Korean J Physiol Pharmacol.  2022 Nov;26(6):447-456. 10.4196/kjpp.2022.26.6.447.

Arctigenin induces caspase-dependent apoptosis in FaDu human pharyngeal carcinoma cells

• Kang KR
• Kim JS
• Lim H
• Seo JY
• Park JH
• Chun HS
• Yu SK
• Kim HJ
• Kim CS
• Kim DK

• KMID: 2534521
• DOI: http://doi.org/10.4196/kjpp.2022.26.6.447

Abstract

The present study was carried out to investigate the effect of Arctigenin on cell growth and the mechanism of cell death elicited by Arctigenin were examined in FaDu human pharyngeal carcinoma cells. To determine the apoptotic activity of Arctigenin in FaDu human pharyngeal carcinoma cells, cell viability assay, DAPI staining, caspase activation analysis, and immunoblotting were performed. Arctigenin inhibited the growth of cells in a dose-dependent manner and induced nuclear condensation and fragmentation. Arctigenin-treated cells showed caspase-3/7 activation and increased apoptosis versus control cells. FasL, a death ligand associated with extrinsic apoptotic signaling pathways, was up-regulated by Arctigenin treatment. Moreover, caspase-8, a part of the extrinsic apoptotic pathway, was activated by Arctigenin treatments. Expressions of anti-apoptotic factors such as Bcl-2 and Bcl-xL, components of the mitochondria-dependent intrinsic apoptosis pathway, significantly decreased following Arctigenin treatment. The expressions of pro-apoptotic factors such as BAX, BAD and caspase-9, and tumor suppressor -53 increased by Arctigenin treatments. In addition, Arctigenin activated caspase-3 and poly (ADP-ribose) polymerase (PARP) induced cell death. Arctigenin also inhibited the proliferation of FaDu cells by the suppression of p38, NF-κB, and Akt signaling pathways. These results suggest that Arctigenin may inhibit cell proliferation and induce apoptotic cell death in FaDu human pharyngeal carcinoma cells through both the mitochondria-mediated intrinsic pathway and the death receptormediated extrinsic pathway.

Keyword

Apoptosis; Arctigenin; Caspase-dependent; Human pharyngeal carcinoma

Figure

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  Fig. 1 Chemical structure of Arctigenin.

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  Fig. 2 Effect of Arctigenin on cell viability in FaDu pharyngeal carcinoma cells. The effect of Arctigenin on the viability of L-929 cells (A) and FaDu cells (B) was accessed by the MTT assay. The percentage of cell viability was calculated as a ratio of A570nms of Arctigenin treated with cells and untreated control cells. Each data point represents the mean ± SEM of four experiments. MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. **p < 0.01 vs. control and ***p < 0.001 vs. control (the control cells measured in the absence of Arctigenin).

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  Fig. 3 Induction of cell death by Arctigenin. (A) L-929 cells and FaDu cell death by Arctigenin. The cells were treated with 0, 50, or 100 μM Arctigenin for 24 h. Arctigenin induced the death of FaDu cells in the dose-dependent manner. FaDu cells emitting green fluorescence are live cells stained by green calcein AM, whereas cells emitting red fluorescence are dead cells stained by ethidium homodimer-1. The arrows indicate dead cells stained red. Scale bar represents 100 µm. (B) Quantitative data of (A) were counted, and calculated as 100%. Each data point represents the mean ± SEM of four experiments. **p < 0.01 vs. control and ***p < 0.001 vs. control (the control cells measured in the absence of Arctigenin).

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  Fig. 4 Apoptotic phenomenon in FaDu pharyngeal carcinoma cells stimulated with Arctigenin. (A) Changes in nuclear morphology by Arctigenin. The cells were treated with 0, 50, 100 μM Arctigenin for 24 h. DAPI staining revealed that the number of FaDu cells with nuclear condensation was increased by Arctigenin. The arrows indicated the apoptotic cells with chromatic condensation characteristics. Scale bar represents 100 µm. (B) TUNEL assay revealed that the number of FaDu cells with apoptotic nuclei are stained dark brown was increased by Arctigenin. The arrows indicate apoptotic nuclei stained dark brown. Scale bar represents 100 µm. (C) Arctigenin induces the caspase-3/-7 activation in FaDu cells. The caspase-3/-7 intracellular activity assay was performed using PhiPhiLux-caspase-3/-7 substrate. Images were observed by fluorescence microscopy (Eclipse TE 2000; Nikon Instruments, Melville, NY, USA). Scale bar represents 100 µm.

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  Fig. 5 Arctigenin-induced FaDu pharyngeal carcinoma cell death is meditated by both intrinsic and extrinsic pathways. (A) Extrinsic death receptor-mediated apoptotic signaling pathway induced by Arctigenin. Arctigenin upregulated the expression level of the death receptor ligand Fas and subsequently activated the extrinsic death receptor-mediated apoptotic signaling pathway through the cleavage of caspase-8 in FaDu cells. (B) Intrinsic mitochondria-dependent apoptotic signaling pathway induced by Arctigenin. Arctigenin downregulated anti-apoptotic factors Bcl-2 and Bcl-xL associated with the intrinsic mitochondria-dependent apoptotic pathway and upregulated the mitochondria-dependent pro-apoptotic factors Bax and Bad in FaDu cells. (C) Extrinsic death receptor-mediated and intrinsic mitochondria-dependent apoptosis signaling pathways via the activation of caspase-3 and PARP induced by Arctigenin. Cleaved caspase-8 and cleaved caspase-9 induced the activation of caspase-3 and PARP in FaDu cells treated with Arctigenin.

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  Fig. 6 Arctigenin-induced apoptosis in FaDu cells was mediated by caspase. (A) Z-VAD-fmk, a pan-caspase inhibitor, reversed Arctigenin induced cell death in FaDu cells. (B) Activation of caspase-3 and PARP in FaDu cells treated with Arctigenin was inhibited in the presence of Z-VAD-fmk. Each data point represents the mean ± SEM of four experiments (**p < 0.01).

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  Fig. 7 Arctigenin reduced the phosphorylation of proteins in the MAPKs, NF-κB, and AKT pathways in FaDu pharyngeal cells. FaDu cells were treated with 0, 50, and 100 μM Arctigenin for 24 h. Thereafter, immunoblotting using specific antibodies against extracellular signal-regulated kinase 1/2 (ERK1/2), p38, NFκB, and AKT was performed to verify potential cellular signaling pathways associated with Arctigenin-induced apoptosis.

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