Thalidomide Reduces Cell Proliferation in Endometriosis Experimentally Induced in Rats

► experimental endometriosis ► cell proliferation ► PCNA ► thalidomide ► rat

Objective To analyze the effect of thalidomide on the progression of endometriotic lesions experimentally induced in rats and to characterize the pattern of cell prolifera- tion by immunohistochemical Proliferating Cell Nuclear Antigen (PCNA) labeling of eutopic and ectopic endometrium.

Fifteen female Wistar rats underwent laparotomy for endometriosis induction by resection of one uterine horn, isolation of the endometrium and fixation of a tissue segment to the pelvic peritoneum. Four weeks after, the animals were divided into 3 groups: control (I), 10mg/kg/day (II) and 1mg/kg/day (III) intraperitoneal thalidomide for 10 days. The lesion was excised together with the opposite uterine horn for endometrial gland and stroma analysis. Eutopic and ectopic endometrial tissue was submitted to immunohistochemistry for analysis of cell proliferation by PCNA labeling and the cell proliferation index (CPI) was calculated as the number of labeled cells per 1,000 cells.

Group I showed a mean CPI of 0.248 /C6 0.0513 in the gland and of 0.178 /C6 0.046 in the stroma. In contrast, Groups II and III showed a signi ficantly lower CPI, that is, 0.088 /C6 0.009 and 0.080 /C6 0.021 for the gland ( p < 0.001) and 0.0945 /C6 0.0066 and 0.075 /C6 0.018 for the stroma ( p < 0.001), respectively. Also, t h em e a nl e s i o na r e ao fG r o u pIw a s6 9 . 2m m 2 ,as i g n ificantly higher value compared with Group II (49.4 mm2, p ¼ 0.023) and Group III (48.6 mm2, p ¼ 0.006). No signi fi- cant difference was observed between Groups II and III.

Thalidomide proved to be effective in reducing the lesion area and CPI of the experimental endometriosis implants both at the dose of 1 mg/kg/day and at the dose of 10 mg/kg/day. Resumo Objetivo Analisar o efeito da talidomida na progressão de lesões endometrióticas induzidas experimentalmente em ratas e cara cterizar o padrão de proliferação celular pela marcação imunohistoquímica de Antígeno Nuclear de Célula Proliferativa (PCNA) no endométrio eutópico e ectópico. received April 18, 2019 accepted October 2, 2019 DOI https://doi.org/ 10.1055/s-0039-3399551. ISSN 0100-7203. Copyright © 2019 by Thieme Revinter Publicações Ltda, Rio de Janeiro, Brazil Original Article THIEME 668 Published online: 2019-11-19

Endometriosis is characterized by the presence of endometrial tissue outside the uterine cavity, causing debilitating symp- toms such as severe chronic pelvic pain and infertility in serious cases. 1 The etiopathogeny of this disease is complex and multifactorial, involving genetic predisposition, environ- mental, anatomical and endocrine factors, and immunological changes. 2 Once endometrial tissue is implanted, a constant stimulus of its development occurs in many cases, in fluenced by multiple factors such as escape from attack by the immuno- logical system, changes in the local concentrations of hor- mones and in flammatory mediators, angiogenesis due to increased activity and levels of vascular endothelial growth factor A (VEGF-A) and proliferation of ectopic cells. 3–6 The estimate is that more than 70 million women all over the world are affected by endometriosis, which causes countless problems for the social, professional and marital life of these patients, with annual costs estimated at US$ 22 billion in the US alone. A reason for this high cost is the lack of efficient treatments of the disease. 3 The treatment of endometriosis consists of conservative or radical surgery and drug therapy, with the former being associated with high rates of recurrence and signi ficant mor- bidity. 7 Different drug treatments are currently being proposed such as hormone therapy, anti-inflammatory agents and com- plementary therapies, although they tend to relieve symptoms rather than curing the disease. Thus, there still is the need to develop new medications for this purpose.8 Thalidomide has a potent antiangiogenic effect based on the negative regulation of vascular endothelial growth factor (VEGF). In addition, thalidomide functions as an immunomod- ulator and as an immunosuppressor and anti-in flammatory agent since it acts on the excessive synthesis of Tumor necrosis factor-alpha (TNF-α) and of other cytokines such as interleukin 6, which participate in the genesis of in flammatory pain.9 In view of this potential, under rigid regulation, thalidomide is currently being used for several diseases including erythema nodosum leprosum, Kaposi sarcoma associated with HIV-1, multiple myeloma, and advanced prostate cancer. 10 Within this context, thalidomide acts in the endometrioses pathophysiological pathways. Thalidomide, although little explored, is a drug with great potential for the treatment of this disease, as it is able to act both in the control of progression and in the recurrence of the disease after the surgical treat- ment. Thus, the objective of the present study was to assess the action of thalidomide on the progression of implants and cell proliferation in endometriosis experimentally induced in rats using immunohistochemical PCNA labeling of eutopic and ectopic endometrium.

The present study was conducted in the sector of experimental surgery of the University Hospital of Ribeirão Preto and in the Oncopathology laboratory of the Department of Pathology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (FMRP-USP). The research project was approved by the Animal Experimentation Committee of FMRP-USP n° 060/2005. Fifteen adult albino Wistar rats weighing /C24 200 g were used. The animals were kept in appropriate cages under conditions of controlled temperature, humidity and lighting for 3 days before surgery, receiving water and foodad libitum. The animals were submitted to general anesthesia with 0.4 ml ketamine in combination with 0.2 ml xylestesin, followed by Métodos Quinze ratas Wistar foram submetidas a laparotomia para indução de endometriose por ressecção de um corno uterino, isolamento do endométrio e fixação de um segmento do tecido ao peritônio pélvico. Após quatro semanas, os animais foram divididos em 3 grupos: controle (I), 10 mg/kg/dia (II) e 1 mg/kg/dia (III) de talidomida intraperitoneal por um período de 10 dias. As lesões foram resseccionadas juntamente com o corno uterino oposto para análise da glândula endometrial e do estroma. O tecido endometrial eutópico e ectópico foi submetido à imunohistoquímica para análise da proliferação celular por marcação com PCNA e o índice de proliferação celular (CPI) foi calculado como o número de células marcadas por 1.000 células. Resultados O grupo I apresentou média de CPI de 0,248 /C6 0,0513 na glândula e de 0,178 /C6 0,046 no estroma. Em contraste, os grupos II e III apresentaram CPI signi fi- cativamente menor, isto é, 0,088 /C6 0,009 e 0,080 /C6 0,021 para a glândula ( p < 0,001) e 0,0945 /C6 0,0066 e 0,075 /C6 0,018 para o estroma ( p < 0,001), respectivamente. Além disso, a área de lesão média do Grupo I foi de 69,2 mm 2,v a l o rs i g n ificativamente maior em relação ao Grupo II (49,4 mm 2, p ¼ 0,023) e Grupo III (48,6 mm 2, p ¼ 0,006). Não houve diferença estatisticamente signi ficante entre os Grupos II e III. Conclusão A talidomida mostrou-se e ficaz na redução da área da lesão e CPI dos implantes de endometriose experimental tanto na dose de 1 mg/kg/dia quanto na dose de 10 mg/kg/dia. Palavras-chave ► endometriose experimental ► proliferação celular ► PCNA ► talidomida ► rata Rev Bras Ginecol Obstet Vol. 41 No. 11/2019 Thalidomide Reduces Cell Proliferation in Endometriosis Lourenço et al. 669 laparotomy for induction of endometriosis. The procedure was performed under strict antisepsis conditions, always by the same investigator. The pelvic cavity was opened by a median longitudinal incision of /C24 2 cm at a distance of 2 cm from the pubis. A segment of /C24 4 cm of the uterine horn was resected and the horn was closed. The resected uterine portion was immersed in 0.9% physiological saline at 4°C for 2 minutes and then incised longitudinally for the removal of two 5 /C2 5m m fragments. The fragments of endometrial tissue were sutured to the peritoneum close to the reproductive tract of the animal using Vicryl 6.0 sutures, with the free endometrial surface facing the abdominal cavity; the surgical abdominal incision was then closed. No hormonal supplementation was adminis- tered before or after the surgery, with the procedure being performed during diestrus. 11 Four weeks after surgery, the animals were divided into 3 groups: Group I (control – receiving an intraperitoneal Dimethyl Sulfoxide (DMSO) solution), Group II (10 mg/kg/day intraperitoneal thalidomide for 10 days), and Group III (1 mg/kg/day intraperitoneal thalidomide for 10 days). The rats were then euthanized, the widest diameters of the implants were measured, the total area of the lesion was calculated, the implants were excised, fixed in 10% formalde- hyde and processed for paraf fine m b e d d i n g ,a n ds l i d e sw e r e mounted and stained with hematoxylin and eosin (H&E) to confirm the presence of endometrial tissue (identi fication of glandular epithelium and/or stroma). 11 Histological sections (4 to 5 µm) were submitted to immu- nohistochemistry by the antigen-antibody reaction and the reaction was developed with a marker visible under the microscope. Deparaffinized and hydrated sections were recov- ered antigenically by incubation in buffered medium in a steam pot for 40 minutes. After cooling, the endogenous tissue per- oxidases were removed by adding hydrogen peroxide, and horse serum was added to prevent nonspecific binding of the primary antibody. The slides were then incubated with primary antibodies obtained from Novocastra Laboratories Ltd. (New- castle-upon-Tyne, UK). Cell proliferation was determined using PCNA (product code NCL-PCNA, PC10 clone) with nuclear labeling at a 1:200 dilution. The material was then incubated with the secondary antibody and submitted to the avidin-biotin step. The reaction was developed by treatment with Diamino- benzidine (DAB) (Sigma-Aldrich Inc., St. Louis, MI, USA) for 50 seconds, the material was counterstained with Harris H&E, and mounted on slides. All of the slides were evaluated by two pathologists experienced in immunohistochemistry who were not aware of the type of tissue to be analyzed. 11 For evaluation of PCNA, which has nuclear labeling, we used a quantitative method, counting immunohistochemically la- beled cells per 1,000 cells counted on the slide, with care taken to count cells in the four quadrants of each slide. Based on this calculation, we obtained the cell proliferation index (number of PCNA-labeled cells per 1,000 cells) for both the glandular and stromal tissues, which were analyzed separately. Data were analyzed statistically with the GraphPad Prism 5.0 32-bit executable software (GraphPad Software Inc., San Diego, CA, USA) using the paired t-test, with the level of significance set at 5%.

Group I (control) had a mean lesion area of 69.2 mm 2, whereas Groups II and III had signi ficantly smaller mean areas of 49.4 mm 2 (p ¼ 0.023) and 48.6 mm 2 (p ¼ 0.006) compared with the control group, respectively, with no significant difference between them ( p ¼ 0.472). The control group also exhibited macroscopically more exuberant vas- cularization compared with the treated group. Cell proliferation was determined using PCNA ( ►Fig. 1 ). Group I (control) had a mean lesion CPI (gland and stroma) of 0.249 /C6 0.051 and 0.179 /C6 0.046, respectively. In contrast, Groups II and III had signi ficantly lower lesion CPI values compared with the control group, of 0.088 /C6 0.009 and 0.095 /C6 0.007 (p < 0.001) for the gland and of 0.080 /C6 0.021 and 0.075 /C6 0.018 (p ¼ 0.01) for the stroma, respectively, with no significant differences between them (p ¼ 0.28) (►Fig. 2 ). A similar effect was observed on topic endometrial tissue, with a significant difference between the control and the two treatment groups. Group I (control) had a mean CPI of endome- trium (gland and stroma) of 0.178/C6 0.066 and 0.124/C6 0.0396, respectively. In contrast, the CPI was signi ficantly reduced in Groups II and III, with values of 0.088/C6 0.010 and 0.076/C6 0.021 (p ¼ 0.008) for the gland and of 0.071/C6 0.039 and 0.077/C6 0.015 (p ¼ 0.010) for the stroma, respectively. No signi ficant differ- ence in the CPI values of topic endometrial tissue were detected between Groups II and III (p ¼ 0.12) (►Fig. 2 ).

In most cases, the treatment of endometriosis involves the inhibition of ovarian function, with all its drawbacks. 12 In view of the physiopathology of the disease and of the anti- inflammatory, immunomodulator and antiangiogenic action of thalidomide, 13 this drug could be an option for the long- term treatment of endometriosis without interference with ovarian function. In the present study, the implants of the treated groups (II and III) had signi ficantly lower CPI and lesion areas than the Fig. 1 PCNA immunostaining of ectopic endometrium treated with a high thalidomide dose (Magni fication: 40x). Rev Bras Ginecol Obstet Vol. 41 No. 11/2019 Thalidomide Reduces Cell Proliferation in Endometriosis Lourenço et al.670 control group. Comparison of these items between Groups II and III did not show signi ficant differences, indicating that, even at low doses (Group III), thalidomide was effective in reducing cell proliferation and, consequently, the size of the implants. Similar results showing the antiproliferative effects of thalidomide was found in pancreatic cancer cell lines. 14 Furthermore, thalidomide dithiocarbamate analogs exhibited signi ficant antiproliferative action on human um- bilical vein endothelial cells and MDA-MB-231 human breast cancer cell lines without causing cytotoxicity. 15 It was also possible to perceive macroscopically that vas- cularization was reduced in the treatment groups, probably as a result of the antiangiogenic effect of thalidomide. This effect was first proven at the beginning of the decade of 1990, 16 and today we know that thalidomide exerts its antiangiogenic effect in more than one manner, with emphasis on depletion of VEGF receptors 17 and on the suppression of VEGF and basic fibroblast growth factor (bFGF) secretion. 18 The growth and invasion of endometriosis lesions is absolutely dependent on neoangiogenesis. Within this context, VEGF-A is hyperex- pressed in endometriosis and plays a fundamental role, inhib- iting apoptosis and increasing the proliferation and migration of endothelial cells, representing an important target for the treatment of this disease. 19 Two other studies conducted on an animal models have also obtained results similar to the present ones. Azimirad et al 20 compared 2 groups of rats: a thalidomide group (n ¼ 9; 22 mg/day) and a control group ( n ¼ 9; 0.5 mL 0.9% saline/day). These investigators also observed a signi ficant reduction in implant volume and in the histopathology scores of the treatment group, in addition to a signi ficant reduction in leukocyte, lymphocyte, VEGF-A and interleukin 6 (IL-6) counts in peritoneal fluid after treatment. Bakacak et al 21 compared a thalidomide group ( n ¼ 8; 100 mg/kg) and a control group ( n ¼ 8; 0.5 mL saline). In addition to observing a signi ficant reduction of implant volume and of histopathology scores in the treatment group, they also detected a sigi ficant reduction of VEGF-A and myeloperoxidase in the peritoneal fluid. A clinical study evaluated, with limitations, the adminis- tration of thalidomide to patients with endometriosis in a controlled pilot study. In that study, 10 women with grade IV endometriosis were treated with a Gonadotropin-releasing hormone (GnRH) analogue and thalidomide (300 mg/day) for at least 6 months, with GnRH being discontinued there- after and only thalidomide being used. The study demon- strated the positive effects of thalidomide on the disease, with remission of pelvic pain and ovarian cysts being ob- served in 8 of the 10 patients. However, the cited study did not include a control group and did not assess the effects of thalidomide on the histopathology of endometriosis. 22 The action of thalidomide was also assessed in cultured endometriotic stromal cells from eight women with moderate to severe endometriosis, with speculation that treatment with thalidomide reduced the expression of interleukin 8 (IL-8) by reducing TNF-α-induced nuclear factor kappa B (NF- κB) acti- vation, with this action being of fundamental importance for the reduction of inflammation. 23 Since inflammation is crucial for the pathogenesis of endometriosis, with emphasis on the TNF-α cytokine, whose expression is increased in the tissues of patients with endometriosis, and which also directly influen- ces the expression of estrogen receptors,24 the interference of thalidomide with this pathway may explain part of the find- ings of the present study. The present results, taken together with those obtained in the few published studies, demonstrate the potential of Fig. 2 Cell proliferation index of topic and ectopic endometrial tissues. Rev Bras Ginecol Obstet Vol. 41 No. 11/2019 Thalidomide Reduces Cell Proliferation in Endometriosis Lourenço et al. 671 thalidomide for the treatment of endometriosis, supporting the hypothesis that this drug causes a regression of the evolution of endometriosis and underscoring the need for further research conducted with due caution in view of the teratogenic history of this drug. 25

Thalidomide proved to be efficient in reducing the lesion area and the CPI of peritoneal endometriotic implants in rats, both at the dose of 1 mg/kg/day and at the dose of 10 mg/kg/day, with the lower dose being as effective as the dose of 10 mg/kg/day, which is known to be teratogenic. Thus, thalidomide should be considered as a potential drug for the treatment of endometri- osis in women. Contributions All of the authors of the present article contributed to the design, data collection, analysis, and interpretation of data, the writing of the article or critical review of the intellectual content and to the final approval of the version to be published. Conflicts of Interests The authors have no con flicts of interests to declare. Acknowledgments The authors acknowledge FAPESP for financial support through the scientific initiation grant (process: 07/01658–9).

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