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[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 PML-RAR␣ (1, 2). In 90% of de novo APL patients, ATRA treatment induces differentiation of leukemic blasts and clinical remission, and Treatment with arsenic trioxide (As O ) by inducing apoptosis and 2 3 70% have been cured by ATRA in combination with chemotherapy partial differentiation of acute promyelocytic leukemia (APL) cells results (3). There is a 30% relapse, especially in APL patients presenting with in clinical remission in APL patients resistant to chemotherapy and III high WBC. However, treatment with As2O3 induces complete remis- all-trans-retinoic acid. As2O3 (iAs ) is methylated in the liver to mono- and dimethylated metabolites, including methylarsonic acid, methylarson- sion in 90% in this group of chemotherapy and ATRA-resistant ous acid, dimethylarsinic acid, and dimethylarsinous acid. Methylated relapsed patients (4–6). This suggests that As2O3 treatment may trivalent metabolites that are potent cytotoxins, genotoxins, and enzyme induce remission in APL patients by a mechanism different from inhibitors may contribute to the in vivo therapeutic effect of iAsIII. There- ATRA or chemotherapy. Indeed, only differentiated leukemia cells fore, we compared the potency of iAsIII and trivalent metabolites using have been observed in APL patients after ATRA treatment (7), but chemical precursors of methylarsonous acid and dimethylarsinous acid to both apoptotic and partially differentiated cells have been observed in induce differentiation, growth inhibition, and apoptosis. Methylarsine APL patients and animal models treated with As O (5, 8). This III 2 3 oxide (MAs O) and to a lesser extent iododimethylarsine were more suggests that As O may induce clinical remission in APL patients by potent growth inhibitors and apoptotic inducers than iAsIII in NB4 cells, 2 3 multiple mechanisms. an APL cell line. This was also observed in K562 human leukemia, lymphoma cell lines, and in primary culture of chronic lymphocytic Biomethylation is the major metabolic pathway for iAs in humans leukemia cells, but not human bone marrow progenitor cells. Apoptosis and many animal species (9). In this pathway iAs undergo metabolic v III was associated with greater hydrogen peroxide accumulation and inhibi- conversion that includes reduction of iAs to iAs with subsequent tion of glutathione peroxidase activity. MAsIIIO, in contrast to iAsIII, did methylation yielding mono- and dimethylated metabolites (10, 11). not induce PML-retinoic acid receptor ␣ degradation, or restore PML The postulated scheme is as follows: iAsv 3 iAsIII 3 MAsV 3 nuclear bodies or differentiation in NB4 cells. In a cocultivation experi- MAsIII 3 DMAsV 3 DMAsIII 3 MAsIII and DMAsIII have been ment, hepatoma-derived HepG2 cells, but not NB4 cells, methylate radio- detected in the urine of humans exposed to inorganic arsenic in III labeled iAs . Methylated metabolites released from HepG2 cells are drinking water (11, 12) and in human hepatoma (HepG2) cells ex- preferentially accumulated by NB4 cells. This experimental model sug- III III III posed to iAs (11). Notably, chemical derivatives of MAs are gests that in vivo hepatic methylation of iAs may contribute to As2O3- III significantly more toxic for cultured cells (13, 14) and laboratory induced apoptosis but not differentiation of APL cells. MAs Oasan v III III apoptotic inducer should be considered in the treatment of other hema- animals (15) than either iAs or iAs . DMAs derivatives are more tologic malignancies like lymphoma. 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 As O INTRODUCTION 2 3 and the effect of its methylated metabolites have not been tested. It is 4 APL is an unique subtype of acute myeloid leukemia typically possible that methylated As2O3 metabolites that form in vivo may carrying the specific reciprocal chromosomal translocation t(15;17) contribute to the therapeutic effect of As2O3 in APL. leading to the expression of the leukemia-generating fusion protein 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 Received 11/1/02; accepted 2/21/03. methylated arsenicals that are chemical precursors of methylated III III The costs of publication of this article were defrayed in part by the payment of page trivalent metabolites of iAs, MAs O and DMAs I. The effects of charges. This article must therefore be hereby marked advertisement in accordance with these arsenicals on H2O2 production, glutathione peroxidase activity, 18 U.S.C. Section 1734 solely to indicate this fact. ␣ 1 Supported in part by NIH 5R01CA85478 and Samuel Waxman Cancer Research caspase activation, PML-RAR protein degradation, and PML nu- Foundation (to S. W. and Y. J.), “one hundred-talent” programs of Chinese Academy of clear body formation were also examined. We also studied the pro- Sciences, and Key projects of Shanghai Committee of Science and Technology of China duction and distribution of iAsIII metabolites in a cocultivation cell (to G-Q. C.), and NIH Grants ES09941 and Clinical Nutrition Research Center Grant DK 56350 (to M. S. and F. W.). culture system containing HepG2, a hepatoma-derived cell line and 2 These authors should be considered equal first authors. NB4 cells. The cytotoxic effect of MAsIIIO and iAsIII was also 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. compared in K562 leukemia cells, lymphoma cells, primary cultures Levy Place, New York, NY 10029. Phone: (212) 241-7995; Fax: (212) 996-5787; E-mail: of CLL cells, and in normal human bone marrow progenitor cells. The [email protected]. III III 4 data indicate that MAs O, and to a lesser extent DMAs I, were more The abbreviations used are: APL, acute promyelocytic leukemia; RAR, retinoic acid III v potent growth inhibitors and apoptotic inducers than iAs in leuke- receptor; As2O3, arsenic trioxide; iA, inorganic arsenical; iAs , pentavalent arsenate; iAsIII, trivalent arsenite; MAsV, methylarsonic acid; MAsIII, methylarsonous acid; mia and lymphoma cells but not in human bone marrow progenitor V III III DMAs , dimethylarsine acid; DMAs , dimethylarsinous acid; MAs O, methylarsine cells. H O accumulation and GPx inhibition but not degradation of oxide; DMAsIIII, iododimethylarsine; CLL, chronic lymphocytic leukemia; ATCC, Amer- 2 2 III ican Type Culture Collection; FACS, fluorescence-activated cell sorter; PI, propidium PML-RAR␣ correlated with greater MAs O-induced apoptosis in iodide; MA, methylarsenic; DMA, dimethylarsenic; MNC, mononuclear cell; CFU, col- NB4 cells. NB4 cells did not methylate iAsIII, but mono- and dim- ony-forming unit; E, erythroid; BFU, burst-forming unit; GM, granulocyte-macrophage; III PARP, poly(ADP-ribose) polymerase; PML, promyelocytic leukemia; PML-RAR␣, pro- ethylated metabolites formed from iAs in HepG2 cells released into myelocytic leukemia-retinoic acid receptor alpha. the medium and were preferentially taken up by NB4 cells. 1853

MATERIALS AND METHODS methanol and antihuman PML monoclonal antibody 5E10 and then with FITC-conjugated secondary antibodies. Fluorescence was observed using a III V Arsenicals and Reagents. iAs and iAs , sodium salts (at least 99% Leica confocal laser microscope with excitation at 488 nm (green). Images V pure), were purchased from Sigma (St. Louis, MO). MAs (sodium salt, 98% were overlaid in PhotoShop. V pure) was obtained from Chem Service (West Chester, PA) and DMAs Metabolic Studies in a Cocultivation Culture System. HepG2 cells were III (98% pure) from Strem (Newburyport, MA). MAs O (CH3AsO) and cultured in monolayers in 12-well culture plates with MEM-supplemented III DMAs I [(CH3)2AsI] were synthesized by Dr. William R. Cullen (University 10% serum, and cells were cultured at 37¡C in a humidified incubator in an of British Columbia, Vancouver, British Columbia, Canada) using methods ϳ ϫ 6 atmosphere of 95% air and 5% CO2 to 90% confluence ( 1.6 10 cells/ described previously (21, 22). Identity and purity of these compounds were well). Before exposure to iAsIII, MEM was replaced with a supplemented confirmed using 1H NMR and mass spectrometry. In aqueous solutions, Williams’ medium E (27) with 10% fetal bovine serum (1.5 ml/well), and MAsIIIO and DMAsIIII form the respective (methylarsonous and dimethyl- cultures were left to equilibrate for 1 h. The supplemented Williams’ medium arsenous) oxyanions. The hydrolysis of oxides and iodides of MAsIII has been E has been shown previously to provide an optimal support for methylation of documented previously by Petrick et al. (15) using 1H NMR and mass arsenite in hepatic cells (27). Carrier-free [73As]iAsIII was added into the spectrometry. Carrier-free [73As]iAsv was purchased from Los Alamos Meson equilibrated HepG2 cultures (1 ␮Ci/well) along with desirable amounts of Production Facility (Los Alamos, NM). [73As]iAsIII was prepared from iAsIII carrier, and plates were returned to a culture incubator for additional 30 [73As]iAsv by reduction with metabisulfite/thiosulfate reagent (23). Yields of min to allow for uptake of iAsIII. Suspension of NB4 cells was introduced into [73As]iAsIII in this reaction as determined by TLC (24) typically exceed 95%. the HepG2 cultures in inserts (Becton Dickinson, Franklin Lake, NJ) with Cell Culture and Treatment. NB4 human APL cells (kindly provided by 0.4-␮m pore size (1 ϫ 106 cells in 0.5 ml Williams’ medium E per insert). Dr. M. Lanotte, Hopõ«tal Saint-Louis, Paris, France; Ref. 17) and the NB4- Individual cultures of HepG2 in monolayers and NB4 cells in inserts were used derived ATRA-resistant R4 line (kindly provided by W. Miller, Jr., Lady Davis in parallel with the combined cultures. The final concentrations of iAsIII in the Institute for Medical Research, Montreal, Canada; Ref. 25) and K562 cells combined cultures reached 0.1 or 1 ␮M. The [73As]iAsIII-treated individual and (obtained from ATCC) were cultured in RPMI 1640 supplemented with 10% combined cultures of HepG2 and NB4 cells were kept in a culture incubator for FCS (JRH BioScience, Lenexa, KS) in a humidified atmosphere of 95% air 48 h. Radiolabeled arsenic metabolites were analyzed separately in each type and 5% CO2 at 37¡C. HepG2 cells (obtained from ATCC) were cultured in of cells and in a combined medium (medium in inserts ϩ medium in well) MEM supplemented with 10% FCS. Jurkat and Namalwa human lymphoma using techniques described previously (24). To release protein-bound metab- cells (obtained from ATCC) were cultured in RPMI 1640 adjusted to contain olites and to facilitate analysis of chemical species, medium and cells were 1.5 g/liter of sodium bicarbonate, 4.5 g/liter of glucose, 10 mM HEPES, 1.0 mM treated with acidic CuCl solution (pH 1.0) at 100¡C (28), and oxidized with sodium pyruvate and 10% FCS. CLL cells were obtained from blood from two hydrogen peroxide. Oxidized CuCl extracts were separated by TLC (24). The patients with consent according to Institutional Review Board regulations. distribution of the radioactivity on the developed TLC plates was analyzed B-cell populations were isolated using RosetteSep B Cell Enrichment mixture with an AMBIS 4000 imaging detector (Ambis, San Diego, CA). Because of (StemCell Technologies, Vancouver, British Columbia, Canada), which used the CuCl treatment and subsequent oxidation of samples used for TLC, this antibodies against CD2, CD3, CD16, CD36, and CD56. This negative selection procedure cannot determine original oxidation states of arsenic in metabolites. system enabled the isolation of B cells that were of Ͼ95% purity by FACS Thus, arsenic metabolites in this series of experiments are generically referred analysis without activating the cells through any surface proteins. Whole blood to as iAs, MAs, and DMAs. was incubated with RosetteSep (50 ␮l of RosetteSep mixture/ml whole blood) Colony-forming Assays of Human Bone Marrow Progenitor Cells. at room temperature for 20 minutes. Samples were then diluted 1:2 with PBS Following informed consent according to Mount Sinai School of Medicine ϩ2% FCS and centrifuged over Ficoll-Hypaque density medium (Amersham Institutional Review Board regulations, bone marrow from three normal adults Biosciences, Piscataway, NJ). B cells were collected from the Ficoll:plasma were collected into syringes containing 50 units of Heparin. To obtain MNCs, interface, washed in PBS, and ready for use. For treatment with arsenicals, bone marrow was diluted 1:1 with ␣ medium, layered onto an equal volume of cells were seeded at 2 ϫ 105 cells/ml and cultured in the above-mentioned Ficoll-Paque, and centrifuged at 450 ϫ g for 30 min at 18¡C. For methylcel- medium with or without the indicated doses of compounds for the indicated lulose cultures, MNCs were cultured according to methods described previ- time. Cell viability was estimated by trypan blue dye exclusion, and cell ously. Each ml of culture contained 300,000 MNCs, 0.8% methylcellulose in numbers were counted by hemacytometer. ␣ medium, 30% fetal bovine serum, 1% BSA (COHN fraction IV), 0.1 Annexin-V Assay. Apoptotic cells were detected by Annexin-V assay. mmol/liter 2-mercaptoethanol, 0.1 units of penicillin, and 0.1 ␮g/ml strepto- Generally 5Ð10 ϫ 105 cells after treatments were washed twice with PBS. mycin. Erythroid cultures contained erythropoietin (2 units/ml). Myeloid cul- Then, cells were labeled by Annexin-V-FITC and PI in binding buffer accord- tures contained GM colony-stimulating factor (2 ng/ml) and interleukin 3 (50 ing to the instruction in the Annexin V-FITC Apoptosis Detection kit provided units/ml). Cultures were performed in triplicate in 0.3-ml volume in Nunc by the manufacturer (Oncogene, Cambridge, MA). Fluorescent signals of FITC four-well dishes (Intermed, Roskilde, Denmark) and incubated at 37¡C in 4% III and PI were detected, respectively, by FL1 (FITC detector) at 518 nm and FL2 CO2 with or without As2O3 or MAs O. Colonies derived from CFU-E were at 620 nm on FACScan (Becton Dickson, San Jose, CA). The log of Annexin- counted on day 7 and those from BFU-E and CFU-GM on day 13 using a V-FITC fluorescence was displayed on the X axis and the log of PI fluores- Stereozoom dissecting microscope (Bausch & Lomb, Rochester, NY). cence 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 intracel- RESULTS lular hydrogen peroxide level was detected as we reported before by using III 5,6-carboxy-2Ј,7Ј-dichloroflurescein-diacetate (Molecular Probe, Eugene, OR; Methylation of iAs Increases Cytotoxity in NB4 Cells. We III Ref. 20). Briefly, 2 h before ending the indicated treatment, 10 ␮M 5,6- compared growth inhibition and cell death by treatment with iAs , carboxy-2Ј,7Ј-dichloroflurescein-diacetate was added into the medium and iAsV, and their monomethylated and dimethylated derivatives in NB4 III continued incubation for2hat37¡C, and the fluorescence intensity was cells at concentrations of 0.5, 1, and 2 ␮M for 1Ð3 days. MAs O III measured by FACScan (Becton Dickinson). treatment was more growth inhibitory (IC ϭ 0.3 ␮M) than iAs , 7 50 Glutathione Peroxidase Activity Assays. Cells (5 ϫ 10 ) were washed III III ϳ ␮ whereas DMAs I was similar to iAs (IC50 1.0 M) at 3-day twice with PBS, resuspended in PBS, sonicated for 10 s, centrifuged at 14,000 treatment (Fig. 1A). There was minimal loss of viability after treat- rpm for 10 min, and the supernatants subjected to enzyme assays. Glutathione III, III III ment with 0.5 ␮M of iAs DMAs I, and MAs O. However, 1 ␮M peroxidase activity was determined using commercial kits (Calbiochem, San III III Diego, CA). One milliunit of enzyme activity was defined as 1 nmol of of MAs O caused 90% loss of viability of NB4 cells, whereas iAs III ␮ NADPH oxidized to NADP per milligram of protein per min. and DMAs I remained at 15% (Fig. 1B). Treatment with up to 2 M V V Immunofluorescence and Confocal Laser Microscopy Analysis. Cells of pentavalent methylated arsenicals (Mas and DMAs ) did not were smeared onto slides and kept on Ϫ80¡C. For immunofluorescence significantly inhibit growth or decrease viability of NB4 cells (data staining of PML, which was described previously (26), cells were fixed in 20% not shown). 1854

Fig. 1. Effects of trivalent arsenicals on the growth of NB4 cells. A, NB4 cells were treated with III III 0.5Ð2.0 ␮M iAs (top), MAs O(middle), and DMAsIIII(bottom) for the indicated days. Total cell number was counted. Each point is the mean of triplicates with Ͻ5% variation (data not shown). B, effects of trivalent arsenicals on viability of NB4 cells. Viable cell numbers were counted by trypan- blue exclusion. All data represent an independent experiment from two repeated tests with similar results. Each point is the mean of triplicates; bars, ϮSD.

III III MAs O and DMAs I Are More Potent Apoptotic Inducers apoptosis (12.5 versus 1.8%) by 48 h of treatment with 1 ␮M of Than iAsIII in NB4 Cells. Induction of apoptosis as measured by MAsIIIO as compared with iAsIII. The extent of apoptosis induced by Annexin-Vϩ/PIϪ assay was greatest after MAsIIIO treatment of NB4 MAsIIIO and DMAsIIII correlated with more effective PARP cleavage III III III cells. One ␮M of iAs , MAs O, and DMAs I induced apoptotic and (Fig. 2B). III necrotic cells to 10, 63, and 21% in NB4 cells after 2 days treatment, Production of H2O2 and Inhibition of GPx Activity by MAs O respectively (Fig. 2A). Similarly, K562 human leukemia cells are Correlates with Higher Apoptotic Activity. We reported previously ␮ III more growth inhibited (IC50 0.7 versus, 2.1 M) and undergo greater that iAs -induced apoptosis followed accumulation of H2O2 and

III III III Fig. 2. Apoptosis induced by arsenicals in NB4 cells. A, NB4 cells were treated with 1 ␮M iAs , MAs O, and DMAs I, 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-Vϩ,PIϪ, and both positive cells. B, Western blot analysis of PARP cleavage. Antibody to PARP was used to detect the PARP cleavage product. 1855

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 III III with 2 ␮M iAs for 3 days. In contrast, 0.5 ␮M MAs O 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.”

a Table 1 Relative H2O2 amount in NB4 cells after arsenical treatment 24 H 48 H Control 0.71 Ϯ 0.15 1.44 Ϯ 0.41 III 1 ␮M iAs 4.24 Ϯ 1.33 8.30 Ϯ 1.44 III 1 ␮M MAs O 37.57 Ϯ 1.15 74.0 Ϯ 2.06 III 1 ␮M DMAs I 24.97 Ϯ 0.45 51.01 Ϯ 0.54 a 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 Fig. 4. Effect of arsenicals on PML-RAR␣ degradation and nuclear body formation. A, ␮ III ␮ III ␣ ␮ III ␮ III NB4 cells were treated with 1 M MAs Oor1 M iAs for 24 h, and PML RAR much greater extent by 0.5 M MAs O than by 2 M iAs in NB4 protein was measured by Western blot analysis. B, PML nuclear bodies were assayed by III III cells (Fig. 3). MAs O(1␮M) treatment for 24 or 48 h caused confocal microscopy using PML antibody after NB4 cells were treated with 1 ␮M iAs III ϳ III and 0.25 ␮M MAs O for 24 h. C, methylated arsenicals do not induce differentiation of 10-fold more H2O2 accumulation than iAs (Table 1) and corre- III III NB4 cells. NB4 cells were treated with 0.25 ␮M iAs or MAs O for 7Ð10 days, and lated with the extent of GPx inhibition. These data support the idea differentiation was measured by expression of CD11b as measured by FACS. III III that MAs O, like As , induces apoptosis in APL cells by a H2O2- mediated 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 immunocytochem- istry using confocal microscopy. NB4 cells treated with 1 ␮M III MAs O 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 Lym- phoma Cells. Some lymphoma cells are also sensitive to low con- III centration of As2O3-induced apoptosis (30). The ability of MAs 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 Fig. 5. Effects of iAsIII and MAsIIIO on human lymphoma cell lines. A, cell growth ␮ III III ␮ III Ͻ inhibition. Jurkat and Namalwa cells were treated with 0.5Ð1.0 M iAs and MAs O for Namalwa cells, because treatment with 1 M iAs resulted in 10%, the indicated days. Total cell number was counted. B, cell viability. Viable cell numbers whereas MAsIIIO resulted in Ͼ50% growth inhibition of both lym- were counted by trypan-blue exclusion. Each point is the mean of triplicates; bars, ϮSD. 1856

Table 2 MAsIIIO is a stronger cytotoxic agent than iAsIII in primary cultured a combined cell culture system (Tables 3 and 4). The methylation a CLL cells patterns of iAs in HepG2 cells resemble those reported in primary % Viable cellsb human hepatocytes (11, 27). Similar to primary cultures of hepato- III Drugs Day 1 Day 2 Day 3 cytes, moderate (micromolar) concentrations of iAs inhibit methy- Control 93.1 Ϯ 1.7 90.4 Ϯ 1.8 89.1 Ϯ 1.4 lation reactions, particularly DMA synthesis, in HepG2 cells yielding MAsIIIO the intermediary metabolite, MA, as the major methylated product 0.5 ␮M 69.7 Ϯ 2.2 47.1 Ϯ 1.4 20.4 Ϯ 4.4 ␮ Ϯ Ϯ Ϯ (Tables 3 and 4). The lack of methylated metabolites in NB4 cultures 1.0 M 65.9 4.3 47.5 3.2 15.8 4.3 ␮ III 2.0 ␮M 59.5 Ϯ 3.8 44.8 Ϯ 3.7 15.7 Ϯ 0.5 exposed to either 0.1 or 1 M iAs suggests that these cells do not iAsIII methylate iAs. In the combined cultures NB4 cells took up and ␮ Ϯ Ϯ Ϯ 0.5 M 92.8 4.1 87.1 1.00 81.2 4.3 accumulated significant portions of methylated metabolites, especially 1.0 ␮M 93.1 Ϯ 1.47 85.7 Ϯ 1.2 68.0 Ϯ 3.3 2.0 ␮M 89.1 Ϯ 1.5 74.1 Ϯ 1.0 60.1 Ϯ 2.9 MAs, produced by HepG2 monolayers. The comparison of the ratios a The data shown are cell viability measured by trypan blue staining. of metabolites in the culture medium and of metabolites retained in b The values are mean Ϯ SD of three samples in each dose from one representative NB4 cells suggests that NB4 cells have a very high affinity for MAs CLL patient. as compared with other arsenic metabolites. The arsenic speciation techniques used in this study could not determine oxidation states Effect of iAsIII and MAsIIIO on Normal Hematopoietic Cells. (valencies) of arsenic metabolites. However, because pentavalent ar- Human bone marrow MNCs (n ϭ 3) grown in methylcellulose were senicals are not effectively taken up by cultured cells (11, 27), MAs treated with iAsIII or MAsIIIO, as reported previously (31). CFU-E-, and DMAs retained in NB4 cells are most likely in the trivalent forms III III BFU-E-, and CFU-GM-derived colony growth was unaffected by of MAs and DMAs . III doses of 0.1-1 ␮M iAs . A similar level of toxicity was observed with Among all of the biologically relevant chemical forms of arsenic, III up to 1 ␮M MAs O treatment (Fig. 6). However, erythroid colonies (CFU-E and BFU-E) but not granulocytic colonies (CFU-GM) were III III more inhibited by 2 ␮M MAs O than 2 ␮M iAs . Metabolism of iAsIII and Distribution of Methylated Metabo- lites 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 73 III 1 ␮M [ As]iAs 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 III medium by HepG2 cells when exposed to 1 ␮M iAs . In contrast, only 8.5% of iAs in the medium was taken up by NB4 cells. In the III combined cultures exposed to 1 ␮M iAs 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.

DISCUSSION Fig. 6. Effect of arsenicals in normal human hematopoietic progenitor cells. Human We established that NB4 cells can take up and accumulate meth- 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 ylated metabolites of iAs produced and released by HepG2 cells using and Methods.”

73 Table 3 As metabolites formed in HepG2 and leukemia cells incubated with 0.1 ␮M As arsenite for 48 h (pmol As/culture, Mean Ϯ SD, n ϭ 3) Medium HepG2 Cells NB4 Cells

Cultured cells iAs MAs DMAs iAs MAs DMAs iAs MAs DMAs HepG2 alone 128.5 Ϯ 6.07 1.0 Ϯ 1.26 32.3 Ϯ 3.79 17.7 Ϯ 1.01 6.8 Ϯ 1.13 38.1 Ϯ 1.52 NB4 alone 183.4 Ϯ 3.11 NDa ND 16.6 Ϯ 3.11 ND ND NB4 Plus HepG2 129.7 Ϯ 3.30 ND 22.9 Ϯ 0.27 14.9 Ϯ 0.72 5.3 Ϯ 0.33 8.7 Ϯ 0.64 12.1 Ϯ 1.25 4.1 Ϯ 0.56 2.3 Ϯ 0.55 a ND, not detected. 1857

73 Table 4 As metabolites formed in HepG2 and leukemia cells incubated with 1 ␮M As arsenite for 48 h (pmol As/culture, Mean Ϯ SD, n ϭ 3) Medium HepG2 Cells NB4 Cells

Cultured cells iAs MAs DMAs iAs MAs DMAs iAs MAs DMAs HepG2 alone 1711.5 Ϯ 23.37 79.8 Ϯ 7.35 15.0 Ϯ 4.75 136.9 Ϯ 16.67 33.9 Ϯ 6.01 19.9 Ϯ 4.53 NB4 alone 1871.0 Ϯ 10.62 NDa ND 129.0 Ϯ 10.62 ND ND NB4 Plus HepG2 1648.2 Ϯ 17.2 44.8 Ϯ 6.82 10.2 Ϯ 6.41 106.9 Ϯ 4.07 32.3 Ϯ 3.80 9.4 Ϯ 3.76 115.9 Ϯ 7.94 32.7 Ϯ 2.27 0.6 Ϯ 1.01 a ND, not detected.

MAsIII derivatives are the most potent enzyme inhibitor, as shown associated with differentiation induction is probably related to iAsIII here for GPx (Fig. 3), and cytotoxin (reviewed in Ref. 32). In addition, (8). Unlike iAsIII, MAsIIIO treatment of NB4 cells does not induce MAsIII and DMAsIII derivatives are considerably more effective than differentiation in NB4 cells (Fig. 4C) and does not cooperate with arsenite in nicking naked DNA in an in vitro system or damaging ATRA to induce differentiation in R4 ATRA-resistant NB4 cells (Ref. DNA in intact cells (33). These methylated trivalent arsenicals are 25; data not shown). Therefore, the in vivo therapeutic contribution of also potent inducers of c-Jun phosphorylation and activator protein MAsIIIO would be the induction of apoptosis, not differentiation. The DNA binding in human cells (14, 34). The results of this study suggest contribution of hepatic derived MAsIII from iAsIII to therapeutic that leukemia cells in vivo may accumulate MAsIII produced and outcome remains to be determined. MAsIIIO released from hepato- III released from the liver of patients treated with As2O3. These obser- cytes after metabolism of iAs is avidly taken up by APL cells (Table III vations suggest that methylated metabolites of As2O3 may contribute 3; Ref. 4) and may contribute more than iAs to the induction of to therapeutic and toxic effects in APL patients. To test this possibil- apoptosis, particularly in APL cells, which have a biochemical phe- ity, we compared the extent of growth inhibition and apoptosis in- notype that renders them sensitive to H2O2-mediated apoptosis (18). duced in NB4 cells and K562 cells by treatment with iAsIII, and The finding of significant growth inhibition and apoptosis by treat- III III III MAs O and DMAs I chemical precursors of methylated trivalent ment with As2O3 (16, 18Ð20), and to a greater extent by MAs Oin metabolites of iAsIII (Figs. 1 and 2). Our data indicate that the lymphoma cells (Fig. 5) and in CLL cells in primary culture (Table 2) III Ͼ III Ͼ III III III apoptosis-induction ability is MAs O DMAs I iAs . MAs O supports the evaluation of As2O3 and MAs O in additional clinical III treatment also inhibits growth and induces apoptosis to a greater trials. MAs O, up to a concentration of 1 ␮M, although more potent extent than iAsIII. This suggests that the amount of MAsIIIO in cells than iAsIII to induce apoptosis in NB4 and death in lymphoma cells, III may mediate the sensitivity to As2O3-induced apoptosis in malignant does not show greater toxicity than iAs in human bone marrow as cells and normal tissues such as bone marrow and liver. The unex- measured by colony assays (Fig. 6). We reported previously that the plained and unusual severe liver toxicity in three Chinese patients combination of ascorbic acid and As2O3, which increases H2O2 ac- treated for APL with As2O3 may relate to aberrant hepatocyte meth- cumulation and apoptosis in NB4 and lymphoma cell lines, does not ylation of As2O3 (8). Studies to characterize hepatic arsenic methyl- enhance As2O3 toxicity in human bone marrow as measured by transferase polymorphism, isoforms, and nutritional cofactors are colony assays (31). This correlates with a greater therapeutic effect of required to better understand As2O3 as a therapeutic agent. As2O3 in combination with ascorbic acid without increasing toxicity We compared the uptake of 73As-arsenite and methylated arsenicals in a mouse lymphoma model (31). This may be related to the obser- metabolites in NB4, which are sensitive to As2O3-induced apoptosis, vation that human bone marrow progenitor cells have high catalase and U937 cells, which are insensitive to As2O3-induced apoptosis levels (36), which may render these cells more resistant to H2O2 73 (20). NB4 and U937 cells have similar accumulation of iAs, do not accumulation after As2O3 treatment. The uptake and conversion of methylate iAsIII, and take up MAsIII and DMAsIII to similar amounts iAsIII to MAsIIIO by HepG2 cells support the use of iAsIII or MAsIIIO in cocultivation with HepG2 cells (Tables 3 and 4; data not shown). in the treatment of patients with hepatoma. There may be an increased Thus, other intracellular factors contribute to the higher sensitivity of potential for toxicity, particularly in liver with MAsIIIO treatment. NB4 cells to MAsIIIO-induced apoptosis. The intracellular content of Thus, animal studies are required to evaluate this derivative before glutathione, and GPx, glutathione S-transferase ␲, and catalase activ- future clinical trials. ities 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 ACKNOWLEDGMENTS such as U937 cells (20). MAsIIIO is a more potent H O inducer than 2 2 We thank Dr. George Acs for critical reading of this manuscript. 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 REFERENCES contribute to the greater H O accumulation (Table 1) and apoptosis. 2 2 1. Pandolfi, P. P. PML. PLZF and NPM genes in the molecular pathogenesis of acute It has been suggested that degradation of PML-RAR␣ and PML promyelocytic leukemia, Haematologica, 81: 472Ð82, 1996. 2. de The, H., Lavau, C., Marchio, A., Chomienne, C., Degos, L., and Dejean, A. The protein is required for As2O3-induced apoptosis in APL cells (18, 19). III III PML-RAR ␣ fusion mRNA generated by the t(15;17) translocation in acute promy- This would predict a greater ability of MAs O than iAs to degrade elocytic leukemia encodes a functionally altered RAR. Cell, 66: 675Ð84, 1991. PML-RAR␣ or PML. However, PML-RAR␣ protein degradation 3. Melnick, A., and Licht, J. D. Deconstructing a disease: RAR␣, its fusion partners, and follows iAsIII but not MAsIIIO treatment in NB4 cells (Fig. 4A). their roles in the pathogenesis of acute promyelocytic leukemia, Blood, 93: 3167Ð III 215, 1999. Moreover, iAs decreased abnormal PML nuclear body distribution 4. Shen, Z. X., Chen, G. Q., Ni, J. H., Li, X. S., Xiong, S. M., Qiu, Q. Y., Zhu, J., Tang, and number as reported before (Fig. 4B), whereas neither MAsIIIO nor W., Sun, G. L., Yang, K. Q., Chen, Y., Zhou, L., Fang, Z. W., Wang, Y. T., Ma, J., DMAsIIII (data not shown) change the abnormal distribution of nu- Zhang, P., Zhang, T. D., Chen, S. J., Chen, Z., and Wang, Z. Y. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical clear bodies in NB4 cells. Therefore, As2O3 triggers apoptosis by a efficacy and pharmacokinetics in relapsed patients, Blood, 89: 3354Ð60, 1997. pathway independent of PML or PML-RAR␣ degradation, thus not 5. Soignet, S. L., Maslak, P., Wang, Z. G., Jhanwar, S., Calleja, E., Dardashti, L. J., Corso, D., DeBlasio, A., Gabrilove, J., Scheinberg, D. A., Pandolfi, P. P., and Warrell, limiting this therapeutic effect to APL. Jr., R. P. Complete remission after treatment of acute promyelocytic leukemia with The clinical response of APL patients to As2O3 treatment that is arsenic trioxide. N. Engl. J. Med., 339: 1341Ð1348, 1998. 1858

6. Cohen, M. H., Hirschfeld, S., Flamm Honig, S., Ibrahim, A., Johnson, J. R., O’Leary, Peschle, C., Gambacorti-Passerini, C., Nervi, C., and Miller, W. H., Jr. Arsenic J. J., White, R. M., Williams, G. A., and Pazdur, R. Drug approval summaries: arsenic trioxide as an inducer of apoptosis and loss of PML/RAR ␣ protein in acute trioxide, tamoxifen citrate, anastrazole, paclitaxel, bexarotene. Oncologist, 6: 4Ð11, promyelocytic leukemia cells. J. Natl. Cancer Inst., 90: 124Ð33, 1998. 2001. 20. Jing, Y., Dai, J., Cahlmers-Redman, R. M. E., Tatton, W., and Waxman, S. arsenic 7. Degos, L. Differentiation therapy in acute promyelocytic leukemia: European expe- trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydro- rience. J. Cell Physiol., 173: 285Ð7, 1997. gen peroxide dependent pathway. Blood, 94: 2102Ð11, 1999. 8. Niu, C., Yan, H., Yu, T., Sun, H. P., Liu, J. X., Li, X. S., Wu, W., Zhang, F. Q., Chen, 21. Cullen, W. R., McBride, B. C., and Reglinski, J. The reaction of methylarsenicals Y., Zhou, L., Li, J. M., Zeng, X. Y., Yang, R. R., Yuan, M. M., Ren, M. Y., Gu, F. Y., with thiols: some biological implications. J. Inorganic Biochem., 21: 179Ð194, 1984. Cao, Q., Gu, B. W., Su, X. Y., Chen, G. Q., Xiong, S. M., Zhang, T. d., Waxman, S., 22. Styblo, M., Serves, S. V., Cullen, W. R., and Thomas, D. J. Comparative inhibition Wang, Z. Y., Chen, Z., Hu, J., Shen, Z. X., and Chen, S. J. Studies on treatment of of yeast glutathione reductase by arsenicals and arsenothiols. Chem. Res. Toxicol., acute promyelocytic leukemia with arsenic trioxide: remission induction, follow-up, 10: 27Ð33, 1997. and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic 23. Reay, P. F., and Asher, C. J. Preparation and purification of 74As-labeled arsenate leukemia patients. Blood, 94: 3315Ð24, 1999. and arsenite for use in biological experiments, Anal. Biochem., 78: 557Ð60, 1977. 9. Aposhian, H. V. Enzymatic methylation of arsenic species and other new approaches 24. Styblo, M., Yamauchi, H., and Thomas, D. J. Comparative in vitro methylation of to arsenic toxicity. Annu. Rev. Pharmacol. Toxicol., 37: 397Ð419, 1997. trivalent and pentavalent arsenicals. Toxicol. Appl. Pharmacol., 135: 172Ð8, 1995. 10. Kitchin, K. T. Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol. Appl. Pharmacol., 172: 25. Rosenauer, A., Raelson, J. V., Nervi, C., Eydoux, P., DeBlasio, A., and Miller, W. H., 249Ð61, 2001. Jr. Alterations in expression, binding to ligand and DNA, and transcriptional activity 11. Del Razo, L. M., Styblo, M., Cullen, W. R., and Thomas, D. J. Determination of of rearranged and wild-type retinoid receptors in retinoid-resistant acute promyelo- trivalent methylated arsenicals in biological matrices. Toxicol. Appl. Pharmacol., cytic leukemia cell lines. Blood, 88: 2671Ð82, 1996. 174: 282Ð93, 2001. 26. Borden, K. L., Boddy, M. N., Lally, J., O’Reilly, N. J., Martin, S., Howe, K., 12. Aposhian, H. V., Gurzau, E. S., Le, X. C., Gurzau, A., Healy, S. M., Lu, X., Ma, M., Solomon, E., and Freemont, P. S. The solution structure of the RING finger domain Yip, L., Zakharyan, R. A., Maiorino, R. M., Dart, R. C., Tircus, M. G., Gonzalez- from the acute promyelocytic leukaemia proto-oncoprotein PML. EMBO J., 14: Ramirez, D., Morgan, D. L., Avram, D., and Aposhian, M. M. Occurrence of 1532Ð41, 1995. monomethylarsonous acid in urine of humans exposed to inorganic arsenic. Chem. 27. Styblo, M., Del Razo, L. M., LeCluyse, E. L., Hamilton, G. A., Wang, C., Cullen, Res. Toxicol., 13: 693Ð7, 2000. W. R., and Thomas, D. J. Metabolism of arsenic in primary cultures of human and rat 13. Styblo, M., Del Razo, L. M., Vega, L., Germolec, D. R., LeCluyse, E. L., Hamilton, hepatocytes. Chem. Res. Toxicol., 12: 560Ð5, 1999. G. A., Reed, W., Wang, C., Cullen, W. R., and Thomas, D. J. Comparative toxicity 28. Styblo, M., Hughes, M. F., and Thomas, D. J. Liberation and analysis of protein- of trivalent and pentavalent inorganic and methylated arsenicals in rat and human bound arsenicals. J Chromatogr B Biomed Appl., 677: 161Ð6, 1996. cells. Arch. Toxicol., 74: 289Ð99, 2000. 29. Jing, Y., Wang, L., Xia, L., Chen, G. Q., Chen, Z., Miller, W. H., and Waxman, S. 14. Styblo, M., Drobna, Z., Jaspers, I., Lin, S., and Thomas, D. J. The role of biomethy- Combined effect of all-trans retinoic acid and arsenic trioxide in acute promyelocytic lation in toxicity and carcinogenicity of arsenic. A research update. Environ. Health leukemia cells in vitro and in vivo. Blood, 97: 264Ð9, 2001. Perspect., 110 Suppl. 5: 767Ð771, 2002. 30. Zhu, X. H., Shen, Y. L., Jing, Y. K., Cai, X., Jia, P. M., Huang, Y., Tang, W., Shi, 15. Petrick, J. S., Jagadish, B., Mash, E. A., and Aposhian, H. V. Monomethylarsonous G. Y., Sun, Y. P., Dai, J., Wang, Z. Y., Chen, s. J., Zhang, T. D., Waxman, S., Chen, acid (MMAIII) and arsenite: LD50 in hamsters and in vitro inhibition of pyruvate Z., and Chen, G. Q. Apoptosis and growth inhibition in malignant lymphocytes after dehydrogenase. Chem. Res. Toxicol., 14: 651Ð656, 2001. treatment with arsenic trioxide at clinically achievable concentrations. J. Natl. Cancer 16. Chen, G. Q., Shi, X. G., Tang, W., Xiong, S. M., Zhu, J., Cai, X., Han, Z. G., Ni, J. H., Inst., 91: 772Ð778, 1999. Shi, G. Y., Jia, P. M., Liu, M. M., He, K. L., Niu, C., Ma, J., Zhang, P., Zhang, T. D., 31. Dai, J., Weinberg, R. S., Waxman, S., and Jing, Y. Malignant cells can be sensitixed Paul, P., Naoe, T., Kitamura, K., Miller, W., Waxman, S., Wang, Z. Y., de The, H., to undergo growth inhibition and apoptosis by arsenic trioxide trough modulation of Chen, S. J., and Chen, Z. Use of arsenic trioxide (As2O3) in the treatment of acute the glutathione redox system. Blood, 93: 268Ð277, 1999. promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL 32. Thomas, D. J., Styblo, M., and Lin, S. The cellular metabolism and systemic toxicity cells. Blood, 89: 3345Ð53, 1997. 17. Lanotte, M., Martin-Thouvenin, V., Najman, S., Ballerini, P., Valensi, F., and Bergen, of arsenic. Toxicol. Appl. Pharmacol., 176: 127Ð44, 2001. R. NB4, a maturation inducible cell line with t(15;17) marker isolated from a human 33. Mass, M. J., Tennant, A., Roop, B. C., Cullen, W. R., Styblo, M., Thomas, D. J., and acute promyelocytic leukemia (M3). Blood, 77: 1080Ð1086, 1991. Kligerman, A. D. Methylated trivalent arsenic species are genotoxic. Chem. Res. 18. Chen, G. Q., Zhu, J., Shi, X. G., Ni, J. H., Zhong, H. J., Si, G. Y., Jin, X. L., Tang, Toxicol., 14: 355Ð61, 2001. W., Li, X. S., Xong, S. M., Shen, Z. X., Sun, G. L., Ma, J., Zhang, P., Zhang, T. D., 34. Drobna, Z., Jaspers, I., Thomas, D. J., and Styblo, M. Differential activation of AP-1 Gazin, C., Naoe, T., Chen, S. J., Wang, Z. Y., and Chen, Z. In vitro studies on cellular in human bladder epithelial cells by inorganic and methylated arsenicals. FASEB J., and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute 17: 67Ð69, 2003. promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of 35. Tappel, A. L. selenium-glutathione peroxidase: properties and synthesis. Curr. Topics Bcl-2 expression and modulation of PML-RAR ␣/PML proteins. Blood, 88: 1052Ð Cell. Regul., 24: 87Ð97, 1984. 61, 1996. 36. Bram, S., Froussard, P., Guichard, M., Jasmin, C., Augery, Y., Sinoussi-Barre, F., and 19. Shao, W., Fanelli, M., Ferrara, F. F., Riccioni, R., Rosenauer, A., Davison, K., Wray, W. Vitamin C preferential toxicity for malignant melanoma cells. Nature Lamph, W. W., Waxman, S., Pelicci, P. G., Lo Coco, F., Avvisati, G., Testa, U., (Lond.), 284: 629Ð31, 1980.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2003 American Association for Cancer Research. Methylated Metabolites of Arsenic Trioxide Are More Potent Than Arsenic Trioxide as Apoptotic but not Differentiation Inducers in Leukemia and Lymphoma Cells

Guo-Qiang Chen, Li Zhou, Miroslav Styblo, et al.

Cancer Res 2003;63:1853-1859.

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