Esculetin, a Coumarin Derivative, Exhibits Anti-proliferative and Pro-apoptotic Activity in G361 Human Malignant Melanoma

Abstract

Background: Although esculetin, a coumarin compound, is known to induce apoptosis in human cancer cells, the effects and molecular mechanisms on the apoptosis in human malignant melanoma (HMM) cells are not well understood yet. In this study, we investigated the anti-proliferative effects of esculetin on the G361 HMM cells.

Methods: We analyzed the anti-proliferative effects and molecular mechanisms of esculetin on G361 cells by a 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, 4',6-diamidino-2-phenylindole staining and Western blotting.

Results: Esculetin exhibited significant anti-proliferative effects on the HMM cells in a dose-dependent manner. Interestingly, we found that esculetin induced nuclear shrinkage and fragmentation, typical apoptosis markers, by suppression of Sp1 transcription factor (Sp1). Notably, esculetin modulated Sp1 downstream target genes including p27, p21 and cyclin D1, resulted in activation of apoptosis signaling molecules such as caspase-3 and PARP in G361 HMM cells.

Conclusions: Our results clearly demonstrated that esculetin induced apoptosis in the HMM cells by downregulating Sp1 protein levels. Thus, we suggest that esculetin may be a potential anti-proliferative agent that induces apoptotic cell death in G361 HMM cells.

Keywords: Apoptosis; Esculetin; Melanoma; Sp1 transcription factor.

Figure 1.

The effect of esculetin on the cell viability in G361 cells. (A) Chemical structure of esculetin. (B) Cell viability effects of esculetin on G361 cells. The G361 cells were treated with esculetin (0, 10, 20, 40, and 80 μg/mL) for 24 hours and 48 hours, and the cell viabilities were measured with an MTS assay. The asterisk indicates a significant difference relative to untreated control cells (*P < 0.05). (C) Changes in the cell morphologies in G361 cells treated or untreated with esculetin (0, 20, 40, and 80 μg/mL) for 48 hours (×40).

Figure 2.

Apoptotic effect induced by esculetin in human malignant melanoma cells. The G361 cells were incubated with esculetin (0 and 80 μg/mL) for 48 hours. (A) Fluorescence microscopy images of the DAPI-stained cells. The white arrows represent DNA fragmentation and chromatin condensation. Scale bars: 20 μm. (B) The DNA fragmentation and chromatin condensation were determined to have a significant difference relative to untreated control cells, as indicated by an asterisk (*P < 0.05).

Figure 3.

The effect of esculetin on Sp1 expression in human malignant melanoma. (A) The G361 cells were treated with different concentrations of esculetin (0, 20, 40, and 80 μg/mL) for 48 hours. The cell lysates were separated by SDS PAGE, and then the membranes were transferred from SDS PAGE gels subjected to Western blot analysis for Sp1. An equal loading protein was confirmed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The histogram showed the ratio of Sp1 to GAPDH expression, and the results were expressed as the average of a triplicate sample from three independent experiments. The asterisk suggested P < 0.05 versus untreated control cells. (B) The G361 cells were treated with 80 μg/mL of esculetin for 0, 12, 24, and 48 hours, and then the cells were evaluated in terms of the expression level of Sp1, procaspase 3 and active caspase 3. Equal loading protein was confirmed using GAPDH.

Figure 4.

The effect of esculetin on the expression of cell cycle arrest- and apoptosis-related proteins in human malignant melanoma. (A) The G361 cells were incubated with esculetin (20, 40, and 80 μg/mL) or without for 48 hours. The cell lysates were determined using Western blot analysis with antibodies against p27, p21, and cyclin D1. Equal loading protein was confirmed using glyceraldehyde-3-phosphate de-hydrogenase (GAPDH). (B) The G361 cells were treated with esculetin (0, 20, 40, and 80 μg/mL) for 48 hours. The cell lysates were then determined via Western blot analysis with antibodies against procaspase 3, active caspase 3, Bax, and PARP. Equal loading protein was confirmed using GAPDH. The values were measured using Image J densitometry, and the data were expressed as a representative of two or three independent experiments.