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Document Type and Number:
WIPO Patent Application WO/2000/021506
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The present invention relates to combination of glutathione modulators with antimony and/or arsenic for cancer therapy. More particularly, the present invention relates to a pharmaceutical composition for the treatment of cancer, which comprises a therapeutical amount of a glutathione (GSH) modulator with antimony and/or arsenic in association with a pharmaceutically acceptable carrier.

Application Number:
Publication Date:
April 20, 2000
Filing Date:
October 12, 1999
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International Classes:
A61K33/24; A61K33/36; A61K38/43; (IPC1-7): A61K31/00
Domestic Patent References:
Foreign References:
Other References:
DAI, JIE ET AL: "Malignant cells can be sensitized to undergo growth inhibition and apoptosis by arsenic trioxide through modulation of the glutathione redox system" BLOOD (1999), 93(1), 268-277 , XP000891685
AKAO, YUKIHIRO ET AL: "Arsenic trioxide induces apoptosis in neuroblastoma cell lines through the activation of caspase 3 in vitro" FEBS LETTERS (1999), 455(1,2), 59-62 , XP000891674
OCHI, TAKAFUMI ET AL: "Dimethylarsinic acid causes apoptosis in HL-60 cells via interaction with glutathione" ARCH. TOXICOL. (1996), 70(12), 815-821 , XP000901249
CHEN, ZHE-SHENG ET AL: "Reversal of heavy metal resistance in multidrug-resistant human KB carcinoma cells" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (1997), 236(3), 586-590 , XP000891680
SNYDER, RONALD D. ET AL: "Thiol involvement in the inhibition of DNA repair by metals in mammalian cells" MOL. TOXICOL. (1989), 2(2), 117-28 , XP000901255
KIM, HO-SHIK ET AL: "Intracellular glutathione level modulates the induction of apoptosis by.DELTA.12-prostaglandin J2" PROSTAGLANDINS (1996), 51(6), 413-425 , XP000891683
LEE, TE CHANG ET AL: "Resistance mechanism of an arsenic -resistant cell line" HO TZU K'O HSUEH (1989), 26(6), 488-96 , XP002134918
SHEN, ZHI-XIANG ET AL: "Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients" BLOOD (1997), 89(9), 3354-3360 , XP000891715 cited in the application
BUDAVARI ET AL (EDS): "The Merck Index" 1996 , MERCK &CO INC , WHITEHOUSE STATION NJ, USA XP002134919 page 252
DE BITTENCOURT, PAULO I. HOMEM, JR. ET AL: "Effects of the antiproliferative cyclopentenone prostaglandin A1 on glutathione metabolism in human cancer cells in culture" BIOCHEM. MOL. BIOL. INT. ( 1998 ), 45(6), 1255-1264 , XP000901409
Attorney, Agent or Firm:
Côté, France (Québec H3A 2Y3, CA)
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1. A pharmaceutical composition for the treatment of cancer, which comprises a therapeutical amount of a glutathione (GSH) modulator with antimony and/or arsenic in association with a pharmaceutically acceptable carrier.
2. The pharmaceutical composition of claim 1, wherein said glutathione (GSH) modulator is GSH depleting agent.
3. The pharmaceutical composition of claim 2, wherein said GSHdepleting agent is buthionine sulfoximine (BSO).
4. The pharmaceutical composition of claim 3, wherein BSO concentration is of about 4 to about 10 mM/kg, antimony concentration is of about 0 to about 20 mg/kg and arsenic concentration is about 0 to about 20 mg/kg, with the proviso that both antimony and arsenic concentrations are not together 0.
5. The pharmaceutical composition of claim 1, wherein said cancer is selected from the group consisting of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), breast cancer, prostate cancer, lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, other adenocarcinomas, squamous cell carcinomas, renal cell carcinoma and melanomas.
6. A method for the treatment of cancer, which comprises administering to a patient in need thereof a therapeutical amount of a glutathione (GSH) modulator with antimony and/or arsenic.
7. The use of a therapeutical amount of a glutathione (GSH) modulator with antimony and/or arsenic for the treatment of cancer.
PHARMACEUTICAL COMPOSITION OF GLUTATHIONE MODULATORS WITH ANTIMONY AND/OR ARSENIC FOR CANCER THERAPY BACKGROUND OF THE INVENTION (a) Field of the Invention The invention relates to a pharmaceutical composition for cancer therapy based on the combination of glutathione modulators with antimony and/or arsenic, method of treatment and use thereof.

(b) Description of Prior Art The effective treatment of human cancer currently presents the single greatest obstacle in Western medicine. As such, the search for novel therapies must remain a priority as our population continues to expand and age. A form of arsenic (As), arsenic trioxide (As203) has recently been proven to provide an effective treatment for the rare myeloid leukemia, acute promyelocytic leukemia (APL).

Acute promyelocytic leukemia (APL) represents about 10% of acute myeloid leukemia (AML) cases. APL is characterized by a specific differentiation block of the myeloid progenitor cells at the promyelocytic stage. At the molecular level, in the vast majority of cases (>95%), APL blasts harbor the balanced t (15; 17) chromosomal translocation which fuses the PML gene located on chromosome 15 to the retinoic acid receptor a (RARa) gene on chromosome 17 (Kakizuka, A et al.

(1991) Cell, 66: 663-674). The resulting PML-RARa fusion protein retains most of the functional domains of the parental PML and RARa proteins. The key role of the chimera in the differentiation block has initially been demonstrated in in vitro (Kakizuka, A et al. (1991) Cell, 66: 663-674) models and its leukemogenic potential has more recently been confirmed in transgenic mice. A unique feature of APL blasts is their ability to undergo terminal differentiation after retinoic acid

(RA) treatment in vitro as well as in vivo. Indeed, oral administration of all-transRA, the natural ligand for RARa, induces complete remission in t (15; 17) APL patients, making APL the first example of differentiation therapy in the treatment of advanced cancer (Warrell, R. P. Jr et al. (1991) N. Engl. J.

Med., 324: 1385). Several clinical studies have conclusively shown that the combination of RA with chemotherapy has improved the survival of patients with APL. Despite the broad success of RA therapy in APL, a significant percentage of patients relapse after initial remission and subsequently develop resistance to RA treatment. The clinical outcome of these patients is poor, as an effective substitute for RA is not yet available.

Arsenic and antimony are metals belonging to group Va of the periodic table of elements. Considered non-essential trace elements, these metals share several chemical and toxicological properties and occur naturally in both trivalent and pentavalent states.

The trivalent state of both arsenic and antimony generally demonstrate more toxic effects than their pentavalent counterparts (Stemmer, K. L. (1976) Pharmaco. Ther. A., 1: 157-160). Despite the cellular and organic damage associated with chronic exposure, however, both arsenic and antimony have found medical applications (Stemmer, K. L. (1976) Pharmaco. Ther. A., 1: 157-160). Antimony has, since the turn of the century, been used as a parasiticide and is still used for the treatment of schistosomiasis and leishmaniasis.

More recently, arsenic has been shown to induce clinical remission, with limited side effects, both in patients having retinoid-sensitive and-resistant acute promyelocytic leukemia (APL). One study reported a 90% success rate in the induction of complete remission in

relapsed patients treated with arsenic alone for a period of 28-41 days (Shen, Z. X. et al. (1997) Blood, 89: 3354-3360). In vitro studies in our laboratory have demonstrated that As203 induces apoptosis in both RA- resistant and sensitive APL cells in culture (Shao, W. et al. (1997) J. Nat. Cancer Ins., 90: 124-133).

Arsenic has since been shown to induce apoptosis in other cancer cell types as well, although to a lesser degree than the more sensitive APL cells.

It would be highly desirable to be provided with a novel approach for the treatment of cancer, including acute myeloid leukemias (AML), such as acute promyelocytic leukemia (APL).

SUMMARY OF THE INVENTION One aim of the present invention is to provide a novel approach for the treatment of cancer, including acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL).

One aim of the present invention is to provide a combination of glutathione modulators with antimony and/or arsenic for cancer therapy.

Our research into the mechanism of action of these compounds has yielded a novel approach that may allow antimony (Sb) and arsenic (As) to be employed against other acute myeloid leukemias and potentially against cancers of other sorts.

In accordance with the present invention, there is provided a pharmaceutical composition for the treatment of cancer, which comprises a therapeutical amount of a glutathione (GSH) modulator with antimony and/or arsenic in association with a pharmaceutically acceptable carrier.

In accordance with the present invention, the glutathione (GSH) modulator may be a GSH-depleting

agent, including, without limitation, buthionine sulfoximine (BSO).

In accordance with a preferred embodiment of the pharmaceutical composition of the present invention, BSO concentration is of about 4 to about 10 mM/kg, antimony concentration is of about 0 to about 20 mg/kg and arsenic concentration is about 0 to about 20 mg/kg, with the proviso that both antimony and arsenic concentrations are not together 0.

In accordance with the present invention, there is provided a method for the treatment of cancer, which comprises administering to a patient in need thereof a therapeutical amount of a glutathione (GSH) modulator with antimony and/or arsenic.

In accordance with the present invention, there is provided the use of a therapeutical amount of a glutathione (GSH) modulator with antimony and/or arsenic for the treatment of cancer.

For the purpose of the present invention the following terms are defined below.

The term"cancer"is intended to mean cancers of any sorts, including, without limitation, acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), breast cancer, prostate cancer, lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, other adenocarcinomas, squamous cell carcinomas, renal cell carcinoma and melanomas.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates the synergistic inhibition of myeloid leukemic cell growth by arsenic and BSO; Fig. 2 illustrates the synergistic inhibition of myeloid leukemic cells by antimony and BSO cotreatement ;

Fig. 3 illustrates the synergistic inhibition of breast cancer cell growth by arsenic and BSO; Fig. 4 illustrates growth response of APL and other malignant cell lines to arsenic and antimony; Fig. 5 illustrates induced expression of PML- RARa in the AML (non-APL) cell line, U937, did not sensitize the cells to AS203 ; Fig. 6 illustrates GSH depletion sensitizes APL and other malignant cell types to arsenic and antimony through the synergistic induction of apoptosis; Fig. 7 illustrates an arsenic-resistant NB4 subclone is cross resistant to antimony, yet can be sensitized to arsenic upon glutathione depletion; Fig. 8 illustrates baseline glutathione levels cannot predict tolerance to arsenic or antimony ; Fig. 9 illustrates dose-dependent GSH depletion by BSO directly correlates with cell death in response to arsenic ; Fig. 10 illustrates heavy metals activate AP-1, but glutathione depletion represses this metal-induced activation.

DETAILED DESCRIPTION OF THE INVENTION Acute promyelocytic leukemia (APL) is characterized by a specific t (15 ; 17) chromosomal translocation that fuses the genes encoding PML and the retinoic acid receptor a (RARa). The PML-RARa protein induces a block in the differentiation of the myeloid progenitor cells, which can be released by retinoic acid (RA) in vitro and in vivo. The RA-induced differentiation of APL blasts is paralleled by the degradation of the fusion protein and the relocation of wild-type PML from aberrant nuclear structures to its normal localization in nuclear bodies. Recently, arsenic trioxide (As203) treatment was proposed as an

alternative therapy in APL, as it can induce complete remission in both RA-sensitive and RA-resistant APL patients. Intriguingly, As203 was also shown to induce degradation of the PML-RARa chimera and to reorganize PML nuclear bodies.

We have new evidence that a similar compound of the related element, antimony (Sb), may also be effective against APL. We have recently discovered that Sb also induces apoptosis in cultured APL, AML, and cervical carcinoma cells (HeLa), and have considerable evidence suggesting that its mechanism of action is similar to that of arsenic.

Based on prior preliminary results, we examined whether biochemical modulation might potentiate the action of arsenic and antimony in other malignant cell lines. A recent abstract suggests that the particular sensitivity of NB4 cells to arsenic may be a result of the low level of reduced glutathione (GSH) present in these cells. Glutathione is a ubiquitously expressed tripeptide that serves as the largest source of non- protein thiol in mammalian cells. In keeping with its role as the cell's most important antioxidant, GSH is a substrate of glutathione peroxidases, which destroy organic and hydrogen peroxides. It also maintains the cell's reducing milieu, and is implicated in a variety of thiol-disulfide interconversions. A further major role for GSH is in the detoxification of endogenous and exogenous toxic compounds, including drugs and many environmental mutagens and carcinogens. In fact, cells resistant to a variety of chemotherapeutic drugs, particularly alkylating agents, often have an increased GSH content or disregulated GSH utilization pathways.

There is also some evidence that GSH can bind free arsenic, forming a transient As (GS) 3 complex and thereby preventing arsenic from attacking its

intracellular target. Antimony, on the other hand, is known to be conjugated to glutathione prior to its excretion from cells.

Therefore, we sought to determine whether biochemical modulation of intracellular GSH levels could potentiate the effects of arsenic and antimony in cancer cells intrinsically more resistant to these compounds than APL cells. We discovered that the relatively resistant, non-APL myeloid leukemic cell line PLB-985 could be rendered sensitive to arsenite when co-treated with the GSH-depleting agent buthionine sulfoximine (BSO), a selective inhibitor of a- glutamylcysteine synthetase, the rate-limiting enzyme in the synthesis of glutathione.

MATERIAL AND METHODS Cell culture and treatment of cells NB4 cells, NB4R4 and PLB-985 cells were cultured in RPMI medium (Gibco, BRL) supplemented with antibiotics, glutamine and 10% fetal calf serum. MCF-7 cells and S30 cells were grown in minimal essential medium alpha supplemented with antibodies and 5% fetal calf serum. The HeLa cell-line stably overexpressing PML (F) were as previously described. The cells were grown at 37°C in 5W C02 in Dulbecco's modified minimal essential medium (Gibco, BRL), supplemented with antibiotics, glutamine, 10% fetal calf serum and with G418 (Geneticin, Gibco, BRL) (750Lg/ml). As203 (Sigma) as a 1mM stock solution in PBS, PAS (Aldrich) and meglumine antimonate Glucantime, Rhone-Poulenc) as a 10pM stock solutions in H2O. Sb203 (Fluka) was dissolved in a minimal volume of HC1 and a 100pM stock solution prepared in 100pM Hepes, pH 7.5. BSO (Sigma) was dissolved in H20 at a concentration of 100 mM. Cells excluding Tryptan blue were counted at the indicated time following treatment initiation with hemocytometer.

Growth Assays NB4 and PLB-985 cells were seeded at 2x105 cells/ml in 6-well plates and HeLa cells at 1x104 cells/well in 24-well plates. Cells were treated with various concentrations of As203 (Sigma, Oakville, Ontario, Canada) or Sb203 (Fluka, Ronkonkoma, NY, USA) for seven days. Viable cells were counted by trypan blue (Gibco) exclusion with a hemacytometer. NB4 and PLB-985 cells were maintained at a density lower than 1x106 cells/ml through dilution as required, and HeLa cell media +/-treatment was replaced on every third day.

TUNEL assay Genomic DNA strand breaks characteristic of apoptosis were labeled in situ by terminal deoxynucleotidyl transferase (TdT) using an in situ cell death detection kit (Boehringer Mannheim, Laval, Quebec, Canada) according to the manufacturer's instructions. Briefly, cytospins containing 100,000 cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate. The cells were then exposed to the TUNEL reaction mixture containing TdT enzyme and fluorescein- labeled nucleotides, washed, and photographed under a fluorescence microscope.

Induction and detection of PML-RARa expression in U937- PR9 cells U937 cells stably transfected with a zinc- inducible PML-RARa expression vector were seeded at 1x103 cells/well in 96-well plates. Cells were treated with 100 VtM ZnS04 (Sigma) for 24 hours prior to the addition of As203 to induce expression of the fusion protein. Nuclear extracts were prepared by first washing lx107 cells twice with PBS and resuspending in 2 ml PTG buffer (5 mM NaH2PO4,1mM DTT, 10% glycerol,

lmM PMSF, 10 pg/ml each aprotinin and leupeptin, pH 7.4) at 4°C. Cells were then homogenized using a Dounce and pestle (pestle B) and the extracts transferred to centrifuge tubes. Following centrifugation for 30 minutes at 800g at 4°C, the nuclear pellet was washed with 1 ml PTG buffer and recentrifuged as above. Nuclear pellets were suspended in ice cold TTGK buffer (10 mM Tris-Cl, pH 8.5,1 mM EDTA, 1mM DTT, 10% glycerol, 0.8 M KC1,1mM PMSF, 10 Fg/ml each aprotinin and leupeptin) at 100 IlL/lx107 cells. The suspension was incubated at 4°C for 1 hour, and was resuspended at 15 minute intervals. Extracts were then centrifuged at 14,000 rpm in a microfuge at 4°C, and supernatants transferred to fresh tubes. To detect PML-RARD, 10 uL of nuclear extract (approximately 1x106 cells) was added to 10 cl sample buffer and run on a 10% SDS-polyacrylamide gel.

Proteins were transferred to nitrocellulose membranes (Bio-Rad, Mississauga, Ontario, Canada) and blocked with 25% fetal bovine serum in PBS containing 0.5% Triton X-100 for 1 hour at room temperature. The membrane was then hybridized overnight at 4°C with an antibody directed against the RARoeF domain (provided by Dr. P. Chambon). Following 3xl5-minute washes with PBS containing 0.5% Triton X-100, PML-RARa was visualized by hybridization with 125I-labelled protein A and subsequent autoradiography.

Toxicity Assays Cells were seeded at 1x103 cells/well/0.1 ml in 96-well plates. 24 hours after seeding, buthionine sulfoximine (Sigma) was added at a final concentration of either 25 M or 50 M to half of the wells from each plate, and media was added to all other wells. Cells were cultured overnight prior to the addition of heavy metal to allow for intracellular GSH depletion. As203

or Sb203 was then added at concentrations ranging from 5 nM to 100 M. Following a 96-hour treatment, cells were pelleted if necessary, fixed with 50% TCA, washed, and stained with sulforhodamine B (Sigma). Protein staining was then measured spectrophotometrically at 630 nm.

GSH assay Intracellular reduced glutathione (GSH) levels were assessed enzymatically with glutathione reductase.

10 Million cells were harvested, washed and lysed in 0.9 ml of 100 mM Tris-Cl by freeze-thaw cycling. Cell lysates were centrifuged and the supernatants transferred to eppendorf tubes containing 0.1 ml of 30% S-sulfosalicyclic acid. Following 15 minutes on ice, cell extracts were centrifuged for 2 minutes at 12,000 rpm and the supernatants transferred once again to new tubes. GSH levels per 100 ul sample were then measured spectrophotometrically by conversation of 5,5'-dithio- bis (2-nitrobenzoic acid) to its colored product upon reduction by GSH-dependent glutathione reductase.

AP-1 Transactivation Assay 2 x 106 HeLa cells stably expressing an AP-1 reporter gene construct, TRE6-TATA-CAT (Konig A et al., Blood 1997; 90 (2): 562-70), were seeded in 10 cm plates and allowed to attach overnight. Cells were then serum starved for 24 hours, and then treated with As203 (Sigma) or Sb203 (Fluka) +/-BSO (Sigma) for 16 hours.

Cell extracts were prepared by 3 freeze-thaw cycles, and CAT activity assessed as previously described (Rosenauer A et al., Blood 1996; 88 (7) : 2671-82).

Briefly, cell extracts standardized for protein content (Bio-Rad) were incubated with cold chloramphenicol (ICN, Costa Mesa, CA, USA) and tritiated acetyl-CoA (Mandel, Montreal, Quebec, Canada) at 37°C for 2 hours.

The generation of acetylated chloramphenicol was then

measured by scintillation counting following its extraction from free acetly-CoA by the organic diffusion method (Rosenauer A et al., Blood 1996; 88 (7): 2671-82).

RESULTS Following a 24 hour treatment, PLB-985 cells treated with both 1 tM arsenic trioxide and 100 p. M BSO had a viability of only 22.9%, while that of cells treated with As203 or BSO alone was 96.9% and 97.1%, respectively (Fig. 1). After 48 hours, the viable cell count in the co-treatment group was only 2.22% that of the untreated control. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis confirmed that this loss of cell viability was due to the induction of apoptosis. Furthermore, a GSH assay revealed that 100 pM BSO alone or in combination with arsenic was sufficient to reduce glutathione levels from 18.6 nmol/mg protein in control cell extracts to undetectable levels after a 24 hour treatment (Table 1).

Table 1 Effect of GSH depletion upon arsenic-induced apoptosis

Cell line Treatment % viability % viability 24 hours 48 hours 1 µM As2O3 79.5 40.7 NB4 100 µM BSO 81.8 80.2 1 µM As2O3 + 100 µM BSO 72.7 5.33 1 µM As2O3 96.6 83.0 PLB-985 100 pM BSO 97. 1 78.4 1 M As203 + 100 PU BSO 22. 9 2.22 1 µM As2O3 83.8 78.1 MCF-7 100 s1M BSO 87. 3 114 1 M As203 + 100 µM BSO 59.2 5.74 1 µM As2O3 61.8 10.5 S30 lOOjuMBSO10486.3 1 M As203 + 100 s1M BSO 18. 4 0.95 Effect of arsenic treatment upon intracellular GSH levels Cell line Treatment % viability % viability 24 hours 48 hours Control 33. 0- NB4 1 µM As2O3 22.9 22.6 lOOMBSO0.186 undetectable 1 µM As2O3 + 100 µM BSO 0.152 undetectable Control 18.6 - PLB-985 1 µM As2O3 20.6 0.200 100 ptM BSO undetectable 0.007 1 µM As2O3 + 100 µM BSO undetectable 0.043 Control 47.8 MCF-7 1 µM As2O3 65.2 34.2 100 RM BSO 20.4 6.16 1 µM As2O3 + 100 µM BSO 12.2 0.733 Control 8.62 S30 1 µM As2O3 18.8 7.05 100 ptM BSO 0. 801 undetectable 1 M As203 + 100 pM BSO undetectable undetectable

A similar result was obtained with antimony and BSO co-treatment, but with a slightly delayed response (Fig. 2). Here, a 72-hour treatment yielded 8% of cells viable in the co-treatment group, while antimony-or BSO-treated cells had 86.9% and 80. 8% viability, respectively (Fig. 2). Recently, this result has also been extended to the more common deadly malignancy, breast cancer (Fig. 3). Here we discovered that both

estrogen receptor-positive and negative breast cancer cell lines, some of which display considerable resistance to arsenic, undergo apoptosis when depleted of intracellular GSH at the time of 1 pM arsenic trioxide treatment (Fig. 3). For example, while MCF-7 cells cultured in the presence of arsenic were 78.1% viable after 48 hours, relative to an untreated control group, co-treated cells had a viability of 5.74% (Fig. 3). In vivo studies aimed at confirming the results of these cell culture experiments in tumor- bearing animals are currently in progress.

Clearly, modulation of glutathione levels provides a powerful means by which to sensitize tumor cells to the action of heavy metals. Because treatment with effective doses of arsenic alone may prove to be too toxic for the majority of malignancies outside of APL, this sensitization may allow for an increase in the anti-cancer activity of this heavy metal without an associated increase in toxicity. This is particularly plausible when one considers that many tumors have a seemingly increased need for glutathione, and thus when depleted of it may be placed at a disadvantage over normal cells. Furthermore, the novel combination of antimony and BSO may prove to be an equally effective treatment against APL and other malignancies, with potentially less toxicity than arsenic.

APL cells are uniquely sensitive to As203 and Sb203- induced apoptosis Our lab and others have previously documented the induction of apoptosis in the APL cell line, NB4, upon treatment with arsenic. In this report, we investigated the induction of apoptosis by As203 and Sb203 in several other hematologic and solid tumor cell lines. As Fig. 4 illustrates, NB4 cells were more sensitive to both arsenic and antimony than any other

cell line examined, including the non-APL myeloid leukemic PLB-985 cell line, and solid tumor-derived HeLa cell line. Cells were treated with the indicated concentration of arsenic trioxide (As203) (Fig. 4A) or antimony trioxide (Sb203) (Fig. 4B) for one week.

Viable cells were assessed by counting trypan blue- excluding cells. Each data point represents the average of triplicate or quadruplicate samples, and standard error bars are shown. As shown, 2 jjM AS203 and Sb203 led to 100% cell death in NB4 cells, while both PLB-985 and HeLa cell numbers continued to increase after 7 days of treatment with 2-3 pM AS203 or 5tM Induced expression of PML-RARa does not sensitize non- APL cells to heavy metals In spite of the increased sensitivity of APL cells to treatment with arsenic and antimony, we hypothesized that PML-RARO. expression is not a major determinant of cellular sensitivity to these agents. To test this hypothesis, we determined the sensitivity of the non- APL, myeloid leukemic U937 cell line, to As203 in the presence and absence of exogenously expressed PML-RARa.

U937-PR9 cells stably expressing the fusion protein under the control of a zinc-inducible promoter and mock-transfected U937-SN4 cells were seeded at lux103 cells/well in 96-well plates, and PML-RARa expression was induced by treatment with 100 pLM ZnS04 for 24 hours. Treatment with ZnS04 did not cause additional toxicity itself as the ICSO value for arsenic in control, mock-transfected SN4 cells was not affected by its presence. Induction of PML-RARa expression in PR9 cells was verified through western blotting, and the level of expression of this protein was found to be comparable to that in NB4 cells (Fig. 5A). 7-PR9 and -SN4 cells were treated for 24 hours with 100 jj. M ZnSO4

to induce the expression of PML-RARa. Western blotting with an antibody directed against RARa was performed to detect PML-RARa induction and to compare its level of expression relative to that of NB4 cells (A). Cells were then treated with As203, at concentrations ranging from 5 nM to 100 jn. M, for four days. The absence of a leftward shift in the toxicity curve for zinc-treated PR9 cells (Fig. 5B) demonstrates that expression of the fusion protein does not change the sensitivity of this non-APL cell line to arsenic. U937-PR9 cells were then treated for four days with As203, and survival was assessed in cells expressing PML-RARa or not (N and , respectively) as a measure of protein concentration per well with the sulforhodamine B assay (B).

Glutathione depletion sensitizes tolerant and sensitive cells to arsenic-and antimony-induced apoptosis We therefore investigated an alternative hypothesis suggested by reports that a lower GSH content in APL and other cells might mediate their sensitivity to arsenic and antimony. We tested the response of a variety of cancer cell types to As203 and Sb203 in combination with BSO, an agent that depletes intracellular GSH. We found that all cell lines examined could be sensitized to As203 and Sb203 by glutathione depletion, regardless of their inherent tolerance or sensitivity to metal alone. As Fig. 6A illustrates, pretreatment with 25 or 50 LM BSO sensitized cells to a four-day treatment with arsenic or antimony. (A) A variety of malignant cell types were treated overnight in the presence or absence of 25 or 50 SM BSO followed by a four day treatment with a range of concentrations of As203 (A, left column) or Sb203 (B, right column). Cell survival was assessed by the SRB assay and expressed as a fraction of untreated controls. The sensitization achieved upon GSH

depletion with BSO ranged from 4.3 to 75-fold for AS203 and 20 to 87.5-fold for Sb203 in the four cell lines examined. These included, in addition to NB4, an ER+ breast cancer cell line, MCF-7, an MCF-7-derived adriamycin-resistant line, NIH-ADR, and the cervical carcinoma cell line HeLa.

Although we have previously reported the induction of apoptosis by As203 in NB4 cells, we were interested in determining whether the synergism between BSO and arsenic or antimony was mediated by a synergistic induction of programmed cell death.

Evidence for the induction of apoptosis was obtained through Annexin V staining of arsenic treated NB4 cells, +/-BSO. Here we found that a 24-hour treament with 1 J. M As20s and 100 tM BSO significantly increased the number of apoptotic Annexin V-staining cells, as compared to either the arsenic or BSO-treated groups alone (Fig. 6B). (B) NB4 cells were pre-treated for 24 hours with 100 pLM BSO, followed by a 48 hour treatment with the indicated concentration of arsenic. Apoptosis was assessed by Annexin V-FITC staining, and apoptotic cells quantitated through flow cytometry. We also investigated the induction of apoptosis in the relatively arsenic-resistant AML cell line, PLB-985.

Fig. 6C shows a clear, time-dependent sensitization to 0.3 or 0.5 M As203 upon GSH depletion. (C) PLB-985 cells were co-treated with 25 M BSO (p) and 0.3 (~) or 0.5 pM As203 (+). Viable cells were counted by trypan blue exclusion with a hemacytometer over a period of five days. TUNEL analysis, shown in Fig. 6D, was performed to confirm that arsenic and BSO synergize in the induction of apoptosis in these cells, as shown for the more sensitive NB4 cell line. (D) Cytospins of 2 x 105 PLB-985 cells were prepared from cells treated for 48 hours with either 25 LM BSO, 1 M As203, or both.

Apoptosis was assessed by terminal nucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and visualized by fluorescence microscopy. Here a 48- hour treatment with 1 pM As203 caused only a slight increase in the number of apoptotic cells over the untreated control. Co-treatment with 25 M BSO, however, sensitized the cells such that a 1 M treatment induced a response similar to that induced by 10 pM As203 alone, corresponding to a roughly 10-fold sensitization by BSO.

Arsenic-resistant NB4 cells can be re-sensitized to heavy metals through GSH depletion Just as we have previously used retinoid resistant subclones of the APL cell line, NB4, to investigate mechanisms of response to RA, we developed arsenic-resistant subclones to address the question of how heavy metals exert their effects. These cells were generated by constant culture in the presence of As203 at concentrations that were slowly increased over time.

Once a population of cells were obtained that could be sustained in 1 pM As203, single clones were selected by plating in methylcellulose. The two clones that were selected for further expansion are now routinely cultured in the presence of 2 LM As203. As Fig. 7 illustrates, AsR3 cells are approximately 10-fold less sensitive to AS203, and they also show significant cross-resistance to Sb203. (Fig. 7A) Arsenic-resistant AsR3 and their sensitive parental NB4 cells were seeded at 1,000 cells/well in 98-well plates (+ and , respectively) and treated with a range of concentrations of arsenic (left panel) or antimony (right panel) for a period of four days. Cell survival was assessed by measuring protein content through the SRB assay, and expressed as a fraction of control groups. (Fig. 7B) Sensitization by GSH depletion was

demonstrated by plating cells as above, then treating for four days with arsenic following a 24 hour pre- treatment with 100 pM BSO. Despite this resistance, these cells could also be rendered sensitive to As203 (Fig. 7) and Sb203 by intracellular GSH depletion with a non-toxic dose of BSO.

Baseline glutathione levels alone cannot predict tolerance or sensitivity to metals, although upregulation of GSH may be a mechanism for resistance in As203-resistant NB4 cells To determine whether low GSH content could explain the unique sensitivity of APL cells to heavy metals, we assessed baseline GSH levels in several cell types and correlated these with their ICso values for a four-day treatment with As203 or Sb203. While we found no correlation between baseline GSH and inherent sensitivity or tolerance to metals among cells of different types (Fig. 8A), we did find that glutathione levels were elevated in the arsenic resistant cell lines, AsR2 and AsR3 (Fig. 8B). (Fig. 8A) Intracellular reduced glutathione levels were assessed in a variety of cancer cell lines having different IC50 values for AS203 and Sb203. GSH levels were measured indirectly by reduction of by GSH-dependent glutathione reductase, and expressed as nmol/mg protein. (Fig. 8B) GSH levels were also assessed in parental, sensitive NB4 cells and compared to those in two subclones of arsenic-resistant cells. GSH values from a representative experiment are shown, and are the mean of triplicate measurements.

Dose-dependent GSH depletion by BSO correlates with response of NB4 cells to As203 and Sb203 In order to determine whether sensitization to heavy metals by BSO results from GSH depletion, we correlated the dose-dependent suppression of GSH levels

in NB4 cells with the growth inhibitory effect by BSO plus As203. Fig. 9A shows a linear relationship between BSO concentration and depletion of intracellular GSH following a three day treatment with doses ranging from 0.1 to 10 pM. (Fig. 9A) NB4 cells were treated for three days with sublethal doses of BSO ranging from 0.1 to 10 pM, after which glutathione content was assessed.

NB4 cells were also co-treated with BSO doses in this range and 1 J. M As203. This dose-dependent depletion of GSH directly correlated with the degree of sensitization conferred by BSO, as shown in Fig. 9B, strongly suggesting that glutathione depletion sensitizes NB4 cells to arsenic and antimony. Cell survival was measured by counting trypan-blue excluding cells, and the values shown represent triplicate measurements (Fig. 9B).

As203 and Sb203 cause a dose-dependent increase in AP-1 activity in HeLa cells, and GSH depletion represses this metal-induced activation Based on previous reports that arsenic can induce phosphorylation of JNK, an activator of the AP-1 transcription factor, we investigated the ability of As203 and Sb203 to induce AP-1 activity. As Fig. 10 illustrates, 16 hour treatment with arsenic or antimony induced a strong dose-dependent activation of AP-1 activity, as measured by chloramphenicol acetyl transferase (CAT) activity in HeLa cells stably expressing a (TRE) 6-TATA-CAT reporter gene construct.

Treatment with 1,5, or 15 M As203 induced a 1.12-, 3.26-, and 22.6-fold induction, respectively. HeLa cells stably expressing the AP-1 reporter gene, TRE6- TATA-CAT, were serum-starved overnight and treated for 16 hours with the indicated dose of arsenic or antimony in the presence or absence of 100 M BSO. CAT activity was measured and normalized for protein content

following cell lysis. Treatment with the same concentrations of Sb203 triggered 1.11-, 2.78-, and 37.7-fold inductions. Because GSH depletion has independently been shown to activate JNK and AP-1, we investigated the ability of coupled glutathione depletion and heavy metal treatment to synergistically induce AP-1 transactivation. Interestingly, we found that pretreatment with 1000M BSO repressed AP-1 activation by both arsenic and antimony. This repression was complete in the case of antimony, while GSH depletion repressed arsenic-induced AP-1 activity to an intermediate level.

Discussion As203 has been reported to be an effective therapy against a specific hematologic malignancy, acute promyelocytic leukemia (APL), which is known to be uniquely responsive in vitro and in vivo to RA.

Recent in vitro results suggest that Sb203 may also have activity against APL cells. To date, however, arsenic and antimony have been less effective in vitro against other cancer cells. Because the PML-RARa fusion protein is the characteristic feature of APL, and since both RA and these metals induce its degradation, we examined whether the exogenous expression of this protein could modulate sensitivity to arsenic or antimony. By inducing the expression of PML-RARa from a zinc-inducible promoter in stably transfected U937 cells, we found that the expression of the fusion protein alone did not measurably increase sensitivity to arsenic. This is consistent with previous reports by our lab and others that a RA-resistant NB4 subclone, NB4.306, which lacks detectable PML-RARD expression, is equally sensitive to arsenic as its parental cell line.

These data suggest that the unique sensitivity of APL cells may result from their biochemical or cellular

background, not merely from the presence of the fusion protein. This leads to our hypothesis that modulation of the biochemical environment of the cell might increase the efficacy of arsenic and antimony against other, non-APL malignancies.

An alternative explanation for the unique sensitivity of APL cells is their reportedly low GSH content, as compared to that of cell lines of other hematological malignancies. GSH confers protection against xenobiotics and several chemotherapeutic agents, including alkylating agents and cisplatin, and plays a major role in maintaining intracellular redox potential. Also, all cancer cells tested, regardless of GSH content or their initial response to As203 or Sb203, could be sensitized by intracellular GSH depletion.

Thus, steady state GSH content appears to be only one factor dictating cellular resistance or sensitivity to heavy metals. The ability to synthesize GSH may be an additional factor in determining cellular tolerance to heavy metals, suggesting an important role for the enzymes in the biosynthetic pathway of GSH.

Also, the expression level of other proteins, including GSH utilizing enzymes (including glutathione S- transferases, GSTs) and those involved in metal ion chelating (metallothionein), free radical scavenging, and peroxide metabolism (GSH peroxidase, catalase) may play important roles. Because arsenic is exported from the cell in an ATP-dependent manner by the multidrug- resistance associated protein (MRP), high expression of this membrane pump could also cause metal resistance.

Each of these proteins has been shown to be inducible by arsenic, or overexpressed in some arsenic-resistant cell systems, and their relative abundance may exert a combined effect on overall As203 or Sb203 tolerance.

Expression of pGp, on the other hand, may not play a

role in arsenic or antimony resistance, as we find that an adriamycin-resistant cell line known to overexpress this membrane pump, NIH ADR, is not cross-resistant to these metals (Fig. 6A). Similarly, a cisplatin- resistant squamous cell carcinoma cell line displayed no increased resistance to arsenic or antimony, and was sensitized by BSO to the same degree as its cisplatin- sensitive parental cell line. These results do suggest that this combined therapy may be effective against tumors resistant to other chemotherapeutic agents.

To analyze the mechanism by which arsenic/antimony and BSO synergize in the induction of apoptosis, we first examined the ability of these drugs to modulate the transcriptional activity of the AP-1 transcription factor. AP-1 activity is induced both by mitogens and cellular stresses including oxidative stress and arsenite. As expected, we observed that both metals induced a dose-dependent activation of AP-1 in HeLa cells. On the other hand, although GSH depletion promotes an oxidative environment and BSO treatment has been shown to activate the SAP kinase JNK, BSO treatment did not increase AP-1 activity in HeLa cells.

This latter observation contrasts with the report that, under conditions of GSH depletion, baseline and tert- butylhydroquinone-induced AP-1 binding activity increased in HepG2 cells. AP-1 activity can be induced by three different MAP/SAP kinase cascades, which can have opposite effects on cell proliferation and apoptosis. It is possible that AP-1 activation in response to heavy metals may reflect a protective response which, when inhibited by glutathione depletion, may leave the cell more vulnerable to their toxic effects. This argument is supported by the finding that GSH depletion repressed the induction of AP-1 activity by antimony to a greater extent than that

by arsenic (Fig. 10), while consistently leading to a greater sensitization to antimony than that by arsenic (Fig. 6A).

Induction of apoptosis of mouse epidermal JB6 cells by high doses of arsenic has recently been shown to proceed through induction of the JNK SAP kinase. On the other hand, the extra cellular signal-regulated kinases (ERKs) are also induced in a delayed fashion, and activation of ERKs was found to mediate arsenic- induced transformation of JB6 cells. It will be of interest to assess which signalling pathways are modulated by BSO treatment. Indeed, BSO may synergize with arsenic or antimony by inhibiting the anti- apoptotic ERK pathway, or by activating pro-apoptotic pathways that do not induce AP-1.

In order for a treatments therapeutic index to be improved, its efficacy must be increased more than its associated toxicity. Although several studies have shown that malignant cells often have significantly elevated glutathione levels, GSH is also an important component of normal cell function. If GSH is depleted and repleted from various normal and tumor tissues with different kinetics, however, it may be possible to establish a protocol that optimizes antitumor efficacy and therapeutic gain. In fact, in KHT sarcoma-bearing mice, Siemann et al found that GSH levels in tumor cells were depleted more slowly than those in bone marrow cells, but also recovered more slowly than the normal marrow. This provided a window of time between the recovery of bone marrow cells and reestablishment of GSH levels in the tumor during which co-treatment with BSO and the alkylating agent, melphalan, enhanced the tumor cell death.

While the invention has been described in con- nection with specific embodiments thereof, it will be

understood that it is capable of further modifications and this application is intended to cover any varia- tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.