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Here is my slide presentation, describing my research of the link between ovarian cancer and diabetes!

Here is my slide presentation, describing my research of the link between ovarian cancer and diabetes!

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Metformin presentation sigma xi Metformin presentation sigma xi Presentation Transcript

  • Ovarian Cancer and Diabetes: Metformin’s Metabolic Effects LACEY GIBSON, SOUTHERN ILLINOIS UNIVERSITY, CLASS OF 2015 DR. BUCK HALES, DEPARTMENT CHAIR OF PHYSIOLOGY, SOUTHERN ILLINOIS UNIVERSITY SCHOOL OF MEDICINE
  • Introduction: Objectives Objectives Our study was undertaken to:  compare the ability of Metformin and flaxseed in reducing hyperglycemic, pro-inflammatory environment  and to set the stage for a long term study of their direct comparison in reducing ovarian cancer incidence and severity, using a hen model.
  • Introduction: How are Ovarian Cancer & Diabetes Related? Ovarian Cancer & Diabetes  Ovarian cancer & type II diabetes are devastating diseases that share a variety of risk factors; individuals diagnosed with type II diabetes are more likely than by chance to be diagnosed with cancer (Giovannuci et al 2010)
  • Introduction: How are Ovarian Cancer & Diabetes Related?  Warburg Effect describes ability of fast-growing cancer cells to metabolize glucose via glycolysis in addition to oxidative phosphorylation; More glucose needed for proliferation (Ladley 2012).  Diabetes provides a nutrient-rich metabolic environment for ovarian cancer cells, where tumor formation and growth is encouraged by free radical-induced DNA damage. (Kellenberger et al 2010).
  • Introduction: What are Metformin and Flaxseed? Flaxseed and Metformin as Mediators of Proliferative Metabolic Environment  Flaxseed and Metformin both may reduce incidence and severity of cancer by reducing hyperglycemic, pro-inflammatory environment that feeds cancer proliferation.
  • Introduction: What are Metformin and Flaxseed? Metformin: Anti-diabetes drug Metformin (1carbamimidamido-N,N-dimethylmethanimidamide, C4H11N5 ) activates fuel sensing enzyme AMPactivated protein kinase.  -> alters expression/location of enzymes/transporters associated with glucose metabolism: glucose-6-phosphatase (G6PC2), glucose family transporter-2 (GLUT-2), histone deacetylase 7 (HDAC7), phosphoenolpyruvate carboxykinase (PCK1), and pyruvate kinase (PKM2) and…  -> glucose homeostasis is improved (Fulgencio et al 2001; Ait-Omar et al 2011; Hardie 2013; Yuan et al 2002).
  • Introduction: What are Metformin and Flaxseed? Flaxseed: Dietary supplement flaxseed rich in ALA, which is converted to EPA and DHA  ->decreases expression of inflammatory markers COX-1 and COX-2  -> reduction in inflammatory markers correlates to reduction in severity of ovarian cancer in hens (Eilati et al 2013). ALA EPA DHA COX
  • Introduction: What Endpoints Can We Use to Compare the Metabolic Effects of Metformin & Flaxseed? Diabetes, Ovarian Cancer, and Metabolic Syndrome  Metabolic Syndrome: Clustering of risk factors for type II diabetes, cardiovascular disease, hypertension, lipid problems, obesity, NAFLD, cancers, and ovarian distress.  Metabolic distress indicated by the following measurements: low albumin; high aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and glutamate dehydrogenase (GLDH), triglycerides, cholesterol, blood glucose.
  • Methods Metformin animal study: 3 week dose finding: Animal management and procedures were reviewed and approved by IACUC at Southern Illinois University Carbondale. 28 hens of 1.5 years were obtained and housed at SIUC vivarium in Life Science II. 7 hens per group were treated daily with either capsules containing 10, 30, or 100 mg/kg of Metformin or a control rice bran pill. On day 21 of treatment, hens were sacrificed. Biochemical serum analysis: 12 hens were bled on day zero, and all hens were bled on day 21 of the study. Blood was spun at 20º C for 10 minutes at 1200 rpm. Serum was collected and shipped on dry ice to University of Illinois Veterinarian Diagnostic Laboratory for analysis of cholesterol, triglycerides, glucose, AST, albumin, GGT, and GLDH. Statistical analysis of data was performed using ANOVA using standard parametric methods to identify significant differences (p<.05). A principal components analysis (PCA) test was then performed to compare variables.
  • Methods Analysis of gene expression: Liver and ovary tissue were collected from all hens during necropsy and stored at -80º C. RNA was isolated from the tissue samples followed by cDNA synthesis. PCR primers were identified by literature review and obtained through a commercial vendor (Eurofins). Fold differences in gene expression were validated through semiquantitative RT-PCR using the BioRad CFX96 PCR system. mRNA levels were normalized to GAPDH and relative expression was calculated using the comparative Ct method. Statistical analysis of data was performed using ANOVA using standard parametric methods to identify significant differences (p<.05). Comparison of data to previous flaxseed findings: Results were compared to biochemical analysis and gene expression of hens fed control or flaxseed diets during a series of long term studies
  • Results: General Health: Metformin General health: Hens treated with 30 mg/kg Metformin lost weight during the trial (Figure 1). Hens of all treatments maintained egg-laying ability, and hens treated with 30 mg/kg Metformin were the most fertile on day 21 compared to hens of other treatments (Figure 2).
  • Results: Serum Analysis: Metformin Biochemical serum analysis: Significant increases in serum glucose and GGT were seen in post-treated hens compared to pre-treated hens (Figures 3, 4). No significant changes in cholesterol, triglycerides, albumin, or GLDH were seen (Table 1). 250 GGT (U/L) Glucose (mg/dL) 200 150 100 50 0 Baseline (n=14) Metformin (n=14) Treatment 18 16 14 12 10 8 6 4 2 0 Baseline (n=14) Metformin (n=14) Treatment Figure 3(left) Mean values for glucose in serum of hens with and without Metformin treatment. Mean values connected with an asterisk are significantly different (P<0.05, ANOVA). Figure 4(right) Mean values for GGT in serum of hens with and without Metformin treatment. Mean values connected with an asterisk are significantly different (P<0.05, ANOVA).
  • Results: Serum Analysis: Metformin Variability in glucose and GGT caused by living conditions, not by Metformin treatment; Variability in cholesterol, triglycerides, and albumin unrelated to treatment or conditions (Figure 5). Figure 5 PCA analysis of biochemical serum variables in hens of all treatments on day 0 (blue points) and day 21 (yellow points) of treatment. Increasing glucose, GGT Increasing Cholesterol, Triglycerides, Albumin
  • Results: Serum Analysis: Flaxseed 300 A 250 Avg AST (U/L) In long-term flaxseed studies, cholesterol, triglycerides, and AST were decreased in hens fed a flaxseed-based diet compared to hens fed a control diet (Figures 6, 7, 8). No significant changes in GGT, albumin, or GLDH were seen (Table 1) 200 150 B B B C 100 50 C C C 0 Control Flax D Contol Flax E Control Flax F Contol Flax G D (n=16) E (n=11) F (n=16) G (n=17) (n=16) (n=12) (n=15) (n=17) Figure 6 Mean AST levels of serum from hens fed flaxseed or control diets of age groups D (youngest, 2.5 years) through G (oldest, 4 years) in a long term study. Mean values sharing the same letter are not significantly different (P<0.05, Tukey’s test).
  • 4000 3500 3000 AB 2500 2000 1500 1000 500 0 250 A AB AB BC BC C C Average Cholesterol (mg/dL) Average Triglyceride Level (mg/dL) Results: Serum Analysis: Flaxseed A AB 200 AB AB 150 B AB AB AB 100 50 0 Group Control Flax D Contol Flax E Control Flax F Contol Flax G D (n=16) E (n=11) F (n=16) G (n=17) (n=16) (n=12) (n=15) (n=17) Group Figure 7 (left) Mean triglyceride levels of serum from hens fed flaxseed or control diets of age groups D (youngest, 2.5 years) through G (oldest, 4 years). Mean values sharing the same letter are not significantly different (P<0.05, Tukey’s test). Figure 8 (right) Mean cholesterol levels of serum from hens fed flaxseed or control diets of age groups D (youngest, 2.5 years) through G (oldest, 4 years). Mean values sharing the same letter are not significantly different (P<0.05, Tukey’s test).
  • Results: Serum Analysis: Summary Flax Metformin Glucose ? -* Cholesterol +* NC Triglycerides +* NC Albumin NC NC AST +* NC GGT NC -* GLDH NC NC Table 2 Summary of the effects of a long-term flaxseed diet or short-term Metformin treatment in hens on biochemical metabolic markers in serum analysis. (- = negative effect, + = positive effect, NC = no change; * = statistical significance: p<.05)
  • Results: Gene Expression: Metformin Gene expression: Liver: Expression of GLUT-2 was significantly increased in hens treated with 30 mg/kg Metformin. No significant changes in expression of COX-2, G6PC2, HDAC7, PCK1, or PKM2 were seen (Figure 9). Figure 9 (left) Relative normalized expression of metaboolic genes in liver tissue of control or Metfromintreated hens. Mean values connected with an asterisk are significantly different (P<0.05, Tukey’s test).
  • Results: Gene Expression: Metformin Gene expression: Ovary: No significant changes in expression of COX-2, HDAC7, or PKM2 were seen in ovary tissue (Figure 10). Figure 10 (left) Relative normalized expression of metaboolic genes in ovary tissue of control or Metfromin-treated hens. Mean values connected with an asterisk are significantly different (P<0.05, Tukey’s test).
  • Results: Gene Expression: Flaxseed Gene expression: Liver: In long-term flaxseed studies, expression of PCK1 was significantly decreased in hens treated with 15 g/kg flaxseed. No changes in expression of COX-2, G6PC2, GLUT-2, HDAC7, or PKM2 were seen (Figure 11, Table 3). Figure 11 (left) Relative normalized expression of metaboolic genes in liver tissue of control or flaxseed-fed hens. Mean values connected with an asterisk are significantly different (P<0.05, Tukey’s test).
  • Results: Gene Expression: Summary Flax Metformin COX-2 NC NC G6PC2 NC NC GLUT-2 NC +* PCK1 +* NC PKM2 NC NC Table 3 Summary of the effects of a long-term flaxseed diet or short-term Metformin treatment in hens on expression of metabolic genes. (- = negative effect, + = positive effect, NC = no change; * = statistical significance: p<.05)
  • Conclusions: Metformin  Flax and Metformin differentially effect biochemical and genetic markers of metabolic syndrome, liver disease, and insulin resistance.  Our short-term Metformin study has shown that Meformin is related to:  up-regulation in GLUT-2. Previous studies have found that Metformin may alter the location and functioning of this transporter (Ait-Omar et al 2011);  Increase in GGT and glucose due to conditions rather than treatment. 3 weeks was not long enough for Metformin to change biochemical serum markers.
  • Conclusion: Metformin  Literature has shown that a single oral dose of 300 mg/kg Metformin can reduce blood glucose and feed intake (Ashwell and McMurty 2003).  However, consistent with our findings, another study has shown that daily diets of Metformin of 250-10,000 mg/kg reduce feed intake and increase lactate in highest doses without reducing plasma glucose (Rosebrough and Ashwell 2005).  Thus, in the hen, Metformin may reduce symptoms of Metabolic Syndrome, thereby reducing cancer risk through an undefined form of feedback inhibition that increases satiety without altering long-term plasma glucose levels
  • Conclusions: Flaxseed  Our long-term flaxseed studies have shown that flax is related to: reduction in cholesterol, triglycerides, AST.  decreased expression of PCK1, consistent with previous studies that have found SDG from flaxseed to reduce expression of this gene (Prasad 2002).  .
  • Conclusions: Future Directions  Despite lack of change in blood glucose levels, our short term study has shown that most positive metabolic health effects (up-regulation of GLUT-2, decrease in body weight, egg laying ability) occur in hens treated with 30 mg/kg. This is therefore our optimal dose for future studies for which this study has laid a foundation.  Future long-term studies must be performed to directly compare the benefits of flaxseed and Metformin in reducing incidence and severity of ovarian cancer as well as its associated pro-inflammatory, hyperglycemic environment.
  • Acknowledgements  I would like to graciously thank the following:      All members of my lab for aiding in data collection and interpretation The REACH program at SIUC for financial support My family for moral support The selected sources below for providing background and discussion information And YOU for your time and attention! Thanks!! Ait-Omar, A., M. Monteiro-Sepulveda, C. Poitou, M. Le Gall, A. Cotillard, J. Gilet, K. Garbin, A. Houllier, D. Chateau, A. Lacombe, N. Veyrie, D. Hugol, J. Tordjman, C. Magnan, P. Serradas, K. Clement, A. Leturque, and E. BrotLaroche. "GLUT2 Accumulation in Enterocyte Apical and Intracellular Membranes: A Study in Morbidly Obese Human Subjects and Ob/ob and High Fat-Fed Mice." Diabetes 60.10 (2011): 2598-607. Print. Ashwell, CM, and JP McMurty. "Hypoglycemia and Reduced Feed Intake in Broiler Chickens Treated with Metformin." Poultry Science 82.1 (2003): 106-10. Print. Eilati, Erfan, Karen Hales, Yan Zhuge, Kristine Ansenberger, Rui Yu, Richard Breeman, and Dale Hales. "Flaxseed Enriched Diet-mediated Reduction in Ovarian Cancer Severity Is Correlated to the Reduction of Prostaglandin E2 in Laying Hen Ovaries."Prostaglandins Leukotrienes and Essential Fatty Acids (2013) Print. Fulgencio, Jean-Pierre, Claude Kohl, Jean Girard, and Jean-Paul Pégorier. "Effect of Metformin on Fatty Acid and Glucose Metabolism in Freshly Isolated Hepatocytes and on Specific Gene Expression in Cultured Hepatocytes." Biochem Pharmacol15.64 (2001): 439-46. Print. Giovannuci, E., Harlan, D. M., Archer, M. C., Bergenstal, R. M, Gapstur, R. M., Habel, L. A., Pollack, M., Regensteiner, J. G., and Yee, Douglas. “Diabetes and Cancer: A Consensus Report.” Diabetes Care, 2010, vol. 33, pp. 16741685. Gonzalez-Periz, A., R. Horrillo, N. Ferre, K. Gronert, B. Dong, E. Moran-Salvador, E. Titos, M. Martinez-Clemente, M. Lopez-Parra, V. Arroyo, and J. Claria. "Obesity-induced Insulin Resistance and Hepatic Steatosis Are Alleviated by -3 Fatty Acids: A Role for Resolvins and Protectins." The FASEB Journal 23.6 (2009): 1946-957. Print. Hardie, D. G. "AMPK: A Target for Drugs and Natural Products with Effects on Both Diabetes and Cancer." Diabetes 62.7 (2013): 2164-172. Print. Kellenberger, L. D., J. E. Bruin, J. Greenaway, N. E. Campbell, R. A. Moorehead, A. C. Holloway, and J. Petrik. "The Role of Dysregulated Glucose Metabolism in Epithelial Ovarian Cancer." Journal of Oncology 2010 (2010): 1-13. Print. Ladley, S. E., The Role of Metabolic Reorigination and Mitochondria in EOC. Diss. Southern Illinois University Carbondale, 2012. Carbondale, 2012. Print. Prasad, Kailash. "Suppression of Phosphoenolpyruvate Carboxykinase Gene Expression by Secoisolariciresinol Diglucoside (SDG), a New Antidiabetic Agent." International Journal of Angiology 11.2 (2002): 107-09. Print. Rosebrough, R., and C. Ashwell. "Dietary Metformin Effects on in Vitro and in Vivo Metabolism in the Chicken." Nutrition Research 25.5 (2005): 491-97. Print. Yuan, L., Zeigler Yuan, and Hamann A. "Inhibition of Phosphoenolpyruvate Carboxykinase Gene Expression by Metformin in Cultured Hepatocytes." Chinese Medical Journal 115.12 (2002): 1843-848. Print.