Samuel Marcos Ribeiro de Noronha

Association between the angiotensin-converting enzyme (insertion/deletion) and angiotensin II type 1 receptor (A1166C) polymorphisms and breast cancer among Brazilian women

Introduction.We evaluated the association between components of the renin-angiotensin system and the development of breast cancer in a case-control study by means of angiotensin-converting enzyme (ACE) insertion/deletion (I/D) and angiotensin II type 1 (AT 1)-receptor A1166C polymorphisms. Methods. Genotyping was performed by PCR-RFLP (restriction fragment length polymorphism) or PCR (polymerase chain reaction) using genomic DNA extracted from buccal cells of subjects with (101 cases) or without (307 controls) breast cancer. Results.The frequencies of genotypes for ACE were: DD, ID and II (in %: cases: 60; 20; 20; controls: 46; 37; 17; p=0.019, χ2); and for AT1receptor were:AA,AC and CC (in %: cases: 65; 30; 5; controls: 51; 44; 5; p=0.114, χ 2).The results suggested that the A1166C polymorphism was not associated with breast cancer risk. On the other hand, for the ACE (I/D), there seemed to be different risks for cancer between cases and controls. Conclusions.The ID genotype was less frequently associated with the disease than were the DD or II; that is, women with the ID genotype were 3.1 times less likely to develop breast cancer than those with the other genotypes.The ID genotype might be protective against breast cancer and the ACE (I/D) polymorphism a possible target for developing genetic markers for breast cancer.

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.

Keratinocyte growth factor, tumor necrosis factor-alpha and interleukin-1 beta gene expression in cultured fibroblasts and keratinocytes from burned patients

PURPOSE To evaluate the gene expression of KGF, TNF-alpha and IL-1 beta in skin fibroblasts and keratinocytes cultured from burned patients. METHODS Three patients with large burns and three patients with small burns, as well as two controls, were included. The cell culture was initiated by the enzymatic method. After extraction and purification of mRNA, qPCR was used to assess the gene expression of KGF, TNF-alpha and IL-1 beta. RESULTS The expression of KGF was increased on average 220-fold in large burns and 33.33-fold in small burns in fibroblasts, and 11.2-fold in large burns and 3.45-fold in small burns in keratinocytes compared to healthy patients (p<0.05). Expression of TNF-alpha was not observed. IL-1 beta is down-regulated in fibroblasts of burned patients, and much more repressed in small burns (687-fold, p<0.05). In keratinocytes, the repression of IL-1 beta expression occurs in patients with small burns (28-fold), while patients with large burns express this gene intensively (15-fold). CONCLUSIONS The study showed a quantitative pattern in the expression of KGF gene, which is more expressed according to the size of the burn. TNF-alpha was not expressed. A qualitative pattern in the expression of IL-1 beta gene was demonstrated.

Association of angiotensin-converting enzyme I gene I/D polymorphism with endometrial but not with ovarian cancer.

Associations have been found between the angiotensin-converting enzyme insertion deletion (I/D) polymorphism (ACE I/D) and endometrial and epithelial ovarian cancer (EC and EOC, respectively). In this study, the following frequencies for each of three ACE polymorphisms, DD, ID, and II, respectively, were observed: in the EC group, 55, 24, and 21% versus the control group 39, 40, and 21% (p = 0.033*); in the EOC group 49, 36, and 15% versus the control group 49, 33, and 18% (p = 0.82). According to these allelic distributions, DD carriers are 2.0 times more likely than individuals carrying the ID or II genotypes to develop EC; therefore, the DD genotype seems to be protective against EC. In contrast, no association was observed between ACE (I/D) polymorphism with EOC. The ACE (I/D) polymorphism might play a role in the pathogenesis of EC and it should be considered when identifying genetic markers for EC.

Genetic Association Study of Angiotensin II Receptor Types 1 (A168G) and 2 (T1247G and A5235G) Polymorphisms in Breast Carcinoma among Brazilian Women

Background: Many types of cancer are associated with polymorphisms of the renin-angiotensin system. Our aim was to assess possible association between single-nucleotide polymorphisms (SNPs) of the angiotensin II receptor types 1 (A168G), and 2 (T1247G and A5235G) with breast cancer. Patients and Methods: 242 participating subjects were genotyped and allocated to case or control groups. Results: Genotype distribution (in %) was: for AGTR1 (A168G): AA, AG, GG = 61, 30, 09 for cases, and 69, 25, 06 for controls (p = 0.55); for AGTR2 (T1247G): TT, TG, GG = 84, 12, 04 for cases, and 81, 17, 02 for controls (p = 0.45); for AGTR2 (A5235G): AA, AG, GG = 32, 67, 01 for cases, and 53, 28, 19 for controls (p < 0.0001). Women carrying genotypes AA/AG in the intronic region of angiotensin II type 2 receptor had an 11-fold higher risk of breast cancer than GG carriers. Conclusions: Many types of cancer have been associated with polymorphisms of the renin-angiotensin system. For SNP A5235G, the GG genotype seems to be protective against breast cancer. The other 2 SNPs showed no association. However, SNPs T1247G and A5235G were associated with at least 1 clinical variable, with G being a predictor of better outcome. The use of SNPs A5235G and T1247G (the latter to a lesser degree) as genetic markers should be considered.

Human chorionic gonadotropin and a 15 amino acid hCG fragment of the hormone induce downregulation of the cytokine IL-8 receptor in normal breast epithelial cells.

Abstract Cytokine receptors are associated with tumor cell growth by increasing proliferation, metastasis and regulating self-renewal of cancer stem cells (SCs). There is a strong association between cytokine IL-8 receptor (CXCR1) over-expression and cells displaying SC characteristics. Human chorionic gonadotropin (hCG) causes differentiation, inhibition of cell proliferation and increased apoptosis of the breast epithelium. hCG receptor (LHCGR) expression in breast tumors and in breast cancer cell lines is undetectable or low. In this study, our objective was to assess and compare the effects of hCG and a 15 amino acid hCG fragment of the hormone on mRNA expression of CXCR1 and LHCGR on normal breast epithelial cells (MCF-10F) by real time RT-PCR after treatment with hCG or a hCG fragment for 15 days. Cell proliferation was also measured. hCG and the hCG fragment decreased cell proliferation in both groups. The compounds upregulated LHCGR expression and downregulated CXCR1 expression. It is possible to postulate that an increase of LHCGR mRNA seems to respond to the decrease of CXCR1 expression. These genes probably act synergistically to reduce the amount of cancer SCs in the mammary gland. Thereby, the use of hCG or the hCG fragment as a therapeutic or preventive tool should be considered.

Toll like receptors gene expression of human keratinocytes cultured of severe burn injury

PURPOSE To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human Primary Epidermal keratinocytes of patients with severe burns. METHODS After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific TLR pathways PCR Arrays plates (SA Biosciences). RESULTS After the analysis of gene expression we found that 21% of these genes were differentially expressed, of which 100% were repressed or hyporegulated. Among these, the following genes (fold decrease): HSPA1A (-58), HRAS (-36), MAP2K3 (-23), TOLLIP (-23), RELA (-18), FOS (-16), and TLR1 (-6.0). CONCLUSIONS This study contributes to the understanding of the molecular mechanisms related to TLR pathways and underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome.

Innate and adaptive immunity gene expression of human keratinocytes cultured of severe burn injury

PURPOSE Evaluate the expression profile of genes related to Innate and Adaptive Immune System (IAIS) of human Primary Epidermal keratinocytes (hPEKP) of patients with severe burns. METHODS After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific IAIS PCR Arrays plates (SA Biosciences). RESULTS After the analysis of gene expression we found that 63% of these genes were differentially expressed, of which 77% were repressed and 23% were hyper-regulated. Among these, the following genes (fold increase or decrease): IL8 (41), IL6 (32), TNF (-92), HLA-E (-86), LYS (-74), CCR6 (- 73), CD86 (-41) and HLA-A (-35). CONCLUSIONS This study contributes to the understanding of the molecular mechanisms underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome.

Keratinocyte growth factor and the expression of wound-healing-related genes in primary human keratinocytes from burn patients

PURPOSE To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients. METHODS Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC. RESULTS Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes. CONCLUSION There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.

Aldefluor protocol to sort keratinocytes stem cells from skin

PURPOSE To investigate the use Aldefluor® and N, N - Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients. METHODS Activated Aldefluor® aliquots were prepared and maintained at temperature between 2 to 8°C, or stored at -20°C. Next, the cells were collected following the standard protocol of sample preparation. RESULTS Best results were obtained with Aldefluor® 1.5µl and DEAB 15 µl for 1 x 106 cells, incubated at 37°C for 15 minutes. Flow cytometer range for keratinocyte stem cells separation was evaluated. There were 14.8% of stem cells separated in one sample of keratinocyte culture used to pattern the protocol. After being defined the ideal concentration, the same test pattern was performed in other keratinocyte samples. We observed a final mean of 10.8%. CONCLUSION Aldefluor® has been shown as a favorable marking of epidermal keratinocyte stem cells for subsequent separation on a flow cytometer, with detection of 10.8% of epidermal keratinocyte stem cells, in this protocol.