Impact of the prolactin levels in breast cancer: a systematic review and meta-analysis.

Prolactin (PRL) acts stimulating the mammary glands development, and its deregulation has been associated to the emergence of several types of tumors, including breast cancer. Breast cancer represents the most prevalent malignancy in women, and the second cause of death in several countries. This tumor can be arise due to several molecular alterations, among them PRL has been the object of increasing interest from researchers worldwide. OBJECTIVE: To assess the association between elevated levels of plasma prolactin and breast cancer development. METHODS: A total of 158 studies were found in search databases (48 from PubMed, 69 from Scopus, 88 from Cochrane, 25 from Embase and 10 retrieved from the gray literature) after removing duplicates. Of these, 104 studies were excluded after title and abstract reading, and 54 studies were then read in full, of which only 14 were selected for this review because they had evaluated the association between PRL and breast cancer. Meta-analysis was carried out using the relative risk (RR), mean and standard deviation, confidence interval (95% CI), and the total number of patients for each study. Fixed- and random-effect models were used as applicable and, for the analysis. RESULTS: The meta-analysis showed a positive association between elevated levels of PRL and breast cancer occurrence (RR 1.26; 95%CI 1.15-1.37). Additionally, the patient sub-group analyses showed a positive association between PRL and invasive breast cancer (1.42; 1.24-1.60), ER+/PR+ (1.49; 1.23-1.75), and post-menopausal status (1.29; 1.16-1.43). CONCLUSION: The results showed a positive association between plasma prolactin levels and breast cancer, especially in women with ER+/PR + tumors, of post-menopausal age and those with invasive cancer.

Does melatonin influence the apoptosis in rat uterus of animals exposed to continuous light?

Melatonin has been described as a protective agent against cell death and oxidative stress in different tissues, including in the reproductive system. However, the information on the action of this hormone in rat uterine apoptosis is low. Our objective was to evaluate the effects of melatonin on mechanisms of cell death in uterus of rats exposed to continuous light stress. Twenty adult Wistar rats were divided into two groups: GContr (vehicle control) and GExp which were treated with melatonin (0.4 mg/mL), both were exposed to continuous light for 90 days. The uterus was removed and processed for quantitative real time PCR (qRT-PCR), using PCR-array plates of the apoptosis pathway; for immunohistochemistry and TUNEL. The results of qRT-PCR of GEXP group showed up-regulation of 13 and 7, pro-apoptotic and anti-apoptotic genes, respectively, compared to GContr group. No difference in pro-apoptotic proteins (Bax, Fas and Faslg) expression was observed by immunohistochemistry, although the number of TUNEL-positive cells was lower in the group treated with melatonin compared to the group not treated with this hormone. Our data suggest that melatonin influences the mechanism and decreases the apoptosis in uterus of rats exposed to continuous light.

Differences in neonatal exposure to estradiol or testosterone on ovarian function and hormonal levels.

Exposure to an excess of androgen or estrogen can induce changes in reproductive function in adult animals that resemble polycystic ovary syndrome in humans. However, considerable differences exist among several types of animal models. Little is known about the molecular features of steroidogenesis and folliculogenesis in the ovaries of rats exposed to different sex steroids as neonates. Here, we evaluated the impact of androgen and estrogen exposure on the ovaries of adult female rats during their neonatal period in the gene expression of Lhr and Cyp17a1, two key players of steroidogenesis. We also assessed hormone levels, folliculogenesis and the theca-interstitial cell population. The study was performed on the second postnatal day in thirty female Wistar rats that were sorted into the following three intervention groups: testosterone, estradiol and vehicle (control group). The animals were euthanized 90 days after birth. The main outcomes were hormone serum levels, ovary histomorphometry and gene expression of Lhr and Cyp17a1 as analyzed via quantitative real-time PCR. We found that exposure to excess testosterone in early life increased the LH and testosterone serum levels, the LH/FSH ratio, ovarian theca-interstitial area and gene expression of Lhr and Cyp17a1 in adult rats. Estrogen induced an increase in the ovarian theca-interstitial area, the secondary follicle population and gene expression of Lhr and Cyp17a1. All animals exposed to the sex steroids presented with closed vaginas. Our data suggest that testosterone resulted in more pronounced reproductive changes than did estrogen exposure. Our results might provide some insight into the role of different hormones on reproductive development and on the heterogeneity of clinical manifestations of conditions such as polycystic ovary syndrome.

ROCK1 as a novel prognostic marker in vulvar cancer.

BACKGROUND: Vulvar carcinoma is an infrequent tumour, accounting for fewer than 3% of all malignant tumours that affect women, but its incidence is rising in the past few decades. In young women, the manifestation of the vulvar carcinoma is often linked to risk factors such as smoking and HPV infection, but most cases develop in women aged over 50 years through poorly understood genetic mechanisms. Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) has been implicated in many cellular processes, but its function in vulvar cancer has never been examined. In this study, we aimed to determine the prognostic value of ROCK1 gene and protein analysis in vulvar squamous cell carcinoma (VSCC). METHODS: ROCK1 expression levels were measured in 16 vulvar tumour samples and adjacent normal tissue by qRT-PCR. Further, 96 VSCC samples were examined by immunohistochemistry (IHC) to confirm the involvement of ROCK1 in the disease. The molecular and pathological results were correlated with the clinical data of the patients. Sixteen fresh VSCC samples were analyzed by array-based comparative genomic hybridization (aCGH). RESULTS: In each pair of samples, ROCK1 levels were higher by qRT-PCR in normal tissue compared with the tumour samples (p = 0.016). By IHC, 100% of invasive front areas of the tumour and 95.8% of central tumour areas were positive for ROCK1. Greater expression of ROCK1 was associated with the absence of lymph node metastasis (p = 0.022) and a lower depth of invasion (p = 0.002). In addition, higher ROCK1 levels correlated with greater recurrence-free survival (p = 0.001). Loss of ROCK1 was independently linked to worse cancer-specific survival (p = 0.0054) by multivariate analysis. This finding was validated by IHC, which demonstrated enhanced protein expression in normal versus tumour tissue (p < 0.001). By aCGH, 42.9% of samples showed a gain in copy number of the ROCK1 gene. CONCLUSIONS: ROCK1 is lower expressed in tumour tissue when compared with adjacent normal vulvar epithelia. In an independent sample set of VSCCs, lower expression levels of ROCK1 correlated with worse survival rates and a poor prognosis. These findings provide important information for the clinical management of vulvar cancer.

Metoclopramide-induced hyperprolactinemia effects on the pituitary and uterine prolactin receptor expression.

UNLABELLED: In this work we have evaluated the gene expression profile of prolactin and prolactin receptor in the pituitary and the uterus of female mice with metoclopramide-induced hyperprolactinemia treated with estrogen and/or progesterone. For this purpose, 49 Swiss female mice were allocated to seven groups. INTERVENTIONS: 50-day treatment with metoclopramide, progesterone and estrogen. Our results showed that in the pituitary, metoclopramide-induced hyperprolactinemia increased prolactin expression. In the castrated animals, progesterone, with or without estrogen, produced an increase in prolactin. Pituitary prolactin receptor and the estrogen and progesterone treatment were responsible for the rise in PRLR-S2. In the uterus, no differences in prolactin expression were found between the different study groups. PRLR-S1 had its expression reduced in all castrated animals as against the castrated group treated with vehicle. In the noncastrated animals, PRLR-S2 rose in the metoclopramide-treated group, and, in the castrated animals, its expression diminished in all groups in relation to the vehicle-treated castrated controls. An increase in PRLR-S3 was found in the oophorectomized animals treated with a combination of estrogen and progesterone. PRLR-L rose in the oophorectomized animals treated with progesterone in isolation or in association with estrogen. These findings suggest that metoclopramide associated to progesterone or estrogen may determine an increase in pituitary prolactin and PRLR-S2 expression. The estrogen-progesterone may enhance the expression of PRLR-S3 and PRLR-L isoform of prolactin receptor.

Polymorphisms of estrogen receptor-α gene in Brazilian women with high breast density after menopause.

UNLABELLED: The association of genetic polymorphism in the estrogen receptor alpha (ERα) gene and risk for diseases including breast cancer (BC) has been the subject of great interest. OBJECTIVE: Checking on women with high breast density after menopause, the frequency of the Pvull and Xbal polymorphisms of the ERα gene and the correlation between them and the known risk factors for breast cancer. METHOD: Observational study with 308 women between 45 and 65 years old with high breast density, without hormonal therapy, menstruation for a year or more, breast and ovarian cancer history. It was characterized in clinical history and physical examination: menarche, menopause, parity, family history of BC, smoking, alcohol intake and body mass index. RESULTS: The allelic and genotypic frequencies for ERα-Pvull and Xbal: p = 43.99%; p = 56.01%; pp = 32.14%; Pp = 47.73% and PP = 20.13%; X = 41.56%; x = 58.44%; xx = 33.44%; Xx = 50.00% and XX = 16.56%, respectively. The most frequent risk factors for BC: menarche before 12 years old (35.38%), nulliparity or first child after 28 years old (41.66%), family history of BC (19.16%) and overweight/obesity (62.01%). CONCLUSION: Allelic and genotypic distribution similar to literature. The risk factors for BC were more prevalent in women with high breast density but without significant associations with these polymorphisms.

Prognostic significance of c-KIT in vulvar cancer: bringing this molecular marker from bench to bedside.

BACKGROUND: Vulvar carcinomas are rare tumors, and there is limited data regarding molecular alterations. To our knowledge there are no published studies on c-KIT and squamous cell carcinomas of the vulva (VSCC). Although there are a significant number of other tumor types which express c-KIT, there remains controversy as to its relationship to patient outcome. Thus, we wished to investigate such controversial findings to determine the prognostic importance of c-KIT by evaluating its protein and mRNA expression in VSCCs, correlating these findings with clinicopathological features and Human Papillomavirus (HPV) infection. METHODS: c-KIT expression was scored by immunohistochemistry (IHC) as positive or negative in 139 formalin-fixed paraffin-embedded (FFPE) cases of vulvar carcinomas arrayed in a tissue microarray (TMA) using the DAKO A4502 rabbit polyclonal c-KIT antibody (diluted 1:100). c-KIT mRNA was evaluated by qRT-PCR in 34 frozen samples from AC Camargo Hospital Biobank (17 tumoral and 17 non-tumoral samples) using TaqMan probes-Applied Biosystems [Hs00174029_m1]. HPV genotyping was assessed in 103 samples using Linear Array® HPV Genotyping Test kit (Roche Molecular Diagnostics, Basel, Switzerland). All results obtained were correlated with clinical and pathological data of the patients. RESULTS: c-KIT protein was positive by immunohistochemistry in 70.5% of the cases and this was associated with a higher global survival (p = 0.007), a higher recurrence-free survival (p < 0.0001), an absence of associated lesions (p = 0.001), lymph node metastasis (p = 0.0053), and HPV infection (p = 0.034). Furthermore, c-KIT mRNA quantitation revealed higher levels of transcripts in normal samples compared to tumor samples (p = 0,0009). CONCLUSIONS: Our findings indicate that those vulvar tumors staining positively for c-KIT present better prognosis. Thus, positivity of c-KIT as evaluated by IHC may be a good predictor for use of more conservative surgery techniques and lymph node dissection in vulvar cancer. So part of the essence of our study is to see the possibility of translating our current results from the bench to the bedside. This will help provide patients a more appropriate, less mutilating treatment, in order to keep the maximum physical and psychic quality as possible to these women.

Evidence that Melatonin Increases Inhibin Beta-A and Follistatin Gene Expression in Ovaries of Pinealectomized Rats.

Melatonin plays an important role in the regulation of ovarian function including oocyte maturation in different mammalian species. Many studies indicate that melatonin has an impact on the ovarian function of a variety of ovarian cells. However, the information on the exact mechanism and involved hormones is low. To evaluate inhibin beta-A (INHBA) and follistatin (FST) expression in the ovaries of pinealectomized rats treated with melatonin, thirty adult female Wistar rats were randomized into three groups of ten animals each: group 1 (GSh), sham-operated controls receiving vehicle; group 2 (GPx), pinealectomized animals receiving vehicle; and group 3 (GPxMe), pinealectomized animals receiving replacement melatonin (1.0 mg/kg body weight. It was assumed that each animal drank 6.5 ± 1.2 ml per night and weighs approximately 300 g.) for 60 consecutive days. The ovaries were collected for mRNA abundance and protein of INHBA and FST by qRT-PCR and immunohistochemical analyses, respectively. Treatment with melatonin resulted in the upregulation of INHBA and FST genes in the ovarian tissue of the melatonin-treated animals (GPxMe), when compared with GPx. These findings were then confirmed by analyzing the expression of protein by immunohistochemical analyses, which revealed higher immunoreactivity of INHBA and FST in GPxMe animals in the follicular cells compared with GSh and GPx rats. Melatonin increases the expression of INHBA and FST in the ovaries of pinealectomized female rats.

Impaired branched-chain amino acid metabolism may underlie the nonalcoholic fatty liver disease-like pathology of neonatal testosterone-treated female rats.

Polycystic ovary syndrome (PCOS) is frequently associated with non-alcoholic fatty liver disease (NAFLD), but the mechanisms involved in the development of NAFLD in PCOS are not well known. We investigated histological changes and metabolomic profile in the liver of rat models of PCOS phenotype induced by testosterone or estradiol. Two-day old female rats received sc injections of 1.25 mg testosterone propionate (Testos; n = 10), 0.5 mg estradiol benzoate (E2; n = 10), or vehicle (control group, CNT; n = 10). Animals were euthanized at 90-94 d of age and the liver was harvested for histological and metabolomic analyses. Findings showed only Testos group exhibited fatty liver morphology and higher levels of ketogenic and branched-chain amino acids (BCAA). Enrichment analysis showed effects of testosterone on BCAA degradation pathway and mitochondrial enzymes related to BCAA metabolism. Testos group also had a decreased liver fatty acid elongase 2 (ELOVL2) activity. E2 group had reduced lipid and acylcarnitine metabolites in the liver. Both groups had increased organic cation transporters (SLC22A4 and SLC16A9) activity. These findings indicate that neonatal testosterone treatment, but not estradiol, produces histological changes in female rat liver that mimic NAFLD with testosterone-treated rats showing impaired BCAA metabolism and dysfunctions in ELOVL2, SLC22A4 and SLC16A9 activity.

Virulence of Paracoccidioides brasiliensis and gp43 expression in isolates bearing known PbGP43 genotype.

Paracoccidioides brasiliensis is the dimorphic fungus responsible for human paracoccidioidomycosis (PCM). We previously observed that P. brasiliensis isolates bearing highly polymorphic PbGP43 of genotype A (Pb2, Pb3 and Pb4) were phylogenetically distant from the others. The PbGP43 gene encodes an immune dominant diagnostic antigen (gp43), and its polymorphism reflects broader genetic diversity in the species. In the present study, we observed that isolates with PbGP43 of genotype A showed low virulence when inoculated in B10.A mice by the intraperitoneal, intratracheal and intravenous routes. In vitro studies detected sharp and prolonged down-regulation of PbGP43 in Pb3 (and not in Pb18 or Pb339) as a result of heat shock at 42 degrees C and temperature shift to prompt mycelium to yeast transition, which was, however, not disturbed. Differences in transcriptional regulation are possibly a consequence of mutations in the PbGP43 promoter region, which we here show to be more polymorphic in genotype A isolates. As opposed to Pb3's rapid adaptation to in vitro culture conditions after isolation from the lung, Pb12, the most aggressive isolate tested here, showed slow growth and phase transition in vitro. Interestingly, animals that were highly infected by Pb12 produced small amounts of anti-gp43 antibodies. That was apparently due to down-regulation in PbGP43 expression. We present the first evidence of transcriptional regulation of gp43 expression, but our results suggest that gene expression is also regulated at the protein and/or secretion levels.

Best practice for PTEN gene and protein assessment in anatomic pathology.

There is a lack of standardization of a best practice protocol for Phosphatase and Tensin Homolog (PTEN) assessment by immunohistochemistry in anatomic pathology routine practice. We performed immunohistochemistry for 19 antibodies against PTEN, eleven of which were excluded during the standardization step. Immunohistochemistry of the remaining eight antibodies was performed on a Tissue Microarray containing 55 prostate and 40 renal carcinoma samples. Fluorescent in situ hybridization (FISH) was used as reference standard for immunohistochemistry specificity evaluation. Concerning nuclear staining, polyclonal (Cat#22034-1-AP); 6H2.1 mMAb (Cat#ABM-2052), Y184 RabMAb (Cat#NB110-57441) and 217702 mMAb antibodies presented the highest agreement with fluorescent in situ hybridization (p<0.001 for all) and with regard to cytoplasmic staining, Y184 RabMAb (Cat#NB110-57441); polyclonal (Cat#22034-1-AP) and 217702 mMAb presented the highest agreement (p<0.001 for all). Our results indicate that several commercially available antibodies do not show reliability of sensitivity and specificity for PTEN evaluation and we propose 6H2.1 mMAb (Cat#ABM-2052) as the antibody of choice for laboratory standardization and best practice in clinical routine, which demonstrated excellent sensitivity for both nuclear and cytoplasmic staining, specificity for PTEN by Western blot and good correlation with PTEN status by FISH with regard to nuclear staining.

GLUT1 expression in malignant tumors and its use as an immunodiagnostic marker.

OBJECTIVE: To analyze glucose transporter 1 expression patterns in malignant tumors of various cell types and evaluate their diagnostic value by immunohistochemistry. INTRODUCTION: Glucose is the major source of energy for cells, and glucose transporter 1 is the most common glucose transporter in humans. Glucose transporter 1 is aberrantly expressed in several tumor types. Studies have implicated glucose transporter 1 expression as a prognostic and diagnostic marker in tumors, primarily in conjunction with positron emission tomography scan data. METHODS: Immunohistochemistry for glucose transporter 1 was performed in tissue microarray slides, comprising 1955 samples of malignant neoplasm from different cell types. RESULTS: Sarcomas, lymphomas, melanomas and hepatoblastomas did not express glucose transporter 1. Forty-seven per cent of prostate adenocarcinomas were positive, as were 29% of thyroid, 10% of gastric and 5% of breast adenocarcinomas. Thirty-six per cent of squamous cell carcinomas of the head and neck were positive, as were 42% of uterine cervix squamous cell carcinomas. Glioblastomas and retinoblastomas showed membranous glucose transporter 1 staining in 18.6% and 9.4% of all cases, respectively. Squamous cell carcinomas displayed membranous expression, whereas adenocarcinomas showed cytoplasmic glucose transporter 1 expression. CONCLUSION: Glucose transporter 1 showed variable expression in various tumor types. Its absence in sarcomas, melanomas, hepatoblastomas and lymphomas suggests that other glucose transporters mediate the glycolytic pathway in these tumors. The data suggest that glucose transporter 1 is a valuable immunohistochemical marker that can be used to identify patients for evaluation by positron emission tomography scan. The function of cytoplasmic glucose transporter 1 in adenocarcinomas must be further examined.

GLUT1 expression in malignant tumors and its use as an immunodiagnostic marker

OBJECTIVE: To analyze glucose transporter 1 expression patterns in malignant tumors of various cell types and evaluate their diagnostic value by immunohistochemistry. INTRODUCTION: Glucose is the major source of energy for cells, and glucose transporter 1 is the most common glucose transporter in humans. Glucose transporter 1 is aberrantly expressed in several tumor types. Studies have implicated glucose transporter 1 expression as a prognostic and diagnostic marker in tumors, primarily in conjunction with positron emission tomography scan data. METHODS: Immunohistochemistry for glucose transporter 1 was performed in tissue microarray slides, comprising 1955 samples of malignant neoplasm from different cell types. RESULTS: Sarcomas, lymphomas, melanomas and hepatoblastomas did not express glucose transporter 1. Fortyseven per cent of prostate adenocarcinomas were positive, as were 29 percent of thyroid, 10 percent of gastric and 5 percent of breast adenocarcinomas. Thirty-six per cent of squamous cell carcinomas of the head and neck were positive, as were 42 percent of uterine cervix squamous cell carcinomas. Glioblastomas and retinoblastomas showed membranous glucose transporter 1 staining in 18.6 percent and 9.4 percent of all cases, respectively. Squamous cell carcinomas displayed membranous expression, whereas adenocarcinomas showed cytoplasmic glucose transporter 1 expression. CONCLUSION: Glucose transporter 1 showed variable expression in various tumor types. Its absence in sarcomas, melanomas, hepatoblastomas and lymphomas suggests that other glucose transporters mediate the glycolytic pathway in these tumors. The data suggest that glucose transporter 1 is a valuable immunohistochemical marker that can be used to identify patients for evaluation by positron emission tomography scan. The function of cytoplasmic glucose transporter 1 in adenocarcinomas must be further examined.

Expression in bacteria of the gene encoding the gp43 antigen of paracoccidioides brasiliensis: immunological reactivity of the recombinant fusion proteins.

gp43 is the major diagnostic antigen of Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis (PCM) in humans. In the present study, cDNA of the gp43 gene (PbGP43) was obtained by reverse transcriptase PCR, inserted into a pGEX vector in frame with the glutathione S-transferase (GST) gene, and expressed in Escherichia coli as inclusion bodies. Immunoblotting showed that all sera from patients with chronic pulmonary and acute lymphatic forms of PCM reacted with the recombinant fusion protein of the mature gp43 (381 amino acids). Reactivity with fusion proteins containing subfragments of the N-terminal, internal, or C-terminal regions occurred eventually, and the C-terminal region was the most antigenic. Lack of reactivity with the subfragments may be due to the conformational nature of the gp43 epitopes. Sera from patients with aspergillosis, candidiasis, and histoplasmosis did not react with the gp43-GST fusion protein. Our results suggest that recombinant gp43 corresponding to the processed antigen can be a useful tool in the diagnosis of PCM.