More than one-third of all pharmaceutical molecules approved by the Food and Drug Administration and by the European Medicines Agency are natural compounds, or their derivatives, and about one-quarter of them specifically originate from plants [1
]. In detail, over 60% of the anticancer drugs are phytochemicals, such as alkaloids and polyphenols, confirming that the plant kingdom is a valuable source of chemopreventive and chemotherapeutic agents [5
Overall, scientific data have documented that this type of metabolites exerts an inhibitory effect on a broad range of mammalian tumor cell lines in in vitro and in vivo systems [8
]. According to the literature, the main mammalian cellular and molecular mechanisms influenced by plant molecules are those that involve the following targets: nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB); protein tyrosine kinases (PTKs); target mitogen-activated protein kinases (MAPKs); cyclooxygenase (COX-2); cyclin-dependent kinases (Cdks); phosphoinositide 3-kinase (PI3K) interactors; activator protein 1 (AP1); and cytoskeleton components [10
Among all biological properties, the free radical scavenging activity seems to be the most validated device employed by phytochemicals to inhibit tumor cell growth, counteracting reactive oxygen species (ROS) production and limiting protein, lipid, and DNA oxidation [23
]. Nevertheless, a growing body of evidence brought this opinion into question, arguing that the antiradical activity of the natural compounds has a potential role in chemoprevention, but it cannot fully explain the relative antitumor effect [24
]. In addition, it is important to underline that a wide number of plant secondary metabolites have also shown unexpected pro-oxidant consequences, including DNA damage and apoptosis, especially at high concentrations and in the presence of transition metal ions [26
L., also known as oregano, is a Mediterranean plant species belonging to the Lamiaceae family which, nowadays, represents one of the most used culinary herbs. However, the application of oregano in several ethnobotanical practices, including folk medicine, dates to ancient times. Regarding its phytotherapic effect, various investigations have been performed, documenting that oregano essential oil possesses antimicrobial, antiviral, antifungal, antioxidant, anti-inflammatory, digestive, expectorant, neuroprotective, antispasmodic, and antidiabetic properties, simultaneously. Moreover, some literature works have also associated a strong anticancer activity to such type of plant extract. For these reasons, O. vulgare
is capturing greatly the attention of the food, cosmetic, and pharmaceutical industries [30
According to all this evidence, the present research aimed at investigating the molecular mechanism underlying the antineoplastic effect of O. vulgare L. ssp. hirtum phytocomplex against murine (B16–F10) and human (A375) melanoma cells.
Among tumors, skin cancer is the most common neoplasia worldwide. In particular, the more aggressive and deadliest form of this pathology is represented by melanoma [69
]. Melanoma is a multi-factorial disease, depending on both environmental and endogenous factors. Indeed, about 90% of melanomas are caused by ultraviolet light exposure [70
], while the remaining 10% has been associated to genetic defects [71
]. Currently, such type of skin cancer is treated by surgical removal, which leads to a high survival rate except in the presence of metastases. In the latter case, a chemotherapeutic approach based on several drugs, such as Dacarbazine (an alkylating agent), Vemurafenib (BRAF kinase inhibitor), Ipilimumab (monoclonal antibody targeting for cytotoxic T-lymphocyte antigen-4), Pemrolizumab (monoclonal IgG4 antibody), and Nivolumab (monoclonal antibody targeting for Programmed Death-1 protein) [72
], is the most efficient strategy to treat melanoma. However, the aggressiveness and the high rate of multi-drug resistance of this pathology highlight the need of new antineoplastic molecules.
Phytochemicals, namely secondary metabolites, produced by plants to protect themselves from environmental stresses and promote their reproduction [73
], have been widely documented to exert a great variety of non-negligible bioactivities even on mammalian systems. Indeed, it has been documented that several plant compounds promote apoptosis and inhibit metastasis and angiogenesis [74
]. For this reason, they have been taken under consideration thanks to their relevant medical and pharmaceutical properties. In this scenario, plant phytocomplexes can represent potential antiproliferative and anti-invasive cocktails for drug-resistant melanomas.
Based on the previous evidence, in the current research, the biological effect of a hydroalcoholic extract from plants of Origanum vulgare
L. ssp. hirtum
(HCOE) was investigated on highly metastatic and drug-resistant murine (B16-F10) and human (A375) melanoma cells. Indeed, although a potential antineoplastic effect has been associated to oregano extracts [30
], the capacity of this herb to contrast the growth of the above-mentioned melanoma lines has never been elucidated in detail. Moreover, as one of the main goals of the cancer research is the discovery of new drugs with limited or without adverse side effects for healthy tissues, oregano extract was also tested on C2C12 myoblasts, which is a non-tumor cell model.
Since plant extract bioactivity cannot be attributed only to its more representative compounds, rather than to the synergic effect of plant molecules present both in high concentration and in trace [78
], first of all, we investigated the biochemical profile of the oregano extract used in this study by chromatographic approaches (HPLC-DAD and GC-MS). A total of 58 metabolites were detected and recognized. Moreover, the amount of total phenols and flavonoids in HCOE was also measured, together with its in vitro antiradical power, to better characterize the plant extract.
The GC-MS chemoprofile obtained in the current research totally was in line with those documented in the literature. In this regard, O. vulgare
essential oil can be classified in four chemotypes, according to its main constituents, especially thymol and carvacrol, and relative ratios. For instance, O. vulgare
essential oil extracted from plants grown in Southern Italy and Northern Greece would seem rich in thymol, while that obtained from plant material propagated in Southern Greece was abundant in carvacrol [81
]. Although purified from plants grown on Mount Athos (Northern Greece), our oregano extract showed a chemoprofile more similar to those of Southern Greece. To explain this phenomenon, it is important to keep in mind that several independent variables, such as plant growth stage and environment conditions, may strongly influence the phytocomplex. On the other hand, the oregano sample here studied revealed a content of phenolics double compared to that reported in the literature [84
As concerns oregano biological activity, the plant phytocomplex determined a great reduction of B16-F10 cell growth, especially after 48 h of exposure with the highest doses (6, 8, 10 mg/mL), while it minimally influenced the myoblast division rate. B16-F10 and C2C12 proliferation curves confirmed the previous MTT outcomes. Moreover, HCOE induced a significant time- and dose-dependent toxicity on murine melanoma cells, whereas a low percentage of Trypan blue positive cells was detected in the case of C2C12. To validate these results, IC50
values and selectivity indexes (SI) for the plant treatments on both cell lines were calculated. According to the literature, a reliable SI value must be equal to or greater than 2 [86
], and HCOE satisfied this requirement. Cell cycle analysis of B16-F10 demonstrated that oregano extract caused an increase of cells in G2/M phase.
This promising evidence encouraged us to check if oregano extract could exert antineoplastic effects also against a human melanoma cell line with the aim to lay the basis for future desirable application in translational medicine. As expected, significant antiproliferative activity, with a high selectivity index, together with a relevant cytotoxic effect of HCOE on A375 cells was confirmed.
Taking into account all previous data, the concentration of 10 mg/mL of oregano sample was selected for further experiments, showing the best antiproliferative effect.
Cytofluorimetric and RT-PCR analyses proved that the plant extract blocked cell division in the G2/M phase, acting on the expression of specific key genes implicated in the inception of the mitotic process (i.e., CDK1, CCNB1, P21, and P27) [53
] such as paclitaxel, which is a well-known plant anticancer drug with antimitotic property.
Before proceeding with the other analyses, as the final objective of the current research was the valorization of an oregano hydroalcoholic extract for potential chemotherapeutic applications, the control of the safety for the plant preparation with reference to mutagenic properties was necessary. For this purpose, the Ames test, recommended by the European Food Safety Authority (EFSA) as a proper assay to assess food safety [87
], was carried out. It provided the proof that HCOE did not have a non-mutagenic effect [88
], as expected. Indeed, O. vulgare
and its derivatives, which are accepted as food ingredients by the U.S. Food and Drug Administration, are listed among the GRAS (Generally Recognized As Safe) substances by the Code of Federal Regulations of the USA and are generally well tolerated by the human body, although gastrointestinal upset and skin allergic reactions have been associated to them [90
]. However, the lack of data regarding oregano genotoxicity [91
] highlights the need for further studies on this topic and valorizes the present preliminary results. The mutagen-protective activity of HCOE was also investigated in order to further analyze the plant extract under a healthy point of view. Unfortunately, at all tested doses, no protective property against known mutagenic compounds was documented.
In the second part of this work, the molecular mechanism underlying the bioactivity of HCOE on melanoma cells was investigated in depth. First of all, the efficiency of the MITF pathway was verified, studying the expression rate of MITF, TYR, and TYRP1 genes. The results revealed an antimelanogenic activity of the oregano extract on A375, as already suggested by the literature [93
At low levels, reactive species play a key role in cell signaling, but their overproduction can lead to mitochondrial alterations (i.e., DNA mutations, respiratory chain damage, membrane permeability loss), oxidative stress, and an inhibition of specific genes, including those related to melanin synthesis [29
]. Therefore, according to the previous results, intracellular ROS and RNS levels were measured in A375 cells, demonstrating that HCOE10 had a remarkable pro-oxidant effect, especially after 24 h of incubation. This evidence suggested that oregano antitumor activity could be based on a reactive species-mediated apoptotic process. Literature data about carvacrol bioactivity, one of the most abundant phenolic monoterpenoids of oregano (as also documented in the present research by GC-MS analysis, see Table 2
), would support this hypothesis. Indeed, several published works showed that this compound induces apoptosis in different tumor cell lines by increasing the ROS amount and disrupting mitochondrial membrane potential [95
All previous considerations suggested that HCOE10 could trigger cell death in A375. For this reason, to clarify which cell death pathway was induced by oregano extract, a pan-caspase inhibitor, Z-VAD-FMK, and an inhibitor of necroptosis, necrostatin-1 [98
], were used in co-treatments with HCOE10 on the melanoma cells. The experiments demonstrated that the decrease of tumor cell proliferation was partially due to both apoptosis and necroptosis induction, as already suggested by Savini et al. [99
] and Rubin et al. [100
]. Indeed, these two phenomena are strongly correlated to each other because they share the same stimuli (such as TNF-α), ligands, and receptors [101
]. The mechanism underlying the activation of apoptosis and/or necroptosis is still under investigation. Nevertheless, Annexin V/propidium iodide staining in the presence of necrostatin-1, as well as the analysis of TNF-α level, could be performed in the future to better clarify the role of these two pathways in oregano-induced cell death [103
Since in MTT assay, only HCOE10 + Z-VAD double treatment rescued A375 viability, taking into account that mitochondria are both generators of and targets for reactive species (whose levels were previously observed to be increased in the presence of HCOE), a mitochondria impairment caused by oregano extract was hypothesized. To validate this theory, cytofluorimetric analyses, based on the use of MitoTracker Green and MitoTracker Red CMX-ROS probes, were performed. The data confirmed a loss of mitochondrial potential after HCOE10 treatment, while no significant change in mitochondrial mass was appreciated. By contrast, CCCP-treated cells (the positive control) showed an increase of mitochondrial mass, which was probably due to mitochondrial fragmentation [105
] associated to an expected membrane depolarization.
As known in the literature, the permeabilization of the mitochondrial outer membrane is an event promoted by the pro-apoptotic protein Bax. Bcl-2, on the other hand, is an anti-apoptotic factor that prevents apoptosis by inhibiting Bax. Therefore, an elevated Bax/Bcl-2 ratio is a feature of apoptotis induction. As a consequence of Bax activation, damaged mitochondria release cytochrome c into the cytoplasm, leading to caspase-3 induction and Parp-1 cleavage [107
]. In view of this, the amount of apoptosis-related proteins (Bax, Bcl-2, Cycs, Casp-3, and Parp-1) were evaluated in A375 after 48 h of incubation with HCOE10. Oregano extract was able to trigger apotosis in the human melanoma cells by increasing Bax and Cycs concentrations and decreasing Bcl-2 and pro Casp-3 amounts. Moreover, as expected, Parp-1 cleavage was evident in oregano-treated cells. HCOE10 + Z-VAD co-treatment reversed HCOE effect, showing protein levels similar to those detected in the respective negative control. All these results confirmed that the plant extract induced, in A375 cells, a caspase-dependent apoptosis, which was mediated by mitochondrial damage. However, considering that both ROS and RNS play an important role also in autophagy and that mitophagy is strictly involved in mitochondrial turnover [109
], it would be interesting in the future to investigate if autophagy/mitophagy is also induced by oregano treatment.
Another important key element in the apoptotic process is the P53 gene, whose protein promotes BAX gene expression by direct activation of its promoter and BCL2 downregulation [111
]. P53 is activated in response to a wide range of genotoxic insults. It is involved in several DNA-repair machineries, such as nucleotide excision repair, for the removal of helix-distorting lesions (typical of UV-damage) and base excision repair (BER) in case of base oxidative modifications [114
]. As known in the literature, plant compounds, especially polyphenols, can cause oxidative DNA strand breakage, alone or in the presence of transition metal ions. Indeed, among all, the copper (that is the most abundant ion of the cell nucleus, together with zinc), after reduction from Cu(II) to Cu(I) form by the action of plant metabolites, is particularly prone to produce ROS (especially the hydroxyl radical) during its re-oxidation, bind chromatin (particularly guanines), and cause DNA breakages [116
The existence of such types of event prompted us to analyze P53 mRNA and protein levels and evaluate DNA damages (detecting γH2AX and 53BP1 foci by IF) upon HCOE10 exposure. Oregano treatment induced in A375 p53 upregulation, at both the transcriptional and translational level, and DNA breakages. In detail, γH2AX is the phosphorylated form of the histone 2AX (H2AX). Its phosphorylation is an early consequence of double and single-strand breakages [118
]; therefore, the detection of γH2AX is widely used as a marker of DNA damage. On the other hand, p53 binding protein 1 (53BP1) locates only to DNA double-strand breaks [122
]. IF analyses highlighted a great number of γH2AX and 53BP1 foci, which often co-localized in HCOE10-treated cells with respect to untreated controls. This evidence, together with Ames test results, pointed out that oregano hydroalcoholic extract acts as a genotoxic but not mutagenic agent in A375 melanoma cells, causing DNA single and double-strand breaks [123
]. Furthermore, taking into account that DNA damage often results from the binding of phytochemical to transition metal ions, such as copper [124
], co-treatments with HCOE10 and a copper chelator (TETA) were performed. Surprisingly, the sequestration of copper by TETA protected A375 cells from HCOE10-induced DNA damage, confirming that this metal ion plays a fundamental role in oregano bioactivity and relative DNA cleavage reaction.
In cancer masses, the concentration of iron and zinc is lower, whereas the copper concentration is usually higher than in healthy tissues [127
]. This feature can explain why plant compounds exert selective cytotoxic activity against tumor cells but not toward non-tumor ones [133
], justifying our data about oregano cytotoxicity on B16–F10 and A375 but not on C2C12.
In conclusion, O. vulgare hydroalcoholic extract, due to its peculiar chemical profile and pro-oxidant effect, inhibits melanogenesis and melanoma cell proliferation. Through in-depth molecular analyses, the antineoplastic activity of the oregano extract was associated to its ability to trigger programmed cell death (apoptosis and necroptosis) in A375 human melanoma cells via mitochondria and DNA damage. As this molecular mechanism was correlated to the intracellular/nuclear concentration of copper ions, oregano phytochemicals appeared to be slightly toxic or non-toxic for non-tumor cells. All this evidence represents a robust starting point for further studied focused on the design of new anti-melanoma natural drugs. Indeed, according to the present data, O. vulgare phytocomplex, working in synergy, represent an excellent candidate as anticancer agent, being highly selective and effective against human melanoma cells.