Generation of oxidative stress by the respiratory chain following treatment with DNA damaging agents

grantor: University of TorontoCancer-associated molecular abnormalities are known to affect the activity of cellular signaling networks. The potential to target activated survival signaling pathways and antioxidant-redox regulation to improve treatment response in pancreatic ductal adenocarcinoma is addressed in this thesis. An activated signaling profile distinguished cancer cells from morphologically normal duct cells in the adjacent normal pancreas as well as non-cancer pancreas surgical resections. A capacity of the PKB/Akt survival pathway to modulate gemcitabine sensitivity was examined using 'in vitro' and ' in vivo' experimental models. Inhibition of PI3K, the principle activator of PKB, or the suppression of total PKB enhanced gemcitabine cytotoxicity. However, a subset of cell lines displayed an inversion of gemcitabine sensitivity ' in vivo' as compared to 'in vitro,' suggesting that additional factors such as antioxidant-redox potential and tumor microenvironment influenced treatment response 'in vivo.' An oxidative stress mediated apoptosis was examined as an alternative therapy using sulforaphane (SFN). The compound was tested on two cell lines that demonstrated resistance to gemcitabine as well as in combination with PI3K inhibition. A lower redox potential in PANC-1 cells was associated with a higher SFN-induced cytotoxicity and tumor growth inhibition. In this thesis, pancreatic cancer was sub-classified based on bio-functional characteristics including transcript and protein levels as well as chemotherapy response. These results give insight into the pathogenesis as well as potential treatment strategies for pancreatic cancer., The generation of mitochondrial reactive oxygen intermediates (ROI) and expression of the antioxidants glutathione and thioredoxin were examined in OCI/AML-2 cells following treatment with the DNA damaging agents cytosine arabinoside, etoposide and ã-radiation. A flow cytometry method was developed for the assessment of mitochondrial ROI and membrane potential in digitonin permeabilized cells under various respiratory conditions. Advantages of this new method are: (1) mitochondrial ROI and respiratory chain activity can be assessed at the single cell level; (2) ROI generation by non-mitochondrial sources are excluded. Cells were permeabilized and assessed for respiratory chain activity 24 hr post-treatment to the cytotoxic agents. Treated cells that were still capable of generating a mitochondrial membrane potential with the respiratory substrates (malate/glutamate, succinate), were observed to have higher ROI generation than untreated, control cells. Associated with the oxidative stress is the increased expression of glutathione and thioredoxin. Thioredoxin regulates redox sensitive protein sulphydryls that are associated with potentiating cell survival. Therefore, mitochondrial ROI generation appears to be involved with the induction of protective mechanisms, in addition to mediating cytotoxicity.M.Sc