Methylated Metabolites of Arsenic Trioxide Are ... - Semantic Scholar

[CANCER RESEARCH 63, 1853–1859, April 15, 2003]

Methylated Metabolites of Arsenic Trioxide Are More Potent Than Arsenic Trioxide as Apoptotic but not Differentiation Inducers in Leukemia and Lymphoma Cells1 Guo-Qiang Chen,2 Li Zhou,2 Miroslav Styblo, Felecia Walton, Yongkui Jing, Rona Weinberg, Zhu Chen, and Samuel Waxman3 Department of Pathophysiology [G-Q. C.] and Shanghai Institute of Hematology [Z. C.], Shanghai Second Medical University, Shanghai, China; Division of Hematology/ Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029 [L. Z., Y. J., R. W., S. W.]; and Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina [M. S., F. W.]

ABSTRACT Treatment with arsenic trioxide (As2O3) by inducing apoptosis and partial differentiation of acute promyelocytic leukemia (APL) cells results in clinical remission in APL patients resistant to chemotherapy and all-trans-retinoic acid. As2O3 (iAsIII) is methylated in the liver to monoand dimethylated metabolites, including methylarsonic acid, methylarsonous acid, dimethylarsinic acid, and dimethylarsinous acid. Methylated trivalent metabolites that are potent cytotoxins, genotoxins, and enzyme inhibitors may contribute to the in vivo therapeutic effect of iAsIII. Therefore, we compared the potency of iAsIII and trivalent metabolites using chemical precursors of methylarsonous acid and dimethylarsinous acid to induce differentiation, growth inhibition, and apoptosis. Methylarsine oxide (MAsIIIO) and to a lesser extent iododimethylarsine were more potent growth inhibitors and apoptotic inducers than iAsIII in NB4 cells, an APL cell line. This was also observed in K562 human leukemia, lymphoma cell lines, and in primary culture of chronic lymphocytic leukemia cells, but not human bone marrow progenitor cells. Apoptosis was associated with greater hydrogen peroxide accumulation and inhibition of glutathione peroxidase activity. MAsIIIO, in contrast to iAsIII, did not induce PML-retinoic acid receptor ␣ degradation, or restore PML nuclear bodies or differentiation in NB4 cells. In a cocultivation experiment, hepatoma-derived HepG2 cells, but not NB4 cells, methylate radiolabeled iAsIII. Methylated metabolites released from HepG2 cells are preferentially accumulated by NB4 cells. This experimental model suggests that in vivo hepatic methylation of iAsIII may contribute to As2O3induced apoptosis but not differentiation of APL cells. MAsIIIO as an apoptotic inducer should be considered in the treatment of other hematologic malignancies like lymphoma.

INTRODUCTION APL4 is an unique subtype of acute myeloid leukemia typically carrying the specific reciprocal chromosomal translocation t(15;17) leading to the expression of the leukemia-generating fusion protein Received 11/1/02; accepted 2/21/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH 5R01CA85478 and Samuel Waxman Cancer Research Foundation (to S. W. and Y. J.), “one hundred-talent” programs of Chinese Academy of Sciences, and Key projects of Shanghai Committee of Science and Technology of China (to G-Q. C.), and NIH Grants ES09941 and Clinical Nutrition Research Center Grant DK 56350 (to M. S. and F. W.). 2 These authors should be considered equal first authors. 3 To whom requests for reprints should be addressed, at Department of Medicine, Division of Hematology/Oncology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-7995; Fax: (212) 996-5787; E-mail: [email protected]. 4 The abbreviations used are: APL, acute promyelocytic leukemia; RAR, retinoic acid receptor; As2O3, arsenic trioxide; iA, inorganic arsenical; iAsv, pentavalent arsenate; III iAs , trivalent arsenite; MAsV, methylarsonic acid; MAsIII, methylarsonous acid; DMAsV, dimethylarsine acid; DMAsIII, dimethylarsinous acid; MAsIIIO, methylarsine oxide; DMAsIIII, iododimethylarsine; CLL, chronic lymphocytic leukemia; ATCC, American Type Culture Collection; FACS, fluorescence-activated cell sorter; PI, propidium iodide; MA, methylarsenic; DMA, dimethylarsenic; MNC, mononuclear cell; CFU, colony-forming unit; E, erythroid; BFU, burst-forming unit; GM, granulocyte-macrophage; PARP, poly(ADP-ribose) polymerase; PML, promyelocytic leukemia; PML-RAR␣, promyelocytic leukemia-retinoic acid receptor alpha.

PML-RAR␣ (1, 2). In 90% of de novo APL patients, ATRA treatment induces differentiation of leukemic blasts and clinical remission, and 70% have been cured by ATRA in combination with chemotherapy (3). There is a 30% relapse, especially in APL patients presenting with high WBC. However, treatment with As2O3 induces complete remission in 90% in this group of chemotherapy and ATRA-resistant relapsed patients (4 – 6). This suggests that As2O3 treatment may induce remission in APL patients by a mechanism different from ATRA or chemotherapy. Indeed, only differentiated leukemia cells have been observed in APL patients after ATRA treatment (7), but both apoptotic and partially differentiated cells have been observed in APL patients and animal models treated with As2O3 (5, 8). This suggests that As2O3 may induce clinical remission in APL patients by multiple mechanisms. Biomethylation is the major metabolic pathway for iAs in humans and many animal species (9). In this pathway iAs undergo metabolic conversion that includes reduction of iAsv to iAsIII with subsequent methylation yielding mono- and dimethylated metabolites (10, 11). The postulated scheme is as follows: iAsv 3 iAsIII 3 MAsV 3 MAsIII 3 DMAsV 3 DMAsIII 3 MAsIII and DMAsIII have been detected in the urine of humans exposed to inorganic arsenic in drinking water (11, 12) and in human hepatoma (HepG2) cells exposed to iAsIII (11). Notably, chemical derivatives of MAsIII are significantly more toxic for cultured cells (13, 14) and laboratory animals (15) than either iAsv or iAsIII. DMAsIII derivatives are more toxic than iAs species in most cell types tested (13, 14). Although As2O3 has been found to induce remission in APL patients and induce apoptosis in cultured APL cells (4, 5, 8, 16), the metabolism of As2O3 and the effect of its methylated metabolites have not been tested. It is possible that methylated As2O3 metabolites that form in vivo may contribute to the therapeutic effect of As2O3 in APL. NB4 cells, a cell line derived from t(15;17) APL (17–20) were used to compare the induction of apoptosis and differentiation by iAsIII to methylated arsenicals that are chemical precursors of methylated trivalent metabolites of iAs, MAsIIIO and DMAsIIII. The effects of these arsenicals on H2O2 production, glutathione peroxidase activity, caspase activation, PML-RAR␣ protein degradation, and PML nuclear body formation were also examined. We also studied the production and distribution of iAsIII metabolites in a cocultivation cell culture system containing HepG2, a hepatoma-derived cell line and NB4 cells. The cytotoxic effect of MAsIIIO and iAsIII was also compared in K562 leukemia cells, lymphoma cells, primary cultures of CLL cells, and in normal human bone marrow progenitor cells. The data indicate that MAsIIIO, and to a lesser extent DMAsIIII, were more potent growth inhibitors and apoptotic inducers than iAsIII in leukemia and lymphoma cells but not in human bone marrow progenitor cells. H2O2 accumulation and GPx inhibition but not degradation of PML-RAR␣ correlated with greater MAsIIIO-induced apoptosis in NB4 cells. NB4 cells did not methylate iAsIII, but mono- and dimethylated metabolites formed from iAsIII in HepG2 cells released into the medium and were preferentially taken up by NB4 cells.

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APOPTOSIS BY AS2O3 METHYLATED METABOLITES IN LEUKEMIA

MATERIALS AND METHODS Arsenicals and Reagents. iAsIII and iAsV, sodium salts (at least 99% pure), were purchased from Sigma (St. Louis, MO). MAsV (sodium salt, 98% pure) was obtained from Chem Service (West Chester, PA) and DMAsV (98% pure) from Strem (Newburyport, MA). MAsIIIO (CH3AsO) and DMAsIIII [(CH3)2AsI] were synthesized by Dr. William R. Cullen (University of British Columbia, Vancouver, British Columbia, Canada) using methods described previously (21, 22). Identity and purity of these compounds were confirmed using 1H NMR and mass spectrometry. In aqueous solutions, MAsIIIO and DMAsIIII form the respective (methylarsonous and dimethylarsenous) oxyanions. The hydrolysis of oxides and iodides of MAsIII has been documented previously by Petrick et al. (15) using 1H NMR and mass spectrometry. Carrier-free [73As]iAsv was purchased from Los Alamos Meson Production Facility (Los Alamos, NM). [73As]iAsIII was prepared from [73As]iAsv by reduction with metabisulfite/thiosulfate reagent (23). Yields of [73As]iAsIII in this reaction as determined by TLC (24) typically exceed 95%. Cell Culture and Treatment. NB4 human APL cells (kindly provided by Dr. M. Lanotte, Hopı´tal Saint-Louis, Paris, France; Ref. 17) and the NB4derived ATRA-resistant R4 line (kindly provided by W. Miller, Jr., Lady Davis Institute for Medical Research, Montreal, Canada; Ref. 25) and K562 cells (obtained from ATCC) were cultured in RPMI 1640 supplemented with 10% FCS (JRH BioScience, Lenexa, KS) in a humidified atmosphere of 95% air and 5% CO2 at 37°C. HepG2 cells (obtained from ATCC) were cultured in MEM supplemented with 10% FCS. Jurkat and Namalwa human lymphoma cells (obtained from ATCC) were cultured in RPMI 1640 adjusted to contain 1.5 g/liter of sodium bicarbonate, 4.5 g/liter of glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and 10% FCS. CLL cells were obtained from blood from two patients with consent according to Institutional Review Board regulations. B-cell populations were isolated using RosetteSep B Cell Enrichment mixture (StemCell Technologies, Vancouver, British Columbia, Canada), which used antibodies against CD2, CD3, CD16, CD36, and CD56. This negative selection system enabled the isolation of B cells that were of ⬎95% purity by FACS analysis without activating the cells through any surface proteins. Whole blood was incubated with RosetteSep (50 ␮l of RosetteSep mixture/ml whole blood) at room temperature for 20 minutes. Samples were then diluted 1:2 with PBS ⫹2% FCS and centrifuged over Ficoll-Hypaque density medium (Amersham Biosciences, Piscataway, NJ). B cells were collected from the Ficoll:plasma interface, washed in PBS, and ready for use. For treatment with arsenicals, cells were seeded at 2 ⫻ 105 cells/ml and cultured in the above-mentioned medium with or without the indicated doses of compounds for the indicated time. Cell viability was estimated by trypan blue dye exclusion, and cell numbers were counted by hemacytometer. Annexin-V Assay. Apoptotic cells were detected by Annexin-V assay. Generally 5–10 ⫻ 105 cells after treatments were washed twice with PBS. Then, cells were labeled by Annexin-V-FITC and PI in binding buffer according to the instruction in the Annexin V-FITC Apoptosis Detection kit provided by the manufacturer (Oncogene, Cambridge, MA). Fluorescent signals of FITC and PI were detected, respectively, by FL1 (FITC detector) at 518 nm and FL2 at 620 nm on FACScan (Becton Dickson, San Jose, CA). The log of AnnexinV-FITC fluorescence was displayed on the X axis and the log of PI fluorescence on the Y axis, and data were analyzed using the CELLQuest (Becton Dickson) program. For each analysis, 10,000 events were recorded. Measure of Intracellular Hydrogen Peroxide Production. The intracellular hydrogen peroxide level was detected as we reported before by using 5,6-carboxy-2⬘,7⬘-dichloroflurescein-diacetate (Molecular Probe, Eugene, OR; Ref. 20). Briefly, 2 h before ending the indicated treatment, 10 ␮M 5,6carboxy-2⬘,7⬘-dichloroflurescein-diacetate was added into the medium and continued incubation for 2 h at 37°C, and the fluorescence intensity was measured by FACScan (Becton Dickinson). Glutathione Peroxidase Activity Assays. Cells (5 ⫻ 107) were washed twice with PBS, resuspended in PBS, sonicated for 10 s, centrifuged at 14,000 rpm for 10 min, and the supernatants subjected to enzyme assays. Glutathione peroxidase activity was determined using commercial kits (Calbiochem, San Diego, CA). One milliunit of enzyme activity was defined as 1 nmol of NADPH oxidized to NADP per milligram of protein per min. Immunofluorescence and Confocal Laser Microscopy Analysis. Cells were smeared onto slides and kept on ⫺80°C. For immunofluorescence staining of PML, which was described previously (26), cells were fixed in 20%

methanol and antihuman PML monoclonal antibody 5E10 and then with FITC-conjugated secondary antibodies. Fluorescence was observed using a Leica confocal laser microscope with excitation at 488 nm (green). Images were overlaid in PhotoShop. Metabolic Studies in a Cocultivation Culture System. HepG2 cells were cultured in monolayers in 12-well culture plates with MEM-supplemented 10% serum, and cells were cultured at 37°C in a humidified incubator in an atmosphere of 95% air and 5% CO2 to 90% confluence (⬃1.6 ⫻ 106 cells/ well). Before exposure to iAsIII, MEM was replaced with a supplemented Williams’ medium E (27) with 10% fetal bovine serum (1.5 ml/well), and cultures were left to equilibrate for 1 h. The supplemented Williams’ medium E has been shown previously to provide an optimal support for methylation of arsenite in hepatic cells (27). Carrier-free [73As]iAsIII was added into the equilibrated HepG2 cultures (1 ␮Ci/well) along with desirable amounts of iAsIII carrier, and plates were returned to a culture incubator for additional 30 min to allow for uptake of iAsIII. Suspension of NB4 cells was introduced into the HepG2 cultures in inserts (Becton Dickinson, Franklin Lake, NJ) with 0.4-␮m pore size (1 ⫻ 106 cells in 0.5 ml Williams’ medium E per insert). Individual cultures of HepG2 in monolayers and NB4 cells in inserts were used in parallel with the combined cultures. The final concentrations of iAsIII in the combined cultures reached 0.1 or 1 ␮M. The [73As]iAsIII-treated individual and combined cultures of HepG2 and NB4 cells were kept in a culture incubator for 48 h. Radiolabeled arsenic metabolites were analyzed separately in each type of cells and in a combined medium (medium in inserts ⫹ medium in well) using techniques described previously (24). To release protein-bound metabolites and to facilitate analysis of chemical species, medium and cells were treated with acidic CuCl solution (pH 1.0) at 100°C (28), and oxidized with hydrogen peroxide. Oxidized CuCl extracts were separated by TLC (24). The distribution of the radioactivity on the developed TLC plates was analyzed with an AMBIS 4000 imaging detector (Ambis, San Diego, CA). Because of the CuCl treatment and subsequent oxidation of samples used for TLC, this procedure cannot determine original oxidation states of arsenic in metabolites. Thus, arsenic metabolites in this series of experiments are generically referred to as iAs, MAs, and DMAs. Colony-forming Assays of Human Bone Marrow Progenitor Cells. Following informed consent according to Mount Sinai School of Medicine Institutional Review Board regulations, bone marrow from three normal adults were collected into syringes containing 50 units of Heparin. To obtain MNCs, bone marrow was diluted 1:1 with ␣ medium, layered onto an equal volume of Ficoll-Paque, and centrifuged at 450 ⫻ g for 30 min at 18°C. For methylcellulose cultures, MNCs were cultured according to methods described previously. Each ml of culture contained 300,000 MNCs, 0.8% methylcellulose in ␣ medium, 30% fetal bovine serum, 1% BSA (COHN fraction IV), 0.1 mmol/liter 2-mercaptoethanol, 0.1 units of penicillin, and 0.1 ␮g/ml streptomycin. Erythroid cultures contained erythropoietin (2 units/ml). Myeloid cultures contained GM colony-stimulating factor (2 ng/ml) and interleukin 3 (50 units/ml). Cultures were performed in triplicate in 0.3-ml volume in Nunc four-well dishes (Intermed, Roskilde, Denmark) and incubated at 37°C in 4% CO2 with or without As2O3 or MAsIIIO. Colonies derived from CFU-E were counted on day 7 and those from BFU-E and CFU-GM on day 13 using a Stereozoom dissecting microscope (Bausch & Lomb, Rochester, NY).

RESULTS Methylation of iAsIII Increases Cytotoxity in NB4 Cells. We compared growth inhibition and cell death by treatment with iAsIII, iAsV, and their monomethylated and dimethylated derivatives in NB4 cells at concentrations of 0.5, 1, and 2 ␮M for 1–3 days. MAsIIIO treatment was more growth inhibitory (IC50 ⫽ 0.3 ␮M) than iAsIII, whereas DMAsIIII was similar to iAsIII (IC50 ⬃1.0 ␮M) at 3-day treatment (Fig. 1A). There was minimal loss of viability after treatment with 0.5 ␮M of iAsIII, DMAsIIII, and MAsIIIO. However, 1 ␮M of MAsIIIO caused 90% loss of viability of NB4 cells, whereas iAsIII and DMAsIIII remained at 15% (Fig. 1B). Treatment with up to 2 ␮M of pentavalent methylated arsenicals (MasV and DMAsV) did not significantly inhibit growth or decrease viability of NB4 cells (data not shown).

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Fig. 1. Effects of trivalent arsenicals on the growth of NB4 cells. A, NB4 cells were treated with 0.5–2.0 ␮M iAsIII (top), MAsIIIO (middle), and DMAsIIII (bottom) for the indicated days. Total cell number was counted. Each point is the mean of triplicates with ⬍5% variation (data not shown). B, effects of trivalent arsenicals on viability of NB4 cells. Viable cell numbers were counted by trypanblue exclusion. All data represent an independent experiment from two repeated tests with similar results. Each point is the mean of triplicates; bars, ⫾SD.

MAsIIIO and DMAsIIII Are More Potent Apoptotic Inducers Than iAsIII in NB4 Cells. Induction of apoptosis as measured by Annexin-V⫹/PI⫺ assay was greatest after MAsIIIO treatment of NB4 cells. One ␮M of iAsIII, MAsIIIO, and DMAsIIII induced apoptotic and necrotic cells to 10, 63, and 21% in NB4 cells after 2 days treatment, respectively (Fig. 2A). Similarly, K562 human leukemia cells are more growth inhibited (IC50 0.7 versus, 2.1 ␮M) and undergo greater

apoptosis (12.5 versus 1.8%) by 48 h of treatment with 1 ␮M of MAsIIIO as compared with iAsIII. The extent of apoptosis induced by MAsIIIO and DMAsIIII correlated with more effective PARP cleavage (Fig. 2B). Production of H2O2 and Inhibition of GPx Activity by MAsIIIO Correlates with Higher Apoptotic Activity. We reported previously that iAsIII-induced apoptosis followed accumulation of H2O2 and

Fig. 2. Apoptosis induced by arsenicals in NB4 cells. A, NB4 cells were treated with 1 ␮M iAsIII, MAsIIIO, and DMAsIIII, respectively, for 24 and 48 h. Annexin-V and PI staining were measured by flow cytometry. X axis, the fluorescent intensity of Annexin-V. Y axis, the fluorescent intensity of PI. Each panel shows the percentages of Annexin-V⫹, PI⫺, and both positive cells. B, Western blot analysis of PARP cleavage. Antibody to PARP was used to detect the PARP cleavage product.

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phoma cell lines after 3 days of treatment. This was reflected by a greater loss of cell viability after MAsIIIO treatment (Fig. 5B). CLL cells in primary cultures are minimally affected by treatment with 2 ␮M iAsIII for 3 days. In contrast, 0.5 ␮M MAsIIIO treatment for 1 day inhibits growth (48%) and decreases viability (25%), and after 3 days there is 85% loss of viability (Table 2).

Fig. 3. Inhibition of GPx activity. NB4 cells were treated with various concentrations of iAsIII and MAsIIIO for 8 and 24 h. GPx activity was measured as described in “Materials and Methods.”

Table 1 Relative H2O2 amount in NB4 cells after arsenical treatmenta

Control 1 ␮M iAsIII 1 ␮M MAsIIIO 1 ␮M DMAsIIII a

24 H

48 H

0.71 ⫾ 0.15 4.24 ⫾ 1.33 37.57 ⫾ 1.15 24.97 ⫾ 0.45

1.44 ⫾ 0.41 8.30 ⫾ 1.44 74.0 ⫾ 2.06 51.01 ⫾ 0.54

H2O2 amount was measured by FACS.

inhibition of GPx activity (20). GPx activity was partially inhibited by iAsIII treatment of NB4 cells in a time- and concentration-dependent manner. However, GPx activity was more rapidly inhibited and to a much greater extent by 0.5 ␮M MAsIIIO than by 2 ␮M iAsIII in NB4 cells (Fig. 3). MAsIIIO (1 ␮M) treatment for 24 or 48 h caused ⬃10-fold more H2O2 accumulation than iAsIII (Table 1) and correlated with the extent of GPx inhibition. These data support the idea that MAsIIIO, like AsIII, induces apoptosis in APL cells by a H2O2mediated pathway. MAsIIIO-induced Apoptosis Is Independent of PML-RAR␣ Protein Degradation. To evaluate the role of PML-RAR␣ protein degradation in MAsIIIO-induced apoptosis, we measured PML-RAR␣ levels by Western blot analysis and PML levels by immunocytochemistry using confocal microscopy. NB4 cells treated with 1 ␮M MAsIIIO for 24 h produced more apoptosis than treatment with 1 ␮M iAsIII (Fig. 2A). However, iAsIII but not MAsIIIO treatment degraded PML-RAR␣ in NB4 cells after 24 h (Fig. 4A). Furthermore, confocal microscopy using PML antibody demonstrated that iAsIII treatment but not MAsIIIO reversed the abnormal microspeckled pattern found in NB4 cells, and partially reformed PML nuclear bodies (Fig. 4B). Thus, these data demonstrate that degradation of PML-RAR␣ protein is not a requirement for the more effective MAsIIIO-induced apoptosis. PML-RAR␣ protein degradation after iAsIII treatment in vitro and in vivo is thought to be a requirement for differentiation induction of ATRA-sensitive and -resistant APL cells (29). As shown in Fig. 4C, iAsIII- but not MAsIIIO-treated NB4 cells undergo partial differentiation as measured by CD11b using FACS analysis. MAsIIIO Is a More Potent Cytotoxic Agent in Human Lymphoma Cells. Some lymphoma cells are also sensitive to low concentration of As2O3-induced apoptosis (30). The ability of MAsIII to induce growth inhibition and cell death was studied in two lymphoma cell lines: Jurkat cells, which are insensitive, and Namalwa cells, which are sensitive to As2O3-induced apoptosis. As shown in Fig. 5A, MAsIIIO was more growth inhibitory than iAsIII in both Jurkat and Namalwa cells, because treatment with 1 ␮M iAsIII resulted in ⬍10%, whereas MAsIIIO resulted in ⬎50% growth inhibition of both lym-

Fig. 4. Effect of arsenicals on PML-RAR␣ degradation and nuclear body formation. A, NB4 cells were treated with 1 ␮M MAsIIIO or 1 ␮M iAsIII for 24 h, and PML RAR␣ protein was measured by Western blot analysis. B, PML nuclear bodies were assayed by confocal microscopy using PML antibody after NB4 cells were treated with 1 ␮M iAsIII and 0.25 ␮M MAsIIIO for 24 h. C, methylated arsenicals do not induce differentiation of NB4 cells. NB4 cells were treated with 0.25 ␮M iAsIII or MAsIIIO for 7–10 days, and differentiation was measured by expression of CD11b as measured by FACS.

Fig. 5. Effects of iAsIII and MAsIIIO on human lymphoma cell lines. A, cell growth inhibition. Jurkat and Namalwa cells were treated with 0.5–1.0 ␮M iAsIII and MAsIIIO for the indicated days. Total cell number was counted. B, cell viability. Viable cell numbers were counted by trypan-blue exclusion. Each point is the mean of triplicates; bars, ⫾SD.

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Table 2 MAsIIIO is a stronger cytotoxic agent than iAsIII in primary cultured CLL cellsa % Viable cellsb Drugs Control MAsIIIO 0.5 ␮M 1.0 ␮M 2.0 ␮M iAsIII 0.5 ␮M 1.0 ␮M 2.0 ␮M

Day 1

Day 2

Day 3

93.1 ⫾ 1.7

90.4 ⫾ 1.8

89.1 ⫾ 1.4

69.7 ⫾ 2.2 65.9 ⫾ 4.3 59.5 ⫾ 3.8

47.1 ⫾ 1.4 47.5 ⫾ 3.2 44.8 ⫾ 3.7

20.4 ⫾ 4.4 15.8 ⫾ 4.3 15.7 ⫾ 0.5

92.8 ⫾ 4.1 93.1 ⫾ 1.47 89.1 ⫾ 1.5

87.1 ⫾ 1.00 85.7 ⫾ 1.2 74.1 ⫾ 1.0

81.2 ⫾ 4.3 68.0 ⫾ 3.3 60.1 ⫾ 2.9

a

The data shown are cell viability measured by trypan blue staining. The values are mean ⫾ SD of three samples in each dose from one representative CLL patient. b

Effect of iAsIII and MAsIIIO on Normal Hematopoietic Cells. Human bone marrow MNCs (n ⫽ 3) grown in methylcellulose were treated with iAsIII or MAsIIIO, as reported previously (31). CFU-E-, BFU-E-, and CFU-GM-derived colony growth was unaffected by doses of 0.1-1 ␮M iAsIII. A similar level of toxicity was observed with up to 1 ␮M MAsIIIO treatment (Fig. 6). However, erythroid colonies (CFU-E and BFU-E) but not granulocytic colonies (CFU-GM) were more inhibited by 2 ␮M MAsIIIO than 2 ␮M iAsIII. Metabolism of iAsIII and Distribution of Methylated Metabolites in a Cocultivation Cell Culture System. To examine the affinity of APL cells toward natural metabolites of iAs, we carried out cocultivation experiments in combined cultures of HepG2 and NB4 cells. Individual cultures of HepG2 and NB4 cells were used as controls. The combined and individual cultures were exposed to 0.1 or 1 ␮M [73As]iAsIII for 48 h (Table 3; Ref. 4). Radiolabeled metabolites of [73As]iAsIII were analyzed in culture medium, HepG2 cells, and/or NB4 cells separately. No methylated metabolites were detected in the individual NB4 cultures regardless of the iAsIII concentration. U937 human leukemia cells like NB4 cells take up but do not methylate [73As]iAsIII (data not shown). In the individual and combined cultures containing HepG2 cells, [73As]iAsIII was methylated to yield radiolabeled MAs and DMAs. DMAs was the major methylated metabolite in HepG2 cultures exposed to 0.1 ␮M arsenite (Table 3). On the other hand, MAs was the predominant methylated metabolite in cultures exposed to 1 ␮M arsenite (Table 4). In the combined cultures, significant portions of MAs and DMAs synthesized by HepG2 cells and released into the medium were found in NB4 cells. MAs retained in NB4 cells represented 73% of this metabolite released in culture medium by HepG2 cells when exposed to 1 ␮M iAsIII. In contrast, only 8.5% of iAs in the medium was taken up by NB4 cells. In the combined cultures exposed to 1 ␮M iAsIII the ratio of iAs:MAs was 10-fold greater in the culture medium (36.8) than in NB4 cells (3.5). DMAs represented only a minor fraction of the total arsenic in NB4 cells regardless of treatment.

a combined cell culture system (Tables 3 and 4). The methylation patterns of iAs in HepG2 cells resemble those reported in primary human hepatocytes (11, 27). Similar to primary cultures of hepatocytes, moderate (micromolar) concentrations of iAsIII inhibit methylation reactions, particularly DMA synthesis, in HepG2 cells yielding the intermediary metabolite, MA, as the major methylated product (Tables 3 and 4). The lack of methylated metabolites in NB4 cultures exposed to either 0.1 or 1 ␮M iAsIII suggests that these cells do not methylate iAs. In the combined cultures NB4 cells took up and accumulated significant portions of methylated metabolites, especially MAs, produced by HepG2 monolayers. The comparison of the ratios of metabolites in the culture medium and of metabolites retained in NB4 cells suggests that NB4 cells have a very high affinity for MAs as compared with other arsenic metabolites. The arsenic speciation techniques used in this study could not determine oxidation states (valencies) of arsenic metabolites. However, because pentavalent arsenicals are not effectively taken up by cultured cells (11, 27), MAs and DMAs retained in NB4 cells are most likely in the trivalent forms of MAsIII and DMAsIII. Among all of the biologically relevant chemical forms of arsenic,

DISCUSSION We established that NB4 cells can take up and accumulate methylated metabolites of iAs produced and released by HepG2 cells using

Fig. 6. Effect of arsenicals in normal human hematopoietic progenitor cells. Human bone marrow MNCs were treated with varying concentrations of iAsIII (A) and MAsIIIO (B), and assayed for CFU-E, BFU-E, and CFU-GM colonies as described in “Materials and Methods.”

Table 3 As metabolites formed in HepG2 and leukemia cells incubated with 0.1 ␮M Medium

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As arsenite for 48 h (pmol As/culture, Mean ⫾ SD, n ⫽ 3)

HepG2 Cells

NB4 Cells

Cultured cells

iAs

MAs

DMAs

iAs

MAs

DMAs

HepG2 alone NB4 alone NB4 Plus HepG2

128.5 ⫾ 6.07 183.4 ⫾ 3.11 129.7 ⫾ 3.30

1.0 ⫾ 1.26 NDa ND

32.3 ⫾ 3.79 ND 22.9 ⫾ 0.27

17.7 ⫾ 1.01

6.8 ⫾ 1.13

38.1 ⫾ 1.52

14.9 ⫾ 0.72

5.3 ⫾ 0.33

8.7 ⫾ 0.64

a

ND, not detected.

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iAs

MAs

DMAs

16.6 ⫾ 3.11 12.1 ⫾ 1.25

ND 4.1 ⫾ 0.56

ND 2.3 ⫾ 0.55


Table 4 As metabolites formed in HepG2 and leukemia cells incubated with 1 ␮M Medium

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As arsenite for 48 h (pmol As/culture, Mean ⫾ SD, n ⫽ 3)

HepG2 Cells

NB4 Cells

Cultured cells

iAs

MAs

DMAs

iAs

MAs

DMAs

HepG2 alone NB4 alone NB4 Plus HepG2

1711.5 ⫾ 23.37 1871.0 ⫾ 10.62 1648.2 ⫾ 17.2

79.8 ⫾ 7.35 NDa 44.8 ⫾ 6.82

15.0 ⫾ 4.75 ND 10.2 ⫾ 6.41

136.9 ⫾ 16.67

33.9 ⫾ 6.01

19.9 ⫾ 4.53

106.9 ⫾ 4.07

32.3 ⫾ 3.80

9.4 ⫾ 3.76

a

iAs

MAs

DMAs

129.0 ⫾ 10.62 115.9 ⫾ 7.94

ND 32.7 ⫾ 2.27

ND 0.6 ⫾ 1.01

ND, not detected.

MAsIII derivatives are the most potent enzyme inhibitor, as shown here for GPx (Fig. 3), and cytotoxin (reviewed in Ref. 32). In addition, MAsIII and DMAsIII derivatives are considerably more effective than arsenite in nicking naked DNA in an in vitro system or damaging DNA in intact cells (33). These methylated trivalent arsenicals are also potent inducers of c-Jun phosphorylation and activator protein DNA binding in human cells (14, 34). The results of this study suggest that leukemia cells in vivo may accumulate MAsIII produced and released from the liver of patients treated with As2O3. These observations suggest that methylated metabolites of As2O3 may contribute to therapeutic and toxic effects in APL patients. To test this possibility, we compared the extent of growth inhibition and apoptosis induced in NB4 cells and K562 cells by treatment with iAsIII, and MAsIIIO and DMAsIIII chemical precursors of methylated trivalent metabolites of iAsIII (Figs. 1 and 2). Our data indicate that the apoptosis-induction ability is MAsIIIO⬎DMAsIIII⬎iAsIII. MAsIIIO treatment also inhibits growth and induces apoptosis to a greater extent than iAsIII. This suggests that the amount of MAsIIIO in cells may mediate the sensitivity to As2O3-induced apoptosis in malignant cells and normal tissues such as bone marrow and liver. The unexplained and unusual severe liver toxicity in three Chinese patients treated for APL with As2O3 may relate to aberrant hepatocyte methylation of As2O3 (8). Studies to characterize hepatic arsenic methyltransferase polymorphism, isoforms, and nutritional cofactors are required to better understand As2O3 as a therapeutic agent. We compared the uptake of 73As-arsenite and methylated arsenicals metabolites in NB4, which are sensitive to As2O3-induced apoptosis, and U937 cells, which are insensitive to As2O3-induced apoptosis (20). NB4 and U937 cells have similar accumulation of 73iAs, do not methylate iAsIII, and take up MAsIII and DMAsIII to similar amounts in cocultivation with HepG2 cells (Tables 3 and 4; data not shown). Thus, other intracellular factors contribute to the higher sensitivity of NB4 cells to MAsIIIO-induced apoptosis. The intracellular content of glutathione, and GPx, glutathione S-transferase ␲, and catalase activities also play an important role in catabolism of H2O2. In NB4, cells with low levels As2O3 treatment induces H2O2 accumulation and apoptosis, and when high, there is less apoptosis in leukemic cells such as U937 cells (20). MAsIIIO is a more potent H2O2 inducer than iAsIII (Table 1) and is known to have a stronger binding affinity to thiol-enzymes (21). GPx ,which has a thiol-like group required for its activation (35), is more inhibited by MAsIIIO (Fig. 3), which may contribute to the greater H2O2 accumulation (Table 1) and apoptosis. It has been suggested that degradation of PML-RAR␣ and PML protein is required for As2O3-induced apoptosis in APL cells (18, 19). This would predict a greater ability of MAsIIIO than iAsIII to degrade PML-RAR␣ or PML. However, PML-RAR␣ protein degradation follows iAsIII but not MAsIIIO treatment in NB4 cells (Fig. 4A). Moreover, iAsIII decreased abnormal PML nuclear body distribution and number as reported before (Fig. 4B), whereas neither MAsIIIO nor DMAsIIII (data not shown) change the abnormal distribution of nuclear bodies in NB4 cells. Therefore, As2O3 triggers apoptosis by a pathway independent of PML or PML-RAR␣ degradation, thus not limiting this therapeutic effect to APL. The clinical response of APL patients to As2O3 treatment that is

associated with differentiation induction is probably related to iAsIII (8). Unlike iAsIII, MAsIIIO treatment of NB4 cells does not induce differentiation in NB4 cells (Fig. 4C) and does not cooperate with ATRA to induce differentiation in R4 ATRA-resistant NB4 cells (Ref. 25; data not shown). Therefore, the in vivo therapeutic contribution of MAsIIIO would be the induction of apoptosis, not differentiation. The contribution of hepatic derived MAsIII from iAsIII to therapeutic outcome remains to be determined. MAsIIIO released from hepatocytes after metabolism of iAsIII is avidly taken up by APL cells (Table 3; Ref. 4) and may contribute more than iAsIII to the induction of apoptosis, particularly in APL cells, which have a biochemical phenotype that renders them sensitive to H2O2-mediated apoptosis (18). The finding of significant growth inhibition and apoptosis by treatment with As2O3 (16, 18 –20), and to a greater extent by MAsIIIO in lymphoma cells (Fig. 5) and in CLL cells in primary culture (Table 2) supports the evaluation of As2O3 and MAsIIIO in additional clinical trials. MAsIIIO, up to a concentration of 1 ␮M, although more potent than iAsIII to induce apoptosis in NB4 and death in lymphoma cells, does not show greater toxicity than iAsIII in human bone marrow as measured by colony assays (Fig. 6). We reported previously that the combination of ascorbic acid and As2O3, which increases H2O2 accumulation and apoptosis in NB4 and lymphoma cell lines, does not enhance As2O3 toxicity in human bone marrow as measured by colony assays (31). This correlates with a greater therapeutic effect of As2O3 in combination with ascorbic acid without increasing toxicity in a mouse lymphoma model (31). This may be related to the observation that human bone marrow progenitor cells have high catalase levels (36), which may render these cells more resistant to H2O2 accumulation after As2O3 treatment. The uptake and conversion of iAsIII to MAsIIIO by HepG2 cells support the use of iAsIII or MAsIIIO in the treatment of patients with hepatoma. There may be an increased potential for toxicity, particularly in liver with MAsIIIO treatment. Thus, animal studies are required to evaluate this derivative before future clinical trials. ACKNOWLEDGMENTS We thank Dr. George Acs for critical reading of this manuscript.

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