Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells

Abstract

The aim of the present study was to determine the effects of Licochalcone D (LD) on the apoptosis and migration and invasion in human melanoma A375 cells. Cell proliferation was determined by sulforhodamine B assay. Apoptosis was assessed by Hoechst 33258 and Annexin V‑FITC/PI staining and JC‑1 assay. Total intracellular reactive oxygen species (ROS) was examined by DCFH‑DA. Wound healing and Transwell assays were used to detect migration and invasion of the cells. The activities of matrix metalloproteinase (MMP‑2 and MMP‑9) were assessed via gelatin zymography. Tumor growth in vivo was evaluated in C57BL/6 mice. RT‑PCR, qPCR, ELISA and western blot analysis were utilized to measure the mRNA and protein levels. Our results showed that LD inhibited the proliferation of A375 and SK‑MEL‑5 cells in a concentration‑dependent manner. After treatment with LD, A375 cells displayed obvious apoptotic characteristics, and the number of apoptotic cells was significantly increased. Pro‑apoptotic protein Bax, caspase‑9 and caspase‑3 were upregulated, while anti‑apoptotic protein Bcl‑2 was downregulated in the LD‑treated cells. Meanwhile, LD induced the loss of mitochondrial membrane potential (ΔΨm) and increased the level of ROS. ROS production was inhibited by the co‑treatment of LD and free radical scavenger N‑acetyl‑cysteine (NAC). Furthermore, LD also blocked A375 cell migration and invasion in vitro which was associated with the downregulation of MMP‑9 and MMP‑2. Finally, intragastric administration of LD suppressed tumor growth in the mouse xenograft model of murine melanoma B16F0 cells. These results suggest that LD may be a potential drug for human melanoma treatment by inhibiting proliferation, inducing apoptosis via the mitochondrial pathway and blocking cell migration and invasion.

Figure 1.

Chemical structure of Licochalcone D.

Figure 2.

Effects of Licochalcone D (LD) on A375 and SK-MEL-5 cell proliferation and survival. (A) The inhibition rate of A375 cell proliferation was determined by SRB assay and the lethal rate was detected by trypan blue exclusion test after treatment with LD (0, 15, 30, 45, 60, 75 and 90 µmol/l) for 24 h. (B) SK-MEL-5 cell viability was determined by SRB assay after 24 h treatment with LD (0, 20, 40, 60 and 80 µmol/l). Data are presented as means ± SD of at least three independent experiments. *P<0.05, **P<0.01 compared with the untreated control group cells.

Figure 3.

Induction of apoptosis in A375 cells by Licochalcone D (LD) treatment. (A) Cell morphological changes were observed by phase-contrast microscopy (magnification, ×200) after treatment with LD (0, 30, 60 and 90 µmol/l) for 24 h. (B) Apoptosis was visualized by the appropriate changes in nuclei stained with Hoechst 33258 (blue) (magnification, ×200). (C) The effects of LD on the induction of A375 cell apoptosis were analyzed by FCM analysis. (D) The apoptosis rate as statistically analyzed. (E) RT-PCR analyses of A375 cells to evaluate mRNA expression of Bcl-2, Bax, caspase-3 and caspase-9. (F) qPCR analyses of A375 cells to evaluate mRNA expression of Bcl-2, Bax, caspase-3 and caspase-9, and relative intensities were normalized by levels of GAPDH. The untreated group level was considered as ‘1.0’. Data are presented as means ± SD of at least three independent experiments. *P<0.05, **P<0.01 as compared with the untreated control group.

Figure 4.

Licochalcone D (LD) treatment decreases the mitochondrial membrane potential and increases ROS production in A375 cells. (A and B) Cells were treated with LD (0, 30, 60 and 90 µmol/l) and then exposed to JC-1 dye solution. Changes in mitochondrial membrane potential ΔΨm in A375 cells were tested by staining with JC-1 dye solution after treatment with LD, and the staining was detected by flow cytometry and fluorescence plate reader. A concentration-dependent reduction in ΔΨm was observed in the LD-treated cells. (C and D) DCF-DA was used as a fluorescence indicator to measure the intracellular ROS level. The ROS levels in the LD-treated cells were significantly higher than that noted in the control. Moreover, ROS scavenger (NAC) was used to determine whether ROS exerted an interference effect against LD-induced A375 cell proliferation. (E and F) ROS production was inhibited obviously with the co-addition of NAC (300 µM). (G) A375 cell survival after a 24 h post-NAC treatment. (H) Effects of NAC on apoptotic rates in A375 cells. All data are presented as the means ± SD of at least three independent experiments; *P<0.05, **P<0.01 as compared with the normal control group. ^#P<0.05 as compared with the LD alone treated group. NAC, N-acetyl-cysteine.

Figure 5.

Licochalcone D (LD) decreases A375 cell migration and invasion. (A) Representative images of the wound-healing assay in A375 cells following treatment with LD (0, 5, 15, 25 µmol/l) for 24 h under a ×200 light microscope (Carl Zeiss). (B) Analysis of the wound closure rate. (C) Transwell migration assay of A375 cells upon treatment of 25 µmol/l LD. (D) Transwell invasion assay of A375 cells upon treatment of 25 µmol/l LD. (E) Activities of MMP-2 and MMP-9 were determined via gelatin zymography. (F) The gray value in the same area for each band was measured by Gel-PRO Analyzer software, and the MMP-2 and MMP-9 activities were expressed as a percentage of the control. All data are presented as the means ± SD of at least three independent experiments; *P<0.05, **P<0.01 as compared with the normal control group.

Figure 6.

Levels of mRNA and protein expression of MMP-2 and MMP-9 in A375 cells treated with Licochalcone D (LD). Transcript levels for MMP-2 and MMP-9 were monitored by (A) quantitative real-time PCR and (B) RT-PCR analysis, and relative intensities were normalized by levels of GAPDH. (C) Protein levels of MMP-2 and MMP-9 were monitored by ELISA analysis. Data are presented as the means ± SD of at least three independent experiments. *P<0.05, **P<0.01 as compared with the untreated control group.

Figure 7.

Effects of low concentrations of Licochalcone D (LD) on A375 cell proliferation. The inhibition rate on A375 cell proliferation was determined by SRB assay after cells were treated with low concentrations of LD (1, 2.5 and 5 µmol/l). Data are presented as means ± SD of at least three independent experiments.

Figure 8.

Licochalcone D (LD) decreases A375 cell migration at low concentrations. (A) Representative image of the wound healing ability of A375 cells treated with low concentrations of LD (1, 2.5 and 5 µmol/l) under a ×200 light microscope (Carl Zeiss). (B) Analysis of the wound closure rate of A375 cells by quantitation. All data are presented as the means ± SD of at least three independent experiments; *P<0.05, **P<0.01 as compared with the normal control group.

Figure 9.

Licochalcone D (LD) inhibits tumor growth in a mouse xenograft model of murine melanoma B16F0 cells. (A) Representative images of the tumor morphology. (B) Data for the tumor inhibition rate of LD on tumor growth. Data are presented as means ± SD of at least three independent experiments. *P<0.05, **P<0.01 as compared with the untreated control group.