Cudraflavone C Induces Apoptosis of A375.S2 Melanoma Cells through Mitochondrial ROS Production and MAPK Activation

Abstract

Melanoma is the most malignant form of skin cancer and is associated with a very poor prognosis. The aim of this study was to evaluate the apoptotic effects of cudraflavone C on A375.S2 melanoma cells and to determine the underlying mechanisms involved in apoptosis. Cell viability was determined using the MTT and real-time cytotoxicity assays. Flow cytometric evaluation of apoptosis was performed after staining the cells with Annexin V-FITC and propidium iodide. The mitochondrial membrane potential was evaluated using the JC-1 assay. Cellular ROS production was measured using the CellROX assay, while mitochondrial ROS production was evaluated using the MitoSOX assay. It was observed that cudraflavone C inhibited growth in A375.S2 melanoma cells, and promoted apoptosis via the mitochondrial pathway mediated by increased mitochondrial ROS production. In addition, cudraflavone C induced phosphorylation of MAPKs (p38, ERK, and JNK) and up-regulated the expression of apoptotic proteins (Puma, Bax, Bad, Bid, Apaf-1, cytochrome C, caspase-9, and caspase-3/7) in A375.S2 cells. Pretreatment of A375.S2 cells with MitoTEMPOL (a mitochondria-targeted antioxidant) attenuated the phosphorylation of MAPKs, expression of apoptotic proteins, and the overall progression of apoptosis. In summary, cudraflavone C induced apoptosis in A375.S2 melanoma cells by increasing mitochondrial ROS production; thus, activating p38, ERK, and JNK; and increasing the expression of apoptotic proteins. Therefore, cudraflavone C may be regarded as a potential form of treatment for malignant melanoma.

Keywords: MAPKs; apoptosis; cudraflavone C; melanoma cells; mitochondria; pro-oxidation.

Figure 1

(A) Chemical structure of cudraflavone C; (B) Inhibition of A375.S2 cell proliferation by cudraflavone C, as determined by the SRB assay at 24 h; (C) Effects of cudraflavone C on cell viability in A375.S2 cells, as determined by the MTT assay at 24 and 48 h; (D) Effects of cudraflavone C on cell viability in human skin fibroblasts(open bars) and human keratinocytes (HaCaT cells) (shard bars), as determined by the MTT assay at 24 h; (E) Effects of cudraflavone C on cell apoptosis in A375.S2 cells, as determined by flow cytometry following AnnexinV-FITC and propidium iodide staining at 24 h. Cells in the right lower quadrant are undergoing early apoptosis; (F) Effects of cudraflavone C on cell apoptosis (determined by DNA fragmentation assay, left panel) and sub-G1 cell cycle arrest (determined by flow cytometry following propidium iodide staining, right panel) in A375.S2 cells at 24 h. (B–F) Results are shown as mean ± SEM of three independent experiments. * p < 0.05 compared to the control group.

Figure 2

(A) Effect of cudraflavone C on mitochondrial membrane potential in A375.S2 cells, as determined by the JC-1 assay at 24; (B) A375.S2 cells were labeled with JC-1 (10 μg/mL) and then stimulated with cudraflavone C for the indicated times (6, 12 and 24 h). The ΔΨm results are representative of three independent experiments; (C) Effect of cudraflavone C on cellular ROS production (determined by flow cytometry after staining with CellROX reagent) in A375.S2 cells as a function of time (upper left panel) and concentration (upper right panel). Effect of cudraflavone C on mitochondrial ROS production (determined by flow cytometry after staining with MitoSOX Red indicator) in A375.S2 cells as a function of time (lower left panel) and concentration (lower right panel); (D) A375.S2 cells were pretreated for 1 h with mitochondria-targeted antioxidant (MitoTEMPOL), antioxidant (NAC), or NADPH oxidase inhibitor (APO) and then treated with cudraflavone C for 240 min, and cellular ROS production was determined by flow cytometry (after staining with CellROX reagent); (E) A375.S2 cells were pretreated for 1 h with MitoTEMPOL, NAC or APO and then treated with cudraflavone C for 360 min, and mitochondrial ROS production was determined by flow cytometry (after staining with MitoSOXRed indicator); (F) Effect of MitoTEMPOL (mitochondria-targeted antioxidant) on cudraflavone C-induced A375.S2 cell apoptosis (determined by flow cytometry following Annexin-V and propidium iodide staining). Cells in the right lower quadrant are undergoing early apoptosis; (A–F) Results are shown as mean ± SEM of three independent experiments. * p < 0.05 compared to the control group.

Figure 3

Effects of cudraflavone C on the phosphorylation status of p38, ERK (p44/p42) and JNK in A375.S2 cells over various time periods (0–6 h), as determined by Western blotting. In addition, cells were pretreated for 1 h with a p38 inhibitor (SB202190), MEK1/ERK inhibitor (U0126), JNK inhibitor (SP600125), and mitochondria-targeted antioxidant (MitoTEMPOL) and then treated with cudraflavone C, and the phosphorylation status of p38, ERK, and JNK was evaluatedover various time periods. GAPDH was used as a loading control. Blots were representative of three independent experiments.

Figure 4

(A) Effects of cudraflavone C on the expression levels of apoptotic proteins Puma, Bax, Bad, Bid, Apaf-1 and cytochrome c in A375.S2 cells over various time periods (0–24 h), as determined by Western blotting; (B) Cells were pretreated with MitoTEMPOL (mitochondria-targeted antioxidant) for 1 h and then treated with cudraflavone C for 16 h, and the expression of apoptotic proteins was determined by Western blotting. GAPDH was used as a loading control. The intensity of the bands was quantified by densitometry, and data are expressed as mean ± SEM of three experiments. * p < 0.05 compared to the control group.

Figure 5

(A) Effects of cudraflavone C on the expression status of caspase-7, caspase-3, and caspase-9 in A375.S2 cells over various time periods (0–24 h), as determined by Western blotting; (B) In addition, cells were pretreated for 1 h with the caspase inhibitor Z-VAD-FMK and then treated with cudraflavone C for 16 h, and the levels of active caspase-7, -3, and -9 were evaluated by Western blotting; (C,D) Cells were treated with cudraflavone C for indicated times. The caspase activity was analyzed by using caspase-3, -7, and -9 colorimetric assay kits; (E) Confluent cells were pre-incubated with or without inhibitors of ERK1/2 (U0126), p38 (SB202190), JNK1/2 (SP600125) and MitoTEMPOL (mitochondria-targeted antioxidant). After incubation for 1 h, cells were treated with cudraflavone C (10 µM) for 24 h. The activities of caspases were analyzed by using caspase-3, -7, and -9 colorimetric assay kits. Results are representative of three independent experiments. * p < 0.05, ^# p < 0.01 compared to the control group.