Artonin E induces p53-independent G1 cell cycle arrest and apoptosis through ROS-mediated mitochondrial pathway and livin suppression in MCF-7 cells

Abstract

Artonin E is a prenylated flavonoid compound isolated from the stem bark of Artocarpus elasticus. This phytochemical has been previously reported to be drug-like with full compliance to Lipinski's rule of five and good physicochemical properties when compared with 95% of orally available drugs. It has also been shown to possess unique medicinal properties that can be utilized in view of alleviating most human disease conditions. In this study, we investigated the cytotoxic mechanism of Artonin E in MCF-7 breast cancer cells, which has so far not been reported. In this context, Artonin E significantly suppressed the breast cancer cell's viability while inducing apoptosis in a dose-dependent manner. This apoptosis induction was caspase dependent, and it is mediated mainly through the intrinsic pathway with the elevation of total reactive oxygen species. Gene and protein expression studies revealed significant upregulation of cytochrome c, Bax, caspases 7 and 9, and p21 in Artonin E-treated MCF-7 cells, while MAPK and cyclin D were downregulated. Livin, a member of the inhibitors of apoptosis, whose upregulation has been noted to precede chemotherapeutic resistance and apoptosis evasion was remarkably repressed. In all, Artonin E stood high as a potential agent in the treatment of breast cancer.

Keywords: Artonin E; apoptosis; breast cancer; cell cycle; livin.

Figure 1

Chemical structure of Artonin E.

Figure 2

Artonin E induces morphological features of apoptosis in MCF-7 cells. Notes: (A) Untreated breast cancer cells, (B) MCF-7 cells treated for 24 h with 3 µM, (C) 10 µM, (D) 30 µM, and (E) quantification of viable, apoptotic, and necrotic cells at both 24 and 48 h. *Means differ significantly from that of control at P<0.05. All values are expressed as mean ± standard deviation of three independent experiments. Abbreviations: BL, cell membrane blebbing; EA, early apoptosis; VC, viable cells; CC, cancer control; LA, late apoptosis; MN, marginated nuclear chromatin; SN, secondary necrosis.

Figure 3

Effect of Artonin E on viability of MCF-7 cells after 24- and 48-h treatment. Notes: Cell population in lower left quadrant is viable, in lower right quadrant is in early apoptosis, in upper right quadrant is at late stage of apoptosis, and in upper left corner is at the necrotic stage. A1, B1, C1, and D1 represent MCF-7 cells treated accordingly. The image is a representative histogram of three independent experiments.

Figure 4

Caspase activity in Artonin E-treated MCF-7 cells after 24-h incubation with different concentrations of Artonin E. The experiment was done in triplicate and the data are expressed as mean ± standard deviation, *P<0.05.

Figure 5

Total reactive oxygen species (ROS) production by MCF-7 cells after 24-h treatment with Artonin E. Notes: (A) Untreated control and (B, C), and (D) are treatment with 3, 10, and 30 µM Artonin E, respectively, (E) The analysis of the flow cytometric data. Values are expressed as mean ± standard deviation of three independent experiments. *Means differ significantly (P<0.05) from that of untreated control. M1 = non-ROS, M2 = ROS-producing cell population.

Figure 6

Artonin E arrests MCF-7 cell cycle after 12-h treatment with (B) 3, (C) 10 µM, and (D) 30 µM Artonin E. (A) Is untreated control. (E) Analysis of cell population in the cycle phases. Values are expressed as mean ± standard deviation. Note: *Means in each cell cycle phase differ significantly (P<0.05) from that of control.

Figure 7

Artonin E arrest MCF-7 cell cycle after 24-h treatment with (B) 3 µM, (C)10 µM, and (D) 30 µM Artonin E. (A) is untreated control. (E) Analysis of cell population in the cycle phases. Values are mean ± standard deviation. Note: *Means in each cell cycle phase differ significantly (P<0.05) from that of control.

Figure 8

Western blot protein expression of apoptosis- and cell cycle-related proteins in Artonin E-treated MCF-7 cells at 24 h. Notes: (A) Western blot and (B) analysis of protein expression. There was an upregulation of Bax and p21 and a downregulation of livin and p53 proteins, *P<0.05.

Figure 9

Mechanism of action of Artonin E on MCF-7 breast cancer cells. Abbreviation: ROS, reactive oxygen species.