BackgroundThe acellular fraction of epithelial ovarian cancer (EOC) ascites promotes de novo resistance of tumor cells and thus supports the idea that tumor cells may survive in the surrounding protective microenvironment contributing to disease recurrence. Levels of the pro-inflammatory cytokines IL-6 and IL-8 are elevated in EOC ascites suggesting that they could play a role in tumor progression.MethodsWe measured IL-6 and IL-8 levels in the ascites of 39 patients with newly diagnosed EOC. Commercially available enzyme-linked immunosorbent assay (ELISA) was used to determine IL-6 and IL-8 ascites levels. Ascites cytokine levels were correlated with clinicopathological parameters and progression-free survival.ResultsMean ascites levels for IL-6 and IL-8 were 6419 pg/ml (SEM: 1409 pg/ml) and 1408 pg/ml (SEM: 437 pg/ml) respectively. The levels of IL-6 and IL-8 in ascites were significantly lower in patients that have received prior chemotherapy before the surgery (Mann-Whitney U test, P = 0.037 for IL-6 and P = 0.008 for IL-8). Univariate analysis revealed that high IL-6 ascites levels (P = 0.021), serum CA125 levels (P = 0.04) and stage IV (P = 0.009) were significantly correlated with shorter progression-free survival. Including these variables in a multivariate analysis revealed that elevated IL-6 levels (P = 0.033) was an independent predictor of shorter progression-free survival.ConclusionElevated IL-6, but not IL-8, ascites level is an independent predictor of shorter progression-free survival.

Ascites are commonly found in ovarian cancer patients with advanced disease and are rich in cellular components and growthpromoting factors. The purpose of this study was to assess the effect of malignant ascites on TRAIL-induced apoptosis. We demonstrate that malignant ascites obtained from women with advanced ovarian cancer protect tumor cells from TRAIL-and FasL-induced apoptosis but not against cisplatin-induced apoptosis. This antiapoptotic effect was consistently found among different malignant ascites while nonmalignant peritoneal fluids or conditioned medium from TRAIL-resistant cells failed to protect tumor cells against TRAIL killing. Malignant ascites strongly inhibits TRAIL-induced caspase-3 activation and PARP cleavage. Furthermore, ascites activate PI3K and its downstream target Akt and increases c-FLIP S protein levels without affecting ERK phosphorylation status. The antiapoptotic effect of malignant ascites is abrogated by the inhibition of PI3K with LY294002, by a specific inhibitor of Akt and by Akt siRNA. We further show that the prosurvival effect of ascites can be suppressed by down-regulation of c-FLIP S . Our data indicate that malignant effusions protect against TRAIL-induced apoptosis by activating the PI3K/Akt pathway. These findings demonstrate that the tumor microenvironment may contribute to the resistance of ovarian cancer cells to death receptor-induced apoptosis. ' 2007 Wiley-Liss, Inc.

Clara cell protein (CC-16, also designated CC-10) is synthesized by the bronchiolar epithelium and has been suggested as an inhibitor of phospholipase A2 (PLA2) activity. Therefore, CC-16 is a candidate for controlling inflammatory events in the lung. Because CC-16 amounts and function may be altered in fibrosing lung diseases in which bronchiolar injury has been reported, it was measured in alveolar fluids and sera. Secretory PLA2 activity in alveolar fluids and the influence of CC-16 on platelet-derived growth factor-induced human fibroblast chemotaxis and cytosolic PLA2 activity were also explored. CC-16 content was decreased in alveolar fluids from idiopathic pulmonary fibrosis (IPF: 1.3 +/- 0.1 mg/L) and bleomycin lung (1.1 +/- 0.2 versus 2.1 +/- 0.2 mg/L in controls, p < 0.05), whereas there was a three- to ninefold increase in secretory PLA2 activity (p < 0.05 versus controls). CC-16 inhibited fibroblast chemotaxis in a dose-dependent manner (90% inhibition at 30 micrograms/ml CC-16). This inhibition was reversed by reducing CC-16. CC-16 was also able to lower fibroblastic cytosolic PLA2 activity by 50% in vitro. In summary, CC-16 is able to inhibit fibroblast chemotaxis in vitro by mechanisms that may be related to a blockage of cytosolic PLA2 activity. It can be postulated that CC-16 deficiency may contribute to fibroblast burden activity in fibrosing lung diseases.

Interactions between ovarian cancer cells and the surrounding tumor microenvironment are not well characterized. We have earlier shown that ovarian cancer ascites induces Akt activation and protect tumor cells from TRAIL-induced apoptosis. Here, we investigated the mechanism by which ascites activates Akt. The ability of ovarian cancer ascites to activate Akt and inhibit TRAILinduced cell death and caspase activity was decreased by heat inactivation, but was retained in ascites fractions 45 kDa. The survival promoting activity of ascites was not affected by inhibitors of growth factor receptor including epidermal growth factor receptor (EGFR), VEGFR, FGFR, Her2/neu, and IGF-R1. However, this activity was inhibited by an avb5 integrin-blocking antibody, but not by blocking antibodies against avb3, b1, or b3 integrins. avb5 integrin-blocking antibodies also inhibited ascites-induced Akt phosphorylation and c-FLIPs up-regulation. Ovarian cancer ascites induced a rapid phosphorylation of focal adhesion kinase (FAK), which closely correlated with the phosphorylation of Akt overtime. FAK phosphorylation was strongly inhibited by avb5 integrin-blocking antibodies. Depletion of FAK content by RNA interference was also associated with inhibition of ascites-mediated Akt activation and survival. These results suggest that ovarian cancer ascites induces FAK and Akt activation in an avb5 integrin-dependent pathway, which confers protection from TRAIL-induced cell death and caspase activation.

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