This presentation features research on the link between ovarian cancer and hyperglycemia, conducted by Lacey Gibson, a sophomore at Southern Illinois University Carbondale.

1. OVARIAN CANCER AND
HYPERGLYCEMIA: CAN
METFORMIN BE USED TO HALT
TUMOR GROWTH AND
PROLIFERATION?
LACEY GIBSON
S O PHO M O R E U N D E RG R A D UAT E PR E S ID E N T IA L S C HO LA R
MENTOR: DR. BUCK HALES
DEPARTMENT OF PHYSIOLOGY
SOUTHERN ILLINOIS UNIVERSITY CARBONDALE

2. RESEARCH OBJECTIVES
• Study was undertaken to answer the following question:
“Can Metformin be used to halt growth and proliferation of ovarian cancer?”
• Hypothesis:
• Treatment of cancerous ovary cells lines will lead to an increased buildup of lactic
acid, representing an inability for these cells to produce glucose via
gluconeogenesis, which in turn causes decreased energy reservoir for tumor
proliferation.
• Metformin halts tumor growth and proliferation, as evident by its ability to block
gluconeogenesis.

3. BACKGROUND INFORMATION: WHAT IS
OVARIAN CANCER?
• Ovarian cancer currently has the leading rate of mortality compared with all other
gynecological cancers.
• It is the fifth leading cause of death in women (Ahn et al).
• Largely lacking in methods of early detection and preventative treatment.

4. BACKGROUND INFORMATION: WHAT IS
HYPERGLYCEMIA?
Hyperglycemia = Type II Diabetes = Insulin Resistance
Insulin Resistance:
 Insulin released to convert blood glucose to glycogen for
long-term storage in muscle and liver cells
 High sugar diet causes high storage of glycogen in cells
 Pancreas pumps excess insulin, but insulin receptors do not
respond, causing glucose to remain in blood.

5. BACKGROUND INFORMATION: INSULIN
RESISTANCE AND OVARIAN CANCER
• Excess insulin pumped by pancreas shown by in vitro studies to cause cell
proliferation and prevent apoptosis (Thune et al)
• Excess insulin also affects synthesis of certain hormones, such as estrogen,
which play a role in cell differentiation and proliferation(Thune et al)
• Recent studies have suggested a role for hyperglycemia in the development of a
number of cancers, including endometrial, liver, and pancreatic cancer (Swerdlow
et al)
• Ovarian cancer and hyperglycemia share a variety of risk factors and individuals
diagnosed with hyperglycemia are more likely than by chance to be diagnosed
with cancer (Giovannuci et al.)

6. BACKGROUND INFORMATION: HYPERGLYCEMIC
ENVIRONMENT & OVARIAN CANCER
• Warburg Effect describes ability of fast-growing cancer cells to metabolize glucose
via anaerobic glycolysis in addition to oxidative phosphorylation; More glucose
needed for proliferation (Ladley).
• Hyperglycemia provides a nutrient-rich, growth signal-rich environment for
epithelial ovarian cancer cells, where tumor formation and growth is encouraged
by free radical-induced DNA damage. (Kellenberger et al)
• If hyperglycemia contributes to tumor growth and progression, then anti-diabetes
drugs, such as Metformin, may also have an important antitumor role.

7. BACKGROUND INFORMATION: WHAT IS
METFORMIN?
• 1-carbamimidamido-N,N-dimethylmethanimidamide, C4H11N5
• World’s most popular anti-diabetes drug
• Activates AMP-activated protein kinase in cancer cells, which may play a role in
inhibiting cellular growth by:
• Lowering sugar output from via inhibition of gluconeogenesis
• Ultimately lowering blood sugar
• Starving the cells of their abundant glucose supply that previously allowed them to
proliferate
• Has shown anti-proliferative effects on cancerous tissue from a variety of cell
lines

8. BACKGROUND INFORMATION: WHAT IS LACTIC
ACIDOSIS?
• Lactic acid produced as a bi-product of fermentation, which occurs in cancerous
cells
• Lactic acidosis = overproduction or underutilization (via glucogenesis inhibition)
of lactic acid.
• Biguanide drugs such as Metformin linked to lactic acidosis:
• Decreases gluconeogenesis to lower blood glucose
2 lactate (C3H6O3) + energy (from 16 ATP) ---> glucose (C6H12O6)
• Lactate uptake decreased-> less glucose produced; Cancer cells starved.
Metformin
X

9. BACKGROUND INFORMATION: WHAT IS LACTIC
ACIDOSIS?
• Although rare, when caused by Metformin, lactic acidosis has mortality rate of
50% (Price)
• BUT: 9 cases lactic acidosis/10,000 Metformin users (no difference compared to
placebo); Risk factors include: “age of >60 yr; decreased cardiac, hepatic, or
renal function; diabetic ketoacidosis; surgery; respiratory failure; ethanol
intoxication; and fasting” (Luft)

10. BACKGROUND INFORMATION: GLUCONEOGENESIS: TESTING
THE METFORMIN’S ANTI PROLIFERATIVE MECHANISM
Gluconeogenesis = generation of glucose from noncarbonhydrate carbon substrates
(i.e. lactic acid)
Gluconeogenesis could occur as an additional glucose source for “greedy” ovarian
cancer cells
• Cancer cells undergo fermentation in addition to aerobic respiration (Warburg
Effect)… why not gluconeogenesis too?
How can we test this?
Certain drugs that inhibit cancer cell proliferation should also inhibit gluconeogenesis:
 If Metformin inhibits gluconeogenesis, a buildup in lactic acid will occur in
Metformin-treated cell lines.
Lactic Acid

11. METHODS: METFORMIN DOSE-RESPONSE
STUDY
Dose-finding study was performed to determine optimal dose of Metformin for cells
during lactic acid assays:
 SKOV3 (cancerous human epithelial ovary) cell lines were treated with media
containing various doses (0-25 mmol/L) of Metformin.
 Cell count was measured after 24 hours incubation at 37°C in each treatment.

12. RESULTS: METFORMIN DOSE-RESPONSE STUDY
Total Cell Count
3.5
Total Cells (10^5 cell/mL)
3
• Observations: 2.5
• Cells Treated with 25 mmol/L Metformin 2
Control
appeared smaller and fewer in number after 1.5
10 mmol/L Metformin
second dose, compared to other treatments 1
25 mmol/L Metformin
0.5
• T-25 Cell Counter detected zero control cells
0
after third dose, but cells in flask appeared 1 2 3
to be growing under microscope the next Dose Number
day; Human error in measurement?
• After third treatment of Metformin, there DID
appear to be more cells in group treated
Live Cell Count
100
with 10 mmol/L Metformin than control. 90
80
70
Live Cells (%)
60
50 Control
40
30 10 mmol/L Metformin
20 25 mmol/L Metformin
10
0
1 2 3
Dose Number

13. CONCLUSION: METFORMIN DOSE-RESPONSE
STUDY
• Conclusions:
• Treatment of SKOV3 cells with 10 mmol/L Metformin does not decrease cell
viability
• 10 mmol/L is optimal treatment for cells in Lactic Acid Assays; 25 mmol/L is too
high.

14. METHODS: LACTIC ACID ASSAYS
Lactic Acid levels were tested in Control and Metformin-treated cancerous (SKOV3)
and noncancerous (IOSE) epithelial ovarian cell lines
 3 Day Procecedure:
 Day 1: Cell lines were passaged and transferred to 96-well plate in normal
media; 24 hours of incubation at 37 C followed.
 Day 2: Media was replaced with treatment media (Control or 10 mmol/L
Metformin Hank’s Balanced Salt Solution); 24 hours of incubation at 37 C
followed.
 Day 3: Lactic acid production was measured with plate reader.

15. RESULTS: LACTIC ACID ASSAY RESULTS
Results
Control
Metformin Metformin
10 mmol/L 25 mmol/L
Lactic Acid
Avg L- Production, Control vs.
Lactate Metformin-Treated SKOV3
(mM) 0.252 0.61 0.437
SEM 0.02 0.06 0.04
0.8 Cells
Average L-Lactate (mM)
0.6
n 16 16 10 0.4
0.2
***
• Cotrol vs. 10 mmol/L Metformin treatment is 0
significant, p<.001 Control Metformin 10 Metformin 25
mmol/L mmol/L
• Lactic acid production increases with Metformin treatment; 25 Treatment
mmol/L Metformin was too high of a dose for cell
survival, causing decrease in lactic acid production.

16. RESULTS: LACTIC ACID ASSAY RESULTS
Lactic Acid Production in SKOV3 and
IOSE Cell Lines
0.8
0.7
0.6
L-lactate (mM)
0.5
0.4
* *
0.3
0.2
0.1
0
Control SKOV3 Metformin Control IOSE Metformin IOSE
SKOV3
Cell Type
Lactic acid increases with Metformin treatment; Results are significant in cancerous
(SKOV3) cells.
Increase in lactic acid levels also significant in cancerous Metformin-treated cells
compared to noncancerous control-treated cells.

17. CONCLUSION: LACTIC ACID ASSAYS
• Lactic acid levels increased significantly in Metformin-treated cancerous epithelial
ovary cells compared to cancerous cells in control media.
• Increase in lactic acid levels was not significant in noncancerous Metformin-
treated cells compared to noncancerous cells in control media.
• This indicates an inhibition of gluconeogenesis in Metformin-treated cancerous
cells.

18. CONCLUSION: EXPLANATION OF METFORMIN’S EFFECT
ON LACTIC ACID PRODUCTION
(O2 present, returns to) Pyruvate (Gluconeogenisis)
Lactic acid production (Metformin treatment
from fermentation in activates
AMPK, decreasing
addition to aerobic gluconeogenisis)
respiration in cancer
cells Glucose
Lactic Acid Buildup
&
Decreased Glucose Production
Decreased reservoir of glucose for cancer cell proliferation
Increased lactic acid production in Metformin-treated
cells indicates an inhibition of gluconeogenesis, which
indicates an decrease in proliferation of ovarian cancer
cells

19. CONCLUSION: WHERE TO NEXT?
Metformin has the ability to decrease energy for proliferation of ovarian cancer cells
via inhibition of gluconeogenesis. This inhibition of gluconeogenesis is shown by a
decrease in lactic acid levels.
Metformin has potential to be used as a drug to treat patients with ovarian cancer
AND patients with hyperglycemia. Wonder drug!
BUT more testing is needed to confirm cancer-treating abilities…

20. CONCLUSION: FUTURE STUDIES
Future studies:
• Test other intermediaries and enzymes related to gluconeogenesis
• Pyruvate, pyruvate kinase activity, glucose
• In vivo testing of Metformin’s ability to suppress ovarian cancer growth and
proliferation
• Study inflammatory genetic markers (i.e. COX-1 and prostaglandin) in tissue
collected from Metformin-treated & control hens.

21. ACKNOWLEDGEMENTS
I am especially grateful for the knowledge that I have gained by working in Dr. Hales’ lab at
Southern Illinois University of Carbondale. I am thankful for the being allowed to learn in the
presence of all the supportive individuals in his facility. I also truly appreciate the support of
the SIUC’s Saluki Scholar Research Opportunity program, the advice of my father, David
Gibson, and the wisdom of the following authors:
Ahn, Suzie E., Jin Choi, Deivendran Rengaraj, Hee Seo, Whasun Lim, Jae Han, and Gwonhwa Song. "Increased Expression of Cysteine Cathepsins in
Ovarian Tissue from Chickens with Ovarian Cancer." Reproductive Biology and Endocrinology8.1 (2010): 100. 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. 1674-1685.
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, Sara E. The Role of Metabolic Reorigination and Mitochondria in EOC. Diss. Southern Illinois University Carbondale, 2012. Carbondale, 2012.
Print.
Luft, F. C. "Lactic Acidosis Update for Critical Care Clinicians." Journal of American Society of Nephrology 12.17 (2001): n. pag. Print.
Swerdlow, A. J., S. P. Laing, Z. Qiao, S. D. Slater, A. C. Burden, J. L. Botha, N. R. Waugh, A. D. Morris, W. Gatling, E. A. Gale, C. C. Patterson, and H. Keen.
"Cancer Incidence and Mortality in Patients with Insulin-treated Diabetes: A UK Cohort Study." British Journal of Cancer 92.11 (2005): 2070-075.
Print.
Thune, I. "Sustained Physical Activity, Energy Balance and Risk of Breast Cancer." European Journal of Cancer Prevention 7.Supplement 1 (1998): S67-
68. Print.