Cytotoxicity Of As2O3 Research Articles

L-Ascorbic Acid and α-Tocopherol Synergistically Triggers Apoptosis Inducing Antileukemic Effects of Arsenic Trioxide via Oxidative Stress in Human Acute Promyelocytic Leukemia Cells.

Chemosensitization is an effective strategy to overcome the drawbacks of arsenic trioxide (As2O3) treatment, which may be possible through the use of dietary supplements in combination. The present investigation evaluates the synergistic mechanism of action of vitamins, such as L-ascorbic acid (L-AA) and α-tocopherol (α-TOC) in As2O3 chemotherapy using human leukemia (HL-60) cells. In vitro assays on the cytotoxicity of As2O3 and vitamins and cellular apoptotic evidences were done; a proteomic investigation with mass spectrometry was also performed. The combination of L-AA and α-TOC potentiates As2O3 cytotoxicity in HL-60 cells, substantiated by depletion in antioxidant status, mitochondrial transmembrane potential, and inhibition of nuclear factor erythroid 2-related factor 2 and B-cell lymphoma 2 transcription factors. Mass spectrometry results showed decreased expression of proteins regulating cell cycle and translation in cells treated with As2O3, L-AA, and α-TOC when compared with As2O3-treated sample. In addition, this combination treatment identified numerous proteins associated with apoptosis and cell stress. HL-60 cells became more prone to As2O3 on exposure to L-AA and α-TOC, indicating that this combination may be a promising approach to increase the outcome of As2O3 chemotherapy.

Arsenic trioxide and tetraarsenic oxide induce cytotoxicity and have a synergistic effect with cisplatin in paclitaxel-resistant ovarian cancer cells

Background: Arsenic compounds (As2O3 and As4O6) have demonstrated anticancer effects in various malignancies. In this study, the cytotoxicity of arsenic compounds on ovarian cancer cell lines and the anticancer activity of the combination of arsenic compounds and cisplatin IN chemoresistant ovarian cancer cells were investigated.Methods: We investigated the cytotoxicity of As2O3 and As4O6 and their combinations with cisplatin in the paclitaxel-sensitive ovarian cancer cell lines SKOV3ip1 and HeyA8 and paclitaxel-resistant ovarian cancer cell lines SKOV3TRip2 and HeyA8-MDR. Growth and apoptosis were evaluated by MTT assay and annexin V assay using flow cytometry, respectively. For detection of apoptotic cells, immunofluorescence was performed using a cleaved caspase-3 antibody. Cell-cycle distribution was determined by propidium iodide staining and flow cytometry.Results: Treatment of each cell line with As2O3 or As4O6 led to a marked dose-dependent inhibition of cell growth. As2O3 and As4O6 treatment induced caspase-3-dependent apoptosis in all cell lines compared to the respective control groups (p < .05). As2O3 and As4O6 induced apoptosis of paclitaxel-sensitive and -resistant cancer cell lines following G2/M cell cycle arrest (p < .05). A synergistic effect was achieved by combining cisplatin with As2O3 or As4O6 in the paclitaxel-resistant ovarian cancer cell lines.Conclusions: As2O3 and As4O6 can inhibit cell growth and induce apoptosis in paclitaxel-sensitive and -resistant ovarian cancer cell lines. Their combination with cisplatin resulted in a synergistic effect in paclitaxel-resistant cancer cell lines. These results suggest that arsenic compounds may be given in monotherapy or combination therapy with cisplatin for treating paclitaxel-resistant ovarian cancer.

Novel Combination of Arsenic Trioxide (As2O3) Plus Resveratrol in Inducing Programmed Cell Death of Human Neuroblastoma SK-N-SH Cells.

Arsenic trioxide (As2O3), known as pi-shuang and the most toxic compound in traditional Chinese medicine, has been used as an antitumor agent for thousands of years. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural phenol that has significant anti-bacterial, anti-fungaI and antiaging activities. Our study aimed to examine the combined anticancer effects of As2O3 and resveratrol against human neuroblastoma SK-N-SH cells, and elucidate the underlying intracellular signaling. SK-N-SH cells were treated with an extremely low-dose (2-4 μM) of As2O3 alone or combined with 75 μg/ml resveratrol for further comparisons. Cell viability, apoptotic signaling as well as synergistic cytotoxic effects were estimated using the MTT assay, microscopy observation, flow cytometric analysis for loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), and typical quantitative western blotting analysis. Student's t-test, and one- and two-way analysis of variance (ANOVA) were used for examination of significant differences. The combined treatment was more effective than single treatment of As2O3 or resveratrol alone in suppressing cell viability, which correlated with the elevation of ROS levels. The intracellular mechanisms of cytotoxicity of As2O3 plus resveratrol were revealed as ROS accumulation and relative decrease of MMP, leading to activation of caspase-3 and -9, but not of caspase-1, -7 and-8. Combination treatment reduced the expression of B-cell lymphoma 2 (BCL2), BH3 interacting domain death agonist (BID), and BCL-x/L. Combined treatment at extremely low concentration of two agents from natural products, As2O3 and resveratrol, has high potential as a cocktail of anticancer drugs for neuroblastoma.

Sensitivity of leukemic T-cell lines to arsenic trioxide cytotoxicity is dependent on the induction of phosphatase B220/CD45R expression at the cell surface.

BackgroundArsenic trioxide (As2O3) is highly effective in treating acute promyelocytic leukemia (APL), but shows more variable therapeutic efficacy for other types of hematological malignancies. Previously, we reported that As2O3 selectively eliminates pathogenic B220-expressing T cells in autoimmune MRL/lpr mice. We investigated herein the relationship between As2O3 sensitivity of leukemic T-cell lines and the expression levels of the B220 isoform of transmembrane tyrosine phosphatase CD45.MethodsGSH content, O2- production, and B220, HSP70, Fas and FasL membrane expression was measured by flow cytometry. Subcellular localization of B220 was determined by imaging flow cytometry. Cell death was analyzed by morphological changes, annexin V and propidium iodide staining, and caspase 8 and 9 activation. B220 mRNA expression was analyzed by RT-PCR. Activated NF-κB p50 was quantified by a DNA binding ELISA.ResultsWe selected human (Jurkat, Jurkat variant J45.01, HPB-ALL) and mouse (EL-4, BW5147, L1210) T-cell lines for their marked differences in As2O3 sensitivity over a large range of doses (1 to 20 μM). Differences in redox status cannot explain the dramatic differences in As2O3 sensitivity observed among the T-cell lines. Unexpectedly, we found that B220 is differentially induced on As2O3-treated T-cell lines. As2O3 treatment for 24 h induced low (HPB-ALL), intermediate (Jurkat) and high (EL-4, BW5147) levels of B220 membrane expression, membrane-bound HSP70 and cell death, but inhibited NF-κB p50 nuclear translocation. When high levels of B220 expression were achieved with low doses of As2O3, the T-cell lines died by apoptosis only. When high doses of As2O3 were required to induce B220 expression, leukemic T cells died by both apoptosis and necrosis.ConclusionsCellular redox status is not essential for As2O3 sensitivity of leukemic T cells, suggesting the existence of additional factors determining their sensitivity to As2O3 cytotoxicity. Phosphatase B220 could be such a factor of sensitivity. As2O3 treatment inhibits NF-κB p50 nuclear translocation, and induces B220 expression and cell death in a dose and time dependent manner. The levels of B220 induction on leukemic T cells strictly correlate with both the extent and form of cell death, B220 might therefore play a checkpoint role in death pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-251) contains supplementary material, which is available to authorized users.

Regulatory role of KEAP1 and NRF2 in PPARγ expression and chemoresistance in human non-small-cell lung carcinoma cells

The nuclear factor-E2-related factor 2 (NRF2) serves as a master regulator in cellular defense against oxidative stress and chemical detoxification. However, persistent activation of NRF2 resulting from mutations in NRF2 and/or downregulation of or mutations in its suppressor, Kelch-like ECH-associated protein 1 (KEAP1), is associated with tumorigenicity and chemoresistance of non-small-cell lung carcinomas (NSCLCs). Thus, inhibiting the NRF2-mediated adaptive antioxidant response is widely considered a promising strategy to prevent tumor growth and reverse chemoresistance in NSCLCs. Unexpectedly, stable knockdown of KEAP1 by lentiviral shRNA sensitized three independent NSCLC cell lines (A549, HTB-178, and HTB-182) to multiple chemotherapeutic agents, including arsenic trioxide (As2O3), etoposide, and doxorubicin, despite moderately increased NRF2 levels. In lung adenocarcinoma epithelial A549 cells, silencing of KEAP1 augmented the expression of peroxisome proliferator-activated receptor γ (PPARγ) and genes associated with cell differentiation, including E-cadherin and gelsolin. In addition, KEAP1-knockdown A549 cells displayed attenuated expression of the proto-oncogene cyclin D1 and markers for cancer stem cells (CSCs) and reduced nonadherent sphere formation. Moreover, deficiency of KEAP1 led to elevated induction of PPARγ in response to As2O3. Pretreatment of A549 cells with PPARγ agonists activated PPARγ and augmented the cytotoxicity of As2O3. A mathematical model was formulated to advance a hypothesis that differential regulation of PPARγ and detoxification enzymes by KEAP1 and NRF2 may underpin the observed landscape changes in chemosensitivity. Collectively, suppression of KEAP1 expression in human NSCLC cells resulted in sensitization to chemotherapeutic agents, which may be attributed to activation of PPARγ and subsequent alterations in cell differentiation and CSC abundance.

Combination of arsenic trioxide and BCNU synergistically triggers redox-mediated autophagic cell death in human solid tumors

Arsenic trioxide (As2O3) is an effective treatment for relapsed or refractory acute promyelocytic leukemia (APL). After the discovery of As2O3 as a promising treatment for APL, several studies investigated the use of As2O3 as a single agent in the treatment of solid tumors; however, its therapeutic efficacy is limited. Thus, the systematic study of the combination of As2O3 with other clinically used chemotherapeutic drugs to improve its therapeutic efficacy in treating human solid tumors is merited. In this study, we demonstrate for the first time, using isobologram analysis, that As2O3 exhibits a synergistic interaction with N,N′-bis(2-chloroethyl)-N-nitrosourea (BCNU). The synergistic augmentation of the cytotoxicity of As2O3 with BCNU is in part through the autophagic cell death machinery in human solid tumor cells. As2O3 and BCNU in combination produce enhanced cytotoxicity via the depletion of reduced glutathione (GSH) and augmentation of reaction oxygen species (ROS) production. Further analysis indicated that the extension of GSH depletion by this combined regimen occurs through the inhibition of the catalytic activity of glutathione reductase. Blocking ROS production with antioxidants or ROS scavengers effectively inhibits cell death and autophagy formation, indicating that redox-mediated autophagic cell death involves the synergism of As2O3 with BCNU. Taken together, this is the first evidence that BCNU could help to extend the therapeutic spectrum of As2O3. These findings will be useful in designing future clinical trials of combination chemotherapy with As2O3 and BCNU, with the potential for broad use against a variety of solid tumors.

Differential augmentative effects of buthionine sulfoximine and ascorbic acid in As2O3-induced ovarian cancer cell death: Oxidative stress-independent and -dependent cytotoxic potentiation

The potential of arsenic trioxide (As2O3) as a novel therapy against ovarian cancer has been progressively recognized. Its prospective usefulness for treatment of this malignancy either alone or in combination with other chemotherapeutic agents has been increasingly explored. In this study, we attempted to enhance the cytotoxicity of As2O3 in ovarian cancer cells through manipulation of cellular glutathione (GSH) levels using either buthionine sulfoximine (BSO) or ascorbic acid (AA). Results from our studies showed that combinatorial therapies using As2O3 with either low dose BSO or only pharmacological doses of AA acted synergistically to enhance the cytotoxicity of As2O3 in ovarian tumor cells. With these regimens, therapeutic selectivity was observed with preferential killing of ovarian tumor cells over normal fibroblast controls. Furthermore, contrary to previous reports, enhancement of As2O3-mediated cell killing by these two agents was propagated through different effects. With BSO, apoptotic and non-apoptotic cell death enhancement were mediated through increased arsenic accumulation and GSH depletion that occurred independently of reactive oxygen species. With pharmacological doses of AA, increase in cell death proceeded through non-apoptotic routes via an oxidative stress-related pathway independent of GSH levels. Taken together, these results indicate that GSH depleting agents or pro-oxidative chemicals have capabilities of improving the utility of As2O3 in ovarian cancer management.

Arsenic Trioxide overcomes cell adhesion-mediated drug resistance through down-regulating the expression of β1-integrin in K562 chronic myelogenous leukemia cell line

The bone marrow microenvironment protects leukemia cells from drug-induced damage, and cell adhesion-mediated drug resistance (CAM-DR) is the main obstacle for the eradication of bone marrow minimal residual disease. In this study, we evaluated the survival response of K562 cells co-cultured with bone marrow stromal cells (BMSCs) derived from patients with chronic myelogenous leukemia (CML). Co-cultured K562 cells had a survival advantage after exposure to cytotoxic drugs compared to suspended K562 cells, but there was no survival difference between the two groups following exposure to As2O3. The cytotoxicity of As2O3 combined with cytotoxic drugs had an additive effect in the suspension group and a synergistic effect in the co-cultured group. Similar apoptotic responses were found using flow cytometry assays. As2O3 treatment caused a dose dependent reduction of the adhesion ability of K562 cells to BMSCs, and Western blot analysis revealed that the treatment of K562 cells with As2O3 led to down-regulation of β1-integrin.

Ascorbic Acid Potentiation of Arsenic Trioxide Anticancer Activity Against Acute Promyelocytic Leukemia.

IntroductionAcute promyelocytic leukemia (APL) is a malignant disorder of the white blood cells. Arsenic trioxide (As2O3) has been used as a therapeutic agent to treat APL and other tumors. Studies suggest that ascorbic acid (AA) supplementation may improve the clinical outcome of As2O3 for APL patients. Our aim was to use human leukemia (HL-60) APL-cells as an in vitro test model to evaluate the effect of physiologic doses of AA on As2O3-induced toxicity and apoptosis of HL-60 cells.MethodsHL-60 cells were treated either with a pharmacologic dose of As2O3 alone and with several physiologic doses of AA. Cell survival was determined by trypan blue exclusion test. The extent of oxidative cell/tissue damage was determined by measuring lipid hydroperoxide concentration by spectrophotometry. Cell apoptosis was measured by flow cytometry using Annexin-V and propidium iodide (PI) staining.ResultsAA treatment potentiates the cytotoxicity of As2O3 in HL-60 cells. Viability decreased from (58 ± 3)% in cells with As2O3 alone to (47 ± 2)% in cells treated with 100 µM AA and 6 µg/mL As2O3 with P < 0.05. There was a significant (P < 0.05) increase in lipid hydroperoxide concentrations in HL-60 cells co-treated with AA compared to As2O3 alone. Flow cytometry assessment (Annexin V FITC/PI) suggested that AA co-treatment induces more apoptosis of HL-60 cells than did As2O3 alone, but this was not statistically significant. Taken together, our experiment indicates that As2O3 induced in vitro cell death and apoptosis of HL-60 cells. Administration of physiologic doses of AA enhanced As2O3-induced cytotoxicity, oxidative cell/tissue damage, and apoptosis of HL-60 cells through externalization of phosphatidylserine.ConclusionsThese suggest that AA may enhance the cytotoxicity of As2O3, suggesting a possible future role of AA/As2O3 combination therapy in patients with APL.

Catalase activity and arsenic sensitivity in acute leukemia

Arsenic trioxide (As2O3) is currently employed as a treatment for relapsed acute promyelocytic leukemia (APL), where it can induce remission in greater than 90% of patients, but is ineffective in patients with non-APL acute myeloid leukemia (AML). As2O3 induces apoptosis in APL cells through mechanisms dependent and independent of the PML-RARα fusion protein. Through PML-RARα fusion-independent mechanisms, As2O3 increases H2O2 production via its effects on glutathione and glutathione peroxidase. Catalase is an alternative mechanism to convert H2O2 to water. Therefore, we explored the relationship between catalase activity and As2O3 sensitivity. In AML and APL cell lines, but not primary patient samples, basal catalase levels matched sensitivity to As2O3. However, the chemical inhibition of catalase did not enhance As2O3-induced cell death. Failure of catalase inhibition to sensitise cells to As2O3 was due to a failure of catalase inhibition to increased levels of reactive oxygen species. Therefore, other strategies should be explored to enhance the cytotoxicity of As2O3 in AML.

Arsenic Trioxide (As2O3) Mediated Cell Death in Hodgkin Lymphoma (HL) and Non-Hodgkin Lymphoma (NHL): Investigation of Reactive Oxygen Species (ROS), Caspase, and MAP Kinase Signaling Pathways.

The cytotoxic activity of As2O3 in leukemia and myeloma has been shown to be mediated in part by ROS. We determined the mechanism of cytotoxicity of As2O3 in HL and NHL cell lines. Ramos, HF-1, SUDHL4 (NHL lines), and L428 (HL line) cells were cultured with increasing clinically relevant As2O3 concentrations (1.0mm-10mm) at 24-72 hours with and without the oxidizing agent, buthionine sulfoxime (BSO, 100mm), and with and without caspase inhibitors Z-VAD-FMK and Z-IETD-FMK. Apoptosis was measured by Annexin-V/propidium iodide (PI) staining and detected by flow cytometry (FACS). ROS was measured by oxidation of 2′7′ dichlorofluorescein diacetate (H2DCFDA) to Dichlorofluorescein (DCF) and analyzed by FACS. Immunoblots were performed for bcl-2, PARP, cleaved caspases 3, 8, 9, and mitogen activated protein kinase (MAPK) pathways (ERK, JNK, and p38 activation). As2O3 alone at 10mM resulted in dose- and time-dependent apoptosis in all cell lines (HL and NHL) with >75% annexin+/PI+ between 48 and 72 hours. In Ramos and L428 cells, a combination of As2O3 (2 mM) and BSO 100mM resulted in >80% annexinV+/PI+, while either agent alone resulted in <15% apoptosis (p=0.001). A four-fold increase in ROS was seen in all four cell lines with As2O3/BSO, but not with As2O3 alone. Both ROS production and apoptosis induced by As2O3/BSO and As2O3 alone were reversible with the anti-oxidant N-acetylcysteine (NAC) (10mM). Furthermore, As2O3/BSO induced PARP cleavage and caspase-3 activation in all NHL cell lines, but not in L428. In Ramos, Z-VAD-FMK and Z-IETD-FMK blocked As2O3-induced apoptosis (suggesting a caspase-dependent mechanism), but had no effect on As2O3/BSO-induced cell death (caspase-independent). To investigate the signaling pathways involved, we performed western blot analysis for phospho-p38 (p-p38), phospho-JNK and phospho-ERK (p-ERK) in Ramos and L428 cells following As2O3 alone and As2O3/BSO. We found up-regulation of p-p38 in Ramos cells, while in L428, p-ERK was activated following As2O3 and As2O3/BSO. Inhibitors of p-38 in Ramos cells resulted in up-regulation of ERK. In summary, As2O3 induced dose- and time-dependent apoptosis in HL and NHL cell lines was significantly enhanced with the addition of BSO, and inhibited by NAC, suggesting that apoptosis was in part ROS-dependent. In Ramos, As2O3 alone resulted in caspase-dependent apoptosis, while addition of BSO resulted in caspase-independent apoptosis. As2O3 alone and combined with BSO resulted in activation of MAPK pathways in HL and NHL lines. These data provide a basis and a mechanism for As2O3-induced cell death in HL and NHL and have therapeutic implications.

Effect of Arsenic Trioxide on E14 Murine Embryonic Stem Cells In Vitro.

The ability of pluripotent murine embryonic stem (muES) cells to differentiate into all cell types makes them a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the growth and differentiation of the ES cells is a pre-requisite for selecting adequately the cell conditions which will be accessible for future use of ES cells in treating hematological malignancies. Pharmacological agents such as arsenic trioxide (As2O3) have shown to be an effective anticancer agent for acute promyelocytic leukemia but its effect on murine embryonic stem cells prior to differentiation and subsequent target organ toxicity is yet to be determined. This study was done to gain insight into the biological effects of As2O3 on muES cell line, E14, with respect to differentiation, proliferation, cytotoxicity and cell cycle status in vitro. MuEs cells were cultured in complete DMEM in the presence of LIF on gelatin coated plates for 24 hours prior to the addition of varying concentrations of As2O3 (0, 0.5,1, 5, and 10 μmol/L, respectively), and the cells were incubated for an additional 48 hours. The differentiation potential of As2O3 was determined by measuring Oct-4 levels by flow cytometry. Proliferation was measured by trypan blue exclusion. Cytotoxicity of As2O3 was determined by MTT assay. Cell cycle status was measured by Propidium Iodide using flow cytometry. Our results demonstrated that As2O3 at different concentrations did not induce differentiation. However, the proliferation of muES cells treated with As2O3 was inhibited in a concentration and time dependent manner. The cell-killing rate of As2O3 on muES cells was both dose and time dependent with an inhibitory concentration (IC50) of ~ 5±0.2 μM and ~3.75±0.7 μM at 24 and 48 hours respectively. Propidium iodide DNA staining revealed that after 48 hour incubation with arsenic, cells in S phase remained relatively constant (~62–67%) at low doses of As2O3 but this decreased by half (33%) at a high dose of 10uM As2O3. The percent of cells in the G2 phase of the cell cycle increased to 41±9% in 10μM treated cells compared to 6±3% in control. In conclusion, As2O3 does not induce the differentiation of muES cells into different lineages, but does inhibit growth and alter the cell cycle kinetics of these cells in a dose dependent manner.

Gene expression alteration during redox-dependent enhancement of arsenic cytotoxicity by emodin in HeLa cells

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) could enhance the sensitivity of tumor cells to arsenic trioxide (As2O3)-induced apoptosis via generation of ROS, but the molecular mechanism has not been elucidated. Here, we carried out cDNA microarray-based global transcription profiling of HeLa cells in response to As2O3/emodin cotreatment, comparing with As2O3-only treatment. The results showed that the expression of a number of genes was substantially altered at two time points. These genes are involved in different aspects of cell function. In addition to redox regulation and apoptosis, ROS affect genes encoding proteins associated with cell signaling, organelle functions, cell cycle, cytoskeleton, etc. These data suggest that based on the cytotoxicity of As2O3, emodin mobilize every genomic resource through which the As2O3-induced apoptosis is facilitated.

Vitamin C protects HL60 and U266 cells from arsenic toxicity

AbstractAlthough there is no compelling evidence that vitamin C has antitumor activity in humans, clinical trials are testing the hypothesis that ascorbic acid (AA) will enhance the efficacy of arsenic trioxide (As2O3) in myeloma. In vitro, AA cytotoxicity depends on its interaction with free transition metal ions in culture media leading to the generation of H2O2 and other reactive oxygen species (ROSs). Therefore, to circumvent the extracellular in vitro pro-oxidant effects of AA, we loaded HL60, U266, and RPMI-8226 cells with vitamin C by incubation with dehydroascorbic acid (DHA). Loading cells in this manner resulted in prominent, dose-dependent protection of As2O3-treated cells as measured by viability, colony formation, and apoptosis assays. Glutathione depletion enhanced cell sensitivity to the cytotoxic effects of As2O3 and vitamin C loading provided protection. AA was found to generate cytotoxic concentrations of H2O2 in culture medium without cells and copper/iron chelators inhibited this reaction. However, AA did not generate H2O2 in simple buffer or human plasma. Direct incubation with AA resulted in increased intracellular ROSs, whereas DHA incubation decreased it. These results clarify an apparent paradox and indicate that vitamin C loading in HL60, U266, and RPMI-8226 cells ameliorates As2O3 cytotoxicity.

Cell cycle arrest and apoptotic cell death in cultured human gastric carcinoma cells mediated by arsenic trioxide

To investigate the effect of arsenic trioxide on human gastric cancer cell line MKN45 with respect to both cytotoxicity and induction of apoptosis in vitro. MKN45 cells were treated with arsenic trioxide (As2O3) at the concentration of 1, 5, and 10 micromol/L, respectively, for three successive days. Cell growth and proliferation were observed by cell counting and trypan blue exclusion. Cytotoxicity of As2O3 was determined by MTT assay. Morphologic changes were studied with light microscopy. Flow cytometry was used to assay cell DNA distribution and apoptotic cells were confirmed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and DNA electrophoresis. The growth of MKN45 cells was significantly inhibited by As2O3 which was confirmed by colony-forming assay. After 7 d of culture with various concentrations of As2O3, colony-forming capacity of MKN45 cells decreased with As2O3 increment in comparison with that of control group. The inhibitory rate of colony-formation was 38.5%, 99.1%, and 99.5% when the concentration of As2O3 was 1, 5, and 10 micromol/L in culture medium, respectively. The cell number of a single colony in drug treatment groups was less than that of control group. The cell-killing rate of As2O3 to MKN45 cells was both dose- and time-dependent with an IC50 of (11.05+/-0.25) micromol/L. After incubation in 10 micromol/L As2O3 for 24 h, the cell-killing rate was 27.1%, and it was close to 50% after 48 h. The results showed that As2O3 induced time- and dose-dependent apoptosis in MKN45 cells, blocked at G2/M phase. The apoptotic peak (sub-G1 phase) appeared and cell apoptotic rate in MKN45 cells was 18.3-32.5% after treatment by 10 micromol/L As2O3 for 48 h. The percentage of G2/M cell of the experimental groups was 2.0-5.0 times than that of the control group. Gel electrophoresis of DNA from cells treated with each concentration of As2O3 for 48 h revealed a "ladder" pattern, indicating preferential DNA degradation at the internucleosomal, linker DNA sections. TUNEL also demonstrated strand breaks in DNA of MKN45 cells treated with As2O3, while control cells showed negative labeling. As2O3 can induce apoptosis of human gastric carcinoma cells MKN45, which is the basis of its effectiveness. It shows great potential in the treatment of gastric carcinoma.

Enhancement of arsenic trioxide-induced apoptosis in renal cell carcinoma cells by L-buthionine sulfoximine.

Arsenic trioxide (As2O3) induces clinical remission in acute promyelocytic leukemic patients and apoptosis in various tumor cells in vitro. To develop As2O3-based combination chemotherapy for renal cell carcinoma (RCC), we investigated the cytotoxic effects of As2O3 in combination with chemotherapeutic agents or L-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor. Cytotoxicity and synergy were assessed by the MTT assay and isobolographic analysis, respectively. Apoptosis was monitored by Hoechst 33342 staining, flow cytometrical analysis, and DNA fragmentation assay. Treatment of ACHN cells with As2O3 in combination with adriamycin, vinblastine, or 5-fluorouracil induced an antagonistic effect. However, combination treatment with As2O3 and BSO resulted in a synergistic cytotoxic effect. Synergy was also obtained in Caki-1, Caki-2, NC65 cells and freshly derived RCC cells from 6 patients. Simultaneous treatment of ACHN cells with As2O3 and BSO caused significantly more cytotoxicity than the As2O3 first BSO second or the reverse treatment. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of As2O3 and BSO was realized by inducing apoptosis. This combination markedly decreased intracellular GSH content and GSH-S-transferase (GST) activity. However, neither the intracellular GSH nor GST was decreased by As2O3 with adriamycin, vinblastine, or 5-fluorouracil. Furthermore, the GSH-increasing agents N-acetylcysteine and lipoic acid significantly inhibited the combined cytotoxicity of As2O3 and BSO. These findings indicate that BSO sensitizes RCC cells to As2O3-induced apoptosis through the down-regulation of the intracellular GSH redox system, suggesting the potential application of a combination of As2O3 and BSO for the treatment of RCC.

Ascorbic acid enhances arsenic trioxide–induced cytotoxicity in multiple myeloma cells

AbstractMultiple myeloma (MM) is a clonal B-cell malignancy characterized by slow-growing plasma cells in the bone marrow (BM). Patients with MM typically respond to initial chemotherapies; however, essentially all progress to a chemoresistant state. Factors that contribute to the chemorefractory phenotype include modulation of free radical scavenging, increased expression of drug efflux pumps, and changes in gene expression that allow escape from apoptotic signaling. Recent data indicate that arsenic trioxide (As2O3) induces remission of refractory acute promyelocytic leukemia and apoptosis of cell lines overexpressing Bcl-2 family members; therefore, it was hypothesized that chemorefractory MM cells would be sensitive to As2O3. As2O3 induced apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and U266/Bcl-xL. The addition of interleukin-6 had no effect on cell death. Glutathione (GSH) has been implicated as an inhibitor of As2O3-induced cell death either through conjugating As2O3 or by sequestering reactive oxygen induced by As2O3. Consistent with this possibility, increasing GSH levels with N-acetylcysteine attenuated As2O3 cytotoxicity. Decreases in GSH have been associated with ascorbic acid (AA) metabolism. Clinically relevant doses of AA decreased GSH levels and potentiated As2O3-mediated cell death of all 4 MM cell lines. Similar results were obtained in freshly isolated human MM cells. In contrast, normal BM cells displayed little sensitivity to As2O3 alone or in combination with AA. Together, these data suggest that As2O3 and AA may be effective antineoplastic agents in refractory MM and that AA might be a useful adjuvant in GSH-sensitive therapies.