Effect of anandamide (AEA) on HBC cell proliferation. (a) Dose-dependent inhibition of EFM-19 and MCF-7 cell proliferation by anandamide; the time-dependent hydrolysis of [¹⁴C]anandamide by EFM-19 cells is shown in the Inset. (b) Effect of 1 and 10 μM anandamide on the growing curve of EFM-19 cells. (c) Dose-dependent effects of anandamide, with or without the fatty acid amide hydrolase inhibitor arachidonoyl-trifluoromethyl-ketone (ATFMK, 5 μM, Biomol) and of (R)-methanandamide (Met-AEA, Biomol) on EFM-19 cell proliferation; (d) Dose-dependent effects of anandamide and arachidonic acid (AA, Sigma) on EFM-19 cell proliferation. The AEA profiles in a, c, and d are from three different experiments conducted in triplicates. Data in a, c, and d are means ± SEM (n ≥ 3) and are expressed as percentages of control cell proliferation (1 − [Control cell number − treated cell number]/[Control cell number − initial cell number] × 100; 100% = no effect, 0% = maximal cytostatic effect). Data in b are means ± SEM (n = 3) and are expressed as percentages of cell proliferation ([Cell number − initial cell number]/initial cell number × 100). AEA also inhibited [³H]thymidine incorporation into EFM-19 and MCF-7 cell DNA (IC₅₀s were 0.65 and 0.70 μM, and maximal inhibition at 1 μM was 75.0 ± 0.6 and 67.0 ± 0.9%, respectively, mean ± SD, n = 3).

A CB1-like cannabinoid receptor mediates anandamide effect on EFM-19 cell proliferation. (a) Dose-related effects of two cannabimimetic compounds, HU-210 and 2-arachidonoylglycerol, and of a non-CB1 agonist, palmitoylethanolamide, on EFM-19 cell proliferation, as compared with anandamide. (b) Effect of two different doses of the CB1 antagonist SR 141716A on the anti-proliferative action of anandamide (1 and 2.5 μM) and arachidonic acid (AA, 1 μM). (c) Displacement of [³H]CP 55,940 from EFM-19 cell membrane preparations by anandamide (AEA), HU-210, 2-arachidonoylglycerol, SR 141716A, and palmitoylethanolamide. In a and b, data are mean ± SD (n = 3) and are expressed as in Fig. 1 a, c, and d. In c, data are expressed as percentages of [³H]CP 55,940 bound to membranes, are means of triplicates, and are representative of three distinct experiments. To avoid confusion, we do not show SD bars. Asterisks indicate statistically significant differences from data without SR 141617A. ∗, P < 0.05; ∗∗, P < 0.01. N.S., not significant.

Anandamide interferes with prolactin action. (a) Dose-related effect of prolactin mAb (Pierce) on EFM-19 cell proliferation in the presence or absence of 1 μM anandamide (AEA). (b) Effect of human (h) prolactin (50 ng/ml) on EFM-19 cell proliferation and its counteraction by low doses of anandamide with or without 0.5 μM SR 141716A. (c) Effect on the levels of the long form (100 kDa) of prolactin receptor of 3-day treatment of EFM-19 cells with anandamide (2.5 μM) in the absence (lanes B and E) or presence (lanes C and F) of SR 141716A (0.5 μM); lanes A and D are from untreated cells. (d) Effect on the levels of the brca1 gene product (220 kDa) of 3-day treatment of EFM-19 cells with anandamide (2.5 μM) in the absence (lane B) or presence (lane C) of SR141716A (0.5 μM). In a, the difference observed between the two sets of data was never statistically significant except for 0 μg/ml prolactin antibody. In a and b, data are mean ± SD (n = 3) and are expressed as described in Fig. 1 a, c, and d. ∗, P < 0.05 vs. h-prolactin + [AEA] = 0; ∗∗, P < 0.05 vs. h-prolactin + [AEA] = 0.5 μM. In a, control experiments were performed by using BSA or a NO synthase III polyclonal antibody instead of prolactin mAb, with no effect on proliferation. In c and d, Western immunoblotting was performed with a monoclonal anti-prolactin receptor antibody (c, lanes A–C), polyclonal anti-phosphotyrosine antibody (c, lanes D–F), or a polyclonal anti-brca1 protein antibody (d, lanes A–C). Proteins immunoprecipitated with a monoclonal anti-prolactin receptor antibody or total proteins (50 μg) were used in c and d, respectively. Control experiments (not shown) did not exhibit the bands at 220 or 100 kDa and were carried out with: (i) no proteins, (ii) no first antibody, and (iii) using, as the first antibody, various antibodies other than the ones mentioned above. The mobility of molecular weight markers is shown. Data are representative of at least three separate experiments. Similar data were obtained with MCF-7 cells. Photographs were taken from films exposed with the enhanced chemiluminescence methodology.