Soft Coral-Derived Dihydrosinularin Exhibits Antiproliferative Effects Associated with Apoptosis and DNA Damage in Oral Cancer Cells

Abstract

Dihydrosinularin (DHS) is an analog of soft coral-derived sinularin; however, the anticancer effects and mechanisms of DHS have seldom been reported. This investigation examined the antiproliferation ability and mechanisms of DHS on oral cancer cells. In a cell viability assay, DHS showed growth inhibition against several types of oral cancer cell lines (Ca9-22, SCC-9, OECM-1, CAL 27, OC-2, and HSC-3) with no cytotoxic side effects on non-malignant oral cells (HGF-1). Ca9-22 and SCC-9 cell lines showing high susceptibility to DHS were selected to explore the antiproliferation mechanisms of DHS. DHS also causes apoptosis as detected by annexin V, pancaspase, and caspase 3 activation. DHS induces oxidative stress, leading to the generation of reactive oxygen species (ROS)/mitochondrial superoxide (MitoSOX) and mitochondrial membrane potential (MitoMP) depletion. DHS also induced DNA damage by probing γH2AX phosphorylation. Pretreatment with the ROS scavenger N-acetylcysteine (NAC) can partly counter these DHS-induced changes. We report that the marine natural product DHS can inhibit the cell growth of oral cancer cells. Exploring the mechanisms of this cancer cell growth inhibition, we demonstrate the prominent role DHS plays in oxidative stress.

Keywords: DNA damage; Dihydrosinularin (DHS); MitoMP; MitoSOX; apoptosis; oral cancer; reactive oxygen species (ROS); soft coral.

Figure 1

DHS kills oral cancer cells. (A) MTS assay (48 h) for DHS-treated oral cancer and non-malignant oral cells. Six oral cancer cell lines (Ca9-22, SCC-9, OECM-1, CAL 27, OC-2, and HSC-3) and one non-malignant oral cell line (HGF-1) were chosen. Cells were treated with DHS (0 (vehicle containing 0.08% DMSO), 0.1, 0.2, 0.4 and 0.8 mM) for 48 h. (B) Recovery of cell viability of DHS-treated Ca9-22 and SCC-9 cells by NAC. Cells were pretreated with NAC (10 mM for 1 h) or not. They were then treated with 0.4 mM DHS or with the vehicle for 0 and 48 h. Data = means ± SDs (n = 3 independent experiments). * and ** indicate significant differences between drug treatments (NAC/DHS) and DHS (p < 0.05 and 0.01, respectively).

Figure 2

DHS effects on subG1 accumulation of oral cancer cells. (A,B) Representative cell cycle patterns of DHS-treated oral cancer cells. Cells (Ca9-22 and SCC-9) were treated with 0.4, 0.6 and 0.8 mM DHS or vehicle (containing 0.08% DMSO) for 48 h. (C,D) Alleviation of subG1 accumulation of DHS-treated oral cancer cells by NAC. Cells were pretreated with NAC (4 mM, 1 h) or not, and then they were treated with 0.6 mM DHS or vehicle for 0, 36 and 48 h. Data = means ± SDs (n = 3 independent experiments). * and ** indicate significant differences between (A,B) DHS and control and (C,D) DHS and NAC/DHS (p < 0.05 and 0.01, respectively). A positive control for subG1 accumulation of oral cancer cells is provided in Supplementary Figure S1.

Figure 2

DHS effects on subG1 accumulation of oral cancer cells. (A,B) Representative cell cycle patterns of DHS-treated oral cancer cells. Cells (Ca9-22 and SCC-9) were treated with 0.4, 0.6 and 0.8 mM DHS or vehicle (containing 0.08% DMSO) for 48 h. (C,D) Alleviation of subG1 accumulation of DHS-treated oral cancer cells by NAC. Cells were pretreated with NAC (4 mM, 1 h) or not, and then they were treated with 0.6 mM DHS or vehicle for 0, 36 and 48 h. Data = means ± SDs (n = 3 independent experiments). * and ** indicate significant differences between (A,B) DHS and control and (C,D) DHS and NAC/DHS (p < 0.05 and 0.01, respectively). A positive control for subG1 accumulation of oral cancer cells is provided in Supplementary Figure S1.

Figure 3

DHS effects on annexin V/7AAD-detected apoptosis of oral cancer cells. (A,B) Representative annexin V/7AAD patterns of DHS-treated oral cancer cells. Cells (Ca9-22 and SCC-9) were treated with 0.4, 0.6 and 0.8 mM DHS or vehicle (containing 0.08% DMSO) for 48 h. (C,D) Alleviation of the annexin V/7AAD changes of DHS-treated oral cancer cells by NAC. Cells were pretreated with NAC (4 mM, 1 h) or not, and then they were treated with 0.6 mM DHS or vehicle for 0, 36 and 48 h. Data = means ± SDs (n = 3 independent experiments). * and ** indicate significant differences between (A,B) DHS and control and (C,D) DHS and NAC/DHS (p < 0.05 and 0.01, respectively). A positive control for annexin V/7AAD-detected apoptosis of oral cancer cells is provided in Supplementary Figure S1.

Figure 3

DHS effects on annexin V/7AAD-detected apoptosis of oral cancer cells. (A,B) Representative annexin V/7AAD patterns of DHS-treated oral cancer cells. Cells (Ca9-22 and SCC-9) were treated with 0.4, 0.6 and 0.8 mM DHS or vehicle (containing 0.08% DMSO) for 48 h. (C,D) Alleviation of the annexin V/7AAD changes of DHS-treated oral cancer cells by NAC. Cells were pretreated with NAC (4 mM, 1 h) or not, and then they were treated with 0.6 mM DHS or vehicle for 0, 36 and 48 h. Data = means ± SDs (n = 3 independent experiments). * and ** indicate significant differences between (A,B) DHS and control and (C,D) DHS and NAC/DHS (p < 0.05 and 0.01, respectively). A positive control for annexin V/7AAD-detected apoptosis of oral cancer cells is provided in Supplementary Figure S1.

Figure 4

DHS effects on caspase-based apoptosis of oral cancer cells. (A,B) Representative pancaspase patterns of DHS-treated oral cancer cells. Cells (Ca9-22 and SCC-9) were treated with vehicle (containing 0.08% DMSO), 0.4, 0.6 and 0.8 mM DHS for 48 h. (+) indicates the high pancaspase activity populations. (C,D) Alleviation of the pancaspase changes of DHS-treated oral cancer cells by NAC. Cells were pretreated with NAC (4 mM, 1 h) or not, and then they were treated with the vehicle and 0.6 mM DHS for 0, 36 and 48 h. (E,F) Representative caspase 3 patterns of DHS-treated oral cancer cells. Data = means ± SDs (n = 3). *, ** and *** indicate significant differences between (A,B) DHS and control and (C,D) DHS and NAC/DHS (p < 0.05, 0.01, and 0.001, respectively). A positive control for pancaspase-detected apoptosis of oral cancer cells is provided in Supplementary Figure S1.

Figure 5

DHS effects on ROS contents of oral cancer cells. (A,B) Representative ROS patterns of DHS-treated oral cancer cells. Cells (Ca9-22 and SCC-9) were treated with vehicle (containing 0.08% DMSO), 0.4, 0.6 and 0.8 mM DHS for 48 h. (+) indicates the high ROS populations. (C,D) Alleviation of ROS changes in DHS-treated oral cancer cells by NAC. Cells were pretreated with NAC (4 mM, 1 h) or not, and then they were treated with 0.6 mM DHS or vehicle for 0, 36 and 48 h. (E) Positive control for ROS induction. Cells (Ca9-22) were treated with H₂O₂ (0, 0.1 and 0.2 mM) for 24 h. Data = means ± SDs (n = 3 independent experiments). *, ** and *** indicate significant differences between (A,B) DHS and control and (C,D) DHS and NAC/DHS (p < 0.05, 0.01 and 0.001, respectively).

Figure 6

DHS effects on MitoSOX contents of oral cancer cells. (A,B) Representative MitoSOX patterns of DHS-treated oral cancer cells. Cells (Ca9-22 and SCC-9) were treated with 0.4, 0.6 and 0.8 mM DHS or vehicle (containing 0.08% DMSO) for 48 h. (+) indicates the high MitoSOX populations. (C,D) Alleviation of the MitoSOX changes of DHS-treated oral cancer cells by NAC. Cells were pretreated with NAC (4 mM, 1 h) or not, and then they were treated with 0.6 mM DHS or vehicle for 0, 36, and 48 h. Data = means ± SDs (n = 3 independent experiments). *, ** and *** indicate significant differences between (A,B) DHS and control and (C,D) DHS and NAC/DHS (p < 0.05, 0.01 and 0.001, respectively). A positive control for MitoSOX generation of oral cancer cells is provided in Supplementary Figure S1.

Figure 7

DHS effects on MitoMP contents of oral cancer cells. (A,B) Representative MitoMP patterns of DHS-treated oral cancer cells. Cells (Ca9-22 and SCC-9) were treated with 0.4, 0.6 and 0.8 mM DHS or vehicle (containing 0.08% DMSO) for 48 h. (−) indicates the low MitoMP populations. (C,D) Alleviation of the MitoMP changes of DHS-treated oral cancer cells by NAC. Cells were pretreated with NAC (10 mM, 1 h) or not, and then they were treated with 0.6 mM DHS or vehicle for 0, 36 and 48 h. Data = means ± SDs (n = 3 independent experiments). * and ** indicate significant differences between (A,B) DHS and control and (C,D) DHS and NAC/DHS (p < 0.05 and 0.01, respectively). A positive control for MitoMP depletion of oral cancer cells is provided in Supplementary Figure S1.

Figure 8

DHS effects on γH2AX contents of oral cancer cells. (A,B) Representative γH2AX patterns of DHS-treated oral cancer cells. Cells (Ca9-22 and SCC-9) were treated with 0.4, 0.6, and 0.8 mM DHS or vehicle (containing 0.08% DMSO) for 48 h. (+) indicates the high γH2AX populations. (C,D) Alleviation of the γH2AX changes of DHS-treated oral cancer cells by NAC. Cells were pretreated with NAC (10 mM, 1 h) or not, and then they were treated with 0.6 mM DHS or vehicle for 0, 36 and 48 h. Data = means ± SDs (n = 3 independent experiments). * and ** indicate significant differences between (A,B) DHS and control and (C,D) DHS and NAC/DHS (p < 0.05 and 0.01, respectively). A positive control for γH2AX phosphorylation of oral cancer cells is provided in Supplementary Figure S1.