Therapeutic implications of disorders of cell death signalling: membranes, micro-environment, and eicosanoid and docosanoid metabolism.

Disruptions of cell death signalling occur in pathological processes, such as cancer and degenerative disease. Increased knowledge of cell death signalling has opened new areas of therapeutic research, and identifying key mediators of cell death has become increasingly important. Early triggering events in cell death may provide potential therapeutic targets, whereas agents affecting later signals may be more palliative in nature. A group of primary mediators are derivatives of the highly unsaturated fatty acids (HUFAs), particularly oxygenated metabolites such as prostaglandins. HUFAs, esterified in cell membranes, act as critical signalling molecules in many pathological processes. Currently, agents affecting HUFA metabolism are widely prescribed in diseases involving disordered cell death signalling. However, partly due to rapid metabolism, their role in cell death signalling pathways is poorly characterized. Recently, HUFA-derived mediators, the resolvins/protectins and endocannabinoids, have added opportunities to target selective signals and pathways. This review will focus on the control of cell death by HUFA, eicosanoid (C20 fatty acid metabolites) and docosanoid (C22 metabolites), HUFA-derived lipid mediators, signalling elements in the micro-environment and their potential therapeutic applications. Further therapeutic approaches will involve cell and molecular biology, the multiple hit theory of disease progression and analysis of system plasticity. Advances in the cell biology of eicosanoid and docosanoid metabolism, together with structure/function analysis of HUFA-derived mediators, will be useful in developing therapeutic agents in pathologies characterized by alterations in cell death signalling.

Calcium-sensitive mitochondrial membrane potential in human platelets and intrinsic signals of cell death.

The mechanisms involved in storage-induced damage in platelets are not well understood, but membrane signalling via Ca2+ ion flux may affect mitochondrial H+ gradients and metabolism and the intrinsic pathways of cell death, platelet survival and function. In this study, the effects of blood bank storage conditions, including reduced plasma concentration and interrupted agitation, were evaluated in platelets from 136 healthy donors. Mitochondrial membrane potential (DeltaPsim), an indicator of intrinsic cell death, and its sensitivity to Ca2+ ionophore A23187, were monitored using JC-1 by flow cytometry and fluorescence microscopy. Platelet survival was examined using lactate dehydrogenase release, annexin V binding and caspase-3/7 activity. Decreased plasma concentration and interrupted agitation affected DeltaPsim and caspase-3/7. Over 7 days in 30% plasma DeltaPsim showed a significant reduction (86.3 +/- 1.1% platelets with polarised mitochondria day 1; 79.9 +/- 2.1% day 5; 75.1 +/- 3.8% day 7, P = 0.01 day 1 vs. day 7). Whilst DeltaPsim in agitated platelets in 100% plasma was unchanged up to day 7, interruption of agitation was associated with a 44% reduction in the proportion of platelets with polarised mitochondria after 5 days (56 +/- 11%). The Ca2+ sensitivity of DeltaPsim changed earlier: 5 microM A23187 caused a 20-30% change in the fraction of platelets with polarised mitochondria by day 5. Ca2+ sensitivity also increased during interrupted agitation and reduced plasma concentration. DeltaPsim also correlated with indicators of platelet death, caspase-3 activity and annexin V binding (correlation coefficients of 0.8). In conclusion, changes in Ca2+-sensitive DeltaPsim are involved in the initiation of storage-induced cell death signals that influence platelet count and function in vivo.

Intracellular oxidation by human glioma cell populations: effect of arachidonic acid.

Arachidonic acid (AA) and Gamma linolenic acid have been shown to limit glioma cell growth, stimulate apoptosis and lipid peroxidation. However, brain tumours are characterised by cellular heterogeneity and responding cell populations have not been identified. Brain tumour samples from patients were disaggregated. In cell preparations from 7 gliomas, reactive oxygen species (ROS), morphology and plasma membrane integrity were monitored +/-18-36 microM AA for 15-120 min using flow cytometry. Basal oxidative activity related to cell size/morphology, small granular cells showed lower activity. AA stimulation of ROS formation depended on cell size/morphology. Large, less granular cells showed greater AA stimulation. In 17 gliomas, GFAP immunofluorescence was demonstrated in larger cell populations. The large GFAP positive cell population with low side scatter was the highest responding cell population, suggesting selective tumour cell sensitivity to AA induced ROS formation. ROS may have a role in AA induced cell death and anti-tumour activity of AA in glioma.

Highly unsaturated fatty acid induced tumour regression in glioma pharmacodynamics and bioavailability of gamma linolenic acid in an implantation glioma model: effects on tumour biomass, apoptosis and neuronal tissue histology.

Highly unsaturated fatty acids (HUFAs) are naturally occurring anti-tumour agents. HUFAs act as intracellular signalling molecules in cell proliferation and death. In human glioma, HUFAs may stimulate tumour regression and apoptosis. An implantation glioma model, using the C6 glioma cell line, was used to investigate the bioactivity of locally infused n-6 HUFA gamma linolenic acid (GLA). Rat brains (15 normal and 37 C6 tumour bearing) were infused with vehicle or GLA 200 microM-2 mM. The most active local concentration of GLA for anti-tumour activity was 2 mM, infused at 1 microl/h over 7 days. Tumour regression, increased apoptosis and decreased proliferation were observed in tumours of rats infused with this concentration of GLA. Little effect on normal neuronal tissue was detected. The intraparenchymal route was an effective method of GLA administration in the treatment of glioma. These studies provide further insights into the potential role of HUFAs as anti-glioma agents.

Antitumour and pro-apoptotic actions of highly unsaturated fatty acids in glioma.

The highly unsaturated fatty acids (HUFA) of the n-6 and n-3 series are involved in cell signalling in normal and transformed cells and have recently been associated with pathways leading to tumour cell death. The antitumour activity of three HUFA (arachidonic acid, gamma linolenic acid and eicosapentaenoic acid) were studied in glioma cells and tissue. Using five glioma models, including primary cell suspensions prepared from 46 human glioma samples and an in vivo rat C6 glioma model, we obtained evidence that, following exposure to HUFA, either administered into the medium surrounding human glioma cells or in 16 preparations of multicellular spheroids derived from human and rodent glioma cell lines (C6, MOG, U87, U373) or administered intra-tumourally by infusion using osmotic mini-pumps in 48 rats, glioma regression and apoptosis were detected. Additionally, synergy between gamma irradiation and HUFA administration was observed in 13 experiments analyzing C6 glioma cell apoptosis in vitro. These pro-apoptotic and antiproliferative activities were observed using both C18 and C20 fatty acids of the n-6 and n-3 series, but not when saturated and monounsaturated C18 and C20 fatty acid preparations were used. In the glioma infusion model, in addition to the apoptosis detected in glioma tissue infused with HUFA for 3-7 days, preservation of normal neural tissue and vasculature in adjacent brain was observed. Also, there was little evidence of acute inflammatory infiltration in regressing tumours. Our findings suggest that intraparenchymal infusion of HUFA may be effective in stimulating glioma regression.

The development of necrosis and apoptosis in glioma: experimental findings using spheroid culture systems.

Cell death in gliomas may occur either by apoptosis, or, in the case of high grade tumours, by necrosis, but questions remain as to the pathogenesis and relationship between these processes. The development of cell death was investigated in multicellular glioma spheroid cultures. Spheroids model the development of cell death due to diffusion gradients in a three-dimensional system without confounding influences of immune response, pressure gradients, etc. Spheroid cultures were established from four malignant glioma cell lines: U87, U373, MOG-G-CCM and A172; harvested from culture at weekly intervals and stained with Haematoxylin and Eosin (H&E), TdT-mediated dUTP-X nick end labelling (TUNEL) and by immunohistochemistry for vimentin, Glial Fibrillary Acidic Protein (GFAP) and Ki67. Annexin V flow cytometry and counts of apoptotic cells on H & E stained sections were performed to assess levels of apoptosis. Modes of cell death were also characterized by electron microscopy. Spatially separate zones of proliferation, differentiation and central cell death developed with increasing spheroid diameter. Central cell death developed at a predictable radius (300-400 microm) for each cell line. Ultrastructural examination showed this to be necrotic in type. Apoptosis was most reliably assayed by morphological counts using H & E. Basal levels of apoptosis were low (< 0.5%), but increased with increasing spheroid diameter (> 2% in U87). In particular, levels of apoptosis rose following development of central necrosis and apoptoses were most abundant in the peri-necrotic zone. There were quantitative differences in the levels of apoptosis and necrosis between glioma cell lines. The predictable onset of necrosis in the spheroids will allow us to investigate the pathogenesis of necrosis and events in prenecrotic cells. There is a relationship between the development of necrosis and apoptosis in this model and these processes can be separately assayed. Further in vitro and genetic studies will enable us to study these events and interactions in greater detail than is possible using other cell culture and in vivo systems.

Acute phase responses following minimal access and conventional thoracic surgery.

OBJECTIVES: Major thoracic surgery is associated with trauma-related immunological changes. These may impair anti-tumour immunity. We hypothesize that the reduced operative trauma associated with a video-assisted thoracic surgery (VATS) approach may decrease acute phase responses and, consequently, lead to better preservation of immune function. This prospective randomized study compared the effects of conventional open thoracic surgery and VATS on acute phase responses in patients undergoing pulmonary lobectomy. METHODS: Acute phase indicators were analyzed in patients undergoing lobectomy for suspected bronchogenic carcinoma. Surgery was prospectively randomized to pulmonary lobectomy by VATS or limited postero-lateral thoracotomy. Blood was taken pre-operatively and at 4, 24, 48, 72, 120 and 168 h post-operatively for analysis of C-reactive protein (CRP; 41 patients: open, n=22; VATS, n=19) interleukin (IL)-6, tumour necrosis factor (TNF) receptors (TNF-sR55, TNF-sR75) and P-selectin (24 patients: open, n=12; VATS, n=12). Samples taken at 48 and 168 h were also analyzed for phagocyte reactive oxygen species (ROS) production (25 patients: open, n=16; VATS, n=19). RESULTS: Surgery increased acute phase responses. VATS was associated with lower CRP and IL-6 levels. In the open surgery group, significant increases in ROS in neutrophils (up to 36% greater than before surgery, n=12, P<0.02-0.05) were detected at 2 days after surgery, but in the VATS group, the increase after surgery (of up to 17%, n=18) did not reach significance. Similarly, monocyte ROS increases of up to 25% in the mean ROS in the open surgery group and of up to 17% in the VATS group were detected on days 2 and 7 after surgery. CONCLUSIONS: VATS pulmonary lobectomy is associated with reduced peri-operative changes in acute phase responses. This finding may have implications for peri-operative tumour immuno-surveillance in lung cancer patients.

Lymphocyte responses following open and minimally invasive thoracic surgery.

BACKGROUND: Immunosuppression associated with surgery may predispose to increased tumour growth or recurrence. Lymphocytes are central components of the immune network, signalling specific and non-specific responses in tumour immunosurveillance. This study was therefore designed to compare the effects of minimally invasive and conventional approaches to major thoracic surgery on lymphocyte populations and oxidative activity. PATIENTS AND METHODS: The effects of conventional and minimally invasive video-assisted thoracic surgery (VATS) on the numbers and types of circulating lymphocytes and on lymphocyte oxidation were compared in a prospective randomized study of 41 patients undergoing lobectomy for peripheral bronchogenic carcinoma. Blood taken pre-operatively and on days 2 and 7 post-operatively was analysed for T (CD4, CD8), B (CD19) and natural killer (NK) (CD56, CD16) cell counts and for lymphocyte oxidative activity. Leucocyte numbers were compared with pre-surgical values and oxidative rate with healthy donor controls. RESULTS: Lymphocyte counts fell after surgery; VATS was associated with less effect on circulating T (CD4) cells at 2 days and on NK lymphocytes at 7 days post-surgery. Lymphocyte oxidation was less suppressed in the VATS group 2 days after surgery. In general, post-surgical changes in key cells of cellular immunity were smaller in the VATS group, and recovery to normal levels was more rapid. CONCLUSION: The degree of invasiveness of thoracic surgery may influence the extent of immunosuppression in patients undergoing pulmonary lobectomy for pulmonary neoplasm.

Effects of N-6 essential fatty acids on glioma invasion and growth: experimental studies with glioma spheroids in collagen gels.

OBJECT: Intracranial infusions of gamma-linolenic acid (GLA), an essential fatty acid, have been used as an adjuvant therapy following malignant glioma resection; however, little is known about the dose response of glioma cells to this therapy. In this in vitro study the authors address this important pharmacological question. METHODS: Glioma spheroids derived from U87, U373, MOG-G-CCM, and C6 cell lines were grown in collagen gel and exposed to a range of GLA concentrations (0-1 mM) for 5 days. The diameter of glioma spheroids was measured, the apoptotic index was assessed using both the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique and cell morphological testing, and the levels of proliferating cell nuclear antigen were also measured. CONCLUSIONS: The dose-response patterns were similar for all four glioma spheroids. Low concentrations of GLA (<100 microM) increased both apoptosis and proliferation with a net increase in tumor growth and invasion, whereas high-dose GLA (>100 microM) significantly impaired spheroid cell growth. The proliferative effects of low-dose GLA could be a hazard in the clinical treatment of malignant glioma; however, because of the low toxicity of GLA against normal cells, local delivery of millimolar doses of GLA could significantly reduce tumor size.