Immunohistochemical evaluation of cell proliferation and apoptosis markers in ovaries and uterus of tamoxifen-treated rats

Authors

  • T. CIRPAN,

    Corresponding author
    1. Departments of *Obstetrics and Gynecology and †Physiology, Ege University Faculty of Medicine, Izmir, Turkey; and ‡Department of Pathology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey
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  • M.C. TEREK,

    1. Departments of *Obstetrics and Gynecology and †Physiology, Ege University Faculty of Medicine, Izmir, Turkey; and ‡Department of Pathology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey
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  • M. ULUKUS,

    1. Departments of *Obstetrics and Gynecology and †Physiology, Ege University Faculty of Medicine, Izmir, Turkey; and ‡Department of Pathology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey
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  • E.C. ULUKUS,

    1. Departments of *Obstetrics and Gynecology and †Physiology, Ege University Faculty of Medicine, Izmir, Turkey; and ‡Department of Pathology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey
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  • L. AKMAN,

    1. Departments of *Obstetrics and Gynecology and †Physiology, Ege University Faculty of Medicine, Izmir, Turkey; and ‡Department of Pathology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey
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  • L. KANIT

    1. Departments of *Obstetrics and Gynecology and †Physiology, Ege University Faculty of Medicine, Izmir, Turkey; and ‡Department of Pathology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey
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Teksin Cirpan, MD, Department of Obstetrics and Gynecology, Ege University Faculty of Medicine, Bornova, Izmir 35100, Turkey. Email: teksin.cirpan@ege.edu.tr

Abstract

The aim of the study was to evaluate the immunohistochemical expression of cell proliferation and apoptosis markers in the ovaries and uterus of tamoxifen-treated rats. Twelve rats (150–200 g) were divided into two equal groups. The study group received daily intraperitoneal injections of tamoxifen dissolved in 5% dimethyl sulfoxide (n= 6). The control group received only the vehicle (n= 6). The rats were sacrificed at the 20th day of injection and were perfused. The ovaries and uterus of the rats were extracted. The sections were immunohistochemically stained with cell proliferation marker Ki-67 and the apoptosis markers PTEN and CD95. The expressions of the markers were quantified by a semiquantitative H-score method in myometrium, endometrial glands, ovarian surface epithelium, ovarian follicles, corpus luteum, and ovarian stroma separately. The mean H-scores of CD95 and PTEN obtained from myometrium, glandular endometrium, ovarian surface epithelium, ovarian follicles, corpus luteum, and ovarian stroma did not show significant difference between the study and the control groups. Proliferative index (Ki-67) of endometrial glands was significantly higher in the study group than in the control group (P < 0.05). In addition, proliferative index (Ki-67) of corpus luteum was significantly higher in the study group than in the control group (P < 0.05). Tamoxifen treatment has a potential to stimulate the cell proliferation of endometrial glands and corpus luteum in tamoxifen-treated rats. Apoptosis markers of PTEN and CD95 did not demonstrate significant difference after the tamoxifen treatment.

Tamoxifen is used for adjuvant therapy of early and metastatic breast cancer both, providing improvement in disease-free survival and reducing the risk of developing cancer in the contralateral breast(1). Tamoxifen belongs to a group of drugs termed selective estrogen receptor modulators that are tissue specific in their actions. The pharmacologic and endocrine effects of tamoxifen are target site specific. Tamoxifen appears to behave primarily as an antiestrogen in the treatment of breast cancer(2). Tamoxifen also has estrogenic effects, as documented in the endometrium(3) and other organs(4,5). There have been several reports of endometrial proliferative lesions, including polyps, hyperplasia, and carcinoma, developing in the setting of tamoxifen therapy(6). In 1998, prophylactic administration of tamoxifen was approved by the Food and Drug Administration for the prevention of estrogen receptor–positive breast cancer in high-risk women based on results from the National Surgical Adjuvant Breast and Bowel Project P-1 study. This double-blind, randomized controlled trial found a 49% decrease in the incidence of invasive breast cancer in the tamoxifen arm versus placebo(6).

Tamoxifen is a selective estrogen receptor–modulating agent with many unknown effects on endometrium and ovarian compartments. The objective of the study was to examine markers of proliferation and apoptosis in the endometrial glands and myometrium given the propensity for tamoxifen treatment to result in endometrial hyperplasia, endometrial carcinoma, adenomyosis, and stromal fibrosis. And given the association of tamoxifen with ovarian cyst development, it is also reasonable to look at these markers in ovarian surface epithelium and perhaps corpus luteum as well as ovarian stroma and follicles because of their intimate relationship for the ovarian cyst formation. We believe that a balance between proliferation and apoptosis in these structures predisposes to the development of ovarian cysts.

Materials and methods

Twelve rats of the similar weight (150–200 g) were used in the study according to the permission obtained from the Animal Ethics Committee of Ege University. They were divided into two equal groups. The study group received daily intraperitoneal injections of tamoxifen dissolved in 5% dimethyl sulfoxide (n= 6). The control group received only the vehicle (n= 6). The rats were sacrificed at the 20th day of injection and were perfused. The ovaries and uterus of the rats were extracted with median incision. The specimens were bathed in 10% formalin solution and embedded in paraffin. The blocks were sectioned at poly-l-lysine-coated slides. Sections were deparaffinized in xylol solution for 20 min and rehydrated in decreasing concentration of alcohol series (96%, 90%, 80%, and 70%). The endogen peroxidase activity was blocked using H2O2 3% for 10 min. Sections were put in a special box including 1 mM ethylenediamine tetraacetic acid tampone solution (pH 8.0), and they were boiled in a microwave three times for 5 min. Then, sections were cooled in a room with 24°C for 20 min and washed with a tris solution (pH 7.2) for 5 min. After, these Ki-67 (dilution 1/25, Neo Markers, Fremont, CA), PTEN (dilution 1/100, Novocastra, Newcastle upon Tyne, UK), and CD95 (dilution 1/40, Neomarkers) were applied on the sections. Sixty minutes later, sections were washed with tris solution three times for 5 min. Then, biotinized antibodies were incubated in sections. After 10 min, sections were washed again with tris solution for 5 min, and streptavidin peroxidase solution was applied on sections. Ten minutes later, the sections were washed with tris solution for 5 min, and then 3,3′-diaminobenzidinetetrachloride (DAKO, Glostrup, Denmark) was incubated and left until brown color was seen. After this step, sections were washed with water, and all sections were stained in Mayer’s hematoxylin to make a counterstain. After 2 min, later sections were again washed with water and applied with increasing concentration of alcohol and incubated in xylol for 20 min. Last, the sections were put on a lamel after applying entellan (Merck, Darmstadt, Germany) on it. The expression of the markers in myometrium, glandular endometrium, ovarian surface epithelium, ovarian follicles, corpus luteum, and ovarian stroma was separately evaluated by H-score method. For each slide, an H-score value was derived by summing the percentages of stained cells at each intensity, multiplied by the score of intensity inline image, where i is the intensity score and Pi is the corresponding percentages of the cells. In each slide, five different areas were evaluated under a microscope and percentage of the cells for each intensity within these areas was determined by a pathologist (E.C.U.) blinded to the groups.

All myometrial, endometrial, ovarian surface epithelium, ovarian follicles, corpus luteum, and ovarian stromal H-score values were normally distributed as tested by Kolmogorov–Smirnoff test. Differences between study and control group samples were analyzed by using t test.

All statistical analyses were performed using Sigmastat for Windows, version 3.0 (Jandel Scientific Corporation, San Rafael, CA). Data are presented as the mean ± SEM. Differences were considered to be significant at P < 0.05.

Results

The mean H-scores of CD95 and PTEN obtained from myometrium, glandular endometrium, ovarian surface epithelium, ovarian follicles, corpus luteum, and ovarian stroma did not show significant difference between the study and the control groups (Tables 1, 2).

Table 1.  The results of H-scores of the control and study groups of the PTEN immunostaining
 Control groupStudy groupP value
Myometrium178.3 ± 24.8190.0 ± 56.90.6
Endometrium (glandular structures)191.6 ± 20.4200.0 ± 0.00.6
Endometrium (stroma)213.3 ± 43.2213.3 ± 43.21.0
Ovary (surface epithelium)350.0 ± 83.6350.0 ± 54.70.8
Ovary (corpus luteum)183.3 ± 40.8200.0 ± 0.00.6
Ovary (follicles)230.0 ± 54.7211.6 ± 74.90.6
Ovary (stroma)138.3 ± 23.1173.3 ± 61.20.2
Table 2.  The results of H-scores of the control and study groups of the CD95 immunostaining
 Control groupStudy groupP value
Myometrium188.3 ± 13.2175.0 ± 24.20.2
Endometrium (glandular structures)331.6 ± 77.5393.3 ± 16.30.2
Endometrium (stroma)225.8 ± 92.2186.6 ± 30.70.3
Ovary (surface epithelium)366.6 ± 51.6316.6 ± 75.20.2
Ovary (corpus luteum)383.3 ± 40.8383.3 ± 40.81.0
Ovary (follicles)281.6 ± 57.4276.6 ± 26.50.5
Ovary (stroma)100.0 ± 0.0106.6 ± 5.10.06

Proliferative index (Ki-67) of endometrial glands was significantly higher in the study group (Fig. 1) than in the control group (Fig. 2), P < 0.05. In addition, proliferative index (Ki-67) of corpus luteum was significantly higher in the study group (Fig. 3) than in the control group (Fig. 4), P < 0.05 (Table 3).

Figure 1.

Endometrial glands of tamoxifen-treated rats with a high Ki-67 proliferative index, scale bar represents 50 μm.

Figure 2.

Endometrial glands of control with a low Ki-67 proliferative index, scale bar represents 50 μm.

Figure 3.

Corpus luteum of tamoxifen-treated rats with a high Ki-67 proliferative index, scale bar represents 50 μm.

Figure 4.

Corpus luteum of control with a low Ki-67 proliferative index, scale bar represents 50 μm.

Table 3.  The results of proliferative index for control and study groups
 Control groupStudy groupP value
Endometrium (glandular structures)0.0 ± 0.020.5 ± 19.30.03
Ovary (corpus luteum)0.0 ± 0.02.6 ± 1.80.01
Ovary (follicles)23.5 ± 11.024.1 ± 12.4>0.05

Discussion

Tamoxifen is a widely used drug for the treatment of all stages of breast cancer. Tamoxifen affects a variety of organ systems other than the breast. One of the most important side effects of tamoxifen appears to be its proliferative effect on the endometrium, which may result in the development of polyps and malignant neoplasms(7). Tamoxifen users have a twofold to sevenfold increased risk of endometrial cancer, and the risk seems to be highest after long-term use(8,9).

Tamoxifen has both agonistic and antagonistic effects on the female genital tract, depending on the ambient estradiol concentration and the menopausal status of the patient. In postmenopausal women, tamoxifen has an estrogen agonistic effect on the vaginal epithelium, the uterine myometrium, and the endometrium. Many studies have suggested an association between the use of tamoxifen in breast carcinoma patients and the subsequent development of endometrial carcinomas(10–12). Silva et al.(13) showed that 10 (77%) of 13 postmenopausal patients who developed endometrial carcinoma after tamoxifen treatment had endometrial polyps, whereas 16 (34%) of 47 patients in a comparable group who did not receive tamoxifen had endometrial polyps. Deligdisch et al.(14) reported that 15 of 33 endometrial carcinomas that developed in postmenopausal breast cancer patients subsequent to adjuvant tamoxifen treatment were found in endometrial polyps.

Mourits et al.(15) evaluated apoptosis and apoptosis-related factors in endometrium in relation to tamoxifen exposure. No differences were observed in the mean apoptotic index in benign endometrium in tamoxifen users (0.17%) versus controls (0.08%) or in tamoxifen-exposed (2.46%) versus nonexposed endometrial cancer (2.28%). However, the ratio of the apoptotic index with the previously reported proliferation index was lower in benign endometrium from tamoxifen users than in controls. In benign endometrium, FasL was more frequently expressed in tamoxifen users than in controls. They conclude that the apoptosis/proliferation ratio in benign endometrium from tamoxifen users is lower than in controls, indicating that the tamoxifen-induced higher proliferation is not compensated for by increased apoptosis. An imbalance between cell proliferation and apoptosis and possibly suppression of the antitumor immune response by FasL overexpression in tamoxifen-exposed endometrium might play a role in the development of endometrial cancer in tamoxifen users. Holtz et al.(16) evaluated PTEN immunohistochemical expression in tamoxifen-associated endometrial cancer. During the study period, 4 of 15 (27%) tamoxifen users expressed PTEN compared with 2 of 13 (15%) of nonusers. They indicated that tamoxifen-associated endometrial cancers are not significantly different from sporadic endometrial cancer with regards to PTEN expression. In the present study, there is no significant difference in apoptosis index of myometrium and glandular endometrium between the study and the control groups. However, proliferative index of endometrial glands was significantly higher in the study group than in the control group.

Little is known about steroid receptor status, somatic alterations in oncogenes and tumor suppressor genes, and inherited susceptibility in endometrial carcinomas associated with tamoxifen use. In contrast to the endometrium, there has been little published regarding the pathologic effects of tamoxifen on other tissues of the female genital tract. Anecdotal reports of pathologic changes in the ovary and fallopian tube during tamoxifen therapy have been reported(4,5).

In premenopausal women, tamoxifen may induce ovarian cysts resulting in high serum estradiol levels. Kazandi et al.(17) investigated the frequency of ovarian cysts in tamoxifen-treated postmenopausal breast cancer patients along with endometrial thickening detected by transvaginal sonography. During the study period, 5 of 38 tamoxifen-treated postmenopausal patients (13.2%) had ovarian cysts. The three patients with ovarian cysts underwent laparotomy revealing simple cysts on histopathologic examination. Seoud et al.(18) reported that a patient with breast cancer has been affected with papillary serous carcinoma of the ovary after tamoxifen use. Metindir et al.(19) evaluated patient-related parameters that determine ovarian cyst formation in women using tamoxifen for breast cancer. Twenty-nine of 150 tamoxifen-treated patients (19.3%) had ovarian cysts. Cysts were detected in 28 of 57 premenopausal women (49.1%) and 1 of 93 postmenopausal women (1.1%). Patients with ovarian cysts had higher serum estradiol levels compared with patients without cysts. They indicated that patients still having a menstrual cycle during tamoxifen had high risk (58.33%) of developing ovarian cysts.

Tamoxifen trials have not reported an increased risk of ovarian cancer(6,20). However, not statistically significant, Metcalfe et al.(21) reported that tamoxifen use was associated with modestly increased risk of ovarian cancer after breast cancer in women who had mutation of BRCA1 or BRCA2. In immunohistochemical study, Ki-67 was a significant survival factor in ovarian malignancies(22) and was related in dissemination of the disease, residual tumor bulk, and poor differentiation(23). In addition, reduced PTEN expression was closely associated with tumorigenesis and pathologic behaviors of ovarian adenocancers, and abnormal expression of PTEN gene was involved in progression of ovarian cancer(24,25). Leng et al.(26) found that droloxifen (a tamoxifen derivative) could induce apoptosis of rat luteal cells in vitro. The immunohistochemical study of ovarian cancer associated with tamoxifen use was limited. Considering the rat menstrual cycle (average 4 days), five cycles of intraperitoneal treatment is sufficient to see changes in the endometrium or the formation of ovarian cysts. The credibility of intraperitoneal injection is always higher despite its difficulty, and the vehicle of tamoxifen is not a proper gavage fluid because of its toxicity.

For this reason, we evaluated immunohistochemical expression of cell proliferation and apoptosis markers not only in rat uterus but also in ovaries. In the present study, the apoptosis index of ovarian surface epithelium, ovarian follicles, corpus luteum, and ovarian stroma did not show significant difference between the study and the control groups. In addition, proliferative index (Ki-67) of corpus luteum was significantly higher in the study group than in the control group.

In conclusion, tamoxifen treatment has a potential to stimulate the cell proliferation in rat uterus and ovary. Apoptosis markers of PTEN and CD95 did not demonstrate significant difference after the tamoxifen treatment. Tamoxifen may have a downregulating effect on Ki-67, which may be contributing to ovarian cyst formation and endometrial pathology.

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