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

Matte I, Lane D, Bachvarov D, Rancourt C, Piché A - BMC Cancer (2014)

Bottom Line: 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).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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Microbiologie et Infectiologie, Faculté de Médecine, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, Québec, J1H 5N4, Canada. alain.piche@usherbrooke.ca.

ABSTRACT

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.

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TRAIL-induced apoptosis in ascites-stimulated HPMCs. (A) Diagram of HPMC-priming assays. Ascites-stimulated or benign fluid-stimulated HPMCs were culture overnight (shown in yellow), washed TWICE and cultured in serum/hormone-free medium for 8 to 24 h to generate HPMC-conditioned medium (shown in pink) that were collected at either 12 h (B) or different time points (C). HPMC-conditioned medium was then added to CaOV3 tumor cells in the presence of TRAIL (25 ng/ml). TRAIL-induced apoptosis was measured in CaOV3 cells incubated with the indicated HPMC-conditioned medium overnight and expressed as fold increased relative to cells that were exposed to HPMC-conditioned medium but not to TRAIL. Data are expressed as means of triplicates from three independent experiments ± SD. * indicate P < 0.01.
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Figure 3: TRAIL-induced apoptosis in ascites-stimulated HPMCs. (A) Diagram of HPMC-priming assays. Ascites-stimulated or benign fluid-stimulated HPMCs were culture overnight (shown in yellow), washed TWICE and cultured in serum/hormone-free medium for 8 to 24 h to generate HPMC-conditioned medium (shown in pink) that were collected at either 12 h (B) or different time points (C). HPMC-conditioned medium was then added to CaOV3 tumor cells in the presence of TRAIL (25 ng/ml). TRAIL-induced apoptosis was measured in CaOV3 cells incubated with the indicated HPMC-conditioned medium overnight and expressed as fold increased relative to cells that were exposed to HPMC-conditioned medium but not to TRAIL. Data are expressed as means of triplicates from three independent experiments ± SD. * indicate P < 0.01.

Mentions: Soluble factors produced by cancer-associated fibroblasts and bone marrow stromal cells have been shown to confer resistance to TRAIL-induced apoptosis in tumor cells [19-21]. We reasoned that malignant ascites-stimulated HPMCs might also secrete soluble factors that could attenuate TRAIL-induced apoptosis. HPMCs were incubated with benign fluids or malignant ascites overnight. The cells were then washed twice and conditioned media (CM) were collected 12 h later. Ovarian cancer CaOV3 cells were treated with TRAIL in presence of CM from HPMCs exposed to either benign fluids or malignant ascites and apoptosis was measured (Figure 3A). As shown in Figure 3B, TRAIL-induced apoptosis was decreased in CaOV3 cells exposed to CM from malignant ascites-exposed HPMCs as compared to CM from benign fluid-exposed HPMCs. These results suggest that ascites-stimulated HPMCs secrete soluble factors that attenuate TRAIL-induced apoptosis. To examine the effect of ascites exposure on the secretion of soluble factors overtime, HPMCs were stimulated with malignant ascites or benign fluids overnight. Cells were then washed twice and CM were collected after 8, 12 and 24 h. Whereas CM from benign fluid-stimulated HPMCs collected at different time did not affect TRAIL-induced apoptosis (OV370, OV401), CM from ascites-stimulated HPMCs significantly reduced apoptosis in CaOV3 cells (Figure 3C). The maximum protection was observed at 12 h.


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

Matte I, Lane D, Bachvarov D, Rancourt C, Piché A - BMC Cancer (2014)

TRAIL-induced apoptosis in ascites-stimulated HPMCs. (A) Diagram of HPMC-priming assays. Ascites-stimulated or benign fluid-stimulated HPMCs were culture overnight (shown in yellow), washed TWICE and cultured in serum/hormone-free medium for 8 to 24 h to generate HPMC-conditioned medium (shown in pink) that were collected at either 12 h (B) or different time points (C). HPMC-conditioned medium was then added to CaOV3 tumor cells in the presence of TRAIL (25 ng/ml). TRAIL-induced apoptosis was measured in CaOV3 cells incubated with the indicated HPMC-conditioned medium overnight and expressed as fold increased relative to cells that were exposed to HPMC-conditioned medium but not to TRAIL. Data are expressed as means of triplicates from three independent experiments ± SD. * indicate P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008408&req=5

Figure 3: TRAIL-induced apoptosis in ascites-stimulated HPMCs. (A) Diagram of HPMC-priming assays. Ascites-stimulated or benign fluid-stimulated HPMCs were culture overnight (shown in yellow), washed TWICE and cultured in serum/hormone-free medium for 8 to 24 h to generate HPMC-conditioned medium (shown in pink) that were collected at either 12 h (B) or different time points (C). HPMC-conditioned medium was then added to CaOV3 tumor cells in the presence of TRAIL (25 ng/ml). TRAIL-induced apoptosis was measured in CaOV3 cells incubated with the indicated HPMC-conditioned medium overnight and expressed as fold increased relative to cells that were exposed to HPMC-conditioned medium but not to TRAIL. Data are expressed as means of triplicates from three independent experiments ± SD. * indicate P < 0.01.
Mentions: Soluble factors produced by cancer-associated fibroblasts and bone marrow stromal cells have been shown to confer resistance to TRAIL-induced apoptosis in tumor cells [19-21]. We reasoned that malignant ascites-stimulated HPMCs might also secrete soluble factors that could attenuate TRAIL-induced apoptosis. HPMCs were incubated with benign fluids or malignant ascites overnight. The cells were then washed twice and conditioned media (CM) were collected 12 h later. Ovarian cancer CaOV3 cells were treated with TRAIL in presence of CM from HPMCs exposed to either benign fluids or malignant ascites and apoptosis was measured (Figure 3A). As shown in Figure 3B, TRAIL-induced apoptosis was decreased in CaOV3 cells exposed to CM from malignant ascites-exposed HPMCs as compared to CM from benign fluid-exposed HPMCs. These results suggest that ascites-stimulated HPMCs secrete soluble factors that attenuate TRAIL-induced apoptosis. To examine the effect of ascites exposure on the secretion of soluble factors overtime, HPMCs were stimulated with malignant ascites or benign fluids overnight. Cells were then washed twice and CM were collected after 8, 12 and 24 h. Whereas CM from benign fluid-stimulated HPMCs collected at different time did not affect TRAIL-induced apoptosis (OV370, OV401), CM from ascites-stimulated HPMCs significantly reduced apoptosis in CaOV3 cells (Figure 3C). The maximum protection was observed at 12 h.

Bottom Line: 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).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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Microbiologie et Infectiologie, Faculté de Médecine, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, Québec, J1H 5N4, Canada. alain.piche@usherbrooke.ca.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus