Korean J Physiol Pharmacol.  2020 May;24(3):223-232. 10.4196/kjpp.2020.24.3.223.

Sesamin induces A549 cell mitophagy and mitochondrial apoptosis via a reactive oxygen species-mediated reduction in mitochondrial membrane potential

• Yang S
• Li X
• Dou H
• Hu Y
• Che C
• Xu D

• KMID: 2502504
• DOI: http://doi.org/10.4196/kjpp.2020.24.3.223

Abstract

Sesamin, a lipid-soluble lignin originally isolated from sesame seeds, which induces cancer cell apoptosis and autophagy. In the present study, has been reported that sesamin induces apoptosis via several pathways in human lung cancer cells. However, whether mitophagy is involved in sesamin induced lung cancer cell apotosis remains unclear. This study, the anticancer activity of sesamin in lung cancer was studied by reactive oxygen species (ROS) and mitophagy. A549 cells were treated with sesamin, and cell viability, migration ability, and cell cycle were assessed using the CCK8 assay, scratch-wound test, and flow cytometry, respectively. ROS levels, mitochondrial membrane potential, and apoptosis were examined by flow cytometric detection of DCFH-DA fluorescence and by using JC-1 and TUNEL assays. The results indicated that sesamin treatment inhibited the cell viability and migration ability of A549 cells and induced G0/G1 phase arrest. Furthermore, sesamin induced an increase in ROS levels, a reduction in mitochondrial membrane potential, and apoptosis accompanied by an increase in cleaved caspase-3 and cleaved caspase-9. Additionally, sesamin triggered mitophagy and increased the expression of PINK1 and translocation of Parkin from the cytoplasm to the mitochondria. However, the antioxidant N-acetyl-L-cysteine clearly reduced the oxidative stress and mitophagy induced by sesamin. Furthermore, we found that cyclosporine A (an inhibitor of mitophagy) decreased the inhibitory effect of sesamin on A549 cell viability. Collectively, our data indicate that sesamin exerts lethal effects on lung cancer cells through the induction of ROS-mediated mitophagy and mitochondrial apoptosis.

Keyword

Apoptosis; Lung cancer; Mitophagy; Reactive oxygen species; Sesamin

Figure

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  Fig. 1 Sesamin inhibits the proliferation and migration of human lung cancer cells. (A) A549 cells were treated with 10, 20, 40, 60, 80, and 100 μM sesamin or 0.1% chloroform (as a control) for 12, 24, 36, and 48 h, and cell viability was examined using CCK-8 assays. Data are means ± standard deviation (SD) (n = 3). (B) Scratch-wound assays were used to assess the effect of sesamin on the migration ability of A549 cells treated with 20 or 40 μM sesamin (or 0.1% chloroform) for 48 h. Quantitative analysis of healing rate in wound-scratch assays. Data are means ± SD (n = 3; **p < 0.01). (C) A549 cells were treated with 40 μM sesamin (or 0.1% chloroform) for 48 h, and then detected the cell cycle by flow cytometry. Data are means ± SD (n = 3; *p < 0.05).

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  Fig. 2 Sesamin triggers the collapse of mitochondrial membrane potential by increasing reactive oxygen species (ROS) levels in A549 cells. (A) A549 cells were treated with 40 μM sesamin and/or 5 mM NAC for 48 h, and then ROS levels were examined by flow cytometric detection of DCFH-DA fluorescence. Data are means ± standard deviation (SD) (n = 3; **p < 0.01). (B) Changes in mitochondrial membrane potential were detected a JC-1 assay kit containing the cationic dye, which undergoes a readily detectable shift from red to green with decreases in membrane potential. The images were obtained at 400× magnification. (C) Quantitative analysis of JC-1 results. Data are means ± SD (n = 3; **p < 0.01). NAC, N-acetyl-L-cysteine.

• 

  Fig. 3 Sesamin induces A549 cell apoptosis via a reactive oxygen species (ROS)-dependent mitochondrial pathway. (A) A549 cells were treated with 40 μM sesamin and/or 5 mM N-acetyl-L-cysteine (NAC) for 48 h, stained with Annexin V-FITC and propidium iodide (PI), and then analyzed using a FACScan flow cytometer. (B) Summary data for Annexin V-FITC/PI staining. Data are means ± standard deviation (SD) (n = 3; **p < 0.01). (C) A549 cells were treated with 40 μM sesamin and/or 5 mM NAC for 48 h, and TUNEL assays were performed according to the manufacturer’s instructions. Samples were analyzed under a fluorescence microscope using standard filter sets for fluorescein (green) and propidium iodide (PI; red). The images were obtained at 200× magnification. (D) Summary data for TUNEL assays. Data are means ± SD (n = 3; ***p < 0.001). (E) Expression of the mitochondrial apoptosis-related proteins, caspase-3, caspase-9, Bax, Bax/B-cell lymphoma-2 (Bcl-2) and cytochrome c in A549 cells treated with 40 μM sesamin and/or 5 mM NAC for 48 h was assessed by Western blot analysis. (F) Quantification of caspase-3, caspase-9, Bcl-2 and cytochrome c. Data are means ± SD (n = 3; *p < 0.05, **p < 0.01).

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  Fig. 4 Sesamin triggers mitophagy through a reactive oxygen species (ROS)-mediated PINK1/Parkin pathway in A549 cells. (A) A549 cells were treated with 40 μM sesamin for 48 h and then observed by TEM (5,000×; arrows indicate autophagosomes). (B) Expression of PINK1 in whole cell and Parkin in cytoplasm or mitochondria of A549 cells treated with 40 μM sesamin and/or 5 mM NAC for 48 h was assessed by Western blotting with anti-PINK1, anti-Parkin, anti-VDAC1 (mitochondria marker), and anti-β-Tubulin (cytoplasm marker) antibodies. (C) Quantification of PINK1 and Parkin. Data are means ± standard deviation (n = 3; **p < 0.01). NAC, N-acetyl-L-cysteine.

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  Fig. 5 Inhibition of mitophagy attenuates the inhibitory effect of sesamin on A549 cell viability. (A) A549 cells were treated with 40 μM sesamin and/or 5 μM cyclosporine A (CsA) for 48 h, and cell viability was measured by CCK-8 assay. Data are means ± standard deviation (SD) (n = 3; **p < 0.01). (B) A549 cells were treated with 40 μM sesamin and/or 5 μM CsA for 48 h, and then expression of PINK1 in whole cell and Parkin in cytoplasm or mitochondria was assessed by Western blotting with anti-PINK1, anti-Parkin, anti-VDAC1 (mitochondria marker), and anti-β-Tubulin (cytoplasm marker) antibodies. (C) Quantification of PINK1 and Parkin. Data are means ± SD (n = 3; **p < 0.01).

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