Anandamide (AEA) induced cell death was partially attenuated by the cyclooxygenase 2 (COX-2) selective inhibitor NS398. Three colorectal carcinoma cell lines, SW480 (A, B), HT29 (C, D) and HCA7/C29 (E, F) were pretreated with 10 μM NS398 for 24 hours followed by treatment with AEA or with AEA alone. Cells were retreated after 72 hours when cells shed into the medium were counted. After a total of six days of treatment with AEA, adherent and shed cells were harvested, based on a method by Diaz and colleagues.32 Adherent cell yield was represented as a percentage of vehicle control (A, C, E). Shed cell numbers were pooled at the 72 hour and six day time points and represented as a proportion of total cell yield (B, D, F). One way ANOVA indicated that NS398 significantly protected against AEA induced cell death in HT29 cells at all three concentrations of AEA, for both adherent (p<0.05) and shed cell data (p<0.001), and HCA7/C29 cells for both adherent and shed cell data (p<0.05) at 10 μM AEA and above. NS398 did not affect the response of SW480 cells to AEA. Data are means (SEM) from three separate experiments conducted in triplicate. Controls included vehicle control, and NS398; for all cell lines there was no significant difference between vehicle and NS398 treated cells.

 Anandamide induced cell death was neither apoptosis nor necrosis. (A) HT29 cells were treated for six days with either vehicle control (control) or 25 μM anandamide (anandamide) and adherent (Ad) and shed (F) cell samples were subjected to DNA extraction and run on an agarose gel. HT29 cells were also treated with 0.5 μg/ml TRAIL for 16 hours as a positive control for apoptosis or 32 mM H₂O₂ as a positive control for necrosis. The pattern of DNA fragmentation for anandamide treated cells appeared to be different from apoptosis but produced a similar pattern to necrosis. (B) Dual parameter blots of propidium iodide (PI) versus annexin V-FITC obtained in (i) vehicle control HT29 shed cells (Spontaneous), (ii) 25 μM anandamide shed cells (Anandamide), or (iii) 32 mM H₂O₂ shed cells (H₂O₂) treated for 72 hours. Percentages shown are means (SD) of proportions of cells distributed within quadrants from at least three separate experiments. Lower left, negative; lower right, annexin V; upper right, annexin V/PI; upper left, PI.

 Inhibition of anandamide hydrolysis to arachidonic acid via inhibition of fatty acid amide hydrolase (FAAH) potentiated anandamide induced cell death. (A) FAAH mRNA expression was detected in colorectal carcinoma (CRC) cell lines by reverse transcriptase-polymerase chain reaction. Lane 1, CaCo2 cells are known to express FAAH mRNA15; lane 2, SW480; lane 3, HT29; lane 4, HCA7/C29. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown in the lower panel. FAAH amplification was confirmed by sequencing. FAAH protein expression was confirmed by western blotting (B). The selective FAAH inhibitor MAFP significantly potentiated both growth inhibition induced by anandamide (AEA 25 μM) (C) and cell death (D) in HT29 cells after 72 hours of treatment. ***p<0.001 versus control by Dunnett’s post hoc t test. Data points shown are means (SEM) from three separate experiments conducted in triplicate. Shed cells were collected from these experiments and subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis for the presence of cleaved PARP (E). Adherent cells samples (Ad) were used as a positive control for intact (116 kDa) and cleaved PARP (89 kDa). PARP cleavage was investigated in shed cell samples, blank control (blank) (2% FBS NUT mix medium), vehicle control (control) (2% FBS NUT mix medium+ethanol), 0.1 μM MAFP (MAFP), 25 μM anandamide (AEA), and 0.1 μM MAFP in combination with 25 μM anandamide (MAFP+AEA). Intact PARP was detected in both AEA alone and in the combination of AEA and MAFP but cell death induced by the combination of AEA and MAFP potentiated the reduction in PARP cleavage resulting in less cleaved PARP compared with anandamide only treated samples. Repeat probing for α-tubulin controls for equal loading and transfer.

 Cyclooxygenase (COX) expression and induction of cell death by anandamide in colorectal carcinoma (CRC) cell lines. (A) Sodium dodecyl sulphate-polyacrylamide gel western blot analysis of COX-1 and COX-2 protein levels in SW480, HT29, and HCA7/C29 cells. HCA7/C29 are known to express high levels of COX-2 protein39 and were used here as a positive control. SW480 express very low levels of COX-2 and in the exposure shown COX-2 protein is not visible in SW480 cells but could be detected in longer exposures. Equal loading was confirmed by reprobing for α-tubulin. (B) Three CRC cell lines were treated with vehicle control or anandamide 25 or 50 μM for a period of 72 hours. Adherent cell yields are represented as a percentage of vehicle control. Anandamide treatment resulted in decreased cell yield in HT29 and HCA7/C29 cells (moderate and high COX-2 expressors, respectively) with a corresponding increase in the proportion of cell shedding (C), representing cell death. The very low COX-2 expressor (SW480) was not affected by anandamide treatment. **p<0.01, ***p<0.001 versus control by Dunnett’s post hoc t test. All data are means (SEM) from three separate experiments, conducted in triplicate. Blank controls (2% fetal bovine serum (FBS) NUT mix medium) were also included but are not shown as there was no significant difference when compared with vehicle control (2% FBS NUT mix medium+ethanol) to show ethanol did not have any stimulatory or inhibitory effect on cell yield.

 Determination of anandamide induced cell death. Shed cells were collected from control and anandamide treated cells and were dual stained with acridine orange (AO) (5 μg/ml) and ethidium bromide (EB) (5 μg/ml). Spontaneous apoptosis was evident in vehicle control cells (A); arrow indicates apoptotic cells with condensation and segregation of chromatin, membrane blebbing, and cellular shrinkage. Early apoptotic cells stain green and late apoptosis/secondary necrosis fluoresce red. (B) HT29 cells treated with 25 μM anandamide (six days) and stained with AO/EB. Anandamide treated cells were predominantly stained with EB, were small in size, and the chromatin had condensed but did not segregate. Examples are indicated with block arrows; apoptosis was also detected in anandamide treated samples (fine arrow) at a rate of ∼1%. (C) H₂O₂ (72 hours) induced necrosis in HT29 cells, represented by the uptake of EB, with swollen nuclei present in all cells within the field. (D) SW480, HT29, and HCA7/C29 cells were treated for six days with anandamide, and adherent and shed cell lysates were collected from experiments shown in fig 2 ▶. Intact (116 kDa) and cleaved PARP (89 kDa) were detected in adherent cell samples (Ad) and cleaved PARP was detected in blank (B; 2% fetal bovine serum (FBS) NUT mix medium) and vehicle control shed cell samples (Veh; 2% FBS NUT mix medium+0.25% ethanol) due to spontaneous apoptosis in colorectal carcinoma cell lines. Anandamide induced significant cell death in both HT29 and HCA7/C29 at 10 μM anandamide and above, which corresponds to a decrease in the amount of cleaved PARP and the appearance of intact PARP (116 kDa). Repeat probing for α-tubulin controls for equal loading and transfer. (E) HT29 cells were treated with vehicle control (Control), or 10 μM or 25 μM anandamide for a total of six days. Shed cells were collected and stained with AO and EB, and 300 cells from randomly selected fields were counted and the proportion of cells with apoptotic, non-apoptotic, and necrotic morphology were calculated.

 Anandamide induced cell death corresponded to an increase in prostaglandin E₂/ prostaglandin E₂-ethanolamide (PGE₂/PGE₂-EA) secretion but did not affect cyclooxygenase 2 (COX-2) protein expression. (A) HT29 cells were treated with vehicle control (control) or the concentration of anandamide indicated, for 72 hours. PGE₂/PGE₂-EA was secreted into the medium and assessed using a PGE₂ enzyme immunoassay (Cayman Chemical) which also detects PGE₂-EA, but cannot distinguish between them. Anandamide 25 μM significantly increased PGE₂/PGE₂-EA secretion (***p<0.001). The lower limit of accurate detection of the assay was 15 pg/ml; †concentrations below this limit. The results shown are means of duplicate measurements. Similar results were obtained in repeat experiments. (B) Adherent cells from the above experiments were collected as cell lysates and subjected to sodium dodecyl sulphate-polyacrylamide gel immunoblotting for COX-2 protein expression. HCA7/C29 cells were used as a positive control for COX-2 protein expression (+ve), vehicle control (control), 1 μM anandamide (1 μM), 10 μM anandamide (10 μM), and 25 μM anandamide (25 μM). COX-2 protein expression was not affected by anandamide treatment over 72 hours (or six days, data not shown). Repeat probing for α-tubulin controls for equal loading and transfer.

 Cyclooxygenase 2 (COX-2) metabolites of anandamide induced apoptosis in colorectal carcinoma cell lines. SW480, HT29, and HCA7/C29 cells were treated with the indicated concentrations of prostaglandin E₂ (PGE₂)-ethanolamide (A, B) and PGD₂-ethanolamide (C, D) for 72 hours. Adherent cell yields are presented as a percentage of vehicle control (A, C). Shed cells are presented as a proportion of total cell yield (B, D). PGE₂-ethanolamide and PGD₂-ethanolamide inhibited cell growth and induced cell death in colorectal carcinoma cells. *p<0.05, **p<0.01, ***p<0.001 versus control, by Dunnett’s post hoc t test. All data are means (SEM) from three separate experiments, conducted in triplicate. Shed cells from these experiments were collected and subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis for the presence of cleaved PARP. The example shown is from HT29 cells treated with PGE₂-ethanolamide (E) and PGD₂-ethanolamide (F). As a positive control for intact and cleaved PARP adherent cells treated with vehicle control were included (Ad), and vehicle control shed cell samples (Control) were included as a positive control for cleaved PARP.