Denis Lane

Prognostic significance of IL-6 and IL-8 ascites levels in ovarian cancer patients

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.

Malignant ascites protect against TRAIL-induced apoptosis by activating the PI3K/AKt pathway in human ovarian carcinoma cells

Ascites are commonly found in ovarian cancer patients with advanced disease and are rich in cellular components and growth‐promoting 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‐FLIPS 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 pro‐survival effect of ascites can be suppressed by down‐regulation of c‐FLIPS. 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.

CA125 (MUC16) tumor antigen selectively modulates the sensitivity of ovarian cancer cells to genotoxic drug-induced apoptosis

OBJECTIVE Little is known about the biological functions of CA125/MUC16 tumor antigen. Here, we examined the role of CA125/MUC16 in regulating the sensitivity of epithelial ovarian carcinoma (EOC) cells to different drugs. METHODS An endoplasmic reticulum targeted single-chain antibody (scFv) was used to down-regulate cell surface expression of CA125/MUC16 in NIH:OVCAR3 cells and the C-terminal domain (CTD) of MUC16 was ectopically expressed in CA125-negative SKOV3 cells. Sensitivity to genotoxic agents and to inhibitors of microtubule depolymerization was examined in NIH:OVCAR3 and SKOV3 cell sublines. Cell viability was determined by XTT assay, apoptosis by propidium iodide staining and caspase activation by Western blot and fluorogenic assay. RESULTS Down-regulation of cell surface MUC16 decreases cisplatin IC(50) by 5-fold in NIH:OVCAR3 cells but does not affect paclitaxel IC(50). We found that the sensitivity to other genotoxic agents such as cyclophosphamide, doxorubicine and etoposide was also increased by down-regulation of MUC16. Caspase-9 and caspase-3 activation also significantly augmented in cisplatin-treated NIH:OVCAR3 cells expressing the anti-MUC16 scFv. Ectopic expression of MUC16 CTD has the opposite effect. Cisplatin sensitivity and caspases activation are decreased by the ectopic expression of MUC16 CTD in SKOV3 cells. CONCLUSIONS CA125/MUC16 selectively modulates the sensitivity of EOC cells to genotoxic agents. The MUC16 CTD appears to be sufficient to promote cisplatin resistance.

Acquired resistance to TRAIL-induced apoptosis in human ovarian cancer cells is conferred by increased turnover of mature caspase-3

Little is known on how cancer cells can acquire resistance to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). In this study, we established TRAIL-resistant cells from the TRAIL-sensitive human ovarian carcinoma cell line OVCAR3 to evaluate the potential mechanisms of acquired resistance to TRAIL. The selected resistant cells were cross-resistant to Fas ligand but remained sensitive to drug-induced apoptosis. Expression of TRAIL receptors was not altered in TRAIL-resistant OVCAR3 cells. Cleavage of caspase-8 and caspase-3 occurred in both TRAIL-resistant and TRAIL-sensitive cells. However, mature caspase-3 fragments were not detected by immunoblot in TRAIL-resistant cells and caspase-3 activity was significantly inhibited in these cells. The addition of proteasome inhibitors significantly increased TRAIL-induced apoptosis in resistant cells and enhanced the accumulation of mature caspase-3 fragments. Pretreatment with cycloheximide showed that active caspase-3 fragments have a high turnover rate in OVCAR3 R350 cells. X-linked inhibitor of apoptosis down-regulation by RNA interference also increased the accumulation of cleaved caspase-3 intermediates and resensitized TRAIL-resistant cells. Our findings show that altered turnover of mature caspase-3 may lead to acquired TRAIL resistance in ovarian cancer cells. Proteasome and X-linked inhibitor of apoptosis inhibitors could have a role in clinical situations to potentiate the cytotoxic effects of TRAIL in resistant tumor cells. [Mol Cancer Ther 2006;5(3):509–21]

Interleukin-2 involvement in early acute respiratory distress syndrome: relationship with polymorphonuclear neutrophil apoptosis and patient survival.

ObjectiveTo determine blood and lung alveolar concentrations of interleukin (IL)-2 in acute respiratory distress syndrome (ARDS) and their relationship with polymorphonuclear neutrophil (PMN) apoptosis and patient survival. DesignProspective cohort study. SettingMedical and surgical intensive care units (ICUs; Canada) and the intensive care department (Belgium). PatientsNineteen consecutive patients with ARDS, 14 non-ARDS ICU patients, and 20 healthy volunteers. InterventionsBlood samples and bronchoalveolar lavages (BAL) obtained via venous puncture and by fiberoptic bronchoscopy in the first 72 hrs after the onset of ARDS. Measurements and Main ResultsOne early point concentration of IL-2 was measured in both blood and BAL fluids of the three groups. In vivo alveolar PMN apoptotic index in BAL fluids and the influence of BAL fluid exposure on normal blood PMN spontaneous apoptosis in vitro were evaluated. Blood IL-2 was significantly lower in patients with ARDS compared with non-ARDS ICU patients and controls. In contrast, IL-2 in BAL fluids of patients with ARDS was dramatically elevated compared with non-ARDS ICU patients and controls. ARDS survivors exhibited lower early IL-2 blood levels than nonsurvivors and generally had a higher IL-2 lung content. Lung alveolar PMN apoptosis in vivo was lower in patients with ARDS in comparison with controls. This apoptotic index was correlated with corresponding IL-2 alveolar levels in patients with ARDS. Exposure of normal blood PMN to BAL fluids from patients with ARDS delayed apoptosis in vitro. Immunodepletions of IL-2, granulocyte-macrophage colony stimulating factor, and a combination of both cytokines from BAL fluids of ARDS patients significantly restored PMN apoptosis. The recovery of PMN apoptosis was more effective when IL-2 was depleted in BAL fluids from ARDS survivors compared with nonsurvivors. ConclusionsOpposite and disproportional concentrations of IL-2 are observed in blood and lung fluids of patients with early ARDS. IL-2 significantly contributes (with granulocyte-macrophage colony stimulating factor) to the inhibition of PMN apoptosis in BAL fluids of patients with ARDS. Early low blood IL-2 and high IL-2-driven inhibition of PMN apoptosis are beneficial to survivors. Thus, IL-2 is a new candidate for monitoring in early ARDS and an interesting indicator of prognosis.

The prosurvival activity of ascites against TRAIL is associated with a shorter disease-free interval in patients with ovarian cancer

BackgroundThe production of ascites is a common complication of ovarian cancer. Ascites constitute a unique tumor microenvironment that may affect disease progression. In this context, we recently showed that ovarian cancer ascites may protect tumor cells from TRAIL-induced apoptosis. In this study, we sought to determine whether the prosurvival effect of ascites affects disease-free intervals.MethodsPeritoneal fluids were obtained from 54 women undergoing intra-abdominal surgery for suspected ovarian cancer (44 cancers and 10 benign diseases). The ability of peritoneal fluids to protect from TRAIL was assessed in the ovarian cancer cell line CaOV3, and IC50 were determined. The anti-apoptotic activity of 6 ascites against cisplatin, paclitaxel, doxorubicin, etoposide and vinorelbine was also assessed in CaOV3 cells, and the prosurvival activity of two ascites was assessed in 9 primary ovarian cancer cultures.ResultsAmong the 54 peritoneal fluids tested, inhibition of TRAIL cytotoxicity was variable. Fluids originating from ovarian cancer were generally more protective than fluids from non-malignant diseases. Most of the 44 ovarian cancer ascites increased TRAIL IC50 and this inhibitory effect did not correlate strongly with the protein concentration in these ascites or the levels of serum CA125, a tumor antigen which is used in the clinic as a marker of tumor burden. The effect of ascites on cisplatin- and paclitaxel-induced cell death was assessed with 4 ascites having inhibitory effect on TRAIL-induced cell death and 2 that do not. The four ascites with prosurvival activity against TRAIL had some inhibitory on cisplatin and/or paclitaxel. Two ovarian cancer ascites, OVC346 and OVC509, also inhibited TRAIL cytotoxicity in 9 primary cultures of ovarian tumor and induced Akt activation in three of these primary cultures. Among a cohort of 35 patients with ascites, a threshold of TRAIL IC50 with ascites/IC50 without ascites > 2 was associated with shorter disease-free interval.ConclusionsThe prosurvival activity of ascites against TRAIL is associated with shorter disease-free interval, which may be explained, at least in part, by ascites-induced cisplatin/paclitaxel resistance. Our findings suggest that ascites may contain prosurvival factors that protect against TRAIL and chemotherapy and consequently affect disease progression.

Ovarian cancer ascites increase Mcl-1 expression in tumor cells through ERK1/2-Elk-1 signaling to attenuate TRAIL-induced apoptosis

BackgroundAscites may affect the progression of ovarian cancer (OC). In particular, soluble factors present in OC ascites can create a protective environment for tumor cells that promote de novo resistance to drug- and death receptor-induced apoptosis. However, the underlying molecular mechanisms responsible for ascites-induced drug resistance are not well characterized.MethodsUsing human OC cell lines and tissues microarrays of human OC biopsies, we assessed the mechanism by which OC ascites increase Mcl-1 expression using Western blots, chemical inhibitors of ERK and small-inhibitory RNA treatments.ResultsIn the present study, we found that both Mcl-1 mRNA and protein levels were upregulated within 2 h upon treatment of OC cells with ascites obtained from women with advanced OC. In contrast, the expression of other Bcl-2 family antiapoptotic members such as Bcl-2 and Bcl-XL was not affected by ascites. An increase of Mcl-1 expression was consistently observed across different ascites from women with advanced serous OC. The knockdown of Mcl-1 significantly blocked ascites-induced Mcl-1 upregulation and ascites-mediated inhibition of TRAIL-induced apoptosis. Ascites induced a rapid phosphorylation of ERK1/2 and Elk-1 transcription factor. Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression. In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression.ConclusionsThese results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis. The ERK1/2/Elk-1/Mcl-1 pathway represents a novel mechanism by which ascites induce de novo TRAIL resistance in OC cells.

Osteoprotegerin (OPG) activates integrin, focal adhesion kinase (FAK), and Akt signaling in ovarian cancer cells to attenuate TRAIL-induced apoptosis

BackgroundResistance to apoptosis is a major problem in ovarian cancer (OC) and correlates with poor prognosis. Osteoprotegerin (OPG) is a soluble secreted factor that acts as a decoy receptor for receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). OPG has been reported to attenuate TRAIL-induced apoptosis in a variety of cancer cells, including OC cells. OPG-mediated protection against TRAIL has been attributed to its decoy receptor function. However, OPG activates integrin/focal adhesion kinase (FAK) signaling in endothelial cells. In OC cells, activation of integrin/FAK signaling inhibits TRAIL-induced apoptosis. Based on these observations, we hypothesized that OPG could attenuate TRAIL-induced apoptosis in OC cells through integrin/FAK signaling.MethodsIn vitro experiments including immunoblots, colony formation assays, and apoptosis measurements were used to assess the effect of OPG on TRAIL-induced apoptosis.ResultsExogenous OPG protected from TRAIL-induced apoptosis in a TRAIL binding-independent manner and OPG protection was αvβ3 and αvβ5 integrin/FAK signaling-dependent. Moreover, OPG-mediated activation of integrin/FAK signaling resulted in the activation of Akt. Inhibition of both integrin/FAK and Akt signaling significantly inhibited OPG-mediated attenuation of TRAIL-induced apoptosis. Although OPG also stimulated ERK1/2 phosphorylation, inhibition of ERK1/2 signaling did not significantly altered OPG protection.ConclusionsOur studies provide evidence, for the first time, that OPG can attenuate TRAIL-induced apoptosis in a TRAIL binding-independent manner through the activation of integrin/FAK/Akt signaling in OC cells.

Osteoprotegerin (OPG) protects ovarian cancer cells from TRAIL-induced apoptosis but does not contribute to malignant ascites-mediated attenuation of TRAIL-induced apoptosis

BackgroundResistance to apoptosis is a major problem in ovarian cancer and correlates with poor prognosis. Osteoprotegerin (OPG) is a secreted factor in malignant ascites and acts as a decoy receptor for receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL promotes apoptosis in ovarian cancer cells. Ovarian cancer ascites attenuate TRAIL-induced apoptosis raising the possibility that OPG contained in ascites may abrogate the anti-tumor activity of TRAIL.MethodsDetermination of OPG levels in ascites was measured by ELISA. Effect of OPG on TRAIL-induced cell death was determined by XTT and colony forming assays in ovarian cancer cell lines and primary tumor cells. Apoptosis was assessed by ELISA.ResultsWe found that recombinant OPG and malignant ascites attenuates TRAIL-induced cell death and apoptosis in a dose-dependent manner in ovarian cancer cell lines and primary ovarian tumor cells. OPG is present at high levels in the ascites of patients with ovarian cancer. We found a positive correlation between the levels of OPG in ascites and the ability of the ascites to attenuate TRAIL-induced cell death. The anti-apoptotic effect of ascites was not reversed by co-incubation with an OPG blocking antibody.ConclusionsOPG and malignant ascites protect ovarian cancer cells from TRAIL-induced apoptosis. Although malignant ascites contain high levels of OPG, OPG is not a critical component that contributes to ascites-mediated attenuation of TRAIL-induced apoptosis.

Antiapoptotic proteins Bcl-2 and Bcl-XL inhibit Clostridium difficile toxin A-induced cell death in human epithelial cells.

ABSTRACT It has been well established that Clostridium difficile toxin A (TcdA) induces cell death in human epithelial cells. However, the mechanism of TcdA-induced cell death remains to be fully characterized. Here, we show that TcdA induces dose-dependent cell death in ovarian carcinoma and colonic carcinoma cell lines. TcdA-mediated cell death, as well as caspase 8 and caspase 3 activation, were specifically abrogated by anti-toxin antibodies. Although caspase 8 and caspase 3 were activated by TcdA in OVCAR3 ovarian carcinoma and T84 colonic cancer cells, pancaspase and caspase 8, 3, and 9 inhibitors did not block TcdA-induced cell death. In contrast, tumor necrosis factor-related apoptosis-inducing ligand-induced cell death was nearly completely blocked by caspase inhibitors in OVCAR3 cells. In these cells, TcdA induces the mitochondrial pathway of apoptosis, as demonstrated by changes in mitochondrial outer membrane permeabilization (MOMP). Furthermore, overexpression of the antiapoptotic proteins Bcl-2 and Bcl-XL significantly inhibited TcdA-induced cell death, as well as TcdA-induced MOMP. Conversely, small interfering RNA-mediated inhibition of Bcl-XL in TcdA-resistant SKOV3ip1 cells enhanced TcdA-induced cell death. Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-XL in T84 cells also inhibited TcdA-induced cell death. Altogether, our data demonstrate that TcdA induces cell death in both ovarian and colonic cancer cells preferentially via the mitochondrial pathway of apoptosis by a death receptor-independent and a caspase-independent mechanism. This process is regulated by antiapoptotic members of the Bcl-2 family.