Pseudolaric acid B induces apoptosis, senescence, and mitotic arrest in human breast cancer MCF-7
Abstract
Aim: The aim of the present study was to investigate the inhibitory effect of pseudolaric acid B (PAB) on human breast cancer MCF-7 cells.
Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, morphological changes, acridine orange staining, and agarose gel electrophoresis were applied to detect apoptosis. The percentage of apoptotic and necrotic cells was calculated by the lactate dehydrogenase activity-based cytotoxicity assay; senescence associated (SA)-β-galactosidase activity was detected to evaluate senescence; flow cytometric analysis of propidium iodide staining was carried out to investigate the distribution of cell cycle, and the protein expression was examined by Western blot analysis.
Results: During apoptosis, the half maximal inhibitory concentration IC50 was 3.4 and 1.35 μmol/L at 36 and 48 h after PAB treatment, respectively. The MCF-7 cells exposed to PAB showed typical characteristics of apoptosis, including the morphological changes and DNA fragmentation. The MCF-7 cells treated with 4 μmol/L PAB for 36 h underwent apoptosis, but not necrosis. The apoptosis induced by PAB was independent of the death receptor pathway. The senescent cells became larger and flatter, and the SA-β-galactosidase staining was positive. PAB induced obvious mitotic arrest and it preceded apoptosis and senescence. The expressions of p21 and p53 was upregulated with PAB treatment, and cyclin B1 was upregulated and transported from the cytoplasm to nuclei, and sustained stable levels.
Conclusion: PAB induced mitotic arrest in the MCF-7 cells and inhibited proliferation through apoptosis and senescence. The apoptosis was independent of the death receptor pathway.
Keywords:
Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, morphological changes, acridine orange staining, and agarose gel electrophoresis were applied to detect apoptosis. The percentage of apoptotic and necrotic cells was calculated by the lactate dehydrogenase activity-based cytotoxicity assay; senescence associated (SA)-β-galactosidase activity was detected to evaluate senescence; flow cytometric analysis of propidium iodide staining was carried out to investigate the distribution of cell cycle, and the protein expression was examined by Western blot analysis.
Results: During apoptosis, the half maximal inhibitory concentration IC50 was 3.4 and 1.35 μmol/L at 36 and 48 h after PAB treatment, respectively. The MCF-7 cells exposed to PAB showed typical characteristics of apoptosis, including the morphological changes and DNA fragmentation. The MCF-7 cells treated with 4 μmol/L PAB for 36 h underwent apoptosis, but not necrosis. The apoptosis induced by PAB was independent of the death receptor pathway. The senescent cells became larger and flatter, and the SA-β-galactosidase staining was positive. PAB induced obvious mitotic arrest and it preceded apoptosis and senescence. The expressions of p21 and p53 was upregulated with PAB treatment, and cyclin B1 was upregulated and transported from the cytoplasm to nuclei, and sustained stable levels.
Conclusion: PAB induced mitotic arrest in the MCF-7 cells and inhibited proliferation through apoptosis and senescence. The apoptosis was independent of the death receptor pathway.