The Effect of Bornyl cis-4-Hydroxycinnamate on Melanoma Cell Apoptosis Is Associated with Mitochondrial Dysfunction and Endoplasmic Reticulum Stress

Bornyl cis-4-hydroxycinnamate, an active compound isolated from Piper betle stems, was investigated in terms of its effects on A2058 and A375 melanoma cell proliferation and protein expression in this study. We used flow cytometric analysis to examine the early stages of apoptosis induced by bornyl cis-4-hydroxycinnamate in the two melanoma cell lines and employed comparative proteomic analysis to investigate the effects of this compound on protein expression in A375 cells. Master maps generated by PDQuest software from two-dimensional electrophoresis (2-DE) analysis of A375 cells showed that the expression levels of 35 proteins were significantly altered, with 18 proteins upregulated and 17 downregulated. The proteomics study identified several proteins that are involved in mitochondrial dysfunction and endoplasmic reticulum stress (ER stress), in addition to apoptosis-associated proteins, including prohibitin, hypoxia-upregulated protein 1, stress 70 protein, 78 kDa glucose-regulated protein (GRP78), and protein deglycase DJ-1 (protein DJ-1) in melanoma cells exposed to bornyl cis-4-hydroxycinnamate. The treatment also resulted in a marked decline of the mitochondrial membrane potential, in cytochrome C release into the cytosol, in the activation of Bcl-2-associated X protein (Bax), Bcl-2-associated death promoter protein (Bad), caspase-3, and caspase-9, and in the decreased expression of p-Bad, B-cell lymphoma 2 (Bcl-2), Bcl-xl, and induced myeloid leukemia cell differentiation protein-1 (Mcl-1), indicating that apoptosis induced by bornyl cis-4-hydroxycinnamate was mediated by the mitochondria through the caspase-dependent pathway. Also, salubrinal (an eukaryotic initiation factor 2α inhibitor; eIF2α inhibitor) was able to protect the cells from bornyl cis-4-hydroxycinnamate-induced apoptosis. Bornyl cis-4-hydroxycinnamate-related cell death also implied that the protein kinase R-like endoplasmic reticulum kinase (PERK)–eIF2α–ATF4–CHOP signal pathways was activated upon bornyl cis-4-hydroxycinnamate treatment. Altogether, our results support the conclusion that bornyl cis-4-hydroxycinnamate-induced apoptosis in melanoma cells is associated with mechanisms correlated with the activation of caspase cascades, mitochondrial dysfunction, and endoplasmic reticulum stress, and indicate that this molecule has the potential to be developed as a chemotherapeutic agent for human melanoma.


Introduction
Melanoma has a relatively low incidence among skin cancers, but also causes the vast majority of skin cancer deaths. In addition, the incidence rates of melanoma worldwide continue to rise rapidly [1]. In 2017, the American Cancer Society estimated that more than 87,000 new melanomas would be diagnosed in the US and approximately 10,000 people would die of melanoma that year [2]. The majority of patients with metastatic melanoma are almost invariably incurable because no effective chemotherapy or immunotherapy are available [3][4][5]. Therefore, currently, surgical resection alone or in combination with other therapies is still the best option to prolong the survival of patients [6,7]. Despite the great numbers of studies and clinical trials that have been performed in recent decades, dacarbazine, the most active single agent against melanoma, can only achieve an approximate 20% objective response rate [8,9], and interleukin (IL)-2 therapy only produces a response rate below 20% [10,11]. Therefore, the development of novel drugs and therapeutic approaches for malignant melanoma is an urgent medical issue.
Piper betle Linn. (Piperaceae), known as the betel vine, is a perennial semi-woody climber with a strong pungent and aromatic flavor that grows widely in Southeast Asia. It is an important medicinal and economical plant in some Asian countries, such as Taiwan and India, and has been proven to exhibit antioxidative, antimicrobial, and anti-hemolytic activities [12][13][14]. Several bioactive constituents such as lignins, polyphenols, alkaloids, steroids, saponins, tannins, and terpenes have been isolated from the leaves and stems of P. betle [15]. Bornyl cis-4-hydroxycinnamate is an active compound isolated from P. betle stem. In this study, we investigated the effects of bornyl cis-4-hydroxycinnamate on the proliferation of melanoma cells and employed a comparative proteomics approach to identify the molecules involved in bornyl cis-4-hydroxycinnamate-induced apoptosis in A2058 and A375 melanoma cells. We aimed to ascertain the underlying mechanism, which may lead to the development of bornyl cis-4-hydroxycinnamate as a new treatment agent or a potential strategy against human melanoma.

Introduction
Melanoma has a relatively low incidence among skin cancers, but also causes the vast majority of skin cancer deaths. In addition, the incidence rates of melanoma worldwide continue to rise rapidly [1]. In 2017, the American Cancer Society estimated that more than 87,000 new melanomas would be diagnosed in the US and approximately 10,000 people would die of melanoma that year [2]. The majority of patients with metastatic melanoma are almost invariably incurable because no effective chemotherapy or immunotherapy are available [3][4][5]. Therefore, currently, surgical resection alone or in combination with other therapies is still the best option to prolong the survival of patients [6,7]. Despite the great numbers of studies and clinical trials that have been performed in recent decades, dacarbazine, the most active single agent against melanoma, can only achieve an approximate 20% objective response rate [8,9], and interleukin (IL)-2 therapy only produces a response rate below 20% [10,11]. Therefore, the development of novel drugs and therapeutic approaches for malignant melanoma is an urgent medical issue.
Piper betle Linn. (Piperaceae), known as the betel vine, is a perennial semi-woody climber with a strong pungent and aromatic flavor that grows widely in Southeast Asia. It is an important medicinal and economical plant in some Asian countries, such as Taiwan and India, and has been proven to exhibit antioxidative, antimicrobial, and anti-hemolytic activities [12][13][14]. Several bioactive constituents such as lignins, polyphenols, alkaloids, steroids, saponins, tannins, and terpenes have been isolated from the leaves and stems of P. betle [15]. Bornyl cis-4-hydroxycinnamate is an active compound isolated from P. betle stem. In this study, we investigated the effects of bornyl cis-4-hydroxycinnamate on the proliferation of melanoma cells and employed a comparative proteomics approach to identify the molecules involved in bornyl cis-4-hydroxycinnamate-induced apoptosis in A2058 and A375 melanoma cells. We aimed to ascertain the underlying mechanism, which may lead to the development of bornyl cis-4-hydroxycinnamate as a new treatment agent or a potential strategy against human melanoma.

Characterization of the Constituents of the Ethyl Acetate (EA) Fraction of P. betle Stems
One monoterpene, bornyl cis-4-hydroxycinnamate, was afforded from the ethyl acetate fraction of P. betle stems, and its chemical structure was confirmed by comparing its physical and spectral data (specific rotation, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy) with the values described in the literature [16]. Bornyl cis-4-hydroxycinnamate (
Differential proteins, such as stress 70, prohibitin, protein DJ-1, GRP78, ATP synthase, and TER ATPase were found to be associated with the induction of apoptosis and acted against cell proliferation. Western blotting was used to validate the changed profiles of these proteins. Altogether, proteomic analysis of 2-DE data and western blotting were in agreement in terms of the trends of changes of these identified proteins ( Figure 4B).

Bornyl cis-4-Hydroxycinnamate Activates the Apoptosis Pathway through the Induction of Mitochondrial Depolarization
As per the aforementioned results showing that bornyl cis-4-hydroxycinnamate induced apoptosis, we also noted that several mitochondrial-related proteins were identified in the bornyl cis-4-hydroxycinnamate-treated cells from 2-DE analysis, including prohibitin, stress 70, and ATP synthase subunit β. These proteins are known to be involved in energy production. We then employed the tetraethylbenzimidazolylcarbocyanine iodide (JC)-1 dye to measure the loss of mitochondrial membrane potential (∆ψm) in A2058 and A375 cells after bornyl cis-4-hydroxycinnamate treatment. Fluorescence microscopy showed that bornyl cis-4-hydroxycinnamate-treated cells had a lower intensity of red fluorescence and a higher intensity of green florescence due to ∆ψm loss ( Figure 5A). The mitochondrial-mediated apoptosis pathway plays an important role in apoptosis. We further analyzed molecules involved in the mitochondrial-mediated apoptosis pathway by examining the expression of related protein markers, including Bcl-2, Bcl-xl, Mcl-1, Bad, p-Bad, Bax, and cytosolic cytochrome C. As shown in Figure 5B, bornyl cis-4-hydroxycinnamate significantly increased the expression levels of Bad, Bax, and cytosolic cytochrome C in A2058 and A375 cells. In contrast, the levels of other marker proteins, such as Bcl-2, Bcl-xl, Mcl-1, and p-Bad, were decreased after bornyl cis-4-hydroxycinnamate treatment.
mitochondrial-mediated apoptosis pathway by examining the expression of related protein markers, including Bcl-2, Bcl-xl, Mcl-1, Bad, p-Bad, Bax, and cytosolic cytochrome C. As shown in Figure 5B, bornyl cis-4-hydroxycinnamate significantly increased the expression levels of Bad, Bax, and cytosolic cytochrome C in A2058 and A375 cells. In contrast, the levels of other marker proteins, such as Bcl-2, Bcl-xl, Mcl-1, and p-Bad, were decreased after bornyl cis-4-hydroxycinnamate treatment.

Bornyl cis-4-Hydroxycinnamate Treatment Induces the Endoplasmic Reticulum (ER) Stress Pathway
Next, we investigated whether the ER stress pathway was involved in bornyl cis-4-hydroxycinnamate-induced apoptosis in melanoma cells. Using western blot analysis, the expression levels of ER-related proteins GRP78 and TER ATPase were found to increase in the melanoma cells after bornyl cis-4-hydroxycinnamate treatment in a dose-dependent manner ( Figure  4B). We then further verified the expression of three ER-resident transmembrane sensor proteins, i.e., endoribonuclease inositol-requiring enzyme 1α (IRE1α), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6), as well as of caspase-12 by

Bornyl cis-4-Hydroxycinnamate Treatment Induces the Endoplasmic Reticulum (ER) Stress Pathway
Next, we investigated whether the ER stress pathway was involved in bornyl cis-4-hydroxycinnamate-induced apoptosis in melanoma cells. Using western blot analysis, the expression levels of ER-related proteins GRP78 and TER ATPase were found to increase in the melanoma cells after bornyl cis-4-hydroxycinnamate treatment in a dose-dependent manner ( Figure 4B). We then further verified the expression of three ER-resident transmembrane sensor proteins, i.e., endoribonuclease inositol-requiring enzyme 1α (IRE1α), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6), as well as of caspase-12 by western blot. The results showed that the expression of IRE1α and caspase-12 was unchanged after bornyl cis-4-hydroxycinnamate treatment. Under ER stress, endogenous ATF6 (p90 ATF6) is cleaved into a 50 KDa fragment transcription factor (p50 ATF6) and enters the nucleus to activate the GRP78 genes [16]. Our results showed that p-PERK and p-eIF2α expression increased after bornyl cis-4-hydroxycinnamate treatment. The level of transcription factor ATF4, a downstream target of PERK in the PERK-eIF2α pathway, was also increased after treatment in A2058 and A375 melanoma cells. Additionally, the expression and nuclear translocation of ER stress-induced CCAAT/enhancer-binding protein (C/EBP)-homologous protein (CHOP) was also increased by bornyl cis-4-hydroxycinnamate treatment ( Figure 7A).
Bornyl cis-4-hydroxycinnamate increased the ER stress-related protein expression levels similar to the two ER stress inducer agents tunicamycin (Tm) and thapsigargin (Tg), used to monitor the ER stress response. These results imply that bornyl cis-4-hydroxycinnamate-triggered cell apoptosis is mediated by the ER stress pathway in A2058 and A375 melanoma cells ( Figure 7B). into a 50 KDa fragment transcription factor (p50 ATF6) and enters the nucleus to activate the GRP78 genes [16]. Our results showed that p-PERK and p-eIF2α expression increased after bornyl cis-4-hydroxycinnamate treatment. The level of transcription factor ATF4, a downstream target of PERK in the PERK-eIF2α pathway, was also increased after treatment in A2058 and A375 melanoma cells. Additionally, the expression and nuclear translocation of ER stress-induced CCAAT/enhancer-binding protein (C/EBP)-homologous protein (CHOP) was also increased by bornyl cis-4-hydroxycinnamate treatment ( Figure 7A).
Bornyl cis-4-hydroxycinnamate increased the ER stress-related protein expression levels similar to the two ER stress inducer agents tunicamycin (Tm) and thapsigargin (Tg), used to monitor the ER stress response. These results imply that bornyl cis-4-hydroxycinnamate-triggered cell apoptosis is mediated by the ER stress pathway in A2058 and A375 melanoma cells ( Figure 7B).  To further demonstrate that bornyl cis-4-hydroxycinnamate-induced apoptosis occurs through ER stress-related pathways as described above, salubrinal (an inhibitor) was tested to examine PERK-activated cell apoptosis upon bornyl cis-4-hydroxycinnamate treatment. An increase in cell viability from 64% to 78% was observed in bornyl cis-4-hydroxycinnamate-treated A2058 and A375 cells following treatment with salubrinal at a concentration of 10 µM (Figure 8). To further demonstrate that bornyl cis-4-hydroxycinnamate-induced apoptosis occurs through ER stress-related pathways as described above, salubrinal (an inhibitor) was tested to examine PERK-activated cell apoptosis upon bornyl cis-4-hydroxycinnamate treatment. An increase in cell viability from 64% to 78% was observed in bornyl cis-4-hydroxycinnamate-treated A2058 and A375 cells following treatment with salubrinal at a concentration of 10 µM (Figure 8).

Discussion
In the current study, we investigated the cytotoxic effect of bornyl cis-4-hydroxycinnamate, isolated from P. betel stems, and studied the signaling pathways involved in the apoptosis induced by bornyl cis-4-hydroxycinnamate in melanoma cells. Our results demonstrate that bornyl cis-4-hydroxycinnamate inhibited cell proliferation ( Figure 2) and increased early apoptosis rates ( Figure 3) in A2058 and A375 melanoma cells, in a concentration-dependent manner. These findings support the conclusion that bornyl cis-4-hydroxycinnamate possesses activity against cell proliferation and may induce apoptosis in melanoma cells.
The molecular mechanism involved in bornyl cis-4-hydroxycinnamate-induced apoptosis in A375 melanoma cells was elucidated from proteomic data, which showed significant changes in the expression of several crucial proteins, including sodium/potassium-transporting ATPase subunit α-1, ATP synthase subunit β, prohibitin, stress 70 protein, dihydrolipoyl dehydrogenase, and 3-hydroxyacyl-CoA dehydrogenase type-2, which are associated with mitochondrial function or apoptosis in melanoma cells. Prohibitin is located in the mitochondria and is known to regulate apoptosis, cellular signaling, cell migration, and cell proliferation and to stabilize mitochondria proteins [17][18][19][20]. The translocation of prohibitin to mitochondria together with p53 has been shown to be highly correlated with the suppression of cancer growth [21,22]. Therefore, the antiproliferative effects of bornyl cis-4-hydroxycinnamate are likely mediated by the mitochondria in A2058 and A375 cells and associated with the enhancement of prohibitin.
The apoptosis induced by bornyl cis-4-hydroxycinnamate is also a good indicator of the potential success of this molecule in the treatment of melanoma, as the development of anticancer drugs has often been based on the apoptosis effect as a potential mechanism of chemotherapy [23]. The mechanisms of apoptosis can be subdivided into two major pathways, i.e., the extrinsic and the intrinsic pathways. The former is also called the death receptor pathway, and the latter is also known as the mitochondrial pathway [24]. A study has suggested that the two pathways are related and influence each other [25], and molecules in the intrinsic pathway are located in either the ER or the mitochondria [26]. In addition, increased expression levels of the pro-apoptotic Bax and Bak

Discussion
In the current study, we investigated the cytotoxic effect of bornyl cis-4-hydroxycinnamate, isolated from P. betel stems, and studied the signaling pathways involved in the apoptosis induced by bornyl cis-4-hydroxycinnamate in melanoma cells. Our results demonstrate that bornyl cis-4-hydroxycinnamate inhibited cell proliferation ( Figure 2) and increased early apoptosis rates ( Figure 3) in A2058 and A375 melanoma cells, in a concentration-dependent manner. These findings support the conclusion that bornyl cis-4-hydroxycinnamate possesses activity against cell proliferation and may induce apoptosis in melanoma cells.
The molecular mechanism involved in bornyl cis-4-hydroxycinnamate-induced apoptosis in A375 melanoma cells was elucidated from proteomic data, which showed significant changes in the expression of several crucial proteins, including sodium/potassium-transporting ATPase subunit α-1, ATP synthase subunit β, prohibitin, stress 70 protein, dihydrolipoyl dehydrogenase, and 3-hydroxyacyl-CoA dehydrogenase type-2, which are associated with mitochondrial function or apoptosis in melanoma cells. Prohibitin is located in the mitochondria and is known to regulate apoptosis, cellular signaling, cell migration, and cell proliferation and to stabilize mitochondria proteins [17][18][19][20]. The translocation of prohibitin to mitochondria together with p53 has been shown to be highly correlated with the suppression of cancer growth [21,22]. Therefore, the antiproliferative effects of bornyl cis-4-hydroxycinnamate are likely mediated by the mitochondria in A2058 and A375 cells and associated with the enhancement of prohibitin.
The apoptosis induced by bornyl cis-4-hydroxycinnamate is also a good indicator of the potential success of this molecule in the treatment of melanoma, as the development of anticancer drugs has often been based on the apoptosis effect as a potential mechanism of chemotherapy [23]. The mechanisms of apoptosis can be subdivided into two major pathways, i.e., the extrinsic and the intrinsic pathways. The former is also called the death receptor pathway, and the latter is also known as the mitochondrial pathway [24]. A study has suggested that the two pathways are related and influence each other [25], and molecules in the intrinsic pathway are located in either the ER or the mitochondria [26]. In addition, increased expression levels of the pro-apoptotic Bax and Bak proteins and decreased expression levels of the anti-apoptotic Bcl-2, Bcl-xl, and Mcl-1 proteins are known to play important roles in the intrinsic pathway and are associated with changes in the mitochondrial membrane potential and with the release of mitochondrial apoptotic factors [27]. The collapse of ∆ψm subsequently leads to cytochrome C release into the cytosol, resulting in caspase-9 activation, and further activates the downstream effector caspase-3. This process then causes poly(ADP-ribose) polymerase cleavage [28]. Existing information and our findings showing decreased ∆ψm and cytochrome C release from the mitochondria ( Figure 5B) suggest that bornyl cis-4-hydroxycinnamate-induced apoptosis in A2058 and A375 cells occurs via the mitochondrial pathway. Additionally, the suppression of the anti-apoptotic Bcl-2, Bcl-xl, and Mcl-1 proteins and the augmented expression of the pro-apoptotic Bad and Bax proteins ( Figure 5B), as well as the activation of caspase-3, caspase-9, and PARP ( Figure 6), indicate that the caspase activation cascades are crucial in bornyl cis-4-hydroxycinnamate-induced apoptosis in A2058 and A375 cells.
The ER plays many important roles in regulating essential cellular functions, including proper protein folding, protein synthesis, and calcium homeostasis [29]. When too many unfolded proteins accumulate in the ER, the homeostasis of the ER is destroyed, causing stress and leading to cells activating a self-rescue program or triggering apoptosis. In the presence of stress, the ER may react in various ways, including the induction of ER-associated degradation (ERAD), of the unfolded protein response (UPR), and of apoptosis [30,31]. Continued and severe ER stress increases the UPR, which will trigger apoptosis [32]. The UPR process is regulated by three ER signaling sensors, named protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol requiring enzyme 1-α (IRE1-α), and activating transcription factor 6 (ATF6). When unfolded proteins accumulate in the ER, chaperones, such as GRP78/GRP94, will release the transmembrane proteins IRE1-α, PERK, and ATF6, resulting in ER dysfunction. The PERK pathway either leads to cell survival controlled by autophagy [33] or to apoptosis by increasing ATF4/CHOP [34]. In the presence of ER stress, the cells activate a self-rescue program to trigger the UPR, which prevents cell death. Persistent ER stress initiates caspase-dependent apoptosis, leading to cell death [35]. When cells are under oxidative stress, unfolded or misfolded proteins accumulate in the ER, causing ER stress, and this imposed ER stress subsequently causes the UPR to alleviate the stress and restore ER homeostasis. The process upregulates chaperone GRP78 and disulfide isomerase protein, which promote protein folding and alleviate protein aggregation, and modulates calreticulin activity for the storing of calcium in the ER [36,37]. Our immunostaining results demonstrated that the levels of GRP78 and calreticulin were increased in the presence of a high concentration of bornyl cis-4-hydroxycinnamate, indicating that these proteins were activated to restore normal protein folding and reduce ER stress. If the UPR is insufficient to relieve cells from ER stress, the PERK, IRE1-α, and ATF6 pathways may activate downstream proteins to induce apoptosis in order to destroy the ER stress-damaged cells [38]. It was observed that PERK activates eIF2α, ATF4, and CHOP, causing apoptosis. The immunostaining results of this study also showed that the levels of p-PERK, p-eIF2α, and downstream ATF4 were increased, as was the expression of ATF6. These responses led to the activation of CHOP and increased its expression to induce apoptosis. The level of caspase-12 expression did not change, suggesting that the apoptotic mechanism in our study was mediated by the PERK-eIF2α-ATF4-CHOP signaling pathways in the ER ( Figure 7A). In order to verify our hypothesis that bornyl cis-4-hydroxycinnamate-induced apoptosis in melanoma cells is associated with ER stress, we used ER stress agonists, namely, Tm and Tg (1 and 3 µM, respectively), to treat the cells. The results showed that the treatment with increasing concentrations of Tm and Tg resulted in trends of the expression of PERK, p-PERK, eIF2α, p-eIF2α, CHOP, ATF4, and ATF6 consistent with those of cells treated with bornyl cis-4-hydroxycinnamate. This finding suggests that bornyl cis-4-hydroxycinnamate-induced apoptosis occurs through a mechanism that involves ER stress ( Figure 7B). We also used salubrinal, an eIF2α inhibitor, to further confirm our hypothesis. An MTT assay showed that the survival rate of cells treated with bornyl cis-4-hydroxycinnamate and salubrinal together was significantly higher than that of cells treated with bornyl cis-4-hydroxycinnamate alone ( Figure 8). In conclusion, the results indicate that bornyl cis-4-hydroxycinnamate initiated ER stress and subsequently induced apoptosis in melanoma cells.
Overall, our results demonstrate that bornyl cis-4-hydroxycinnamate-induced apoptosis in A2058 and A375 cells is mediated by a dysfunction of the mitochondrial pathway, the activation of caspase cascades, and ER stress.

General Instrumental Operation for the Isolation and Identification of Compounds
NMR spectra were analyzed in CDCl 3 at room temperature using a Varian Mercury plus 400 NMR spectrometer, and the solvent resonance was used as the internal shift reference (tetramethyl silane [TMS] as standard). EI-MS were recorded on a SX-102A mass spectrometer (JEOL USA, Inc., Peabody, MA, USA). Thin-layer chromatography was used on silica gel 60 F254 plates (Merck KGaA, Darmstadt, Germany), and the spots were visualized by spraying with 10% H 2 SO 4 solution. Silica gels (230-400 mesh ASTM, Merck KGaA) were used for column chromatography. Semi-preparative HPLC was performed using LiChrosorb Si 60 column, (7 µm, 250 × 10 mm; (Merck KGaA) on a LDC Analytical-III system.

Source, Extraction, Fractionation, and Purification of P. betle Stem Compounds
Stems of P. betle were collected in Pingtung County, Taiwan. Air-dried stems of P. betle (3 kg) were ground using an electric high-speed grinder until a fine powder that passed through a 10-mesh sieve was obtained, and the powder was extracted with methanol (solid/solvent ratio = 1:5) at room temperature three times (7 d each). The filtrates were pooled and dried under reduced pressure to allow the solvent to evaporate. The dry crude residue was then suspended in water before being successively partitioned with ethyl acetate and n-butanol.

Cell Lines and Cell Culture Conditions
A2058 and A375 melanoma cells were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). A2058 and A375 melanoma cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 1 mM sodium pyruvate, 4 mM L-glutamine, 100 µg/mL streptomycin and 100 U/mL penicillin in a humidified incubator at 37 • C with 5% CO 2 .

Cytotoxicity Assessment
A2058 and A375 cells were seeded in 96-well plates at a density of 1 × 10 5 /well with 200 µL of Dulbecco's modified Eagle medium (DMEM) and 10% fetal bovine serum in a humidified, 5% CO 2 atmosphere at 37 • C. The cells in each well were treated with bornyl cis-4-hydroxycinnamate (at concentrations of 0, 3, 6, 9, 12, 15, and 18 µM) for 24 h. Cells treated with DMSO without bornyl cis-4-hydroxycinnamate were used as a control. After a 24 h incubation period, 50 µL of MTT (0.5 mg/mL stock) was added to the cells, followed by incubation for 2 h. The medium was then removed, and DMSO (200 µL) was added to dissolve the formazan. The optical density (OD) was determined at 595 nm on an ELISA reader (Bio-Rad, Hercules, CA, USA) to provide a relative estimate of cell viability. All experiments were performed in at least triplicate to confirm their reproducibility.

Flow Cytometric Analysis
To analyze apoptosis in A2058 and A375 melanoma cells after bornyl cis-4-hydroxycinnamate treatment, the cells were stained with an annexin V-FITC apoptosis staining kit (Pharmingen, San Diego, CA, USA) and analyzed as described in previous reports [39,40]. Briefly, 1 × 10 6 cells were seeded into 5 cm Petri dishes and treated with different concentrations of bornyl cis-4-hydroxycinnamate (3, 6, and 12 µM) for 24 h. Then, the cells were harvested and stained with annexin V-FITC/propidium iodide (PI) in a cell culture incubator for 30 min. Samples were then assessed using a FACScan flow cytometer (Becton-Dickinson, Mansfield, MA, USA), and the data were analyzed using FlowJo software v10.4.2 (TreeStar, Inc., Ashland, OR, USA).

Two-Dimensional Gel Electrophoresis and Differential Proteomic Analyses
For two-dimensional gel electrophoresis (2-DE), protein samples (200 µg) from the control cells and the cells treated with 12 µM bornyl cis-4-hydroxycinnamate were analyzed using a GE Healthcare Ettan IPGphor 3. For second-dimension electrophoresis, the equilibrated strip was placed onto the top of a 12.5% SDS-PAGE gel and run on an SE 600 Ruby Vertical electrophoresis system (Hoefer, Holliston, MA, USA). Each sample was run in triplicate, and the spots on the 2-DE gels were visualized by silver staining. The gels were scanned and then analyzed using PDQuest image analysis software v8.0.1 (Bio-Rad). Each triplicate sample was normalized prior to statistical analysis. The protein spots with an intensity difference between the control and bornyl cis-4-hydroxycinnamate-treated cells greater than 1.5-fold were identified, and statistically significant differences in 2-DE between 11-epi-sinulariolide acetate-treated HA22T cells and the control [41] were identified. The protein spots of interest were excised from the 2-DE gels, and in-gel digestion was performed with trypsin. The digested samples were analyzed by LC/MS/MS using a QTRAP 5500Q mass spectrometer (AB Sciex, Framingham, MA, USA). MS scanning ranged from m/z 100 to1000. The raw MS data were converted into the text file format WIFF using Analyst 1.5.1 (AB Sciex, Framingham, MA, USA).

Assessment of the Mitochondrial Membrane Potential (∆ψm)
The mitochondrial membrane potential (∆ψm) was determined using cells stained with the cationic dye JC-1. Briefly, A2058 and A375 cells at 1 × 10 5 cells/well in a 12-well plate were treated with different concentrations of bornyl cis-4-hydroxycinnamate (0, 3, 6, and 12 µM). The treated cells were collected and washed twice with PBS, incubated with 70 µL of JC-1 staining solution, and placed in a cell culture incubator for 30 min. Following washing with a buffer, the cells were then directly observed under a fluorescence microscope [42].

Statistical Analysis
All experiments were repeated at least in triplicate. Data analysis was performed using Student's t test (Sigma-Stat 2.0, San Rafael, CA, USA); p values < 0.05 were considered significant.

Conclusions
Our results established that bornyl cis-4-hydroxycinnamate extracted from P. betel stems possesses the ability to induce apoptosis in A2058 and A375 melanoma cells. A differential proteomic analysis identified several important mitochondrial and endoplasmic reticulum proteins and proved that mitochondrial dysfunction and the activation of endoplasmic reticulum stress pathways are key molecular mechanisms in the apoptosis induced by bornyl cis-4-hydroxycinnamate (Figure 9). Our findings provide evidence that bornyl cis-4-hydroxycinnamate has the potential to be developed as new therapeutic agent for melanoma treatment.