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ANTICANCER RESEARCH 25: 991-998 (2005)

Arsenic Trioxide Circumvents Multidrug Resistance Based on Different Mechanisms in Human Lines

TAMAMI SEO1, YOSHIMASA URASAKI2, HARUYUKI TAKEMURA1 and TAKANORI UEDA1

1First Department of Internal Medicine and 2Division of Transfusion Medicine, Faculty of Medical Sciences, University of Fukui, Fukui, 910-1193, Japan

Abstract. To determine the antitumor effect of cells (7-9). As2O3 affects numerous intracellular signal (As2O3) on multidrug-resistant cells, we applied 3 human transduction pathways and causes many alternations in leukemia cell lines: (DNR)-resistant cell line cellular function. These actions of As2O3 may cause the K562/D1-9, which overexpresses p-glycoprotein (Pgp); DNR induction of , the inhibition of growth and and 1-‚-D-arabinofuranosylcytosine (Ara-C) double-resistant angiogenesis and the promotion of differentiation. cell line HL60/AD, which overexpresses multidrug resistance- The clinical efficiency of anticancer is frequently associated (MRP1); and Bcl-2-transfected pre-B lineage limited by the emergence of a variety of mechanisms of leukemia cell line 697/Bcl-2. Interestingly, K562/D1-9 showed resistance in tumor cells. One of these mechanisms is collateral sensitivity. Only HL60/AD showed small cross related to the increased expression of efflux pumps, resistance, but 697/Bcl-2 had no resistance to As2O3. An including P-glycoprotein (Pgp) and multidrug resistance- intracellular content of glutathione (GSH) played a critical role associated protein (MRP1). Both of them are members of in sensitivity to As2O3. Buthionine-sulfoximine (BSO), which the ABC superfamily of transporters and Zhou et al. reduces the GSH content, not only increased the As2O3 reported both of these gene expressions could play an sensitivity but also conquered the MRP1- related cross important role in the outcome of of acute resistance in HL60/AD. In conclusion, As2O3 was effective in leukemia (10). We examined the effect of As2O3 on all 3 cell lines, suggesting that As2O3 may be a promising agent multidrug-resistant cell lines which exhibit over-expression for the treatment of multidrug-resistant leukemia. of Pgp or MRP1 genes. We also examined the sensitivity to As2O3 in the apoptosis-resistant leukemia cell line which Arsenic trioxide (As2O3), a trivalent arsenical salt, has been carries the transfected Bcl-2 gene. used for many decades in traditional Chinese medicine for the treatment of various human diseases including tumors. Materials and Methods During the last decade, the efficacy of As O in both newly 2 3 Cell culture, cell treatment and materials. Human leukemia cell lines diagnosed and relapsed patients with acute promyelocytic K562 and its daunorubicin (DNR)-resistant cell line overexpressing leukemia (APL) has been reported (1-3). Even at low Pgp, K562/D1-9 (11), human myelogenous leukemia cell line HL60 concentrations, As2O3 induced a high rate of clinical and its DNR and 1-‚-D-arabinofuranosylcytocine (Ara-C) double- remission, causing few adverse effects and only minimal resistant cell line which overexpresses MRP1, HL60/AD (12), and human pre-B lineage leukemia cell line 697/Neo and its Bcl-2- bone marrow suppression. In patients with APL, As2O3 has been shown to cause degradation of the aberrant PML- transfected cells (697/Bcl-2), which is dexamethasone resistance,(13) were cultured and passaged in RPMI 1640 (Sigma retinoic acid receptor · fusion protein, resulting in complete Chemical Co, St. Louis, MO, USA) supplemented with 10% fetal remission (4-6). Recently, experimental studies have bovine serum(FBS, Sigma-Aldrich. Co.Ltd., St. Louis, MO, USA) provided further evidence of the antiproliferative properties in a 5% CO2 incubator at 37ÆC. of As2O3 towards malignant lymphoid cells and solid tumor Reagents. As2O3 was obtained from Sigma; DNR from Meiji Seika Co.(Tokyo, Japan); (VP-16), from Nippon Kayaku Co.(Tokyo, Japan); (VCR), from Shionogi Seiyaku Co. Correspondence to: Yoshimasa Urasaki, Division of Transfusion Ltd.(Osaka, Japan); (MIT), from Takeda Co. Medicine, Faculty of Medical Sciences, University of Fukui, 23-3 (Osaka, Japan); Ara-C from Nippon Shinyaku Co.(Kyoto, Japan); Shimoaizuki, Matuoka-cho, Yoshida-gun, Fukui 910-1193, Japan. MK571 from ALEXIS (San Diego, CA, USA); buthionine sulfoximine (BSO, a selective inhibitor of Á-glutamyl cysteine Key Words: Arsenic trioxide, glutathione, multidrug-resistance, synthetase) from Nacalai Tesque (Kyoto, Japan). Other chemicals buthionine-sulfoximine, reactive oxygen species. were obtained from commercial sources. As2O3 was dissolved in a

0250-7005/2005 $2.00+.40 991 ANTICANCER RESEARCH 25: 991-998 (2005)

Table I. Growth inhibition effect of As2O3 and other antileukemic agents in 3 leukemic cell lines.

Cell line

Drugs K562 K562/D1-9 HL60 HL60/AD 697/Neo 697/Bcl-2

As2O3 2.37±0.14 0.84±0.12* 1.40±0.34 4.54±0.10** 1.53±0.18 2.57±0.75 DNR 0.084±0.013 8.63±1.63** 0.030±0.014 0.32±0.13* ND*** ND VP-16 0.85±0.13 97.5±17.1** 0.11±0.047 6.33±1.53** ND ND VCR 0.047±0.13 1.73±0.41** ND ND ND ND MIT 0.053±0.02 3.13±1.96* 0.010±0.003 0.28±0.10* ND ND Ara-C 0.021±0.007 0.025±0.011 0.047±0.021 0.42±0.07* ND ND

4 IC50 values (mM) were determined using the MTT assay. Cells (1x10 ) were seeded and exposed to As2O3 and other antileukemic agents for 72 h. Values are means±SD of at least three independent experiments performed in triplicate. Significantly different from the parental cells (*p<0.05, **p<0.01 by Student’s t-test,*** Not determined). DNR, daunorubicin; VP-16, etoposide; VCR, vincristine; MIT, mitoxantrone

small amount of 1.0N NaOH and then diluted to 1 mM with Morphological assessment. As2O3-treated or -untreated cells were phosphate-buffered saline (PBS) as the stock solution. centrifuged onto slides using Cytospin and observed using May- Giemsa staining under a light microscope. Cytotoxicity. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine sensitivity. In brief, Annexin V analysis. Annexin-V assay was performed according to 1x104 cells were seeded as a suspension (100 Ìl/well) in 96-well the instructions provided in the Annexin-V-FLOUS Staining Kit microtiter plates. The cells were incubated at 37ÆC in the continuous (Roche, Penzberg, Germany). Briefly, cells (1x106) were collected presence of drug for 72 h. Cell viability was assayed by adding 50 Ìg by centrifugation at 200 xg for 5 min and washed with ice-cold PBS of MTT dye (in PBS). After a 4-h incubation, during which activated at 200 xg for 5 min at 4ÆC. The obtained cells were suspended with cells reduced the yellow MTT salt to purple formazan, the stain was 100 Ìl incubation buffer containing 2 Ìl annexin-V and incubated eluted into the medium by the addition of 100 Ìl 2-propanol for 15 min at room temperature. Fluorescent intensities were (containing 0.04N HCl). Optical densities were measured at 595 nm. determined on a FACScan. Determinations for all experiments were made in triplicate. Results were expressed as means and SD. ROS analysis. After a 120-min incubation with or without BSO, cells were incubated with dihydrorhodamine 123 Measurement of total intracellular GSH level. Total intracellular hydrochloride(DHR)and As2O3 for 60 min at 37ÆC. ROSs in the GSH content was assayed using the total Glutathione cells were determined on a FACScan as fluorescent intensities. Quantification Kit according to the manufacturer's instructions (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Results Briefly, cells (1x106) were washed with cold PBS, suspended in 10 mmol/l HCl, and two freeze-thaw cycles were performed. Five As O induced growth inhibition. Table I shows the IC for percent 5-sulfosalicylic acid was added and centrifuged at 8000 xg 2 3 50 for 10 min at 4ÆC. The supernatant was assayed for GSH content As2O3 and other anticancer agents in the parental and according to the manufacturer's instructions. The intracellular resistant cell lines. K562/D1-9, which over-expresses Pgp, GSH content was expressed in nmol/1x106 cells. showed about 100-fold resistance to DNR and cross resistance to VCR, VP-16 and MIT. Interestingly, K562/D1-9 Flow cytometric analysis of cell-cycle status. Cells (1x106) were showed about 3-fold collateral sensitivity to As2O3 which collected by centrifugation at 200 xg for 5 min, and washed twice was significantly different from the parental cell K562. with ice-cold PBS at 200 xg for 5 min at 4ÆC. The obtained cells HL60/AD, which showed MRP1 over-expression and were suspended in 1 ml PBS and 2.5 ml of 99% ethanol was added while mixing. The cells were incubated at 4ÆC for more than 30 min, 10-fold resistance to both DNR and Ara-C, was significantly washed with PBS, suspended in 200 Ìl PBS, and 100 Ìl of RNase (5 about 3-fold more resistant to As2O3 compared with the mg/ml) was added. Incubation was continued at 37ÆC for 20 min, parental cell HL60. 697/Bcl-2 did not show resistance to followed by centrifugation at 200 xg for 5 min at 4ÆC, and washing As2O3 compared with 697/Neo. with ice-cold PBS at 200 xg for 5 min at 4ÆC. The cellular DNA was then stained by applying 500 Ìl of propidium iodide (50 Ìg/ml). The Role of GSH on collateral sensitivity to As O in K562/D1-9. stained cells were analyzed on a FACScan (Becton Dickinson, 2 3 Franklin Lakes, NJ, USA). Cells with DNA content less than the We examined the relationship between the As2O3-induced cells in the G1-phase (sub-G1) were considered as apoptotic cells growth inhibition and the intracellular level of GSH. To (14). The percentage of apoptotic cells was estimated by counting reduce intracellular GSH, we exposed the cells to 3 ÌM the cell number in the subG1 fraction. BSO at the concentration which inhibits GSH synthetase, in

992 Seo et al: Arsenic Trioxide Can Overcome Multidrug Resistance

Table II. Sensitivity to As2O3 in leukemic cell lines.

Cell line

Drugs K562 K562/D1-9 HL60 HL60/AD

BSO– 2.37±0.14 0.84±0.12* 1.40±0.34 4.54±0.10** BSO+ 0.38±0.10 0.27±0.14 0.51±0.07 0.26±0.05**

4 IC50 values (mM) were determined using the MTT assay. Cells (1x10 ) were seeded and exposed to As2O3 for 72 h with (BSO+) and without (BSO-) 3 mM BSO. Values are means±SD of at least three independent experiments performed in triplicate. Significantly different from the parental cells (*p<0.05, **p<0.01 by Student’s t-test).

Figure 1. Effect of BSO on sensitivity to As2O3 in K562 and K562/D1-9. different cells were incubated in the absence or presence of The cells were incubated at 37ÆC in the continuous presence of drug for As2O3 (1, 2, 5 ÌM) at 37ÆC for 72 h. There was a dose- 72 h with or without 3 ÌM of BSO. Cell viability was assayed by adding dependent increase in the sub-G1-phase in all these cell 50 Ìg of MTT dye and 4-h incubation. K562 treatment without BSO, ●; with BSO, ●; K562/D1-9 without BSO, ▲; with BSO, ▲. lines. As shown in Figure 4, 5ÌM As2O3 induced apoptosis in K562/D1-9 more strongly than in K562, while such induction was not observed in HL60/AD. These results were consistent with the growth inhibition effect of As2O3 by combination with As2O3 for 72 h. After exposure, IC50 MTT assay. Secondly, the effect of BSO was examined in values were determined using MTT assay. At this HL60 and HL60/AD by flow cytometric analysis. Three concentration BSO alone did not affect the survival in any micromoles BSO alone did not induce apoptosis. However, cell lines. The BSO enhanced the sensitivity to As2O3 in as shown in Figure 4, the treatment with 0.5 ÌM As2O3 plus both K562 and K562/D1-9 and in the presence of BSO there 3 ÌM BSO caused apoptosis in HL60/AD but not in HL60. were no significant differences in IC50 between K562 and Treatment with 1 ÌM As2O3 plus 3 ÌM BSO caused K562/D1-9 (Figure 1, Table II). The intracellular GSH level apoptosis in HL60 (data not shown). These results in K562/D1-9 was significantly lower compared with K562. suggested that HL60/AD showed a collateral sensitivity to The addition of BSO significantly reduced the intracellular the combination of As2O3 and BSO. GSH levels in both K562 and K562/D1-9 and there were no Apoptosis induced by As2O3 was confirmed by cell significant differences between K562 and K562/D1-9 in this morphology and Annexin V assay. After treatment with condition with 3 ÌM BSO (Figure 2). 1 ÌM As2O3 for 6 h, nuclear fragmentation was observed, 4.8% in As2O3-treated HL60 cells and less than 0.1% in Role of MRP1 on resistance to As2O3 in HL60/AD cells. untreated HL60 cells, respectively(Figure 5). Additionally, HL60/AD was found to be about 3-fold more resistant to annexin V-positive cells were increased (Figure 6). As2O3 than the parental HL60 cells. GSH has been shown to play a major role in the MRP1-mediated export of various Role of BSO on ROS production in HL60 and HL60/AD. To compounds (15). We, therefore, studied the effects of BSO determine the mechanism of collateral sensitivity to As2O3 and MK571, a potent inhibitor of the MRP1 efflux pump with BSO in HL60/AD, we measured the ROS level in HL60 (16). The addition of 3 ÌM BSO enhanced the sensitivity to and HL60/AD. In HL60, ROS were increased slightly by As2O3 in both HL60 and HL60/AD. HL60/AD showed a addition of As2O3 and BSO. In HL60/AD, by simultaneous collateral sensitivity to As2O3 by the addition of BSO treatment with As2O3 and BSO, the ROS level increased (p=0.001) (Figure 3A, Table III). The addition of 10 ÌM more than 2-fold compared with As2O3 treatment. MK571 enhanced the sensitivity to As2O3 which showed no significant difference between HL60 and HL60/AD (Figure Discussion 3B, Table III). BSO increased the cytotoxic effect of As2O3 in HL60/AD more strongly than MK571 (0.26±0.05 ÌM vs. In this study, we demonstrated that exposure to As2O3 at 0.44±0.17 ÌM in IC50, respectively, p=0.011). the clinically achievable concentration (1-5 ÌM) inhibited cell growth in three different types of multidrug-resistant As2O3 induced apoptosis. The effect of As2O3 on the cell (MDR) leukemia cell lines. cycle was determined in K562, HL60 and their resistant cells Pgp is an energy-dependent efflux pump and related to using flow cytometric analysis of DNA content. The by decreasing intracellular drug accumulation.

993 ANTICANCER RESEARCH 25: 991-998 (2005)

Figure 2. The intracellular GSH level with or without BSO in K562 and K562/D1-9. After preincubation with or without 3 mM BSO for 72 h, the GSH content was assayed. The intracellular GSH content is expressed in nmol/1x106 cells. Open bar, without BSO; closed bar, with BSO. The values are presented as means±SD of three separate experiments. *p<0.05, **p<0.01 by Student’s t-test.

The MDR leukemia cells overexpressing Pgp were not resistant to the growth inhibition and apoptosis induced by As2O3 (17, 18). In our study, K562/D1-9 cells overexpressing Pgp showed about 100-fold more resistance to DNR and cross resistance to VCR, VP-16 and MIT. There was no significant cross resistance to As2O3 in K562/D1-9 cells Figure 3. Effect of BSO or MK571 on sensitivity to As2O3 in HL60 and HL60/AD. The cells were incubated at 37ÆC in the continuous presence of inversely about 3-fold more sensitive to As2O3 collaterally, compared with parental K562 cells. Moreover, Salerno et al. drug for 72 h with or without 3 ÌM BSO or 10 ÌM MK571. Cell viability was assayed by MTT assay. (A) HL60 treatment without BSO, ●; with (19) showed, using atomic absorption spectrometry, that BSO, ●; HL60/AD without BSO, ▲; with BSO, ▲. (B) HL60 treatment there was no difference in arsenic uptake by both K562 and without MK571, ●; with MK571, ●; HL60/AD without MK571, ▲; with ▲ K562/ADR overexpressing Pgp. Thus, As2O3 is not the MK571, . substrate of Pgp and cannot be effluxed by Pgp like VCR, or VP-16. Recently, Wei et al. (20) reported that K562/ADM cells overexpressing Pgp were more sensitive to As2O3 than parental K562 cells. Moreover, As2O3 inhibited Table III. Sensitivity of HL60 and HL60/AD to As2O3, with and without Pgp expression and cell growth and induced apoptosis in BSO or MK571. K562/ADM cells. K562/D1-9 cells are also known to be resistant to DNR due Cell line to topoisomerase II activity reduction (11). Topoisomerase II is a specific target for certain anticancer agents, such as DNR Drugs HL60 HL60/AD p value or VP-16, and decrease of topoisomerase II is known as the 1.40±0.34 4.54±0.10 <0.001 mechanism of atypical MDR (21). Therefore, As2O3 may BSO+ 0.51±0.07 0.26±0.05 <0.001 overcome atypical MDR. MK571+ 0.38±0.14 0.44±0.17 0.65 As2O3 has been shown to cause apoptosis in vitro through 4 the modulation of PML-RAR· protein and down- IC50 values (mM) were determined using the MTT assay. Cells (1x10 ) were seeded and exposed to As2O3 for 72 h with (BSO+, MK571+) regulation of Bcl-2 in NB4 cells of APL having the and without 3 ÌM BSO or 10 ÌM MK571. Values are means±SD of at t(15;17)(q22;q11) chromosome translocation (4). The least three independent experiments performed in triplicate. P value overexpression of Bcl-2 protein in colon cell lines did indicates the significance between HL60 and HL60/AD.

994 Seo et al: Arsenic Trioxide Can Overcome Multidrug Resistance

Figure 4. Effect of As2O3 on the using flow cytometry analysis of DNA content in K562, K562/D1-9, HL60 and HL60/AD. Cells were incubated in the absence or the presence of 5 ÌM As2O3, or 3 ÌM BSO alone, and 0.5 ÌM As2O3 plus 3 ÌM BSO for 72 h. The DNA contents were analyzed by flow cytometry. The percentage of sub-G1 cells at each condition was indicated.

not prevent As2O3-induced apoptosis (22). The Bcl-2- from attacking its intracellular targets by the formation of a transfected pre-B lineage leukemia cell line 697/Bcl-2 was transient As(GS)3 complex (28). Therefore, the intracellular about 1000-fold more resistant to dexamethasone and cross GSH content tended to be inversely correlated with the resistant to VCR, VP-16 and Ara-C (13)compared with sensitivity of the cells to As2O3 (22, 25, 29). In the present control virus-infected 697cells (697/Neo). In the present study, the decreased intracellular GSH level in K562/D1-9 study, 697/Bcl-2 did not show resistance to As2O3, was considered to be the reason for the collateral sensitivity compared with 697/Neo, which suggested that the Bcl-2 to As2O3 compared with the parental cell K562. gene did not confer resistance to As2O3-induced apoptosis. As mentioned above, the intracellular GSH level itself acts Several studies have reported that multidrug-resistant cells as one of the MDR factors. Ruiz-Gomez reported that Pgp over-expressing MRP1 are resistant to growth inhibition by expression, GSH level and glutathione S-transferase (GST) As2O3 (19, 23). MRP1 over-expressed human lung tumor cells activity increased simultaneously after exposure to colchicine were about 10-fold more resistant to As2O3 and 20 ÌM in a human colon cancer cell line (30). However, Benderra et MK571 almost totally reversed this resistance. The al. suggested that intracellular GSH in Pgp-mediated intracellular As content in resistant cells was decreased resistance dose not play an important role in contrast to that compared with parental cells (23). When MRP1 was in MRP1-mediated resistance (31). Increase of the GSH transfected without the GSH level increasing, MRP1 was not level appeared not to be indispensable to acquire drug able to efflux As (24). From these results, MRP1 was resistance. The decreased intracellular GSH level in suggested to mediate outwardly-directed transport of drugs K562/D1-9 may have occurred independently of over- and the intracellular GSH level might affect MRP1-mediated expressing Pgp and led to the collateral sensitivity to As2O3. anion transport (15). In the present study, the resistance to BSO is known to decrease the intracellular GSH level As2O3 in HL60/AD seemed to be caused by MRP1 over- through the inhibition of Á-glutamyl cysteine synthase activity. expression and increased intracellular GSH content (12). Two micromoles BSO enhanced As2O3-induced apoptosis Because the cross resistance was limited at 3-fold, As2O3 could and increased the production of ROS in hepatocellular be used for overcoming the MRP1-related drug resistance. carcinoma cells (32). A low concentration of 3 ÌM BSO Recently, it was reported that the GSH redox system resulted in about 50% depletion of the GSH level for 24 h in modulates both the antiproliferative and the apoptotic effects a rat epithelial cell line (33). This low concentration of of As2O3 (25, 26). Intracellular GSH scavenges reactive BSO can be safely administered to patients. Therefore we oxygen species (ROS) such as hydrogen peroxide and exposed each cell to 3 ÌM BSO and As2O3 simultaneously for hydroxyl radicals, protecting mammalian cells from ROS- 72 h. In the present study, 3 ÌM BSO enhanced the sensitivity mediated apoptosis. Elevation of the intracellular GSH to As2O3 in K562 and HL60 both parental and resistant cells. content is closely associated with drug resistance in some With the addition of BSO, the collateral sensitivity between malignant cell types (12, 27). GSH can prevent arsenic acid K562 and K562/D1-9 disappeared. This result supports the

995 ANTICANCER RESEARCH 25: 991-998 (2005)

Figure 5. Changes in the morphology induced by As2O3 in HL60. HL60, cultured with or without 1 ÌM As2O3 for 6 h, was subjected to May-Giemsa staining (x400). Nuclear fragmentation is indicated by arrows. A, HL60 treatment without As2O3; B, HL60 treatment with As2O3.

Figure 6. Flow cytometric analysis of apoptosis as assessed by Annexin V binding. HL60 was treated with or without 1 ÌM As2O3 for 6 h. Thin solid line, without As2O3; thick solid line, with As2O3. Figure 7. ROS were increased in HL60/AD compered to parent cells. After 120-min incubation with 3 ÌM BSO, HL60 and HL60/AD cells were treated with or without 1 ÌM or 10 ÌM As2O3 and DHR for 60 min. ROSs conclusion that As2O3 sensitivity is related to GSH levels. in the cells were determined on a FACScan as fluorescent intensities. Since the depletion of GSH can inhibit the MRP1 efflux pump and reduce drug resistance in HL60/AD similarly to MK571, one of the reversing agents of MRP, BSO appears to also be useful for circumvention of drug resistance. -resistant NTU-B1/P14 over-expressing GST- were A recent study reported that raising the intracellular ROS cross-resistant to As2O3 but, by the addition of 10 ÌM BSO, by depletion of GSH after treatment with BSO resulted in these resistant cells and parental cells became more sensitive the down-regulation of intrinsic Pgp expression (34). to As2O3 (29). In the present study, HL60/AD over- Furthermore, BSO pretreatment enhanced the - expressing MRP1 also showed a collateral sensitivity to induced DNA protein cross-links and potentiated the activity As2O3 by the addition of BSO. In HL60/AD, on simultaneous of camptothecin (35). In vivo, BSO potentiated more the treatment with As2O3 and BSO, the ROS level was increased therapeutic efficacy of doxorubicin against MRP1-over- 2-fold (Figure 7). This result seems to be elucidative that expressing tumor than parent tumor (36). Moreover, BSO induced the increase of As2O3 cytotoxicity in HL60/AD, etoposide-resistant MCF-7/VP over-expressing MRP1 and but not in HL60 cells.

996 Seo et al: Arsenic Trioxide Can Overcome Multidrug Resistance

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