{"paper_id":"d21ef696-7c5d-4d3c-909a-0e96dbdfe91c","body_text":"Thalidomide Reduces Cell Proliferation in\nEndometriosis Experimentally Induced in Rats\nA talidomida induz diminuição da proliferação celular\nem endometriose experimentalmente induzida em ratas\nLuana Grupioni Lourenço Antônio 1 Julio Cesar Rosa-e-Silva 1 Deborah Juliani Machado 1\nAndrezza Telles Westin 1 Sergio Britto Garcia 1 Francisco José Candido-dos-Reis 1\nOmero Benedicto Poli-Neto 1 Antonio Alberto Nogueira 1\n1 Faculty of Medicine of Ribeirão Preto, Universidade de São Paulo,\nRibeirão Preto, SP, Brazil\nRev Bras Ginecol Obstet 2019;41:668 –672.\nAddress for correspondence Julio Cesar Rosa-e-Silva, PhD, Faculty of\nMedicine of Ribeirão Preto, Universidade de São Paulo, Av.\nBandeirantes, 3900, 14049-900, Ribeirão Preto, SP , Brazil\n(e-mail: juliocrs@usp.com.br).\nKeywords\n► experimental\nendometriosis\n► cell proliferation\n► PCNA\n► thalidomide\n► rat\nAbstract Objective To analyze the effect of thalidomide on the progression of endometriotic\nlesions experimentally induced in rats and to characterize the pattern of cell prolifera-\ntion by immunohistochemical Proliferating Cell Nuclear Antigen (PCNA) labeling of\neutopic and ectopic endometrium.\nMethods Fifteen female Wistar rats underwent laparotomy for endometriosis induction\nby resection of one uterine horn, isolation of the endometrium and fixation of a tissue\nsegment to the pelvic peritoneum. Four weeks after, the animals were divided into 3\ngroups: control (I), 10mg/kg/day (II) and 1mg/kg/day (III) intraperitoneal thalidomide for\n10 days. The lesion was excised together with the opposite uterine horn for endometrial\ngland and stroma analysis. Eutopic and ectopic endometrial tissue was submitted to\nimmunohistochemistry for analysis of cell proliferation by PCNA labeling and the cell\nproliferation index (CPI) was calculated as the number of labeled cells per 1,000 cells.\nResults Group I showed a mean CPI of 0.248 /C6 0.0513 in the gland and of\n0.178 /C6 0.046 in the stroma. In contrast, Groups II and III showed a signi ficantly lower\nCPI, that is, 0.088 /C6 0.009 and 0.080 /C6 0.021 for the gland ( p < 0.001) and\n0.0945 /C6 0.0066 and 0.075 /C6 0.018 for the stroma ( p < 0.001), respectively. Also,\nt 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\nwith Group II (49.4 mm2, p ¼ 0.023) and Group III (48.6 mm2, p ¼ 0.006). No signi fi-\ncant difference was observed between Groups II and III.\nConclusion Thalidomide proved to be effective in reducing the lesion area and CPI of\nthe experimental endometriosis implants both at the dose of 1 mg/kg/day and at the\ndose of 10 mg/kg/day.\nResumo Objetivo Analisar o efeito da talidomida na progressão de lesões endometrióticas\ninduzidas experimentalmente em ratas e cara cterizar o padrão de proliferação celular\npela marcação imunohistoquímica de Antígeno Nuclear de Célula Proliferativa (PCNA)\nno endométrio eutópico e ectópico.\nreceived\nApril 18, 2019\naccepted\nOctober 2, 2019\nDOI https://doi.org/\n10.1055/s-0039-3399551.\nISSN 0100-7203.\nCopyright © 2019 by Thieme Revinter\nPublicações Ltda, Rio de Janeiro, Brazil\nOriginal Article\nTHIEME\n668\nPublished online: 2019-11-19\n\nIntroduction\nEndometriosis is characterized by the presence of endometrial\ntissue outside the uterine cavity, causing debilitating symp-\ntoms such as severe chronic pelvic pain and infertility in\nserious cases.\n1 The etiopathogeny of this disease is complex\nand multifactorial, involving genetic predisposition, environ-\nmental, anatomical and endocrine factors, and immunological\nchanges.\n2\nOnce endometrial tissue is implanted, a constant stimulus\nof its development occurs in many cases, in fluenced by\nmultiple factors such as escape from attack by the immuno-\nlogical system, changes in the local concentrations of hor-\nmones and in flammatory mediators, angiogenesis due to\nincreased activity and levels of vascular endothelial growth\nfactor A (VEGF-A) and proliferation of ectopic cells.\n3–6\nThe estimate is that more than 70 million women all over\nthe world are affected by endometriosis, which causes\ncountless problems for the social, professional and marital\nlife of these patients, with annual costs estimated at US$ 22\nbillion in the US alone. A reason for this high cost is the lack of\nefficient treatments of the disease.\n3\nThe treatment of endometriosis consists of conservative or\nradical surgery and drug therapy, with the former being\nassociated with high rates of recurrence and signi ficant mor-\nbidity.\n7 Different drug treatments are currently being proposed\nsuch as hormone therapy, anti-inflammatory agents and com-\nplementary therapies, although they tend to relieve symptoms\nrather than curing the disease. Thus, there still is the need to\ndevelop new medications for this purpose.8\nThalidomide has a potent antiangiogenic effect based on the\nnegative regulation of vascular endothelial growth factor\n(VEGF). In addition, thalidomide functions as an immunomod-\nulator and as an immunosuppressor and anti-in flammatory\nagent since it acts on the excessive synthesis of Tumor necrosis\nfactor-alpha (TNF-α) and of other cytokines such as interleukin\n6, which participate in the genesis of in flammatory pain.9 In\nview of this potential, under rigid regulation, thalidomide is\ncurrently being used for several diseases including erythema\nnodosum leprosum, Kaposi sarcoma associated with HIV-1,\nmultiple myeloma, and advanced prostate cancer.\n10\nWithin this context, thalidomide acts in the endometrioses\npathophysiological pathways. Thalidomide, although little\nexplored, is a drug with great potential for the treatment of\nthis disease, as it is able to act both in the control of progression\nand in the recurrence of the disease after the surgical treat-\nment. Thus, the objective of the present study was to assess the\naction of thalidomide on the progression of implants and cell\nproliferation in endometriosis experimentally induced in rats\nusing immunohistochemical PCNA labeling of eutopic and\nectopic endometrium.\nMethods\nThe present study was conducted in the sector of experimental\nsurgery of the University Hospital of Ribeirão Preto and in the\nOncopathology laboratory of the Department of Pathology,\nFaculty of Medicine of Ribeirão Preto, University of São Paulo\n(FMRP-USP). The research project was approved by the Animal\nExperimentation Committee of FMRP-USP n° 060/2005.\nFifteen adult albino Wistar rats weighing /C24 200 g were\nused. The animals were kept in appropriate cages under\nconditions of controlled temperature, humidity and lighting\nfor 3 days before surgery, receiving water and foodad libitum.\nThe animals were submitted to general anesthesia with 0.4 ml\nketamine in combination with 0.2 ml xylestesin, followed by\nMétodos Quinze ratas Wistar foram submetidas a laparotomia para indução de\nendometriose por ressecção de um corno uterino, isolamento do endométrio e fixação\nde um segmento do tecido ao peritônio pélvico. Após quatro semanas, os animais\nforam divididos em 3 grupos: controle (I), 10 mg/kg/dia (II) e 1 mg/kg/dia (III) de\ntalidomida intraperitoneal por um período de 10 dias. As lesões foram resseccionadas\njuntamente com o corno uterino oposto para análise da glândula endometrial e do\nestroma. O tecido endometrial eutópico e ectópico foi submetido à imunohistoquímica\npara análise da proliferação celular por marcação com PCNA e o índice de proliferação\ncelular (CPI) foi calculado como o número de células marcadas por 1.000 células.\nResultados O grupo I apresentou média de CPI de 0,248 /C6 0,0513 na glândula e de\n0,178 /C6 0,046 no estroma. Em contraste, os grupos II e III apresentaram CPI signi fi-\ncativamente menor, isto é, 0,088 /C6 0,009 e 0,080 /C6 0,021 para a glândula ( p < 0,001) e\n0,0945 /C6 0,0066 e 0,075 /C6 0,018 para o estroma ( p < 0,001), respectivamente. Além\ndisso, a área de lesão média do Grupo I foi de 69,2 mm\n2,v a l o rs i g n ificativamente maior\nem relação ao Grupo II (49,4 mm 2, p ¼ 0,023) e Grupo III (48,6 mm 2, p ¼ 0,006). Não\nhouve diferença estatisticamente signi ficante entre os Grupos II e III.\nConclusão A talidomida mostrou-se e ficaz na redução da área da lesão e CPI dos\nimplantes de endometriose experimental tanto na dose de 1 mg/kg/dia quanto na dose\nde 10 mg/kg/dia.\nPalavras-chave\n► endometriose\nexperimental\n► proliferação celular\n► PCNA\n► talidomida\n► rata\nRev Bras Ginecol Obstet Vol. 41 No. 11/2019\nThalidomide Reduces Cell Proliferation in Endometriosis Lourenço et al. 669\n\n\nlaparotomy for induction of endometriosis. The procedure was\nperformed under strict antisepsis conditions, always by the\nsame investigator. The pelvic cavity was opened by a median\nlongitudinal incision of /C24 2 cm at a distance of 2 cm from the\npubis. A segment of /C24 4 cm of the uterine horn was resected\nand the horn was closed. The resected uterine portion was\nimmersed in 0.9% physiological saline at 4°C for 2 minutes and\nthen incised longitudinally for the removal of two 5 /C2 5m m\nfragments. The fragments of endometrial tissue were sutured\nto the peritoneum close to the reproductive tract of the animal\nusing Vicryl 6.0 sutures, with the free endometrial surface\nfacing the abdominal cavity; the surgical abdominal incision\nwas then closed. No hormonal supplementation was adminis-\ntered before or after the surgery, with the procedure being\nperformed during diestrus.\n11\nFour weeks after surgery, the animals were divided into\n3 groups: Group I (control – receiving an intraperitoneal\nDimethyl Sulfoxide (DMSO) solution), Group II (10 mg/kg/day\nintraperitoneal thalidomide for 10 days), and Group III\n(1 mg/kg/day intraperitoneal thalidomide for 10 days). The\nrats were then euthanized, the widest diameters of the\nimplants were measured, the total area of the lesion was\ncalculated, the implants were excised, fixed in 10% formalde-\nhyde and processed for paraf fine m b e d d i n g ,a n ds l i d e sw e r e\nmounted and stained with hematoxylin and eosin (H&E) to\nconfirm the presence of endometrial tissue (identi fication of\nglandular epithelium and/or stroma).\n11\nHistological sections (4 to 5 µm) were submitted to immu-\nnohistochemistry by the antigen-antibody reaction and the\nreaction was developed with a marker visible under the\nmicroscope. Deparaffinized and hydrated sections were recov-\nered antigenically by incubation in buffered medium in a steam\npot for 40 minutes. After cooling, the endogenous tissue per-\noxidases were removed by adding hydrogen peroxide, and\nhorse serum was added to prevent nonspecific binding of the\nprimary antibody. The slides were then incubated with primary\nantibodies obtained from Novocastra Laboratories Ltd. (New-\ncastle-upon-Tyne, UK). Cell proliferation was determined using\nPCNA (product code NCL-PCNA, PC10 clone) with nuclear\nlabeling at a 1:200 dilution. The material was then incubated\nwith the secondary antibody and submitted to the avidin-biotin\nstep. The reaction was developed by treatment with Diamino-\nbenzidine (DAB) (Sigma-Aldrich Inc., St. Louis, MI, USA) for\n50 seconds, the material was counterstained with Harris H&E,\nand mounted on slides. All of the slides were evaluated by two\npathologists experienced in immunohistochemistry who were\nnot aware of the type of tissue to be analyzed.\n11\nFor evaluation of PCNA, which has nuclear labeling, we used\na quantitative method, counting immunohistochemically la-\nbeled cells per 1,000 cells counted on the slide, with care taken\nto count cells in the four quadrants of each slide. Based on this\ncalculation, we obtained the cell proliferation index (number\nof PCNA-labeled cells per 1,000 cells) for both the glandular\nand stromal tissues, which were analyzed separately.\nData were analyzed statistically with the GraphPad Prism\n5.0 32-bit executable software (GraphPad Software Inc., San\nDiego, CA, USA) using the paired t-test, with the level of\nsignificance set at 5%.\nResults\nGroup I (control) had a mean lesion area of 69.2 mm 2,\nwhereas Groups II and III had signi ficantly smaller mean\nareas of 49.4 mm 2 (p ¼ 0.023) and 48.6 mm 2 (p ¼ 0.006)\ncompared with the control group, respectively, with no\nsignificant difference between them ( p ¼ 0.472). The control\ngroup also exhibited macroscopically more exuberant vas-\ncularization compared with the treated group.\nCell proliferation was determined using PCNA ( ►Fig. 1 ).\nGroup I (control) had a mean lesion CPI (gland and stroma) of\n0.249 /C6 0.051 and 0.179 /C6 0.046, respectively. In contrast,\nGroups II and III had signi ficantly lower lesion CPI values\ncompared with the control group, of 0.088 /C6 0.009 and\n0.095 /C6 0.007 (p < 0.001) for the gland and of 0.080 /C6 0.021\nand 0.075 /C6 0.018 (p ¼ 0.01) for the stroma, respectively, with\nno significant differences between them (p ¼ 0.28) (►Fig. 2 ).\nA similar effect was observed on topic endometrial tissue,\nwith a significant difference between the control and the two\ntreatment groups. Group I (control) had a mean CPI of endome-\ntrium (gland and stroma) of 0.178/C6 0.066 and 0.124/C6 0.0396,\nrespectively. In contrast, the CPI was signi ficantly reduced in\nGroups II and III, with values of 0.088/C6 0.010 and 0.076/C6 0.021\n(p ¼ 0.008) for the gland and of 0.071/C6 0.039 and 0.077/C6 0.015\n(p ¼ 0.010) for the stroma, respectively. No signi ficant differ-\nence in the CPI values of topic endometrial tissue were detected\nbetween Groups II and III (p ¼ 0.12) (►Fig. 2 ).\nDiscussion\nIn most cases, the treatment of endometriosis involves the\ninhibition of ovarian function, with all its drawbacks. 12 In\nview of the physiopathology of the disease and of the anti-\ninflammatory, immunomodulator and antiangiogenic action\nof thalidomide,\n13 this drug could be an option for the long-\nterm treatment of endometriosis without interference with\novarian function.\nIn the present study, the implants of the treated groups (II\nand III) had signi ficantly lower CPI and lesion areas than the\nFig. 1 PCNA immunostaining of ectopic endometrium treated with a\nhigh thalidomide dose (Magni fication: 40x).\nRev Bras Ginecol Obstet Vol. 41 No. 11/2019\nThalidomide Reduces Cell Proliferation in Endometriosis Lourenço et al.670\n\n\ncontrol group. Comparison of these items between Groups II\nand III did not show signi ficant differences, indicating that,\neven at low doses (Group III), thalidomide was effective in\nreducing cell proliferation and, consequently, the size of the\nimplants. Similar results showing the antiproliferative\neffects of thalidomide was found in pancreatic cancer cell\nlines.\n14 Furthermore, thalidomide dithiocarbamate analogs\nexhibited signi ficant antiproliferative action on human um-\nbilical vein endothelial cells and MDA-MB-231 human breast\ncancer cell lines without causing cytotoxicity. 15\nIt was also possible to perceive macroscopically that vas-\ncularization was reduced in the treatment groups, probably as\na result of the antiangiogenic effect of thalidomide. This effect\nwas first proven at the beginning of the decade of 1990,\n16 and\ntoday we know that thalidomide exerts its antiangiogenic\neffect in more than one manner, with emphasis on depletion\nof VEGF receptors\n17 and on the suppression of VEGF and basic\nfibroblast growth factor (bFGF) secretion. 18 The growth and\ninvasion of endometriosis lesions is absolutely dependent on\nneoangiogenesis. Within this context, VEGF-A is hyperex-\npressed in endometriosis and plays a fundamental role, inhib-\niting apoptosis and increasing the proliferation and migration\nof endothelial cells, representing an important target for the\ntreatment of this disease.\n19\nTwo other studies conducted on an animal models have\nalso obtained results similar to the present ones. Azimirad\net al 20 compared 2 groups of rats: a thalidomide group\n(n ¼ 9; 22 mg/day) and a control group ( n ¼ 9; 0.5 mL 0.9%\nsaline/day). These investigators also observed a signi ficant\nreduction in implant volume and in the histopathology\nscores of the treatment group, in addition to a signi ficant\nreduction in leukocyte, lymphocyte, VEGF-A and interleukin\n6 (IL-6) counts in peritoneal fluid after treatment.\nBakacak et al\n21 compared a thalidomide group ( n ¼ 8;\n100 mg/kg) and a control group ( n ¼ 8; 0.5 mL saline). In\naddition to observing a signi ficant reduction of implant\nvolume and of histopathology scores in the treatment group,\nthey also detected a sigi ficant reduction of VEGF-A and\nmyeloperoxidase in the peritoneal fluid.\nA clinical study evaluated, with limitations, the adminis-\ntration of thalidomide to patients with endometriosis in a\ncontrolled pilot study. In that study, 10 women with grade IV\nendometriosis were treated with a Gonadotropin-releasing\nhormone (GnRH) analogue and thalidomide (300 mg/day)\nfor at least 6 months, with GnRH being discontinued there-\nafter and only thalidomide being used. The study demon-\nstrated the positive effects of thalidomide on the disease,\nwith remission of pelvic pain and ovarian cysts being ob-\nserved in 8 of the 10 patients. However, the cited study did\nnot include a control group and did not assess the effects of\nthalidomide on the histopathology of endometriosis.\n22\nThe action of thalidomide was also assessed in cultured\nendometriotic stromal cells from eight women with moderate\nto severe endometriosis, with speculation that treatment with\nthalidomide reduced the expression of interleukin 8 (IL-8) by\nreducing TNF-α-induced nuclear factor kappa B (NF- κB) acti-\nvation, with this action being of fundamental importance for\nthe reduction of inflammation.\n23 Since inflammation is crucial\nfor the pathogenesis of endometriosis, with emphasis on the\nTNF-α cytokine, whose expression is increased in the tissues of\npatients with endometriosis, and which also directly influen-\nces the expression of estrogen receptors,24 the interference of\nthalidomide with this pathway may explain part of the find-\nings of the present study.\nThe present results, taken together with those obtained in\nthe few published studies, demonstrate the potential of\nFig. 2 Cell proliferation index of topic and ectopic endometrial tissues.\nRev Bras Ginecol Obstet Vol. 41 No. 11/2019\nThalidomide Reduces Cell Proliferation in Endometriosis Lourenço et al. 671\n\n\nthalidomide for the treatment of endometriosis, supporting\nthe hypothesis that this drug causes a regression of the\nevolution of endometriosis and underscoring the need for\nfurther research conducted with due caution in view of the\nteratogenic history of this drug.\n25\nConclusion\nThalidomide proved to be efficient in reducing the lesion area\nand the CPI of peritoneal endometriotic implants in rats, both at\nthe dose of 1 mg/kg/day and at the dose of 10 mg/kg/day, with\nthe lower dose being as effective as the dose of 10 mg/kg/day,\nwhich is known to be teratogenic. Thus, thalidomide should be\nconsidered as a potential drug for the treatment of endometri-\nosis in women.\nContributions\nAll of the authors of the present article contributed to the\ndesign, data collection, analysis, and interpretation of\ndata, the writing of the article or critical review of the\nintellectual content and to the final approval of the\nversion to be published.\nConflicts of Interests\nThe authors have no con flicts of interests to declare.\nAcknowledgments\nThe authors acknowledge FAPESP for financial support\nthrough the scientific initiation grant (process: 07/01658–9).\nReferences\n1 Hickey M, Ballard K, Farquhar C. Endometriosis. BMJ 2014;348:\ng1752. Doi: 10.1136/bmj.g1752\n2 Giudice LC, Kao LC. Endometriosis. Lancet 2004;364(9447):1789-\n–1799. Doi: 10.1016/S0140-6736(04)17403-5\n3 Nothnick W, Alali Z. Recent advances in the understanding of\nendometriosis: the role of in flammatory mediators in disease\npathogenesis and treatment. 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