The impact of telomere length on prostate cancer aggressiveness, genomic instability and health disparities.

The telomere repetitive TTAGGG motif at the ends of chromosomes, serves to preserve genomic integrity and chromosomal stability. In turn, genomic instability is a hallmark of cancer-implicating telomere disturbance. Prostate cancer (PCa) shows significant ancestral disparities, with men of African ancestry at the greatest risk for aggressive disease and associated genomic instability. Yet, no study has explored the role of telomere length (TL) with respect to ancestrally driven PCa health disparities. Patient- and technically-matched tumour-blood whole genome sequencing data for 179 ancestrally defined treatment naïve PCa patients (117 African, 62 European), we assessed for TL (blood and tumour) associations. We found shortened tumour TL to be associated with aggressive PCa presentation and elevated genomic instabilities, including percentage of genome alteration and copy number gains, in men of African ancestry. For European patients, tumour TL showed significant associations with PCa driver genes PTEN, TP53, MSH2, SETBP1 and DDX11L1, while shorter blood TL (< 3200 base pairs) and tumour TL (< 2861 base pairs) were correlated with higher risk for biochemical recurrence. Concurring with previous studies linking TL to PCa diagnosis and/or prognosis, for the first time we correlated TL differences with patient ancestry with important implications for future treatments targeting telomere dysfunction.

Exploring the frequency of a TP53 polyadenylation signal variant in tumor DNA from patients diagnosed with lung adenocarcinomas, sarcomas and uterine leiomyomas.

The TP53 3'UTR variant rs78378222 A>C has been detected in different tumor types as a somatic alteration that reduces p53 expression through modification of polyadenylation and miRNA regulation. Its prevalence is not yet known in all tumors. Herein, we examine tumor tissue prevalence of rs7837822 in Brazilian cohorts of patients from south and southeast regions diagnosed with lung adenocarcinoma (LUAD, n=586), sarcoma (SARC, n=188) and uterine leiomyoma (ULM, n=41). The minor allele (C) was identified in heterozygosity in 6/586 LUAD tumors (prevalence = 1.02 %) and none of the SARC and ULM samples. Additionally, next generation sequencing analysis revealed that all variant-positive tumors (n=4) with sample availability had additional pathogenic or likely pathogenic somatic variants in the TP53 coding regions. Among them, 3/4 (75 %) had the same pathogenic or likely pathogenic sequence variant (allele frequency <0.05 in tumor DNA) namely c.751A>C (p.Ile251Leu). Our results indicate a low somatic prevalence of rs78378222 in LUAD, ULM and SARC tumors from Brazilian patients, which suggests that no further analysis of this variant in the specific studied regions of Brazil is warranted. However, these findings should not exclude tumor molecular testing of this TP53 3'UTR functional variant for different populations.

African-specific molecular taxonomy of prostate cancer.

Prostate cancer is characterized by considerable geo-ethnic disparity. African ancestry is a significant risk factor, with mortality rates across sub-Saharan Africa of 2.7-fold higher than global averages1. The contributing genetic and non-genetic factors, and associated mutational processes, are unknown2,3. Here, through whole-genome sequencing of treatment-naive prostate cancer samples from 183 ancestrally (African versus European) and globally distinct patients, we generate a large cancer genomics resource for sub-Saharan Africa, identifying around 2 million somatic variants. Significant African-ancestry-specific findings include an elevated tumour mutational burden, increased percentage of genome alteration, a greater number of predicted damaging mutations and a higher total of mutational signatures, and the driver genes NCOA2, STK19, DDX11L1, PCAT1 and SETBP1. Examining all somatic mutational types, we describe a molecular taxonomy for prostate cancer differentiated by ancestry and defined as global mutational subtypes (GMS). By further including Chinese Asian data, we confirm that GMS-B (copy-number gain) and GMS-D (mutationally noisy) are specific to African populations, GMS-A (mutationally quiet) is universal (all ethnicities) and the African-European-restricted subtype GMS-C (copy-number losses) predicts poor clinical outcomes. In addition to the clinical benefit of including individuals of African ancestry, our GMS subtypes reveal different evolutionary trajectories and mutational processes suggesting that both common genetic and environmental factors contribute to the disparity between ethnicities. Analogous to gene-environment interaction-defined here as a different effect of an environmental surrounding in people with different ancestries or vice versa-we anticipate that GMS subtypes act as a proxy for intrinsic and extrinsic mutational processes in cancers, promoting global inclusion in landmark studies.

Genome-wide interrogation of structural variation reveals novel African-specific prostate cancer oncogenic drivers.

BACKGROUND: African ancestry is a significant risk factor for advanced prostate cancer (PCa). Mortality rates in sub-Saharan Africa are 2.5-fold greater than global averages. However, the region has largely been excluded from the benefits of whole genome interrogation studies. Additionally, while structural variation (SV) is highly prevalent, PCa genomic studies are still biased towards small variant interrogation. METHODS: Using whole genome sequencing and best practice workflows, we performed a comprehensive analysis of SVs for 180 (predominantly Gleason score ≥ 8) prostate tumours derived from 115 African, 61 European and four ancestrally admixed patients. We investigated the landscape and relationship of somatic SVs in driving ethnic disparity (African versus European), with a focus on African men from southern Africa. RESULTS: Duplication events showed the greatest ethnic disparity, with a 1.6- (relative frequency) to 2.5-fold (count) increase in African-derived tumours. Furthermore, we found duplication events to be associated with CDK12 inactivation and MYC copy number gain, and deletion events associated with SPOP mutation. Overall, African-derived tumours were 2-fold more likely to present with a hyper-SV subtype. In addition to hyper-duplication and deletion subtypes, we describe a new hyper-translocation subtype. While we confirm a lower TMPRSS2-ERG fusion-positive rate in tumours from African cases (10% versus 33%), novel African-specific PCa ETS family member and TMPRSS2 fusion partners were identified, including LINC01525, FBXO7, GTF3C2, NTNG1 and YPEL5. Notably, we found 74 somatic SV hotspots impacting 18 new candidate driver genes, with CADM2, LSAMP, PTPRD, PDE4D and PACRG having therapeutic implications for African patients. CONCLUSIONS: In this first African-inclusive SV study for high-risk PCa, we demonstrate the power of SV interrogation for the identification of novel subtypes, oncogenic drivers and therapeutic targets. Identifying a novel spectrum of SVs in tumours derived from African patients provides a mechanism that may contribute, at least in part, to the observed ethnic disparity in advanced PCa presentation in men of African ancestry.

The role of FASL, BCL-2 and BAX polymorphisms in brazilian patients with prostate cancer and benign prostatic hyperplasia.

BACKGROUND: Prostate cancer (PCA) is one of the leading causes of death among men, being related to several factors, including the aging male population, like benign prostatic hyperplasia (BPH), a histopathological and hyperplastic alteration associated to prostate aging. The FASL, BCL-2 and BAX genes are involved in cell apoptosis regulation and can be related to the development of both cancer and hyperplasia. This study aimed to investigate the association of FASL - 844 (rs763110), BCL-2 -938 (rs2279115) and BAX - 248 (rs4645878) polymorphic variants in Southern Brazilian PCA and BPH patients and healthy controls. METHODS AND RESULTS: 348 samples were analyzed, being 123 from PCA patients, 143 BPH patients and 82 healthy controls, using PCR-RFLP techniques. The results of genotyping analysis were adjusted by age, and compared with PSA levels and prostate volume. The analyzes of genotype frequencies according to PCA, HPB and controls, were performed by logistic regression corrected by age, and showed that the FASL CC genotype can be a risk factor for PCA patients, when compared to controls (p = 0.041). The clinical data investigation indicated higher PSA levels in PCA patients with FASL CC genotype, as compared to TC genotype carriers (p = 0.044), higher PSA levels for healthy individuals with BCL-2 AA genotype, comparing with CC genotype (p = 0.027) and higher PSA levels in BPH group with FASL CC genotype, as compared to TC genotype (p = 0.044). CONCLUSIONS: Our data indicate the FASLCC genotype as a risk factor for prostate pathologies, whileBCL-2 CC can act as a protective genotype.

SARS-CoV-2 RNA in dental biofilms: Supragingival and subgingival findings from inpatients in a COVID-19 intensive care unit.

BACKGROUND: Saliva, salivary glands, gingival crevicular fluid, and supragingival biofilms may harbor SARS-CoV-2 RNA. This observational study aimed to investigate the presence and load of SARS-CoV-2 RNA in supragingival, and subgingival biofilms obtained from intensive care unit (ICU) patients. METHODS: A convenience sample, composed of 52 COVID-19+ participants (48.6 ± 14.8 years, 26.9% females), were evaluated for pre-existing comorbidities, number of teeth, and periodontal data [visible plaque (VPI), bleeding on probing (BOP), periodontal probing depth (PPD), and attachment loss (AL)]. Supragingival and subgingival samples (SubDeep: four sites with the deepest PPD; SubRemain: remaining shallower sites) were analyzed by RT-qPCR with corresponding cycle quantification (Cq). Statistical analyses considered the individual (P = 5%). RESULTS: Twenty-six participants tested positive for dental biofilms (Biofilm+) with 96.2% of them being positive for subgingival samples. Pre-existing comorbidities, number of teeth examined, VPI, PPD, AL, and BOP were similar between Biofilm+ and Biofilm-. SubDeep PPD (3.72 ± 0.86), AL (4.34 ± 1.33), and % of BOP (66.0 ± 31.1) values were significantly greater compared to SubRemain values (2.84 ± 0.48, 3.37 ± 0.34, and 20.4 ± 24.1, respectively). Biofilm+ Cqs showed no association with the periodontal condition. Cqs from Nasopharynx/Oropharynx (Naso/Oro; n = 36) were similar between Biofilm+ and Biofilm- participants. Length of time since ICU intake, last Naso/Oro RT-qPCR readings, onset of COVID-19 symptoms, and biofilm samplings were greater for Biofilm-. CONCLUSIONS: ICU patients harbored SARS-CoV-2 RNA in supragingival and subgingival biofilms, irrespective of the periodontal condition, and systemic viral load. The high number of positive patients highlights the need to better understand this habit to provide adequate oral care.

Stanniocalcin-1 protein expression profile and mechanisms in proliferation and cell death pathways in prostate cancer.

Prostate cancer (PCa) is one of the most prevalent male tumours. Stanniocalcin-1 (STC1) is a glycoprotein and, although the role of STC1 in human cancer is poorly understood, it is suggested to be involved in the development and progression of different neoplasms. This study investigated the protein expression profile of STC1 in PCa and benign prostatic hyperplasia (BPH) samples and STC1 signalling during cell proliferation and cell death in vitro using cell lines. We found higher levels of STC1 in PCa when compared to BPH tissue and that STC1 inhibited forskolin stimulation of cAMP in PC-3 cells. A monoclonal antibody against STC1 was effective in reducing cell proliferation, in promoting cell cycle arrest, and in increasing apoptosis in the same cells. Since STC1 acts as a regulator of prostatic tissue signalling, we suggest that this protein is a novel candidate biomarker for prostate tumour clinical progression and a potential therapeutic target.

PSA secretion and cell proliferation are affected by NCoR1 silencing in prostate cancer cells

Introduction: The androgen receptor (AR) plays an important role in normal development of the prostate gland, as well as in prostatic neoplasms. Transcriptional regulation by AR is modulated by its interaction with co-activators or co-repressors, such as NCoR1 (nuclear receptor co-repressor 1), which is involved in reducing AR activity over the target gene transcription. Methods: To identify the role of NCoR1 in the prostate cancer androgen independence in a cell line model, we aimed to evaluate the effects of silencing NCoR1 on prostate-specific antigen (PSA) gene expression, the proliferative response and PSA secretion on the supernatant of C4-2B and LNCaP cells that were submitted to small interfering RNAs (siRNAs) transfection, and to treatments with different androgen dosages. Results: In LNCaP and C4-2B cells with no dihydrotestosterone (DHT) treatment, a decrease in PSA mRNA expression was observed 48 hours and 72 hours after gene silencing in the siNCoR group when compared to the control and siNC groups. The LNCaP and C4-2B cells showed a biphasic pattern in response to dihydrotestosterone treatment in transfected groups (siNCoR and siNC) as well as in the control condition (without transfection). The secretion of PSA in cell supernatant of LNCaP and C4-2B cells was higher in the siNCoR group, and, in relation to hormonal treatment, higher in the 10-8 M DHT group. Conclusions: A reduction in the NCoR1 levels seems to have a double influence on the activity of AR in PCa cells. These results suggest that NCoR may act as an AR co-repressor depending upon hormonal stimulation.(AU)

Lapatinib Inhibits Amphiregulin-induced BeWo Choriocarcinoma Cell Proliferation by Reducing ERK1/2 and AKT Signaling Pathways.

BACKGROUND: Human choriocarcinoma is the most aggressive type of gestational trophoblastic neoplasia. The expression of epidermal growth factor receptor (EGFR) in choriocarcinomas is significantly higher than those of trophoblastic cells in healthy placentas. Lapatinib is a potent EGFR and human epidermal growth factor receptor 2 (HER2) inhibitor that inhibits cell proliferation and induces apoptosis in various human cancer cells. Amphiregulin (AREG) is the most abundant EGFR ligand in amniotic fluid during human pregnancy. AIM: To explore the role of AREG in human choriocarcinoma cell proliferation. MATERIALS AND METHODS: The effect of lapatinib and AREG on cell proliferation was examined by the MTT assay. Western blots were used to investigate EGFR and HER2 expression, and the activation of caspase-3, extracellular signal-regulated kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase /protein kinase B (PI3K/AKT) signaling pathways. RESULTS: Treatment with lapatinib reduced BeWo cell proliferation by inducing apoptosis. Moreover, AREG treatment stimulated BeWo cell proliferation by activating ERK1/2 and PI3K/AKT signaling pathways, which was blocked by lapatinib. CONCLUSION: Targeting EGFR/HER2 might be a useful therapeutic strategy for human choriocarcinoma.

FMR1 expression in human granulosa cells increases with exon 1 CGG repeat length depending on ovarian reserve.

BACKGROUND: Fragile-X-Mental-Retardation-1- (FMR1)-gene is supposed to be a key gene for ovarian reserve and folliculogenesis. It contains in its 5'-UTR a triplet-base-repeat (CGG), that varies between 26 and 34 in general population. CGG-repeat-lengths with 55-200 repeats (pre-mutation = PM) show instable heredity with a tendency to increase and are associated with premature-ovarian-insufficiency or failure (POI/POF) in about 20%. FMR1-mRNA-expression in leucocytes and granulosa cells (GCs) increases with CGG-repeat-length in PM-carriers, but variable FMR1-expression profiles were also described in women with POI without PM-FMR1 repeat-length. Additionally, associations between low numbers of retrieved oocytes and elevated FMR1-expression levels have been shown in GCs of females with mid-range PM-CGG-repeats without POI. Effects of FMR1-repeat-lengths-deviations (n < 26 or n > 34) below the PM range (n < 55) on ovarian reserve and response to ovarian stimulation remain controversial. METHODS: We enrolled 229 women undergoing controlled ovarian hyperstimulation for IVF/ICSI-treatment and devided them in three ovarian-response-subgroups: Poor responder (POR) after Bologna Criteria, polycystic ovary syndrome (PCO) after Rotterdam Criteria, or normal responder (NOR, control group). Subjects were subdivided into six genotypes according to their be-allelic CGG-repeat length. FMR1-CGG-repeat-length was determined using ALF-express-DNA-sequencer or ABI 3100/3130 × 1-sequencer. mRNA was extracted from GCs after follicular aspiration and quantitative FMR1-expression was determined using specific TaqMan-Assay and applying the ΔΔCT method. Kruskall-Wallis-Test or ANOVA were used for simple comparison between ovarian reserve (NOR, POR or PCO) and CGG-subgroups or cohort demographic data. All statistical analysis were performed with SPSS and statistical significance was set at p ≤ 0.05. RESULTS: A statistically significant increase in FMR1-mRNA-expression-levels was detected in GCs of PORs with heterozygous normal/low-CGG-repeat-length compared with other genotypes (p = 0.044). CONCLUSION: Female ovarian response may be negatively affected by low CGG-alleles during stimulation. In addition, due to a low-allele-effect, folliculogenesis may be impaired already prior to stimulation leading to diminished ovarian reserve and poor ovarian response. A better understanding of FMR1 expression-regulation in GCs may help to elucidate pathomechanisms of folliculogenesis disorders and to develop risk-adjusted treatments for IVF/ICSI-therapy. Herewith FMR1-genotyping potentially provides a better estimatation of treatment outcome and allows the optimal adaptation of stimulation protocols in future.

Androgenic modulation of AR-Vs.

PURPOSE: The importance of androgen receptor variants (AR-Vs) is recognized in prostate cancer. AR-Vs have been the focus of many studies. Expression of AR-Vs has been proposed as a biomarker for resistance to androgen deprivation therapy for metastatic disease. Herein, we show dynamic changes in AR-Vs expression in response to androgen modulation. METHODS: The C4-2B cell line was exposed to low (10-13 M) and high (10-8 M) androgen (dihydrotestosterone, DHT) levels, with or without flutamide. mRNA and protein expression levels were assessed by qPCR and immunohistochemistry, respectively. RESULTS: We demonstrated that high levels of DHT downregulate AR-FL and AR-Vs. Even though AR-Vs did not present ligand-binding domain, thus were not capable of binding to DHT, they present dynamic changes under androgen treatment. Treatment with flutamide alone or in association with low levels of DHT stimulates growth of prostatic cells. CONCLUSIONS: Importantly, we provide evidence that AR-Vs respond differently to androgenic modulation. These findings have implications for a better understanding of the role of AR-Vs in prostate carcinogenesis.

Androgen receptor isoforms expression in benign prostatic hyperplasia and primary prostate cancer.

The role of molecular changes in the androgen receptor (AR) as AR variants (AR-Vs) is not clear in the pathophysiology of benign prostatic hyperplasia (BPH) and hormone-naïve PCa. The aim of the current work was to identify the presence of AR isoforms in benign tissue and primary PCa, and to evaluate the possible association with tumor aggressiveness and biochemical recurrence in primary PCa. The mRNA levels of full length AR (AR-FL) and AR-Vs (AR-V1, AR-V4 and AR-V7) were measured using RT-qPCR. The protein expression of AR-FL (AR-CTD and AR-NTD) and AR-V7 were evaluated by the H-Score in immunohistochemistry (IHC). All investigated mRNA targets were expressed both in BPH and PCa. AR-FL mRNA levels were similar in both groups. AR-V4 mRNA expression showed higher levels in BPH, and AR-V1 and AR-V7 mRNA expression were higher in PCa. The AR-V7 protein showed a similar H-Score in both groups, while AR-CTD and AR-NTD were higher in nuclei of epithelial cells from BPH. These results support the assumption that these constitutively active isoforms of AR are involved in the pathophysiology of primary PCa and BPH. The role of AR-Vs and their possible modulation by steroid tissue levels in distinct types of prostate tumors needs to be elucidated to help guide the best clinical management of these diseases.

Selection of developmentally competent human oocytes aspirated during cesarean section.

PURPOSE: To evaluate the efficiency/safety of Brilliant Cresyl Blue (BCB) staining as a selection method of developmentally competent immature human oocytes. MATERIALS AND METHODS: Immature oocytes of 32 pregnant women were recovered during cesarean section (CS). After retrieval, 92 oocytes were randomly divided into two groups: control (directly disposed to in vitro maturation - IVM) and treated - exposed to BCB 26 µM during 60 min. After staining, the treated group was classified as cytoplasm coloration, BCB positive (blue) or negative (colorless), and then disposed to IVM. Nuclear status was checked after 24 and 48 h of IVM. Nuclear maturation (polar body extrusion), meiosis resumption (absence of germinal vesicle) and degeneration rates were evaluated among the three groups (control, BCB positive and BCB negative) using Generalized Estimating Equations, followed by Bonferroni's correction for multiple comparisons. RESULTS: Nuclear maturation was higher in BCB positive compared to BCB negative, after 24 and 48 h of IVM (p = .004 and p = .032). The control group was equal to BCB positive. There was no difference among groups analyzing meiosis resumption and degeneration rates. CONCLUSION: The BCB test can be a good marker in pre-selection procedures of developmentally competent human oocytes aspirated during CS.

FMR1 and AKT/mTOR signalling pathways: potential functional interactions controlling folliculogenesis in human granulosa cells.

Granulosa cells (GCs) play a major role in folliculogenesis and are crucial for oocyte maturation and growth. In these cells, the mTOR/AKT signalling pathway regulates early folliculogenesis by maintaining the dormancy of primordial follicles, while FSH induces their further differentiation and maturation. Because changes in number of CGG triplets in FMR1 exon 1 (below or beyond normal values of 26-34 triplets) affect ovarian reserve and pre-mutations containing >54 CGG triplets represent a known risk factor for premature ovarian insufficiency/failure, we investigated in the human GC model (COV434) how FMR1/FMRP and mTOR/AKT are expressed and potentially interact during GC proliferation. As FMR protein (FMRP) is expressed mainly in human ovarian GCs, we used these after inducing their proliferation using recombinant FSH (rFSH) and the repression of the mTOR/AKT signalling pathway. We showed that AKT and mTOR expression levels significantly increase after stimulation with rFSH, while S6K and FMR1 expression decrease. After inhibiting mTOR and AKT, FMR1 and S6K expression significantly increased. Only AKT inhibition led to decreased FMRP levels, as expected due to the known FMR1/FMRP negative feedback loop. But rFSH and the mTOR inhibition increased them, indicating a decoupling of this FMR1/FMRP negative feedback loop in our model system.

Ovarian steroid hormones modulate the expression of progesterone receptors and histone acetylation patterns in uterine leiomyoma cells.

Uterine leiomyomas are the most common benign smooth muscle cell tumors in women. Estrogen (E2), progesterone (P4) and environmental factors play important roles in the development of these tumors. New treatments, such as mifepristone, have been proposed. We evaluated the gene expression of total (PRT) and B (PRB) progesterone receptors, and the histone acetyltransferase (HAT) and deacetylase (HDAC) activity after treatment with E2, P4 and mifepristone (RU486) in primary cell cultures from uterine leiomyoma and normal myometrium. Compared to myometrium, uterine leiomyoma cells showed an increase in PRT mRNA expression when treated with E2, and increase in PRB mRNA expression when treated with E2 and P4. Treatment with mifepristone had no significant impact on mRNA expression in these cells. The HDAC activity was higher in uterine leiomyoma compared to myometrial cells after treatment with E2 and E2 + P4 + mifepristone. HAT activity was barely detectable. Our results suggest that ovarian steroid hormones modulate PR, and mifepristone was unable to decrease PRT and PRB mRNA. The higher activity of HDAC leiomyoma cells could be involved in transcriptional repression of genes implicated in normal myometrium cell function, contributing to the maintenance and growth of uterine leiomyoma.

Brilliant cresyl blue staining does not present cytotoxic effects on human luteinized follicular cells, according to gene/protein expression, as well as to cytotoxicity tests.

In vitro oocyte maturation presents many advantages and its success is related to the selection of fully grown oocytes. In animal models, staining of cumulus-oocyte complexes (COCs) with Brilliant Cresyl Blue (BCB) is widely used for this selection. However, a lack of information about the safety of BCB makes its applicability in humans questionable. Because granulosa and cumulus cells have a close relationship with the oocyte and taking into account that follicular cells are also exposed to BCB when this pre-selection method is applied, we aimed to evaluate the effects of BCB on human follicular cells exposed to BCB. Cytotoxicity tests (Sulforhodamine B and Neutral Red Uptake) and gene and protein expression of elements related to the cell cycle (BAX, BCL2, TP53 and CDKN1A), as well as to cell death and metabolism (GAPDH), glucose consumption, and estradiol and progesterone secretion, were examined in granulosa and cumulus cells with and without exposure to BCB. Regardless estradiol secretion and glucose consumption, all other evaluations presented similar results between control and treated (BCB) groups, independently of cell type. Therefore, our results demonstrate convincingly that BCB seems to be safe for use in humans and it should encourage future studies to evaluate the development of embryos derived from human oocytes selected by BCB staining.

Proliferation and metastatic potential of endometrial cancer cells in response to metformin treatment in a high versus normal glucose environment.

In order to improve our understanding of the potential preventive and therapeutic role of metformin, the present study aimed to investigate the capability of low-dose metformin in the efficient inhibition of cancer development and the reduction of the metastasis of endometrial adenocarcinoma type I and primary endometrial epithelial cells (eEPs), with the drug acting as a treatment in a hyperinsulinemic environment exposed to high and normal glucose conditions. The Ishikawa endometrial adenocarcinoma cell line and primary eEPs were exposed to an environment with high (17 mM) or normal glucose (5 mM) and treated with insulin, low-dose metformin (0.1 mM) or a combined treatment. Metastatic potential was assessed by migration and invasion assays, and relative cell proliferation was determined. Metformin at a low dose potently inhibited the insulin action, decreasing the ability of the endometrial cancer (EC) cell line to migrate and invade in a high and normal glucose environment, and decreasing the migration ability of the primary eEPs. In the EC cell line, the insulin treatment increased the proliferation, without any subsequent reduction of proliferation by the addition of 0.1 mM metformin; however, relative cell proliferation sensitivity to metformin was observed in the range between 1 and 5 mM regardless of the glucose concentration present. Overall, metformin at 0.1 mM is not efficient enough to decrease the proliferation in an EC cell line. However, at this concentration, metformin can inhibit the insulin action in endometrial epithelial cancer cells, demonstrating an anti-metastatic effect in high and normal glucose environments.

Effect of Helicobacter pylori on NFKB1, p38α and TNF-α mRNA expression levels in human gastric mucosa.

Helicobacter pylori infects ~50% of the world population, causing chronic gastritis and other forms of cellular damage. The present study assessed the influence of H. pylori on the mRNA expression levels of nuclear factor-κB1 (NFKB1), p38α and tumor necrosis factor-α (TNF-α) in human gastric mucosa in a southern Brazilian population. Human gastric tissue was collected by upper endoscopy and H. pylori diagnosis was performed using a rapid urease test and histological analysis. Total RNA was extracted and purified for subsequent cDNA synthesis and analysis by quantitative polymerase chain reaction (qPCR). The gastric tissue samples were divided into four groups as follows: Normal, inactive chronic gastritis, active chronic gastritis and intestinal metaplasia. The SDHA gene was classified as the most stable when compared with ACTB, GAPDH, B2M and HPRT1 genes, and was therefore selected as the reference gene for qPCR data normalization. TNF-α mRNA expression was significantly higher in samples that were positive for H. pylori and with active chronic gastritis. However, no difference was detected in the mRNA expression levels of NFKB1 and p38α between the groups. The present study concluded that the presence of H. pylori is associated with TNF-α upregulation in human gastric mucosa, but had no effect on NFKB1 and p38α mRNA expression levels.

Safety of brilliant cresyl blue staining protocols on human granulosa and cumulus cells.

The selection of human immature oocytes destined for in vitro maturation (IVM) is performed according to their cumulus-oocyte complex (COC) morphology. In animal models, oocyte pre-selection with brilliant cresyl blue (BCB) staining improves fertilization and blastocyst rates and even increases the number of calves born. As the granulosa cells and cumulus cells (GCs and CCs) have a close relationship with the oocyte and are available in in vitro fertilization (IVF) programs, applying BCB staining to these cells may help to elucidate whether BCB shows toxicity to human oocytes and to determine the safest protocol for this dye. GCs and CCs were isolated from 24 patients who underwent controlled ovarian stimulation. After 48 h, cells were exposed to: Dulbecco's Modified Eagle Medium (DMEM) with or without phenol red, DPBS and mDPBS for 60 min; 13, 20 and 26 µM BCB for 60 min; and 60, 90 or 120 min to 13 µM BCB. Cellular viability was tested using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. The 20 and 26 µM BCB exposures resulted in lower cell viability, similar to when cells were exposed to BCB for 90 or 120 min. GCs and CCs viabilities were equal among control group and 13 µM BCB group after 60 min. BCB staining was not toxic to GCs and CCs when the regime of 13 µM BCB for 60 min was used. Due to the close molecular/biochemical relationship between these cells and the gamete, we propose that it is unlikely that the use of BCB could interfere with the viability/health of human oocytes.