Molecular Human Reproduction, Vol. 9, No. 10, 569-575,
October 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Increased expression of tissue inhibitor of metalloproteinase-2 in clear cell carcinoma of the ovary
Submitted on March 8, 2003; accepted on May 20, 2003
1 Department of Obstetrics and Gynaecology, Chukyo Hospital, 1-1-10, Sanjo, Minami-ku, Nagoya 457-8510 and 2 Department of Obstetrics and Gynaecology, Nagoya University School of Medicine, Nagoya, Japan
3 To whom correspondence should be addressed. e-mail: kmottm8{at}hotmail.com
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Abstract |
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The matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) have been associated with ovarian tissue remodelling and development of ovarian tumours. With respect to ovarian cancer, the majority of previous studies were performed on serous and mucinous tumours, and little is known about clear cell carcinoma, which shows unique characteristics among ovarian cancers. In the present study, we assessed the differences in the levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 in the normal ovary and ovarian tumours of different histology, including clear cell carcinoma, using specific enzyme-linked immunosorbent assays. In malignant tumours, a prominent increase in pro-MMP-9 levels was observed compared with those of normal ovary and benign tumours, and pro-MMP-2 and TIMP-1 levels were moderately increased. In contrast, TIMP-2 levels were markedly decreased in malignant tumours compared with normal ovary with the exception of clear cell carcinoma, in which they were significantly elevated. Similar results were obtained by the organ culture of carcinoma tissue and normal ovary as well as in the cyst fluids of the tumours. Increased expression of TIMP-2 in clear cell carcinoma was also confirmed by Western blot analysis. Immunohistochemistry showed that TIMP-2 immunoreactivity was localized predominantly in epithelial cancer cells in clear cell carcinoma, while it was present mainly in stromal cells in the other histological types. Taken together, the present study shows that TIMP-2 expression is markedly increased in clear cell carcinoma of the ovary, suggesting a role of TIMP-2 in its unique characteristics among ovarian cancers.
Key words: clear cell carcinoma/MMP/ovarian cancer/TIMP
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Introduction |
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The ovary undergoes remarkable morphological and functional changes during the reproductive cycle, and the highly regulated events such as follicular development, ovulation, formation and regression of the corpus luteum involve remodelling of the extracellular matrix (ECM). The regulation of ECM homeostasis is crucial for the maintenance of the integrity of tissues, and uncontrolled degradation of ECM is one of the essential processes in invasion and distant metastasis by tumour cells.
Turnover of the ECM is regulated by several enzyme systems, including matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). MMPs are grouped by their ability to degrade different types of ECM components, including collagen. Type IV collagen, which is a major component of the basement membrane and constitutes an important barrier to tumour cell invasion (Liotta et al., 1986), is degraded by both MMP-2 (72 kDa, gelatinase-A) and MMP-9 (92 kDa, gelatinase-B). Some data, originating from in-vitro studies as well as from studies using clinical specimens, have documented the presence of MMPs, especially MMP-2 and MMP-9, in ovarian tumours and their contribution to the invasive phenotype (Campo et al., 1992; Naylor et al., 1994; Afzal et al., 1996; Nictolis et al., 1996; Fishman et al., 1997; Davidson et al., 2000; Huang et al., 2000; Lengyel et al., 2001; Wu et al., 2002). However, most data were from serous and mucinous tumours, and clear cell carcinoma was rarely, if ever, investigated, probably due to its relatively infrequent occurrence (
5–10%) (Kennedy et al., 1989; Omura et al., 1991). Little information is therefore available concerning MMPs in clear cell carcinoma.
In contrast to other histological types of ovarian cancer, clear cell carcinoma shows distinctive clinical characteristics such as frequent presentation as a large pelvic mass, association with endometriosis, and resistance to platinum-based chemotherapy, resulting in poor prognosis even in early stage disease (Jenison et al., 1989; Kennedy et al., 1989; Goff et al., 1996; Behbakht et al., 1998; Sugiyama et al., 2000). To date, however, there are a limited number of studies with respect to molecular or genetic changes that might be related to biological characteristics of clear cell carcinoma (Shimizu et al., 1999; Sancho-Torres et al., 2000; Ho et al., 2001).
In the present study, we performed quantitative analysis of MMP-2, MMP-9, TIMP-1 and TIMP-2 to characterize their expression pattern in ovarian tumours of different histological subtypes, including clear cell carcinoma. We found that TIMP-2 expression was uniquely increased in clear cell carcinoma.
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Materials and methods |
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Tissue collection and preparation
A total of 92 ovarian tumours were obtained by surgery, including 14 benign tumours (five serous and nine mucinous), seven borderline malignant tumours (three serous and four mucinous), and 71 malignant tumours. Of the 71 malignant cases, 24 were serous, 10 mucinous, nine endometrioid, 18 clear cell, five yolk sac tumours, all of which were primary tumours, and five metastatic cancers to the ovary (three gastric and two breast); 30 were stage I, four stage II, 23 stage III, and nine stage IV cases. None of the patients had received pre-operative chemotherapy or radiotherapy. Thirty-six normal ovaries were obtained at the time of the above-mentioned surgery (i.e. normal contralateral ovary) or from women who underwent laparotomy for benign conditions such as uterine leiomyomata, and 18 of them were in a pre-menopausal state and the remainder were post-menopausal. A total of 35 cyst fluid specimens were collected from 17 benign tumours (nine serous and eight mucinous tumours), and 18 carcinomas (two serous, three mucinous, five endometrioid, eight clear cell). All histopathological diagnoses were confirmed by a gynaecological pathologist, and all cases were surgically staged according to FIGO (International Federation of Gynecologists and Obstetricians) criteria. Tissue samples were collected immediately at surgery, snap-frozen in liquid nitrogen and stored at –80°C. Some of the samples were fixed by immersion in 10% neutral buffered formalin for 12–24 h and processed for paraffin embedding by standard methods. Informed consent for tissue collection was obtained from all women in accordance with the institutional guidelines. The study was also conducted in accordance with the provisions of the Declaration of Helsinki.
The frozen tissue samples were homogenized with a motor-driven Teflon pestle for 10 min on ice in 1 ml of extraction buffer (150 mmol/l NaCl, 20 mmol/l Tris, pH 7.5, 1% Nonidet P-40) per 100 mg tissue wet weight, and the tissue homogenates were obtained after centrifugation at 4000 g for 20 min at 4°C. Protein concentrations were determined with the BCA kit (Pierce, USA).
Organ culture of normal ovary and tumour tissues was performed by the method described previously (Udagawa et al., 1998). Briefly, pieces of fresh tissue were minced into small pieces (0.1–0.5 g), rinsed in phosphate-buffered saline (PBS) and cultured in a 6-well Transwell-COL chamber (Coster, USA). Incubation was carried out in the culture medium (Dulbecco’s minimal essential medium supplemented with 10% fetal calf serum) at 37°C in humidified 5% CO2. Medium covering the tissue was renewed after the first 15 min of culture and the culture was continued in serum-free medium. The culture media were then harvested 1, 2, 4 or 24 h later. Each experiment was performed in triplicate.
Quantification of MMP-2, MMP-9, TIMP-1 and TIMP-2
Concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 in the tissue homogenates and the culture media were measured by the corresponding enzyme-linked immunosorbent assays obtained from Amersham Pharmacia Biotech (UK). For the assays, 100 µl of each sample (corresponding to 10 mg protein of the tissue homogenates) were used. The MMP-2 assay detects both the free form of pro-MMP-2 and the complexed form of pro-MMP-2 with TIMP-2, but not active MMP-2. The MMP-9 assay detects the free form of pro-MMP-9 and the complexed form of pro-MMP-9 with TIMP-1. The TIMP-1 assay measures free TIMP-1 and that complexed with MMP. The TIMP-2 assay detects free TIMP-2 and the complexed form of TIMP-2 with active MMP, but not the complexed form with pro-MMP-2. According to the manufacturer, the within- and between-assay coefficients of variation were 5.7 and 10.0% for pro-MMP-2, 5.2 and 8.8% for pro-MMP-9, 9.9 and 13.6% for TIMP-1, and 3.9 and 4.8% for TIMP-2 respectively. All assays were performed in duplicate. Data were calculated with the value of the lower limit of sensitivity of each assay when the concentration of the proteinase was below the detection threshold.
Gelatin zymography
Analysis of MMP-2 and MMP-9 in the tissue homogenates was performed using sodium dodecyl sulphate (SDS)–polyacrylamide gel zymography. Samples (15 µg of proteins per lane) were electrophoresed on SDS–polyacrylamide gels containing 0.3% gelatin. As a positive control, supernatant of HT 1080 human fibrosarcoma cell line (obtained from the American Type Culture Collection, USA) was used. Following electrophoresis, gels were washed three times for 30 min in 2.5% Triton X-100 to remove SDS. After 12 h of incubation in reaction buffer (50 mmol/l Tris–HCl, pH 7.4, containing 5 mmol/l CaCl2 and 0.02% NaN3), gels were stained with Coomassie brilliant blue and destained in 20% methanol and 10% acetic acid. Proteinase activity was observed as a clear band of digested gelatin. Duplicate gels were incubated in reaction buffer containing 10 mmol/l 1,10-phenanthroline (MMP inhibitor) to confirm specificity of activity detected (Herron et al., 1986).
Western blot analysis of TIMP-2
Expression of TIMP-2 in the tissue homogenates was analysed by Western blot. Samples (50 µg of proteins per lane) were separated on 12.5% SDS–polyacrylamide gels and proteins were transferred to the Nitrocellulose Transfer membranes (Amersham Pharmacia Biotech) using a Semi-dry Electrophoretic Transfer Cell system (Trans-Blot® SD BioRad, USA). Membranes were blocked overnight in 5% skim milk in PBS containing 0.1% Tween-20 (T-PBS). Monoclonal mouse anti-TIMP-2 primary antibody (IgG1; Fuji Chemical Industries, Japan) was used at a 1:500 concentration diluted in T-PBS. After 1 h incubation with the primary antibody, membranes were rinsed, exposed for 1 h to a 1:5000 dilution of horseradish peroxidase-linked anti-mouse antibody (Jackson Immuno-Research Lab. Inc., USA). Positive bands were visualized by enhanced chemiluminescence (ECL, Amersham) and exposure to Fuji film. Recombinant hTIMP-2 (Fuji Chemical Industries) was used as the positive control. Incubation with non-immune mouse IgG1, instead of anti-TIMP-2 primary antibody, was also performed to confirm specificity of the bands detected.
Immunohistochemistry of TIMP-2
Formalin-fixed, paraffin-embedded tissue sections were cut 4 µm thick, deparaffinized in xylene, and dehydrated through graded ethanols. Endogenous peroxidase activity was blocked by a 5 min treatment with 3% hydrogen peroxide. Sections were preincubated with 10% normal rabbit serum to minimize non-specific staining. The slides were then incubated with the primary anti-TIMP-2 mouse monoclonal antibody (Fuji Chemical Industries) at 4°C overnight, washed with PBS and incubated with biotinylated rabbit antimouse immunoglobulin G (10 µg/ml; Nichirei, Japan) at room temperature for 30 min. After washing with PBS, the slides were incubated with streptavidin–biotin–peroxidase complex (100 µg/ml; Nichirei). Diaminobenzidine was used as a chromogen for colour development. The slides were counterstained with haematoxylin. Negative control staining was performed using the primary antibody pretreated with an excess of recombinant TIMP-2.
Statistical analysis
Kruskal–Wallis analysis of variance was used to determine the statistical significance of differences of MMP and TIMP levels among the groups, with the Scheffé’s F-test used for between-group comparisons. Mann–Whitney U-test was also performed when appropriate. Findings of P < 0.05 were considered significant.
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Results |
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Proteinase levels in the tissue homogenates
Levels of pro-MMP-2, pro-MMP-9, TIMP-1, and TIMP-2 were determined and compared in the tissue homogenates of normal ovarian tissue, benign ovarian tumour, ovarian borderline malignant tumour, and ovarian malignant tumour of various histological types. All data were separately related to the tissue weight and tissue protein measured in the same sample. There were strong correlations between these ratios, and similar patterns were observed. Therefore, only data related to tissue weight were used for further calculations.
First, the levels were compared between normal ovaries from pre-menopausal women and normal ovaries from post-menopausal women. No statistically significant difference was observed between the two groups (data not shown). Therefore, the data were combined to create one normal group irrespective of the menopausal status of the women.
The levels of pro-MMP-2 were not significantly increased in most of the malignant tumours compared with those in the normal ovary and benign tumours, except for serous cystadenocarcinoma (Figure 1A). On the other hand, pro-MMP-9 levels were markedly increased in most of the malignant tumours (10–40-fold increase in the mean value; Figure 1B). TIMP-1 levels were also increased in ovarian tumours, including benign tumours, as compared with those in the normal ovary (3.2–7.1-fold increase; Figure 2A). In contrast, TIMP-2 levels were decreased in malignant tumours compared with those in the normal ovary (2.7–3.2-fold decrease) except for clear cell carcinoma, in which the levels were significantly increased (1.7-fold increase compared with the normal ovary and >5-fold increase compared with the other malignant tumours; Figure 2B). No statistical difference was observed when TIMP-2 levels in the malignant tumours (except clear cell carcinoma and metastatic tumour) were compared between stages I–II and stages III–IV, indicating that this feature of TIMP-2 levels is independent of clinical stages.
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Proteinase levels in the cyst fluids
Concentrations of pro-MMP-2, pro-MMP-9, TIMP-1 and TIMP-2 in the cyst fluids were compared among benign tumours, serous/mucinous/endometrioid carcinomas, and clear cell carcinomas. As shown in Figure 3, cyst fluids from carcinoma contained higher concentrations of pro-MMP-2 and pro-MMP-9 compared with those from benign tumours (
3-fold and 30-fold increases respectively). Also, TIMP-1 concentration tended to be higher in the cyst fluids from carcinoma. There was no significant difference in TIMP-2 concentration between benign tumour and serous/mucinous/endometrioid carcinoma, while cyst fluids from clear cell carcinoma contained higher TIMP-2 concentration (5-fold increase), which was consistent with the results obtained from the tissue homogenates.
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Proteinase levels in the culture media
To investigate the secretion potential of pro-MMP-2, pro-MMP-9, TIMP-1 and TIMP-2 by the carcinoma tissue, the amounts of these proteinases were measured in the culture media and they were compared with those of the normal ovary. According to the time course study, the proteinase levels in the culture media increased until 2–4 h of incubation, after which they reached a plateau (data not shown). Thus, the incubation time was set at 4 h. As shown in Figure 4, levels of pro-MMP-9 in the samples from carcinoma tissue were significantly higher than those in the normal ovary (
13-fold increase). Unlike pro-MMP-9, no significant difference in pro-MMP-2 and TIMP-1 levels was seen between normal ovary and carcinoma. Concerning TIMP-2, the levels tended to be decreased in serous/mucinous/endometrioid carcinoma compared with the normal ovary, while they were increased in clear cell carcinoma, which was in accordance with the data obtained by the tissue homogenates and cyst fluids.
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Zymography
Gelatin zymography analysis for the tissue homogenates showed MMP-2 and MMP-9 proteins, and the active forms as well as the pro-forms of both gelatinases were observed (representative data shown in Figure 5A). Activities of MMP-9 were markedly higher in carcinoma tissue than in the normal ovary, while they were not elevated in benign tumours, which was in agreement with the results by the quantitative analyses. In contrast, no marked difference in the activities of MMP-2 was seen between the normal ovary and the tumours. Incubating the gels in reaction buffer containing the MMP inhibitor 1,10-phenanthroline resulted in almost complete inhibition of gelatinolytic activities, thus confirming the identity of gelatinase activities as MMPs (Figure 5B).
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Western blot analysis of TIMP-2
To confirm a marked increase in TIMP-2 levels in clear cell carcinoma, Western blot analysis was performed. As shown in Figure 6A, an immunoreactive band (22 kDa) corresponding to TIMP-2 was identified in each sample, and its level was increased in clear cell carcinoma.
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Immunohistochemistry of TIMP-2
In clear cell carcinoma, immunoreactivity for TIMP-2 was localized predominantly in the epithelial cancer cells (Figure 7A). In serous and mucinous carcinomas, on the other hand, TIMP-2 immunoreactivity was seen mainly in the stromal cells, and epithelial cancer cells were scarcely positive for TIMP-2 (Figure 7C, D).
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Discussion |
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It has been generally accepted that MMP-2 and MMP-9 play major roles in tumour invasion by degrading type IV collagen, the main component of the basement membrane. In addition to invasion, MMPs are now known to contribute to multiple steps of tumour progression, including tumour promotion, angiogenesis, and the establishment and growth of metastatic lesions in distant organ sites (Coussens et al., 2002). With respect to ovarian cancer, several reports have emphasized the importance of MMP-2 in its invasiveness and metastasis (Campo et al., 1992; Afzal et al., 1996; Nictolis et al., 1996; Fishman et al., 1997; Wu et al., 2002). The present study demonstrated that normal ovarian tissue had high secretion potential of MMP-2, and that the active form as well as the pro- form of MMP-2 existed even in normal ovary, suggesting that high MMP-2 expression may be a constitutive feature of ovarian tissue. It may not be surprising, therefore, that the increase in MMP-2 levels was relatively modest in malignant ovarian tumours compared with those in normal ovary. In contrast, levels of MMP-9 were markedly increased in malignant tumours compared with those in normal ovary and benign tumours. It was also demonstrated that ovarian cancer tissue had enhanced potential of MMP-9 secretion compared with normal ovary. Consistent with our results, Huang et al. (2000) reported that MMP-9 expression in tumour cells was significantly enhanced in serous and mucinous ovarian carcinomas compared with benign and borderline tumours. The importance of MMP-9 for the invasiveness of ovarian cancer was also reported by other investigators (Naylor et al., 1994; Lengyel et al., 2001).
Proteolytic activity of MMP is physiologically counterbalanced by TIMP, the major local inhibitors of MMP, by binding 1:1 to MMP to inhibit specifically their activities (Mignatti and Rifkin, 1993). TIMP-1 inhibits the active form of all MMP and pro-MMP-9 and is the more widely distributed TIMP (Goldberg et al., 1992). TIMP-2 binds to both forms of MMP-2 while its inhibitory effect over the other MMP is significantly lower (Stetler-Stevenson et al., 1989). The present study showed that TIMP-2 levels were markedly decreased in malignant ovarian tumours compared with normal ovary and benign tumours (except for clear cell carcinoma). This phenomenon seems to support the suggestion that TIMP-2 may act as a housekeeping gene, maintaining the structural integrity of the ovary during the constant tissue remodelling (Simpson et al., 2001). Thus, even though the increase in MMP-2 levels is minimal, the imbalance of MMP-TIMP production, shifted toward greater MMP-2 activity, occurs in malignant ovarian tumours, which is likely to result in enhanced tumour invasiveness. In this regard, there are several reports indicating that the balance of MMP-2 and TIMP-2 may be a critical factor affecting tumour invasion and metastasis. In an experimental study, higher MMP-2:TIMP-2 ratios of renal cell carcinoma cell sublines were directly correlated with more aggressive phenotype through enhancement of their invasive and metastatic potentials (Miyake et al., 1999). Significance of the balance between MMP-2 and TIMP-2 was also pointed out using clinical cancer specimens such as renal cell carcinoma (Kugler et al., 1998), hepatocellular carcinoma (Giannelli et al., 2002), cervical cancer (Nuovo et al., 1995), and choriocarcinoma (Okamoto et al., 2002). In contrast to TIMP-2, on the other hand, TIMP-1 levels in malignant tumours were increased compared with those in normal ovary, which is in agreement with the result by Huang et al. (2000). Considering that the magnitude of increase in MMP-9 levels of malignant tumours compared with normal ovary was much greater than that of TIMP-1, MMP-9–TIMP-1 balance also shifted in favour of MMP-9. A similar phenomenon was also reported in renal cell carcinoma (Lein et al., 2000). Thus, it could be concluded that net gelatinolytic activities of both MMP-2 and MMP-9 are elevated in malignant ovarian tumours (except clear cell carcinoma) in a dual manner by decreasing TIMP-2 expression relative to MMP-2 and increasing the level of MMP-9 itself.
When compared with tumours of other organs, ovarian tumour growth is characterized by cyst formation, which is accompanied by fluid accumulation originating from a relatively homogeneous compartment surrounded by an epithelial cyst wall. Pathophysiological changes in the cyst wall thus lead to alterations of the metabolic composition of cyst fluid, which could be related to cyst wall histology, and determination of metabolites in cyst fluid may provide additional information on the biology of the tumour. In the present study, we investigated levels of MMPs and TIMPs in the cyst fluids as well as those in the tissue homogenates. With respect to pro-MMP-9, a similar tendency was found between these two different types of specimens. It may be curious, however, that modest but increased levels of pro-MMP-2 were observed in cyst fluids of malignant tumours compared with benign tumours although they were not significantly increased in the tissue homogenates of most of the malignant tumours. The rate of degradation of pro-MMP-2 in cyst fluid may be low, or the rate of resorption from cyst fluid into the tissue may be reduced, and only minimal increase in pro-MMP-2 production by the tissue could lead to its increased levels in cyst fluid.
Unique among ovarian cancers, clear cell carcinoma presents distinct clinical characteristics such as a high incidence of stage I tumours, frequent presentation as a large pelvic mass, association with endometriosis, hypercalcaemia and vascular thrombotic events, and resistance to platinum-based chemotherapy (Yoonessi et al., 1984; Jenison et al., 1989; Kennedy et al., 1989; Goff et al., 1996; Behbakht et al., 1998; Sugiyama et al., 2000). Moreover, clear cell carcinoma has increased in prevalence and now accounts for 18.5% of all ovarian cancers in Japan (Ueki et al., 2001). In the present study, we showed for the first time that TIMP-2 levels are markedly increased in clear cell carcinoma compared with other histological types. Moreover, unlike in the other histological types, TIMP-2 immunoreactivity was present mainly in carcinoma cells, not in stromal cells. These features may contribute to the propensity for clear cell carcinoma to remain localized until it becomes a large pelvic mass, possibly through stabilizing the structure of the ECM. In support of this hypothesis, Ohkawa et al. (1977) reported that cancer cells originating from clear cell tumours seeded on a mesothelial cell monolayer remained attached to the mesothelial cells without invading for longer periods than cells originating from serous tumours. Nevertheless, recurrence rate is higher than other histological types, even though presenting at earlier stages (Behbakht et al., 1998; Sugiyama et al., 2000), which is inconsistent with the concept of TIMPs, as functional inhibitors of MMPs. This may be related to the fact that TIMP-2 could stimulate cell proliferation, at least in vitro, via cAMP production (Corcoran and Stetler-Stevenson, 1995; Chambers and Matrisian, 1997) or a tyrosine kinase-dependent signalling pathway (Yamashita et al., 1996; Saika et al., 1998). In fact, high levels of TIMP-2 have been reported to be associated with poor outcome in breast cancer (Ree et al., 1997; Remacle et al., 2000), bladder cancer (Grignon et al., 1996), and tongue cancer (Yoshizaki et al., 2001). Alternatively, high TIMP-2 levels might confer cancer cells more resistance to apoptosis, resulting in higher recurrence rate. In an experimental model using melanoma cell lines, Valente et al. (1998) reported that clones overexpressing TIMP-2 showed reduction in invasion while they were more resistant to apoptosis. In any case, the current findings warrant further studies to elucidate TIMP-2 roles in biological behaviour of clear cell carcinoma.
Taking into account that TIMP-2 levels are elevated in clear cell carcinoma, it may be questionable if synthetic inhibitors against MMP-2 and/or MMP-9 could inhibit tumour growth or metastasis in such cell types, which supports the argument that patients with clear cell carcinoma should be separated from other epithelial ovarian cancers when performing therapeutic trials.
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