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A., Kolesnick R. 6 transfection reagent was from Roche, and Maxiprep kits were from Qiagen. Horseradish peroxidase- and Cy3-conjugated secondary immunoglobulins and enhanced chemiluminescence (ECL) Western blotting detection kit were obtained from Amersham. Anti-Bid and cytochrome c antibodies were from BD Pharmingen. Anti-LC3 antibody was from Santa Cruz Biotechnology. Human LC3 TruClone cDNA was purchased from Origene. Pan-caspase inhibitor zVAD-fmk and a mammary epithelial growth medium (MEGM) kit were purchased from Lonza Corp. Oligonucleotides and transfection reagents used in short-interfering RNA (siRNA) studies were purchased from Dharmacon. A mitochondria isolation kit was from Pierce. All other chemicals were from either Sigma or Fisher Scientific. C6-pyridinium ceramide (LCL29) and C6-ceramide were synthesized by the Lipidomics Core facility at Medical University of South Carolina as previously described and were dissolved in dimethyl sulfoxide (24). Cell culturing MCF7, MDA-MB-157, and MDA-MB-231 cells were cultured in DMEM supplemented with 10% FBS. MCF10A cells were cultured by using a MEGM kit supplemented with 10% FBS. All cells were maintained in 5% Co2 at 37C. Development of MCF7 cells stably expressing green Ginsenoside Rg3 fluorescent protein-LC3 Human LC3 cDNA was amplified by PCR and cloned into 0.05; **, 0.01; ***, 0.001 versus the control. In addition to examining effects on MCF7 cells, we also compared the effects of these two compounds on the growth of MCF10A, MDA-MB-157, and MD-MB-231 cells by using the MTT assay. Figure 4A shows that increasing concentrations of C6-pyridinium ceramide significantly enhanced the inhibition of cell growth. The IC50s of these compounds were found to be 52 M, 4 M, and 48 M, respectively, for the above-listed cells. On the other hand, the growth of these cells remained static following C6-ceramide treatment. We also monitored cell growth inhibition over time in the presence of 10 M of these compounds. As shown in Fig. 4B, cell growth inhibition and most likely a decrease in cell viability were evident over time for all three cell lines upon C6-pyridinium ceramide treatment, compared with that with C6-ceramide treatment. Open in a separate window Fig. 4. C6-pyridinium ceramide (C6-Pyr-Cer) inhibited growth of various types of cell lines in a dose- and time-dependent manner. MCF10A, MDA-MB-157, and MDA-MB-231 cells were plated onto 6-well plates and treated with different concentrations (0C80 M) of C6-pyridinium ceramide or C6-ceramide (C6-Cer) for 24 h (A) or with 10 M of each of these compounds for various time periods Rabbit Polyclonal to OR4D6 (B). At specified time points, MTT assays were performed. Results were normalized to values from the control cells at the initial time. All results were averaged from three independent Ginsenoside Rg3 studies. As C6-pyridinium ceramide treatment induces cell death as well as autophagy, we then examined whether inhibition or promotion of autophagy enhances cell survival. MCF7 cells were treated with the Ginsenoside Rg3 autophagy inhibitor 3-methyl adenine or the autophagy promoter tamoxifen, in the presence or absence of C6-pyridinium ceramide for 24 h. Cells were then stained with Hoechst dye to detect apopt-otic nuclei. As shown in Fig. 5, the presence of tamoxifen reduced the number of apoptotic nuclei following C6-pyridinium ceramide treatment. This result suggests Ginsenoside Rg3 that in MCF7 cells, autophagy may serve as a protective mechanism against C6-pyridinium ceramide-mediated mitochondrial damage. Open in a separate window Fig. 5. Effect of Ginsenoside Rg3 autophagy regulators on C6-pyridinium ceramide (C6-Pyr-Cer)-mediated cell death. GFP-Bax-stable MCF7 cells were treated with or without 10 M C6-pyridinium ceramide either in the presence or absence of 40 M tamoxifen (TAM) or 3 mM 3-methyl adenine (3-MA) for 24 h. Cells were then stained with Hoechst dye, and percentages of apoptotic nuclei were quantitated from three separate visual fields. Results were averaged from three independent studies. Values are means SEM. *, 0.05; **, .