To determine the potential combined effects of aspirin and anti–HER-2/neu or heregulin, cultures were exposed to 2-mmol/L aspirin, as this concentration induces approximately 50% growth inhibition. As shown in Table 4, combined exposure to heregulin and aspirin induced 66% suppression of cell growth, whereas exposure to anti–HER-2/neu and aspirin induced 82% growth inhibition, relative to 52% inhibition of OVCAR-3 proliferation in cultures exposed to aspirin alone (P < .05). In cultures treated with a combination of aspirin, anti– HER-2/neu, and heregulin, 72% growth inhibition was observed (Table 3).
Aspirin and other nonsteroidal anti-inflammatory drugs have been shown to decrease the formation of colon carcinomas in animal models2 and to induce the regression of adenomas in patients with familial adenomatous polyposis.17 Epidemiological studies suggest a decreased incidence of ovarian adenocarcinoma in patients regularly taking aspirin.10 Our laboratory has previously demonstrated aspirin-induced inhibition of endometrial carcinoma cells in vitro.13 Previous work in this area by our laboratory and others prompted us to investigate the effects of aspirin on ovarian adenocarcinoma cell lines in vitro and to explore possible mechanisms involved in growth inhibition.
This study demonstrated dose-dependent growth inhibition of OVCAR-3 ovarian carcinoma cells in vitro by aspirin. Possible explanations for this decreased proliferation include apoptosis, inhibition of cyclooxygenase, or some other heretofore unrecognized mechanism. Sulindac and its metabolite sulindac sulfide are aspirin-like compounds that have previously been found to induce apoptosis in HT-29 colon carcinoma cell lines.18 Apoptosis was evaluated in this study but does not appear to be involved in the growth inhibition of OVCAR-3 ovarian carcinoma cells in our system.
Cyclooxygenase controls the rate-limiting step in the conversion of arachidonic acid to prostaglandin endoperoxide (prostaglandin H2).19,20 The inhibition of cyclooxygenase by aspirin and other nonsteroidal anti-inflammatory drugs was first reported by Vane in 1971.21 Subsequent studies showing a decreased incidence of colon cancer in patients regularly taking aspirin spurred investigation into the prostaglandin content of various tumors. Increased levels of cyclooxygenase 1 and 2 have been found in colon, breast, and gastric carcinomas.22–25 The inhibition of prostaglandin formation by aspirin and other nonsteroidal anti-inflammatory drugs has been postulated as the explanation for the reported decreased incidence of these tumors in patients known to take such medications. The prostaglandin content of ovarian neoplasms has not been well studied, although a recent study found that cyclooxygenase-2 expression was increased in ovarian cancer.26 The role of prostaglandins could be further investigated, as we have shown a decrease in cellular proliferation by aspirin, a known inhibitor of cyclooxygenase.
In contrast to the paucity of data concerning cyclooxygenase and its possible role in ovarian carcinoma, the presence of HER-2/neu has been more fully investigated. The protooncogene HER-2/neu encodes a transcytoplasmic receptor with tyrosine kinase activity and has been found to be elevated in 30% of ovarian carcinomas.27 This protooncogene has also been demonstrated in breast and endometrial adenocarcinomas.28–30 Over-expression of HER-2/neu has been found to have prognostic significance in patients with carcinoma of the breast; however, the data are mixed in relation to ovarian carcinoma. Some investigators have shown a decrease in progression-free survival among ovarian carcinoma patients whose tumors strongly express HER-2/neu, whereas others have not shown any such association.31–33 We found high expression of HER-2/neu in the untreated OVCAR-3 tumor cell line, and this expression decreased with increasing doses of aspirin.
Previous work has demonstrated that treatment with anti–HER-2/neu monoclonal antibody affects tumor cell proliferation. Hancock et al34 showed that anti–HER-2/neu antibodies enhanced the cytotoxicity of cis-diaminedichloroplatinum against breast and ovarian tumor cells. Baselga et al35 showed a similar effect on breast cancer xenografts when anti–HER-2/neu antibodies were added with paclitaxel or doxorubicin. Similarly, our study showed a significantly greater growth inhibition when OVCAR-3 ovarian carcinoma cells were treated with anti–HER-2/neu monoclonal antibody and aspirin relative to cells treated with aspirin alone.
In addition to the effect of anti–HER-2/neu antibody alone and in combination with aspirin, we also examined the effect of heregulin, a known ligand for the HER-2/neu receptor. Heregulin has been shown to cause proliferation of breast cancer cells in vitro.28 The current study found that OVCAR-3 cells treated with heregulin alone resulted in different responses depending on the dose of heregulin. Lower doses resulted in cellular proliferation, whereas higher doses of the ligand inhibited growth.
Previous studies have reported conflicting findings in cellular responses to heregulin. Some studies have shown growth stimulation by heregulin, whereas others have reported growth inhibition or cellular differentiation. This differential response to heregulin has been attributed to several mechanisms, including the level of HER-2/neu expression by various cell lines, endogenous expression of heregulin by differing cell populations, and characteristics of individual media in which the cells are grown.
The addition of aspirin, anti–HER-2/neu antibody, and heregulin to OVCAR-3 cells resulted in overall growth inhibition. This study has shown that aspirin inhibits the growth of OVCAR-3 ovarian carcinoma cells in vitro. This inhibition is accentuated by the addition of anti–HER-2/neu monoclonal antibody. The exact mechanism for this inhibition has not yet been elucidated and is the subject of ongoing investigation.
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