Melanoma is a malignant tumor of cutaneous melanocytes. Incidence of melanoma is usually lower than other skin cancers such as basal cell cancer and squamous cell cancer. However, melanoma is more invasive and has a higher lethality than other skin cancers [1]. Melanoma incidence has been estimated to reach 70,230 cases in 2011 in the US [2]. In the regions with sunny climates like Australia, New Zealand, North America, Latin America, and northern Europe melanoma occurs the most [3]. The metastases of melanoma are clinically crucial in diagnosed patients since most systemic treatments for metastatic melanoma exert non-obvious effects [4,5]. Surgery is still the most effective measure for malignant melanoma patients with small and thin neoplastic lesions [6]. The survival rate is dependent on the diagnostic stage of melanoma [7]. Other medical treatments such as chemotherapy (with dacarbazine) and immunotherapy (with interleukin-2 or interferon) have also been investigated [8,9,10]. However, their achievements clinically applied in metastatic melanoma are still limited [11]. Therefore, it is necessary to investigate and discover new effective drugs and therapies against human malignant melanoma.

The induction of cell death by apoptosis critically benefits cancer therapy development [12,13]. Apoptotic processes can be triggered either via extrinsic pathways or intrinsic pathways [14,15]. Recent studies have implied that intrinsic pathways are initiated by association with stress in the mitochondria or endoplasmic reticulum (ER) [16]. Processes of mitochondrial dysregulation have been shown as major events during apoptosis while Bcl-2 family like Bax and Bak as well as alteration of mitochondrial membrane potential result in the release of mitochondrial apoptotic factors [17]. Cytochrome C release from mitochondrial inter-membrane spaces initiates subsequent activation of caspase-9 as the downstream effector caspase-3 is further activated [17,18]. ER is responsible for synthesis of cellular regulatory proteins, protein folding and intracellular calcium homeostasis [19]. The abnormality of protein folding or calcium imbalance in ER is able to trigger ER stress and this subsequently induces self-rescuing or destruction responses in cells [20].

Some chemical compounds isolated from soft coral in a recent study were found to possess cytotoxic activities in tumor cells [21,22,23,24]. These compounds derived from marine products were shown to exert apoptosis-induced effects against human hepatocellular carcinoma [25], hormone-resistant prostate cancer cells [26], oral squamous cell carcinoma [27] and bladder cancer carcinoma [23]. Herein we examined the anti-tumor and apoptosis-inducing effects of 11-dehydrosinulariolide on melanoma cells using MTT assay, cell migration assay and flow cytometric analysis. The potential pathways of apoptosis induced by 11-dehydrosinulariolide in melanoma cells were determined by mitochondrial membrane potential measurement, apoptotic inhibition test and immunoblotting. We revealed crucial data on the cytotoxic activities and several apoptotic pathways of 11-dehydrosinulariolide in melanoma cells in vitro.

In this study, the cytotoxic effects and the potential mechanisms of apoptosis induced by 11-dehydrosinulariolide in melanoma cells were examined by MTT assay, cell migration assay, flow cytometry and immunoblotting. We have previously shown that the same compound from soft coral Sinularia leptoclados exerts anti-cancer activities against oral squamous cell carcinoma by anti-proliferative, anti-migratory and proteomic analysis [27]. Similarly, the results in this study indicated that 11-dehydrosinulariolide possesses anti-proliferative and anti-migratory effects against melanoma cells (Figure 1), suggesting broad anti-tumor activities exerted by the soft coral isolated compound 11-dehydrosinulariolide. In the current work, the flow cytometric results indicated that 11-dehydrosinulariolide induces both early and late apoptosis (Figure 2A,B). Apoptosis plays important roles in removing DNA-damaged cells from surrounding normal cells and aims to control the development of multicellular organisms and maintain tissue homeostasis [28]. The carcinogenesis and neoplastic progression of various human cancers are primarily due to deregulation or/and dysfunction of apoptotic signal pathways [29]. Therefore, novel apoptotic inducers indeed provide effective and promising therapies against human cancers.

The mitochondria-dependent apoptotic pathway has been characterized by pro-apoptotic factors including the release of cytochrome C, activation of caspase-3/9 and Bax, as well as suppression of the anti-apoptotic proteins like Bcl-2 and Bcl-xL [18,30]. Our western blotting results found both time- and dose-dependent up-regulation of pro-apoptotic proteins such as cytosolic cytochrome C, cleaved-caspase-3, cleaved-caspase-9, and Bax while pro-caspase-3, pro-caspase-9 and anti-apoptotic factors Bcl-2 as well as Bcl-xL were down-regulated in 11-dehydrosinulariolide-treated melanoma cells (Figure 3C). These data suggested potential involvement of the caspase 3-, caspase 9- and mitochondria-dependent pathway in 11-dehydrosinulariolide-induced apoptosis in melanoma cells. Interestingly, these findings are quiet similar to the possible mechanisms of apoptosis induced by another soft coral extracted compound 13-acetoxysarcocrassolide in bladder cancer cells in our previous study, implicating some common cytotoxic and apoptosis-inducing characteristics exerted by the natural marine products against different tumor/cancer cells [23].

Additionally, evidence showed that the over-expression of pro-survival Bcl-2 or deficiency of pro-apoptotic Bax conferred protection against mitochondrial dysregulation [31,32]. The mitochondrial dysregulation is characterized by the loss of mitochondrial membrane potential and caspase-3/9 is subsequently activated [33]. In the current study mitochondrial membrane potential in melanoma cells was attenuated within 12 h under 11-dehydrosinulariolide treatment (Figure 3A,B). These results are rationally linked with the western blotting data of these mitochondrial apoptotic events such as enhancement of Bax expression, simultaneous inhibition of Bcl-2/Bcl-xL, subsequent Bax insert into mitochondria outer membrane, release cytochrome C and cleavage of caspase-3/9 in 11-dehydrosinulariolide-induced apoptosis in melanoma cells.

Another apoptotic hallmark protein, cleaved-PARP-1, also plays an important role in triggering apoptosis via a caspase-independent mechanism [34]. Investigation indicated that cleaved-PARP-1 initiates apoptosis-inducing factor (AIF)-mediated cell death through a mechanism requiring Bax and calpains but not cathepsins or caspases [35]. Our data showed that exposure to 11-dehydrosinulariolide results in cleavage of PARP-1 in A2058 melanoma cells in a time- and dose-dependent manner (Figure 3C). These results suggested that the cleaved-PARP-1 apoptotic event may be associated with mitochondria-mediated apoptosis through both caspase-dependent and caspase-independent pathways.

The apoptotic events after 11-dehydrosinulariolide treatment in melanoma cells were also found to be correlated with ER stress. The onset of ER stress is due to accumulation and aggregation of unfolded or misfolded proteins in the lumen of ER which initiates unfolded protein response (UPR) and ER-associated protein degradation (ERAD). The UPR and ERAD can restore normal ER function, whereas they are able to trigger apoptosis when the stress is prolonged or the adaptive responses fail [16,36]. The UPR is mediated via three ER transmembrane sensors, PERK, ATF6 and IRE1, which are capable of triggering self-rescuing or apoptotic responses through concerted and complex signaling pathways, respectively [37]. Transcriptional induction of ERAD components have been shown to depend on the IRE1-XBP1 pathway in mammals [38]. Therefore, ER stress sensors such as PERK and ATF6 were also analyzed to verify the involvement of ER stress in the 11-dehydrosinulariolide-induced apoptosis in this study. After 11-dehydrosinulariolide treatment, PERK and eIF2α were phosphorylated and resulted in elevation of p-PERK and p-eIF2α levels in a time- and dose-dependent manner. Transcription factor ATF4, a downstream signal of PERK-eIF2α pathway was also enhanced after 11-dehydrosinulariolide treatment (Figure 4). ATF4 can trigger a self-rescuing response by inducing the genes correlated with amino acid metabolism, redox reaction, stress response and protein secretion [39]. This factor was also shown to induce the pro-apoptotic transcription factor CHOP, which is an ER stress-mediated apoptotic executor [40].

The cleaved and activated form of another transcription factor ATF6, ATF6-f, was also increased under the same conditions of treatment (Figure 4A). Similarly, previous work by Yamamoto et al. (2007) found that activated ATF6 triggers a self-rescuing response via inducing ER chaperones (i.e., GRP78, GRP94, PDI) and XBP1 [41] as ATF6 can also induce CHOP expression [42]. The up-regulation of CHOP has been revealed to cause growth arrest and apoptosis in typical ER stress events after tunicamycin and thapsigargin treatment [43,44]. Our immunoblotting results displayed that CHOP expression is up-regulated in the time-course and dose-dependent test (Figure 4). The ER stress inhibitor salubrinal suppresses dephosphorylation of p-eIF2α by targeting the specific phosphatase of eIF2α [20]. In the current study salubrinal partially abrogated 11-dehydrosinulariolide-induced cell death (Figure 5). Since more than one possible apoptotic pathway of 11-dehydrosinulariolide-induced cytotoxicity against melanoma cells was found in the current study, it is qute reasonable that melanoma cell viability recovery rate after salubrinal treatment is around 20%. Combining the data on ER stress-associated factors with the restorative result of ER stress-mediated apoptosis, it is highly indicated that 11-dehydrosinulariolide is able to induce ER stress-mediated apoptosis in melanoma cells, and the PERK/eIF2α/ATF4/CHOP as well as the ATF6/CHOP pathways are very likely to be involved in the apoptotic events.

Our western blotting data also showed that up-regulations of ER chaperones (GRP78, GRP94, calnexin, CALR, and PDI) are found after 11-dehydrosinulariolide treatment in a dose- and time-dependent manner (Figure 4B). These ER chaperones have been indicated to be relevant to ER stress and the ER stress-mediated apoptotic factors shown above. GRP78 is a key indicator of ER stress responses and is able to control the activation of PERK, ATF6 and IRE1 through a binding-release mechanism [45] as GRP 94 is another indicator of ER stress (Little et al, 1994). Furthermore, investigation has revealed that the up-regulation of GRP78/GRP94 is mediated by single or combined actions of IRE1-XBP1 and ATF6 in ER stress responses [38,46,47]. Immunoblotting results of both ER-stress apoptotic mediators and ER chaperones GRP78/GRP94 were up-regulated in the current study, implying that 11-dehydrosinulariolide initiates ER stress responses and may activate the transcription of GRP78/GRP94 promoters. Other ER-resident chaperones such as calnexin, CALR and PDI constitute a chaperon system in ER, which has been shown to retain nascent unfolded N-linked glycoprotein and further processes them for subsequent folding and assembling [48]. It has been shown that elevated calnexin levels protect cells from apoptosis [49]. Meanwhile, calnexin over-expression has been found in ER stress-induced apoptosis in a fission yeast model [50] as this protein has also been found to regulate ER-stress-mediated apoptosis by a B-cell receptor-associated protein 31 (Bap31) binding in a dependent manner but independent of its chaperone function [51]. On the other hand, in the Caenorhabditis elegans model tunicamycin induced expression of CALR, which requires IRE1-dependent splicing of XBP1 mRNA but not ATF6 and PERK [52]. Investigation on the correlation between PDI expression and UPR has shown that the PDI level is regulated via activation of ATF6 and splicing of XBP1 mRNA [53,54]. Hence, it is proposed that the up-regulations of the ER chaperones calnexin, CALR and PDI after exposure to 11-dehydrosinulariolide are likely self-rescuing responses of the tumor cells or the onset of ER stress-mediated apoptosis induced by the treatment in the melanoma cells.

Taken together, in the current study we found that the natural marine compound 11-dehydrosinulariolide possesses anti-tumor and apoptosis-inducing effects on A2058 melanoma cells. Mitochondrial and ER stress-mediated pathways of the apoptosis were examined and partially revealed. The results in the current study suggest that 11-dehydrosinulariolide is an effective compound against formidable melanoma cells in vitro. The potentially dual apoptotic pathways were found in the cytotoxic activities exerted by 11-dehydrosinulariolide and the finding should be one of the essential clues for the development of melanoma therapies. Thus, the compound 11-dehydrosinulariolide is worthy of further investigation in animal model tests. Future studies on the cross-talk between ER stress- and mitochondria-mediated apoptosis induced by 11-dehydrosinulariolide in melanoma cells will be helpful to uncover its anti-tumor mechanism and also benefit the pharmaceutical development of anti-melanoma drugs.

In the present study, 11-dehydrosinulariolide induced apoptosis in A2058 cells was validated and further investigated. Further functional studies demonstrated that 11-dehydrosinulariolide induced dysregulation of mitochondria and ER stress pathway. This is the first report describing the effect of 11-dehydrosinulariolide on ER stress and mitochondrial dysfunction. Our results indicated that the involvement of 11-dehydrosinulariolide in inducing cytotoxicity in A2058 cells is through both PERK/eIF2α/ATF4/CHOP and ATF6/CHOP pathways, which suggests that 11-dehydrosinulariolide could be a potent anticancer drug for melanoma cancer treatment.