Drake, Janet G. MD; Becker, Jeanne L. PhD
Epithelial ovarian adenocarcinoma often causes few symptoms early in the course of the disease and therefore is often diagnosed in its advanced stages. Unfortunately, no reasonably sensitive or specific tests exist to make routine screening cost‐effective for early detection or prevention. All of these reasons make chemoprevention an attractive concept for carcinoma of the ovary.
Animal and human studies with aspirin and other nonsteroidal anti‐inflammatory drugs have shown a chemopreventive role for these drugs in carcinoma of the colon.1 Formation of colon tumors is decreased in those average‐risk patients who regularly take aspirin.2 The data are mixed concerning the role of aspirin or nonsteroidal anti‐inflammatory drugs and breast cancer incidence. Several rodent models suggest nonsteroidal anti‐inflammatory drugs reduce the formation of breast tumors.3–6 Some studies have also shown a lower incidence of breast cancer than expected in rheumatoid arthritis patients who routinely take aspirin.7,8 Egan et al9 analyzed data prospectively from the Nurses' Health Study and did not find a decreased risk of breast cancer in participants who regularly consumed aspirin. However, epidemiological studies have suggested that regular users of nonsteroidal anti‐inflammatory drugs have a lower incidence of ovarian adenocarcinoma.10 This prompted us to investigate whether aspirin inhibited the growth of ovarian adenocarcinoma in vitro.
Another emerging area of interest in cancer research is that of oncogenes. The protooncogene HER‐2/neu encodes a glycoprotein that resembles the receptor for epidermal growth factor. Over‐expression of HER‐2/neu has been found in breast, endometrial, and ovarian carcinomas. However, the prognostic significance of HER‐2/neu in ovarian carcinoma is uncertain.11 We wished to determine if HER‐2/neu was expressed in the ovarian cancer cell line studied in this report and whether its expression was affected by treatment with aspirin.
MATERIALS AND METHODS
The OVCAR‐3 human ovarian adenocarcinoma cell line was obtained from the American Type Culture Collection (Rockville, MD). The cells were grown in culture media, termed “M‐GTSF,” consisting of a 1:1 mixture of phenol red‐free Dulbecco's Modified Eagle Medium and Ham's Nutrient F‐12 medium (GIBCO‐BRL, Grand Island, NY) supplemented with 20% heat‐inactivated (56C, 30 minutes), charcoal‐adsorbed fetal bovine serum (Hyclone, Logan, UT), 0.5% minimum essential medium vitamins, 0.5% insulin‐transferrin‐selenium, 0.034% NaCO3, 200‐mmol/L L‐glutamine, 8‐mg/mL tylosine, 10‐mmol/L HEPES buffer, 20‐μg/mL gentamycin (all obtained from GIBCO‐BRL), 48‐μg/mL inositol, and 130‐μg/mL fructose (Sigma Chemical Co., St. Louis, MO). Monolayer cultures were incubated at 37C and grown to confluency in a humidified atmosphere of 5% CO2 in 95% air. Confluent cultures were harvested by exposure to 0.1% trypsin for 5 minutes at 37C.
A 1‐mol/L acetylsalicylic acid (Sigma Chemical Co.) stock solution was prepared in absolute ethanol and stored at room temperature. Stock aspirin solutions were diluted in phosphate‐buffered saline (Sigma Chemical Co.) and added to tissue culture medium to achieve final concentrations of 1–5 mmol/L in treated cultures. The aspirin concentrations used were similar to those used in previous in vitro studies performed on colorectal and endometrial cancer cells.12,13 Murine antihuman monoclonal antibody to HER‐2/neu was obtained from Oncogene Research Products (Cambridge, MA). Lyophilized antibody was resuspended in sterile phosphate‐buffered saline to obtain a 100‐μg/mL stock solution from which 0.1‐, 1.0‐, and 5.0‐μg/mL dilutions were prepared. Human recombinant heregulin protein (Oncogene Research Products) was prepared as a 50.0‐μg/mL stock by reconstituting the lyophilized protein in sterile phosphate‐buffered saline containing 0.1% bovine serum albumin (Fraction V, Sigma Chemical Co). Concentrations of heregulin ranging from 0.01 to 50.0 ng/mL were then prepared in phosphate‐buffered saline from the 50.0‐μg/mL stock solution and added to cell cultures.
HER‐2/neu expression was determined by flow cytometry as described previously.14 Briefly, a 0.5‐ml suspension containing approximately 106 cells was incubated with 2.5 ng of the HER‐2/neu monoclonal antibody at 0C for 20 minutes. The cells were washed twice by centrifugation in phosphate‐buffered saline, and the cell pellet was resuspended with phosphate‐buffered saline and incubated with phycoerythrin‐labeled rat antimouse immunoglobulin (Becton Dickinson, San Jose, CA). After centrifugation, the cell pellet was again washed with phosphate‐buffered saline and then resuspended in 1% paraformaldehyde (Sigma Chemical Co.). The percentages of cells exhibiting positive staining were determined utilizing a FACScan flow cytometer and the Lysis II software program (Becton Dickinson).
Cellular proliferation was measured using the Promega Cell Titer 96 Non‐Radioactive Proliferation Assay (Promega, Madison, WI). Cultured flasks of OVCAR‐3 cells were harvested by trypsinization, resuspended in culture media, and counted by hemocytometer. Cells were then seeded into flat‐bottom 96‐well microtiter plates (Becton Dickinson, Lincoln Park, NJ) at concentrations ranging from 1.5 × 103 to 2.5 × 103 cells per well, determined to be optimal based upon pilot studies in which cells were exposed to vehicle versus heregulin at starting densities of 500 to 1.5 × 104 cells per well. Cells were grown at 37C with vehicle (1% absolute alcohol), 1–5‐mmol/L aspirin, 0.1–5.0‐μg/mL anti–HER‐2/neu monoclonal antibody, or 0.01–50.0‐ng/ml heregulin either alone or in combination. Cells were seeded onto 96‐well plates. Proliferation was determined spectophotometrically by the incorporation of tetrazolium dye; optical density at 590 nm was then determined using an enzyme‐linked immunosorbent reader (Titertek Multiscan Plus; Flow Laboratories, McLean, VA). The intra‐ and interassay coefficients of variation were less than 5% and 6%, respectively. Optical densities were compared between controls and treatment groups to obtain the percentage of inhibition or proliferation.
Apoptosis was detected by use of the Promega Apoptosis Detection System with fluorescein as described previously.15 The percentages of cells showing fluorescein labeling of DNA fragments, indicative of apoptosis, were detected with the Becton Dickinson FACScan flow cytometer using Lysis II software. Green fluorescence of fluorescein–12‐deoxyuridine triphosphate at 520 nm and red fluorescence of propidium iodide at 620 nm were measured. Measurements obtained from the apoptosis assays were compared using the Student t test.
Statistical analysis was performed with SigmaStat software (Jandel Scientific, Palo Alto, CA). Cell proliferation data were expressed as percent inhibition of the control culture proliferation. Percent inhibition was calculated by dividing the mean growth in the experimental group by the growth in the control group. Standard errors were within 10% of the mean of replicate wells. Sample size was calculated to detect a difference in the means of 20% with an α of .05 and a power of 0.800. Data were analyzed by one‐way analysis of variance and significant differences determined by the Dunnett test; a P value of less than .05 was considered significant. Dose‐dependent relations were determined by multiple linear regression.
Treatment with aspirin inhibited OVCAR‐3 tumor cell growth in a dose‐dependent fashion (Table 1). Relative to controls, cultures exposed to 5‐mmol/L aspirin exhibited nearly 70% inhibition of growth. The results shown in Table 2 illustrate that exposure of the cells to aspirin also resulted in a dose‐dependent decrease in the expression of HER‐2/neu.
Apoptosis assays were performed on cells treated with 3‐ and 5‐mmol/L aspirin in an attempt to determine the mechanism of growth inhibition. Cultures exposed to 3‐mmol/L aspirin exhibited 5% apoptosis, whereas those treated with 5‐mmol/L aspirin showed less than 1% apoptosis (P > .05).
Exposure of OVCAR‐3 cells to anti‐HER‐2/neu monoclonal antibody resulted in growth inhibition in a dose‐dependent fashion. Treatment with 100‐ng/mL heregulin protein inhibited proliferation by 25%; this response did not significantly differ in the presence of combined treatment with the antibody and heregulin (Table 3). Inhibition of growth by heregulin was surprising, as other in vitro studies have shown cellular proliferation with exposure to the HER‐2/neu receptor ligand.16 For this reason, studies were done to determine if heregulin's effect on cell growth was dose dependent. We found that lower doses of heregulin were growth stimulatory, whereas higher dosages resulted in growth inhibition (Table 4).
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.
Xu et al36 found that heregulin inhibited the growth of SKOV‐3 ovarian cancer, a cell line known to over‐express HER‐2/neu, but stimulated the growth of OVCAR‐3 cells, in which HER‐2 expression was lower. These same cell lines transfected to cause either up‐ or downregulation of HER‐2/neu resulted in inhibition of OVCAR‐3 cell growth and stimulation of SKOV‐3 cells. Xu et al found treatment of OVCAR‐3 cells with 10‐ng/mL heregulin resulted in 400% growth, whereas the current study found only 1% growth in the same cell line treated with a similar dosage. This disparity in growth might be attributed to different growth media used in the two studies. We used growth media supplemented with 20% fetal bovine serum, whereas Xu et al used a 10% serum. Lewis et al37 showed cellular proliferation of MCF‐7 breast cancer cells in response to heregulin when grown in 1% fetal bovine serum, but no effect when grown in 10% serum concentration. Our results are in keeping with those of Pegues et al,38 who found that higher doses of heregulin were associated with growth inhibition, whereas lower doses revealed no or minimal growth stimulation.
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.
1. Shiff SJ, Rigas B. Nonsteroidal anti-inflammatory drugs and colorectal cancer: Evolving concepts of their chemopreventive actions. Gastroenterology 1997;113:1992–8.
2. Kune G, Kune S, Watson L. Colorectal cancer risk, chronic illnesses, operations, and medications: Case-control results from the Melbourne Colorectal Cancer Study. Cancer Res 1988;48:4399–404.
3. Thompson HJ, Briggs S, Paranka NS, Piazza GA, Brendel K, Gross PH, et al. Inhibition of mammary carcinogenesis in rats by sulfone metabolite of sulindac. J Natl Cancer Inst 1995;87:1259–60.
4. Carter CA, Milholland RJ, Shea W, Ip MM. Effect of the prostaglandin synthetase inhibitor indomethacin on 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in rats fed different levels of fat. Cancer Res 1983;43:3559–62.
5. McCormick DL, Madigan MJ, Moon RC. Modulation of rat mammary carcinogenesis by indomethacin. Cancer Res 1985;45:1803–8.
6. McCormick DL, Wilson AM. Combination chemoprevention of rat mammary carcinogenesis by indomethacin and butylated hydroxytoluene. Cancer Res 1986;46:3907–11.
7. Gridley G, McLaughlin JK, Ekbom A, Klareskog L, Adami HO, Hacker DG, et al. Incidence of cancer among patients with rheumatoid arthritis. J Natl Cancer Inst 1993; 85:307–11.
8. Laasko M, Mutru O, Isomaki H, Koota K. Cancer mortality in patients with rheumatoid arthritis. J Rheumatol 1988;15:1319–22.
9. Egan KM, Stampfer MJ, Giovannucci E, Rosner BA, Colditz GA. Prospective study of regular aspirin use and the risk of breast cancer. J Natl Cancer Inst 1996;88:988–93.
10. Cramer DW, Harlow BL, Titus-Ernstoff L, Bohlke K, Welch WR, Greenberg ER. Over-the-counter analgesics and risk of ovarian cancer. Lancet 1998;351:104–7.
11. Cirisano FD, Karlan BY. The role of the HER-2/neu oncogene in gynecologic cancers. J Soc Gynecol Invest 1996;3:99–105.
12. Elder DJ, Hague A, Hicks DJ, Paraskeva C. Differential growth inhibition by the aspirin metabolite salicylate in human colorectal tumor cell lines: Enhanced apoptosis in carcinoma and in vitro-transformed adenoma relative to adenoma cell lines. Cancer Res 1996;56:2273–6.
13. Arango HA, Icely S, Roberts WS, Cavanagh D, Becker JL. Aspirin effects on endometrial cancer cell growth. Obstet Gynecol 2001;97:423–7.
14. Becker JL, Papenhausen PR, Widen RH. Cytogenetic, morphologic, and oncogene analysis of a cell line derived from a heterologous mixed mullerian tumor of the ovary. In Vitro Cell Dev Biol 1997;33:325–31.
15. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493–501.
16. Holmes WE, Sliwkowski MX, Akita RW, Henzel WJ, Lee J, Park JW, et al. Identification of heregulin: A specific activator of p185erbB2. Science 1992;256:1205–10.
17. Waddell WR, Loughry RW. Sulindac for polyposis of the colon. J Surg Oncol 1983;24:83–7.
18. Shiff SJ, Qiao L, Tsai L, Rigas B. Sulindac sulfide, an aspirin-like compound, inhibits proliferation, causes cell cycle quiescence, and induces apoptosis in HT-29 colon adenocarcinoma cells. J Clin Invest 1995;96:491–503.
19. Bjorkman DJ. The effect of aspirin and nonsteroidal anti-infammatory drugs on prostaglandins. Am J Med 1998; 105(1B):8S–12S.
20. Dubois RN, Abramson SB, Crofford L, Gupta RA, Simon LS, Van De Putte LBA, et al. Cyclooxygenase in biology and disease. FASEB J 1998;12:1063–73.
21. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature 1971;231:232–5.
22. Eberhart CE, Coffee RJ, Radhika A, Giardello FM, Ferrenbach S, Dubois RN. Up-regulation of cyclooxygenase-2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 1994;107:1183–8.
23. Bennett A, McDonald AM, Stamford IF, Charlier EM, Simpson JS, Zebro T. Prostaglandins and breast cancer. Lancet 1977;2:624–6.
24. Hwang D, Scollard D, Byrne J, Levine E. Expression of cyclooxygenase-1 and cyclooxygenase-2 in human breast cancer. J Natl Cancer Inst 1998;90:455–60.
25. Uefuji K, Ichikura T, Mochizuki H. Expression of cyclooxygenase-2 in human gastric adenomas and adenocarcinomas. J Surg Oncol 2001;76:26–30.
26. Matsumoto Y, Iskiko O, Deguchi M, Nakagawa E, Ogita S. Cyclooxygenase-2 expression in normal ovaries and epithelial ovarian neoplasms. Int J Mol Med 2001;8:31–6.
27. Goff BA, Muntz HG, Greer BF, Tamimi HK, Gown AM. Oncogene expression: Long-term compared with short-term survival in patients with advanced epithelial ovarian cancer. Obstet Gynecol 1998;92:88–93.
28. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER-2/neu protooncogene in human breast and ovarian cancer. Science 1989;244:707–12.
29. Hetzel DJ, Wilson TO, Keeney GL, Roche PC, Cha SS, Podratz KC. HER-2/neu expression: A major prognostic factor in endometrial cancer. Gynecol Oncol 1992;47:179–85.
30. Rosen PP, Lesser ML, Arroyo CD, Cranor M, Borgen P, Norton L. Immunohistochemical detection of Her-2/neu in patients with axillary lymph node negative breast cancer. Cancer 1995;75:1320–6.
31. Tanner B, Kreutz E, Weikel W, Meinert R, Oesch F, Knapstein PG, et al. Prognostic significance of c-erbB-2 mRNA in ovarian carcinoma. Gynecol Oncol 1996;62:268–77.
32. Berchuck A, Rodriguez GC, Kamel A, Dodge RK, Soper JT, Clarke-Pearson DL, et al. Epidermal growth factor receptor expression in normal ovarian epithelium and ovarian cancer. I. Correlation of receptor expression with prognostic factors in patients with ovarian cancer. Am J Obstet Gynecol 1991;164:669–74.
33. Rubin SC, Finstad CL, Federici MG, Scheiner L, Lloyd KO, Hoskins WJ. Prevalence and significance of HER-2/neu expression in early epithelial ovarian cancer. Cancer 1994;73:1456–9.
34. Hancock MC, Langton BC, Chan T, Toy P, Monahan JJ, Mischak RP. A monoclonal antibody against the c-erbB-2 protein enhances the cytotoxicity of cis-diamminedichloro-platinum against human breast and ovarian tumor cell lines. Cancer Res 1991;51:4575–80.
35. Baselga J, Norton L, Albanell J, Young-Mee K, Mendel-sohn J. Recombinant humanized anti-HER-2/neu (Herceptin™) enhances the antitumor activity of paclitaxel and doxorubicin against HER-2/neu overexpressing human breast cancer xenografts. Cancer Res 1998;58:2825–31.
36. Xu F, Yu Y, Le X, Boyer C, Mills GM, Bast RC. The outcomes of heregulin-induced activation of ovarian cancer cells depends on the relative levels of HER-2/neu and HER-3 expression. Clin Cancer Res 1999;5:3653–60.
37. Lewis GD, Lofgren JA, McMurtrey AE, Nuijens A, Fendly BM, Bauer KD, et al. Growth regulation of human breast and ovarian tumor cells by heregulin: Evidence for the requirement of erbB2 as a critical component in mediating heregulin responsiveness. Cancer Res 1996;56:1457–65.
38. Pegues JC, Kannan B, Stromberg K. ErbB receptor expression and growth response to heregulin β1 in five ovarian carcinoma lines. Int J Oncol 1999;14:1169–76.