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

1. 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

2. 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.

3. 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)

4. 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).

5. 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.

6. 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).

7. 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

8. 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.

9. 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.

10. 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

11. 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).

12. 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).

13. 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

14. 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).

15. 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).

16. 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)

17. 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).

18. 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).

19. 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).

20. 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)

21. 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.

22. 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

23. 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).

.

24. 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.

25. 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
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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.
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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.