Maternal Low-Protein Diet Deregulates DNA Repair and DNA Replication Pathways in Female Offspring Mammary Gland Leading to Increased Chemically Induced Rat Carcinogenesis in Adulthood

Studies have shown that maternal malnutrition, especially a low-protein diet (LPD), plays a key role in the developmental mechanisms underlying mammary cancer programming in female offspring. However, the molecular pathways associated with this higher susceptibility are still poorly understood. Thus, this study investigated the adverse effects of gestational and lactational low protein intake on gene expression of key pathways involved in mammary tumor initiation after a single dose of N-methyl-N-nitrosourea (MNU) in female offspring rats. Pregnant Sprague–Dawley rats were fed a normal-protein diet (NPD) (17% protein) or LPD (6% protein) from gestational day 1 to postnatal day (PND) 21. After weaning (PND 21), female offspring (n = 5, each diet) were euthanized for histological analysis or received NPD (n = 56 each diet). At PND 28 or 35, female offspring received a single dose of MNU (25 mg/kg body weight) (n = 28 each diet/timepoint). After 24 h, some females (n = 10 each diet/timepoint) were euthanized for histological, immunohistochemical, and molecular analyses at PDN 29 or 36. The remaining animals (n = 18 each diet/timepoint) were euthanized when tumors reached ≥2 cm or at PND 250. Besides the mammary gland development delay observed in LPD 21 and 28 groups, the gene expression profile demonstrated that maternal LPD deregulated 21 genes related to DNA repair and DNA replication pathways in the mammary gland of LPD 35 group after MNU. We further confirmed an increased γ-H2AX (DNA damage biomarker) and in ER-α immunoreactivity in mammary epithelial cells in the LPD group at PND 36. Furthermore, these early postnatal events were followed by significantly higher mammary carcinogenesis susceptibility in offspring at adulthood. Thus, the results indicate that maternal LPD influenced the programming of chemically induced mammary carcinogenesis in female offspring through increase in DNA damage and deregulation of DNA repair and DNA replication pathways. Also, Cidea upregulation gene in the LPD 35 group may suggest that maternal LPD could deregulate genes possibly leading to increased risk of mammary cancer development and/or poor prognosis. These findings increase the body of evidence of early-transcriptional mammary gland changes influenced by maternal LPD, resulting in differential response to breast tumor initiation and susceptibility and may raise discussions about lifelong prevention of breast cancer risk.Fil: Zapaterini, Joyce R.. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Fonseca, Antonio R. B.. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Bidinotto, Lucas T.. Barretos Cancer Hospital; Brasil. Barretos School of Health Sciences; BrasilFil: Colombelli, Ketlin T.. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Rossi, André L. D.. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Kass, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; ArgentinaFil: Justulin, Luis A.. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Barbisan, Luis F.. Universidade Estadual Paulista Julio de Mesquita Filho; Brasi

Finasteride treatment alters MMP-2 and-9 gene expression and activity in the rat ventral prostate

P>The safety of using finasteride as a prevention of prostate cancer is still under debate. In this study, we investigated the effects of finasteride on the location, gene expression and activities of matrix metalloproteinases -2 and -9, which are involved in the degradation of extracellular matrix components during tissue remodelling and prostate cancer progression, invasion and metastasis. Ventral prostates (VP) from Wistar rats treated with finasteride (25 mg/kg/day) for 7 and 30 days and age-matched controls were evaluated using histology, immunohistochemistry, semi-quantitative RT-PCR and gelatin zymography. Finasteride treatment reduced the epithelial immunostaining of MMP-2 but increased MMP-9 immunostaining in the epithelial cells and in the stroma. The mRNA expression of both MMP-2 and MMP-9 were significantly increased on day 7 of finasteride treatment, mainly for MMP-9 and returned to the control levels by day 30. However, gelatin zymography showed that MMP-9 activity was significantly increased on day 7 of finasteride treatment and remained elevated on day 30 (p < 0.05), while MMP-2 activity was reduced after 30 days of treatment. Finasteride increases MMP-9 and reduces MMP-2 activities in the prostate, which may affect negatively and positively both normal and tumoural prostatic cell behaviour during the treatment. Studies on expression of MMPs in the prostate during different androgen manipulation or cancer chemoprevention strategies can contribute to understand the tissue's overall response and clinical data.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Doxazosin reduces cell proliferation and increases collagen fibers in rat prostatic lobes

We investigated the effects of doxazosin (Dox), an alpha-adrenoceptor antagonist used clinically for the treatment of benign prostatic hyperplasia (BPH), on the rat prostatic complex by assessing structural parameters, collagen fiber content, cell proliferation, and apoptosis. Adult Wistar rats were treated with Dox (25 mg/kg per day), and the ventral (VP), dorsolateral, and anterior prostate (AP) regions of the prostate complex were excised at 3, 7, and 30 days after treatment. At 24 h before being killed, the rats were injected once with 5-bromodeoxyuridine (BrdU; thymidine analog) to label mitotically active cells. The prostates were weighed and processed for histochemistry, morphometry-stereology, immunohistochemistry for BrdU, Western blotting for proliferating cell nuclear antigen (PCNA), and the TUNEL reaction for apoptosis. Dox-treated prostate lobes at day 3 presented increased weight, an enlarged ductal lumen, low cubical epithelial cells, reduced epithelial folds, and stretched smooth muscle cells. However, at day 30, the prostates exhibited a weight reduction of ∼20% and an increased area of collagen and reticular fibers in the stromal space. Dox also reduced epithelial cell proliferation and increased apoptosis in the three prostatic lobes. Western blotting for PCNA confirmed the reduction of cell proliferation by Dox, with the AP and VP being more affected than the dorsal prostate. Thus, Dox treatment alters epithelial cell behavior and prostatic tissue mechanical demand, inducing tissue remodeling in which collagen fibers assume a major role. © 2007 Springer-Verlag

Tissue inhibitor of metalloproteinase-2 (TIMP-2) location in the ventral, lateral, dorsal and anterior lobes of rat prostate by immunohistochemistry

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a major role in extracellular matrix component degradation in several normal and abnormal tissue situations; they are also found in human seminal plasma. MMPs have been found in rat prostate secretions and are nearly lobe specific in expression pattern. The aim of this study was to evaluate whether TIMP-2, like other semen components, is expressed differently from different rat prostatic lobes. Immunohistochemical staining was performed in both young and adult rat ventral (VP), lateral (LP), dorsal (DP), and anterior (AP) prostatic lobes and confirmed by western blotting. TIMP-2 expression was found in the epithelial cells in the following sequence: LP > AP > DP > VP, in both young and adult rats. In this study, 100% of adult LP presented histological signs of prostatitis, where TIMP-2 immunostaining was positive in normal epithelium even with intraluminal neutrophils, but was reduced or absent in the epithelium with intraepithelial leukocytes or with periductal stroma disorganization associated with mononuclear cell infiltration. However, TIMP-2 expression in LP was not induced by prostatitis, since younger rat LPs were also strongly TIMP-2 positive. The distal and intermediate VP regions were TIMP-2 negative, but the proximal regions were strongly stained. Western blotting results confirmed the high TIMP-2 expression in the LP lobe. Thus, TIMP-2 is expressed differently between the prostatic lobes and is another nearly lobe-specific protein, which plays a role in the regulation of MMP activity in seminal plasma and glandular homeostasis. TIMP-2 is also another regional ductal variation of VP. Further studies should address whether TIMP-2 expression is related to the highest incidence of rat LP prostatitis and adenocarcinoma. © 2006 International Federation for Cell Biology

Editorial: Early Life Epigenetic Programming of Health and Disease through DOHaD Perspective

Peer reviewed: TrueAcknowledgements: The authors are thankful to the contributors to this Research Topic as well as the Editorial support of the Journal

Epithelial-stromal transition of MMP-7 immunolocalization in the rat ventral prostate following bilateral orchiectorny

Epithelial cells from involuting rat ventral prostate (VP) express Matrilysin (MMP-7) mRNA. Herein, we investigated by immunohistochemistry the NIMP-7 protein location and its association with tissue changes following castration in the VP. Normal and castrated adult male Wistar rats were sacrificed at different times after surgery. VP was examined by immunocytochemistry and immunoprecipitation. Castration promoted a shrinking of prostate ducts with an extensive stromal remodeling. In the VP from normal rats, MMP-7 immunoreactivity was found in epithelial secretory granules. Three days after castration, immunostaining for MMP-7 was found in both the epithelial secretory granules and in the stroma just below the epithelium, mainly at the distal ductal tips. At seven and 21 days after castration, the immunostaining for MMP-7 was found only in the stromal space. Immunoprecipitation confirmed the specificity of the primary antibody by rescuing a pro-enzyme form (28 kDa) in the prostate extracts. The present results suggest that MMP-7 participates in the epithelial-stromal interface remodeling of the ventral prostate during the involution achieved by castration, probably in the degradation of components of the epithelial basement membrane. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved

Impact of maternal and postnatal zinc dietary status on the prostate of pubescent and adult rats

Zinc is important for cell physiology and alteration of its levels during development can modulate a series of biological events. The aim of this study was to investigate whether dietary zinc deficiency or supplementation during morphogenesis and early postnatal development could interfere in prostate maturation. Pregnant rats were exposed to a standard diet (NZ: 35 mg Zn/kg chow), low- zinc diet (LZ: 3 mg of Zn/kg chow) and zinc- supplemented diet (HZ: 180 mg/Kg chow) from gestational day 10 (GD10) through postnatal day 21 (PND21). After weaning, male offspring were divided into three groups that were submitted to the same food conditions as their mothers until PND53. The animals were euthanized at PND53 and PND115. The ventral prostate was removed, weighed and its fragments were subjected to histological, western blot and zymography analysis. PND53: body and prostate weight were lower in LZ compared to NZ; the epithelial compartment was reduced while the stromal compartment was increased in LZ compared to NZ; there was an increase in the amount of collagen and reduction in AR and SIRT1 expression in LZ compared to NZ. PND115: body weight was lower in LZ compared to NZ and prostate weight was similar among the groups; peripheral physiological hyperplasia was observed, as well as an increased epithelial proliferation index and reduced PAR4 expression in LZ and HZ compared to NZ. Zinc deficiency during prostate morphogenesis and differentiation is potentially harmful to its morphology, however, by restoring the standard dietary environment, the gland responds to the new microenvironment independent of the previous dietary condition.FAPESP-Sao Paulo State Research FoundationSao Paulo State Univ Unesp, Inst Biosci, Dept Morphol, Botucatu, SP, BrazilUniv Fed Sao Paulo, UNIFESP, Dept Sci Sea, Santos, SP, BrazilUniv Fed Sao Paulo, UNIFESP, Dept Sci Sea, Santos, SP, BrazilFAPESP: 2014/25932-6Web of Scienc

Combined effect of the finasteride and doxazosin on rat ventral prostate morphology and physiology

P>Finasteride (Fin) and Doxazosin (Dox), alone or in combination, have been widely used in treatment of benign prostatic hyperplasia (BPH) symptoms and recently have been suggested as potential drugs for prostate cancer (PCa)prevention and treatment. However, little is known about the effects of the combination therapy on prostate tissue morphology, physiology and matrix metalloproteinases (MMPs) activity, a special set of enzymes closely related to PCa progression and metastasis. In this study, adult Wistar rats were treated with Fin + Dox (25 mg/kg per day) and the ventral prostate (VP) was excised at days 3 and 30 of treatment to evaluate morphology, cell proliferation, death, transforming growth factor-beta1 (TGF-beta 1) protein expression, MMP-2, MMP-9 activities and MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA expression. Fin + Dox treatment induced a transient increase in testosterone (T) plasma concentration and a permanent reduction in dihydrotestosterone (DHT). The VP and epithelial cell proliferation were reduced and the stromal collagen fibre volume fraction and apoptosis of the epithelial cell were increased. Fin + Dox treatment also increased the TGF-beta 1 immunoreaction in the epithelium and in the stroma. The mRNAs for MMP-2, TIMPs-1 and -2 expressions after 30 days of treatment were decreased. The mRNA for MMP-9 was not detected in any of the groups analysed. Fin + Dox treatment for 30 days promoted a decrease in gelatinolytic activity of MMP-2 and an increase in MMP-9. In conclusion, combined treatment with Fin and Dox interferes in the epithelial cell behaviour and in the MMPs activity, potentially via TGF-beta 1-mediated and androgen pathways. Our results contribute to a better understanding of the clinical data and also of the molecular mechanisms behind isolated or combined Fin and Dox long-term treatment.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Cadmium exposure inhibits MMP2 and MMP9 activities in the prostate and testis

AbstractMatrix metalloproteinases (MMPs) are zinc (Zn2+) and calcium (Ca2+) dependant endopeptidases, capable of degradation of numerous components of the extracellular matrix. Cadmium (Cd2+) is a well known environmental contaminant which could impair the activity of MMPs. In this sense, this study was conducted to evaluate if Cd2+ intake inhibits these endopeptidases activities at the rat prostate and testicles and if it directly inhibits the activity of MMP2 and MMP9 at gelatinolytic assays when present in the incubation buffer. To investigate this hypothesis, Wistar rats (5 weeks old), were given tap water (untreated, n = 9), or 15 ppm CdCl2 diluted in drinking water, during 10 weeks (n = 9) and 20 weeks (n = 9). The animals were euthanized and their ventral prostate, dorsal prostate, and testicles were removed. These tissue samples were processed for protein extraction and subjected to gelatin zymography evaluation. Additionally, we performed an experiment of gelatin zymography in which 5 μM or 2 mM cadmium chloride (CdCl2) was directly dissolved at the incubation buffer, using the prostatic tissue samples from untreated animals that exhibited the highest MMP2 and MMP9 activities in the previous experiment. We have found that CdCl2 intake in the drinking water led to the inhibition of 35% and 30% of MMP2 and MMP9 (p < 0.05) at the ventral prostate and testis, respectively, in Cd2+ treated animals when compared to controls. Moreover, the activities of the referred enzymes were 80% and 100% inhibited by 5 μM and 2 mM of CdCl2, respectively, even in the presence of 10 mM of CaCl2 within the incubation buffer solution. These important findings demonstrate that environmental cadmium contamination may deregulate the natural balance in the extracellular matrix turnover, through MMPs downregulation, which could contribute to the toxic effects observed in prostatic and testicular tissue after its exposure

Impact of gestational low protein diet and postnatal bisphenol A exposure on chemically induced mammary carcinogenesis in female offspring rats

This study evaluated the effect of gestational low protein diet (LPD) and/or postnatal bisphenol A (BPA) exposure on mammary gland development and carcinogenesis in female offspring. Pregnant Sprague-Dawley rats were fed a normal protein diet (NPD, 17% protein) or LPD (6% protein). At weaning, female offspring were distributed in four groups (NPD, LPD, NPD + BPA, and LPD + BPA) and received vehicle or BPA in drinking water (0.1%), during postnatal day (PND) 21 to 51. On PND 51, some female offspring were euthanized or received a single dose of 7,12-dimethylbenzoanthracene (DMBA, 30 mg/kg, i.g.) and were euthanized on PND 250. On PND 51, neither gestational LPD nor postnatal BPA exposure, individually or in combination, significantly altered the development of mammary gland tree, mean number of terminal structures or estrogen receptor beta (ER-β), proliferating cell nuclear antigen (PCNA) or caspase-3 protein expression in the mammary tissue. A significant reduction in mammary epithelial area (%) was observed in both LPD groups and a significant increase in ER-α protein expression was detected only in LPD group. In LPD + BPA group was observed a significant increase in both fat pad area (%) and in mean number of mammary epithelial cells positive for progesterone receptor (PR). On PND 250, the groups that received BPA presented lower latency and higher tumor incidence and tumor multiplicity and LPD + BPA group more aggressive tumors. These findings suggest that postnatal BPA exposure associated with gestational LPD is able to induce morphological changes in the mammary gland and increase susceptibility to mammary carcinogenesis.Fil: Varuzza, Muriele B.. Univer. Estadual Paulista Botucatu Biosciences Inst.;Fil: Zapaterini, Joyce R.. Univer. Estadual Paulista Botucatu Biosciences Inst.;Fil: Colombelli, Ketlin T.. Univer. Estadual Paulista Botucatu Biosciences Inst.;Fil: Barquilha, Caroline N.. Univer. Estadual Paulista Botucatu Biosciences Inst.;Fil: Justulin, Luis A.. Univer. Estadual Paulista Botucatu Biosciences Inst.;Fil: Muñoz de Toro, Monica Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; ArgentinaFil: Kass, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; ArgentinaFil: Barbisan, Luis F.. Univer. Estadual Paulista Botucatu Biosciences Inst.