CCL18 from ascites promotes ovarian cancer cell migration through proline-rich tyrosine kinase 2 signaling.

BACKGROUND: Ovarian cancer (OC) ascites consist in a proinflammatory tumor environment that is characterized by the presence of various cytokines, chemokines and growth factors. The presence of these inflammatory-related factors in ascites is associated with a more aggressive tumor phenotype. CCL18 is a member of CCL chemokines and its expression has been associated with poor prognosis in some cancers. However, its role in OC progression has not been established. Therefore, the aim of the current study was to elucidate the role of ascites CCL18 in OC progression. METHODS: ELISA and tissue microarrays were used to assess CCL18 in ascites and phospho-Pyk2 expression in cancer tissues respectively. Cell migration was assessed using Boyden chambers. CCL18 and ascites signaling was examined in ovarian cancer cells utilizing siRNA and exogenous gene expression. RESULTS: Here, we show that CCL18 levels are markedly increased in advanced serous OC ascites relative to peritoneal effusions from women with benign conditions. Ascites and CCL18 dose-dependently enhanced the migration of OC cell lines CaOV3 and OVCAR3. CCL18 levels in ascites positively correlated with the ability of ascites to promote cell migration. CCL18 blocking antibodies significantly attenuated ascites-induced cell migration. Ascites and CCL18 stimulated the phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) in CaOV3 and OVCAR3 cells. Most importantly, the expression of phosphorylated Pyk2 in serous OC tumors was associated with shorter progression-free survival. Furthermore, enforced expression of Pyk2 promoted tumor cell migration while siRNA-mediated downregulation of Pyk2 attenuated cell migration. Downregulation of Pyk2 markedly inhibited ascites and CCL18-induced cell migration. CONCLUSIONS: Taken together, our findings establish an important role for CCL18, as a component of ascites, in the migration of tumor cells and identify Pyk2 as prognostic factor and a critical downstream signaling pathway for ascites-induced OC cell migration.

Ovarian cancer ascites enhance the migration of patient-derived peritoneal mesothelial cells via cMet pathway through HGF-dependent and -independent mechanisms.

Ovarian cancer ascites consist of a proinflammatory environment that is characterized by the presence of abundant human peritoneal mesothelial cells (HPMCs). Cytokines and growth factors in ascites modulate cell activities of tumor cells. The expression of proinflammatory cytokines in ascites is associated with a more aggressive tumor phenotype. The effect of ascites on HPMCs is for the most part unknown but this interplay is thought to be important for epithelial ovarian cancer (EOC) progression. Here, we examine the components of ascites, which stimulate patient-derived HPMC migration, from women with advanced EOC. We show that ovarian cancer ascites enhanced the migration of HPMCs. This effect was inhibited by heat treatment, hepatocyte growth factor (HGF) blocking antibodies and a HGF receptor (cMet) inhibitor. In ovarian cancer ascites, HGF is present at high concentration compared to benign fluids. Ascites-mediated activation of cMet was associated with Akt and EKR1/2 phosphorylation. This response was partly inhibited by heat treatment and cMet inhibitor. Ascites-induced migration and a cMet phosphorylation were strongly inhibited by epidermal growth factor receptor (EGFR) inhibitor PD153035, suggesting the transactivation of cMet by EGFR. Our study suggests that HGF and ligands of EGFR are factors that mediate ovarian cancer ascites-mediated migration of HPMCs by activating cMet and possibly downstream ERK1/2 and Akt pathways. The study provides evidence for the first time that ascites not only support tumor growth but also enhance the migratory potential of cancer-associated mesothelial cells, which in turn may support cancer progression.

Inflammation-regulating factors in ascites as predictive biomarkers of drug resistance and progression-free survival in serous epithelial ovarian cancers.

BACKGROUND: Platinum-based combination therapy is the standard first-line treatment for women with advanced serous epithelial ovarian carcinoma (EOC). However, about 20 % will not respond and are considered clinically resistant. The availability of biomarkers to predict responses to the initial therapy would provide a practical approach to identify women who would benefit from a more appropriate first-line treatment. Ascites is an attractive inflammatory fluid for biomarker discovery as it is easy and minimally invasive to obtain. The aim of this study was to evaluate whether six selected inflammation-regulating factors in ascites could serve as diagnostic or drug resistance biomarkers in patients with advanced serous EOC. METHODS: A total of 53 women with stage III/IV serous EOC and 10 women with benign conditions were enrolled in this study. Eleven of the 53 women with serous EOC were considered clinically resistant to treatment with progression-free survival<6 months. Ascites were collected at the time of the debulking surgery and the levels of cytokines were measured by ELISA. The six selected cytokines were evaluated for their ability to discriminate serous EOC from benign controls, and to discriminate platinum resistant from platinum sensitive patients. RESULTS: Median ascites levels of IL-6, IL-10 and osteoprotegerin (OPG) were significantly higher in women with advanced serous EOC than in controls (P≤0.012). There were no significant difference in the median ascites levels of leptin, soluble urokinase plasminogen activator receptor (suPAR) and CCL18 among serous EOC women and controls. In Receiver Operator curve (ROC) analysis, IL-6, IL-10 and OPG had a high area under the curve value of 0.905, 0.832 and 0.825 respectively for distinguishing EOC from benign controls. ROC analysis of individual cytokines revealed low discriminating potential to stratify patients according to their sensitivity to first-line treatment. The combination of biomarkers with the highest discriminating potential was with CA125 and leptin (AUC=0.936, 95% CI: 0.894-0.978). CONCLUSION: IL-6 was found to be strongly associated with advanced serous EOC and could be used in combination with serum CA125 to discriminate benign and EOC. Furthermore, the combination of serum CA125 and ascites leptin was a strong predictor of clinical resistance to first-line therapy.

Role of malignant ascites on human mesothelial cells and their gene expression profiles.

BACKGROUND: Malignant ascites is often present at diagnostic in women with advanced ovarian cancer (OC) and its presence is associated with a worse outcome. Human peritoneal mesothelial cells (HPMCs) are key components of malignant ascites. Although the interplay between HPMCs and OC cells is believed to be critical for tumor progression, it has not been well characterized. The purpose of this study was to assess the effect of ascites on HPMCs and clarify the role of HPMCs in OC progression. METHODS: Human OC ascites and benign peritoneal fluids were assessed for their ability to stimulate HPMC proliferation. Conditioned medium from ascites- and benign fluid-stimulated HPMCs were compared for their ability to attenuate apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). We conducted a comparative analysis of global expression changes in ascites-stimulated HPMCs using Agilent oligonucleotide microarrays. RESULTS: As compared to benign peritoneal fluids, malignant ascites stimulated the proliferation of HPMCs. TRAIL-induced apoptosis was attenuated in OC cells exposed to conditioned medium from ascites-stimulated HPMCs as compared to OC cells exposed to conditioned medium from benign fluid-stimulated HPMCs. A total of 649 genes were differentially expressed in ascites-stimulated HPMCs. Based on a ratio of more than 1.5-fold and a P < 0.05, 484 genes were up-regulated and 165 genes were down-regulated in ascites-exposed HPMCs. Stimulation of HPMCs with OC ascites resulted in differential expression of genes mainly associated with the regulation of cell growth and proliferation, cell death, cell cycle and cell assembly and organization, compared to benign peritoneal fluids. Top networks up-regulated by OC ascites included Akt and NF-κB survival pathways whereas vascular endothelial growth factor (VEGF) pathway was down-regulated. CONCLUSIONS: The results of this study not only provide evidence supporting the importance of the interplay between cancer cells and HPMCs but also define the role that the tumor environment plays in these interactions.

MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression.

BACKGROUND: MUC16 (CA125) is a large transmembrane mucin protein (> 200 kDa) aberrantly expressed in approximately 80% of human epithelial ovarian cancers (EOC). MUC16 expression in EOC cells is associated with increased tumorigenesis and inhibiton of genotoxic drug-induced apoptosis. However, the mechanism by which MUC16 mediates these effects is unknown. In the present study, we investigated the mechanisms by which MUC16 attenuates TRAIL-induced apoptosis. METHODS: MUC16 expression was down-regulated by stably expressing an anti-MUC16 single-chain antibody (scFv) targeted to the endoplasmic reticulum (ER), which prevents cell surface localization of MUC16 in OVCAR3 cells. We also generated a MUC16 C-terminal domain (MUC16CTD) construct that was stably expressed in MUC16 negative SKOV3 cells. RESULTS: We show that MUC16 attenuates apoptosis, activation of caspase-8 and mitochondria activation in EOC cells in response to TRAIL. MUC16 decreases TRAIL receptor R2 (DR5) expression and inhibits pro-caspase-8 activation at the death-inducing signaling complex (DISC). MUC16CTD expression is sufficient to attenuate the TRAIL signaling cascade. MUC16 knockdown decreases caspase-8 inhibitor cFLIP mRNA levels, increases cFLIP degradation, and consequently increases TRAIL-induced apoptosis. Down-regulation of cFLIP following treatment of MUC16-expressing OVCAR3 cells with cFLIP siRNA also increases TRAIL-induced apoptosis. CONCLUSIONS: These findings indicate that MUC16 protects EOC cells against TRAIL-induced apoptosis through multiple mechanisms including the blockade of TRAIL R2 expression and the regulation of cFLIP expression at both the transcriptional and the protein level.

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

BACKGROUND: Ascites 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. METHODS: Using 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. RESULTS: In 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. CONCLUSIONS: These 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.

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

BACKGROUND: The 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. METHODS: We 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. RESULTS: Mean 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. CONCLUSION: Elevated IL-6, but not IL-8, ascites level is an independent predictor of shorter progression-free survival.

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

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-X(L) significantly inhibited TcdA-induced cell death, as well as TcdA-induced MOMP. Conversely, small interfering RNA-mediated inhibition of Bcl-X(L) in TcdA-resistant SKOV3ip1 cells enhanced TcdA-induced cell death. Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-X(L) 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.

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.

Ascites IL-10 Promotes Ovarian Cancer Cell Migration.

Ovarian cancer (OC) ascites is an inflammatory and immunosuppressive tumor environment characterized by the presence of various cytokines, chemokines and growth factors. The presence of high concentrations of these cytokines/chemokines in ascites is associated with a more aggressive tumor phenotype. IL-10 is an immunosuppressive cytokine for which high expression has been associated with poor prognosis in some cancers. However, its role on OC tumor cells has not been explored. Therefore, the aim of the current study was to elucidate the role of ascites IL-10 on the proliferation, migration and survival of OC cell lines. Here, we show that IL-10 levels are markedly increased in patients with advanced serous OC ascites relative to serous stage I/II ascites and peritoneal effusions from women with benign conditions. Ascites and IL-10 dose-dependently enhanced the proliferation and migration of OC cell lines CaOV3 and OVCAR3 but had no effect on cell survival. IL-10 levels in ascites positively correlated with the ability of ascites to promote cell migration but not proliferation. Depletion of IL-10 from ascites markedly inhibited ascites-induced OC cell migration but was not crucial for ascites-mediated cell proliferation. Taken together, our findings establish an important role for IL-10, as a component of ascites, in the migration of tumor cells.

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.

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

BACKGROUND: Resistance 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. METHODS: In vitro experiments including immunoblots, colony formation assays, and apoptosis measurements were used to assess the effect of OPG on TRAIL-induced apoptosis. RESULTS: Exogenous OPG protected from TRAIL-induced apoptosis in a TRAIL binding-independent manner and OPG protection was αvß3 and αvß5 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. CONCLUSIONS: Our 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.

BACKGROUND: Resistance 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. METHODS: Determination 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. RESULTS: We 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. CONCLUSIONS: OPG 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.

Profiling of cytokines in human epithelial ovarian cancer ascites.

BACKGROUND: The behavior of tumor cells is influenced by the composition of the surrounding tumor environment. The importance of ascites in ovarian cancer (OC) progression is being increasingly recognized. The characterization of soluble factors in ascites is essential to understand how this environment affects OC progression. The development of cytokine arrays now allows simultaneous measurement of multiple cytokines per ascites using a single array. METHODS: We applied a multiplex cytokine array technology that simultaneously measures the level of 120 cytokines in ascites from 10 OC patients. The ascites concentration of a subset (n = 5) of cytokines that was elevated based on the multiplex array was validated by commercially available ELISA. The ascites level of these 5 cytokines was further evaluated by ELISA in a cohort of 38 patients. Kaplan-Meier analysis was used to assess the association of cytokine expression with progression-free survival (PFS) in this cohort. RESULTS: We observed a wide variability of expression between different cytokines and levels of specific cytokines also varied in the 10 malignant ascites tested. Fifty-three (44%) cytokines were not detected in any of the 10 ascites. The level of several factors including, among others, angiogenin, angiopoietin-2, GRO, ICAM-1, IL-6, IL-6R, IL-8, IL-10, leptin, MCP-1, MIF NAP-2, osteprotegerin (OPG), RANTES, TIMP-2 and UPAR were elevated in most malignant ascites. Higher levels of OPG, IL-10 and leptin in OC ascites were associated with shorter PFS. IL-10 was shown to promote the anti-apoptotic activity of malignant ascites whereas OPG did not. CONCLUSION: Our data demonstrated that there is a complex network of cytokine expression in OC ascites. Characterization of cytokine profiles in malignant ascites may provide information from which to prioritize key functional cytokines and understand the mechanism by which they alter tumor cells behavior. A better understanding of the cytokine network is essential to determine the role of ascites in OC progression.

Targeted ovarian cancer treatment: the TRAILs of resistance.

Ovarian cancer (OC) is the leading cause of death from gynecological malignancies. Although most patients respond to the initial therapy when presenting with advanced disease, only 10-15% maintain a complete response following first-line therapy. Recurrence defines incurable disease in most cases. Despite improvements with conventional chemotherapy combinations, the overall cure rate remained mostly stable over the years. Increased long-term survival in OC patients will only be achieved through a comprehensive understanding of the basic mechanisms of tumor cell resistance. Such knowledge will translate into the development of new targeted strategies. In addition, because OC is considered to be a heterogeneous group of diseases with distinct gene expression profiles, it is likely that different approaches to treatment for distinct sub-types will be required to optimize response. One of the new promising anti-cancer therapies is the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL has the ability to selectively induce apoptosis in tumor cells with little toxicity to normal cells. Death receptor ligands such as TRAIL rely on the activation of the apoptotic signaling pathway to destroy tumor cells. TRAIL induces the formation of a pro-apoptotic death-inducing signaling complex (DISC) via its death receptors, TRAIL receptor 1 (TRAIL R1) and TRAIL receptor 2 (TRAIL R2). The formation of the DISC activates caspase-8 which requires further signal amplification through the mitochondrial pathway for an efficient activation of effector caspases in OC cells. The initial enthusiasm for TRAIL has been hampered by accumulating data demonstrating TRAIL resistance in various tumor types including OC cells. There is, therefore, a need to identify markers of TRAIL resistance, which could represent new hits for targeted therapy that will enhance TRAIL efficacy. In addition, the identification of patients that are more likely to respond to TRAIL therapy would be highly desirable. In this review, we discuss the different molecular and cellular mechanisms leading to TRAIL resistance in OC. In particular, we address the mechanisms involved in intrinsic, acquired and environment-mediated TRAIL resistance, and their potential implication in the clinical outcome.

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

BACKGROUND: The 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. METHODS: Peritoneal 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 IC(50 )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. RESULTS: Among 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 IC(50 )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 IC(50 )with ascites/IC(50 )without ascites > 2 was associated with shorter disease-free interval. CONCLUSIONS: The 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.

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-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 pro-survival 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.

Bcl-2 decreases cell proliferation and promotes accumulation of cells in S phase without affecting the rate of apoptosis in human ovarian carcinoma cells.

OBJECTIVES: The Bcl-2 protein is an important regulator of the apoptotic cascade and promotes cell survival. Bcl-2 can also delay entry into the cell cycle from quiescence. In the present study, we used two isogenic human ovarian carcinoma cell lines, which expressed differential levels of Bcl-2 proteins, to demonstrate that Bcl-2 may regulate the growth rates of adenocarcinoma cells. METHODS: The growth rates of two isogenic ovarian cancer cell lines were determined by XTT assays and flow cytometry combined with PI staining. Bcl-2-overexpressing SKOV3 cells were modified to express a doxycycline-inducible anti-Bcl-2 single-chain antibody and the effects of Bcl-2 protein inhibition on cell proliferation and apoptosis were assessed. RESULTS: We demonstrate that Bcl-2 promotes the accumulation of proliferating carcinoma cells in S phase. The Bcl-2-overexpressing SKOV3 cell line proliferates markedly faster and shows delayed progression to G2M phase compared to its low Bcl-2-expressing counterpart SKOV3.ip1 cell line. Single-chain antibody-mediated inhibition of Bcl-2 in SKOV3 cells was associated with increased growth rates and more rapid cell cycle progression. Treatment with cisplatin resulted in more cells accumulating in S phase in Bcl-2-overexpressing SKOV3 cells, while the inhibition of Bcl-2 abolished delayed entry into G2M phase without affecting cisplatin-induced apoptosis. CONCLUSIONS: Our results suggest that, in ovarian cancer cells, Bcl-2 delays cell cycle progression by promoting accumulation of cells in S phase without affecting the rate of apoptosis. Thus, in addition to its known role at the G0/G1 checkpoint, we demonstrate for the first time that Bcl-2 also regulates the S phase.

Role of IFN-gamma and IL-2 in rat lung epithelial cell migration and apoptosis after oxidant injury.

In the present study, IFN-gamma exposure to primary cultures of rat type II epithelial cells (TIIP) upregulated membrane expression of the common gamma-chain of the IL-2 receptor (approximately 2.5- to 4-fold increase) and redistributed receptor affinity in TIIP, as assessed by Western blot, cell, and tissue histochemistry and Scatchard analysis. As for restitution processes of the lung epithelium, functionality of IL-2R on TIIP was conditional to IFN-gamma exposure: 1) IFN-gamma priming promoted a fivefold increase of IL-2-driven TIIP locomotion (P < 0.05 vs. control at 100 U/ml) and 2) IFN-gamma coincubation with IL-2 reduced bleomycin-induced TIIP apoptosis in vitro by 25% (caspase-3 activity) and by approximately 70% (TdT-mediated dUTP nick end labeling/4',6'-diamidino-2-phenylindole assay) as well as in vivo by approximately 90% (caspase-3 activity; P < 0.05 vs. control). Sustained p42/44 extracellular signal-regulated kinase activity played a protective role in this process, whereas specific inhibition by PD-98059 (50 microM) significantly reversed bleomycin-induced TIIP apoptosis (P < 0.05 vs. control). From these in vitro and in vivo data, it is proposed that combinations of IFN-gamma and IL-2 can drive repair activity of TIIP by stimulating migration and preventing programmed cell death, both of which are speculated to be very fast restitution events after oxidant-induced acute lung injury.

Differential induction of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand in human ovarian carcinoma cells.

OBJECTIVES: In this study, we examine the sensitivity of a panel of ovarian carcinoma cells, which includes four primary ovarian cancer cell samples, and four normal ovarian epithelium samples to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We also examine the intracellular regulation of TRAIL-mediated apoptosis. METHODS: The sensitivity to TRAIL was determined by short-term survival assays on seven ovarian carcinoma cell lines, four primary samples of ovarian cancer, and four normal ovarian epithelium samples. We assessed the activation of the apoptotic pathway in TRAIL-resistant and -sensitive tumor cells. The expression of TRAIL receptors was determined by flow cytometry. The protein expression of FADD, XIAP, caspase-8, caspase-3, BAX, and c-FLIP were determined by immunoblot analyses. RESULTS: We show that ovarian cancer cells display variable sensitivity to TRAIL-induced apoptosis although most cell lines have similar sensitivity to cisplatin. Normal ovarian epithelium samples were mostly sensitive to TRAIL. In sensitive cells, TRAIL induced caspase-8-dependent apoptosis, which subsequently led to activation of caspase-3. Both sensitive and resistant cells expressed caspase-8, caspase-3, FADD, XIAP, and c-FLIP at similar levels. A significant enhancement in cell death was observed in TRAIL-resistant cells when c-FLIP(L) levels were downregulated by RNA interference. CONCLUSIONS: These data suggest that sensitivity to TRAIL and chemotherapy does not necessarily correlate in human ovarian cancer cells. Cancerous cells isolated from patients with ovarian cancer show variable sensitivity to TRAIL but most normal ovarian epithelial cells are sensitive. In human ovarian cancer cells, c-FLIP(L) may participate to the regulation of the TRAIL signaling cascade.