRC cells, which may lay the groundwork for the potential combinational chemotherapy of CRCs. According to the results, we found that ATO and AA effectively inhibited the viability of human CRC cells in a synergistic manner. AA and ATO corporately activated caspase-3 to trigger apoptosis and upregulated the expression of caspase-1 and promoted formation of inflammasomes to induce pyroptosis. Furthermore, the stimulation of reactive oxygen species (ROS) overproduction was demonstrated as a subcellular mechanism for apoptosis and pyroptosis induced by ATO/AA combination treatment. Our findings suggest that ATO combination with a conventional dosage of AA offers an advantage for killing CRC cells. The synergistic action of ATO/AA combination might be considered a plausible strategy for the treatment of CRC and perhaps other solid tumors as well.

Double-Sided Personality: Effects of Arsenic Trioxide on Inflammation.

In 1992, arsenic trioxide (As2O3, ATO) was demonstrated to be an effective therapeutic agent against acute promyelocytic leukemia (APL), rekindling attention to ATO applications in U.S. Food and Drug Administration clinical trials for the treatment of cancers, such as leukemia, lymphomas, and solid tumors. ATO is a potent chemotherapeutic drug that can also be used to treat other diseases, such as autoimmune diseases, because it affects multiple pathways including apoptosis induction, differentiation stimulation, and proliferation inhibition. As inflammation is a critical component of disease progression, ATO is a feasible treatment option based on its ability to protect against inflammation. However, ATO is also a well-known carcinogen because of its pro-inflammatory effect. This review will focus on the double-sided effects of ATO on inflammation as well as the relevant mechanisms underlying these effects, aiming to provide a rational understanding of how ATO effects the immune system. We especially aim to provide a comprehensive overview of our current knowledge of how ATO influences inflammation.

Chemical metabolomics for investigating the protective effectiveness of Acanthopanax senticosus Harms leaf against acute promyelocytic leukemia.

Recent advances in the study of high-throughput metabolomics combined with high-resolution mass spectrometry have accelerated our understanding of the efficacy, mechanisms, and application of natural products. In this study, we have used chemical metabolomics to investigate and discover small molecule metabolites for the potential mechanism of Acanthopanax senticosus Harms leaf (ASL) against acute promyelocytic leukemia (APL). Based on high-throughput metabolomics, the underlying biomarker was found by combining chromatography coupled with quadrupole time-of-flight mass spectrometry with multivariate data analysis. The protective effect of ASL was dissected using biochemical indicators, pathology sections, immunohistochemistry, and multivariate analysis. Furthermore, 13 potential biomarkers associated with the pathway of sugar metabolism, amino-acid metabolism, nucleotide metabolism, and the metabolism of arachidonic acid were identified from serum samples. This study would help to understand chemical metabolomics for investigating the anti-APL effectiveness of ASL.

Arsenic Trioxide Potentiates Extracellular Traps-Formation Cell Death in Acute Promyelocytic Leukemia through mTOR-Regulated Autophagy

Background:Emerging clinical data shows that arsenic trioxide (ATO) exerts selective cytotoxicity against acute promyelocytic leukemia (APL) without severe side effects that mainly ascribed to nonspecific induction of apoptosis. It is attractive to speculate whether other uncovered APL cell death exists which can be specifically sparked by ATO administration. We have recently demonstrated that APL cells can undergo a previously unrecognized pathway for death by releasing extracellular DNA traps (ETs), termed ETosis (Ma R et al, Cell Death Dis 2016). However, besides apoptosis, whether ATO induces this APL-specific ETotic cell death remains to be explored. We wereto investigated the effects of a wide range of concentrations of ATO on ETotic death in APL cells and elucidate the underlying molecular mechanisms.Methods: Bone marrow samples were obtained from sixteen APL patients before and after the continuous administration of ATO for two weeks. APL cells were isolated and cultured in the presence and absence of ATRA for 3 days. We used confocal microscopy to image ET formation by APL cells and the percentage of ETotic cells was simultaneously quantified. ELISA was used to measure the concentration of myeloperoxidase (MPO)-DNA complexes in the supernant. We also assessed the effects of a wide range of concentrations (0.1, 0.25, 0.5, 0.75, 1.0, 2.0 μM) of ATO treatment for 24, 48 and 72 hours on ETosis in APL-cell line NB4 cells in vitro. Autophagy activation and leukemia-initiating cell (LIC) activity were evaluated by immunoblotting and imaged by immunostaining. LICs were analyzed using colony-forming unit (CFU) assays, and identified and quantified by flow cytometry.Results: APL cells isolated from ATO-treated APL patients underwent significantly increased spontaneous (P = 0.005) and ATRA-stimulated (P = 0.002) ETosis compared to those from newly diagnosed patients (n = 16). In vitro ATO treatment showed that the percentage of ETotic NB4 cells dramatically increased at 0.5 μM (8 ± 1.6%), peaked at 0.75 μM (15 ± 2.4%) and was gradually suppressed at higher concentrations. The concentration of MPO-DNA complexes, an indirect marker of ETosis, parallelled the dose-dependent change in the percentage of ETotic cells. Interestingly, inhibition experiments indicated that ATO caused concentration-dependent APL cell death: ATO primarily triggered ETosis at moderate concentrations (0.5 to 0.75 μM) and switched to apoptosis at relatively high doses (1.0 to 2.0 μM). Furthermore, We found that ATO induced ETosis through mammalian target of rapamycin (mTOR)-regulated autophagy. Surprisingly, inhibition of ETosis spared LIC activity from ATO reduction, while combined treatment with ATO and rapamycin further increased ETosis-mediated LIC loss (~3.5-fold).Conclusions: This is the first study to show that ATO potentiates ETotic death in APL cells through mTOR-regulated autophagy. Importantly, further investigation suggests that ATO specifically targets the APL LICs to ETosis. This implies that, in combination with ATO, therapy-triggered ETosis by targeting the corresponding signaling pathways could be a novel and effective strategy to improve a long relapse-free survival through LIC clearance, avoid lethal apoptosis-related complications and overcome apoptosis resistance.Conflict of interest statement: None. DisclosuresNo relevant conflicts of interest to declare.

Rosmarinic acid potentiates ATRA-induced macrophage differentiation in acute promyelocytic leukemia NB4 cells

Rosmarinic acid (RA, an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid) has a number of biological activities, but little is known about anti-leukemic activities of RA combined with all-trans retinoic acid (ATRA) against acute promyelocytic leukemia (APL) cells. We examined the differentiation marker, CD11b, in bone marrow cells (BMC) of an APL patient, in NB4 cells (APL cell line), and in normal BMC and peripheral blood mononuclear cells (PBMC) of healthy subjects by flow cytometric analysis. ATRA/RA induced expression of CD11b in the BMC of the APL patient and in NB4 cells, but not in normal BMC or PBMC. Therefore, we realized that RA potentiated ATRA-induced macrophage differentiation in APL cells. Further characterization of the induced macrophages showed that they exhibited morphological changes and were able to phagocytose and generate reactive oxygen species. Th also had typical expression of C–C chemokine receptor type 1 (CCR1), CCR2, and intercellular adhesion molecule-1 (ICAM-1). Moreover, the expression of CD11b+ and CD14+ cells depended on ERK-NF-κB axis activation. Together, these results indicate that RA potentiates ATRA-induced macrophage differentiation in APL cells. Thus, RA may play an important role as an appurtenant differentiation agent for functional macrophage differentiation in APL. Additionally, the differentiated macrophages might have a normal life span and, they could die. These data indicate that co-treatment with RA and ATRA has potential as an anti-leukemic therapy in APL.

Real-life experience of a brief arsenic trioxide-based consolidation chemotherapy in the management of acute promyelocytic leukemia: favorable outcomes with limited anthracycline exposure and shorter consolidation therapy.

Anthracyclines have activity against acute promyelocytic leukemia (APL) but can cause cardiac toxicity and secondary malignancy. The all-trans retinoic acid (ATRA)-arsenic trioxide (ATO) combination is an effective noncytotoxic approach for APL. However, its efficacy against high-risk APL (white blood cell count > 10,000/μL) has not been documented. Also, it requires ≥ 8 months to complete therapy. We report a retrospective analysis of 63 patients with APL given one cycle of ATO-based consolidation chemotherapy. The 5-year overall survival, event-free survival, and leukemia-free survival was 93% (95% confidence interval [CI], 82%-97%), 89% (95% CI, 77%-95%), and 92% (95% CI, 80%-97%), respectively. These data have confirmed that an abbreviated ATO-based chemotherapy regimen is an effective consolidation therapy for APL, including high-risk APL, and can be completed within 4 months.

Polymeric micelles for GSH-triggered delivery of arsenic species to cancer cells

Arsenic trioxide (ATO), dissolved in water as arsenous acid or inorganic arsenite (AsIII), is an effective chemotherapeutic agent against acute promyelocytic leukemia (APL). It has been under investigation as a potential treatment for a variety of solid tumors although with much poorer efficacy than for APL. The toxicity of AsIII and its derivatives is a common concern that has limited its use. The objective of the current study was to develop a polymeric micelle drug delivery system for efficient and controlled delivery of trivalent arsenicals to solid tumor cells. A polymeric micelle-based drug delivery system can potentially extend the duration of drug circulation in blood, restrict access of encapsulated drug to normal tissues, achieve tumor targeted drug delivery, enhance drug accumulation in the tumor area, and trigger drug release at tumor sites if designed properly. These, in turn, can lead to an improved therapeutic index for the polymeric micellar formulation of arsenic species compared to their free form. Towards this goal, a biodegradable block copolymer with pendent thiol groups on the hydrophobic block, i.e., methoxy poly(ethylene oxide)-block-poly[α-(6-mecaptohexyl amino)carboxylate-ε-caprolactone] [PEO-b-P(CCLC6-SH)], was synthesized and used for conjugation of a trivalent arsenical, phenylarsine oxide (PAO), to free thiol groups on the polymer backbone. PAO-loaded micelles had refined size distribution with an average diameter of 150 nm as evidenced by dynamic light scattering (DLS) in water. Prepared polymeric micelles were characterized for the level of PAO conjugation using inductively coupled plasma mass spectrometry (ICP-MS). The results showed 65% of total free thiols were conjugated to PAO providing an arsenic/polymer loading content of ∼2.5 wt%. In vitro release study suggests prolonged release of PAO from its polymeric micellar carrier, which was accelerated in the presence of glutathione (GSH). Cytotoxicity studies against MDA-MB-435 cells show that the IC50 of PEO-b-P(CCLC6-S-PAO) is not significantly different from that of free PAO. The results indicate that PEO-b-P(CCLC6-SH) is a promising carrier for successful arsenic delivery for cancer therapy.

L-Ascorbic acid and α-Tocopherol to protect against arsenic trioxide induced oxidative stress in H9c2 cardiomyocytes

Arsenic has been used as a therapeutic agent against various diseases right from the historical periods. Arsenic trioxide (As2O3), a first line chemotherapeutic against Acute Promyelocytic Leukemia (APL), is well known for its cardiotoxicity. We hypothesized that combination of drug and protective molecules can be used for reducing the side effects of chemotherapeutics. The present study evaluates the protective role of L- ascorbic acid (L-AA) and α-Tocopherol (α-TOC) against oxidative stress in H9c2 cardiomyocytes induced by As2O3. Clinically relevant concentration of arsenic trioxide induces cytotoxicity in cardiac cells. Treatment with antioxidant vitamins significantly reduced the cell death and apoptosis in H9c2 cells. The activity of antioxidant enzymes such as catalase and the concentration of reduced glutathione, which was lowered due to As 2O3 treatment, were found to be increased in cells co treated with L-AA and α-TOC. The treatment with antioxidant vitamins also reduced lactate dehydrogenase release in cells. The overall results clearly indicate the protective potential of L-AA and α-TOC against arsenic induced chemotherapeutic stress on cardiomyocytes. Keywords: Arsenic trioxide, Cardiotoxicity, H9c2 cardiomyocytes, L-Ascorbic acid, Oxidative stress, α- Tocopherol

Enhanced suppression of tumor growth by concomitant treatment of human lung cancer cells with suberoylanilide hydroxamic acid and arsenic trioxide

The efficacy of arsenic trioxide (ATO) against acute promyelocytic leukemia (APL) and relapsed APL has been well documented. ATO may cause DNA damage by generating reactive oxygen intermediates. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, modulates gene and protein expression via histone-dependent or -independent pathways that may result in chromatin decondensation, cell cycle arrest, differentiation, and apoptosis. We investigated whether ATO and SAHA act synergistically to enhance the death of cancer cells. Our current findings showed that combined treatment with ATO and SAHA resulted in enhanced suppression of non-small-cell lung carcinoma in vitro in H1299 cells and in vivo in a xenograft mouse model. Flow cytometric analysis of annexin V+ cells showed that apoptotic cell death was significantly enhanced after combined treatment with ATO and SAHA. At the doses used, ATO did not interfere with cell cycle progression, but SAHA induced p21 expression and led to G1 arrest. A Comet assay demonstrated that ATO, but not SAHA, induced DNA strand breaks in H1299 cells; however, co-treatment with SAHA significantly increased ATO-induced DNA damage. Moreover, SAHA enhanced acetylation of histone H3 and sensitized genomic DNA to DNase I digestion. Our results suggest that SAHA may cause chromatin relaxation and increase cellular susceptibility to ATO-induced DNA damage. Combined administration of SAHA and ATO may be an effective approach to the treatment of lung cancer.

Abstract 2632: SAHA loosens chromatin structure and increases DNA damage in human lung cancer cells induced by arsenic trioxide

Abstract The efficacy of arsenic trioxide (ATO) against acute promyelocytic leukemia (APL) and relapsed APL has been well-documented. ATO may cause DNA damage through the generation of reactive oxygen intermediates. Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), modulates gene or protein expression via histone dependent or independent pathway that may result in chromatin decondensation, cell cycle arrest, differentiation, and apoptosis. The objective of this study is to investigate whether ATO and SAHA could synergistically enhanced cell killing to cancer cells. The present studies showed that combined treatment with ATO and SAHA results in enhanced suppression of non-small cell lung carcinoma (NSCLC) H1299 cells in vitro and in xenograft mouse model. By analyzing the annexin V positive cells, apoptotic cell death is significantly enhanced in combined treatment. At the doses used, ATO does not interfere with the cell cycle progression, whereas SAHA induces the expression of p21WAF1/CIP1 and leads to G1 arrest. By the aid of Comet assay, we demonstrated that ATO induces DNA breaks in H1299 cells, but not SAHA. However, co-treatment with SAHA and ATO significantly increases ATO-induced DNA damage. Moreover, SAHA enhances acetylation of histone H3 and sensitize genomic DNA to DNase I digestion. Our present results suggest that SAHA may result in chromatin relaxation, and lead to cells become more susceptible to ATO for causing DNA damage. Therefore, combination of SAHA with ATO may provide effective approach to treatment of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2632. doi:10.1158/1538-7445.AM2011-2632

Abstract 5360: Differential induction of the anti-oxidant response by arsenic trioxide and darinaparsin in leukemic cells

Abstract Darinaparsin (DAR) is a significantly more potent arsenical than arsenic trioxide (ATO) at mediating apoptosis in various malignant cell lines and is highly active against Acute Promyelocytic Leukemia (APL) cells (NB4). The mechanisms behind DAR's increased efficacy versus ATO remain to be elucidated. We previously reported that DAR triggers apoptosis by inducing cellular responses that do not completely overlap with ATO (Diaz et al. Leukemia). Oxidative damage has been postulated to be a key mechanism by which arsenicals initiate the apoptotic process. Consistent with its increased cytotoxicity, we observed that DAR induces superoxide (O2·−) and hydrogen peroxide (H2O2), as shown respectively by hydorethidine and dichloro-dihydro-fluorescein staining quantified by flow cytometry. In response to oxidative stress, cells induce an arsenal of protective proteins of which HO-1 (heme oxygenase-1), NQO1 (NAD(P)H:quinone oxidoreductase), TrxR1 (thioredoxin-reductase-1) and γGCS (glutamyl cysteine synthetase) are well recognized members. We therefore studied the expression of these stress response proteins after ATO and DAR treatment. As expected, we observed a significantly higher increase in NQO1, TrxR1 and γGCS mRNA and protein levels as assessed by Western Blot and quantitative real PCR, with DAR than with ATO. However, HO-1 expression was not induced by DAR. Nrf2 is a transcription factor that mediates the expression of key protective enzymes through antioxidant response elements (AREs). AREs have been identified in the regulatory regions of genes encoding detoxification enzymes and other cytoprotective proteins, including NQO1, TrxR1, γGCS, and HO-1. Nucleo-cytoplasmic fractionation followed by Western Blot show that both compounds induce Nrf2 translocation to the nucleus. Transcriptional regulation of these detoxifying enzymes involves a competition between the activator Nrf2 and the repressor Bach1 for interaction with their AREs. Accordingly, ChIP assays demonstrate that Bach1 is released and Nrf2 is recruited to the HO-1 ARE in ATO treated cells but not in cells treated with DAR. HO-1's widely reported cytoprotective effects in vitro and in vivo have been related to the generation of the antioxidants biliverdin and bilirubin. In order to elucidate whether the lack of HO-1 expression contributes to the increased efficacy of DAR, we examined the response to ATO and DAR in co-treatment with OB-24, a recently developed HO-1 inhibitor. As indicated by Propidium Iodide staining followed by flow cytometry analysis, pharmacological inhibition of HO-1 increased ATO toxicity but has no significant effect on DAR induced apoptosis. We therefore hypothesize that the increased efficacy of DAR versus ATO might be partially due to the absence of HO-1 up-regulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5360. doi:10.1158/1538-7445.AM2011-5360

Abstract C178: Enhanced suppression of tumor growth by concomitant treatment of human lung cancer cells with SAHA and arsenic trioxide

Abstract The efficacy of arsenic trioxide (ATO) against acute promyelocytic leukemia (APL) and relapsed APL has been well-documented. ATO may cause DNA damage through the generation of reactive oxygen intermediates. Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), modulates gene or protein expression via histone dependent or independent pathway that may result in chromatin decondensation, cell cycle arrest, differentiation, and apoptosis. The objective of this study is to investigate whether ATO and SAHA could synergistically enhanced cell killing to cancer cells. The present studies showed that combined treatment with ATO and SAHA results in synergistic cytotoxicity in non-small cell lung carcinoma H1299 cells. By analyzing the annexin V positive cells, apoptotic cell death is significantly enhanced in combined treatment. By aid of Comet assay, we demonstrated that ATO induces DNA breaks in H1299 cells, but not SAHA. However, co-treatment with SAHA and ATO significantly increases ATO-induced DNA damage. At the doses used, ATO does not interfere with the cell cycle progression, whereas SAHA induces the expression of p21WAF1/CIP1 and leads to G1 arrest. Our present results suggest that SAHA may result in G1 arrest, which becomes more susceptible to ATO for causing DNA damage. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C178.

Arsenic derivatives in hematologic malignancies: a role beyond acute promyelocytic leukemia?

The importance of arsenic trioxide (As2O3) has been underscored over the last decade due to its efficacy against acute promyelocytic leukemia (APL), a disease in which this agent has been associated with complete hematologic and molecular remission rates of 87% and 83%, respectively. The different molecular mechanisms of action of As2O3 suggest its applicability in hematologic malignancies other than APL. However, responses obtained thus far have consisted of improvements in signs and symptoms without the elimination of a given disease. Toxicities derived from As2O3 are significant but manageable and reversible. However, the risk/benefit ratio of As2O3 in hematologic malignancies other than APL is still unclear. The development of new generations of orally bioavailable inorganic, as well as new organic, arsenic compounds with improved toxicity profiles may bolster the therapeutic application of arsenic derivatives in hematologic malignancies such as leukemia, multiple myeloma and myelodysplastic syndromes.

In Vitro Activity of a Novel Organic Arsenical (S-Dimethylarsino-Glutathione, ZIO-101) Against Multiple Myeloma.

Arsenic Trioxide (ATO) has been shown to be highly active against acute promyelocytic leukemia (APL) and has activity in several other diseases including multiple myeloma. While initial clinical trials in both APL and myeloma have suggested that melarsoprol results in greater dose-limiting toxicities than ATO, it is generally accepted that organic arsenicals are less toxic than inorganic arsenicals. Consequently, organic arsenicals that kill myeloma cells could be clinically more effective than ATO. Recently, several organic arsenicals were synthesized that have EC50s similar to ATO against cell line panels but are > 10-fold less toxic. One such compound, ZIO-101 is in phase I studies. Therefore we compared the ability of ZIO-101 and ATO to kill four myeloma cell lines that display differential sensitivity to ATO. The RPMI 8226 and U266 are less sensitive than the KMS11 and MM.1s lines. When dose response curves were generated comparing ATO and ZIO-101 at 24, 48 and 72 hrs we found that the U266, KMS11 and MM.1s lines were consistently 1–3 fold less sensitive to ZIO-101 than to ATO. However if one considers the number of atoms of elemental arsenic/molecule of drug, these data would suggest that the ability of these drugs to kill MM cell lines is similar. In contrast the 8226 line was more sensitive to ZIO-101. Additionally we have previously reported that ATO induces caspase-dependent and -independent cell death in a cell specific fashion in these lines. We found a similar pattern of caspase dependence with ZIO-101 where BocD-FMK, a caspase inhibitor, completely blocks ZIO-101-induced killing of U266, partially blocks killing of MM.1s and KMS11 and has no effect no killing of 8226. These data suggest that the downstream components of the death signaling pathway induced by ZIO-101 and ATO are similar. In contrast, initial responses to these drugs differ. We and others have reported that glutathione (GSH) is a critical regulator of ATO-induced cell death and have utilized ascorbic acid (AA) as a GSH depleting agent both in vitro as well as clinically. We therefore tested the effects of GSH depletion on ZIO-101 induced cell death in MM cell lines. Using concentrations of ATO and ZIO-101 that had similar activity, we determined the effects of both an inhibitor of GSH synthesis (BSO) as well as AA that can transient deplete GSH. BSO sensitized all 4 cell lines to both agents, however it was much more effective at sensitizing cells to ATO than to ZIO-101. Moreover while AA could sensitize cells to ATO, it actually protected cells from cell death induced by ZIO-101. Taken together these data suggest that ZIO-101 has activity against myeloma cells although factors that determine the potency of this compound are different than those for ATO. This may reflect differences in either metabolism or mechanism of action. Thus resistance to one form of arsenic does not preclude the use of another. A phase I/II study of ZIO-101 in myeloma is planned.

Membrane-associated cytotoxicity induced by realgar in promyelocytic leukemia HL-60 cells

Realgar has been shown to have a therapeutic effect against acute promyelocytic leukemia (APL) by inducing apoptosis. However, there is little data about the effects of it on plasma membrane. In the present study, the cytotoxicity of realgar to HL-60 cells including its inhibiting cell growth, inducing apoptosis and bringing about membrane toxicity was investigated. It was suggested that realgar could significantly suppress the proliferation of HL-60 cells in a dose-dependent manner by 3-(4,5-dimethylthiazol-2-diphenyl-tetrazolium bromide (MTT) assay and the IC 50 value was 5.67 μM. Flow cytometric analysis revealed that treatment with realgar resulted in increased percentages of apoptotic cells in a dose-dependent manner. On the other hand, membrane lipid peroxidation level, lactate dehydrogenase (LDH) leakage and membrane surface topography alterations were investigated to assess the membrane toxicity induced by realgar. Treatment with realgar at different concentrations accelerated membrane lipid peroxidation, potentiated LDH leakage, which was consistent with enhanced disorganization of membrane surface observed by atomic force microscopy (AFM). These results suggested that such membrane toxicity induced by realgar might play an important role in the process of apoptotic induction and could be considered as one of mechanisms underlying the cytotoxicity of realgar.

Arsenic Trioxide Therapy for Relapsed Acute Promyelocytic Leukemia: an Useful Salvage Therapy

Arsenic trioxide (As203) was recently identified as a very potent agent against acute promyelocytic leukemia (APL). Intravenous infusion of 10 mg As203 daily for one to two months can induce significant complete remission (CR) of APL, and there is no cross drug-resistance between As203 and other antileukemic agents, including all-trans retinoic acid (ATRA). The CR rate of relapsed and/or refractory APL patients who received As203 treatment ranged from 52.3% to 93.3%. The median duration to CR ranged from 38 to 51 days, with accumulative As203 dosage of 340-430 mg. Although most adverse reactions of As203 treatment were tolerable, certain infrequent but severe toxicities related to As203 were observed, including renal failure, hepatic damage, cardiac arrhythmia and chronic neuromuscular degeneration, which should be monitored carefully. As203 can induce partial differentiation and subsequent apoptosis of APL cells through degradation of wild type PML and PML/RAR a chimeric proteins and possible anti-mitochondrial effects. Like the treatment of ATRA in APL, early relapses from As203 treatment within a few months were not infrequently seen, indicating that rapid emerging resistance to As203 can occur. Nevertheless, the PML/RAR a fusion protein was reported to disappear in some APL patients who received As203, and who might earn long-survival. However, the follow-up is still too short to draw the conclusion. Intriguingly, it has been shown that As203 can also induce apoptosis of other non-APL tumor cells with clinical achievable concentrations. However, the detailed molecular mechanisms are not yet fully understood. Further studies regarding to the pharmacological characters, clinical efficacies, toxicities, apoptogenic mechanisms, and spectrum of anti-tumor activity of As203 are warranted.

Effective salvage therapy using all-trans retinoic acid for relapsed and resistant acute promyelocytic leukemia

All-trans retinoic acid (ATRA) has been shown to be active against acute promyelocytic leukemia (APL). Six patients with APL, either in relapse or resistant to initial chemotherapy were reinduced with ATRA 100 g/m2/day for 6 weeks. Complete remission was achieved in all six of them. Side effects were seen in two of them. ATRA appears to provide a relatively safe and reliable means to induce a complete remission in patients with refractory or relapsed APL.