Mechanism of Lakoochin A Inducing Apoptosis of A375.S2 Melanoma Cells through Mitochondrial ROS and MAPKs Pathway

Abstract

Malignant melanoma is developed from pigment-containing cells, melanocytes, and primarily found on the skin. Malignant melanoma still has a high mortality rate, which may imply a lack of therapeutic agents. Lakoochin A, a compound isolated from Artocarpus lakoocha and Artocarpus xanthocarpus, has an inhibitory function of tyrosinase activity and melanin production, but the anti-cancer effects are still unclear. In the current study, the therapeutic effects of lakoochin A with their apoptosis functions and possible mechanisms were investigated on A375.S2 melanoma cells. Several methods were applied, including 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), flow cytometry, and immunoblotting. Results suggest that lakoochin A attenuated the growth of A375.S2 melanoma cells through an apoptosis mechanism. Lakoochin A first increase the production of cellular and mitochondrial reactive oxygen species (ROSs); mitochondrial ROSs then promote mitogen-activated protein kinases (MAPKs) pathway activation and raise downstream apoptosis-related protein and caspase expression. This is the first study to demonstrate that lakoochin A, through ROS-MAPK, apoptosis-related proteins, caspases cascades, can induce melanoma cell apoptosis and may be a potential candidate compound for treating malignant melanoma.

Keywords: MAPKs; apoptosis; lakoochin A; melanoma cells; mitochondria; pro-oxidation.

Figure 1

(A) The chemical structure of lakoochin A. (B) The inhibitory effect of lakoochin A on A375.S2 cell proliferation, as determined by the SRB assay at 24 h. (C) Dose and time effects of lakoochin A on A375.S2 cell viability, as determined by the 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay at 24 and 48 h. (D) The effects of lakoochin A on human skin fibroblast and keratinocytes as determined by the MTT assay at 24 h. The cell apoptosis effects of lakoochin A on A375.S2 cells, as (E) presented by the morphology and (F) determined by flow cytometry with AnnexinV-Fluorescein isothiocyanate (FITC) and propidium iodide staining at 24 h. The right lower quadrant indicates early apoptosis. (G) Effects of lakoochin A on cell apoptosis (left panel) and sub-G1 cell cycle arrest (right panel) were determined by DNA fragmentation assay and flow cytometry, with propidium iodide stainingon A375.S2 cells at 24 h, respectively. Results (B–G) expressed as mean ± S.E.M. from three individual experiments. * p < 0.05 and # p < 0.01 compared to the control group.

Figure 2

(A) The dose effect of lakoochin A at 24 h on the mitochondrial membrane potential (∆Ψm) of A375.S2 cells, as determined by flow cytometry staining with JC-1. (B) The time effects of lakoochin A on the ∆Ψm of A375.S2 cells pre-labeled with 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide (JC-1) (10 μg/mL) for the indicated times (0.5–16 h). (C) Effect of lakoochin A on mitochondrial reactive oxygen species (ROS) production (determined by flow cytometry after staining with MitoSOX Red indicator) in A375.S2 cells. (D) Effect of lakoochin A on cellular ROS production (determined by flow cytometry after staining with H2DCFDA reagent). (E) The cellular ROS production of several antioxidants and lakoochin A, determined by flow cytometry staining with H2DCFDA reagent. The A375.S2 cells were pretreated for 1 h with mitochondria-targeted antioxidant (MitoTEMPOL), antioxidant (NAC), or nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (DPI) and then treated with lakoochin A for 4 h. (F) Effect of MitoTEMPOL, NAC, and DPI on lakoochin A-induced A375.S2 cell apoptosis (determined by MTT assay). The data were collected from at least three individual experiments and expressed as mean ± S.E.M. * p < 0.05, # p < 0.01 compared to the control group.

Figure 3

(A) Effects of different time course of lakoochin A, with or without being pre-treated for 1 h with a p38 inhibitor (SB202190), MEK1/ERK inhibitor (U0126), JNK inhibitor (SP600125), and mitochondria-targeted antioxidant (MitoTEMPOL), on expressions of the phosphorylation status of p38, ERK (p44/p42), and JNK in A375.S2 cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Blots were representative of three independent experiments. (B) Effect of U1026, SP600125, and MitoTEMPOL on lakoochin A-induced A375.S2 cell apoptosis (determined by MTT assay). The data were collected from at least three individual experiments and expressed as mean ± S.E.M. * p < 0.05 as compared to the control group.

Figure 4

(A) Effects of lakoochin A on the expression levels of apoptosis-related proteins Puma, Bax, Bad, Bid, Apaf-1, and cytochrome c in A375.S2 cells, over various time periods (0–24 h) (B) The cells were pre-treated with 1 and 10 μM MitoTEMPOL for 1 h followed by treatment of lakoochin A for 16 h. Expression of apoptosis-related proteins was determined by Western blotting. GAPDH was used for a loading control. The intensity of the bands was quantified by densitometry, and the data were collected from three individual experiments and expressed as mean ± S.E.M. * p < 0.05, compared to the control group.

Figure 5

(A) Effects of lakoochin A (10 μM) on the expression status of caspase-7, caspase-3, and caspase-9 in A375.S2 cells in a serial time-period (0–24 h). (B) Effects of caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) on lakoochin A-induced increases in caspase-3, -7, and -9. Cells were pre-treated for 1 h with Z-VAD-FMK (1 or 10 μM) followed by treatment with lakoochin A for 16 h. The levels of caspase-3, -7, and -9 were evaluated by immunoblotting. (C) Effect of Z-VAD-FMK (1 or 10 μM) on lakoochin A-induced A375.S2 cell apoptosis (determined by MTT assay). The data were collected from at least three individual experiments and expressed as mean ± S.E.M. * p < 0.05, as compared to the control group.