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25-Hydroxycholesterol exerts both a cox-2-dependent transient proliferative effect and cox-2-independent cytotoxic effect on bovine endothelial cells in a time- and cell-type-dependent manner.

Nguyen VP, Chen SH, Pizzuto K, Cantarutti A, Terminesi A, Mendonca C, Dumont DJ - J Angiogenes Res (2010)

Bottom Line: These results suggest that some effects of 25-OHC on cells may be dependent on Cox-2 enzymatic activity.Cox-2 dependent elevating effects of 25-OHC on endothelial cell proliferation was transient.The lack of uniform response by the three endothelial cell types examined suggests that our model system of primary cultures of bmLECs, bmVECs, and bmAECs may aid the evaluation of celecoxib in inhibiting proliferation of different types of tumour-associated endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular and Cellular Biology Research, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON, M4N 3M5, Canada. dan.dumont@sri.utoronto.ca.

ABSTRACT

Background: 25-hydroxycholesterol (25-OHC) is a product of oxidation of dietary cholesterol present in human plasma. 25-OHC and other oxidized forms of cholesterol are implicated in modulating inflammatory responses involved in development of atherosclerosis and colon carcinogenesis.

Methods: Primary lymphatic, venous and arterial endothelial cells isolated from bovine mesentery (bmLEC, bmVEC, bmAEC) were treated with 25-OHC and tested for several different cellular parameters.

Results: We found 25-OHC to be a potent inducer of cyclooxygenase-2 (Cox-2, prostaglandin G-H synthase-2) expression in bovine mesenteric lymphatic, venous, and arterial endothelial cells. The induction of Cox-2 expression in endothelial cells by 25-OHC led to an initial increase in cellular proliferation that was inhibited by the Cox-2 selective inhibitor celecoxib (Celebrex). Prolonged exposure to 25-OHC was cytotoxic. Furthermore, endothelial cells induced to express Cox-2 by 25-OHC were more sensitive to the effects of the Cox-2 selective inhibitor celecoxib (Celebrex). These results suggest that some effects of 25-OHC on cells may be dependent on Cox-2 enzymatic activity.

Conclusions: Cox-2 dependent elevating effects of 25-OHC on endothelial cell proliferation was transient. Prolonged exposure to 25-OHC caused cell death and enhanced celecoxib-induced cell death in a cell-type dependent manner. The lack of uniform response by the three endothelial cell types examined suggests that our model system of primary cultures of bmLECs, bmVECs, and bmAECs may aid the evaluation of celecoxib in inhibiting proliferation of different types of tumour-associated endothelial cells.

No MeSH data available.


Related in: MedlinePlus

48 hour exposure to 25-OHC sensitized Cox-2-negative cells to effects of Celecoxib. A/ Celecoxib concentrations of 5 and 10 μM caused the same reduction in viable cell numbers of bmLECs whether or not 25-OHC was present. Unlike bmLECs, viable numbers of bmVECs not treated with 25-OHC did not change as a result of treatment with celecoxib until the concentration was above 5 μM. Mean of fold differences between EtOH-treated (vehicle, 25-OHC untreated) bmLECs and bmVECs in the presence of 5 μM celecoxib analysed by t-test generated p-value of 0.0005 (n = 4). B/ Unlike either bmVECs or bmLECs, even 5 μM of celecoxib statistically significantly reduced viable bmAEC cell counts slightly (p = 0.012, n = 4). Cell count was reduced to a greater extent in the presence of 25-OHC. Mean fold differences between 25-OHC treated and 25-OHC untreated bmAECs at 10 μM analysed by t-test generated p-value of 0.0004 (n = 4). C/ Low doses of celecoxib also increased reduction in viable cell counts of colorectal carcinoma cells HCT-116 (previously shown to be Cox-2 deficient) in the presence of 25-OHC. Unlike ECs, viable HCT-116 cell numbers did not differ from vehicle control in the absence of 25-OHC. Mean fold difference between cell counts of HCT-116 treated and not treated with 25-OHC analysed by t-test generated p-value of 0.029 at 5 μM celecoxib, and a p-value of 0.0006 at 15 μM (n = 3).
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Figure 4: 48 hour exposure to 25-OHC sensitized Cox-2-negative cells to effects of Celecoxib. A/ Celecoxib concentrations of 5 and 10 μM caused the same reduction in viable cell numbers of bmLECs whether or not 25-OHC was present. Unlike bmLECs, viable numbers of bmVECs not treated with 25-OHC did not change as a result of treatment with celecoxib until the concentration was above 5 μM. Mean of fold differences between EtOH-treated (vehicle, 25-OHC untreated) bmLECs and bmVECs in the presence of 5 μM celecoxib analysed by t-test generated p-value of 0.0005 (n = 4). B/ Unlike either bmVECs or bmLECs, even 5 μM of celecoxib statistically significantly reduced viable bmAEC cell counts slightly (p = 0.012, n = 4). Cell count was reduced to a greater extent in the presence of 25-OHC. Mean fold differences between 25-OHC treated and 25-OHC untreated bmAECs at 10 μM analysed by t-test generated p-value of 0.0004 (n = 4). C/ Low doses of celecoxib also increased reduction in viable cell counts of colorectal carcinoma cells HCT-116 (previously shown to be Cox-2 deficient) in the presence of 25-OHC. Unlike ECs, viable HCT-116 cell numbers did not differ from vehicle control in the absence of 25-OHC. Mean fold difference between cell counts of HCT-116 treated and not treated with 25-OHC analysed by t-test generated p-value of 0.029 at 5 μM celecoxib, and a p-value of 0.0006 at 15 μM (n = 3).

Mentions: Low doses of celecoxib (5-10 μM) have been shown to induce G2/M cell cycle arrest through inhibition of Cox-2 dependent prostaglandin E2 production [21]. In order to compare the effects of celecoxib on cells with undetectable basal levels of Cox-2 and cells induced to express detectable levels of Cox-2, we performed cell counts with trypan blue after growing cells for a period of 48 hours in the presence of low doses of celecoxib. We chose 48 hours because exposure of bmECs to 25-OHC over this period of time was not long enough to result in toxic effects leading to massive cell death as measured in Figure 3. Furthermore the initial growth advantage conferred on cells by 25-OHC induction of Cox-2 shown in Figure 1 was no longer detectable after 48 hours due to contact inhibition. Low dose celecoxib caused the same reduction in viable bmLEC cell numbers whether or not 25-OHC was present (Figure 4a). Unlike bmLECs, viable numbers of bmVECs not treated with 25-OHC did not change as a result of treatment with celecoxib until the concentration was above 5 μM (Figure 4a). Unlike either bmVECs or bmLECs, even 5 μM of celecoxib reduced viable bmAEC cell counts, which was reduced to a greater extent in the presence of 25-OHC (Figure 4b). Treatment of bmAECs with 50 μM of celecoxib in the presence of 25-OHC caused massive cell death reducing viable cell numbers to below one-fifth of vehicle control. These results indicate that each bmEC type exhibited different levels of sensitivity to celecoxib with or without 25-OHC treatment.


25-Hydroxycholesterol exerts both a cox-2-dependent transient proliferative effect and cox-2-independent cytotoxic effect on bovine endothelial cells in a time- and cell-type-dependent manner.

Nguyen VP, Chen SH, Pizzuto K, Cantarutti A, Terminesi A, Mendonca C, Dumont DJ - J Angiogenes Res (2010)

48 hour exposure to 25-OHC sensitized Cox-2-negative cells to effects of Celecoxib. A/ Celecoxib concentrations of 5 and 10 μM caused the same reduction in viable cell numbers of bmLECs whether or not 25-OHC was present. Unlike bmLECs, viable numbers of bmVECs not treated with 25-OHC did not change as a result of treatment with celecoxib until the concentration was above 5 μM. Mean of fold differences between EtOH-treated (vehicle, 25-OHC untreated) bmLECs and bmVECs in the presence of 5 μM celecoxib analysed by t-test generated p-value of 0.0005 (n = 4). B/ Unlike either bmVECs or bmLECs, even 5 μM of celecoxib statistically significantly reduced viable bmAEC cell counts slightly (p = 0.012, n = 4). Cell count was reduced to a greater extent in the presence of 25-OHC. Mean fold differences between 25-OHC treated and 25-OHC untreated bmAECs at 10 μM analysed by t-test generated p-value of 0.0004 (n = 4). C/ Low doses of celecoxib also increased reduction in viable cell counts of colorectal carcinoma cells HCT-116 (previously shown to be Cox-2 deficient) in the presence of 25-OHC. Unlike ECs, viable HCT-116 cell numbers did not differ from vehicle control in the absence of 25-OHC. Mean fold difference between cell counts of HCT-116 treated and not treated with 25-OHC analysed by t-test generated p-value of 0.029 at 5 μM celecoxib, and a p-value of 0.0006 at 15 μM (n = 3).
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Figure 4: 48 hour exposure to 25-OHC sensitized Cox-2-negative cells to effects of Celecoxib. A/ Celecoxib concentrations of 5 and 10 μM caused the same reduction in viable cell numbers of bmLECs whether or not 25-OHC was present. Unlike bmLECs, viable numbers of bmVECs not treated with 25-OHC did not change as a result of treatment with celecoxib until the concentration was above 5 μM. Mean of fold differences between EtOH-treated (vehicle, 25-OHC untreated) bmLECs and bmVECs in the presence of 5 μM celecoxib analysed by t-test generated p-value of 0.0005 (n = 4). B/ Unlike either bmVECs or bmLECs, even 5 μM of celecoxib statistically significantly reduced viable bmAEC cell counts slightly (p = 0.012, n = 4). Cell count was reduced to a greater extent in the presence of 25-OHC. Mean fold differences between 25-OHC treated and 25-OHC untreated bmAECs at 10 μM analysed by t-test generated p-value of 0.0004 (n = 4). C/ Low doses of celecoxib also increased reduction in viable cell counts of colorectal carcinoma cells HCT-116 (previously shown to be Cox-2 deficient) in the presence of 25-OHC. Unlike ECs, viable HCT-116 cell numbers did not differ from vehicle control in the absence of 25-OHC. Mean fold difference between cell counts of HCT-116 treated and not treated with 25-OHC analysed by t-test generated p-value of 0.029 at 5 μM celecoxib, and a p-value of 0.0006 at 15 μM (n = 3).
Mentions: Low doses of celecoxib (5-10 μM) have been shown to induce G2/M cell cycle arrest through inhibition of Cox-2 dependent prostaglandin E2 production [21]. In order to compare the effects of celecoxib on cells with undetectable basal levels of Cox-2 and cells induced to express detectable levels of Cox-2, we performed cell counts with trypan blue after growing cells for a period of 48 hours in the presence of low doses of celecoxib. We chose 48 hours because exposure of bmECs to 25-OHC over this period of time was not long enough to result in toxic effects leading to massive cell death as measured in Figure 3. Furthermore the initial growth advantage conferred on cells by 25-OHC induction of Cox-2 shown in Figure 1 was no longer detectable after 48 hours due to contact inhibition. Low dose celecoxib caused the same reduction in viable bmLEC cell numbers whether or not 25-OHC was present (Figure 4a). Unlike bmLECs, viable numbers of bmVECs not treated with 25-OHC did not change as a result of treatment with celecoxib until the concentration was above 5 μM (Figure 4a). Unlike either bmVECs or bmLECs, even 5 μM of celecoxib reduced viable bmAEC cell counts, which was reduced to a greater extent in the presence of 25-OHC (Figure 4b). Treatment of bmAECs with 50 μM of celecoxib in the presence of 25-OHC caused massive cell death reducing viable cell numbers to below one-fifth of vehicle control. These results indicate that each bmEC type exhibited different levels of sensitivity to celecoxib with or without 25-OHC treatment.

Bottom Line: These results suggest that some effects of 25-OHC on cells may be dependent on Cox-2 enzymatic activity.Cox-2 dependent elevating effects of 25-OHC on endothelial cell proliferation was transient.The lack of uniform response by the three endothelial cell types examined suggests that our model system of primary cultures of bmLECs, bmVECs, and bmAECs may aid the evaluation of celecoxib in inhibiting proliferation of different types of tumour-associated endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular and Cellular Biology Research, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON, M4N 3M5, Canada. dan.dumont@sri.utoronto.ca.

ABSTRACT

Background: 25-hydroxycholesterol (25-OHC) is a product of oxidation of dietary cholesterol present in human plasma. 25-OHC and other oxidized forms of cholesterol are implicated in modulating inflammatory responses involved in development of atherosclerosis and colon carcinogenesis.

Methods: Primary lymphatic, venous and arterial endothelial cells isolated from bovine mesentery (bmLEC, bmVEC, bmAEC) were treated with 25-OHC and tested for several different cellular parameters.

Results: We found 25-OHC to be a potent inducer of cyclooxygenase-2 (Cox-2, prostaglandin G-H synthase-2) expression in bovine mesenteric lymphatic, venous, and arterial endothelial cells. The induction of Cox-2 expression in endothelial cells by 25-OHC led to an initial increase in cellular proliferation that was inhibited by the Cox-2 selective inhibitor celecoxib (Celebrex). Prolonged exposure to 25-OHC was cytotoxic. Furthermore, endothelial cells induced to express Cox-2 by 25-OHC were more sensitive to the effects of the Cox-2 selective inhibitor celecoxib (Celebrex). These results suggest that some effects of 25-OHC on cells may be dependent on Cox-2 enzymatic activity.

Conclusions: Cox-2 dependent elevating effects of 25-OHC on endothelial cell proliferation was transient. Prolonged exposure to 25-OHC caused cell death and enhanced celecoxib-induced cell death in a cell-type dependent manner. The lack of uniform response by the three endothelial cell types examined suggests that our model system of primary cultures of bmLECs, bmVECs, and bmAECs may aid the evaluation of celecoxib in inhibiting proliferation of different types of tumour-associated endothelial cells.

No MeSH data available.


Related in: MedlinePlus