Browsing by Author "Isherwood, J."
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Item Open Access MON-P095: The Effect of Fish Oil-Supplemented Gemcitabine Treatment on Leukotriene B4 Levels in Pancreatic Cancer(Elsevier, 2017-09-18) Martin, N.; Isherwood, J.; Madden, M.; Narayanan, V.; Mustafa, N.; Bhangal, C.; Farah, S.; Jones, S.E.; Runau, F.; Chung, W.Y.; Arshad, A.; Dennison, A.R.Pancreatic cancer (PC) is an aggressive cancer and is the 5th leading cause of cancer death in Western Europe and unfavourable prognosis is attributed to advanced disease at diagnosis and lack of effective therapy. The tumour microenvironment is an inflammatory one, and immune-mediators, such as Leukotriene B4 (LTB4), might contribute to the growth and spread of PC. The aim of this study is to determine the potential anti-inflammatory effects of omega-3 fatty acids in patients being treated for PC. Blood samples were taken from PC patients undergoing Gemcitabine treatment (n=8, CON) and from patients undergoing Gemcitabine treatment supplemented (n=17, O-3) with omega-3 fatty acids (Lipidem, BBraun, Germany), for up to a maximum of 6 months where possible. Plasma was isolated from blood and stored at -80⁰C until analysis for baseline LTB4 levels (baseline). A further aliquot of blood was incubated with 1mg/ml Zymosan for 30 min at RT then 30 min at 37⁰C, samples were centrifuged and plasma was collected and stored until analysis (stimulated). All samples were analysed using human LTB4 commercial ELISA kits according to the manufacturers’ instructions (Invitrogen, USA and R&D Systems, UK). There was an overall reduction in plasma LTB4 levels in O-3 patients (CON 3.5±0.3 (mean±SEM) ng/ml vs O-3 2.8±0.1 ng/ml, p=0.02). The stimulated levels of LTB4 in O-3 patients was also significantly reduced (p<0.05). There were no differences in baseline of CON or O-3 patients or stimulated CON patient LTB4 levels of low or high Progression Free Survival (PFS) patients. However, there was a significant reduction in LTB4 levels of stimulated high PFS O-3 patients (O-3 low PFS 4.5±0.3 ng/ml vs O-3 high PFS 3.6±1.1 ng/ml, p=0.01) and in patients at later TNM stage (O-3 stage 3 4.8±0.2 ng/ml vs stage 4 2.9±0.2 ng/ml, p<0.0001). Our results show that although the baseline inflammatory profile of PC patients is unaffected by omega-3 supplementation, the stimulated leukocyte response is reduced and is associated with increased patient PFS and later disease stage. These data indicate that omega-3 supplementation affects an anti-inflammatory profile in these patients, being more effective at later disease stages and may improve survival. Further work is required to assess the effects of omega-3 on the inflammatory profiles in PC.Item Open Access Reduced Pro-coagulant Phenotype of Microparticles in Pancreatic Cancer Patients on Omega-3-Supplemented Gemcitabine Treatment(2018) Martin, N.; Hackney, A.B.; Isherwood, J.; Runau, F.; Arshad, A.; Chung, W.Y.; Dennison, A.R.Pancreatic cancer (PC) is an aggressive cancer associated with advanced disease at diagnosis and lack of effective therapy. Microparticles (MP), biologically active nanoparticles shed from cells, are novel markers of inflammation and might contribute to the growth and spread of PC. The aim of this study is to determine the effects of omega-3 supplementation in PC patients. MP were isolated from blood taken from PC patients undergoing Gemcitabine treatment (n=9, CON), patients undergoing treatment supplemented with omega-3 fatty acids (n=18, Ω-3) and from healthy controls (n=6, HC). MP were stained with AnV (PS-MP) and anti-CD142 to identify those expressing tissue factor (TF), acquired and analysed using flow cytometry. Circulating MP in both groups of PC patients was significantly greater than HC (CON 84±17X105/ml (P<0.03), Ω-3 105±22x105/ml (P<0.05) vs HC 31±10x105/ml), these MP were also significantly more pro-coagulant than HC MP (CON 13795RFU and Ω-3 11232RFU vs HC 7587RFU, P<0.05). MP were significantly greater in number and size at later disease stage in CON (63±19x105/ml stage 3 vs 128±20x105/ml stage 4, P<0.03; HiFSC 156±15 stage 3 vs 533±213 stage 4, P<0.03), and significantly fewer in Ω-3 patients at early disease stage (136±36x105/ml, P<0.03). This effect on size of MP might be explained by in vitro experiments where incubation with rHuTF increased the size of MP released by a PANC1 cells (P<0.05). The percentage of PS-MP was significantly decreased in Ω-3 patients (6.48% vs HC 13.48%, P<0.03). Significant increases in CD142-MP (P<0.05) and the increases in smaller CD142-MP at later disease stage (41±6 stage 3 vs 107±34 stage 4, P<0.03) seen in CON were not seen in Ω-3 patients, but overall Ω-3 patients had significantly greater numbers of smaller CD142-MP (117±24, P<0.03) at earlier disease stage. Our results show that omega-3 supplementation affects the pro-coagulant nature and size of MP in PC patients. Further work is required to assess the effects of omega-3 and the role of MP in PC.Item Metadata only Stearoyl-CoA desaturase 1 inhibitor supplemented with gemcitabine treatment reduces the viability and fatty acid content of pancreatic cancer cells in vitro(Wolters Kluwer, 2021-12) Hackney, A. B.; Chung, W. Y.; Isherwood, J.; Dennison, A. R.; Martin, N.Objective: Pancreatic cancer (PC) is an aggressive cancer with ineffective treatment. Inhibition of stearoyl-CoA desaturase 1 (SCD1) suppresses cancer proliferation and might act as a novel chemotherapy supplement, but this has not been investigated in PC. Here, the effects of SCD1 inhibitor CAY10566 supplemented with gemcitabine treatment (gemcitabine+CAY10566) on PC cell viability, apoptosis, phenotype, fatty acid content, platelet-derived growth factor release, and cell size were investigated. Methods: Human PC cell line (PANC-1) was treated with SCD1 inhibitor CAY10566 with or without gemcitabine. Cell viability was assayed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide and apoptosis and phenotype were determined using flow cytometry. Fatty acid content and platelet-derived growth factor release were measured by enzyme-linked immunosorbent assay. Cell size was determined using scanning electron microscopy. Results: Half-maximal inhibitory concentration of gemcitabine or CAY10566 significantly reduced PANC-1 viability compared to gemcitabine alone (P<.0001). No significant differences in the phenotype of phosphatidylserine, tissue factor or basigin expression were detected at therapeutic doses (P>.05). Apoptosis was significantly increased following incubation with CAY10566 (P<.05). Fatty acid content of cells was significantly higher following gemcitabine treatment compared to CAY10566 alone or gemcitabine +CAY10566 (P<.05). Platelet-derived growth factor released by gemcitabine-treated cells was significantly increased compared to 142nM CAY10566 alone or gemcitabine+CAY10566 (P<.01). CAY10566 did not affect the size of isolated tumor cells but gemcitabine+CAY10566 significantly increased the size compared to the control (P<.05). Cell viability decreased significantly after the treatment with gemcitabine+CAY10566 compared with CAY10566 alone (P<.05) and gemcitabine alone (P<.01). However, when cycles of chemotherapy were mimicked and treatment was removed, the number of cell viability was significantly reduced (P<.05). Conclusion: This study suggests that CAY10566 may be a suitable supplement for gemcitabine chemotherapy for PC.Item Metadata only Stearoyl-CoA-Desaturase 1 inhibition induces apoptosis in Pancreatic Cancer cells(2018-06) Isherwood, J.; Runau, F.; Arshad, A.; Martin, N.; Hackney, A. B.; Chung, W. Y.; Dennison, A. R.Pancreatic cancer (PC) is an aggressive cancer with advanced disease at diagnosis and lack of effective therapy. PC tumours carry a mutated KRAS gene essential to their growth and spread, which plays a key role in deregulating cell metabolism. Stearoyl-CoA-Desaturase 1 (SCD1) is a highly regulated enzyme responsible for desaturating fatty acids, converting Stearic acid to Oleic acid. The expression of SCD1 is known to be upregulated in PC, indicating that it represents a metabolic bottleneck for cancer cell metabolism and might contribute to the growth and spread of PC. The aim of this study is to determine the effects of inhibiting SCD1 activity on a PC cell line (PANC1) in vitro. PANC1 cells were incubated for 48 hours with various concentrations of CAY10566, a selective SCD1 inhibitor. The cells were trypsinized, stained with Annexin V (AnV) and Propidium Iodide (PI) and analysed using flow cytometry. SCD1 inhibition (INHIB) significantly decreases viable PANC1 cells (AnV-, PI-) compared to a medium-only control (CON) (INHIB 48.76% (± 0.03), CON 60.71% (±0.01), P<0.05) and significantly increased apoptosis (AnV +, PI-) (INHIB 31.56% (±0.07), CON 25.32% (±0.02), P<0.05. Microscopically, the membrane of these cells appears less defined after treatment with the SCD inhibitor, indicating changes in composition may occur. Our results show that SCD1 inhibition significantly affects PC cell death and apoptosis. Further work is required to combine SCD1 inhibition with the standard PC chemotherapy Gemcitabine, and assess if there is a synergistic effect of the two drugs. This supplemented treatment could improve TMN (tumour metastasis node) and progression free survival, both clinical markers of patient outcome, in PC patients.