Comparative Evaluation of Anticancer Activity of Natural Methoxylated Flavones Xanthomicrol and Eupatilin in A375 Skin Melanoma Cells

Abstract

Melanoma is a skin cancer caused by the malignant transformation of melanocytes and cutaneous melanoma represents the most aggressive and deadliest type of skin cancer with an increasing incidence worldwide. The main purpose of the present research was to evaluate the anticancer effects of the natural bioactive compounds xanthomicrol (XAN) and eupatilin (EUP) in human A375 malignant skin melanoma cells, a cell line widely used as an in vitro model of cutaneous melanoma. XAN and EUP are lipophilic methoxylated flavones with antioxidant, anti-inflammatory, and antitumor properties. The effects of XAN and EUP on cell viability, morphology, lipid profile, oxidative status, apoptosis, and mitochondrial membrane polarization were determined and compared in A375 cells. At 24 h-incubation (MTT assay), XAN significantly reduced viability at the dose range of 2.5-200 μM, while EUP showed a significant cytotoxicity from 25 μM. Moreover, both methoxylated flavones induced (at 10 and 25 μM, 24 h-incubation) marked cell morphological alterations (presence of rounded and multi-nucleated cells), signs of apoptosis (NucView 488 assay), and a noteworthy mitochondrial membrane depolarization (MitoView 633 assay), coupled to a marked lipid profile modulation, including variations in the ratio of phospholipid/cholesterol and a decrease in the oleic, palmitic, and palmitoleic acid amounts. Moreover, a remarkable time-dependent ROS generation (2',7'-dichlorodihydrofluorescein diacetate assay) was observed during 3 h-incubation of A375 cancer cells in the presence of XAN and EUP (10 and 25 μM). Our results confirm the potential antitumor effect of natural EUP and XAN in cutaneous melanoma by the activation of multiple anticancer mechanisms.

Keywords: apoptosis; cancer cells; eupatilin; lipid profile modulation; methoxylated flavones; xanthomicrol.

Figure 1

Chemical structure of flavones (1), eupatilin (EUP, 2), xanthomicrol (XAN, 3), and quercetin (QRC, 4).

Figure 2

Values of viability, expressed as % of the control (0), induced by 24 h-incubation with different concentrations (2.5–200 μM) of xanthomicrol (XAN), eupatilin (EUP), the reference anticancer flavone quercetin (QRC), and the maximal non-toxic vehicle dose (DMSO 2%) in cancer A375 cells (MTT assay). All data are presented as mean (n = 12) and standard deviation. Statistical significance of differences was assessed by One-way ANOVA and Bonferroni post Test. For each series: *** = p < 0.001 versus control. For each concentration group: °°° = p < 0.001, °° = p < 0.01, ° = p < 0.05 versus XAN-treated cells; ^§§§ = p < 0.001, ^§ = p < 0.05 versus EUP-treated cells.

Figure 3

Phase contrast images of control A375 cancer cells, vehicle-treated cells (DMSO 2%, 24 h-incubation), and cells incubated for 24 h with different amounts (2.5–200 μM) of xanthomicrol (XAN), eupatilin (EUP), and quercetin (QRC). Bar = 100 μm.

Figure 4

Values of viability, expressed as % of the control (0), induced by 24 h-incubation with different concentrations (2.5–100 μM) of xanthomicrol (XAN), eupatilin (EUP), and the maximal non-toxic vehicle dose (DMSO 1%) in normal HaCaT keratinocytes (MTT assay). All data are presented as mean (n = 9) and standard deviation. For each series: *** = p < 0.001, ** = p < 0.01 versus control (One-way ANOVA and Bonferroni post Test). For each concentration group: °°° = p < 0.001, °° = p < 0.01 versus XAN-treated cells (Student’s unpaired t-test with Welch’s correction).

Figure 5

ROS-induced fluorescence, expressed as % of the control (0), measured at different time points in melanoma A375 cells (A) and normal HaCaT keratinocytes (B) during 3 h of incubation with eupatilin (EUP) and xanthomicrol (XAN) 10 and 25 μM. All data are presented as mean (n = 9) and standard deviation. At each time point: *** = p < 0.001, ** = p < 0.01, * = p < 0.05 versus control (0); ^§§§ = p < 0.001, ^§§ = p < 0.01, ^§ = p < 0.05 versus cells treated with EUP 10 μM; °°° = p < 0.001, °° = p < 0.01, ° = p < 0.05 versus cells treated with EUP 25 μM. Statistical significance of differences was evaluated by One-way ANOVA and Bonferroni post Test.

Figure 6

(A) Chromatographic profile obtained by HPLC-ELSD analysis of free cholesterol (FC), polyunsaturated phospholipids (P-PL), and saturated/monounsaturated phospholipids (S/M-PL), measured in control A375 cells (Ctrl) and cells treated for 24 h with eupatilin (EUP) and xanthomicrol (XAN) 10 and 25 μM. The chromatographic region of each lipid class was assigned using a mixture of standard saturated/monounsaturated (PC: 16:0/16:0, 18:1/18:1, 16:0/18:1, 18:1/16:0) and polyunsaturated phosphatidylcholines (PC: 16:0/18:2, 16:0/20:4, 18:2/18:2; 20:5/20:5). (B) Values (expressed as % controls) of PL and FC measured in A375 control cells and cells treated with EUP and XAN (10 and 25 μM). All data are presented as mean (n = 6) and standard deviation. For each data series: *** = p < 0.001, ** = p < 0.01, * = p < 0.05 versus Ctrl; °°° = p < 0.001, °° = p < 0.01 for EUP and XAN 25 μM versus the respective 10 μM-treated cells (One-way ANOVA and Bonferroni post Test).

Figure 7

Values (expressed as μg/plate) of the main saturated and unsaturated fatty acids measured in control A375 melanoma cells (Ctrl) and cells treated for 24 h with eupatilin (EUP) and xanthomicrol (XAN) 10 and 25 μM (A) and the chromatographic profile of the control A375 cells measured by DAD (at 200 nm) and ELSD detection (B). Values of the ratios among the main FA measured in A375 control cells and cells treated with EUP and XAN, with each stacked bar chart representing the sum of FA ratios determined in each cell sample (C). Values of 16:1 n-7/16:0 and 18:1 n-9/18:0 ratios measured in control and treated A375 cells (D). All data are presented as mean (n = 6) and standard deviation. *** = p < 0.001, ** = p < 0.01, * = p < 0.05 versus Ctrl; °°° = p < 0.001, ° = p < 0.05 versus cells treated with EUP 10 μM. Statistical significance of differences was assessed by One-way ANOVA and Bonferroni post Test.

Figure 8

Phase contrast and red emission images as revealed by MitoView 633 fluorescence of control A375 cells (Ctrl) and melanoma cells 24 h-treated with eupatilin (EUP) and xanthomicrol (XAN) 10 and 25 μM (bar = 100 μm) (A). Mitochondrial membrane potential variations (intensity of red emission fluorescence expressed as % controls) after EUP and XAN treatment as resulted after image analysis (B). White arrows display multinucleated figures in red emission images measured in cells after 24 h-treatment with 25 μM EUP and XAN (C). All data are presented as mean (n = 6) and standard deviation. *** = p < 0.001 versus Ctrl; °°° = p < 0.001 versus cells treated with EUP 10 μM. Statistical significance of differences was assessed by One-way ANOVA and Bonferroni post Test.

Figure 9

Phase contrast and green emission images (bar = 100 μm) as revealed by NucView 488 fluorescence of control A375 cells (Ctrl) and melanoma cells 24 h-treated with eupatilin (EUP) and xanthomicrol (XAN) 10 and 25 μM (A). Apoptosis induction (intensity of green emission fluorescence expressed as % controls) after EUP and XAN treatment as resulted after image analysis (B). All data are presented as mean (n = 6) and standard deviation. *** = p < 0.001, ** = p < 0.01 versus Ctrl; °°° = p < 0.001, °° = p < 0.01 versus cells treated with EUP 10 μM; ^§§ = p < 0.01 versus cells treated with EUP 25 μM. Statistical significance of differences was assessed by One-way ANOVA and Bonferroni post Test.