THE ROLE OF METABOLIC REORGANIZATION AND MITOCHONDRIA IN EOC

Author

Sara Ladley, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2012

Degree Name

Master of Science

Department

Molecular Cellular and Systemic Physiology

First Advisor

Hales, Dale

Abstract

Ovarian cancer is the most lethal of gynecological malignancies and is referred to as a `silent killer.' Usually, there are concealed symptoms in the earlier stages of ovarian cancer but it becomes detectable at stages III and IV; therefore, it is crucial to find early detections markers to improve therapeutic intervention. The hypothesis of this study was to determine if a metabolic alteration occurs while ovarian cells are transitioning from an epithelial to mesenchymal phenotype; thus, these findings will give us insight if a dysregulated glucose metabolism governs ovarian cancer and if a Warburg effect is contributes to the metabolic switch. The Warburg Effect was hypothesized in the 1930s and postulates that when normal cells transform into cancerous cells, there will be a change in metabolic preference from oxidative phosphorylation (OXPHOS) to aerobic glycolysis. Normal differentiated cells favor OXHPOS for ATP production. In contrast, tumor cells prefer ATP energy through the glycolysis pathway. The most intriguing variable of the Warburg effect is that even when oxygen is abundant, tumor cells still favor glycolysis (aerobic glycolysis). An important difference between OXPHOS and aerobic glycolysis is the rate of glucose consumption and ATP production. The Warburg effect is operative in lung and gastric cancers and leukemia, which all exhibit a dysregulated glucose metabolism due to a mitochondrial defect. In order to test these predictions, the cell's morphology, lactic acid secretion, ATP production and mitochondrial membrane potential were investigated. The human ovarian cancer cells lines used during this study: IOSE, TOV112D, SkOv3, and HEYC2. In order to determine if cell had an epithelial or mesenchymal histotype, immunocytochemistry (ICC) was done to determine E-cadherin expression. Then, E-cadherin and SNAI2 expression was examined via q-PCR to support the ICC results. E-cadherin is unique in EOC since it is not normally expressed in noncancerous phenotype; however, there is an upregulation of the gene during cancer transformation. SNAI2 is an epithelial to mesenchymal transition (EMT) marker and a transcriptional inhibitor of the E-box and will downregualate E-cadherin for EMT activation. High E-cadherin and low SNAI2 expressions represent an epithelial phenotype. However, a high expression of SNAI2 and low E-cadherin expression, represent a mesenchymal phenotype. Thus, these two genes have an inverse relationship during EMT. These results would establish a study model for understanding ovarian cancer development along an EMT and give insight about the type of metabolites that control these phenotypes during EMT. Identifying morphology phenotypes gave us direction in understanding the prospective metabolites that support the cell's viability. Cancer cells have an accelerated uptake of glucose for there increase demands to proliferate; therefore, there will be a proportional increase of lactic acid secretion. Thus, lactic acid secretion was a way to measure the aggressiveness of the cell; thus, high lactic acid production is proportional to aggressiveness. Next, ATP production was inspected because ATP levels should be related to lactic acid section and aggressiveness of the cell. Aerobic glycolysis generates ATP a hundred times faster than OXPHOS. To determine if an ATP synthesis is via OXPHOS, there cells were treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to selectively inhibit OXPHOS. In contrast, 2-deoxy-D-glucose (2DG) is a glucose isomer that inhibits glycolysis. The last idea inspected was mitochondria function via TMRE uptake and CCCP treatments to determine mitochondrial membrane potential ΔΨm and if a Warburg effect is operative in EOC. We found that the more mesenchymal phenotype (SkOv3) produced more lactic acid and ATP than the more epithelial phenotype (TOV112D). Also, that the CCCP treatment had a bigger effect decreasing ATP levels in the more epithelial phenotype than the more mesenchymal phenotype; thus, these cells were favoring OXPHOS for their bioenergetics. The 2DG treatment had a bigger effect decreasing lactic acid secretion and ATP levels in the more mesenchymal phenotype than the more epithelial phenotype; thus, these cells favor a glycolysis pathway. We also found that more mesenchymal phenotype had a higher uptake of TMRE and this suggested they did not have impaired mitochondrial which supported the Warburg effect is not operative in EOC. Lastly, we found that the mesenchymal cells did have a ΔΨm, which supported the prior results that a Warburg effect is not operative in the cancer cells. Also, that both glycolysis and OXPHOS are providing ATP to the cancer cells.