Effects Ofthe Combination Of Mlt And Ra On Breast Cancer

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Recent studies have suggested that MLT may be a ligand for the ROR receptors (13,57), and that the RORa and RARa receptors may cross-talk at the level of the hormone response element (70). We initiated a series of studies to examine the possible additive or synergistic effects of combined treatment with MLT and RA on breast cancer. As shown in Figure 1, ER-positive MCF-7 breast tumor cells showed significant growth-suppression after five days of treatment with either 10-9 M MLT (64% of control) or 10-9 M atRA (62% of control). Surprisingly, the simultaneous treatment of the cells with MLT and atRA had no inhibitory effect on cell proliferation. However, a sequential regimen of MLT (10-9 M) followed 24 h later by atRA (10-9 M) resulted in a cytocidal effect, decreasing the cell number to below the initial plating density after 5 days of treatment. Similar results were seen with the ER-positive T47D cell line in which both MLT and atRA, when used alone, inhibited cell proliferation by 60% and 61 % of control, respectively. However, in this cell line, the simultaneous administration of MLT and atRA also inhibited cell growth (54% of control). The specificity of the cytocidal effect was demonstrated by the fact that the sequential MLT and atRA regimen had no effect on ER-negative MDA-MB-231 breast cancer cells.

The cytotoxic effects of the sequential MLT and RA treatment could be the result of either cellular necrosis or apoptosis. Apoptosis can be differentiated from cellular necrosis by a unique series of ultrastructural changes, including chromosomal and cytoplasmic condensation, nuclear fragmentation, membrane blebbing, an increased number of lysosomal bodies, and the formation of membrane-bound apoptotic bodies. The pattern of DNA oligomerization in MCF-7 tumor cells treated with the sequential regimen of MLT followed by atRA demonstrated the development over time of a ladder of nucleosomal oligomers (Figure 2). This laddering is characteristic of many cell types undergoing apoptosis. It should also be noted that there was no evidence of complete DNA degradation, which would be expected if the cells were undergoing

Figure 1. Effects of MLT and atRA on the proliferation of (A) MCF-7, (B) T47D, and (C) MDA-MB-231 cells.MCF-7 and MDA-MB-231 cells were seeded at a density of 2 x 106cells/ml andT47D at 5 x 105 cells/ml in Costar 6-well dishes in IDMEM medium supplemented with 10% CS-FBS. Five hours after seeding (Day 0), MLT or atRA, both MLT and atRA, or MLT followed 24h later by atRA were added as 1000-fold concentrates to the appropriate wells. Ethanol vehicle was added to the control plates such that the final concentration was 0.001%. On Days 1,3, and 5, cells were harvested by brief trypsinization, and viable cells were counted on a hemacytometer using the trypan blue dye exclusion method. Each point represents the mean cell count ± SEM from six plates containing either the vehicle (Veh), 10-9 M MLT (Mel), 10-9 M atRA (RA), MLT plus atRA (M + R), or MLT followed 24h later by atRA (M24 + R); * p < 0.05, ** p < 0.01 vs vehicle-treated controls.

Figure 1. Effects of MLT and atRA on the proliferation of (A) MCF-7, (B) T47D, and (C) MDA-MB-231 cells.MCF-7 and MDA-MB-231 cells were seeded at a density of 2 x 106cells/ml andT47D at 5 x 105 cells/ml in Costar 6-well dishes in IDMEM medium supplemented with 10% CS-FBS. Five hours after seeding (Day 0), MLT or atRA, both MLT and atRA, or MLT followed 24h later by atRA were added as 1000-fold concentrates to the appropriate wells. Ethanol vehicle was added to the control plates such that the final concentration was 0.001%. On Days 1,3, and 5, cells were harvested by brief trypsinization, and viable cells were counted on a hemacytometer using the trypan blue dye exclusion method. Each point represents the mean cell count ± SEM from six plates containing either the vehicle (Veh), 10-9 M MLT (Mel), 10-9 M atRA (RA), MLT plus atRA (M + R), or MLT followed 24h later by atRA (M24 + R); * p < 0.05, ** p < 0.01 vs vehicle-treated controls.

Figure 2. Electrophoretic analysis of DNA isolated from MCF-7 cells grown in IDMEM supplemented with 10% FBS was performed after treatment with the timed regimen of MLT and atRA. The molecular weight marker in this figure is a combination of X DNA digested with Hind III and $ 174 digested with Hae III. MCF-7 cells were treated for 1, 3, or 5 days with the sequential regimen of melatonin and atRA (M + R), after which high molecular weight DNA was isolated as described in Materials and Methods. Twenty micrograms of DNA were run on a 2.0% agarose gel. This is a representative picture of three separate experiments.

cellular necrosis. The onset of apoptosis was further substantiated by morphological criteria such as membrane blebbing, increased lysosomal formation, perinuclear chro-matin condensation, and the formation of apoptotic bodies (71).

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