Apoptotic effect of the pure antiestrogen ICI 182 781 in combination with bis(ethyl)norspermine involves down-regulation of spermidine and spermine

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We studied the effects of ICI 182 780 and the polyamine analogue bis(ethyl)norspermine (BE-3-3-3) on two estrogen receptor (ER)-positive human breast cancer cell lines, MCF-7 and T-47D. Combinations of BE-3-3-3 and ICI 182,780 showed increased efficacy in inhibiting cell growth, compared to the effects of these agents individually. Apoptosis of treated cells was determined 4 days after the treatment. Individual treatment with 100 nM ICI 182 780 or 5 μM BE-3-3-3 resulted in 9.1 ± 0.1% and 35.1 ± 4.5% apoptosis, respectively, as determined by the Apo-BrdU assay. However, when ICI 182 780 and BE-3-3-3 were used in combination, the percentage of apoptosis was 60.6 ± 3.8%. In order to understand the mechanism of action of these agents in combination, we determined the changes in intracellular polyamine levels. Changes in putrescine, spermidine and spermine levels were determined by a HPLC procedure, after pre-column derivatization. At the 24 h time point, there was no significant change in putrescine and spermine levels, but spermidine levels decreased up to 50% in combination groups. At the 48 h time point, combination treatments caused no change in putrescine levels, but spermidine and spermine were down-regulated significantly. These changes were higher than that produced by BE-3-3-3 and ICI 182,780 as individual agents. After 72 h of treatment, 2.5 μM BE-3-3-3 caused a 50% decrease in spermidine and spermine levels and 100 nM ICI 182,780 produced about 25% decrease. Combination group showed 80% decrease in these polyamine levels. Similar results were obtained at the 96 h time point also. These results show that the pure antiestrogen ICI 182,780 exerted moderate effect on polyamine pathway and its effects are significantly augmented by co-treatment with a polyamine analogue. Since putrescine levels were not altered by individual or combination treatments, these effects might be exerted by changes in polyamine pathway other than ornithine decarboxylase. These studies show polyamine biosynthetic and/or degradative pathway are remarkably altered by ICI 182,780 when it is combined with BE-3-3-3. This observation will be significant in further developing this combination for pre-clinical and clinical studies.