Diagnostic and Prognostic Performance of Metabolic Signatures in Pancreatic Ductal Adenocarcinoma: The Clinical Application of Quantitative NextGen Mass Spectrometry.

With 64,050 new diagnoses and 50,550 deaths in the US in 2023, pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of all human malignancies. Early detection and improved prognostication remain critical unmet needs. We applied next-generation metabolomics, using quantitative tandem mass spectrometry on plasma, to develop biochemical signatures that identify PDAC. We first compared plasma from 10 PDAC patients to 169 samples from healthy controls. Using metabolomic algorithms and machine learning, we identified ratios that incorporate amino acids, biogenic amines, lysophosphatidylcholines, phosphatidylcholines and acylcarnitines that distinguished PDAC from normal controls. A confirmatory analysis then applied the algorithms to 30 PDACs compared with 60 age- and sex-matched controls. Metabolic signatures were then analyzed to compare survival, measured in months, from date of diagnosis to date of death that identified metabolite ratios that stratified PDACs into distinct survival groups. The results suggest that metabolic signatures could provide PDAC diagnoses earlier than tumor markers or radiographic measures and offer insights into disease severity that could allow more judicious use of therapy by stratifying patients into metabolic-risk subgroups.

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.

Gastrointestinal Transcriptomic Response of Metabolic Vitamin B12 Pathways in Roux-en-Y Gastric Bypass.

OBJECTIVES: Vitamin B12 (B12) deficiency after Roux-en-Y gastric bypass (RYGB) is highly prevalent and may contribute to postoperative complications. Decreased production of intrinsic factor owing to gastric fundus removal is thought to have a major role, but other components of B12 metabolism may also be affected. We evaluated changes in the expression levels of multiple B12 pathway-encoding genes in gastrointestinal (GI) tissues to evaluate the potential roles in contributing to post-RYGB B12 deficiency. METHODS: During double-balloon enteroscopy, serial GI biopsies were collected from 20 obese women (age, 46.9±6.2 years; body mass index, 46.5±5.3 kg/m2) with adult-onset type 2 diabetes (fasting plasma glucose ≥126 mg/dl; hemoglobin A1c≥6.5%) before and, at the same site, 3 months after RYGB. Gene expression levels were assessed by the Affymetrix Human GeneChip 1.0 ST microarray. Findings were validated by real-time quantitative PCR (RT-qPCR). RESULTS: Gene expression levels with significant changes (P≤0.05) included: transcobalamin I (TCN1) in remnant (-1.914-fold) and excluded (-1.985-fold) gastric regions; gastric intrinsic factor (GIF) in duodenum (-0.725-fold); and cubilin (CUBN) in duodenum (+0.982-fold), jejunum (+1.311-fold), and ileum (+0.685-fold). Validation by RT-qPCR confirmed (P≤0.05) observed changes for TCN1 in the remnant gastric region (-0.132-fold) and CUBN in jejunum (+2.833-fold). CONCLUSIONS: RYGB affects multiple pathway-encoding genes that may be associated with postoperative B12 deficiency. Decreased TCN1 levels seem to be the main contributing factor. Increased CUBN levels suggest an adaptive genetic reprogramming of intestinal tissue aiming to compensate for impaired intestinal B12 delivery.

Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer.

BACKGROUND: Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and -4 are highly expressed, but PAR-3 shows low expression and unclear functions. METHODS: Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFß monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. RESULTS: We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and -4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFß in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. CONCLUSIONS: Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.

Epigenetic modulations in activated cells early after HIV-1 infection and their possible functional consequences.

Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either, replicate and produce a viable progeny or be driven to latency. The purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4+ cells after HIV-1 infection analyzing three approaches: (i) global DNA- methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. The analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array, we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. In addition, activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2, IFN-γ and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely, based on epigenetic markers studied here, non-stimulated cells infected by HIV-1, showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover, non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations, it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection.

Prognostic scores after surgical treatment for cervical intraepithelial neoplasia: a proposed model and possible implications for post-operative follow-up.

OBJECTIVE: To develop a prognostic model for women who underwent surgical treatment for cervical intraepithelial neoplasia. DESIGN: Cohort study. Patient inclusion and follow-up occurred retrospectively and prospectively. SETTING: Barretos Cancer Hospital, Barretos, São Paulo, Brazil. POPULATION: Women (n = 242) diagnosed with cervical intraepithelial neoplasia who were submitted to conization. METHODS: Immediately prior to surgical treatment, a cervical cytology sample was collected from each individual included in the study by endocervical brushing and stored in a preservative solution with methanol. A human papilloma virus-DNA test was conducted using an aliquot of the endocervical brushings. The surgical specimens were subjected to immunohistochemical analysis of p16 (immunohistochemical analysis 4a) protein expression. MAIN OUTCOME MEASURES: Two-year disease-free survival rates calculated for each study variable. Identified variables in the multivariate Cox model were used for elaboration of prognostic scores. RESULTS: Variables associated with outcome included age (p = 0.033), tobacco use (p < 0.001), final histopathological diagnosis (p = 0.007), surgical margins (p < 0.001), high-risk human papilloma virus status (p = 0.008), human papilloma virus-16 status (p < 0.001) and immunoexpression of p16 in the cytoplasm (p = 0.049). By the Cox model, independent risk factors for disease recurrence/persistence were: tobacco use (hazard risk = 3.0; 95% confidence interval 1.6-5.6), positive surgical margins (hazard risk = 3.2; 95% confidence interval 1.6-6.1), human papilloma virus-16 (hazard risk = 3.3; 95% confidence interval 1.6-6.9) and age over 45 years (hazard risk = 2.7; 95% confidence interval 1.1-6.6). CONCLUSIONS: Establishment of a prognostic score can represent a valuable tool for determining the risk of cervical intraepithelial neoplasia recurrence after conization. The use of clinical (age and tobacco use), pathological (surgical margins) and molecular (human papilloma virus-16 genotyping) factors can facilitate more appropriate patient follow up according to risk stratification.

A simple strategy for the purification of native recombinant full-length human RPL10 protein from inclusion bodies.

The L10 ribosomal protein (RPL10) plays a role in the binding of the 60 S and 40 S ribosomal subunits and in mRNA translation. The evidence indicates that RPL10 also has multiple extra-ribosomal functions, including tumor suppression. Recently, the presence of RPL10 in prostate and ovarian cancers was evaluated, and it was demonstrated to be associated with autistic disorders and premature ovarian failure. In the present work, we successfully cloned and expressed full-length human RPL10 (hRPL10) protein and isolated inclusion bodies containing this protein that had formed under mild growth conditions. The culture produced 376mg of hRPL10 protein per liter of induced bacterial culture, of which 102.4mg was present in the soluble fraction, and 25.6mg was recovered at approximately 94% purity. These results were obtained using a two-step process of non-denaturing protein extraction from pelleted inclusion bodies. We studied the characteristics of this protein using circular dichroism spectroscopy and by monitoring the changes induced by the presence or absence of zinc ions using fluorescence spectrometry. The results demonstrated that the protein obtained using these non-conventional methods retained its secondary and tertiary structure. The conformational changes induced by the incorporation of zinc suggested that this protein could interact with Jun or the SH3 domain of c-yes. The results suggested that the strategy used to obtain hRPL10 is simple and could be applied to obtaining other proteins that are susceptible to degradation.

Gene expression profile of cytokines and receptors of inflammation from cultured keratinocytes of burned patients.

INTRODUCTION: At all stages of wound healing, growth factors and cytokines play a particularly important role in the interaction with keratinocytes cellular receptors. Keratinocytes have received little attention about their potential to act as a source and target of cytokines. Changes in the cytokine levels after the burning occur prior to the metabolic abnormalities. Thus, it may be possible to develop therapeutic interventions that can mitigate the acute inflammatory response and modulating expression of these cytokines. The objective was to evaluate the expression of 84 genes mediators of the inflammatory response by using PCR array in a primary human epidermal cultured keratinocytes from patients with burns. METHODS: Keratinocytes cultured from normal skin around injury from small and large burn patient were treated for DNA synthesis. The samples were analyzed by the PCR Superarray(®) assay and curve analyses were performed for 84 relevant human genes and their involvement in the inflammatory cytokines pathway and receptors. These genes were checked for the up or down regulation. And it was used MetaCore™ for the analysis of networks and Gene Ontology (GO) processes. RESULTS: Chemokines of the CXC family were more expressed in the large burn group, except CXCL12. The C, CC and CX3C chemokine family were downregulated, especially in the small burn group. The interleukins IL8 and IL1B were more expressed in large burn than in small burn; except IL13RA1, IL13 and IL5RA that were downregulated, mainly in the small burn group. CONCLUSIONS: The cytokine profile showed some important differences between the large and small burn patients, and from this original database, we can create new interventional trials in acute inflammation in burns.

Gene network reconstruction reveals cell cycle and antiviral genes as major drivers of cervical cancer.

Although human papillomavirus was identified as an aetiological factor in cervical cancer, the key human gene drivers of this disease remain unknown. Here we apply an unbiased approach integrating gene expression and chromosomal aberration data. In an independent group of patients, we reconstruct and validate a gene regulatory meta-network, and identify cell cycle and antiviral genes that constitute two major subnetworks upregulated in tumour samples. These genes are located within the same regions as chromosomal amplifications, most frequently on 3q. We propose a model in which selected chromosomal gains drive activation of antiviral genes contributing to episomal virus elimination, which synergizes with cell cycle dysregulation. These findings may help to explain the paradox of episomal human papillomavirus decline in women with invasive cancer who were previously unable to clear the virus.

17ß-Estradiol and steady-state concentrations of H2O2: antiapoptotic effect in endometrial cells from patients with endometriosis.

Increased levels of hydrogen peroxide (H2O2) can initiate protective responses to limit or repair oxidative damage. However, H2O2 signals also fine-tune responses to growth factors and cytokines controlling cell division, differentiation, and proliferation. Because 17ß-estradiol (E2) also plays important roles in these processes, and is considered a major risk factor in the development and progression of endometriosis, this study evaluated whether E2 has an antiapoptotic effect on oxidative stress in endometrial cells in combination with steady-state H2O2 levels ([H2O2]ss). Endometrial stromal cells were prepared from the eutopic endometrium of 18 women with and without endometriosis to produce primary cells. These cells were stimulated with E2 for 20h, exposed to [H2O2]ss, and examined for cell viability, proliferation, and apoptosis. The endometrial cells from women with endometriosis maintained the steady state for 120min at high H2O2 concentrations. When they were pretreated with E2 and exposed to [H2O2]ss, a decrease in apoptosis level was observed compared to the control cells (p<0.01). The endometrial cells from patients with endometriosis subjected to both E2 and [H2O2]ss showed increased ERK phosphorylation. These findings suggested that H2O2 is a signaling molecule that downregulates apoptosis in endometrial cells, supporting the fact that endometriosis, albeit a benign disease, shares some features with cancer such as decreased catalase levels. These results link the E2 effects on [H2O2]ss to resistance to apoptosis and progression of endometriosis.

Unraveling the antifungal activity of a South American rattlesnake toxin crotamine.

Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the ß-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 µg/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 µg/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 µg/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 µg/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. The peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications.

Intrafollicular soluble receptor for advanced glycation end products (sRAGE) and embryo quality in assisted reproduction.

The developmental potential of human embryos has important implications in assisted reproduction and depends, among other factors, on oocyte competency. The receptor for advanced glycation end products (RAGE) is a member of the superfamily of immunoglobulin cell-surface molecules that are constitutively expressed during embryonic development. RAGE is down-regulated in homeostasis in adult life. This study measured the concentration of soluble RAGE (sRAGE) in follicular fluid obtained from the leading follicle after ovarian stimulation of 54 women undergoing intracytoplasmic sperm injection. Corresponding embryos and sRAGE concentrations in follicular fluid were evaluated and correlations were investigated by multi-adjusted regression analysis. High intrafollicular sRAGE concentrations predicted poor-quality embryos (n=45, OR=0.986; P=0.026), adjusted for patient age, body mass index and oocyte quality, showing an inverse association between intrafollicular sRAGE concentrations and embryo development. The developmental potential of human embryos has important implications in assisted reproduction, and it depends, among other factors, on oocyte competency. The receptor for advanced glycation end products (RAGE) is a molecule constitutively expressed during embryonic development, but it is down-regulated in adult life. RAGE is frequently associated with pro-inflammatory responses, and it is implicated as an underlying condition in immune disorders, cardiovascular diseases, diabetes, Alzheimer's disease and cancer. In addition to activating the pro-inflammatory responses, RAGE down-regulates cellular defence mechanisms. The present study measured the concentrations of soluble RAGE (sRAGE) in follicular fluid samples obtained from leading follicles of women undergoing intracytoplasmic sperm injection (ICSI). This prospective cohort study included 54 patients undergoing ICSI, and follicular fluid samples were obtained from the leading follicle after ovarian stimulation. The corresponding embryos were evaluated and correlations with intrafollicular sRAGE concentrations were investigated using multi-adjusted regression analysis. We observed that high intrafollicular concentrations of sRAGE predicted poor embryo quality. Our findings suggest an association between high concentrations of intrafollicular sRAGE and poor embryo development following ovarian stimulation for ICSI.

Spectroscopic and structural analysis of somatic and N-domain angiotensin I-converting enzyme isoforms from mesangial cells from Wistar and spontaneously hypertensive rats.

Angiotensin I-converting enzyme (ACE) plays a key role in the renin-angiotesin aldosterone cascade. We analysed the secondary structure and structural organization of a purified 65kDa N-domain ACE (nACE) from Wistar rat mesangial cells, a 90 kDa nACE from spontaneously hypertensive rats and a 130 kDa somatic ACE. The C-terminal alignment of the 65 kDa nACE with rat ACE revealed that the former was truncated at Ser(482), and the sequence of the 90 kDa nACE ended at Pro(629). Protein's secondary structure consisted predominantly of alpha-helices. The 90 and 65 kDa isoforms were the most stable in guanidine and at low pH, respectively. Enzymatic activity decreased with loss in secondary structure, except in the case of guanidine HCl where the 90 kDa fragment loses its secondary structure faster than its enzymatic activity. We identified and characterized the activity and stability of these isoforms and these findings would be helpful on the understanding of the role of nACE isoforms in hypertension.

The V109G polymorphism in the p27 gene is associated with endometriosis.

OBJECTIVE: To investigate the prevalence of the p27 gene polymorphism in women with endometriosis. STUDY DESIGN: Transversal case-control study. Genomic DNA was extracted from cells collected from buccal swabs. The p27 V109G polymorphism was investigated using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in a hospital-based Brazilian population. RESULTS: We analysed the 104 patients and 109 control subjects. The distribution of genotype and allele frequencies of p27 V109G polymorphism was significantly different between the endometriosis cases and healthy women (p=0.016 and 0.002). Women who had at least one mutated allele presented twofold chances for endometriosis development (OR=1.9; 95% CI, 1.120-3.343). CONCLUSION: The polymorphic variant at codon 109 of the p27 gene seems to be associated with higher risk of endometriosis development.

Endometrial claudin-4 and leukemia inhibitory factor are associated with assisted reproduction outcome.

BACKGROUND: Claudin-4 (CLDN4) is one of several proteins that act as molecular mediators of embryo implantation. Recently, we examined immunolabeling of leukemia inhibitory factor (LIF) in the endometrial tissue of 52 IVF patients, and found that LIF staining intensity was strongly correlated with successful pregnancy initiation. In the same set of patients, we have now examined endometrial CLDN4 expression, to see how expression intensity may vary with LIF. We examined CLDN4 in the luteal phase of the menstrual cycle, immediately preceding IVF treatment. Our aim was to compare expression of LIF and CLDN4 in the luteal phase, and document these patterns as putative biomarkers for pregnancy. METHODS: Endometrial tissue was collected from women undergoing IVF. Endometrial biopsies were obtained during the luteal phase preceding IVF, and were then used for tissue microarray (TMA) immunolabeling of CLDN4. Previously published LIF expression data were then combined with CLDN4 expression data, to determine CLDN4/LIF expression patterns. Associations between successful pregnancy after IVF and combined CLDN4/LIF expression patterns were evaluated. RESULTS: Four patterns of immunolabeling were observed in the endometrial samples: 16% showed weak CLDN4 and strong LIF (CLDN4-/LIF+); 20% showed strong CLDN4 and strong LIF (LIF+/CLDN4+); 28% showed strong CLDN4 and weak LIF (CLDN4+/LIF-); and 36% showed weak CLDN4 and weak LIF (CLDN4-/LIF-). Successful implantation after IVF was associated with CLDN4-/LIF+(p = 0.003). Patients showing this endometrial CLDN4-/LIF+ immunolabeling were also 6 times more likely to achieve pregnancy than patients with endometrial CLDN4+/LIF- immunolabeling (p = 0.007). CONCLUSION: The combined immunolabeling expression of CLDN4-/LIF+ in endometrial tissue is a potential biomarker for predicting successful pregnancy in IVF candidates.

Isoflavone regulates vascular endothelial growth factor expression in urinary tract of castrated rats.

OBJECTIVE: The purpose of this study was to investigate Vascular Endothelial Growth Factor Expression (VEGF) gene regulation by isoflavone in urinary tract tissues of castrated adult rats. DESIGN: Forty-five adult rats, 90 days old, weighting 200 g were used, receiving a soy-free ration. The animals were castrated for drug administration for 30 days (125 microg genisteine/g body weight/day) and sacrificed, divided into three groups: Group I-control; Group II-started isoflavone administration on the 5th day after castration; Group III-started isoflavone administration on the 28th day after castration. RNA was isolated from each bladder and urethra. Determination of VEGF gene regulated by isoflavone was obtained using a semiquantitative RT-PCR and immunohistochemistry of total RNA isolated from bladder and urethra. RESULTS: Our results demonstrate that isoflavone was able to upregulate mRNA level of the VEGF gene in the lower urinary tract of rats in Group II, where isoflavone administration was started at an early phase of estrogen deprivation, while in Group III, where isoflavone administration was started in the late phase of hypoestrogenism, did not show alteration of bladder and urethra VEGF gene expression, compared to placebo, maintaining the same level of the castrated rats without treatment. CONCLUSIONS: The data indicate that VEGF expression in rats is also regulated by isoflavone in early phase of hypoestrogenism.

Correlation between MMPs and their inhibitors in breast cancer tumor tissue specimens and in cell lines with different metastatic potential.

BACKGROUND: The metastatic disease rather than the primary tumor itself is responsible for death in most solid tumors, including breast cancer. The role of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in the metastatic process has previously been established. However, in all published studies only a limited number of MMPs/MMP inhibitors was analyzed in a limited number of cell lines. Here, we propose a more comprehensive approach by analyzing the expression levels of several MMPs (MMP-2, MMP-9 and MMP-14) and MMP inhibitors (TIMP-1, TIMP-2 and RECK) in different models (five human breast cancer cell lines, 72 primary breast tumors and 30 adjacent normal tissues). METHODS: We analyzed the expression levels of MMP-2, MMP-9 and MMP-14 and their inhibitors (TIMP-1, TIMP-2 and RECK) by quantitative RT-PCR (qRT-PCR) in five human breast cancer cell lines presenting increased invasiveness and metastatic potential, 72 primary breast tumors and 30 adjacent normal tissues. Moreover, the role of cell-extracellular matrix elements interactions in the regulation of expression and activity of MMPs and their inhibitors was analyzed by culturing these cell lines on plastic or on artificial ECM (Matrigel). RESULTS: The results demonstrated that MMPs mRNA expression levels displayed a positive and statistically significant correlation with the transcriptional expression levels of their inhibitors both in the cell line models and in the tumor tissue samples. Furthermore, the expression of all MMP inhibitors was modulated by cell-Matrigel contact only in highly invasive and metastatic cell lines. The enzyme/inhibitor balance at the transcriptional level significantly favors the enzyme which is more evident in tumor than in adjacent non-tumor tissue samples. CONCLUSION: Our results suggest that the expression of MMPs and their inhibitors, at least at the transcriptional level, might be regulated by common factors and signaling pathways. Therefore, the multi-factorial analysis of these molecules could provide new and independent prognostic information contributing to the determination of more adequate therapy strategies for each patient.

High levels of granzyme B expression in invasive cervical carcinoma correlates to poor response to treatment.

The present study assessed, in cervical carcinoma, expression levels of seven immune response genes and sought correlation to response to treatment. The expression levels of CD28, CTLA4, ICOS, ICOSL, CD80 and CD86 and granzyme B genes were assessed by real-time RT-PCR in pre-treatment tumor fragments. During the six-month follow-up after treatment, 8 patients presented tumor and 10 survived free of tumor. The only gene whose expression levels were higher in patients with poor outcome (p = 0.03) was granzyme B. Further evaluation, in adequately powered prospective studies is warranted to confirm the data and to translate this observation to the clinical setting.

Progesterone receptor (PROGINS) polymorphism and the risk of ovarian cancer.

The present case-control study evaluates the role of the progesterone receptor (PR) polymorphism known as PROGINS as a risk factor for ovarian cancer development and investigates the association between these genetic variants and clinical/pathologic variables of ovarian cancer. PROGINS polymorphism was examined, by polymerase chain reaction, in a total of 80 patients with ovarian cancer and 282 control subjects. The frequencies of PROGINS polymorphism T1/T1, T1/T2, and T2/T2 were 71.3, 15.0 and 13.8% in ovarian cancer patients and 78.37, 21.63 and 0% in controls, respectively. The chi(2)-test showed a higher incidence of the T2/T2 genotype (P=0.001) in the ovarian cancer group. In addition, women carrying a mutated allele (T2) showed approximately 2.2 times higher risk of ovarian cancer development as compared to women who have a variant allele (odds ratio (OR)=2.2; 95% CI=1.80-3.54). Regarding the clinical and pathologic findings observed within the cancer group, there was a significant correlation between PROGINS polymorphism and patients with a familial history (chi(2)=6.776; P=0.009; Fischer exact test, P=0.01). In this regard, patients with familial antecedents have a 4.7 times higher likelihood to have at least one risk allele (T2) as compared with patients without familial antecedents (OR=4.69; 95% CI=1.38-15.87). No correlations were observed among the other variables. These data suggest that the PROGINS polymorphism T2/T2 genotype might be associated with an increased risk of ovarian cancer.

New approach reveals CD28 and IFNG gene interaction in the susceptibility to cervical cancer.

Cervical cancer is a complex disease with multiple environmental and genetic determinants. In this study, we sought an association between polymorphisms in immune response genes and cervical cancer using both single-locus and multi-locus analysis approaches. A total of 14 single nucleotide polymorphisms (SNPs) distributed in CD28, CTLA4, ICOS, PDCD1, FAS, TNFA, IL6, IFNG, TGFB1 and IL10 genes were determined in patients and healthy individuals from three independent case/control sets. The first two sets comprised White individuals (one group with 82 cases and 85 controls, the other with 83 cases and 85 controls) and the third was constituted by non-white individuals (64 cases and 75 controls). The multi-locus analysis revealed higher frequencies in cancer patients of three three-genotype combinations [CD28+17(TT)/IFNG+874(AA)/TNFA-308(GG), CD28+17(TT)/IFN+847(AA)/PDCD1+7785(CT), and CD28 +17(TT)/IFNG+874(AA)/ICOS+1564(TT)] (P < 0.01, Monte Carlo simulation). We hypothesized that this two-genotype [CD28(TT) and IFNG(AA)] combination could have a major contribution to the observed association. To address this question, we analyzed the frequency of the CD28(TT), IFNG(AA) genotype combination in the three groups combined, and observed its increase in patients (P = 0.0011 by Fisher's exact test). The contribution of a third polymorphism did not reach statistical significance (P = 0.1). Further analysis suggested that gene-gene interaction between CD28 and IFNG might contribute to susceptibility to cervical cancer. Our results showed an epistatic effect between CD28 and IFNG genes in susceptibility to cervical cancer, a finding that might be relevant for a better understanding of the disease pathogenesis. In addition, the novel analytical approach herein proposed might be useful for increasing the statistical power of future genome-wide multi-locus studies.