Annals of Clinical & Laboratory Science 34:271-276 (2004)
© 2004 Association of Clinical Scientists
Arsenic Trioxide (As2O3) Sensitivity of Carcinoma Cell Lines and Cancer Cells from Patients with Carcinomatosis Peritonei
Yeonsook Moon1,
Gyeongsin Park2,
Yonggoo Kim2,
Myungshin Kim2,
Jihyang Lim2,
Soo Hwan Pai1,
Eun Jung Lee2,
Chang Suk Kang2 and
Kyungja Han2
1 Department of Clinical Pathology, Inha University Medical College, Seoul, Korea 2 Department of Clinical Pathology, Catholic University Medical College, Seoul, Korea
Address correspondence to Kyungja Han, M.D., Department of Clinical Pathology, Catholic University Medical College, St. Mary’s Hospital, Youngdeungpo-gu, Youido-dong 62, Seoul, Korea (South) 150-713; tel 82 2 3779 1297; fax 82 2 783 6648; e-mail: hankja{at}catholic.ac.kr.
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Abstract
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The effectiveness of As2O3 treatment was studied in 3 carcinoma cell lines, LoVo, OVCAR-3, and PA-1, and in cancer cells obtained from ascites fluids of 8 patients with carcinomatosis peritonei. LoVo, OVCAR-3, and PA-1 cell lines, and cancer cells from the patients were cultured in As2O3 gradient media; As2O3 sensitivity was evaluated by trypan blue dye exclusion and by morphologic examination after Wright staining. PA-1 was the most sensitive cell line to As2O3; OVCAR-3 and LoVo were resistant to As2O3. Cancer cells from 2 of 8 patients were sensitive to As2O3. The in vivo tumoricidal effect of As2O3 (100 µg/day, ip) was studied in 30 BALB/c nude mice following ip implantation of PA-1 tumor cells. The 17 As2O3-injected mice died of extensive intratumoral hemorrhage, necrosis, and hemorrhagic ascites within 48 hr after initial treatment. In 10 As2O3-untreated tumor-bearing control mice, only focal intratumoral hemorrhage and necrosis were noted. In summary, solid tumor cell lines and cancer cells from patients showed various As2O3 sensitivities in vitro, and As2O3 had a marked tumoricidal effect on PA-1 cells in vivo. These results suggest that As2O3 treatment might possibly be beneficial in patients with carcinomatosis peritonei who are resistant to conventional therapy and whose tumors show in vitro sensitivity to As2O3. However, to minimize the life-threatening tumor lysis effect, it would be better to administer As2O3 after removal of the peritoneal tumor masses.
(received 12 May 2004; accepted 24 May 2004)
Keywords: As2O3, carcinoma cell lines, carcinomatosis peritonei, cancer chemotherapy
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Introduction
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Arsenic compounds have been shown to be comutagens and cocarcinogens by epidemiological studies [1], but they also have a long history of therapeutic use in traditional Chinese medicine, as well as in ancient Greece and Rome. Arsenic trioxide (As2O3) has been shown to have a therapeutic effect in refractory or relapsed acute promyelocytic leukemia (APL) [2,3]. Evidence is growing about the therapeutic efficacy of As2O3 in hematopoietic malignancies [4–7], but evidence is scanty regarding the effectiveness of As2O3 in non-hematopoietic malignancies.
The majority of cancer deaths result from solid cancers rather than leukemia. Many cases of solid cancers progress to carcinomatosis peritonei, especially in terminal stage patients with carcinomas of the stomach, ovary, colon, and pancreas. Most of these patients show resistance to conventional anticancer therapies. There is no effective therapeutic modality in such cases. Recently, As2O3 has been reported to be effective in certain solid tumor cell lines (eg, gastric, esophageal, prostate, transitional cell, and ovarian carcinomas) [8–2]. There is no report about tumoricidal effects of As2O3 in cancer cells directly obtained from such patients, at doses corresponding to the clinically achievable plasma As levels observed during As2O3 treatment of APL patients [6,13].
To investigate the efficacy of As2O3 treatment of solid cancers, we studied the sensitivities to As2O3 of 3 carcinoma-derived cell lines, LoVo, OVCAR-3, and PA-1, and of cancer cells from ascites fluid of 8 patients with peritoneal carcinomatosis. We also performed an in vivo tumoricidal assay of As2O3 in nude mice after ip implantation of PA-1 cells.
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Materials and Methods
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Cancer cell line preparation.
Three human cancer cell lines, LoVo, OVCAR-3, and PA-1, were grown for 3 days in McCoy 5A medium supplemented with 10% (v/v) fetal bovine serum (GIBCO BRL, Grand Island, NY), 2 mmol/L L-glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin under an atmosphere of 95% air and 5% CO2 at 37°C in a humidified incubator. The cancer cells (1 x 105 cell/ ml) of each line were incubated for 24 and 48 hr in the same media containing 1 x 10–6, 5 x 10–6, 10 x 10–6, or 50 x 10–6 mol/L As2O3 (Sigma Chemical Co, St. Louis, MO), under the same atmospheric conditions. For control groups, each cell line was incubated in the same medium and conditions, but without As2O3.
Preparation of cancer cells from ascites of peritoneal carcinomatosis patients.
Ascites fluid samples from patients with peritoneal carcinomatosis were examined, and 8 samples that contained adequate numbers and populations of cancer cells (>10% nucleated elements) were selected for study. The primary lesions of the 8 cases were gastric carcinoma (n = 5), colon carcinoma (n = 1), ovarian carcinoma (n = 1), and endometrial carcinoma (n = 1). The nucleated elements of the ascites fluid samples were spun down and resuspended (1 x 105 cells/ml) in McCoy 5A media with graded concentrations of As2O3, as described above. The cells were incubated in each medium for 48 hr under an atmosphere of 95% air and 5% CO2 at 37°C in a humidified incubator.
As2O3 sensitivity assay.
After incubation, the cell suspensions were mixed with an equal volume of phosphate buffered saline solution (PBS) containing 0.4% trypan blue dye (Hayashi Pure Chemical Industries) and examined under a microscope. Dead cells stained blue and live cells were unstained (Fig. 1
); the percentages of dead and live cells in >200 cells in different microscopic fields were counted [14]. The morphologic changes indicative of apoptotic cells were examined by light microscopy after Wright staining (Fig. 2). The cancer cells were considered to be As2O3-sensitive cells when 90% died at an As2O3 concentration of 5 x 10 –6 mol/L after 48 hr incubation, which corresponds to a level that is clinically achievable with administration of 10 mg/day As2O3 to humans [6,13].
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Fig. 1 (top, left). Trypan blue dye exclusion test of PA-1 cell line, after incubation for 24 hr in medium that contained As2O3 (1 x 10–6mol/L), shows a few dead cells (stained) admixed with viable cells (unstained)(magnification x400).
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Fig. 2 (top, right). Adenocarcinoma cells in peritoneal fluid from case #1, following incubation for 48 hr in medium that contained As2O3 (5 x 10–6mol/L), shows morphologic changes typical of apoptosis, including cytoplasmic blebs and shrunken nuclei with a homogenous chromatin pattern. A normal appearing macrophage with reniform nucleus and normal chromatin pattern is also present (magnification x400).
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Assay of in vivo tumoricidal effect of As2O3.
Thirty healthy BALB/c nude mice (20 - 25 g, body wt) were purchased from Atsugi Breeding Center (Charles River Japan Inc, Tokyo, Japan). The mice were acclimatized for 1 week prior to the experiments in a room with an air-conditioned barrier-system and specific pathogen-free conditions. The mice were housed in polysulfone cages (5 mice/cage; Daejeong E&C, Korea) with autoclaved wood-shavings; they were fed 
-ray irradiated laboratory mouse food (A04-10, UAR Quality Assurance, Orge, France) and water containing minocycline.
The most sensitive cell line, PA-1, was selected for this study. The cultured tumor cells were suspended (5 x 106 cells/ml) in McCoy medium containing 2 mmol/L L-glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin. The cell viability was >95% by trypan blue dye exclusion test. One ml of tumor cell suspension (5 x 106 cells) was injected ip in each mouse. After 2 weeks, 27 mice showed ip masses and/or large amount of ascites; 3 mice did not have any evidence of tumor. Starting 5 days after the detection of peritoneal tumor, As2O3 (100 µg) was administered ip daily to 17 mice. When the mice died, autopsies were performed and the lung, liver, heart, stomach, kidney, and gonads were examined grossly and microscopically. All surviving mice were killed at 4 weeks after the injection of tumor cells.
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Results
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As2O3 sensitivity of cancer cell lines.
In all of the cancer cell lines, the percentages of dead cells were greater after exposure to As2O3 for 48 hr than after 24 hr and were incrementally increased according to the As2O3 exposure levels (Table 1). Of the 3 cell lines, PA-1 was the most sensitive to As2O3. Thus, 90% of PA-1 cells died after 24 hr exposure to an As2O3 concentration of 10 x 10–6 mol/L; 90% of the cells died after 48 hr exposure to an As2O3 concentration of 5 x 10–6 mol/L. Apoptotic cells showing nuclear or cytoplasmic shrinkage, nuclear fragmentation, cytoplasmic blebbing, and/or apoptotic bodies were frequently observed (Fig. 2
) when the proportions of dead cells were 90% in the trypan blue exclusion tests.
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Table 1. As2O3 sensitivity of 3 cell lines that were incubated for 24 and 48 hr in media that contained specified concentrations of As2O3.
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As2O3 sensitivity of cancer cells from ascites of patients with carcinomatosis peritonei.
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As indicated in Table 2, cancer cells from ascitic fluid samples of the 8 patients with carcinomatosis peritonei showed variable sensitivities to As2O3 by trypan blue dye exclusion tests (Fig. 3). After exposure to As2O3 concentration of 5 x10–6 mol/L, 1 of 5 gastric carcinoma cases and 1 colon carcinoma case showed high mortality (ie, 90% of cells dead at 48 hr). At 10 x 10–6 mol/L of As2O3, 3 gastric carcinomas and 1 colon carcinoma showed 90% dead cells at 48 hr. At 50 x 10–6 mol/L As2O3, 7 of the 8 carcinomas showed 90% dead cells at 48 hr.
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Table 2. As2O3 sensitivity of adenocarcinoma cells in the ascites of 8 patients with peritoneal carcinomatosis. The proportions of dead cells (%) were determined by trypan blue dye exclusion test after incubation for 48 hr in media that contained the specified concentrations of As2O3.
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Fig. 3 (bottom, left). Trypan blue dye exclusion test of adenocarcinoma cells in peritoneal fluid from case # 2, following incubation for 48 hr in medium that contained As2O3 (10 x 10–6mol/L), shows 90% dead carcinoma cells admixed with many erythrocytes (magnification x100).
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In vivo tumoricidal effect of As2O3.
In controls that did not receive As2O3 treatment, 2 of the 10 nude mice that developed ip tumors died between 2 to 3 weeks after the injection of AP-1 tumor cells. The remaining 8 mice were sacrificed at 4 weeks after tumor cell injection. At autopsy, a large amount of ascites and multiple tumor masses were found, The masses involved intra- and extra-abdominal organs, including the stomach, liver, pancreas, gonads, abdominal wall, heart, and lung. The tumor masses showed only focal areas of hemorrhage and necrosis (Fig. 4A); hemorrhagic ascites fluid was not found.
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Fig. 4 (bottom, right). Panel A: intact tumor cells in a tumor mass involving the liver of a control nude mouse (without As2O3 injection) sacrificed at 4 weeks after tumor cell injection. Panel B: tumor mass in the liver of a nude mouse that died 24 hr after As2O3 injection, showing extensive hemorrhage and tumor cell necrosis.
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All 17 nude mice that received daily ip As2O3 injections after tumor development died within 48 hr after the first As2O3 injection. At autopsy, a large amount of hemorrhagic ascites was found. Multiple tumor masses involved intra- and extra-abdominal organs, including the stomach, liver, pancreas, gonads, heart, and lung were also found. Extensive hemorrhage and necrosis were evident in the tumor masses (Fig. 4B).
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Discussion
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Arsenic compounds have long been used for medicinal purposes. They are known to predispose people to a variety of diseases, including cancers of the skin, bladder, lung, and liver [15], and also to cause neurotoxicity [16,17].
Recently, As2O3 has emerged as an anticancer agent for hematopoietic malignancies. As2O3 can provide an additional mode of treatment, especially for refractory and relapsed APL, and it has the advantage as an anticancer drug of having antidotes [18,19]. In theory, a sufficient dose of As2O3 could be administered to the patient to kill the tumor cells and after some time the remaining As2O3 could be excreted by antidotes. More patients with solid cancers suffer from resistance to conventional therapy than those with hematopoietic malignancies, but there is no appropriate alternative therapy for these patients. Previous reports suggested that As2O3 showed a growth inhibitory effect for cell lines of some solid cancers, such as esophageal, prostate, ovarian, and bladder carcinomas [9–12]. In the present study, not only the cancer cell lines, but also cancer cells directly obtained from ascites of some patients with carcinomatosis peritonei, were killed by As2O3 at concentrations reported to be clinically acceptable [6,13].
The sensitivities of As2O3 varied according to he cell line types. Among the 3 cancer cell lines, PA-1 was most sensitive to As2O3. An ovarian cancer cell line, OVCAR-3, was relatively sensitive, and a colon carcinoma cell line, LoVo, was resistant to As2O3. Zhang et al [8] also showed that As2O3 sensitivities and its growth inhibitory effects varied according to the cell lines. The tumoricidal effects of As2O3 for the 3 cancer cell lines and for the cancer cells from patients were both time- and dose-dependent, as in previous reports [8,20]. Cancer cells from 1 of 5 patients with carcinomatosis peritonei from gastric carcinomas showed As2O3 sensitivity at the concentration of 5 x 10–6 mol/L As2O3, those from 3 of 5 were killed at the concentration of 10 x 10–6 mol/L As2O3, and those from the other 2 patients were resistant to As2O3 at these in vitro exposure levels. This suggests that As2O3 sensitivity varies according to the individual case rather than the cancer cell type. According to previous reports, cancer cell lines showed various As2O3 sensitivities, but the mechanisms responsible for the variations in sensitivity to As2O3 have not been fully explained [8,14,21–23]. In hematopoietic malignancies, As2O3 may induce apoptosis by altering apoptosis gene expression, affecting the glutathione oxidation-reduction system, and interfering with microtubule function [5,24–26].
As2O3 exerts dose-dependent dual effects, triggering apoptosis at higher concentrations and inducing partial differentiation at lower concentrations [13,27]. In the present study, As2O3 triggered apoptosis of adenocarcinoma cells at various tested concentrations and the As2O3 exposures were related to the proportions of killed cells.
All 17 tumor-bearing nude mice that were injected with As2O3 died within 48 hr after the As2O3 injection, because of massive hemorrhage and necrosis in the tumor masses and/or bleeding into the peritoneal cavity. These results suggest that As2O3 caused a life-threatening tumor lysis effect in the tumor masses.
In conclusion, As2O3 showed various effects on 3 solid tumor cell lines and on cancer cells obtained from 8 patients, and also showed marked in vivo tumoricidal effect. Much more experimentation is necessary, but these results suggest that As2O3 treatment might possibly be beneficial in patients with carcinomatosis peritonei who are resistant to conventional therapy and whose tumors show in vitro sensitivity to As2O3. However, to minimize the life-threatening tumor lysis effect, it would be better to administer As2O3 after removal of the main tumor masses.
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Abstract
Introduction
