Korean J Physiol Pharmacol.  2023 Jul;27(4):383-398. 10.4196/kjpp.2023.27.4.383.

Dihydroaustrasulfone alcohol induces apoptosis in nasopharyngeal cancer cells by inducing reactive oxygen speciesdependent inactivation of the PI3K/AKT pathway

• Tan KT
• Shih YH
• Gong JY
• Zhang X
• Huang CY
• Su JH
• Sheu JH
• Lin CC

• KMID: 2544140
• DOI: http://doi.org/10.4196/kjpp.2023.27.4.383

Abstract

Dihydroaustrasulfone alcohol (DA), the synthetic precursor of a natural compound (austrasulfone) isolated from the coral species Cladiella australis, has shown cytotoxic effects against cancer cells. However, it is unknown whether DA has antitumor effects on nasopharyngeal carcinoma (NPC). In this study, we determined the antitumor effects of DA and investigated its mechanism of action on human NPC cells. The MTT assay was used to determine the cytotoxic effect of DA. Subsequently, apoptosis and reactive oxygen species (ROS) analyses were performed by using flow cytometry. Apoptotic and PI3K/AKT pathway-related protein expression was determined using Western blotting. We found that DA significantly reduced the viability of NPC-39 cells and determined that apoptosis was involved in DA-induced cell death. The activity of caspase-9, caspase-8, caspase-3, and PARP induced by DA suggested caspase-mediated apoptosis in DA-treated NPC-39 cells. Apoptosis-associated proteins (DR4, DR5, FAS) in extrinsic pathways were also elevated by DA. The enhanced expression of proapoptotic Bax and decreased expression of antiapoptotic BCL-2 suggested that DA mediated mitochondrial apoptosis. DA reduced the expression of pPI3K and p-AKT in NPC-39 cells. DA also reduced apoptosis after introducing an active AKT cDNA, indicating that DA could block the PI3K/AKT pathway from being activated. DA increased intracellular ROS, but N-acetylcysteine (NAC), a ROS scavenger, reduced DA-induced cytotoxicity. NAC also reversed the chances in pPI3K/AKT expression and reduced DA-induced apoptosis. These findings suggest that ROSmediates DA-induced apoptosis and PI3K/AKT signaling inactivation in human NPC cells.

Keyword

Apoptosis; Dihydroaustrasulfone alcohol; Nasopharyngeal carcinoma; Proto-oncogene proteins c-akt; Reactive oxygen species

Figure

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  Fig. 1 The effect of dihydroaustrasulfone alcohol (DA) on cell viability and colony formation in NPC-39 and NPC-BM cells. Cells were treated with 0.1% DMSO (0 μM) or DA for 24 h. (A) Chemical structure of DA. (B) Cell viability was examined by MTT assays. **p < 0.01, ***p < 0.001 vs. 0.1% DMSO group. Colony formation by (C) NPC-39 and (D) NPC-BM cells following treatment with DA for one week. The results are presented as the mean ± standard deviation, and all samples were measured independently in triplicate. Significant differences compared to the DMSO-treated control group are indicated by *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 2 The effect of dihydroaustrasulfone alcohol (DA) on apoptosis in NPC-39 cells. Cells were treated with 0.1% DMSO (0 μM) or DA for 24 h (A, C). Cell cycle distribution was determined by propidium iodide (PI) staining and flow cytometry. The means ± SEM of experimental triplicates are shown in the bar graph at the bottom. (B, D) Phosphatidylserine externalization and DNA integrity were determined by FITC-annexin-V and PI, respectively. The lower-right quadrant (annexin-V+/PI−) represents early apoptosis, while the upper-right quadrant (annexin V+/PI+) indicates late apoptosis and necrosis. (E) Expression levels of cleaved poly (ADP-ribose) polymerase (PARP) were investigated by Western blotting using GAPDH as a loading control. (F) Western blot signal intensity quantification. The results are presented as the mean ± standard deviation, and all samples were measured independently in triplicate. Significant differences compared to the DMSO-treated control group are indicated by **p < 0.01, ***p < 0.001.

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  Fig. 3 The effects of dihydroaustrasulfone alcohol (DA) on caspase-3, caspase-8, and caspase-9 activities in NPC-39 cells. (A) NPC-39 cells were treated with different concentrations of DA for 24 h, and the activities of (B) caspase-9, (C) caspase-8, and (D) caspase-3 were determined via flow cytometry. Black line: unstained NPC-39 cells, different colored lines: cells treated with different doses of DA. ***p < 0.001 indicates significant differences compared to the DMSO-treated control group. (E) The viability of NPC-39 cells after treatment with a pancaspase inhibitor (Z-VAD-FMK) and DA. The results are presented as the mean ± standard deviation, and all samples were measured independently in triplicate. ***p < 0.001 vs. the 25 μM DA-treated group, as determined by Student’s t-test.

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  Fig. 4 The effects of dihydroaustrasulfone alcohol (DA) on mitochondrial dysfunction in NPC-39 cells. Cells were treated with different concentrations of DA for 24 h. (A) Mitochondrial membrane potential (Δψm) and (B) cytochrome c release were determined by JC-1 fluorescent dye staining or anti-cytochrome c–FITC antibodies and flow cytometry. The means ± standard deviation of the experimental triplicates are presented in the bar graph showing (C) JC-1 aggregates and (D) cytochrome c release. All data presented are representative of three independent experiments with similar results. The data are the mean ± standard deviation of three experiments, each performed in triplicate. ***p < 0.001 indicates significant differences compared to the DMSO-treated control group.

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  Fig. 5 The effects of dihydroaustrasulfone alcohol (DA) on the expression of the Bcl-2 family in NPC-39 cells. Cells were treated with different concentrations of DA for 24 h. (A) Bcl-2, Bax, and truncated BH3 interacting-domain death agonist (tBid) were examined by Western blot analysis. GAPDH was used as a loading control. (B) Quantitation of the Western blot signal intensities. The results are presented as the mean ± standard deviation, and all samples were measured independently in triplicate. *p < 0.05 and ***p < 0.001 indicate significant differences compared to the DMSO-treated control group.

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  Fig. 6 The effects of dihydroaustrasulfone alcohol (DA) on the expression of the death receptors DR5, DR4, and FAS in NPC-39 cells. (A) Flow cytometry was used to evaluate the activity of DR5, DR4, and FAS after the cells were exposed to varying doses of DA for 24 h. (B–D) The bars represent the mean ± standard deviation, and all samples were measured independently in triplicate. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences compared to the DMSO-treated control group. MFI, mean fluorescence intensity.

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  Fig. 7 The effects of dihydroaustrasulfone alcohol (DA) on the inactivation of the PI3K/AKT signaling pathway in NPC-39 cells. NPC-39 cells were treated for 24 h with various doses of DA. (A) Cell lysates were collected, and the expression of phosphorylated or nonphosphorylated PI3K and AKT proteins was determined by Western blot analysis. GAPDH was used as a loading control. (B) Western blot signal intensity quantification *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences compared to the DMSO-treated control group. (C) Active AKT cDNA-transfected cells were lysed, and the protein extracts were subjected to protein gel blot analysis using antibodies against the appropriate protein after being treated with 25 µM DA for 24 h. GAPDH was used as a loading control. (D, E) Annexin V-FITC/propidium iodide (PI) binding was investigated using flow cytometry. (F) The MTT assay was used to determine cell viability. The results are presented as the mean ± standard deviation, and all samples were measured independently in triplicate. The difference between the DA and active AKT+ DA groups was **p < 0.01 (Student’s t-test).

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  Fig. 8 The effects of dihydroaustrasulfone alcohol (DA) on reactive oxygen species (ROS) production in NPC-39 cells. NPC-39 cells were treated with different concentrations of DA for 1 h, and ROS levels determined by (A, B) flow cytometry. (C) Images were obtained by fluorescence microscopy (original magnification ×200). The results are presented as the mean ± standard deviation, and all samples were measured independently in triplicate. ***p < 0.001 indicates significant differences compared to the DMSO-treated control group.

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  Fig. 9 The effects of dihydroaustrasulfone alcohol (DA) on ROS-dependent inactivation of the PI3K/AKT pathway and apoptosis in NPC-39 cells. After pretreatment with 10 mM NAC for 1 h and then treatment with 25 μM DA for 24 h, cell viability was measured by (A) MTT assays. (B, C) The percentages of apoptotic annexin V+ cells and cell viability were determined by flow cytometry. (D) Cellular proteins were prepared, and the protein expression of pAKT and AKT was evaluated by Western blot analysis and quantitation of the Western blot signal intensities. GAPDH was used as a loading control. The results are presented as the mean ± standard deviation, and all samples were measured independently in triplicate. *p < 0.05, ***p < 0.001 (Student’s t-test) indicates the difference between the DA and NAC + DA groups. ROS, reactive oxygen species; NAC, N-acetylcysteine; PI, propidium iodide.

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  Fig. 10 Proposed model of the mechanism by which dihydroaustrasulfone alcohol induces apoptosis in NCP-39 cancer cells. ROS, reactive oxygen species; NAC, N-acetylcysteine; tBid, truncated BH3 interacting-domain death agonist; PARP, poly (ADP-ribose) polymerase.

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