Lambri, Nico (2018) Oleic acid induced alterations in pancreatic cancer cell proliferation, a consequence of a pre-programmed dependence on lipid synthesis. (PhD thesis), Kingston University, .
Abstract
Cancer cells demonstrate elevated levels of de novo lipogenesis (DNL), which represent a component of the reprogramming of tumour metabolism, driving uncontrolled cell growth. In vitro inhibition of this pathway results in reduced cell proliferation, viability and tumour size, however therapeutic attempts at successfully targeting this pathway are limited. Following an initial phenotypic characterization of a panel of pancreatic cell lines (BxPC-3, AsPC-1, Capan-1 and MiaPaca-2), along with a well characterized hepatic cell line (HepG2), cells were exposed to long term incubation (120 hours) with 300µM oleic acid (OA) and effects on cell proliferation were determined. BxPC-3 cells demonstrated a significant reduction of 16% in cell proliferation (p<0.05) as demonstrated by a live cell analysis system. These results were confirmed using an independent cell tracking staining assay which showed a 14% decrease in proliferation at 48 hours. Several approaches were utilised to determine the mechanisms responsible for this reduced proliferation. On supplementation with OA, all pancreatic cancer cell lines showed a reduction in glucose derived carbon contribution towards palmitate enrichment, however only BxPC-3 cells showed an increase in labelled glutamine derived palmitic acid enrichment when compared to MiaPaca-2. Extensive comparative studies were performed between MiaPaca-2 and BxPC-3 cells, since MiaPaca-2 cells are Kras positive, whereas BxPC-3 are Kras negative, which consequently results to distinct metabolic phenotypes between the two cell lines. Crosstalk between DNL and the pentose phosphate pathway (PPP) was investigated by measuring glucose-6-phosphate dehydrogenase activity and shown to be reduced in BxPC-3 cells (~50%) following the addition of OA, thus perturbing a major NADPH generating pathway for this cell line. However, although NADPH/NADP ratios did not seem to be affected by the addition of OA for the pancreatic cancer cell lines, NADPH labelling by [3-2H] glucose and its utilization by DNL through the measurement of labelled palmitate showed reduced levels (-20%) of labeled palmitate following the addition of OA in BxPC-3 and MiaPaca-2 cells. The dependency of the pancreatic cell lines to β-oxidation was indirectly measured by incorporating the carnitine palmitoyltransferase 1 (CPT1) inhibitor etomoxir with the OA supplementation experiments. Results showed growth arrest could be rescued with the addition of OA to all cell lines although levels of the β-oxidation enzymes ACADVL and ACADM were expressed at much lower levels in BxPC-3 compared to MiaPAca-2 cells (2-3 fold lower), possibly indicating less reliance on the β-oxidation pathway in this cell line. This study has potentially identified a metabolic weakness of BxPC-3 cells based on their metabolic phenotype. The data reveals the interdependency of DNL on the PPP pathway and the key role of maintaining a homeostatic balance regarding NADP+/NADPH levels. Further understanding and exploitation of these metabolic weakness may provide novel therapeutic strategies that can be developed for targeting cell survival.
Actions (Repository Editors)
 |
Item Control Page |