Dawn Ann Bradbury

Vascular Endothelial Growth Factor Induction by Prostaglandin E2 in Human Airway Smooth Muscle Cells Is Mediated by E Prostanoid EP2/EP4 Receptors and SP-1 Transcription Factor Binding Sites

Prostaglandin E2 (PGE2) can increase thelial vascular endogrowth factor A (VEGF-A) production but the mechanisms involved are unclear. Here we characterized the transcriptional mechanisms involved in human airway smooth muscle cells (HASMC). PGE2 increased VEGF-A mRNA and protein but not mRNA stability. PGE2 stimulated the activity of a transiently transfected 2068-bp (–2018 to +50) VEGF-A promoter-driven luciferase construct. Functional 5′ deletional analysis mapped the PGE2 response element to the 135-bp sequence (–85/ +50) of the human VEGF-A promoter. PGE2-induced luciferase activity was reduced in cells transfected with a 135-bp VEGF promoter fragment containing mutated Sp-1 binding sites but not in cells transfected with a construct containing mutated EGR-1 binding sites. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed binding of Sp-1 to the VEGF promoter. PGE2 increased phosphorylation of Sp-1 and luciferase activity of a transfected Sp-1 reporter construct. PGE receptor agonists EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) mimicked the effect of PGE2, and reverse transcription-PCR, Western blotting, and flow cytometry confirmed the presence of EP2 and EP4 receptors. VEGF protein release and Sp-1 reporter activity were increased by forskolin and isoproterenol, which increase cytosolic cAMP, and the cAMP analogue, 8-bromoadenosine-3′,5′-cyclophosphoric acid. These studies suggest that PGE2 increases VEGF transcriptionally and involves the Sp-1 binding site via a cAMP-dependent mechanism involving EP2 and EP4 receptors.

Cyclooxygenase (COX) inhibitors induce apoptosis in non-small cell lung cancer through cyclooxygenase independent pathways.

Cyclooxygenase (COX) inhibitors are chemopreventive in many tumours but the role of COX inhibition in their effects is contentious. Here we determined if COX inhibitors influenced apoptosis in two non-small cell lung cancer cells one which over expresses COX-2 (MOR-P) and one which expresses neither isoform (H-460). NS398, a selective COX inhibitor, and indomethacin, a non-selective COX inhibitor, were cytotoxic in both cell lines, independently of their COX-2 expression. Furthermore, the cytotoxic concentrations were far greater than the concentrations required to inhibit COX. As indomethacin was more effective we used it in mechanistic studies. Indomethacin induced apoptotic cell death assessed as cytochrome c and apoptotic inducing factor (AIF) release, caspase activation, PARP, lamin B and gelsolin cleavage, chromatin condensation and nuclear fragmentation. The pan-caspase inhibitor, z-VAD, attenuated cell death, and blocked caspase activation, PARP cleavage and nuclear fragmentation without preventing cytochrome c release, suggesting that cytochrome c release is upstream of caspase activation. These observations suggest that COX inhibitors induce apoptosis in non-small lung cancer cells through cytochrome c and AIF release, and subsequent caspase activation, independently of COX-2 expression and prostaglandin production.

Cytokines upregulate vascular endothelial growth factor secretion by human airway smooth muscle cells: Role of endogenous prostanoids

Here, we report that vascular endothelial growth factor (VEGF)‐A secretion by human airway smooth muscle cells was increased by interleukin 1 beta (IL‐1β) and transforming growth factor beta (TGFβ). IL‐1β and TGFβ induced cyclo‐oxygenase (COX)‐2 protein and increased prostaglandin E2 (PGE2). Both IL‐1β and TGFβ increased VEGF‐A165 mRNA and VEGF promoter luciferase construct activity, in addition VEGF‐A protein was inhibited by actinomycin D suggesting transcriptional regulation. The COX inhibitors indomethacin and NS398 inhibited IL‐1β but not TGFβ mediated VEGF‐A production. Furthermore, the effect of the COX inhibitors was overcome by adding exogenous PGE2. In conclusion, IL‐1β increases VEGF‐A secretion by COX‐2 derived PGE2 production whereas TGFβ uses COX‐independent pathways.

Camptothecin-induced apoptosis in non-small cell lung cancer is independent of cyclooxygenase expression

Recent observations show a positive correlation between the expression of cyclooxygenase (COX), especially COX-2), and cancer development. Here we tested the hypothesis that expression of COX-2 could influence apoptosis in lung cancer cell lines. To address this question, we determined the effects of camptothecin-induced apoptosis on three lung cancer cell lines which over express COX-1 (CORL23), COX-2 (MOR-P) and neither isoform (H-460), and determine if these effects were prostaglandin mediated. We also compared the effects of non-selective and isoenzyme selective COX-2 inhibitors on camptothecin-induced apoptosis in these three cell lines. Camptothecin induced apoptosis in all three cell lines independently of COX-1 or COX-2 expression. Indomethacin, a non-selective COX inhibitor and NS398, a selective COX-2 inhibitor had no effect on camptothecin-induced apoptosis at concentrations that abolished prostaglandin production. In conclusion, these finding suggest that the COX pathway is not involved in camptothecin-induced apoptosis of non-small cell lung cancer cell lines.

Determination of the Effects of Camptothecin on Cell Cycling and Apoptosis in Human Leukaemic Cell Lines

INTRODUCTION. The measurement of ATP is a recognised method for determination of proliferation or the effect of toxic environments on the cell. More recently, the relationship between ATP and ADP has been shown to be an important factor in determining the apoptotic or necrotic state of the cell (1). The ratio of ADP to ATP shows significant correlation with the degree of apoptosis, as measured by the estimation of the sub-G0 by propidium iodide staining of the nuclei and flow cytometry. Camptothecin is a topoisomerase inhibitor and as such, has effects on the cell cycle prior to inducing apoptosis. This compound has also been used as a chemotherapeutic agent in the treatment of a number of malignancies including leukemias. We have investigated these effects in a number of human leukaemic cell lines, concentrating on U937 and HL-60 cells, together with blast cells isolated from the peripheral blood of patients with acute myeloid leukemia. METHOD. HL-60 cells were incubated for 4 hours, while with the U937 cells a 24-hour incubation was required for induction of apoptosis, with increasing concentrations of camptothecin. The AML blast cells, from 10 patients were incubated with increasing concentrations of camptothecin, Ara-C and etoposide for 4 to 24 hours. The extent of apoptosis was shown by measurement of the ratio of ADP:ATP using the ApoGlow Kit (LumiTech (UK) Ltd). The presence of apoptotic events within the cell population was confirmed using Annexin V, JC-1, and propidium iodide (PI) staining and flow cytometry. The PI staining was also used to determine the relative percentage of cells in different phases of the cell cycle. RESULTS. The HL-60 and U-937 cells showed a concentration dependent effect on apoptosis induction with concentrations of camptothecin from 100 to 500nM. At the highest concentration of camptothecin used (1000nM) there was a significant reduction (n=7, p<0.0001) in the ratios compared with the 500nM sample. Comparison with the PI data revealed emergence of the G0/G1 peak showing that a subpopulation of cells was beginning to cycle again. With HL-60 cells at 1000nM of the drug, there was an increase in the percentage viable cells as shown by exclusion of PI, this correlated with a reduction in the level of ADP relative to ATP with the emergence of a resistant viable population. Of the AML cells investigated there was a marked difference in the response of the patient cells to the different drugs investigated, with respect to induction of apoptosis and drug resistance. DISCUSSION. The U937 cells displayed variations in responses according to the concentration of camptothecin used. The cells arresting in S-phase at low concentrations, could