Chalcone-Acridine Hybrid Suppresses Melanoma Cell Progression via G2/M Cell Cycle Arrest, DNA Damage, Apoptosis, and Modulation of MAP Kinases Activity

This study was focused on investigating the antiproliferative effects of chalcone hybrids in melanoma cancer cells. Among seven chalcone hybrids, the chalcone-acridine hybrid 1C was the most potent and was selected for further antiproliferative mechanism studies. This in vitro study revealed the potent antiproliferative effect of 1C via cell cycle arrest and apoptosis induction. Cell cycle arrest at the G2/M phase was associated with modulation of expression or phosphorylation of specific cell cycle-associated proteins (cyclin B1, p21, and ChK1), tubulins, as well as with the activation of the DNA damage response pathway. Chalcone 1C also induced apoptosis accompanied by mitochondrial dysfunction evidenced by a decrease in mitochondrial membrane potential, increase in Bax/Bcl-xL ratio and cytochrome c release followed by caspase 3/7 activation. In addition, increased phosphorylation of MAP kinases (Erk1/2, p38 and JNK) was observed in chalcone 1C-treated melanoma cells. The strong antiproliferative activities of this chalcone-acridine hybrid suggest that it may be useful as an antimelanoma agent in humans.


Introduction
The incidence of melanoma worldwide is increasing at a greater rate than other types of cancer (1.7% of global cancer diagnoses) [1]. The prevalence of skin melanoma differs among populations. The disease occurs mostly in white-skinned Caucasian populations in Australia and New Zealand [2]. In Europe, melanoma annually claims more than 20,000 lives, and it is a significant public health burden [3]. The therapy of melanoma depends on the stage of disease but generally includes surgical excision, treatment with immune checkpoint inhibitors, targeted therapy, radiotherapy or chemotherapy [4]. Although mortality rates have fallen over the past decade with the approval of new targeted therapies such as BRAF and MEK inhibitors and immune checkpoint inhibitors, many of the current anti-melanoma drugs are expensive and toxic [5][6][7]. Furthermore, despite the substantial therapeutic outcome of modern therapy, some patients acquire drug resistance and melanoma recurrence. For this reason, the development of novel, less toxic therapeutics for patients with melanoma remains essential.
In the last few years, plant-derived natural compounds have been extensively studied for their anti-proliferative and anti-cancer effects [8][9][10][11][12][13]. Phytochemicals are predominantly attractive because of their availability, low toxicity and absence of serious adverse reactions [14,15]. Furthermore, several clinical trials have reported that natural compounds tested either as single agent or in combination with standard chemotherapeutic drugs 2 of 25 improve sensitivity to chemotherapy and radiotherapy along with the survival of patients [16][17][18][19][20].
In the present study, we investigated the mechanism of acridine chalcone 1C on the induction of apoptosis in A2058 and BLM melanoma cells. Our results indicate that the antiproliferative effect of chalcone 1C is associated with the induction of an intrinsic pathway of apoptosis, G2/M cell cycle arrest, DNA damage and the modulation of selected signaling pathways. To the best of our knowledge, this is the first study displaying the antiproliferative activity of the chalcone-acridine hybrid against melanoma cancer cell lines.

MTT Screening Assay
The effect of synthetic chalcone derivatives on the metabolic activity of selected melanoma and healthy cell lines was determined using the MTT assay. Chalcone derivates suppressed cell metabolism with IC50 values ranging from 7.96 to >100 µmol/L (Table 1). For further experiments, the most potent acridine chalcone 1C was selected using a concentration of 1C 10 µmol/L for A2058 and 20 µmol/L for the BLM melanoma cell line. DMSO was used as negative control with no effect on melanoma cell growth. Results are presented as the mean ± SD of three independent experiments.

BrdU Cell Proliferation Assay
The BrdU Cell Proliferation Assay is based on the detection of BrdU (5-bromo-2deoxyuridine) incorporated into the newly synthesized DNA during the replication process in proliferating cells. The results showed that chalcone 1C suppressed proliferation of melanoma cells ranging from 5 to 20 µmol/L compared with DMSO control (Figure 1). The IC50 values were 8.40 ± 0.05 µmol/L (A2058) and 16.51 ± 0.01 µmol/L (BLM) after 72 h of incubation. The comparison of IC50 from MTT and BrdU assay is shown in Table 2. of melanoma cells ranging from 5 to 20 μmol/L compared with DMSO control (Figure 1). The IC50 values were 8.40 ± 0.05 μmol/L (A2058) and 16.51 ± 0.01 μmol/L (BLM) after 72 h of incubation. The comparison of IC50 from MTT and BrdU assay is shown in Table 2.

AO/PI Apoptosis Analysis
Staining with acridine orange (AO) and propidium iodide (PI) is a method for dividing cells into populations according to whether they are living, apoptotic or necrotic. AO is a dye binding to living and dead cells while PI only stains cells with lost membrane integrity. As shown in Figure 2, the chalcone 1C caused a significant decrease in proliferation and viability of A2058 and BLM melanoma cells. The number of apoptotic (yellow and orange) cells increased with a time-dependent trend simultaneously with the increased detachment of cells as a result of lost adhesion. The results of AO/PI staining, annexin V/PI staining and cell cycle analysis show that chalcone 1C induces the apoptotic cell death of melanoma cells. Melanoma cells were exposed to chalcone 1C at concentrations ranging from 5 to 20 µmol/L for 72 h. The data show the mean ± SD values of three independent experiments. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO. Results are presented as the mean ± SD of three independent experiments. The selectivity index was calculated on the basis of MTT or BrdU results.

AO/PI Apoptosis Analysis
Staining with acridine orange (AO) and propidium iodide (PI) is a method for dividing cells into populations according to whether they are living, apoptotic or necrotic. AO is a dye binding to living and dead cells while PI only stains cells with lost membrane integrity. As shown in Figure 2, the chalcone 1C caused a significant decrease in proliferation and viability of A2058 and BLM melanoma cells. The number of apoptotic (yellow and orange) cells increased with a time-dependent trend simultaneously with the increased detachment of cells as a result of lost adhesion. The results of AO/PI staining, annexin V/PI staining and cell cycle analysis show that chalcone 1C induces the apoptotic cell death of melanoma cells.

Cell Cycle Analysis
To determine whether cell cycle arrest is related to the inhibition of cell proliferation by chalcone 1C in A2058 and BLM melanoma cells, the cell cycle progression was examined by flow cytometry analysis. As demonstrated in Table 3 and Figure 3, treatment with chalcone 1C increased the number of melanoma cells in the G2/M phase at all exposition times (24, 48 and 72 h). Furthermore, a significant increase in the subG0 population (the marker of apoptosis) was observed in both melanoma cell lines with the highest peak after 72 h of treatment with chalcone 1C. The results suggest that the antiproliferative effect of chalcone 1C in A2058 and BLM melanoma cells can be associated with G2/M cell cycle arrest and the induction of apoptosis in a time-dependent pattern.

Cell Cycle Analysis
To determine whether cell cycle arrest is related to the inhibition of cell proliferation by chalcone 1C in A2058 and BLM melanoma cells, the cell cycle progression was examined by flow cytometry analysis. As demonstrated in Table 3 and Figure 3, treatment with chalcone 1C increased the number of melanoma cells in the G2/M phase at all exposition times (24, 48 and 72 h). Furthermore, a significant increase in the subG0 population (the marker of apoptosis) was observed in both melanoma cell lines with the highest peak after 72 h of treatment with chalcone 1C. The results suggest that the antiproliferative effect of chalcone 1C in A2058 and BLM melanoma cells can be associated with G2/M cell cycle arrest and the induction of apoptosis in a time-dependent pattern.   The data show the mean ± SD values of three independent experiments. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO (vehicle).

Apoptosis Detection
Phosphatidylserine (PS) is an anionic phospholipid located on the cytoplasmic surface of the plasma membrane in cells with intact membranes. The externalization of PS from the inner to outer surface of the lipid bilayer of plasma membrane is a typical marker of apoptosis in the early stages. Annexin V/PI double staining is a method for dividing cells into populations of living (An − /PI − Q3), early apoptotic (An + /PI − Q4), late apoptotic/necrotic (An + /PI + Q2) and dead (An − /PI + Q1). The analysis showed the significant increase of early apoptotic and late apoptotic/necrotic A2058 and BLM melanoma cells after 48 and 72 h of treatment with chalcone 1C. Furthermore, the concomitant decrease of living cells and the increased number of dead cells were also observed in a time-dependent pattern (Table 4, Figure 4). The p53 is an important tumor suppressor protein activated when cells experience stress stimuli such as DNA damage and other genetic alterations. Phosphorylation of p53 protein disrupts the binding with its negative regulator Mdm2, which results in either cell cycle arrest, DNA repair or apoptosis. WB analysis showed significant increase in the amount of total and phosphorylated (maximum at 72 h) form of p53 protein in A2058 melanoma cells after treatment with chalcone 1C ( Figure 5, Supplement Figure S1A,B). In BLM melanoma cells the total p53 was maintenanted on basic levels as DMSO treated controls, with little decrease at 24 h. In comparison with A2058, in BLM cells phosphorylation increased late on 72 h.

p21 Protein
The p21Waf1/Cip1 is a tumor suppressor protein involved in cell cycle regulation. The inhibition of cyclin-CDK complexes by p21 protein leads to the blockade of cell cycle progression in the G1/S and G2/M phase. The increased expression of p21 protein is subsequently followed by phosphorylation of p53 protein as a response to DNA damage. A western blot analysis revealed a significant increase in the expression of p21 protein in A2058 melanoma cells after treatment with chalcone 1C at concentration 10 µmol/L after

p21 Protein
The p21Waf1/Cip1 is a tumor suppressor protein involved in cell cycle regulation. The inhibition of cyclin-CDK complexes by p21 protein leads to the blockade of cell cycle progression in the G1/S and G2/M phase. The increased expression of p21 protein is subsequently followed by phosphorylation of p53 protein as a response to DNA damage. A western blot analysis revealed a significant increase in the expression of p21 protein in A2058 melanoma cells after treatment with chalcone 1C at concentration 10 μmol/L after 24 and 48 h. In contrast with A2058 cells, BLM melanoma cells showed an increase and maintained a trend in the expression of p21 protein after treatment with tested chalcone at concentrations of 20 μmol/L ( Figure 6, Supplement Figure S2A,B).

p21 Protein
The p21Waf1/Cip1 is a tumor suppressor protein involved in cell cycle regulation The inhibition of cyclin-CDK complexes by p21 protein leads to the blockade of cell cycle progression in the G1/S and G2/M phase. The increased expression of p21 protein is subsequently followed by phosphorylation of p53 protein as a response to DNA damage. A western blot analysis revealed a significant increase in the expression of p21 protein in A2058 melanoma cells after treatment with chalcone 1C at concentration 10 μmol/L after 24 and 48 h. In contrast with A2058 cells, BLM melanoma cells showed an increase and maintained a trend in the expression of p21 protein after treatment with tested chalcone at concentrations of 20 μmol/L ( Figure 6, Supplement Figure S2A,B).

Chk1 Kinase
Chk1 is a serine/threonine kinase involved in cellular response to DNA damage and activation of cell cycle checkpoints. Chk1 is phosphorylated by activated ATR kinase and is restricted to S and G2 phases. Results showed that chalcone 1C significantly induced the phosphorylation of Chk1 in A2058 melanoma cells after 24, 48 (maximum) and 72 h of

Chk1 Kinase
Chk1 is a serine/threonine kinase involved in cellular response to DNA damage and activation of cell cycle checkpoints. Chk1 is phosphorylated by activated ATR kinase and is restricted to S and G2 phases. Results showed that chalcone 1C significantly induced the phosphorylation of Chk1 in A2058 melanoma cells after 24, 48 (maximum) and 72 h of treatment. Analysis confirmed a consilient trend of Chk1 phosphorylation in BLM melanoma cells ( Figure 6, Supplement Figure S2C,D).

Cyclin B1
Cyclin B1 is a regulatory protein expressed predominantly during the G2/M phase. The activated cyclin B1-Cdk1 complex is involved in early events of mitosis, such as chromosome condensation, breakdown of the nuclear envelope and the assembly of the spindle apparatus. The analysis showed significant downregulation of phosphorylated cyclin B1 in A2058 and BLM melanoma cells after treatment with chalcone 1C with maximum after 72 h in both cell lines ( Figure 6, Supplement Figure S2E,F).

Effect on Tubulins
Microtubules, key components of the cytoskeleton, are polymers of αand β-tubulins responsible for many cellular processes including cell division, cell migration and maintenance of cell structure. The dysregulation of tubulins can lead to G2/M cell cycle arrest or apoptosis. Western blot analysis revealed significant alterations in expression of α-, α1Cand β-tubulin in A2058 and BLM melanoma cells after treatment with chalcone 1C. As shown in Figure 6 and Supplement Figure S2G-L, the significant upregulation of α-, α1Cand β-tubulin was observed in the A2058 cell line with a maximum after 48 and 72 h. In the BLM cell line, results showed significant downregulation of αand β-tubulin in a time-dependent pattern and the upregulation of α1Ctubulin with a maximum after 72 h of treatment.

Cytochrome c Release
Cytochrome c is essential for controlling the energy metabolism of cells and apoptosis. As a result of apoptotic stimulus, cytochrome c is released from the mitochondria into cytosol, which activates programmed cell death. Results showed the significant release of cytochrome c in A2058 and BLM melanoma cells after 24, 48 and 72 h of treatment with chalcone 1C (Figure 8).

Mitochondrial Membrane Potential (MMP)
The mitochondria play a crucial role in the regulation of cell survival and the induction of apoptotic cell death. One of the key indicators of mitochondrial activity is mitochondrial membrane potential (MMP). The loss of MMP due to mitochondrial dysfunction may lead to apoptosis. As shown in Figure 9, the treatment of A2058 and BLM melanoma cells with chalcone 1C caused a significant increase in the percentage of cells with reduced MMP at all exposition times (24, 48 and 72 h).

Cytochrome c Release
Cytochrome c is essential for controlling the energy metabolism of cells and apoptosis. As a result of apoptotic stimulus, cytochrome c is released from the mitochondria into cytosol, which activates programmed cell death. Results showed the significant release of cytochrome c in A2058 and BLM melanoma cells after 24, 48 and 72 h of treatment with chalcone 1C (Figure 8).

Cytochrome c Release
Cytochrome c is essential for controlling the energy metabolism of cells and apoptosis. As a result of apoptotic stimulus, cytochrome c is released from the mitochondria into cytosol, which activates programmed cell death. Results showed the significant release of cytochrome c in A2058 and BLM melanoma cells after 24, 48 and 72 h of treatment with chalcone 1C (Figure 8).  The mitochondria play a crucial role in the regulation of cell survival and the induction of apoptotic cell death. One of the key indicators of mitochondrial activity is mitochondrial membrane potential (MMP). The loss of MMP due to mitochondrial dysfunction may lead to apoptosis. As shown in Figure 9, the treatment of A2058 and BLM melanoma cells with chalcone 1C caused a significant increase in the percentage of cells with reduced MMP at all exposition times (24, 48 and 72 h).   The mitochondria play a crucial role in the regulation of cell survival and the induction of apoptotic cell death. One of the key indicators of mitochondrial activity is mitochondrial membrane potential (MMP). The loss of MMP due to mitochondrial dysfunction may lead to apoptosis. As shown in Figure 9, the treatment of A2058 and BLM melanoma cells with chalcone 1C caused a significant increase in the percentage of cells with reduced MMP at all exposition times (24, 48 and 72 h).

Cleavage of PARP
Poly (ADP-ribose) polymerase (PARP) plays an important role in DNA repair and programmed cell death. The activation of caspases leads to cleavage of PARP resulting in DNA repair inhibition. As shown in Figure 11 and Supplement Figure S4, the levels of cleaved PARP (89 kDa) significantly increased in A2058 and BLM melanoma cells after treatment with chalcone 1C at all exposition times (24, 48 and 72 h).

Cleavage of PARP
Poly (ADP-ribose) polymerase (PARP) plays an important role in DNA repair and programmed cell death. The activation of caspases leads to cleavage of PARP resulting in DNA repair inhibition. As shown in Figure 11 and Supplement Figure S4, the levels of cleaved PARP (89 kDa) significantly increased in A2058 and BLM melanoma cells after treatment with chalcone 1C at all exposition times (24, 48 and 72 h).

ATM Kinase
The ATM (ataxia-telangiectasia mutated) serine/threonine kinase phosphorylates several key proteins that regulate DNA repair, cell cycle checkpoint control and apoptosis. The phosphorylation of ATM is activated by DNA double-strand breaks. As demonstrated in Figure 12A  The ATM (ataxia-telangiectasia mutated) serine/threonine kinase phosphorylates several key proteins that regulate DNA repair, cell cycle checkpoint control and apoptosis. The phosphorylation of ATM is activated by DNA double-strand breaks. As demonstrated in Figure 12A,B, chalcone 1C significantly induced phosphorylation of ATM in A2058 and BLM melanoma cells in a time-dependent manner (24, 48 and 72 h).

SMC1 Protein
The SMC1 (structural maintenance of chromosomes 1) protein is a member of key proteins regulating DNA repair and the cohesion of chromosomes during the cell cycle. SMC1 activation is mediated by ATM as a response to DNA damage. Analysis showed the significant phosphorylation of SMC1 in A2058 and BLM melanoma cells after 24, 48 and 72 h of treatment with chalcone 1C (Figure 12C,D).

Histone HA2.X
The Histone H2A.X is a histone variant essential for DNA repair. The phosphorylation of H2A.X occurs due to double-strand DNA breaks induced by genotoxic stress resulting in DNA repair, cell cycle arrest or apoptosis. Treatment of A2058 and BLM melanoma cells with chalcone 1C significantly induced phosphorylation of H2A.X after 24, 48 and 72 h ( Figure 12E,F).

Chalcone 1C Modulates Signalling Pathways/Changes in Expression and Phosphorylation of MAPK Proteins
Mitogen-activated protein kinases (MAPKs) are a family of proteins which modulate a series of vital signalling pathways involved in the regulation of cell proliferation, differentiation, survival and apoptosis. Each signalling pathway is initiated by external stimuli and leads to the activation of particular MAPK. The mammalian MAPKs are grouped into three families including extracellular signal-regulated kinases (ERKs), c-jun N-terminal kinases (JNKs) and p38s. Results from a WB analysis revealed that treatment of A2058 melanoma cells with chalcone 1C significantly increased the phosphorylation of ERK1/2 with a maximum after 48 h. Analysis also confirmed a consilient trend of ERK1/2 phosphorylation in BLM melanoma cells. In both melanoma cell lines, chalcone 1C significantly increased the phosphorylated form of p38 MAPK mainly after 48 h of incubation and phospho-JNK in all tested timepoints (Figure 13, Supplement Figure S5).

SMC1 Protein
The SMC1 (structural maintenance of chromosomes 1) protein is a member of key proteins regulating DNA repair and the cohesion of chromosomes during the cell cycle. SMC1 activation is mediated by ATM as a response to DNA damage. Analysis showed three families including extracellular signal-regulated kinases (ERKs), c-jun N-terminal kinases (JNKs) and p38s. Results from a WB analysis revealed that treatment of A2058 melanoma cells with chalcone 1C significantly increased the phosphorylation of ERK1/2 with a maximum after 48 h. Analysis also confirmed a consilient trend of ERK1/2 phosphorylation in BLM melanoma cells. In both melanoma cell lines, chalcone 1C significantly increased the phosphorylated form of p38 MAPK mainly after 48 h of incubation and phospho-JNK in all tested timepoints (Figure 13, Supplement Figure S5).

Discussion
As was mentioned above, numerous pharmacological activities (including antiproliferative and anticancer) have effects that have been attributed to chalcones [54,55]. It is well known that chalcone moiety is an effective template for the discovery of new anticancer drugs and its hybridization with other anticancer agents represents a promising approach to develop novel agents with high anticancer activity [56].
Acridine-based agents represent a family of heterocyclic compounds which are currently undergoing significant research because of their potential anticancer activity. The pleiotropic mechanism of the antiproliferative effect of acridine derivatives, including topoisomerase II inhibition [57], cell cycle arrest in S phase [58] and G2/M phase [59], tubulin polymerization inhibition [60], DNA damage [61] and apoptosis induction [58] have been documented.

Discussion
As was mentioned above, numerous pharmacological activities (including antiproliferative and anticancer) have effects that have been attributed to chalcones [54,55]. It is well known that chalcone moiety is an effective template for the discovery of new anticancer drugs and its hybridization with other anticancer agents represents a promising approach to develop novel agents with high anticancer activity [56].
Acridine-based agents represent a family of heterocyclic compounds which are currently undergoing significant research because of their potential anticancer activity. The pleiotropic mechanism of the antiproliferative effect of acridine derivatives, including topoisomerase II inhibition [57], cell cycle arrest in S phase [58] and G2/M phase [59], tubulin polymerization inhibition [60], DNA damage [61] and apoptosis induction [58] have been documented.
On the other hand, the antiproliferative effect of chalcone-acridine hybrids has been studied only minimally. Recently, we described the antiproliferative effect of the chalconeacridine hybrid in colorectal cancer cells [36]. At micromolar concentrations, this chalcone inhibited cell proliferation associated with G2/M block, and dysregulated of tubulin expression, apoptosis induction as well as the modulation of several signalling pathways associated with cell life and death. We later showed that the antiproliferative effect of this acridine chalcone is closely related to the generation of reactive oxygen species [37]. Moreover, it may overcome drug resistance as we observed growth suppression in P-glycoproteinexpressing cancer cells [62]. Furthermore, in the most recent study we documented the proapoptotic effect of the novel chalcone-acridine hybrid in breast cancer cells. In addition to the induction of mitochondrial apoptosis, DNA studies demonstrated that it interacts with DNA through bimodal binding mode, i.e., intercalation and groove-binding [38]. Besides this, some in silico studies also suggest the anticancer potential of chalcone-acridine hybrids [63,64].
In the present work, we studied possible mechanisms of action of either acridine or indole chalcone derivatives in vitro using a melanoma cancer model. Among the tested chalcones, a chalcone-acridine hybrid (1C) showed the highest antiproliferative potency and was selected for the next study.
In our recent article [41], we mentioned pleiotropic mechanisms of the antiproliferative effect of chalcones in in vitro cancer models. Among others, chalcones caused cell cycle arrest, mostly at the G2/M phase [36,65,66]. In the present paper, the exposure of melanoma cells to chalcone 1C caused cell cycle arrest at the G2/M phase with simultaneous increase in cell number with sub-G0/G1 DNA content. Moreover, chalcone 1C also affected the expression as well as the phosphorylation of specific cell cycle-associated proteins including cyclin B1, p21, and Chk1. Our results indicated that the suppression of cell proliferation and arrest at the G2/M phase of cell cycle in chalcone 1C-treated melanoma cells may be related to the modulation of cycle-associated protein activity.
Furthermore, cell cycle arrest at the G2/M phase is the consequence of DNA damage and it is the last chance for cell repair prior to entering mitosis [67]. In response to DNA damage, the DNA damage response (DDR) pathway is activated [68], and the main components of DDR, ATM and ATR (ATM and Rad3-related) kinases subsequently phosphorylate several components involved in the cell cycle, DNA replication, DNA repair or apoptosis [69]. Our results showed that chalcone 1C-induced a significant increase in ATM phosphorylation followed by its downstream molecules phosphorylation including histone H2A.X at Ser139 (γ-H2A.X), p53 and p21 proteins, SMC1, indicating that this chalcone has DNA toxicity. Recently, different chalcone hybrids have been reported to have an antiproliferative effect associated with DNA damage [48,70]. In addition, DDR is also associated with the activation of PARP, an enzyme involved in DNA repair [71]. Our experiments showed that chalcone 1C induced the cleavage of PARP. After cleavage, PARP loses its function, resulting in the suppression of DNA repair [72].
Irreparable DNA damage is often associated with the induction of cell death, including apoptosis [73]. As was mentioned above, chalcone 1C induced the increase in cell numbers with sub-G0/G1 DNA content, which is the result of internucleosomal DNA fragmentation and is considered a marker of apoptosis. This result prompted us to (i) confirm apoptosis, and (ii) study molecular mechanisms of 1C-induced apoptosis in melanoma cells.
The translocation of PS from the cytoplasmic surface of the plasma membrane on the outer leaflet of the plasma membrane is one of the first events of apoptosis. Once externalized, PS can be visualised by annexin V staining [74]. In the present paper, we observed the significant increase in the number of apoptotic cells after 48 and 72 h of treatment in A2058 cells. In BLM melanoma cells, chalcone 1C increased the number of apoptotic cells after 24-72 h of incubation. Furthermore, apoptosis has also been proven by acridine orange and propidium iodide staining.
Today, the mechanisms of the apoptosis-inducing effect of 1C in melanoma cancer cells is not known. In the present study, we tried to discover the mechanism by which this chalcone induces apoptosis.
Mitochondria play a key role in cell life and death. Mitochondrial membrane potential (∆Ψm, MMP) is critical for maintaining the mitochondrial physiological function and its loss is often considered as an early event in apoptosis [75]. The decrease in MMP is closely related to the permeabilization of the mitochondrial outer membrane with the subsequent release of several proapoptotic proteins including cytochrome c, Smac/DIABLO or apoptosis-inducing factor [76]. In our study, chalcone 1C significantly increased the number of cells with dissipated MMP after 24, 48 and 72 h of incubation and, concurrently, increased the cytosolic concentration of cytochrome c in both melanoma cell lines. Once cytochrome c is released, it interacts with apoptosis protease-activating factor 1, forming apoptosome, which mediates the activation of an initiator caspase-9 [77] followed by the activation of its downstream executioner caspases-3/7 [78]. In this context, our results showed the significant activation of caspases 3/7 in both melanoma cell lines after 24-72 h of incubation.
In addition, the integrity of the mitochondrial membrane is strictly regulated by the members of the Bcl-2 protein family [79]. The presented results showed the significant effect of chalcone 1C on different members of these proteins. We found that the exposition of both melanoma cell lines to chalcone 1C led to upregulation of the proapoptotic Bax protein and the downregulation of the antiapoptotic Bcl-xL protein. It has been documented that an increased Bax/Bcl-xL ratio supports apoptosis due to cytochrome c release with subsequent activation of caspases, as mentioned earlier [80]. Similar results were obtained with either synthetic or natural chalcones in different cancer cells [41,81]. Furthermore, we observed the increased phosphorylation of antiapoptotic Bcl-2 and proapoptotic Bad proteins. It is well known that the phosphorylation of the Bcl-2 family proteins is a key regulator of its function [82,83]. However, phosphorylation can be modulated by several factors, such as kinases involved in phosphorylation or target sites on Bcl-2 proteins resulting to either proapoptotic or antiapoptotic activity [84]. Several lines of evidence indicate that Bcl-2 phosphorylation induced by microtubule damaging agents led to the inactivation of its antiapoptotic function [85][86][87]. Furthermore, it has been documented that Bad phosphorylation is associated with the loss of its proapoptotic activity [88,89]. On the other hand, several experimental works showed that microtubule damaging agents-induced Bad phosphorylation promoted apoptosis [90,91]. As mentioned above, chalcone 1C induced cell arrest at the G2/M phase of the cell cycle and simultaneously modulated tubulin expression, which indicated its potentially microtubule damaging activity. Recently, Liu and co-workers [92] have reviewed the ability of several chemically different chalcones to interfere with tubulin and to disturb the dynamic balance of microtubules. On the basis of the above-mentioned facts, we suggest that the modulation of the expression/phosphorylation of the Bcl-2 protein family is involved in 1C-induced apoptosis in melanoma cells.
It is known that several protein kinases are involved in cell survival and death. Among them, MAPK is a key pathway related to apoptosis [93]. Of the main components of MAPK, ERK1/2 is mostly involved in cell survival, while the phosphorylation of JNK and p38 promote apoptosis [94].
A large number of studies have demonstrated that JNK and p38 phosphorylation is associated with apoptosis induced by various compounds such as taxanes [95], vinblastine [96] or doxorubicin [97]. In the present study, we documented increased JNK and p38 phosphorylation in both 1C-treated melanoma cells. Our results agreed with previous studies where the association between JNK and p38 phosphorylation and chalconeinduced apoptosis was documented [40,41,98,99]. Surprisingly, we also found the activation (i.e., phosphorylation) of Erk1/2 which, generally, prevents the apoptosis via either downregulation of proapoptotic or the upregulation of antiapoptotic proteins [93]. On the other hand, the opposite activity has also been documented [100]. The activation of Erk1/2 may lead to mitochondrial membrane disruption with subsequent cytochrome c release [101], modulation of Bcl-2 family protein expression [102], or the suppression of the PI3K/Akt pathway [103]. Furthermore, Erk phosophorylation in anticancer drug-induced apoptosis has also been noted [104,105]. . The structure of compounds was confirmed by using 1 H, 13 C nuclear magnetic resonance (NMR), infrared (IR) spectroscopy and mass spectrometry (MS), with 97% purity based quantitative NMR [106]. The studied compounds were dissolved in dimethyl sulfoxide (DMSO) with the final concentration of <0.2% in the culture medium. DMSO exhibited no cytotoxicity on cultured cells.

Tested Compounds
(NMR Laboratory, Institute of Chemistry, Faculty of Science, P.J. Košice). The structure of compounds was confirmed by using 1 H, 13 C resonance (NMR), infrared (IR) spectroscopy and mass spectrometry rity based quantitative NMR [106]. The studied compounds were dis sulfoxide (DMSO) with the final concentration of <0.2% in the cultur exhibited no cytotoxicity on cultured cells.

MTT Viability Assay
The half-maximal inhibitory concentration values (IC50) and metabolism inhibition of tested synthetic chalcone derivatives were determined by MTT (3-(4,5-di-methylthiazol-2yl)2,5-diphenyltetrazolium bromide) colorimetric assay (Sigma-Aldrich Chemie, Steinheim, Germany). Tested cell lines were seeded at a density of 5 × 10 3 cells/well in 96-well culture plates. After 24 h, the tested chalcones in concentrations of 100, 50 and 10 µmol/L were added and incubation proceeded for the next 72 h. In the next step, 10 µL of MTT (5 mg/mL) was added to each well containing cells and incubated for another 4 h at 37 • C during which MTT was metabolized to insoluble formazan in cells. After 4 h, 100 µL of a 10% sodium dodecyl sulphate (SDS) was added to each well and another 24 h were allowed for the formazan crystals to dissolve. The metabolic activity of cells was evaluated by measuring the absorbance at wavelength 540 nm using the automated Cytation™ 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). Three independent analyses were performed.

BrdU (5-Bromo-2 -deoxyuridine) Cell Proliferation Assay
The A2058 (5 × 10 3 /well) and BLM (5 × 10 3 /well) melanoma cells were plated in a 96-well culture plate in 80 µL suitable medium. Twenty-four hours after cell seeding, different concentrations of the chalcone 1C were added ranging from 5-20 µmol/L. After 48 h of treatment, BrdU labelling solution was added to melanoma cells and incubated for another 24 h at 37 • C followed by fixation and incubation with anti-BrdU peroxidase conjugate solution for an additional 90 min in the dark at room temperature. Cells were then washed 3× with washing solution and incubated with TMB (tetramethylbenzidine) substrate solution (all Roche, Basel, Switzerland) for 5 to 30 min according to colour intensity. Finally, the stop solution (25 µL 1 M H 2 SO 4 ) was added, and the incorporated BrdU was detected with an automated Cytation™ 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA) at 450 nm. Three independent analyses were performed.

AO/PI Viability Assay
The A2058 and BLM melanoma cells were seeded at a density of 5 × 10 4 /well into 6-well culture plates. Twenty-four hours after seeding, A2058 cells were treated with chalcone 1C at 10 µmol/L concentration, BLM cells at 20 µmol/L concentration and both melanoma cell lines were treated with vehicle (DMSO) at the same concentrations. At 24, 48 and 72 h after treatment, the culture medium was removed, the cells were washed with washing buffer (PBS) and fixed with 4% paraformaldehyde (pH 7.2) for 30 min. In the next step, the paraformaldehyde was removed, the cells were washed with PBS and the staining solution (10 µg/mL acridine orange and 10 µg/mL propidium iodide, Sigma-Aldrich) was added to each well for 1 h incubation at room temperature in the dark. Finally, the staining solution was removed, the cells were washed with PBS and apoptosis was observed using an automated Cytation™ 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA).

Cell Cycle Analysis
The A2058 and BLM melanoma cells were seeded at a density of 1 × 10 5 /dish in a Petri dish. Twenty-four hours after incubation, A2058 cells were treated with chalcone 1C at 10 µmol/L concentration, BLM cells at 20 µmol/L concentration and both melanoma cell lines were treated with DMSO as the negative control at the same concentrations. For flow cytometric analysis of the cell cycle, adherent and floating A2058 and BLM melanoma cells were harvested in three different times (24, 48 and 72 h) after treatment with chalcone 1C and DMSO, washed in cold washing buffer (PBS), fixed in cold 70% ethanol and stored at −20 • C at least overnight. Prior to analysis, cells were washed with PBS, resuspended in staining solution (Triton X-100 final concentration 0.1%, ribonuclease A final concentration 0.5 mg/mL and propidium iodide final concentration 0.025 mg/mL, all Sigma), and incubated for 30 min in the dark at room temperature. Stained melanoma cells were analysed using a BD FACSCalibur TM Flow Cytometer (Becton Dickinson, San Jose, CA, USA). Three independent analyses were performed.

Apoptosis Detection
To perform apoptosis detection, A2058 and BLM melanoma cells were seeded in Petri dishes at a density of 1 × 10 5 /dish and treated with chalcone 1C and DMSO as the negative control at the same concentrations for 24, 48 and 72 h. In the next step, adherent and floating melanoma cells were harvested, centrifuged and pellets were resuspended in washing buffer (PBS). Resuspended melanoma cells were stained with Annexin V-Alexa Fluor ® 647 antibody (Thermo Scientific, Rockford, IL, USA) in binding buffer for 20 min in the dark at room temperature. Finally, melanoma cells were washed, stained with 1 µL of propidium iodide (final concentration 25 µg/mL) for 5 min and analysed using a BD FACSCalibur TM Flow Cytometer (Becton Dickinson, San Jose, CA, USA). Three independent analyses were performed.

Flow Cytometric Analyses
The A2058 and BLM melanoma cells were seeded 1 × 10 6 in Petri dishes with complete growth medium and cultivated for 24 h. After cell cultivation, the A2058 cells were treated with chalcone 1C at 10 µmol/L concentration, BLM cells at 20 µmol/L concentration and both melanoma cell lines were treated with DMSO as the negative control at the same concentrations for 24, 48 and 72 h. Adherent and floating cells were harvested, pelleted by centrifugation at 1200 rpm for 5 min. Pellets were resuspended in washing buffer (PBS) and divided for a particular analysis. Afterwards, cells were prepared according to assay kit protocols or fixed with cold 4% paraformaldehyde (15 min) and permeabilized with 90% methanol (10 min on ice) with washing steps (PBS) and stained prior to analysis ( Table 5) for 15 min in the dark at room temperature. Fluorescence was detected using a BD FACSCalibur TM Flow Cytometer (Becton Dickinson, San Jose, CA, USA).

Western Blot Analyses
Melanoma cells (A2058 and BLM) were treated with the tested chalcone 1C (10 µM and 20 µM) for 24, 48 and 72 h. Protein lysates from melanoma cells were prepared using a Laemmle lysis buffer containing glycerol, 1M Tris/HCl (pH 6.8), 20% sodium dodecyl sulfate (SDS), deionized H20, phosphatase and protease inhibitors (Sigma-Aldrich) and a sonication process. The protein concentrations were determined by the Pierce ® BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) and measured by an automated Cytation™ 3 Cell Imaging Multi-Mode Reader (Biotek) at a wavelength of 570 nm. Proteins (25-40 µg of sample per well) were separated on SDS-PAA gel (12%) at 100 V for 3 h and transferred to a polyvinylidene difluoride (PVDF) membrane using the iBlot™ 2 Dry Blotting System (Invitrogen, Carlsbad, CA, USA). Membranes with transferred proteins were blocked in 5% BSA (bovine serum albumin; SERVA, Heidelberg, Germany) or 5% non-fat dry milk (Cell Signaling Technology ® , Danvers, MA, USA) in TBS-Tween (pH 7.4) for 1 h at room temperature to minimise non-specific binding. Blocking was followed by incubation with primary antibodies (Table 6) overnight at 4 • C. The next day, membranes were washed in TBS-Tween (3 × 5 min) and incubated with the corresponding horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibody for 1 h at room temperature. After incubation, membranes were washed again in TBS-Tween (3 × 5 min) and the expression of proteins was detected using the MF-ChemiBIS 2.0 Imaging System (DNR BIO-Imaging Systems, Jerusalem, Israel) with chemiluminescent ECL substrate (Thermo Scientific, Rockford, IL, USA). A densitometric analysis of Western Blot (WB) results was performed using the Image Studio™ Lite Software (LI-COR Biosciences, Lincoln, NE, USA). Equal loading was verified by using the β-actin antibody. Three independent analyses were performed.

Statistical Analyses
Results are expressed as mean ± standard deviation (SD). Statistical analyses of the data were performed using standard procedures with one-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparison test. Differences were considered significant when p < 0.05. Throughout this paper * indicates p < 0.05, ** p < 0.01, *** p < 0.001 versus vehicle (DMSO).

Conclusions
Overall, our results demonstrate that the chalcone-acridine hybrid 1C is a strong suppressor of melanoma cell survival. It induced G2/M cell cycle arrest by modulation of the p53, p21, cyclin B1, and ChK1 expression. Moreover, we also observed that chalcone 1C promoted apoptosis by disruption of mitochondrial functions as proved by the decrease of MMP, the modulation of the Bcl-2 protein family functions and cytochrome c release with subsequent caspase activation. Furthermore, the activation of several MAP kinases may also have an important role in the antiproliferative and pro-apoptotic effect of this chalcone. Taken together, the presented results showed that this chalcone-acridine hybrid may be a promising agent for melanoma treatment.