Graziela Rosa Ravacci

Complementarity of Subjective Global Assessment (SGA) and Nutritional Risk Screening 2002 (NRS 2002) for predicting poor clinical outcomes in hospitalized patients

BACKGROUND & AIMS We evaluated the ability of Nutritional Risk Screening 2002 (NRS 2002) and Subjective Global Assessment (SGA) to predict malnutrition related to poor clinical outcomes. METHODS We assessed 705 patients at a public university hospital within 48 h of admission. Logistic regression and number needed to screen (NNS) were calculated to test the complementarity between the tools and their ability to predict very long length of hospital stay (VLLOS), complications, and death. RESULTS Of the patients screened, 27.9% were at nutritional risk (NRS+) and 38.9% were malnourished (SGA B or C). Compared to those patients not at nutritional risk, NRS+, SGA B or C patients were at increased risk for complications (p=0.03, 0.02, and 0.003, respectively). NRS+ patients had an increased risk of death (p=0.03), and SGA B and C patients had an increased likelihood of VLLOS (p=0.008 and p<0.0001, respectively). Patients who were both NRS+ and SGA C had lower estimates of NNS than patients who were NRS+ or SGA C only, though their confidence intervals did overlap. CONCLUSIONS The concurrent application of SGA in NRS+ patients might enhance the ability to predict poor clinical outcomes in hospitalized patients in Brazil.

Lipid raft disruption by docosahexaenoic acid induces apoptosis in transformed human mammary luminal epithelial cells harboring HER-2 overexpression.

In HER-2-overexpressing breast cells, HER-2 receptors exist on the cell surface as monomers, homodimers and heterodimers. For signal activation and transduction to occur, HER-2 must be localized to lipid rafts. Therefore, we hypothesized that the amount of lipid rafts on the cell membrane would be a factor in HER-2 signaling. To test this, we used HB4a (an untransformed human mammary epithelial cell line) and HB4aC5.2 cells. HB4aC5.2 cells are HB4a derivatives that have been transfected with five copies of pJ5E.c-ErbB-2 and express approximately 900 times more HER-2 than HB4a cells. In these cells, HER-2 overexpression was accompanied by increased lipid rafts in cell membranes, a hyperactivation of downstream Akt and ERK1/2 proteins, and an increased rate of cell growth compared to HB4a. In addition, HER-2 overexpression was associated with an increased activation of FASN, a key enzyme involved in cellular lipogenesis. Its final product, palmitate, is frequently used to synthesize lipid rafts. We further hypothesized that treatment with docosahexaenoic acid (DHA), an omega-3 fatty acid, would disrupt the lipid rafts and lead to a growth arrest. In HB4aC5.2 cells, but not HB4a cells, we found that DHA treatment disrupted lipid raft; inhibited HER-2 signaling by decreasing activation of Akt, ERK1/2 and FASN proteins; and induced apoptosis. Although little is known about lipid rafts, our data support the idea that disturbances in these microdomains induced by DHA may represent a useful tool for controlling the signaling initiated by HER-2 receptors and its therapeutic potential in the treatment of HER-2 positive breast cancer.

Phase angle obtained by bioelectrical impedance analysis independently predicts mortality in patients with cirrhosis

AIM To evaluate the prognostic value of the phase angle (PA) obtained from bioelectrical impedance analysis (BIA) for mortality prediction in patients with cirrhosis. METHODS In total, 134 male cirrhotic patients prospectively completed clinical evaluations and nutritional assessment by BIA to obtain PAs during a 36-mo follow-up period. Mortality risk was analyzed by applying the PA cutoff point recently proposed as a malnutrition marker (PA ≤ 4.9°) in Kaplan-Meier curves and multivariate Cox regression models. RESULTS The patients were divided into two groups according to the PA cutoff value (PA > 4.9°, n = 73; PA ≤ 4.9°, n = 61). Weight, height, and body mass index were similar in both groups, but patients with PAs > 4.9° were younger and had higher mid-arm muscle circumference, albumin, and handgrip-strength values and lower severe ascites and encephalopathy incidences, interleukin (IL)-6/IL-10 ratios and C-reactive protein levels than did patients with PAs ≤ 4.9° (P ≤ 0.05). Forty-eight (35.80%) patients died due to cirrhosis, with a median of 18 mo (interquartile range, 3.3-25.6 mo) follow-up until death. Thirty-one (64.60%) of these patients were from the PA ≤ 4.9° group. PA ≤ 4.9° significantly and independently affected the mortality model adjusted for Model for End-Stage Liver Disease score and age (hazard ratio = 2.05, 95%CI: 1.11-3.77, P = 0.021). In addition, Kaplan-Meier curves showed that patients with PAs ≤ 4.9° were significantly more likely to die. CONCLUSION In male patients with cirrhosis, the PA ≤ 4.9° cutoff was associated independently with mortality and identified patients with worse metabolic, nutritional, and disease progression profiles. The PA may be a useful and reliable bedside tool to evaluate prognosis in cirrhosis.

Tissue-specific methylation profile in obese patients with type 2 diabetes before and after Roux-en-Y gastric bypass

Eating habits, lifestyles, and exposure to specific environmental factors can greatly impact the risk of developing type 2 diabetes (T2D), influence the genome epigenetically, and affect the expression of genes, including genes related to glycemic control, at any stage of life. The epigenetic mechanism underlying obesity and T2D pathogenesis remains poorly understood. Conventional strategies for the treatment of obesity and its comorbidities often have poor long-term adherence, and pharmacological interventions are limited. Bariatric surgery is the most effective current option to treat severe obesity, and Roux-en-Y gastric bypass (RYGB) is the most applied technique worldwide. Epigenetic changes differ depending on the approach used to treat obesity and its associated comorbidities (clinical or surgical). Compared to primary clinical care, bariatric surgery leads to much greater loss of body weight and higher remission rates of T2D and metabolic syndrome, with methylation profiles in promoter regions of genes in obese individuals becoming similar to those of normal-weight individuals. Bariatric surgery can influence DNA methylation in parallel with changes in gene expression pattern. Changes in clinical biomarkers that reflect improvements in glucose and lipid metabolism after RYGB often occur before major weight loss and are coordinated by surgery-induced changes in intestinal hormones. Therefore, the intestine methylation profile would assist in understanding the mechanisms involved in improved glycemic control after bariatric surgery. The main objectives in this area for the future are to identify epigenetic marks that could be used as early indicators of metabolic risk, and to develop treatments able to delay or even reverse these epigenetic changes. Studies that provide the “human epigenetic profile” will be of considerable value to identify tissue-specific epigenetic signatures and their role in the development of chronic diseases. Further studies should apply methods based on global analysis of the genome to identify methylated sites associated with disease and epigenetic marks associated with the remodeling response to bariatric surgery. This review describes the main epigenetic alterations associated with obesity and T2D and the potential role of RYGB in remodeling these changes.

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.

Docosahexaenoic Acid Modulates a HER2-Associated Lipogenic Phenotype, Induces Apoptosis, and Increases Trastuzumab Action in HER2-Overexpressing Breast Carcinoma Cells.

In breast cancer, lipid metabolic alterations have been recognized as potential oncogenic stimuli that may promote malignancy. To investigate whether the oncogenic nature of lipogenesis closely depends on the overexpression of HER2 protooncogene, the normal breast cell line, HB4a, was transfected with HER2 cDNA to obtain HER2-overexpressing HB4aC5.2 cells. Both cell lines were treated with trastuzumab and docosahexaenoic acid. HER2 overexpression was accompanied by an increase in the expression of lipogenic genes involved in uptake (CD36), transport (FABP4), and storage (DGAT) of exogenous fatty acids (FA), as well as increased activation of “de novo” FA synthesis (FASN). We further investigate whether this lipogenesis reprogramming might be regulated by mTOR/PPARγ pathway. Inhibition of the mTORC1 pathway markers, p70S6 K1, SREBP1, and LIPIN1, as well as an increase in DEPTOR expression (the main inhibitor of the mTOR) was detected in HB4aC5.2. Based on these results, a PPARγ selective antagonist, GW9662, was used to treat both cells lines, and the lipogenic genes remained overexpressed in the HB4aC5.2 but not HB4a cells. DHA treatment inhibited all lipogenic genes (except for FABP4) in both cell lines yet only induced death in the HB4aC5.2 cells, mainly when associated with trastuzumab. Neither trastuzumab nor GW9662 alone was able to induce cell death. In conclusion, oncogenic transformation of breast cells by HER2 overexpression may require a reprogramming of lipogenic genetic that is independent of mTORC1 pathway and PPARγ activity. This reprogramming was inhibited by DHA.

Gene expression profile in response to doxorubicin-rapamycin combined treatment of HER-2-overexpressing human mammary epithelial cell lines

HER-2–positive breast cancers frequently sustain elevated AKT/mTOR signaling, which has been associated with resistance to doxorubicin treatment. Here, we investigated whether rapamycin, an mTOR inhibitor, increased the sensitivity to doxorubicin therapy in two HER-2–overexpressing cell lines: C5.2, which was derived from the parental HB4a by transfection with HER-2 and SKBR3, which exhibits HER-2 amplification. The epithelial mammary cell line HB4a was also analyzed. The combined treatment using 20 nmol/L of rapamycin and 30 nmol/L of doxorubicin arrested HB4a and C5.2 cells in S to G2–M, whereas SKBR3 cells showed an increase in the G0–G1 phase. Rapamycin increased the sensitivity to doxorubicin in HER-2–overexpressing cells by approximately 2-fold, suggesting that the combination displayed a more effective antiproliferative action. Gene expression profiling showed that these results might reflect alterations in genes involved in canonical pathways related to purine metabolism, oxidative phosphorylation, protein ubiquitination, and mitochondrial dysfunction. A set of 122 genes modulated by the combined treatment and specifically related to HER-2 overexpression was determined by finding genes commonly regulated in both C5.2 and SKBR3 that were not affected in HB4a cells. Network analysis of this particular set showed a smaller subgroup of genes in which coexpression pattern in HB4a cells was disrupted in C5.2 and SKBR3. Altogether, our data showed a subset of genes that might be more robust than individual markers in predicting the response of HER-2–overexpressing breast cancers to doxorubicin and rapamycin combination. Mol Cancer Ther; 11(2); 464–74. ©2011 AACR.

Abstract P4-21-29: HER2-associated lipogenic phenotype as a potential therapeutical target in breast cancer patients

Lipogenic phenotype (genetic program activation involved inde novo fatty acid –FA-synthesis and key metabolic regulators) is associated to oncogenic transformation and malignancy. We hypothesize that in HER2 overexpressing breast cancer cells, HER2 function is affected by cell membrane lipid profile, mainly lipid rafts, necessary to HER2 location and activation within the cell membrane. Therefore, metabolic lipid imbalance could be a potential factor and marker associated to HER2 expression. To evaluate if cell lipogenesis and membrane lipid composition are affected by HER2 overexpression, we used an oncogenic transformed cell line engineered to overexpress HER2 receptor (HB4aC5.2), but identical to their parental strain (HB4a) in all other aspects. Several lipid related molecules including synthesis-FASN, uptake-CD36, transport-FABP4 and FA storage-DGAT by RT-PCR; and lipogenic regulatory pathways (mTOR, DEPTOR, SREBP1 and PPARγ) were evaluated in both cells. Lipogenic contribution to lipid rafts formation was evaluated by gas chromatography and confocal microscopy. The influence of HER2 overexpression and lipogenic phenotype on proteins activated by HER2 (AKT, ERK1/2 and FASN) was analyzed by western blot. Next, HB4a and HB4aC5.2 cells were treated, alone or in combination with DHA (omega-3 polyunsaturated fatty acid), by Trastuzumab (anti-HER2), and GW9662 (anti-PPARγ) for 72h, and all above experiments were repeated. Cell death was analyzed by flow cytometry. In vitro results were confirmed in Primary tumor and adjacent tissue samples from a cohort of 182 European breast cancer patients through metabolomics analysis. Statistical analysis was performed by MetaboAnalyst software (p Citation Format: Ravacci GR, Santos JR, Brentani MM, Tortelli T, Dale I, Logullo AF, Waitzberg DL. HER2-associated lipogenic phenotype as a potential therapeutical target in breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-21-29.

Abstract P1-08-08: Reduction of HER2-associated lipogenic phenotype by docosahexaenoic acid (DHA) induces apoptosis in breast tumor cells harboring HER2 overexpression

The lipogenic phenotype is associated with oncogenic transformation and malignancy in the cancer setting. Our hypothesis is that the oncogenic nature of lipogenesis depends on the expression of HER2 oncogene, and its modulation by docosahexaenoic acid-DHA, might induce apoptosis in breast tumor cells with HER2 overexpression. To evaluate if the lipogenic phenotype might be induced by HER2 overexpression, we used a oncogenic transformation cell model in which, cells were engineered to overexpress HER-2 receptor (HB4aC5.2), but are identical to their parental strain (HB4a) in all other aspects, permitting an specific analysis of enhanced HER-2 effects. Toward this end the lipogenesis profiling was characterized, evaluating several molecular features, including synthesis (FASN), uptake(CD36), transport(FABP4) and storage(DGAT) of FA by RT-PCR and lipogenic regulatory pathways (mTOR, DEPTOR, SREBP1 and PPARγ) in both cells. Lipogenic contribution to lipid rafts formation, which is necessary to HER2 receptor location and activation in the cell membrane, was evaluated by gas chromatography and confocal microscopy. The influence of HER2 overexpression and lipogenic phenotype on proteins activated by HER-2 (AKT, ERK1/2 and FASN) was analyzed by western blot. Next, HB4a and HB4aC5.2 cells were treated, alone or in combination, with DHA, Trastuzumab (anti-HER2), and GW9662 (PPARγ inhibitor) for 72h, and the above experiments were repeated. Cell death was analyzed by flow cytometry and confocal microscopy. Results: In HB4aC5.2 cells, the oncogenic transformation by HER2 overexpression was associated with a lipogenic phenotype, which contributed to increase of lipid rafts formation, activation of survival and proliferation signals, as compared to HB4a normal cells (p Citation Format: Graziela R Ravacci, Maria M Brentani, William Festuccia, Tharcisio Tortelli, Angela F Waitzberg, Dan L Waitzberg. Reduction of HER2-associated lipogenic phenotype by docosahexaenoic acid (DHA) induces apoptosis in breast tumor cells harboring HER2 overexpression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-08-08.

Abstract 5424: Docosahexaenoic acid (DHA) induces gene expression of RASSF1A through epigenetics mechanisms in breast cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Epigenetic changes, such as DNA methylation and post-translational histones modifications have an important role in mammary tumorigenesis. Changes in DNA methylation include global hypomethylation and hypermethylation of promoter regions of specific genes. Several genes are hypermethylated in breast cancer, especially the tumor suppressor gene RASSF1A, that have potential prognostic value. Epigenetic events are important as therapeutic targets due to its reversible feature. Dietary compounds can modulate the epigenetic processes in cancer. Experimentally, docosahexaenoic acid (DHA), a member of the omega-3 fatty acids, can reduce proliferation, induce apoptosis and decrease the invasive potential of breast tumor cells. However, the antitumor mechanisms of DHA and its ability to modulate epigenetic events are not yet fully elucidated. Objectives: To verify DHA action on RASSF1A expression and promoter region methylation in different human breast cancer cell lines. Material and Methods: Three different breast cell lines (MCF-7, MDA-MB-231, SKBR-3) were treated or not with 100 μM of DHA for 72 hours. Real time quantitative PCR were performed for gene expression quantification of RASSF1A. DNA methylation of the promoter region of RASSF1A was evaluated by methylation specific PCR. The western blot will be held for analyze of the histone 3 lysine 9 acetylated (H3K9ac) protein. Moreover, we will evaluate the phases of the cell cycle and apoptosis by flow cytometry. Results and Conclusion: Compared to control cells (not treated), DHA increased RASSF1A expression on MCF-7, but not in MDA-MB and in SKBR-3. These three transformed breast cells lines show methylation in specific region of RASSF1A promoter, indicating its role as a tumour supressing gene. DHA treatment increased RASSF1A promoter region hypomethylation in MCF-7, but not in MDA-MB and in SKBR-3. From this perspective, we can suggest that DHA could act by epigenetic mechanisms and reactivate RASSF1A, previously silenced. This action occured specifically in MCF-7, a less aggressive breast tumor cell line. It is expected that the present results promote better understanding of DHA role as a potential anti-oncogenic compound of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5424. doi:1538-7445.AM2012-5424