Apoptosis Pathways Triggered by a Potent Antiproliferative Hybrid Chalcone on Human Melanoma Cells

Abstract

The World Health Organization reported that approximately 324,000 new cases of melanoma skin cancer were diagnosed worldwide in 2020. The incidence of melanoma has been increasing over the past decades. Targeting apoptotic pathways is a potential therapeutic strategy in the transition to preclinical models and clinical trials. Some naturally occurring products and synthetic derivatives are apoptosis inducers and may represent a realistic option in the fight against the disease. Thus, chalcones have received considerable attention due to their potential cytotoxicity against cancer cells. We have previously reported a chalcone containing an indole and a pyridine heterocyclic rings and an α-bromoacryloylamido radical which displays potent antiproliferative activity against several tumor cell lines. In this study, we report that this chalcone is a potent apoptotic inducer for human melanoma cell lines SK-MEL-1 and MEL-HO. Cell death was associated with mitochondrial cytochrome c release and poly(ADP-ribose) polymerase cleavage and was prevented by a non-specific caspase inhibitor. Using SK-MEL-1 as a model, we found that the mechanism of cell death involves (i) the generation of reactive oxygen species, (ii) activation of the extrinsic and intrinsic apoptotic and mitogen-activated protein kinase pathways, (iii) upregulation of TRAIL, DR4 and DR5, (iv) downregulation of p21^Cip1/WAF1 and, inhibition of the NF-κB pathway.

Keywords: apoptosis; caspases; cytotoxicity; extracellular signal-regulated kinases; hybrid chalcones; melanoma; mitogen-activated protein kinase; reactive oxygen species.

Figure 1

The hybrid chalcone (HY-CHAL) reduced the viability of human SK-MEL-1 and MEL-HO melanoma cells. (a) Chemical structure of the synthetic HY-CHAL; (b) Dose-response study on MTT reduction (means ± SEs; * indicates significant difference from the control at 0.05 significance level). Cells were incubated in the presence of increasing concentrations of HY-CHAL for 72 h, and thereafter mitochondrial respiratory function was determined by the MTT assay; (c) Cells were incubated with 3 μM HY-CHAL for different time periods and images were obtained with an inverted phase-contrast microscope; original magnification 20×.

Figure 2

HY-CHAL induced apoptosis in melanoma cells. (a) Cells were incubated with increasing concentrations of HY-CHAL for 24 h, stained with bisbenzimide trihydrochloride, and images of representative fields were obtained with a fluorescence microscopy; (b) Representative histograms of flow cytometry study. Cells were incubated with 3 µM HY-CHAL, fixed and analyzed by flow cytometry after propidium iodide labeling. Hypodiploid cells (i.e., apoptotic cells) are shown in the region marked with a double edge arrow; (c) Cells were treated with increasing concentrations of HY-CHAL and the percentage of hypodiploid cells was determined by flow cytometry. Bars represent the means ± SEs of three independent experiments each were performed in triplicate. * p < 0.05, significantly different from untreated control; (d) Cells were incubated with the specified concentrations of HY-CHAL for 24 h, stained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. Data shown are representative of three independent experiments with similar results.

Figure 3

HY-CHAL induced caspase cascade in SK-MEL-1 melanoma cells. (a) Cells were treated with 3 μM HY-CHAL for the indicated times and caspase activation was determined using specific colorimetric substrates; (b) Cells were treated with the specified concentrations of HY-CHAL and poly(ADP-ribose) polymerase (PARP) cleavage and caspase processing was determined by immunoblotting. The membrane was stained with Ponceau S before antibody detection to control equal protein loading; (c) Cells were pretreated with 100 µM z-VAD-fmk for 1 h and then incubated in the absence or in the presence of 3 µM HY-CHAL for 24 h and images were obtained with an inverted phase-contrast microscope; (d) Cells were treated as in (c) and hypodiploid cells (i.e., apoptotic cells) were analyzed by flow cytometry. Bars represent the mean ± SE of three independent experiments each performed in triplicate. * Indicates p < 0.05 for comparison with untreated control. ^# Indicates p < 0.05 for comparison with HY-CHAL treatment alone; (e) Cells were treated as in (c) and cell viability was determined by the trypan blue exclusion method using a TC counter; (f) Cells were pretreated with the selective caspase inhibitors z-DEVD-fmk (iC3, 50 µM), z-IETD-fmk (iC8, 50 µM) and z-LEHD-fmk (iC9, 50 µM) for 1 h before the addition of 3 µM HY-CHAL and apoptotic cells were quantified as in (d).

Figure 4

Role of the mitochondria, TRAIL and Death Receptors in the apoptotic cell death triggered by HY-CHAL in SK-MEL-1 melanoma cells. (a) Cells were treated with the specified concentrations of HY-CHAL for the indicated times and whole cell lysates were probed with antibodies raised against the indicated Bcl-2 family proteins, TRAIL, and death receptors by immunoblotting. Equal protein loading was controlled by staining the membranes with Ponceau S before the incubation with antibodies (a representative section of the stained membrane is shown); (b) Cells were treated with 3 µM HY-CHAL for the indicated times and cytosolic fractions prepared, separated by SDS-PAGE and cytochrome c was detected by immunoblotting. Equal protein loading was controlled by staining the membranes with Ponceau S; (c) Cells were incubated with the indicated concentrations of HY-CHAL for 6 h and ΔΨ_m analyzed by flow cytometry after staining with the JC-1 probe as described in the Experimental Section; (d) Cells were incubated in control conditions or in the presence of the indicated concentrations of HY-CHAL for the specified times, and the percentage of cells with reduced ΔΨ_m was quantified by flow cytometry using the fluorescent probe JC-1. * Indicates p < 0.05 for comparison with untreated control.

Figure 5

Effect of HY-CHAL on MAPK and AKT pathways. (a) Time-dependent phosphorylation of ERK1/2, JNK/SAPK and p38^MAPK and (b) of AKT and GSK-3β by HY-CHAL. Cells were incubated with the concentrations specified for the time periods shown. Lysates were analyzed on Western blots probed with specific antibodies to ascertain the phosphorylation of MAPKs, AKT, and GSK-3β. Membranes were stripped and reprobed with specified antibodies as loading controls. Equal protein loading was also controlled by staining the membrane with Ponceau S; (c) Effect of MAPKs and PI3K inhibitors on HY-CHAL-induced cell death. Cells were preincubated with the p38^MAPK inhibitor SB203580 (2 µM), the JNK/SAPK inhibitor SP600125 (10 µM), the indicated concentrations of the MEK1/2 inhibitors U0126 and PD98059 or the PI3K inhibitor LY294002 (20 µM) and then treated with 3 µM HY-CHAL for 24 h. The percentages of hypodiploid cells were determined by flow cytometry after propidium iodide staining. Bars represent means ± SEs of two independent experiments performed in triplicate. * Indicates p < 0.05 for comparison with untreated control. ^# Indicates p < 0.05 for comparison with HY-CHAL treatment alone.

Figure 6

HY-CHAL downregulated β-catenin, c-myc and p21^Cip1/WAF1, and inhibited NF-κB. (a,b) Cells were treated with the specified concentrations of HY-CHAL for the indicated times and cell lysates were probed with the specified antibodies. Equal loading was controlled by staining the membrane with Ponceau S.

Figure 7

Role of ROS in HY-CHAL-induced cell death. (a) Representative histogram obtained by flow cytometry of the fluorescence of oxidized H₂DCF after treatment of SK-MEL-1 with 3 μM HY-CHAL for 1 h; (b) Cells were preincubated with catalase (500 units/mL), N-acetyl-L-cysteine (5 mM), or glutathione (5 mM) and then treated with HY-CHAL (3 µM) for 24 h and thereafter the percentage of hypodiploid cells was determined by flow cytometry; (c) Fluorescence obtained by the oxidation of H₂DCF of cells preincubated with glutathione (5 mM) for 2 h and then incubated with HY-CHAL (3 µM) as in (a). * Indicates p < 0.05, significantly different from untreated control. ^# Indicates p < 0.05, significantly different from HY-CHAL.