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Arsenic Trioxide-mediated Cytotoxicity and Apoptosis in Prostate and Ovarian Carcinoma Cell Lines

Departments of Medical Oncology and Hematology [R. U., U. A. S., C. S., B. K., S. B. O., E. G.], and Department of Immunology [E. T.], Ege University School of Medicine, 35100, Izmir, Turkey

	ABSTRACT

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We studied the effect of arsenic trioxide (As₂O₃) on prostate and ovarian carcinoma cell lines. As₂O₃ has been shown to be effective in leukemia, and acute promyelocytic leukemia in particular, both in vitro and in vivo. As model cell lines, we used DU145 and PC-3 for prostate cancer and MDAH 2774 for ovarian cancer. New modalities of treatment are essential in these kinds of cancers, which produce a high death toll. The 3-(4,5-dimethyl-thiazoyl-2-yl)-2,5diphenyl-tetrazolium bromide assay was used to evaluate cytotoxicity. Flow cytometric analysis and mono-oligo nucleosome detection-based ELISA were used to determine the apoptosis. Isobologram analysis was used to evaluate synergism and/or the additive effects of As₂O₃ and conventional chemotherapeutic agents. We clearly demonstrated that As₂O₃ has significant cytotoxic effect on both prostate and ovarian carcinoma cell lines. The dose range of As₂O₃ in all three cell lines was 10^-6 M. The mechanism underlying cytotoxicity of As₂O₃ was shown to be apoptosis. The experiments by butylated hydroxyanisole showed that the cytotoxic effect of As₂O₃ was not through superoxide generation. There was no synergism, but the additive effects of As₂O₃ were demonstrated with cisplatin, adriamycin, and etoposide. We strongly suggest that As₂O₃ alone or in combination with conventional chemotherapeutic agents be evaluated further as a new agent for the treatment of prostate and ovarian cancers.

	INTRODUCTION

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Ovarian and prostate cancers are commonly diagnosed malignities in humans, with a high death rate and poor prognosis, especially in refractory cases (1 , 2) . Tumor cells very often develop resistance to chemotherapy (3) . Whereas increased dosages of chemotherapeutic agents or high-dose ablative regimens are frequently more effective at tumor cell cytoreduction, progressive dose escalation of these agents is ultimately limited by their nonspecific end-organ toxicity.

Ovarian cancer is a model disease to investigate chemotherapeutic resistance because of both its intrinsic and acquired resistance to drugs. Approximately 75% of patients have surgically incurable disease at the time of diagnosis. Aggressive treatment of these patients with platinum-based combination chemotherapy usually results in severe adverse effects, and it is rarely curative (4) . Therefore, new effective and subtoxic therapeutic modalities are needed.

Prostate cancer is the most commonly diagnosed cancer in men, with an estimated 41,000 Americans dying annually from this disease (2) . Hormonal ablation is the main treatment for disseminated prostate cancer (5) . However, after a short period of time, prostate cancer commonly recurs, and the patients relapse with hormonally independent tumors. Furthermore, androgen-independent tumors also become resistant to a wide variety of cytotoxic drugs. Currently there is no therapy that has been shown to prolong the survival of these patients (6) . Therefore, new therapeutic approaches need to be developed for metastatic, hormone-refractory prostate cancer.

Arsenic agents have been used as anticancer agents in traditional Chinese medicine. In the 1970s, the effective component in the remedy was identified as As₂O₃.² Long-term clinical trials have indicated that As₂O₃ is very effective in the treatment of several types of leukemia, including APL (7) . It has been shown that As₂O₃ can induce clinical remission in patients with APL, including in those who have relapsed after retinoic acid treatment (8) . However, the mechanism whereby As₂O₃ targets the tumor cells is not clearly understood. Several studies indicated that As₂O₃ may be an oxidative agent that induces damage to DNA and causes DNA mutations (9) . Ex vivo studies on the APL cell line NB4 demonstrated that As₂O₃ down-regulates bcl-2 expression and induces apoptosis in the absence of apparent differentiation (10) . Moreover, a recent study showed that As₂O₃ has a specific effect on APL tumor cells by inducing the degradation of the nuclear receptor for retinoic acid fusion protein (10) . Although As₂O₃ may display a specific biological action on APL tumor cells, the apoptotic effect of As₂O₃ in leukemic cells has led investigators to propose that As₂O₃ may also induce apoptosis in other types of cancer cells.

The apoptotic effect of As₂O₃ on other myeloid leukemic cell lines (11) , megakaryositic leukemic cell lines (12) , and esophageal carcinoma cell lines (13) have been reported. In this study, we examined the cytotoxic and apoptotic effects of As₂O₃ on prostate and ovarian carcinoma cell lines. For this purpose, we used the ovarian carcinoma cell line MDAH 2774 and hormone-independent prostate carcinoma cell lines PC-3 and DU145. The rationale underlying the selection of ovarian and prostate carcinoma cell lines as model systems are the characteristic patterns of recurrences of these types of carcinomas. Ovarian cancer very often causes disseminated peritoneal metastasis, and prostate cancer recurs as an advanced local disease. Studying As₂O₃ cytotoxicity on these cell lines would allow the clinical investigators to try locoregional use of As₂O₃ in these tumors, which may cause less systemic toxicity. As₂O₃ is also foreseen as an optional treatment with these special indications, e.g., i.p. treatment. As₂O₃ is tolerated as a less systemically toxic drug by leukemia patients; hence, it may be an easy method of chemoprotection as well. With these factors in mind, we performed experiments using As₂O₃ in combination with other chemotherapeutic agents and alone. We also tried to bring to light the role of superoxide radical generation in As₂O₃-induced cytotoxicity by using BHA, an antioxidant, in these cancer cells.

	MATERIALS AND METHODS

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Tumor Cells
The human hormone-independent and drug-resistant prostatic carcinoma cell lines DU145 and PC-3 were purchased from American Type Culture Collection (Manassas, VA). The human ovarian carcinoma cell line MDAH 2774 was generously supplied by Dr. E. Aktar, Istanbul University, Istanbul, Turkey. These tumor cell lines were maintained in culture as adherent cells and cultured in RPMI 1640 (Sigma Chemical Co., St. Louis, MO) plus 10% heat inactivated FCS (Sigma Chemical Co.) added to 1% L-glutamine (Sigma Chemical Co.), 1% nonessential amino acids (Sigma Chemical Co.), 10,000 units/ml penicillin (Sigma Chemical Co.), and 10 mg/ml streptomycin (Sigma Chemical Co.). All lines were grown in a humidified atmosphere at 37°C in 5% CO₂. When the tumor cell lines were used as target cells, they were treated with trypsin-EDTA (Sigma Chemical Co.), washed, and resuspended in complete medium.

Reagents
Cisplatin, adriamycin, etoposide, BHA, MTT, DMSO, and PBS were purchased from Sigma Chemical Co. As₂O₃ was kindly supplied by F. Lermioglu, Ege University, Izmir, Turkey. It is a lyophilized, 99.5% pure, inorganic compound corresponding to the reported extracted drug used in Chinese herbal medicine. CycleTest-Plus DNA reagent KIT was purchased from Becton Dickinson (Mountain View, CA). Cell Death Detection ELISA^plus KIT was purchased from Boehringer Mannheim (Mannheim, Germany). Stock solution of As₂O₃, was prepared in distilled water. Stock solutions of cisplatin, adriamycin, and etoposide were prepared in DMSO (Sigma Chemical Co.); the DMSO concentration in the assay did not exceed 0.1% and was not cytotoxic to the tumor cells.

Cytotoxicity Assay
The MTT assay was used to determine drug-mediated cytotoxicity, as described previously (14 , 15) . For MTT assay, briefly, target tumor cells were resuspended in medium at 1 x 10⁵ cells/ml after verifying cell viability by trypan blue dye (Sigma Chemical Co.) exclusion assay. One hundred µl of cell suspension were distributed into each well of 96-well flat-bottomed microtiter plate (Greiner Labortechnik, Frickenhausen, Germany), and each plate was incubated for 24 h at 37°C and 5% CO₂ atmosphere. After the incubations, 100-µl reagent solutions or media at the desired concentrations were distributed into each well. The well containing only media served as a positive control. Treatments were performed in triplicate. Two hundred µl of the medium alone without cells and reagent were used as a negative control. The microtiter plate was incubated for the desired period of time. Thereafter, 20 µl of the MTT dye (5 mg/ml) were added into each well. The unreactive supernatants in the well were carefully aspirated and replaced with 100 µl of isopropanol (Sigma Chemical Co.) supplemented with 0.05 N HCl to solubilize the reactive dye. The absorbance (A) values of each well at 540 nm were read using an automatic multiwell spectrophotometer (Bio-Rad-Coda, Richmond, CA). The negative control well was used for zeroing absorbance. The percentage of cytotoxicity was calculated using the background-corrected absorbance as follows:

Experiments were performed at least three times with representative data presented.

Analysis of DNA Fragmentation
Flow Cytometric Analysis.
Commercially available CycleTest-Plus DNA reagent KIT (Becton Dickinson, Mount View, CA) was used to stain the cells by propidium iodide. Fluorescence data related to the DNA content of the cells in different cell cycles was collected with the Becton Dickinson FACSCalibur flow cytometer. Apoptotic DNA was gated and evaluated in relation to the cytotoxicity assay. Assays were performed at least three times, and data shown are representative of those assays.

DNA Fragment Detection by ELISA.
A cell death detection ELISA kit (Cell Death Detection ELISA^plus; Boehringer Mannheim) was used according to the manufacturer’s instructions for DNA fragment detection (16) . The DNA fragments are discrete multiples of a 180-bp subunit, which is detected as a DNA ladder on agarose gel. The enrichment of mono- and oligonucleosomes in the cytoplasm of the apoptotic cells is attributable to the fact that DNA degradation occurs several hours before plasma membrane breakdown. The principle on which this test is based is the detection of mono- and oligonucleosomes in the cytoplasmic fractions of cell lysates by using biotinylated antihistone- and peroxidase-coupled anti-DNA antibodies. The enrichment of mono- and oligonucleosomes released into the cytoplasm is calculated as absorbance of sample cells/absorbance of control cells. The enrichment factor was used as a parameter of apoptosis and is shown on the Y axis as mean ± SD of triplicates. Assays were performed at least three times, and data shown are representatives of those assays.

	Statistical Analysis

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All assays were set up in triplicate, and the results were expressed as the mean ± SD. Statistical analysis was determined by Student’ s t test. For synergy, isobolograph analysis was performed according to Berenbaum (17) . The isobole has been used to evaluate the presence of synergism or antagonism in many fields. It requires experimental data for agents used alone and in different dose combinations at equieffective levels. These data are plotted on isoeffective graphs with the axis representing the doses of each agent. If two agents do not interact, the line forming the point corresponding to the combination of those on the axis representing doses isoeffective with the combination will be a straight line. When agents in combination are more effective than what might be expected from their dose-response curves (synergy), smaller amounts will be needed to produce the effect under consideration, and a concave-up isobole results. On the other hand, when agents in combination are less effective than expected (antagonism), greater doses than expected will be needed to produce the same effect, and a concave-down isobole is generated. The fraction of inhibitory concentrations was calculated as the percentage of each treatment to effect a fixed level of cytotoxicity.

	RESULTS

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Sensitivity of Human Prostate Carcinoma Cell Lines and Human Ovarian Carcinoma Cell Line to As₂O₃.
Two hormone- and drug-resistant human prostate carcinoma cell lines, DU145 and PC-3, and an ovarian carcinoma cell line, MDAH 2774, were selected for the study. They were treated with different concentrations of As2O₃, and cytotoxicity was determined by the MTT assay. Cytotoxicity was determined every 24 h for three days. All cell lines were sensitive to As₂O₃-mediated cytotoxicity, and maximum cytotoxicity was achieved at 72 h. With the increasing concentrations of As₂O₃, cell death increased. At 5 x 10^-6 M concentrations of As₂O₃, more than 50% of the cells were dead in all cell lines at 72 h, as shown in Fig. 1 . The experiments clearly showed that As₂O₃ induces cytotoxicity in DU145 and PC-3 prostatic carcinoma cell lines and in the MDAH 2774 ovarian carcinoma cell line in direct proportion to the time and concentration.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Cytotoxicity of tumor cells treated with different concentrations of As2O3. Cytotoxicity was assessed by MTT assay after a 72-h culture. The data represent the mean of three different experiments with a SD not exceeding 5%.

We further investigated whether the cytotoxic effect was by apoptosis. For this purpose, cell suspensions with deferring concentrations of As₂O₃ were stained with propidium iodide at 72 h and evaluated by flow cytometry. As seen in the flow cytometric photographs in Fig. 2 , representative of the three cell lines, in contrast to the control, there was a clear shift in the degraded DNA with a nonspecific cell cycle phase pattern. Also, the mono- and oligonucleosomes were evaluated at 24 h by ELISA. The reason for the different time points between flow cytometry and ELISA was the emergence of nucleosomes approximately 24 h before the complete DNA fragmentation, which can be detected by flow cytometry. With ELISA, the increasing concentrations of As₂O₃ demonstrated an increased enrichment factor, indicating the increased mono- and oligonucleosomes with the incubation time. Fig. 3 presents the data related to MDAH 2774 and is representative of the apoptosis also occurring in prostatic cancer cell lines DU145 and PC-3.

View larger version (71K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. As2O3 induces apoptosis of human ovarian carcinoma cell line MDAH 2774. Cells were treated with 5 x 10-6 M As2O3 for 72 h, and As2O3mediated apoptosis was determined by flow cytometric analysis. a, control; b, As2O3, 5 x 10-6 M).

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. As2O3-induced apoptosis in human ovarian carcinoma cell line MDAH 2774. Cells were treated with different concentrations of As2O3 for 24 h. Apoptosis was examined by determining nucleosomal DNA fragmentation using an apoptosis determination kit. Rate of apoptosis is reflected by enrichment of nucleosomes in the cytoplasm shown on the Y axis. Data presented show one representative of three independently performed experiments.

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. The effect of BHA on As2O3-mediated cytotoxicity against DU145 cancer cell line. Cytotoxicity was determined in a 72-h MTT assay. The results are derived from three different experiments (SD < 5).

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Cytotoxicity and isobolograph analysis of treatment with combination of As2O3 and cis-diamminedichloroplatinum (II); of PC-3 cells. Cytotoxicity was determined by MTT assay in a 72-h culture. The results are expressed as the mean of three different experiments (SD < 5).

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Cytotoxicity and isobolograph analysis of treatment of PC-3 cells with combination of As2O3 and adriamycin. Cytotoxicity was determined by MTT assay in a 72-h culture. The results are expressed as the mean of three different experiments (SD < 5).

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. Cytotoxicity and isobolograph analysis of treatment of PC-3 cells with combination of As2O3 and etoposide. Cytotoxicity was determined by MTT assay in a 72-h culture. The results are expressed as the mean of three different experiments (SD <5).

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Fig. 8. Effect on cytotoxicity of sequential treatment with either As2O3 or cisplatin. DU145 tumor cells were pretreated with medium or with the desired concentrations of As2O3 for 24 h and thereafter, the medium was aspirated, and the tumor cells were washed twice with RPMI 1640 and subsequently incubated with medium or with the desired concentrations of cisplatin for 48 h; cytotoxicity was assessed by MTT assay (a). Then, DU145 cells were pretreated with medium or with the desired concentrations of cisplatin for 24 h and thereafter, the medium was aspirated, and the tumor cells were washed twice with RPMI 1640 and subsequently incubated with medium or the desired concentrations As2O3 for 48 h; cytotoxicity was assessed by MTT assay (b). The data represent the mean of three different experiments with a SD not exceeding 5%.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Fig. 9. Effect of sequential treatment with either As2O3 or adriamycin on cytotoxicity. DU145 tumor cells were pretreated with medium or with the desired concentrations of As2O3 for 24 h and thereafter, the medium was aspirated, and the tumor cells were washed twice with RPMI 1640 and subsequently incubated with medium or desired concentrations adriamycin for 48 h; cytotoxicity was assessed by MTT assay (a). Then, DU145 cells were pretreated with medium or with the desired concentrations of adriamycin for 24 h and thereafter, the medium was aspirated, and the tumor cells were washed twice with RPMI 1640 and subsequently incubated with medium or with the desired concentrations of As2O3 for 48 h; cytotoxicity was assessed by MTT assay (b). The data represent the mean of three different experiments with a SD not exceeding 5%.

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Fig. 10. Effect of sequential treatment with either As2O3 or etoposide on cytotoxicity. The DU145 tumor cells were pretreated with medium or with the desired concentrations of As2O3 for 24 h and thereafter, the medium was aspirated, and the tumor cells were washed twice with RPMI 1640 and subsequently incubated with medium or with the desired concentrations of etoposide for 48 h; cytotoxicity was assessed by MTT assay (a). Then, DU145 cells were pretreated with medium or with the desired concentrations of etoposide for 24 h and thereafter, the medium was aspirated, and the tumor cells were washed twice with RPMI medium and subsequently incubated with medium or with the desired concentrations of As2O3 for 48 h; cytotoxicity was assessed by the MTT assay (b). The data represent the mean of three different experiments with a SD not exceeding 5%.

	DISCUSSION

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The mechanisms underlying cytotoxicity and differentiation seem to be completely different from each other. At higher concentrations with a considerable cytotoxicity, apoptosis appeared to be the main phenomenon resulting in significant cell death. In our experiments, we have clearly demonstrated that apoptosis was induced both in prostate and ovarian carcinoma cell lines with significant cytotoxicity. It is not known how As₂O₃ induces apoptosis in these cell lines. In previous reports, it was shown that superoxide generation was the main of mechanism of apoptosis by As₂O₃ (9 , 23 , 24) . However, our experiments using BHA, which is a strong antioxidant agent (18) , demonstrated that BHA could not prevent cytotoxicity by As₂O_3, indicating that in prostate and ovarian carcinoma cell lines, apoptosis is induced by a different mechanism, rather than by superoxide generation. For example, it has been shown that in certain leukemia cell lines, thiol diester bonds were lysed by As₂O₃, and that was the main mechanism underlying cytotoxicity (7) . As₂O₃, may also induce its apoptotic effects by means of its interaction with the cytoskeleton and tubuli system of the cell, which has been reported in leukemic cell lines (25) .

In APL, it has been shown that As₂O₃ degrades abnormal PML-RAR fusion protein and induces bcl-2 down-regulation (10) . There is the possibility that in prostate and ovarian carcinoma cell lines, bcl-2 down-regulation can be induced by As₂O₃.

At the beginning of our study, we thought that As₂O₃ would interact with conventional chemotherapeutic agents synergistically, but we could not demonstrate any synergism with the cisplatin, adriamycin, and etoposide. However, the additive effect demonstrated with the isobologram analysis opens a window for new combined modality approaches to achieve augmented cytotoxicity in treating prostate and ovarian carcinomas. But these combinations may result in greater toxicity, and thus, before strong recommendation or use with patients, they should be tried in animal models. Also, we showed that treatment of cancer cells with As₂O₃ and the other chemotherapeutic agents, whether sequential or nonsequential, did not cause a significant difference in the total cytotoxicity. This suggests ease of use of different chemotherapeutic agents with As₂O₃ resulting in the desired total net effect but with fewer adverse effects.

Ovarian and prostate cancers are very common and account for a considerable death toll. Although life expectancy has increased thanks to conventional chemotherapeutic drugs, the number of patients unresponsive to such treatments is also increasing. New modalities of treatment are essential for these groups of patients. As₂O₃ is a new option for these types of cancers, as has been shown in our study. A second use for As₂O₃ may be the i.p. local administration in these desperate patients. Because most patients have disease limited to the i.p. cavity, using As₂O₃ i.p. may lessen systemic toxicity while increasing cytotoxicity against ovarian carcinoma. The high molecular weight of As₂O₃ provides the opportunity for this type of administration. Additional pharmacokinetic studies are warranted.

In conclusion, As₂O₃ is a very promising new drug for use in prostate and ovarian cancers.

	ACKNOWLEDGMENTS

 
We thank Drs. F. Buyukkececi, G. Saydam, and C. Cal for their generous support of our study.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Ege University School of Medicine, Department of Medical Oncology, 35100 Bornova, Izmir, Turkey. Fax: 90-232-374-7321; E-mail: uslu{at}alpha.med.ege.edu.tr

² The abbreviations used are: As₂O_3, arsenic trioxide; APL, acute promyelocytic leukemia; MTT, 3-(4,5-dimethyl-thiazoyl-2-yl)-2,5-diphenyl-tetrazolium bromide; BHA, butylated hydroxyanisole.

Received 6/27/00; revised 9/26/00; accepted 9/26/00.

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