Interferon alpha is an immune cytokine with a molecular weight between 10,000 and 50,000 daltons. It has been recently used in various experimental models as an antitumor agent both in vitro and in human clinical studies. Recombinant interferon α-2b has been shown to inhibit the proliferation of fibroblasts in vitro.1 This was found to be due to a reduction in the RNA coding for collagen by collagen-producing cells, as well as an increase in the production of collagenase by fibroblasts.2 Other studies also demonstrated that the combination of interferon α-2b and retinoic acid has a synergistic effect leading to cell growth inhibition and apoptosis in human carcinoma cells in vivo.3 Interferon alpha and transretinoic acid were shown to inhibit the growth of various cervical cancer cell lines in vitro.4 Also, interferon α-2b was used for ophthalmic surgery to treat conjunctival and corneal neoplasia successfully.5
Recently, interferon α-2b was used in the rat model intraperitoneally and subcutaneously to treat experimental endometriosis. The results demonstrated a reduction in the size of the implants, suggesting antiproliferative activity on endometriosis.6 Laparoscopic intraperitoneal injection of human interferon α-2b in women with various stages of endometriosis resulted in an alleviation of symptoms and a reduction in the staging of the disease.7
The present study examined the effect of interferon α-2b at various concentrations on the growth of endometrioma cell lines in vitro. In addition, the study evaluated the effect of interferon α-2b on new DNA production by measurement of thymidine uptake.
MATERIALS AND METHODS
One to two centimeters of endometriomas surgically removed from four patients were used for culture in our laboratory. Patients signed an informed consent document approved by the Institutional Review Board. Our study consisted of 72 observations, 18 observations from each of four different endometrioma cell lines. These were all tumors that fit the histopathologic criteria of endometrioma, taken from all these patients entered in the study. The cell lines grown in vitro from each of these tumors were used in this study to evaluate the effect of interferon α-2b on endometrioma cell growth. The cells were from either the first or second passage of four separate endometrioma stromal cell lines. They were seeded at 3.0 × 104 viable cells per well into six-well plates coated with fibronectin-like protein polymer plus (Sigma-Aldrich, St. Louis, MO). The cells were plated in M199 medium with antibiotic/antimycotic, L-glutamine, endothelial cell growth factor (Sigma-Aldrich), and 10% fetal bovine serum (Hyclone, Logan, UT). Plated cells were incubated at 37C in 5% carbon dioxide and given 24 hours to adhere to the plate. At 24 hours, the medium was exchanged for experimental media containing increasing levels of interferon α-2b (Intron-A, Schering-Plough, Madrid, Spain) as follows: 0 (control), 50, 100, 500, 1000, and 2000 U/mL. Cells were counted by cytometry on days 0, 3, 6, and 9 in quadruplicate, and the counts were averaged for each condition.
In a separate experiment, cells were given interferon α-2b at 4000 U/mL for a 24-hour period and then replaced with M199 without interferon α-2b to evaluate whether the effect on cell growth was transient or permanent. The control consisted of 0 and 4000 U/mL interferon α-2b. The cells were counted on days 3, 6, and 9.
Cells from two endometriomas were plated in quadruplicate at 1 × 104 viable cells per well in plates (96 wells, flat bottom) for 72 hours. On day 3, a concentration of 1000 and 4000 U/mL interferon α-2b was added and simultaneously run with the control media. 3H-thymidine at a concentration of 1 μCi per well (New England Nuclear, Boston, MA) was added to each condition for 24 and 48 hours of incubation at 37C. 3H-thymidine uptake was measured by washing with phosphate-buffered saline and lifting cells off the well with trypsin/ethylenediaminetetra-acetic acid onto Whatman grade 934-AH fiberglass filters. The filters were rinsed five times with distilled water, air dried, and placed in counting vials with scintillation fluid. 3H-thymidine uptake was counted using the Wallac 1410 liquid scintillation counter. The counts were averaged for each condition.
Statistical analyses were performed using Student t tests, analysis of variance, and multiple regression analysis. Student t tests were used to make pairwise comparisons of the effect of the dose on cell growth on a given day. Multiple regression analysis was used to analyze the effects of the dose and time on the cell growth, controlling for patients. Analysis of variance was used to compare the effect of the dose and uptake of 3H-thymidine.
Endometrioma cell growth in vitro was suppressed in proportion to the concentration of interferon α-2b (50–2000 U/mL) compared with the control (0 U/mL). The higher the concentration of interferon α-2b, the lower the cell count (Table 1).
The multiple regression results showed that the higher the dose of interferon α-2b, the lower the cell count, adjusting for time and cell lines. A positive association was found between time and cell growth, controlling for dose and cell lines (Table 2).
When interferon α-2b was added for 24 hours and then replaced with M199, the cell growth rate increased but not to the extent of the growth with the control (0 U/mL). The other control sustained with 4000 U/mL of interferon α-2b continued to decrease (Figure 1).
Interferon α-2b decreased the rate of 3H-thymidine uptake compared with the control at a concentration of 1000 U/mL and 4000 U/mL interferon α-2b, respectively, after 24 and 48 hours' incubation. 3H-thymidine incorporation was reduced significantly relative to the dosage of interferon α-2b (Figure 2, Table 3).
Medical treatment of endometriosis has been based on several concepts. The use of birth control pills and medroxyprogesterone acetate has been well established in producing pseudopregnancy and atrophy in the ectopic endometrium. The use of danazol and GnRH agonists has been used for medical treatment of endometriosis by producing a pseudomenopausal status.8,9 All these medical lines of treatment are associated with side effects on many systems of the body, which may not be acceptable to some patients. Interferon α also has some side effects, including leukopenia, somnolence, tachycardia, hypertension, and some flu-like symptoms. However, some patients may develop a tolerance to these symptoms.10
Interferon α-2b has been tried in vitro and in vivo to suppress the proliferative activity of various tumors.1–5 Recently, interferon α-2b was shown to reduce the size of endometriosis implants in the peritoneal cavity of the rat model. In addition, instillation of interferon α-2b laparoscopically into the peritoneal cavity of patients with endometriosis resulted in the alleviation of the symptoms and a reduction of endometriosis.6,7 It has been suggested that interferon may activate macrophages that help in the suppression of endometriosis growth.11
Previous studies from our laboratory established cell lines in vitro that were derived from endometrioma tissues. These cell lines were evaluated with regard to the effect of estrogen, progesterone, and transforming-growth factor β.12 These cell lines are very helpful in evaluating the effect of interferon α-2b on the growth of endometrioma cells.
The present study demonstrated that interferon α-2b has a direct effect on endometrioma cells in culture. Interferon α-2b has been shown to inhibit the growth of these cells in relation to the concentration and time of exposure. Furthermore, this growth inhibition was demonstrated to be a result of the inhibition of the synthesis of DNA by the cells. This was shown by the marked decrease in 3H-thymidine uptake by the endometrioma cells in vitro as a result of the use of interferon α-2b at varying concentrations. The effect of interferon α-2b on DNA synthesis appears to be a temporary phenomenon. This was evident from the results in this study that revealed the regrowth of the cells after the removal of interferon α-2b.
Some studies have demonstrated that interferon α causes apoptosis for hemangioma and lymphoma cells.13,14 It is not clear from the results of the present study whether interferon α-2b treatment leads to apoptosis of cultured endometrioma cells. This question may be answered with long-term studies. It is also possible that the endometrioma cells' response to interferon is different from other tissues.
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© 2001 by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.
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