OVARIAN CANCER AND HYPERGLYCEMIA: CAN METFORMIN BE USED TO HALT TUMOR GROWTH AND PROLIFERATION?

L A C E Y G I B S O N

S O P H O M O R E U N D E R G R A D U A T E P R E S I D E N T I A L S C H O L A R

M E N T O R : D R . B U C K H A L E S

D E P A R T M E N T O F P H Y S I O L O G Y

S O U T H E R N I L L I N O I S U N I V E R S I T Y C A R B O N D A L E

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.

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.

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.

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.)

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.

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

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.

XMetformin

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)

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

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.

RESULTS: METFORMIN DOSE-RESPONSE STUDY

• Observations:• Cells Treated with 25 mmol/L Metformin

appeared smaller and fewer in number after second dose, compared to other treatments

• T-25 Cell Counter detected zero control cells after third dose, but cells in flask appeared to be growing under microscope the next day; Human error in measurement?

• After third treatment of Metformin, there DID appear to be more cells in group treated with 10 mmol/L Metformin than control.

1 2 30

0.5

1

1.5

2

2.5

3

3.5

Total Cell Count

Control10 mmol/L Metformin25 mmol/L Metformin

Dose Number

Tota

l C

ells (

10^

5 c

ell/m

L)

1 2 30

102030405060708090

100

Live Cell Count

Control10 mmol/L Metformin25 mmol/L Metformin

Dose Number

Liv

e C

ells (

%)

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.

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.

Control Metformin 10 mmol/L

Metformin 25 mmol/L

00.20.40.60.8

Lactic Acid Produc-tion, Control vs. Met-

formin-Treated SKOV3 Cells

TreatmentAvera

ge L

-Lacta

te (

mM

)

RESULTS: LACTIC ACID ASSAY RESULTS

• Cotrol vs. 10 mmol/L Metformin treatment is significant, p<.001

• Lactic acid production increases with Metformin treatment; 25 mmol/L Metformin was too high of a dose for cell survival, causing decrease in lactic acid production.

***

ControlMetformin 10 mmol/L

Metformin 25 mmol/L

Avg L-Lactate (mM) 0.252 0.61 0.437SEM 0.02 0.06 0.04

n 16 16 10

Results

RESULTS: LACTIC ACID ASSAY RESULTS

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.

Control SKOV3

Metformin SKOV3

Control IOSE Metformin IOSE

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Lactic Acid Production in SKOV3 and IOSE Cell Lines

Cell Type

L-lacta

te (

mM

)

**

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.

CONCLUSION: EXPLANATION OF METFORMIN’S EFFECT ON LACTIC ACID PRODUCTION

(O2 present, returns to) Pyruvate (Gluconeogenisis)

Glucose

Lactic acid production from fermentation in addition to aerobic respiration in cancer cellsLactic Acid Buildup

(Metformin treatment activates AMPK, decreasing gluconeogenisis)

& 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

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…

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.

ACKNOWLEDGEMENTSI 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.