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THE METABOLIC PHENOTYPE OF PANCREATIC CANCER AND ITS LINK TO CYTOSOLIC CALCIUM HOMEOSTASIS AND SURVIVAL

Chan, Anthony

[Thesis]. Manchester, UK: The University of Manchester; 2013.

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Abstract

THE METABOLIC PHENOTYPE OF PANCREATIC CANCER AND ITS LINK TO CYTOSOLIC CALCIUM HOMEOSTASIS AND SURVIVAL IntroductionPancreatic ductal adenocarcinoma (PDAC) is an insidious and aggressive cancer characterised by poor survival rates. In cancer, there is a pathological switch in metabolism from mitochondrial oxidative phosphorylation to glycolysis, known as the Warburg effect. Cells depend on an ATP-driven plasma membrane Ca¬2+ pump (PMCA) to maintain a low resting cytosolic Ca2+ concentration ([Ca2+]i), high levels of which can produce cytotoxicity and cell death. The reliance of PDAC on glycolysis can be targeted by selective metabolic inhibitors. We hypothesize that disrupting the glycolytic ATP supply will impair PMCA, and its ability to maintain a low resting [Ca2+]i that prevents cell death.Methods & MaterialsTo measure the effect of glycolytic and mitochondrial inhibitors, we utilised an in situ PMCA activity Fura-2 assay to measure [Ca2+]i clearance in the PDAC cell line, Panc-1. We also measured the effects of the inhibitors on intracellular ATP levels using bioluminescence assays, and cell necrosis rates using cell death assays.ResultsWe firstly show that selective inhibition of glycolysis using 3-bromopyruvate (3-BP) induces cell necrosis in Panc-1 cells whereas inhibition of mitochondrial metabolism using carbonyl cyanide m-chlorophenyl hydrazone (CCCP) has no effect. Furthermore, we show that glycolytic inhibitors 3-BP and iodoacetate inhibits PMCA, and impairs the ability of the Panc-1 cells to maintain Ca2+ homeostasis. Mitochondrial inhibitors CCCP and oligomycin has no effect on Ca2+ clearance. Finally, we show that inhibition of glycolysis, but not mitochondrial metabolism, causes a significant reduction in intracellular ATP levels in Panc-1 cells.DiscussionOur data suggests that an inhibition of the glycolytic ATP supply to PMCA in PDAC is an effective therapeutic target that could represent a new strategy for selectively killing PDAC cells and sparing normal, healthy cells.

Layman's Abstract

THE METABOLIC PHENOTYPE OF PANCREATIC CANCER AND ITS LINK TO CYTOSOLIC CALCIUM HOMEOSTASIS AND SURVIVALCancer of the pancreas is one of the most aggressive cancers responsible for over 250,000 deaths worldwide every year. It is usually diagnosed late, and as a result it is often too late to remove the tumour because either surgery is too dangerous or because it has already spread. As a result, only 1-3% of patients survive more than 5 years. All cells in the body generate energy using one of two mechanisms – mitochondrial oxidative phosphorylation or glycolysis. Normally, mitochondria generate up to 90% of a normal cell’s energy requirements. In cancer however, there is a shift whereby glycolysis takes over and becomes the major source of energy. This is known as the Warburg effect. One of the uses of this energy is to maintain calcium at very low levels by using a calcium pump, which moves calcium across the cell membrane and out of the cell. If this pump is inhibited, the cell becomes overloaded with calcium, which is very toxic, and eventually leads to cell death.The aim of this project is to test if inhibiting glycolysis in a pancreatic cancer cell has an effect on the calcium pump and the levels of calcium inside the cell. We also look at the effects of inhibiting glycolysis on cell death rates and on how it affects energy levels in the cell.We found that inhibiting glycolysis profoundly affects the calcium pump, and the cell’s ability to maintain a low resting calcium level. Furthermore, by inhibiting mitochondrial oxidative phosphorylation, there was no effect on the pump. We also found that glycolytic inhibitors increased the rate of cell death, and significantly reduced the energy levels in the pancreatic cancer cells. Our results suggest that inhibiting the main ATP supply in cancer (glycolysis) is an effective target that selectively kills pancreatic cancer cells, but at the same time sparing normal healthy cells that do not rely on glycolysis.

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Bibliographic metadata

Type of resource:
Content type:
Form of thesis:
Type of submission:
Degree programme:
MPhil Medicine (Cardiovascular Sciences)
Publication date:
Location:
Manchester, UK
Total pages:
42
Abstract:
THE METABOLIC PHENOTYPE OF PANCREATIC CANCER AND ITS LINK TO CYTOSOLIC CALCIUM HOMEOSTASIS AND SURVIVAL IntroductionPancreatic ductal adenocarcinoma (PDAC) is an insidious and aggressive cancer characterised by poor survival rates. In cancer, there is a pathological switch in metabolism from mitochondrial oxidative phosphorylation to glycolysis, known as the Warburg effect. Cells depend on an ATP-driven plasma membrane Ca¬2+ pump (PMCA) to maintain a low resting cytosolic Ca2+ concentration ([Ca2+]i), high levels of which can produce cytotoxicity and cell death. The reliance of PDAC on glycolysis can be targeted by selective metabolic inhibitors. We hypothesize that disrupting the glycolytic ATP supply will impair PMCA, and its ability to maintain a low resting [Ca2+]i that prevents cell death.Methods & MaterialsTo measure the effect of glycolytic and mitochondrial inhibitors, we utilised an in situ PMCA activity Fura-2 assay to measure [Ca2+]i clearance in the PDAC cell line, Panc-1. We also measured the effects of the inhibitors on intracellular ATP levels using bioluminescence assays, and cell necrosis rates using cell death assays.ResultsWe firstly show that selective inhibition of glycolysis using 3-bromopyruvate (3-BP) induces cell necrosis in Panc-1 cells whereas inhibition of mitochondrial metabolism using carbonyl cyanide m-chlorophenyl hydrazone (CCCP) has no effect. Furthermore, we show that glycolytic inhibitors 3-BP and iodoacetate inhibits PMCA, and impairs the ability of the Panc-1 cells to maintain Ca2+ homeostasis. Mitochondrial inhibitors CCCP and oligomycin has no effect on Ca2+ clearance. Finally, we show that inhibition of glycolysis, but not mitochondrial metabolism, causes a significant reduction in intracellular ATP levels in Panc-1 cells.DiscussionOur data suggests that an inhibition of the glycolytic ATP supply to PMCA in PDAC is an effective therapeutic target that could represent a new strategy for selectively killing PDAC cells and sparing normal, healthy cells.
Layman's abstract:
THE METABOLIC PHENOTYPE OF PANCREATIC CANCER AND ITS LINK TO CYTOSOLIC CALCIUM HOMEOSTASIS AND SURVIVALCancer of the pancreas is one of the most aggressive cancers responsible for over 250,000 deaths worldwide every year. It is usually diagnosed late, and as a result it is often too late to remove the tumour because either surgery is too dangerous or because it has already spread. As a result, only 1-3% of patients survive more than 5 years. All cells in the body generate energy using one of two mechanisms – mitochondrial oxidative phosphorylation or glycolysis. Normally, mitochondria generate up to 90% of a normal cell’s energy requirements. In cancer however, there is a shift whereby glycolysis takes over and becomes the major source of energy. This is known as the Warburg effect. One of the uses of this energy is to maintain calcium at very low levels by using a calcium pump, which moves calcium across the cell membrane and out of the cell. If this pump is inhibited, the cell becomes overloaded with calcium, which is very toxic, and eventually leads to cell death.The aim of this project is to test if inhibiting glycolysis in a pancreatic cancer cell has an effect on the calcium pump and the levels of calcium inside the cell. We also look at the effects of inhibiting glycolysis on cell death rates and on how it affects energy levels in the cell.We found that inhibiting glycolysis profoundly affects the calcium pump, and the cell’s ability to maintain a low resting calcium level. Furthermore, by inhibiting mitochondrial oxidative phosphorylation, there was no effect on the pump. We also found that glycolytic inhibitors increased the rate of cell death, and significantly reduced the energy levels in the pancreatic cancer cells. Our results suggest that inhibiting the main ATP supply in cancer (glycolysis) is an effective target that selectively kills pancreatic cancer cells, but at the same time sparing normal healthy cells that do not rely on glycolysis.
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Non-digital content not deposited electronically:
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Thesis main supervisor(s):
Thesis co-supervisor(s):
Thesis advisor(s):
Language:
en

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University researcher(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:202693
Created by:
Chan, Anthony
Created:
27th July, 2013, 18:41:41
Last modified by:
Chan, Anthony
Last modified:
14th November, 2013, 13:22:28

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