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Arsenic trioxide (As2O3) inhibits peritoneal invasion of ovarian carcinoma cells in vitro and in vivo
Gynecologic Oncology 103 (2006) 199 – 206 www.elsevier.com/locate/ygyno
Arsenic trioxide (As2O3) inhibits peritoneal invasion of ovarian carcinoma cells in vitro and in vivo Jingjing Zhang ⁎, Bo Wang Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan 250012, Shandong, China Received 21 December 2005 Available online 19 April 2006
Abstract Objectives. To study the role of arsenic trioxide (As2O3) in regulating peritoneal invasive activity of ovarian carcinoma cells in vitro and in vivo. Methods. The effects of As2O3 on human ovarian cancer cell lines (3AO, SW626 and HO-8910PM) migration, invasion and adhesion with tumor cells and human peritoneal mesothelial cells (HPMC) were observed by means of cell migration test, cell invasion test and cell adhesion test. The effects of As2O3 on MMP-2, MMP-9, TIMP-1 and TIMP-2 gene expressions and protein expressions of tumor cells were determined by RT-PCR and ELISA, respectively. In animal experiments, ovarian tumor cells were implanted into abdominal cavity of nude mice and then the nude mice were treated by intraperitoneal injection of different doses As2O3. The foci on the surface of peritoneum were counted. Results. As2O3 inhibited tumor cells migration, invasion and adhesion with HPMC in a dose-dependent manner, while the same treatment enhanced tumor cell–tumor cell interactions. As2O3 inhibited mRNA and protein expressions of MMP-2, MMP-9 and TIMP-2 of tumor cells. In contrast, As2O3 increased mRNA and protein expressions of TIMP-1. As2O3 could reduce tumor cells peritoneal metastasis in nude mice. Conclusion. As2O3 inhibits in vitro and in vivo peritoneal invasive activity of ovarian carcinoma cells in a dose-dependent manner. Its antiinvasive activity may be the results of reduced cell motility, inhibited attachment of tumor cells to HPMC and enhanced tumor cell–tumor cell interaction, as well as down-regulation of MMP-2 and MMP-9 levels and up-regulation of TIMP-1 level. © 2006 Elsevier Inc. All rights reserved. Keywords: Arsenic trioxide; Ovarian neoplasms; Peritoneum; Metastasis
Introduction Ovarian cancer is the second most frequent gynecological cancer. In 60–70% of patients, ovarian tumors are diagnosed at advanced stages only, since symptoms are absent and a specific tumor marker is lacking. Ovarian cancer, which originates in the surface epithelium in about 97% of the cases, spreads prevalently by direct extension of the tumor to adjacent tissues and by shedding of tumor cells from primary tumor into the peritoneal cavity. Although peritoneal disseminated metastasis is often associated with malignant ascites and a poor prognosis, no effective therapy exists at present. As2O3 is an active ingredient of a traditional Chinese medicine that has been used successfully for treating acute promyelocyte ⁎ Corresponding author. Present address: Department of Obstetrics and Gynecology, Qingdao Municipal Hospital, Medical College of Qingdao University, Qingdao 266011, Shandong, China. E-mail address: [email protected] (J. Zhang). 0090-8258/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2006.02.037
leukemia(APL) [1] and has been shown to be an effective inducer of apoptosis in some solid cancer cells, such as esophageal, prostate and ovarian carcinoma [2,3]. However the effects of As2O3 on the tumor cell peritoneal invasiveness has seldom been reported. The steps required for ovarian carcinoma metastasis include the following: (1) separation of cells from the primary tumor mass on the ovarian surface; (2) passive movement of the tumor cells through the peritoneal fluid; (3) adherence of the cells to the mesothelium, which lines the peritoneal cavity; (4) migration of tumor cells past the mesothelium into the stroma; (5) enzymatic digestion or remodeling of the underlying stroma by proteinases; (6) growth of the newly established tumor cells in their new location. So adhesion, motility and proteolysis are important factors involved in ovarian carcinoma cells peritoneal invasion. Matrix metalloproteinases (MMPs) are a family of structurally related neutral metalloproteinases, which together can degrade all the components of the extracellular matrix proteins (ECM) and are involved in tumor invasion and metastasis. Among the MMPs, the 72 kDa gelatinase A (or
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MMP-2) and 92 kDa gelatinase B (or MMP-9) are believed to be the critical enzymes for degrading type IV collagen, a major component of basement membrane, which lies under peritoneal mesothelial cells, thus playing a critical role in cancer cell invasion and metastasis. Their catalytic activity is tightly controlled by endogenous inhibitors, including tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. In the present study, we investigated the effect of As2O3 on adhesion, motility, expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2, and peritoneal invasive activity of ovarian carcinoma cells in vitro and in vivo, to clearly demonstrate the anti-invasive activity of As2O3 and to provide evidence for exploring drugs for controlling the peritoneal metastasis of ovarian tumor cells. Materials and methods Cell culture Human ovarian cancer cell lines 3AO and SW626 cells were obtained from the American Type Culture Collection (Rockville, MD, USA), and HO-8910PM cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Science. Tumor cells were maintained in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a cell culture incubator at 37°C in 5% CO2. Human peritoneal mesothelial cells (HPMC) were isolated from the greater omentum in patients operated on for a nonperitonitis cause and without disseminated cancer and cultured as previously reported with minor modification [4]. Human omental tissue was obtained from surgically resected specimens from patients with informed consent. The omentum was washed in several changes of sterile phosphate-buffered saline (PBS), and finely divided into approximately 1-mm2 segments. These were washed in PBS twice to remove any contaminating red blood cells. The tissue was then treated with a 0.25% trypsin-EDTA solution for about 20 min. The cell suspension was centrifuged at 1000 rpm for 10 min, and collected cells were cultured in RPMI1640 medium supplemented with 20% FBS, 0.4% dermacort and 10 μg/ml insuline at 37°C in 5% CO2. HPMC were confirmed by immunohistochemistry with antibodies against cytokeratin, vimentin, CD45 antigen and factor VIII (Signet, Dedham, MA, USA). Passage 2 cultures were used for experiments.
Tumor cells were labeled with calcein as described above. The labeled tumor cells were washed to remove free dye and incubated with indicated concentrations (0.5 μM, 1.0 μM, 2.0 μM) of As2O3 for 24 h. Mesothelial and tumor monolayers were established in a 96-well plate. The calcein-labeled, As2O3 -treated tumor cells (2.5 × 104) were seeded additionally. After plates were incubated at 37°C for 60 min, the medium of each well was removed and washed twice with RPMI-1640 medium. The remaining fluorescence per well was measured on a Perkin Elmer plate reader, using 485 excitation and 530 emission filters. On each plate, a standard was prepared by adding different numbers of labeled tumor cells to the wells. The amount of adherent tumor cells was determined by calibrating the measured fluorescence of the experimental wells in relation to the standard.
Migration assays To determine the effect of As2O3 on the migration of tumor cells, a woundmigration assay was performed. Tumor cells were cultured on a 12-well plate. When cells were 80% confluent, monolayers were wounded with a 200 μl micropipette tip, washed twice with PBS, and incubated for 24 h with serum-free medium in the presence or absence of indicated concentrations (0.5 μM, 1.0 μM, 2.0 μM) of As2O3. After incubation, cells were washed with PBS. Migration was quantified by counting the number of cells that migrated from the wound edge into the denuded area for a distance of 1 cm.
Cell invasion assay An invasion assay was performed according to the method reported by Hirashima et al. [6]. A millicell (Sigma) with an 8-μm porosity cell-permeable polycarbonate filter was covered with recombinant basement membrane Matrigel (Sigma) and was placed on a 24-cell culture plate. HPMC were cultured fully on Matrigel, and washed, and 2 × 105 tumor cells pretreated with or without As2O3 were then added into the millicell. As a chemoattractant, NIH3T3 fibroblast conditioned medium was used outside of the millicell. After cultivation for 12 h, the millicell was immersed in 100% methanol for 1 min for fixation, and all cells were then stained by hematoxylin. The cells remaining on the top surface of the filter were completely removed with a cotton swab, and the filter was removed from the millicell and mounted on a glass slide. These preparations were examined under a microscope at ×400 magnification. The number of infiltrating tumor cells was counted in five regions selected at random, and the extent of invading tumor cells was determined by the mean count.
Cytotoxicity assays
RT-PCR
In vitro cytotoxic effects of As2O3 (Sigma) on tumor cells was determined by using CellTiter 96 Non-Radioactive Cell Proliferation Assay kit (Promega, Madison, WI). Briefly, cells growing in plates were dispersed in 0.05% trypsin solution and resuspended in RPMI-1640 containing 10% FBS. Approximately 5000 cells were added to each well of 96-well plate and incubated for 18 h. After changing the media with or without 10% serum, various concentrations (0.5 μM, 1.0 μM, 2.0 μM) of As2O3 were added to each well. After 48 h of incubation, cell cytotoxicity was estimated by MTT based assay following the supplier's instructions.
Total RNA was extracted from tumor cells, pretreated with or without indicated concentrations of As2O3 for 24 h, by Trizol (Invigen) according to the manufacturer's protocol. Reverse transcription of RNA was performed in a final reaction volume of 20 ml containing 5 μg of total RNA in Moloney murine leukemia virus (MMLV) reverse transcriptase buffer (Promega, Madison, WI, USA). A 1 μl portion of the reaction mixture was then amplified by PCR with the pairs of primers (Table 1). A total of 30 cycles were performed, with each cycle comprising 1 min at 94°C, 1 min at 50–60°C depending on each gene, and 1.5 min at 72°C with a final extension of 8 min at 72°C. Human β-actin reactions were amplified for 25 cycles. The reaction products were separated on 2% agarose gel, stained with 1 μg/ml ethidium bromide, and visualized using a Digital imaging system. Semi-quantitative analysis was performed using the ratio of the genes tested and β-actin.
Calcein-AM solution and incubation According to Van Rossen et al. [5], the dye solution, calcein-AM, used to quantify tumor cell adhesion, was prepared by dissolving 50 μg of calcein (Molecular Probes, Leiden, The Netherlands) in 5 μl of anhydrous dimethyl sulphoxide (Sigma) and adding this solution to 5 ml of RPMI medium supplemented with 0.5% bovine serum albumin (Sigma). Trypsinized tumor cells (1 × 106 cells/ml) were incubated in RPMI/0.5% BSA at 37°C for 45 min, with occasional mixing.
Enzyme-linked immunosorbent assays (ELISA) After tumor cells were pretreated with or without As2O3 for 24 h, the supernatant was collected. The levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 were measured in the supernatants using the ELISA kit (R&D Systems) according to the manufacturer's instructions.
Adhesion assay Animal experiments To quantify tumor cells adhesion to the tumor cells (homotypic adhesion) and peritoneal mesothelial cells (heterotypic adhesion), a standardized cell adhesion assay was developed according to methods described previously [5].
Male, 4- to 6-week-old BALB/C nu/nu mice (provided by Animal research center, Chinese Academy of Medical Sciences, SCXT11-00-0006) were used.
J. Zhang, B. Wang / Gynecologic Oncology 103 (2006) 199–206 Table 1 Sequences of PCR primers and length of PCR product Genes
PCR primers
Length of PCR product (bp)
MMP-2
5′-CTGAGATCTGCAAACAGGACATTGT-3′ 5′-CCGCCCTGCAGGTCCACGACGGCAT-3′ 5′-GACTCGGTCTTTGAGGAGCC-3′ 5′-GAACTCACGCGCCAGTAGAA-3′ 5′-ATCCTGTTGTTGCTGTGGCTG-3′ 5′-GACTGGAAGCCCTTTTCAGA-3′ 5′-CTCGCTGGACGTCGTTCGAGGAAAGAA-3′ 5′-AGCCCATCTGGTACCTGTGGTTCA-3′ 5′-GCATGGAGTCCTGTGGCAT-3′ 5′-GGTGGCGTTTACGAAGATC-3′
481
MMP-9 TIMP-1 TIMP-2 β-actin
350
201
cells added. As shown in Fig. 2A, pre-incubation of 3AO, SW626 and HO-8910PM cells with As2O3 resulted in decreased tumor cell adhesion to HPMC (heterotypic adhesion) (P < 0.01). These effects were dose-dependent (P < 0.01). On the other hand, As2O3 treatment dose-dependently increased tumor cell–tumor cell adhesion (homotypic adhesion) (P < 0.05) (Fig. 2B).
520 155 320
Tumor injections containing 4 × 106 (0.2 ml) tumor cells were made i.p. into abdominal cavity. The animals were randomized into one of four groups: (1) normal saline (control); (2) 0.5 mg/kg As2O3; (3) 1.0 mg/kg As2O3; (4) 2.0 mg/ kg As2O3. At 4 days after implantation, normal saline or various concentrations of As2O3 was administered i.p. once a day for 7 days. Another course of treatment was given 3 weeks later. The animals were killed (n = 8 per group). The peritoneum was stained with H and E, and the metastasis foci were counted.
Statistics Data are expressed as mean ± standard deviation (x ¯ ± SD). Statistical evaluation of the data was performed with analysis of variance and chi-square test. P < 0.05 was considered statistically significant.
Results Effect of As2O3 on cytotoxicity of tumor cells 3AO, SW626 and HO-8910PM cells were cultured and cytotoxicity of As2O3 on cells in the presence or absence of serum was measured. As shown in Fig. 1, significant cytotoxic effects were observed in the serum-supplemented media following the As2O3 treatment for 48 h. In the serumsupplemented condition, 2 μM As2O3 exhibited approximately 40% growth inhibition of 3AO, SW626 and HO8910PM cells, respectively (P < 0.01) (Figs. 1A–C). There was no significant difference of growth inhibition between different cell lines (P > 0.05). In the serum-free condition, 2 μM As2O3 did not affect 3AO cell population (P > 0.05), and significant reduction of the cell population was observed at high concentration of As2O3 (10 μM) (P < 0.01) (Fig. 1A). Likewise, As2O3 in absence of serum did not inhibit growth of SW626 and HO-8910PM cells until the concentration reached 10 μM (P < 0.01) (Figs. 1B and C). Therefore noncytotoxic concentrations of As2O3 (below 2 μM) in absence of serum were used in the following in vitro experiments. Effects of As2O3 on cell–cell adhesion Adhesion of tumor cells was calculated by ratio of the number of adhesive tumor cells to the total amount of tumor
Fig. 1. Effect of As2O3 on the cytotoxicity of tumor cells. Exponentially growing 3AO (A), SW626 (B) and HO-8910PM cells (C) were treated with indicated concentrations of As2O3 for 2 days in the presence or absence of serum. Cytotoxicity was determined by MTT-based assay as described.
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level, As2O3 markedly dose-dependently reduced protein levels of MMP-2, MMP-9, and TIMP-2 of tumor cells (P < 0.05), but increased TIMP-1 levels in a dose-dependent manner (P < 0.01) (Figs. 4D–F). Effects of As2O3 on peritoneal metastasis in vivo Furthermore, this study examined the effect of As2O3 on tumor peritoneal metastasis by the animal models. 3AO, SW626 and HO-8910PM cells were injected into abdominal cavity of mice, respectively, then different concentrations of As2O3 were used i.p. During or after the administration, the mice did not show abnormality, such as anorexy, loss of weight or nervous system disorder. Fig. 5 demonstrated that the injection of As2O3 to abdominal cavity reduced the number of foci on the peritoneum dose-dependently (P < 0.05), indicating that As2O3 may control peritoneal metastasis of ovarian carcinoma cells. Discussion In vitro growth inhibition of As2O3 has previously been reported in various human hematologic cancers and solid tumors. According to Park et al. [7], the cytotoxicity of As2O3 Fig. 2. Effects of As2O3 on the heterotypic and homotypic adhesion of tumor cells. Labeled tumor cells incubated with or without various concentrations of As2O3 were seeded onto the 96-well plates coated with HPMC(A) or tumor cells (B). After 60 min, the remaining fluorescence per well was measured.
Effects of As2O3 on migration and invasion of tumor cells in vitro The results showed that As2O3 not only reduced the migration ability of three kinds of tumor cells (P < 0.01), but also significantly inhibited tumor peritoneal invasion potential using the invasion assay in vitro (P < 0.01), and the inhibitive effects were both dose-dependent (P < 0.01) (Figs. 3A and B). Effects of As2O3 on the expression of proteases and their inhibitors This study next explored whether As2O3 regulated the expressions of MMP-2, MMP-9, which were involved in creating and maintaining an environment that contributes peritoneal metastasis of tumor cells, and their inhibitors (TIMP-2 and TIMP-9) in tumor cells. RT-PCR was performed to determine whether the proteases and their inhibitors were modulated by As2O3 at mRNA levels. ELISA was used to determine the protein levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 in the supernatants of tumor cells before or after the stimulation of As2O3. As shown in Figs. 4A–C, 3AO, SW626 and HO-8910PM cells expressed MMP-2, MMP-9, TIMP-1 and TIMP-2. As2O3 significantly reduced mRNA levels of MMP-2, MMP-9, and TIMP-2 of all three kinds of tumor cells dose-dependently (P < 0.05), but it increased TIMP-1 levels in a dose-dependent manner (P < 0.01). Consistent with the mRNA
Fig. 3. Effects of As2O3 on the migration and peritoneal invasion of tumor cells in vitro. (A) Cell migration was assessed by counting the number of cells that migrated into the denuded area over a 1 cm distance along the wounded edge. (B) Invasion through a layer of Matrigel covered by HPMC monolayer was determined by a Boyden chamber method.
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Fig. 4. Effects of As2O3 on the expression of various proteases and their inhibitors in tumor cells. 3AO (A), SW626 (B) and HO-8910PM cells (C) were exposed to indicated concentrations of As2O3 for 24 h. Total RNA of the cells was extracted and RT-PCR was used for semiquantitation. The PCR products (MMP-2, MMP-9, TIMP-1, TIMP-2) and β-actin were separated by 2% agarose gel electrophoresis. B: 3AO (D), SW626 (E) and HO-8910PM cells (F) were pretreated with indicated concentrations of As2O3 for 24 h. The levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 in the supernatant were determined using the ELISA kit.
was influenced by the presence of serum. Likewise, remarkable in vitro inhibition of cell growth was induced in all three ovarian cancer cell lines at low or high doses As2O3 in the presence of serum. It may be the results of that some ingredient in serum may influence the action of As2O3. But in the absence of serum, only high dose As2O3 had pronounced cytotoxic effects on tumor cells. In order to avoid the possibility that As2O3 may reduce tumor invasiveness by influencing the growth of tumor cells, noncytotoxic dosage of As2O3 in absence of serum was used in vitro experiments. In order for the ovarian tumor cells to spread to peritoneum, they need to separate from the primary site and attach to HPMC
which cover the surface of peritoneum, involving the loss of tumor cell–tumor cell adhesion (homotypic adhesion) and gain of tumor cell–host cell (HPMC) adhesion (heterotypic adhesion). Our present study showed that As2O3 enhanced homotypic adhesion and inhibited heterotypic adhesion dosedependently. Homotypic adhesion of epithelial carcinoma is mediated for the most part by the E-cadherin/catenin complex [8]. In most primary foci of advanced ovarian carcinoma, the expression of E-cadherin is down-regulated and instable, which looses the adherent junctions and benefits for the tumor cells to detach from each other [9]. Whether As2O3 enhances tumor cells homotypic adhesion through changing the expression of
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Fig. 5. Effects of As2O3 on peritoneal metastasis of tumor cells in vitro. Tumor cells were injected into the abdominal cavity of nude mice and the mice were treated with various concentrations of As2O3 as indicated. The animals were killed. The peritoneum was stained with H&E, and the foci were counted.
E-cadherin/catenin remains further research. It has been reported that the CD44 molecule, which is a major receptor for hyaluronic acid, is frequently expressed by ovarian cancer cells and is partly responsible for mediating mesothelial binding through recognition of mesothelial-associated hyaluronic acid [10]. Liu et al. found that As2O3 induced decrease of the expression of CD44 in stomach cancer cells [11]. As2O3 may inhibit heterotypic adhesion of ovarian tumor cells with HPMC by the same mechanisms. Our results suggest that down- and up-regulation of heterotypic and homotypic adhesion, respectively, by As2O3 may be a crucial event in the As2O3-induced suppression of invasion of ovarian tumor cells. Tumor cell motility is required for the cells to invade and metastasize [12]. The tumor cells with high invasive ability usually have active locomotivity. Although many factors can influence cellular motility through different signal transduction cascades, cellular movement requires reorganization of cytoskeleton at last, such as microtubule and microfilament. The factors which can affect the reorganization of cytoskeleton may influence cell motility. It has been found that microtubuleaffecting agents, such as Paclitaxel, Vincristine and Vindesine at non-cytotoxic doses can inhibit the migration of tumor cells [13]. It has been reported that As2O3 markedly inhibits GTPinduced polymerization and microtubule formation in vitro in myeloid leukemia cells [14]. So it may have some effects on migration of tumor cells. Our results proved that As2O3 inhibited migration of ovarian carcinoma cells in vitro. It may be the results of the action on ovarian tumor cell's cytoskeleton, such as microtubule formation. For cancer cells to invade the interstitial tissue, various proteinases such as MMPs are required. MMP-2 and MMP-9 are well known as decomposing enzymes of type IV collagen, which are important components of basement membrane underlying HPMC. Tumor cells can dissolve type IV collagen and penetrate basement membrane of peritoneum to form peritoneal metastasis foci through secreting MMP-2 and MMP9, and the contents of MMP-2 and MMP-9 of tumor cells are related to their peritoneal invasive ability. TIMP-2 and TIMP-1 are tissue inhibitors of MMP-2 and MMP-9, respectively, which can modulate activity of MMP-2 and MMP-9. Keeping dynamic balance of MMPs/TIMPs expressions and maintaining
integrity of basement membrane are important mechanisms to anti-metastasis therapy [15–17]. So we detect whether As2O3 has influence on the expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2 of ovarian tumor cells. This study demonstrated that As2O3 decreased MMP-2 and MMP-9 expressions at mRNA and protein levels. As2O3 also decreased mRNA and protein expression levels of TIMP-2, but it increased TIMP-1 expression at mRNA and protein levels. The increase of TIMP1 expression inhibits the enzyme activity of MMP-9. Therefore, the down-regulation of MMP-2 and MMP-9, and up-regulation of TIMP-1 by As2O3 attenuated the dissolution and destruction of basement membrane and inhibited the peritoneal invasion ability of ovarian carcinoma cells. The disagree of expression of TIMP-1 and TIMP-2 may be the results of different regulations in cells. TIMP-2 mRNA transcript levels were differently regulated from TIMP-1 levels, both in cell culture as evidenced by studies using 12-O-tetradecanoylphorbol-13-acetate (TPA) and transforming growth factor beta1 and in vivo as evidenced by comparison of transcript levels for these inhibitors in human colon adenocarcinoma tissue and adjacent normal colonic mucosa [18]. Additional evidence supporting independent regulation of TIMP-1 and TIMP-2 has been provided by experiments showing their differential responses to cytokines or other agents. For example, in macrophages, lipopolysaccharides have been reported to down-regulate TIMP-1 and up-regulate TIMP-2 [19]. TIMP-1 was up-regulated by interleukin-1 and interleukin-6, whereas TIMP-2 was not affected by both agents [20]. With characterization and comparison of the structure of the genes, differences and divergence of these genes accounted for the fact that although there was a marked homology between TIMP-1 and TIMP-2 at the protein level, they were differently regulated [18,21]. MMPs and TIMPs expressions are regulated by several transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), because their promoter sequences contain the putative binding sites for the transcription factors. Although MMP-2 promoter itself does not contain a NF-κB binding site, several reports show that NF-κB can augment MMP-2 activation [22,23]. It was reported that As2O3 blocked the promoter stimulating activity and DNA binding activity of NF-κB and suppressed the transcription of MMP-2 and MMP-9 genes [7]. Moreover, arsenite inhibited IκB kinase, which was essential for NF-κB activation [24]. So As2O3 may inhibit the expression of MMP-2 and MMP-9 by influencing the function of NF-κB. In vitro invasive assay was used to verify the effects of As2O3 on tumor cell peritoneal invasive potential. It was found that when all three kinds of ovarian tumor cells were pretreated with As2O3, the number of filter-infiltrating cells was decreased, demonstrating that As2O3 inhibited the tumor cell peritoneal invasion ability in vitro. Recent clinical studies have shown that As2O3 given intravenously is effective and relatively safe in the treatment of APL [25]. However, the clinical use of As2O3 for treatment of ovarian cancer as well as tumor peritoneal metastasis by ways of abdominal administration has not been reported. Here we observed the effect of As2O3 given i.p. on ovarian cancer peritoneal metastasis using animal experiments to provide basis
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for further clinical application. LD50 of As2O3 for abdominal administration in mice is 12 mg/kg [26]. In previous reports, it was found that large doses As2O3 (4 mg/kg, 8 mg/kg, 16 mg/kg) could cause brain necrosis, liver necrosis, loss of appetite, high excitability and even death of mice in seven days, but therapeutic doses As2O3 (1 mg/kg, 2 mg/kg) did not damage the brain, liver or kidney [27]. Dosages chosen in our in vivo experiments did not cause abnormal reaction of mice, showing safety of these dosages too. Such dosages were also applied by other investigators [28,29]. Our results revealed that when mice were treated with various concentrations of As2O3 after tumor injections, the number of colonies formed on peritoneum reduced in a dose-dependent mode, which was agree with results in vitro and proved As2O3 could inhibit the peritoneum metastasis of ovarian carcinoma. Huang et al. also found that As2O3 reduced the 3AO cell tumor formation rate in nude mice and concluded As2O3 could inhibit the abdomino-plantation of human ovarian carcinoma in nude mice [28], which was agree with our results. When different doses As2O3 were injected into abdominal cavity of nude mice, the appearance of peritoneal mesothelial cells on the surface of organs in abdominal cavity did not change, indicating that As2O3 could act on carcinoma cells by choice but did not damage the normal host cells, which proved the safety of As2O3 [30]. Ovarian cancer commonly spreads within the peritoneal cavity. Combined chemotherapy based on platinum analogue is important therapy at present. But drug-resistance, toxic and side effects restrict the effects of chemotherapy. As a major component of a traditional Chinese medicine, As2O3 has wide anticancer spectra, significant anticancer effects and little adverse reaction in experiment and clinic. The most important is that drug-resistance or cross drug-resistance with other antitumor drugs has not been found. So As2O3 shows favorable perspective in the future. This study showed that As2O3 inhibited the peritoneal invasion of human ovarian carcinoma cells in vitro and in vivo, mainly by suppressing migration, heterotypic adhesion with HPMC and down-regulating MMP-2 and MMP-9 expressions as well as enhancing homotypic adhesion with tumor cells and up-regulating TIMP-1 expression. The rationale for intraperitoneal administration of As2O3 is that tumor cells in abdominal cavity receive sustained exposure to high concentrations of agents while normal tissues, such as the bone marrow, are relatively spared. With more research in pharmacodynamics and clinic application of As2O3, it might be considered as a possible agent for controlling the peritoneal metastasis of ovarian cancer. References [1] Gallagher RE. Arsenic: new life for an old potion. N Engl J Med 1998; 339:1389–91. [2] Shen ZY, Shen WY, Chen MH, Shen J, Cai WJ, Zeng Y. Mitochondria, calcium and nitric oxide in the apoptotic pathway of esophageal carcinoma cells induced by As2O3. Int J Mol Med 2002;9:385–90. [3] Uslu R, Sanli UA, Sezgin C, Karabulut B, Terzioglu E, Omay SB, et al. Arsenic trioxide-mediated cytotoxicity and apoptosis in prostate and ovarian carcinoma cell lines. Clin Cancer Res 2000;6:4957–64. [4] Mizutani K, Kofuji K, Shirouzu K. The significance of MMP-1 and MMP-2
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Arsenic trioxide (As2O3) inhibits peritoneal invasion of ovarian carcinoma cells in vitro and in vivo Jingjing Zhang ⁎, Bo Wang Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan 250012, Shandong, China Received 21 December 2005 Available online 19 April 2006
Abstract Objectives. To study the role of arsenic trioxide (As2O3) in regulating peritoneal invasive activity of ovarian carcinoma cells in vitro and in vivo. Methods. The effects of As2O3 on human ovarian cancer cell lines (3AO, SW626 and HO-8910PM) migration, invasion and adhesion with tumor cells and human peritoneal mesothelial cells (HPMC) were observed by means of cell migration test, cell invasion test and cell adhesion test. The effects of As2O3 on MMP-2, MMP-9, TIMP-1 and TIMP-2 gene expressions and protein expressions of tumor cells were determined by RT-PCR and ELISA, respectively. In animal experiments, ovarian tumor cells were implanted into abdominal cavity of nude mice and then the nude mice were treated by intraperitoneal injection of different doses As2O3. The foci on the surface of peritoneum were counted. Results. As2O3 inhibited tumor cells migration, invasion and adhesion with HPMC in a dose-dependent manner, while the same treatment enhanced tumor cell–tumor cell interactions. As2O3 inhibited mRNA and protein expressions of MMP-2, MMP-9 and TIMP-2 of tumor cells. In contrast, As2O3 increased mRNA and protein expressions of TIMP-1. As2O3 could reduce tumor cells peritoneal metastasis in nude mice. Conclusion. As2O3 inhibits in vitro and in vivo peritoneal invasive activity of ovarian carcinoma cells in a dose-dependent manner. Its antiinvasive activity may be the results of reduced cell motility, inhibited attachment of tumor cells to HPMC and enhanced tumor cell–tumor cell interaction, as well as down-regulation of MMP-2 and MMP-9 levels and up-regulation of TIMP-1 level. © 2006 Elsevier Inc. All rights reserved. Keywords: Arsenic trioxide; Ovarian neoplasms; Peritoneum; Metastasis
Introduction Ovarian cancer is the second most frequent gynecological cancer. In 60–70% of patients, ovarian tumors are diagnosed at advanced stages only, since symptoms are absent and a specific tumor marker is lacking. Ovarian cancer, which originates in the surface epithelium in about 97% of the cases, spreads prevalently by direct extension of the tumor to adjacent tissues and by shedding of tumor cells from primary tumor into the peritoneal cavity. Although peritoneal disseminated metastasis is often associated with malignant ascites and a poor prognosis, no effective therapy exists at present. As2O3 is an active ingredient of a traditional Chinese medicine that has been used successfully for treating acute promyelocyte ⁎ Corresponding author. Present address: Department of Obstetrics and Gynecology, Qingdao Municipal Hospital, Medical College of Qingdao University, Qingdao 266011, Shandong, China. E-mail address: [email protected] (J. Zhang). 0090-8258/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2006.02.037
leukemia(APL) [1] and has been shown to be an effective inducer of apoptosis in some solid cancer cells, such as esophageal, prostate and ovarian carcinoma [2,3]. However the effects of As2O3 on the tumor cell peritoneal invasiveness has seldom been reported. The steps required for ovarian carcinoma metastasis include the following: (1) separation of cells from the primary tumor mass on the ovarian surface; (2) passive movement of the tumor cells through the peritoneal fluid; (3) adherence of the cells to the mesothelium, which lines the peritoneal cavity; (4) migration of tumor cells past the mesothelium into the stroma; (5) enzymatic digestion or remodeling of the underlying stroma by proteinases; (6) growth of the newly established tumor cells in their new location. So adhesion, motility and proteolysis are important factors involved in ovarian carcinoma cells peritoneal invasion. Matrix metalloproteinases (MMPs) are a family of structurally related neutral metalloproteinases, which together can degrade all the components of the extracellular matrix proteins (ECM) and are involved in tumor invasion and metastasis. Among the MMPs, the 72 kDa gelatinase A (or
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MMP-2) and 92 kDa gelatinase B (or MMP-9) are believed to be the critical enzymes for degrading type IV collagen, a major component of basement membrane, which lies under peritoneal mesothelial cells, thus playing a critical role in cancer cell invasion and metastasis. Their catalytic activity is tightly controlled by endogenous inhibitors, including tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. In the present study, we investigated the effect of As2O3 on adhesion, motility, expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2, and peritoneal invasive activity of ovarian carcinoma cells in vitro and in vivo, to clearly demonstrate the anti-invasive activity of As2O3 and to provide evidence for exploring drugs for controlling the peritoneal metastasis of ovarian tumor cells. Materials and methods Cell culture Human ovarian cancer cell lines 3AO and SW626 cells were obtained from the American Type Culture Collection (Rockville, MD, USA), and HO-8910PM cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Science. Tumor cells were maintained in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a cell culture incubator at 37°C in 5% CO2. Human peritoneal mesothelial cells (HPMC) were isolated from the greater omentum in patients operated on for a nonperitonitis cause and without disseminated cancer and cultured as previously reported with minor modification [4]. Human omental tissue was obtained from surgically resected specimens from patients with informed consent. The omentum was washed in several changes of sterile phosphate-buffered saline (PBS), and finely divided into approximately 1-mm2 segments. These were washed in PBS twice to remove any contaminating red blood cells. The tissue was then treated with a 0.25% trypsin-EDTA solution for about 20 min. The cell suspension was centrifuged at 1000 rpm for 10 min, and collected cells were cultured in RPMI1640 medium supplemented with 20% FBS, 0.4% dermacort and 10 μg/ml insuline at 37°C in 5% CO2. HPMC were confirmed by immunohistochemistry with antibodies against cytokeratin, vimentin, CD45 antigen and factor VIII (Signet, Dedham, MA, USA). Passage 2 cultures were used for experiments.
Tumor cells were labeled with calcein as described above. The labeled tumor cells were washed to remove free dye and incubated with indicated concentrations (0.5 μM, 1.0 μM, 2.0 μM) of As2O3 for 24 h. Mesothelial and tumor monolayers were established in a 96-well plate. The calcein-labeled, As2O3 -treated tumor cells (2.5 × 104) were seeded additionally. After plates were incubated at 37°C for 60 min, the medium of each well was removed and washed twice with RPMI-1640 medium. The remaining fluorescence per well was measured on a Perkin Elmer plate reader, using 485 excitation and 530 emission filters. On each plate, a standard was prepared by adding different numbers of labeled tumor cells to the wells. The amount of adherent tumor cells was determined by calibrating the measured fluorescence of the experimental wells in relation to the standard.
Migration assays To determine the effect of As2O3 on the migration of tumor cells, a woundmigration assay was performed. Tumor cells were cultured on a 12-well plate. When cells were 80% confluent, monolayers were wounded with a 200 μl micropipette tip, washed twice with PBS, and incubated for 24 h with serum-free medium in the presence or absence of indicated concentrations (0.5 μM, 1.0 μM, 2.0 μM) of As2O3. After incubation, cells were washed with PBS. Migration was quantified by counting the number of cells that migrated from the wound edge into the denuded area for a distance of 1 cm.
Cell invasion assay An invasion assay was performed according to the method reported by Hirashima et al. [6]. A millicell (Sigma) with an 8-μm porosity cell-permeable polycarbonate filter was covered with recombinant basement membrane Matrigel (Sigma) and was placed on a 24-cell culture plate. HPMC were cultured fully on Matrigel, and washed, and 2 × 105 tumor cells pretreated with or without As2O3 were then added into the millicell. As a chemoattractant, NIH3T3 fibroblast conditioned medium was used outside of the millicell. After cultivation for 12 h, the millicell was immersed in 100% methanol for 1 min for fixation, and all cells were then stained by hematoxylin. The cells remaining on the top surface of the filter were completely removed with a cotton swab, and the filter was removed from the millicell and mounted on a glass slide. These preparations were examined under a microscope at ×400 magnification. The number of infiltrating tumor cells was counted in five regions selected at random, and the extent of invading tumor cells was determined by the mean count.
Cytotoxicity assays
RT-PCR
In vitro cytotoxic effects of As2O3 (Sigma) on tumor cells was determined by using CellTiter 96 Non-Radioactive Cell Proliferation Assay kit (Promega, Madison, WI). Briefly, cells growing in plates were dispersed in 0.05% trypsin solution and resuspended in RPMI-1640 containing 10% FBS. Approximately 5000 cells were added to each well of 96-well plate and incubated for 18 h. After changing the media with or without 10% serum, various concentrations (0.5 μM, 1.0 μM, 2.0 μM) of As2O3 were added to each well. After 48 h of incubation, cell cytotoxicity was estimated by MTT based assay following the supplier's instructions.
Total RNA was extracted from tumor cells, pretreated with or without indicated concentrations of As2O3 for 24 h, by Trizol (Invigen) according to the manufacturer's protocol. Reverse transcription of RNA was performed in a final reaction volume of 20 ml containing 5 μg of total RNA in Moloney murine leukemia virus (MMLV) reverse transcriptase buffer (Promega, Madison, WI, USA). A 1 μl portion of the reaction mixture was then amplified by PCR with the pairs of primers (Table 1). A total of 30 cycles were performed, with each cycle comprising 1 min at 94°C, 1 min at 50–60°C depending on each gene, and 1.5 min at 72°C with a final extension of 8 min at 72°C. Human β-actin reactions were amplified for 25 cycles. The reaction products were separated on 2% agarose gel, stained with 1 μg/ml ethidium bromide, and visualized using a Digital imaging system. Semi-quantitative analysis was performed using the ratio of the genes tested and β-actin.
Calcein-AM solution and incubation According to Van Rossen et al. [5], the dye solution, calcein-AM, used to quantify tumor cell adhesion, was prepared by dissolving 50 μg of calcein (Molecular Probes, Leiden, The Netherlands) in 5 μl of anhydrous dimethyl sulphoxide (Sigma) and adding this solution to 5 ml of RPMI medium supplemented with 0.5% bovine serum albumin (Sigma). Trypsinized tumor cells (1 × 106 cells/ml) were incubated in RPMI/0.5% BSA at 37°C for 45 min, with occasional mixing.
Enzyme-linked immunosorbent assays (ELISA) After tumor cells were pretreated with or without As2O3 for 24 h, the supernatant was collected. The levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 were measured in the supernatants using the ELISA kit (R&D Systems) according to the manufacturer's instructions.
Adhesion assay Animal experiments To quantify tumor cells adhesion to the tumor cells (homotypic adhesion) and peritoneal mesothelial cells (heterotypic adhesion), a standardized cell adhesion assay was developed according to methods described previously [5].
Male, 4- to 6-week-old BALB/C nu/nu mice (provided by Animal research center, Chinese Academy of Medical Sciences, SCXT11-00-0006) were used.
J. Zhang, B. Wang / Gynecologic Oncology 103 (2006) 199–206 Table 1 Sequences of PCR primers and length of PCR product Genes
PCR primers
Length of PCR product (bp)
MMP-2
5′-CTGAGATCTGCAAACAGGACATTGT-3′ 5′-CCGCCCTGCAGGTCCACGACGGCAT-3′ 5′-GACTCGGTCTTTGAGGAGCC-3′ 5′-GAACTCACGCGCCAGTAGAA-3′ 5′-ATCCTGTTGTTGCTGTGGCTG-3′ 5′-GACTGGAAGCCCTTTTCAGA-3′ 5′-CTCGCTGGACGTCGTTCGAGGAAAGAA-3′ 5′-AGCCCATCTGGTACCTGTGGTTCA-3′ 5′-GCATGGAGTCCTGTGGCAT-3′ 5′-GGTGGCGTTTACGAAGATC-3′
481
MMP-9 TIMP-1 TIMP-2 β-actin
350
201
cells added. As shown in Fig. 2A, pre-incubation of 3AO, SW626 and HO-8910PM cells with As2O3 resulted in decreased tumor cell adhesion to HPMC (heterotypic adhesion) (P < 0.01). These effects were dose-dependent (P < 0.01). On the other hand, As2O3 treatment dose-dependently increased tumor cell–tumor cell adhesion (homotypic adhesion) (P < 0.05) (Fig. 2B).
520 155 320
Tumor injections containing 4 × 106 (0.2 ml) tumor cells were made i.p. into abdominal cavity. The animals were randomized into one of four groups: (1) normal saline (control); (2) 0.5 mg/kg As2O3; (3) 1.0 mg/kg As2O3; (4) 2.0 mg/ kg As2O3. At 4 days after implantation, normal saline or various concentrations of As2O3 was administered i.p. once a day for 7 days. Another course of treatment was given 3 weeks later. The animals were killed (n = 8 per group). The peritoneum was stained with H and E, and the metastasis foci were counted.
Statistics Data are expressed as mean ± standard deviation (x ¯ ± SD). Statistical evaluation of the data was performed with analysis of variance and chi-square test. P < 0.05 was considered statistically significant.
Results Effect of As2O3 on cytotoxicity of tumor cells 3AO, SW626 and HO-8910PM cells were cultured and cytotoxicity of As2O3 on cells in the presence or absence of serum was measured. As shown in Fig. 1, significant cytotoxic effects were observed in the serum-supplemented media following the As2O3 treatment for 48 h. In the serumsupplemented condition, 2 μM As2O3 exhibited approximately 40% growth inhibition of 3AO, SW626 and HO8910PM cells, respectively (P < 0.01) (Figs. 1A–C). There was no significant difference of growth inhibition between different cell lines (P > 0.05). In the serum-free condition, 2 μM As2O3 did not affect 3AO cell population (P > 0.05), and significant reduction of the cell population was observed at high concentration of As2O3 (10 μM) (P < 0.01) (Fig. 1A). Likewise, As2O3 in absence of serum did not inhibit growth of SW626 and HO-8910PM cells until the concentration reached 10 μM (P < 0.01) (Figs. 1B and C). Therefore noncytotoxic concentrations of As2O3 (below 2 μM) in absence of serum were used in the following in vitro experiments. Effects of As2O3 on cell–cell adhesion Adhesion of tumor cells was calculated by ratio of the number of adhesive tumor cells to the total amount of tumor
Fig. 1. Effect of As2O3 on the cytotoxicity of tumor cells. Exponentially growing 3AO (A), SW626 (B) and HO-8910PM cells (C) were treated with indicated concentrations of As2O3 for 2 days in the presence or absence of serum. Cytotoxicity was determined by MTT-based assay as described.
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level, As2O3 markedly dose-dependently reduced protein levels of MMP-2, MMP-9, and TIMP-2 of tumor cells (P < 0.05), but increased TIMP-1 levels in a dose-dependent manner (P < 0.01) (Figs. 4D–F). Effects of As2O3 on peritoneal metastasis in vivo Furthermore, this study examined the effect of As2O3 on tumor peritoneal metastasis by the animal models. 3AO, SW626 and HO-8910PM cells were injected into abdominal cavity of mice, respectively, then different concentrations of As2O3 were used i.p. During or after the administration, the mice did not show abnormality, such as anorexy, loss of weight or nervous system disorder. Fig. 5 demonstrated that the injection of As2O3 to abdominal cavity reduced the number of foci on the peritoneum dose-dependently (P < 0.05), indicating that As2O3 may control peritoneal metastasis of ovarian carcinoma cells. Discussion In vitro growth inhibition of As2O3 has previously been reported in various human hematologic cancers and solid tumors. According to Park et al. [7], the cytotoxicity of As2O3 Fig. 2. Effects of As2O3 on the heterotypic and homotypic adhesion of tumor cells. Labeled tumor cells incubated with or without various concentrations of As2O3 were seeded onto the 96-well plates coated with HPMC(A) or tumor cells (B). After 60 min, the remaining fluorescence per well was measured.
Effects of As2O3 on migration and invasion of tumor cells in vitro The results showed that As2O3 not only reduced the migration ability of three kinds of tumor cells (P < 0.01), but also significantly inhibited tumor peritoneal invasion potential using the invasion assay in vitro (P < 0.01), and the inhibitive effects were both dose-dependent (P < 0.01) (Figs. 3A and B). Effects of As2O3 on the expression of proteases and their inhibitors This study next explored whether As2O3 regulated the expressions of MMP-2, MMP-9, which were involved in creating and maintaining an environment that contributes peritoneal metastasis of tumor cells, and their inhibitors (TIMP-2 and TIMP-9) in tumor cells. RT-PCR was performed to determine whether the proteases and their inhibitors were modulated by As2O3 at mRNA levels. ELISA was used to determine the protein levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 in the supernatants of tumor cells before or after the stimulation of As2O3. As shown in Figs. 4A–C, 3AO, SW626 and HO-8910PM cells expressed MMP-2, MMP-9, TIMP-1 and TIMP-2. As2O3 significantly reduced mRNA levels of MMP-2, MMP-9, and TIMP-2 of all three kinds of tumor cells dose-dependently (P < 0.05), but it increased TIMP-1 levels in a dose-dependent manner (P < 0.01). Consistent with the mRNA
Fig. 3. Effects of As2O3 on the migration and peritoneal invasion of tumor cells in vitro. (A) Cell migration was assessed by counting the number of cells that migrated into the denuded area over a 1 cm distance along the wounded edge. (B) Invasion through a layer of Matrigel covered by HPMC monolayer was determined by a Boyden chamber method.
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Fig. 4. Effects of As2O3 on the expression of various proteases and their inhibitors in tumor cells. 3AO (A), SW626 (B) and HO-8910PM cells (C) were exposed to indicated concentrations of As2O3 for 24 h. Total RNA of the cells was extracted and RT-PCR was used for semiquantitation. The PCR products (MMP-2, MMP-9, TIMP-1, TIMP-2) and β-actin were separated by 2% agarose gel electrophoresis. B: 3AO (D), SW626 (E) and HO-8910PM cells (F) were pretreated with indicated concentrations of As2O3 for 24 h. The levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 in the supernatant were determined using the ELISA kit.
was influenced by the presence of serum. Likewise, remarkable in vitro inhibition of cell growth was induced in all three ovarian cancer cell lines at low or high doses As2O3 in the presence of serum. It may be the results of that some ingredient in serum may influence the action of As2O3. But in the absence of serum, only high dose As2O3 had pronounced cytotoxic effects on tumor cells. In order to avoid the possibility that As2O3 may reduce tumor invasiveness by influencing the growth of tumor cells, noncytotoxic dosage of As2O3 in absence of serum was used in vitro experiments. In order for the ovarian tumor cells to spread to peritoneum, they need to separate from the primary site and attach to HPMC
which cover the surface of peritoneum, involving the loss of tumor cell–tumor cell adhesion (homotypic adhesion) and gain of tumor cell–host cell (HPMC) adhesion (heterotypic adhesion). Our present study showed that As2O3 enhanced homotypic adhesion and inhibited heterotypic adhesion dosedependently. Homotypic adhesion of epithelial carcinoma is mediated for the most part by the E-cadherin/catenin complex [8]. In most primary foci of advanced ovarian carcinoma, the expression of E-cadherin is down-regulated and instable, which looses the adherent junctions and benefits for the tumor cells to detach from each other [9]. Whether As2O3 enhances tumor cells homotypic adhesion through changing the expression of
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Fig. 5. Effects of As2O3 on peritoneal metastasis of tumor cells in vitro. Tumor cells were injected into the abdominal cavity of nude mice and the mice were treated with various concentrations of As2O3 as indicated. The animals were killed. The peritoneum was stained with H&E, and the foci were counted.
E-cadherin/catenin remains further research. It has been reported that the CD44 molecule, which is a major receptor for hyaluronic acid, is frequently expressed by ovarian cancer cells and is partly responsible for mediating mesothelial binding through recognition of mesothelial-associated hyaluronic acid [10]. Liu et al. found that As2O3 induced decrease of the expression of CD44 in stomach cancer cells [11]. As2O3 may inhibit heterotypic adhesion of ovarian tumor cells with HPMC by the same mechanisms. Our results suggest that down- and up-regulation of heterotypic and homotypic adhesion, respectively, by As2O3 may be a crucial event in the As2O3-induced suppression of invasion of ovarian tumor cells. Tumor cell motility is required for the cells to invade and metastasize [12]. The tumor cells with high invasive ability usually have active locomotivity. Although many factors can influence cellular motility through different signal transduction cascades, cellular movement requires reorganization of cytoskeleton at last, such as microtubule and microfilament. The factors which can affect the reorganization of cytoskeleton may influence cell motility. It has been found that microtubuleaffecting agents, such as Paclitaxel, Vincristine and Vindesine at non-cytotoxic doses can inhibit the migration of tumor cells [13]. It has been reported that As2O3 markedly inhibits GTPinduced polymerization and microtubule formation in vitro in myeloid leukemia cells [14]. So it may have some effects on migration of tumor cells. Our results proved that As2O3 inhibited migration of ovarian carcinoma cells in vitro. It may be the results of the action on ovarian tumor cell's cytoskeleton, such as microtubule formation. For cancer cells to invade the interstitial tissue, various proteinases such as MMPs are required. MMP-2 and MMP-9 are well known as decomposing enzymes of type IV collagen, which are important components of basement membrane underlying HPMC. Tumor cells can dissolve type IV collagen and penetrate basement membrane of peritoneum to form peritoneal metastasis foci through secreting MMP-2 and MMP9, and the contents of MMP-2 and MMP-9 of tumor cells are related to their peritoneal invasive ability. TIMP-2 and TIMP-1 are tissue inhibitors of MMP-2 and MMP-9, respectively, which can modulate activity of MMP-2 and MMP-9. Keeping dynamic balance of MMPs/TIMPs expressions and maintaining
integrity of basement membrane are important mechanisms to anti-metastasis therapy [15–17]. So we detect whether As2O3 has influence on the expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2 of ovarian tumor cells. This study demonstrated that As2O3 decreased MMP-2 and MMP-9 expressions at mRNA and protein levels. As2O3 also decreased mRNA and protein expression levels of TIMP-2, but it increased TIMP-1 expression at mRNA and protein levels. The increase of TIMP1 expression inhibits the enzyme activity of MMP-9. Therefore, the down-regulation of MMP-2 and MMP-9, and up-regulation of TIMP-1 by As2O3 attenuated the dissolution and destruction of basement membrane and inhibited the peritoneal invasion ability of ovarian carcinoma cells. The disagree of expression of TIMP-1 and TIMP-2 may be the results of different regulations in cells. TIMP-2 mRNA transcript levels were differently regulated from TIMP-1 levels, both in cell culture as evidenced by studies using 12-O-tetradecanoylphorbol-13-acetate (TPA) and transforming growth factor beta1 and in vivo as evidenced by comparison of transcript levels for these inhibitors in human colon adenocarcinoma tissue and adjacent normal colonic mucosa [18]. Additional evidence supporting independent regulation of TIMP-1 and TIMP-2 has been provided by experiments showing their differential responses to cytokines or other agents. For example, in macrophages, lipopolysaccharides have been reported to down-regulate TIMP-1 and up-regulate TIMP-2 [19]. TIMP-1 was up-regulated by interleukin-1 and interleukin-6, whereas TIMP-2 was not affected by both agents [20]. With characterization and comparison of the structure of the genes, differences and divergence of these genes accounted for the fact that although there was a marked homology between TIMP-1 and TIMP-2 at the protein level, they were differently regulated [18,21]. MMPs and TIMPs expressions are regulated by several transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), because their promoter sequences contain the putative binding sites for the transcription factors. Although MMP-2 promoter itself does not contain a NF-κB binding site, several reports show that NF-κB can augment MMP-2 activation [22,23]. It was reported that As2O3 blocked the promoter stimulating activity and DNA binding activity of NF-κB and suppressed the transcription of MMP-2 and MMP-9 genes [7]. Moreover, arsenite inhibited IκB kinase, which was essential for NF-κB activation [24]. So As2O3 may inhibit the expression of MMP-2 and MMP-9 by influencing the function of NF-κB. In vitro invasive assay was used to verify the effects of As2O3 on tumor cell peritoneal invasive potential. It was found that when all three kinds of ovarian tumor cells were pretreated with As2O3, the number of filter-infiltrating cells was decreased, demonstrating that As2O3 inhibited the tumor cell peritoneal invasion ability in vitro. Recent clinical studies have shown that As2O3 given intravenously is effective and relatively safe in the treatment of APL [25]. However, the clinical use of As2O3 for treatment of ovarian cancer as well as tumor peritoneal metastasis by ways of abdominal administration has not been reported. Here we observed the effect of As2O3 given i.p. on ovarian cancer peritoneal metastasis using animal experiments to provide basis
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for further clinical application. LD50 of As2O3 for abdominal administration in mice is 12 mg/kg [26]. In previous reports, it was found that large doses As2O3 (4 mg/kg, 8 mg/kg, 16 mg/kg) could cause brain necrosis, liver necrosis, loss of appetite, high excitability and even death of mice in seven days, but therapeutic doses As2O3 (1 mg/kg, 2 mg/kg) did not damage the brain, liver or kidney [27]. Dosages chosen in our in vivo experiments did not cause abnormal reaction of mice, showing safety of these dosages too. Such dosages were also applied by other investigators [28,29]. Our results revealed that when mice were treated with various concentrations of As2O3 after tumor injections, the number of colonies formed on peritoneum reduced in a dose-dependent mode, which was agree with results in vitro and proved As2O3 could inhibit the peritoneum metastasis of ovarian carcinoma. Huang et al. also found that As2O3 reduced the 3AO cell tumor formation rate in nude mice and concluded As2O3 could inhibit the abdomino-plantation of human ovarian carcinoma in nude mice [28], which was agree with our results. When different doses As2O3 were injected into abdominal cavity of nude mice, the appearance of peritoneal mesothelial cells on the surface of organs in abdominal cavity did not change, indicating that As2O3 could act on carcinoma cells by choice but did not damage the normal host cells, which proved the safety of As2O3 [30]. Ovarian cancer commonly spreads within the peritoneal cavity. Combined chemotherapy based on platinum analogue is important therapy at present. But drug-resistance, toxic and side effects restrict the effects of chemotherapy. As a major component of a traditional Chinese medicine, As2O3 has wide anticancer spectra, significant anticancer effects and little adverse reaction in experiment and clinic. The most important is that drug-resistance or cross drug-resistance with other antitumor drugs has not been found. So As2O3 shows favorable perspective in the future. This study showed that As2O3 inhibited the peritoneal invasion of human ovarian carcinoma cells in vitro and in vivo, mainly by suppressing migration, heterotypic adhesion with HPMC and down-regulating MMP-2 and MMP-9 expressions as well as enhancing homotypic adhesion with tumor cells and up-regulating TIMP-1 expression. The rationale for intraperitoneal administration of As2O3 is that tumor cells in abdominal cavity receive sustained exposure to high concentrations of agents while normal tissues, such as the bone marrow, are relatively spared. With more research in pharmacodynamics and clinic application of As2O3, it might be considered as a possible agent for controlling the peritoneal metastasis of ovarian cancer. References [1] Gallagher RE. Arsenic: new life for an old potion. N Engl J Med 1998; 339:1389–91. [2] Shen ZY, Shen WY, Chen MH, Shen J, Cai WJ, Zeng Y. Mitochondria, calcium and nitric oxide in the apoptotic pathway of esophageal carcinoma cells induced by As2O3. Int J Mol Med 2002;9:385–90. [3] Uslu R, Sanli UA, Sezgin C, Karabulut B, Terzioglu E, Omay SB, et al. Arsenic trioxide-mediated cytotoxicity and apoptosis in prostate and ovarian carcinoma cell lines. Clin Cancer Res 2000;6:4957–64. [4] Mizutani K, Kofuji K, Shirouzu K. The significance of MMP-1 and MMP-2
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