Novel Aza-podophyllotoxin derivative induces oxidative phosphorylation and cell death via AMPK activation in triple-negative breast cancer

摘要

a Chemical structure of SU212. b The growth-inhibitory effect SU212 was evaluated using MTT assay. In total, 5000 cells from TNBC cell lines (MDA-MB-231, MDA-MB-468 and 4T1), normal breast cell lines (MCF10A and MCF12A) and neural cell lines (SN-SY5Y and N27) cells were plated per well in a 96-well plate, and the next day, treated with vehicle (DMSO) alone or 0.01–10 µM of SU212 in fresh medium. After 48 h of these treatments, cell viability was recorded using MTT assay. c, d Effect of SU212 on cell- cycle distribution and cell-cycle regulators in human TNBC cells. c MDA-MB-468 and MDA-MB-231 cells were treated with vehicle or 0.1–0.5 µM concentrations of SU212 for 6 and 12 h. At the end of treatment, cells were collected and analysed for cell-cycle-phase distribution as detailed in the “Methods” section. d Percentage of mitotic cells in MDA-MB-468 and -231 cell cultures treated for 12 h with vehicle or 0.1–0.5 µM of SU212. At the end of treatment, cells were stained with Alexa Fluor 488 p-Histone H3 and PI and analysed using flow cytometry. e TNBC cells were treated with SU212 for 6 h, and total cell lysates were prepared as described in the “Methods” section. SDS-PAGE and western blot analysis were performed for cell-cycle and autophagic markers. Membranes were stripped and re-probed with anti-beta-actin antibody to ensure equal protein loading. f The apoptotic effect of SU212 on MDA-MB-468, MDA-MB-231 and MCF12A cells. Cells were treated with 0.25 or 0.5 µM SU212 for 12, 24 and 48 h. At the end of treatment, all cells were collected and stained with annexin V/PI and analysed for apoptotic cell population as mentioned in “Methods”. g TNBC cells were treated with SU212 for 12 h, and total cell lysates were prepared as described in “Methods”. SDS-PAGE and western blot analysis were performed for apoptotic cell death markers. Membranes were stripped and re-probed with anti-beta-actin antibody to ensure equal protein loading. Numbers on top of the bands represent changes in protein levels as determined by densitometric analysis of the immunoreactive bands, and corrected for beta-actin loading control. The cell number data are shown as mean ± SD of three independent plates; each sample was counted in duplicate. Data were analysed using a one-way ANOVA Dunnett’s test. * indicates P < 0.05.