Dual effects of 1,15-bis(ethylamino)4,8,12-triazapentadecane (BE-3-3-3-3) on MCF-7 cells in the presence and absence of estradiol: Role of Beclin 1 and autophagy Free

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Polyamines are ubiquitous cellular cations with multiple functions in cell proliferation and cell death. Bis(ethyl) polyamine analogues have been synthesized and tested as chemotherapeutic agents for different subsets of cancer. A major problem impeding the progress of these compounds to the clinic appears to be their lack of efficacy at low concentrations, and delayed action in suppressing cell growth. Recent studies indicate that programmed cell death can occur through different pathways, apoptosis (Type I), autophagic cell death (Type II) or a combination of both. Autophagy is also a cell survival mechanism to utilize long-lived proteins when cells are under nutritional or other stress. Controversy exists over whether autophagy assists the growth or death of cancer cells. There have been recent reports indicating that autophagy sensitizes cancer cells for radiation therapy. Beclin 1 is the first identified mammalian gene associated with autophagy. In order to examine the role of autophagy in the mechanism of action of the polyamine analogue, BE-3-3-3-3, in breast cancer cell growth, we first examined its effects on estrogen receptor alpha (ERa)-positive MCF-7 cells in the presence and absence of estradiol (E2). There was a concentration-dependent decrease of cell growth, with an IC50 of 1.25 µM, as measured by [3H]-thymidine incorporation and trypan blue exclusion assays. Surprisingly, we found that BE-3-3-3-3 could increase [3H]-thymidine incorporation of estrogen deprived MCF-7 cells by 5-fold. Confocal microscopic studies showed autophagic vacuole formation of cells treated with BE-3-3-3-3 both in the presence and absence of E2. Western blot analysis showed a 2-fold increase in Beclin 1 protein levels and an increase in autophagy-associated MAP LC-3 protein when MCF-7 cells were treated with 1.25 to 10 µM BE-3-3-3-3 for 48 h in the presence of E2. Re-probing of the blots with b-actin antibody showed comparable levels of protein in control and treatment groups. BE-3-3-3-3 also induced the degradation of ERa. Immunocytochemical staining with confocal microscopy showed that BE-3-3-3-3 treatment induced a movement of Beclin 1 from cytoplasm toward the membrane. In addition, we found that stable transfection of Beclin 1 sensitized MCF-7 cells to BE-3-3-3-3, with 90% cell death by day 8 of treatment at 1.25 ѵM concentration. These results provide a novel mechanistic pathway for the action of polyamine analogues on breast cancer cell growth. Beclin 1 and autophagy might play an important role in the dual action of these compounds. Therapeutic agents specifically targeted for modulating autophagy might be useful in combination with polyamine analogues in breast cancer.