Reactive oxygen species mediate soft corals-derived sinuleptolide-induced antiproliferation and DNA damage in oral cancer cells

Figures & data

Figure 1 NAC effect on cell viabilities of sinuleptolide-treated oral cancer and normal cells.

Notes: (A) Cell viabilities of oral cancer cells. (B) Cell viabilities of oral normal cells. With or without 4 mM NAC pretreatment for 1 h, oral cancer cells (Ca9-22) or oral normal cells (HGF-1) were incubated with 3, 6, 12, and 24 µg/mL of sinuleptolide for 24 h. Cell viability was measured by the MTS assay. Data, mean ± SD (n=6). **P-value of <0.002 for the significance between data with or without NAC pretreatment.
Abbreviation: NAC, N-acetylcysteine.

Figure 2 NAC effect on cell morphology of sinuleptolide-treated oral cancer and oral normal cells.

Notes: NAC pretreatment condition was 4 mM for 1 h. No NAC pretreatment was kept in culture medium for 1 h. Cells were incubated with 0, 3, 6, 12, and 24 µg/mL of sinuleptolide for 24 h. (A and B) Cell morphology of sinuleptolide-treated oral cancer cells (Ca9-22) and oral normal cells (HGF-1). Cell morphology was observed under 100× magnification (scale bar is 100 µm).
Abbreviation: NAC, N-acetylcysteine.

Figure 3 NAC effect on ROS generation of sinuleptolide-treated oral cancer cells and oral normal cells.

Notes: NAC pretreatment condition was 4 mM for 1 h. No NAC pretreatment was kept in culture medium for 1 h. Oral cancer cells (Ca9-22) were incubated with 0, 12, and 24 µg/mL of sinuleptolide for 6 h. (A and B) Typical ROS patterns of sinuleptolide treated Ca9-22 and HGF-1 cells. The right side of each flow cytometry panel indicates the ROS positive (+%) region. (C and D) Statistics of relative mean intensity of ROS pattern (in A and B). Data, mean ± SD (n=3). **P-value of <0.002 for the significance between data with or without NAC pretreatment.
Abbreviations: NAC, N-acetylcysteine; ROS, reactive oxygen species.

Figure 4 NAC effect on mitochondrial (mt) superoxide generation of sinuleptolide-treated oral cancer cells.

Notes: NAC pretreatment condition was 4 mM for 1 h. No NAC pretreatment was kept in culture medium for 1 h. Oral cancer cells (Ca9-22) were incubated with 0, 12, and 24 µg/mL of sinuleptolide for 1 h. (A and B) Typical mt superoxide patterns of sinuleptolide treated Ca9-22 and HGF-1 cells. The right side of each flow cytometry panel indicates the mt superoxide positive (+%) region. (C and D) Statistics of relative mean intensity of mt superoxide pattern (in A and B). Data, mean ± SD (n=3). **P-value of <0.002 for the significance between data with or without NAC pretreatment.
Abbreviation: NAC, N-acetylcysteine.

Figure 5 NAC effect on γH2AX-based DNA damage in sinuleptolide-treated oral cancer and oral normal cells.

Notes: NAC pretreatment condition was 4 mM for 1 h. No NAC pretreatment was kept in culture medium for 1 h. Oral cancer cells (Ca9-22) were incubated with 0, 6, 12, and 24 µg/mL of sinuleptolide for 24 h. (A and B) Typical γH2AX pattern of sinuleptolide-treated Ca9-22 and HGF-1 cells. Dashed lines of each flow cytometry panel indicate the γH2AX positive (+%) regions. (C and D) Statistics of mean intensity of γH2AX-based DNA damage pattern (in A and B). Data, mean ± SD (n=3). **P-value of <0.002 for the significance between data with or without NAC pretreatment.
Abbreviations: NAC, N-acetylcysteine; PI, propidium iodide.

Figure 6 Overview of the hypothesized mechanism of sinuleptolide-induced killing of human oral cancer cells (Ca9-22) involving oxidative stress.

Notes: The role of oxidative stress was demonstrated by pretreatment with NAC that scavenges ROS and prevents mitochondrial superoxide generation. NAC consequently inhibits DNA damage and cell death.
Abbreviations: NAC, N-acetylcysteine; ROS, reactive oxygen species.