Abstract
Background/Aim: Estrogen receptor α (ERα) and Ki-67 are strong prognostic and predictive markers in woman breast cancer. This study aimed to evaluate the immunoexpression of prognostic factor markers ERα, Ki-67 proliferation index (Ki-67 PI) and the mitotic activity index (MAI) in mammary tumors induced by 1-methyl-1-nitrosourea (MNU) in female Sprague-Dawley rats submitted to lifelong exercise training. Materials and Methods: Thirty female rats were injected with MNU and randomly divided into two groups: sedentary and exercised. Results: All neoplasms from both groups were ERα-positive with an H-score ≥20. Statistically significant differences were not found in the ERα H-score, Ki-67 PI and MAI between groups. The absolute value of ERα H-score was higher in the exercised group, while the Ki-67 PI and MAI were higher in the sedentary group. Conclusion: Tumors from the exercised group were less proliferative and more differentiated, suggesting that long-term exercise training had positive effects on mammary carcinogenesis.
Breast cancer affects approximately one out of ten women in the world and it has been demonstrated that chemically-induced mammary tumors in female rats histopathologically resemble those developed by humans (1). When exposed to the carcinogen agent 1-methyl-1-nitrosourea (MNU), female Sprague-Dawley rats develop a high number of mammary tumors (2-4). Additionally, the mammary tumors induced by this chemical carcinogen behave as hormone-dependent and they are characterized by the expression of estrogen receptors (ERs) (5-8).
ERs are nuclei receptors that regulate cell growth, differentiation and homeostasis. Until now, two ERs isoforms are known: ERα and ERβ. The steroid hormone estradiol, which is necessary for sexual differentiation, fertility and development, acts by binding with these two isoforms (9, 10). The ERα expression is a well-established marker of cell proliferation and its high expression is considered a good prognostic and predictive marker related with increase of survival and delay of tumor recurrence after hormone therapy in human mammary cancer (11, 12).
Another important marker of cellular proliferation in mammary cancer is the protein Ki-67, which is involved in cellular proliferation and expressed in different phases of the cell cycle, such as G1, S, G2 and M, but not in the resting phase G0. Furthermore, the expression of Ki-67 is associated with a ribosomal pathway for RNA transcription (13-15). Although Ki-67 is not included as a routine biological marker, it is recommended as a prognostic and predictive marker in human mammary cancer (15).
Researchers who have conducted studies in this field, using the female Sprague-Dawley rat model of mammary cancer have observed positive effects of physical exercise on these tumors, including the rate of tumors per animal and the type of tumors in terms of their histopathological grade (16-19). This work aimed to evaluate the effects of lifelong exercise training on the immunoexpression of ERα, Ki-67 and the mitotic activity index (MAI) in MNU-induced mammary tumors in female Sprague-Dawley rats.
Materials and Methods
Animals and experimental design. All procedures followed the biosecurity standards specified for the studies using animal models and were approved by Direção Geral de Alimentação e Veterinária (Approval n° 008961). Thirty outbred female Sprague-Dawley rats were divided into two experimental groups as follows: sedentary (n=15) and exercised (n=15). At 50th day of age, all animals received an intraperitoneal injection (50 mg/kg body weight) of the carcinogen agent MNU (ISOPAC®, lot 100 M1436V; Sigma Chemical Co., Madrid, Spain). The day of the carcinogen administration was defined as the first day of the experiment.
A Treadmill Control® LE 8710 (Panlab, Harvard Apparatus; Holliston, MA, USA) was used for the exercise training. Animals were subjected to the exercise training during 35 weeks (60 min/day, at a speed of 20 m/min, 5 days a week with a rest of 2 days). Both mammary gland chains of all animals were weekly palpated.
Sacrifice and necropsy of animals. All animals were sacrificed by exsanguination by cardiac puncture under deep anesthesia (ketamine and xylazine), as indicated by the Federation of European Laboratory Animal Science Associations (20). Mammary tumors were excised and immediately fixed in 10% phosphate buffered-formaldehyde during 24 hours.
Histopathology and immunohistochemistry. The fixated tissues were cut, processed, embedded in paraffin and 2-μm-thick cut sections were stained with hematoxylin and eosin (H&E) for histopathological evaluation by three independent pathologists. The tumors were histologically classified and categorized according to their histogenesis and biological behavior, according to Russo and Russo (21), taking into consideration only the concordance type of lesion or lesions with a high proportion in each tumor section. For immunohistochemistry, the NovolinkTM Polymer Detection System (Leica®, Newcastle, UK) was used according to the instructions provided by the manufacturer. The sections were incubated with the primary antibody for ERα (clone 6F11, mouse monoclonal anti-human; Leica®) at a dilution of 1:50 for one and half hour at room temperature and with the primary antibody for Ki-67 (clone MIB-5, mouse monoclonal anti-rat; DAKO®, Glostrup, Denmark) at a dilution of 1:50, overnight at 4°C. The antigen retrieval was performed with thermic treatment by microwave using 3 cycles for ERα and 4 cycles for Ki-67 of 5 minutes each one in citrate buffer solution. The tissues were counterstained with hematoxylin.
The H-score method was used for assessment of the ERα immunoexpression in a total of 1,000 neoplastic cells producing a score range from 0 to 300, as previously described by Kinsel et al. (12). An H-score of more than 1 was considered as positive. Normal mammary tissue with and without the primary antibody was used as positive and negative control, respectively.
The Ki-67 was scored as the percentage of nuclei-stained cells in a total of 1,000 neoplastic cells, independently of invasiveness of the tumor, using a 40× objective for high power fields (HPF) and denoted as Ki-67 proliferation index (Ki-67 PI). The tumor tissue without the primary antibody was used as negative control and the hair follicles present in the histological cut that looked a correct intensity of nuclear staining were used as positive control.
The MAI was evaluated in 10 HPF of the most proliferative areas of the tumor, i.e. tumor periphery excluding necrosis and apoptosis, using a 40× objective (LEICA DM500 diagnostic microscope, 0.45 mm diameter; Leica®). The MAI was scored taking into account the mitotic figures per microscopic field and denoted as: low (0-6), intermediate (7-14) or high (≥15) (22).
ER H-score, Ki-67 PI and MAI were evaluated by two independent pathologists.
Statistical analysis. Descriptive statistics, such as mean, standard deviation (SD) and proportion in base to percentage were used to express the presence and classification of mammary tumors in both experimental groups. For the comparison between groups, the ANOVA and Tukey-Kramer honestly significant difference (HSD) tests for all pairs were used. The statistical analysis was performed using the JMP starter 5.0.1. program (SAS Institute Inc., Cary, NC, USA). p-Values lower than 0.05 were considered statistically significant.
Results
General findings. Eight animals died during the experiment: four animals from the sedentary group and four from the exercised one. Additionally, one animal from the exercised group did not adapt to the exercise training and was excluded from the experiment. At the end of the experiment, the sedentary group had 11 animals and the exercised group had 10 animals. All animals from both groups, exercised and sedentary, developed mammary tumors (100% of incidence).
Histopathological evaluation of mammary tumors. Considering the predominant histological patterns in each mammary tumor, a total of 32 mammary lesions (all neoplasms) in the exercised group and 37 mammary lesions (neoplasms and preneoplastic lesions) in the sedentary group were identified (Figure 1A and 1B). Papillary carcinoma was the histological pattern most frequently identified in both groups, followed by the cribriform carcinoma pattern.
The grade of malignancy of mammary tumors was higher in sedentary group than in exercised one; the majority of the mammary neoplasms identified in both groups were classified as epithelial malignant lesions (p>0.05) (Figure 1B). Additionally, the animals from sedentary group developed two preneoplastic lesions (intraductal proliferation (IDP)) and two comedo carcinomas that were not found in exercised group (Figures 1A and 2A).
Immunohistochemical evaluation of mammary tumors. All mammary lesions from both groups were ERα-positive with an H-score ≥20. Concerning the ERα H-score in each histological pattern, the papillary pattern in exercised group exhibited the highest mean and fibroadenoma the lowest mean, with these values being statistically different (p<0.05) (Table I). However, the differences of Ki-67 PI among histological patterns did not reach the level of statistical significance (p>0.05). In general, the MAI was low (<5). The comedo carcinoma in the sedentary group was the histological pattern with the highest MAI (5.35±0.64), followed by cribriform carcinoma pattern in the same group; the differences between these two histological patterns were considered statistically significant (p<0.05) (Table I). Statistically significant differences were also found between the comedo carcinoma and the remaining histological patterns from both groups, exercised and sedentary (p<0.05) (Table I).
Histological patterns of mammary tumors. (A) Number and percentage of each histological pattern and (B) histological lesions classified according to their histogenesis in both groups, exercised and sedentary. IDP, Intraductal proliferation.
Comedo carcinoma pattern. (A) H&E staining, atypical mitosis figures; (B) ERα immunoexpression, (C) Ki-67 immunoexpression. Objective 40×.
ERα H-score, Ki-67 PI and MAI of each histological pattern of rat mammary tumors developed by animals from exercised and sedentary groups. Mean±S.D.
ERα H-score, Ki-67 PI and MAI in different histological types of rat mammary tumors classified according to their histogenesis and biological behavior in exercised and sedentary groups. Mean±S.D.
In both groups, the highest H-score was identified in epithelial malignant neoplasms with a statistically significant difference between this histological type and epithelial-stromal benign neoplasm from exercised group (p<0.05) (Table II, Figure 2B). Statistically significant differences were not found in Ki-67 PI between groups (p>0.05); the epithelial malignant lesions were the histological type with the highest PI in both groups (Table II, Figure 2C). Statistically significant differences in MAI were not found among the histological types in both groups (p>0.05) and, similarly to the Ki-67 PI, the epithelial malignant lesions were the histological type with the highest MAI (Table II).
Comparison of ERα H-score, Ki-67 PI and MAI between groups. Regardless of the histological type or pattern of the mammary lesions, statistically significant differences were not found in the means of ERα H-score, Ki-67 PI and MAI between groups (p>0.05). However, the absolute mean value of ERα H-score was higher in the exercised group, while the Ki-67 PI and MAI were higher in the sedentary group (Table III).
Means' comparison of ERα H-score, Ki-67 PI and MAI between exercised and sedentary groups. Mean±S.D.
Discussion
The mammary tumors identified in this study were similar to those previously described by Russo and Russo (21) who further reported that the papillary carcinoma is the most typical and frequent pattern observed in MNU-induced mammary tumors. Concerning the effects of exercise training on chemically-induced mammary tumorigenesis, a beneficial effect was observed, i.e. decrease of the number of lesions and their malignancy, which is in accordance with previous reports (17-19).
It has been proved that the semi-quantitative method to quantify the expression of ERα in breast cancer through immunohistochemical markers is especially powerful to predict the response of women's breast cancer to endocrine therapy. The high expression of ER is an indicator of better response to antiestrogen therapy and good prognostic in breast cancer patients; while its low expression indicates the need to apply chemotherapy in addition to endocrine therapy (23). In these results, despite the fact that the number of malignant lesions was higher in sedentary animals, the ERα H-score was higher in the exercised group. Away from the fact that the exercise training did not decrease the expression of ERα, as expected, higher expression of ER in epithelial malignant lesions from exercised group suggests that these animals exhibited more differentiated mammary malignant lesions. This is in accordance with the findings of Qiu et al. (24) and Chan et al. (25) who reported that the rats' chemically-induced mammary carcinomas show papillary and cribriform carcinoma patterns with a greater cellular differentiation and higher ERα positive expression. These data are also in agreement with those described by McCormick et al. (26) and Russo and Russo (6) who reported that high levels of steroid hormones and their precursors can be inhibitors of chemically-induced carcinogenesis in rats by the induction of the differentiation of the mammary parenchyma.
Ki-67 expression has been recently identified as a predictive and prognostic factor in breast cancer. Its high expression is associated with poor prognosis and can be used to monitor the tumors' response to treatment (13, 27-29). In a previous study, it was reported that Sprague-Dawley rats have an elevation of Ki-67 expression in line with an altered ribosomal pathway, which could be related to deregulated protein synthesis machinery and the promotion of malignant tumor progression in this strain (30). In this work, the Ki-67 PI exhibited low values in both groups, exercised and sedentary. According to Yerushalmi et al. (15), Ki-67 PI <14 indicates a low risk of neoplasms' recurrence and a better response to therapy. The lower Ki-67 PI in exercised than in sedentary group is an indicator of better prognosis in exercised animals. These results were different from those observed by Malicka et al. (31) who, in a similar study using an exercised model of chemically-induced mammary cancer in female rats, did not observe any differences in Ki-67 expression. However, these authors did not present the values obtained for the Ki-67 immunoexpression. Westerlind et al. (32) evaluated the expression of proliferating cell nuclear antigen (PCNA) in exercised and sedentary animals under a protocol of chemical carcinogenesis. They observed that the expression of PCNA was higher in exercised animals. These results cannot be compared with ours because PCNA is a less specific marker detecting not only cellular proliferation but also cellular reparation. Additionally, these different results may be due to the different period of exercise training employed between studies (12 weeks in the aforementioned study and 35 weeks in the present work).
Mitotic count is primordial in studying cancer and evaluated histopathologically (33). In the last years, this proliferation index was considered to be a strong prognostic factor in breast cancer and employed as a routine component of histopatological studies with clinical applicability (34). Despite the limitations for their estimation, mainly due to the variability of scoring and the standardization of immunohistochemical diagnosis (35), nowadays, mitotic counts in conjunction with Ki-67 PI are important predictors for mammary cancer. In this study, a low MAI was inclusively obtained in the most aggressive histological pattern, the comedo carcinoma. Despite the fact that the comedo carcinoma is a malignant neoplasia, highly cellular, with a solid and expansive growth, the MNU-induced comedo carcinomas of the present study exhibited low proliferation (MAI was <6 and PI<14).
These results denoted that the MNU-induced mammary tumors have, in general, a low rate of proliferation similar to that reported in human mammary cancer where the ER-positive mammary tumors showed a lower rate of proliferation (lower KI-67 PI and MAI) and better prognosis than the ER-negative ones (34). In relation to the effect of the exercise, a positive effect was observed, since both proliferation indexes were lower in the exercise than in the sedentary group.
In this study, the comparison between ERα H-score, Ki-67 PI and MAI was not statistical significant but, taking into account their mean values, interesting results were observed: (i) ERα H-score was higher in exercised group and (ii) Ki-67 PI and MAI were higher in sedentary group denoting the positive effects of exercise training in the development of mammary cancer in rats, thus suggesting a better response to hormonal therapy on mammary tumorigenesis.
Acknowledgements
This work was supported by European Investment Funds by FEDER/COMPETE/POCI - Operational Competitiveness and Internationalization Program, under Project POCI-01-0145-FEDER-006958 and Portuguese Foundation for Science and Technology (FCT), under the project UID/AGR/04033/2013, the project PTDC/DES/114122/2009 and post-graduation grant SFRH/BD/102099/2014.
Footnotes
Conflicts of Interests
None to declare.
- Received February 22, 2016.
- Revision received April 1, 2016.
- Accepted April 4, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
