Melanoma originates in the skin and although it represents one of the rarer forms of skin cancer, it underlies the majority of skin cancer-related deaths [1
] whose incidence has more than doubled in the last 10 years. About 50–60% of melanomas show the BRAFV600E
] where a valine is replaced by either an arginine (V600K) or glutamic acid (V600E). This mutation implies the constitutive activation of extracellular signal-regulated kinases (ERK) signaling that leads to the increase of proliferation and transformation [2
]. Although this mutation commonly occurs in melanoma, it should be noted that the mutation itself is not sufficient to cause cancer since it is also found in benign melanocytic lesions [1
]. Likewise, the phosphoinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of the rapamycin (mTOR) pathway is also involved in melanoma genesis, and its activation often leads to increased cell survival, proliferation, and motility [9
]. Current protocol for melanoma treatment is dependent on the condition of the tumor at the time of disease detection; if diagnosed early, melanoma may be removed using surgery, but if it spreads to the lymph nodes, surgery will be more invasive and chemo- and immunotherapy will be associated with the treatment. Unfortunately, these therapies may undergo patient resistance and generate host tissue damage. In particular, intrinsic resistance to apoptosis of melanoma cells is one of the main causes of anticancer therapy failure. Until recently, the prognosis for advanced malignant melanoma was poor, but the discovery of the major pathways involved in melanoma progression and resistance prompted the use of new therapeutic agents.
Therefore, new strategies targeting melanoma cells, which also reduce resistance and patient side effects, need to be developed and a combination of conventional treatment with biological agents (the so-called complementary therapy) might be an important breakthrough.
The Mediterranean diet is considered an important preventive instrument against chronic diseases, such as neurodegenerative and cardiovascular ones, and cancer [10
]. In particular, epidemiological studies indicate that the dietary consumption of extra virgin olive oil (EVOO) has a protective effect in Mediterranean populations [13
]. EVOO is a functional food with a high level of monounsaturated fatty acids and a minor level of highly bioactive multiple components, including polyphenols, to which have been mainly attributed the beneficial effects [17
]. The phenol composition of olive oil includes the phenolic alcohols, hydroxytyrosol (HT, 3,4dihydroxyphenylethanol, 3,4-DHPEA, DOPET), and tyrosol (p-hydroxyphenylethanol, p-HPEA) together with their secoiridoid precursors. The main secoiridoid in olive oil is 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA), whose glycated form is also known as Oleuropein (Ole), the main agent responsible for the bitter taste of olive leaves and drupes. Ole has been reported to have many pharmacological properties, among which are antioxidant, anti-inflammatory, cardioprotective, neuroprotective, and hepatoprotective properties [20
]. Recently, the accumulating in vitro and in vivo experiments, together with epidemiological and clinical data, have provided support to the anti-tumor properties of Ole toward different tumor histotypes, such as breast, colon, and lung cancer [22
]. Of translational importance is that Ole was found to be a powerful sensitizer of the Doxorubicin-mediated killing of prostate and breast cancer cells [24
]. In fact, it lowers the cytotoxic dose of Doxorubicin, while producing an anti-proliferative effect in cancer cells.
The aim of our work is to verify both the Ole cytotoxic effect and its cooperation with the current drugs used in BRAF melanoma. We have found that Ole promotes cytotoxicity of Dacarbazine (DTIC), a guanine methylating agent, whose treatment, was approved by the Food and Drug Administration (FDA) and of the mTOR inhibitor Everolimus (RAD001), and that its combination with RAD001 was also an effective strategy in treating Vemurafenib (PLX4032)-resistant BRAF melanoma cells, where Vemurafenib (PLX4032) is a BRAF inhibitor. Overall, these findings disclose the wide possibility of using Ole in the complementary therapy of melanoma.
2. Materials and Methods
2.1. Cell Lines and Culture Conditions
In this study, we used A375 human melanoma cell lines, obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). In some experiments we used also the human melanoma cell lines WM266-4 (from ATCC) and M21 (kindly provided by Dr. Antony Montgomery, The Scripps Research Institute, La Jolla, CA, USA). Melanoma cells were cultivated in Dulbecco’s Modified Eagle Medium high glucose (DMEM 4500, EuroClone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, Boerhinger Mannheim, Binger Strasse, Ingelheim am Rhein, Germany), at 37 °C in a humidified atmosphere containing 90% air and 10% CO2
. Viability of the cells was determined using a trypan blue exclusion test. Cultures were periodically monitored for mycoplasma contamination using Chen’s fluorochrome test [26
A375 melanoma cells resistant to PLX4032 were kindly provided by Laura Poliseno from University of Pisa and they were obtained as explained in Reference [27
]. PLX4032-resistant A375 melanoma cells were maintained without PLX4032 overnight before the start of the experiment.
According to the experiments, cells were treated with Oleuropein glucoside (purity ≥ 90%) (Extrasynthese S.A., Lyon, Nord-Genay, France), DTIC (Sigma Aldrich, Milan, Italy), RAD001 (MedChem Express, Stockholm, Sweden) or PLX4032 (MedChem Express, Stockholm, Sweden).
2.2. MTT Assay
Cell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay (Sigma Aldrich, Milan, Italy). Cells were plated into 96-multiwell plates in complete medium without red phenol. The treatment was added to the medium culture at different doses and times, according to the experiment. Then the MTT reagent was added to the medium and plates were incubated at 37 °C. After 2 h, MTT was removed and the blue MTT-formazan product was solubilized with dimethyl sulfoxide (DMSO) (Sigma Aldrich, Milan, Italy). The absorbance of the formazan solution was read at 595 nm using the microplate reader (Bio-Rad Via Cellini, Segrate (Milan), Italy).
2.3. Sample Preparation for Mass Spectrometry Analysis
Cells were washed with ice-cold phosphate buffered saline (PBS) containing 1 mM Na4VO3, scraped in PBS, centrifuged for 5 min at 1200 rpm and lysed with ice-cold water. Cells were sonicated three times for 5 min and supernatants were collected for mass spectrometry analysis.
The samples were measured using analytical High Performance Liquid Chromatography (HPLC) coupled to API 4000 (AB SCIEX, Toronto, ON, Canada) equipped with the TurboIonSpray source operated in negative ion mode, as previously described [28
] with slight modifications. Briefly, the capillary voltage of the mass spectrometer was set to −4500 V, the “turbo” gas flow was 10 L/min of air heated at 400 °C. The following transitions were monitored in MRM mode (multiple reaction monitoring): m
153.1 > 123.1 for HT; 377.4 > 307.3 for oleuropein aglycone; 539.5 > 275.3 for Ole. Optimal CE (collision energy) and CXP (collision cell exit potential) were found at −18 V and −8 V for HT; −16 V and −6 V for both Oleuropein aglycone and Ole, respectively. The resulting DP (declustering potential) was −70 V. The chromatographic experiments were undertaken by using a Series 1290 Infinity LC System (Agilent Technologies, Waldbronn, Germany) HPLC Capillary Pump coupled to an Agilent Micro ALS autosampler, both being fully controlled from the API 4000 data system. Liquid chromatography was performed using a Zorbax eclipse C18 3 × 150 mm, 3.5 µm HPLC column (Agilent Techonologies, Waldbronn, Germany). Column flow was 0.4 mL/min using a water/acetonitrile (50:50) and 0.05% formic acid in an isocratic elution system. The eluent from the column was directed to the TurboIonSpray probe without a split ratio.
2.4. Evaluation of Apoptosis
Apoptosis was measured using flow cytometry, using the Annexin V staining. Cells were washed once with PBS, detached with Accutase (Euroclone, Milan, Italy), resuspended in 100 mL of 1× Annexin-binding buffer at the concentration of 1 × 106
cells/mL, stained with 5 mL of Annexin V FITC-conjugated (ImmunoTools, Friesoythe, Germany) and 1 mL of 100 mg/mL propidium iodide (PI) working solution and incubated at 4 °C in the dark condition for 15 min. Then, 400 mL of 1× Annexin Binding Buffer was added to each sample and cells were analyzed using flow cytometry (BD-FACS Canto) to find out the viability (annexin V and PI negative, Q3), early apoptosis (annexin V positive and PI negative, Q4), or late apoptosis (annexin V and PI positive, Q2). A minimum of 10,000 events were collected, as previously described [26
2.5. Cell Cycle Analysis
Cell cycle distribution was analyzed via the DNA content using the PI staining method. Cells were centrifugated and stained with a mixture of 50 µg/mL PI (Sigma-Aldrich, St. Louis, MO, USA), 0.1% trisodium citrate and 0.1% NP40 (or triton x-100) (Sigma-Aldrich, St. Louis, MO, USA) in the dark at 4 °C for 30 min. The stained cells were analyzed via flow cytometry (BD-FACS Canto, BD Biosciences, Franklin Lakes, NJ, USA) using red propidium-DNA fluorescence [26
2.6. Invasion Assay
Cells invasion was performed using a polycarbonate cell culture insert with a pore size of 8.0 µm (Sigma-Aldrich) coated with Matrigel (0.25 µg/µL; BD Biosciences, Franklin Lakes, NJ, USA). Cells suspended in 200 µL of their own growth medium were seeded in the upper compartment, while in the lower chamber, fresh complete medium was added as chemo attractant.
Cells were incubated for 6 h at 37 °C, 10% CO2
in air, and 25 µM Ilomastat was used as a control for metalloprotease inhibition (Millipore, Billerica, MA, USA). After incubation, filters were removed and the non-invading cells on the upper surface were wiped off mechanically with a cotton swab. Cells on the lower side of the filters were fixed overnight in ice-cold methanol, then stained using a DiffQuick kit (BD Biosciences, Franklin Lakes, NJ, USA) and pictures of randomly chosen fields were taken, as previously reported [26
2.7. Plate Colony Forming Assay
Approximately 100 cells/mL surviving the different treatments were selected using the trypan blue exclusion test, seeded in fresh medium, and incubated for 10 days at 37 °C. Cells were washed with PBS, fixed in cold methanol, and stained using a Diff Quik kit (BD Biosciences). The stained colonies were photographed with a digital camera and the number of colonies in each well was counted.
2.8. Western Blotting Analysis
Cells were lysed and separated using electrophoresis as previously described [26
]. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and, subsequently, the membrane was probed at 4 °C overnight with primary antibodies diluted in a solution of 1:1 Odyssey blocking buffer (LI-COR®
Bioscience, Lincoln, NE, USA)/Tween (Sigma-Aldrich, St. Louis, MO, USA) -PBS buffer. The primary antibodies were: rabbit anti poly (ADP-ribose) polymerase (PARP)1 and cleaved PARP1 (1:1000, Cell signaling Technology, Danvers, MA, USA), rabbit anti-cleaved caspase 3 (1:1000, IDT, Tema Ricerca, Bologna, Italy), rabbit anti pAKT (1:1000, Cell signaling Technology, Danvers, MA, USA), rabbit anti AKT (1:1000, Cell signaling Technology, Danvers, MA, USA), rabbit anti pERK (1:1000, Cell signaling Technology, Danvers, MA, USA), and rabbit anti ERK (1:1000, Cell signaling Technology, Danvers, MA, USA). The membrane was washed in T-PBS buffer, incubated for 1 h at room temperature with goat anti-rabbit IgG Alexa Flour 750 antibody or with goat anti-mouse IgG Alexa Fluor 680 antibody (Invitrogen, Monza, Italy), and then visualized using an Odyssey Infrared Imaging System (LI-COR®
Bioscience, Lincoln, NE, USA). Mouse anti-β-Tubulin monoclonal antibody (1:1000, Cell signaling Technology, Danvers, MA, USA) was used to control for equal protein loading.
The focus for cancer treatment has been shifted toward strategies of complementary therapy that are able to overcome the limitation of a single-agent treatment. Rational combination approaches are strongly preferred in order to improve the overall patient progression-free survival, overcome or delay the development of drug resistance, and reduce the incidence of side effects. This is of special importance in the treatment of melanoma, particularly of the advanced metastatic form, often resistant to most of the current drugs used in the clinic. Further, due to different transcription pathway activation in melanoma cells, several other mechanisms of resistance to BRAF inhibition have been identified. Melanoma tumors bearing wild-type BRAF are intrinsically resistant to PLX4032 or Dabrafenib. Targeting mitogen-activated protein kinase kinase (MEK) was considered a potential mechanism to overcome BRAF resistance [35
], although the most favorable treatment schedule and sequence is still to be defined. Indeed, multiple levels of cross-talk among mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways and the possibility that ERK can be phosphorylated by the pAKT pathway are demonstrated [36
]. Thus, it is required to also inhibit the AKT pathway in melanoma cells resistant to MAPK inhibitors [38
]. The activation of the PI3K/AKT/mTOR pathway represents one of the major mechanisms of acquired resistance to both targeted BRAF inhibitors and DTIC [39
]. We have found that BRAF melanoma cells exposed to a low extracellular pH medium acquired a resistance to both BRAF and MEK inhibitors but were still sensitive to the inhibition of the AKT/mTOR pathway induced by RAD001 [26
]. It is possible, on the other hand, that during a prolonged mTOR inhibition, PI3K would be able to promote an MAPK pathway through RAS activation. Overall, from these findings emerge the need to use a treatment involving the simultaneous inhibition of the MAPK and AKT pathways in order to have a better drug efficiency and reduce drug resistance.
Although this anti-cancer approach appeared very promising, it had not been as successful as once believed. Indeed, most combined therapies are based on the combination of toxic compounds, leading to toxicity and unexpected side effects.
Given our previous evidence for the protective role of Ole in neurodegenerative and cardiovascular diseases [41
], we have decided to investigate whether Ole might exert some role in melanoma treatment. The existing studies indicate that Ole expresses a well-demonstrated protective role against many types of cancer [22
]. Most of the studies have investigated the anticancer effects of Ole on breast cancer, disclosing that the polyphenol may not only decrease cell viability and proliferation [45
] and synergize with Doxorubicin in in vitro and in vivo models [25
], but also may reverse resistance toward the chemotherapeutic agent, Trastuzumab [47
]. In addition, Ole is effective in reducing cell proliferation by increasing apoptosis in human colorectal cancer cells [48
] and is able to sensitize the Doxorubicin-mediated killing of prostate cancer cells [24
]. Ole also reduces the cell viability of hepatocarcinoma, pancreatic, thyroid, neuroblastoma, mesothelioma, and glioblastoma cancer cells [22
]. Still to be clarified are the effects of Ole on BRAF human melanoma cells. Here, we demonstrate for the first time that Ole treatment represents a new non-toxic anti-cancer agent against BRAF melanoma cells as a suitable promoter of two major agents used in BRAF-resistant melanoma cells, such as RAD001 and DTIC. In addition, the particular approach of Ole to potentiate RAD001 was found appropriate to overcome PLX4032 resistance as demonstrated by the use of special PLX4032-resistant A375 melanoma cells developed in cultures. This finding discloses a complementary approach to the therapy of BRAF inhibitor-resistant melanoma that harbors hyperactivation of AKT. Until now, only one study reported the reversing effect of Ole on chemotherapy-induced resistance [47
]. Thus, these findings open up the chance to use Ole as a therapeutic molecule to improve the anticancer effects of current chemotherapeutics, due to its low toxicity in normal cells as previously reported [49
A375 melanoma cells, used in our study, like most of human solid cancers (prostate, breast, and colon cancer), express the glucose transporter proteins GLUT1 and GLUT3 mRNA and protein, which may likely promote Ole uptake. Hamdi et al. [49
] found that the antiproliferative activity of Ole in normal fibroblasts was reduced by removing the glucose moiety by β-glycosidase. Furthermore D-glucose and Ole compete for the GLUTs, as it was demonstrated by the co-incubation of human melanoma cells with an excess of D-glucose, so it is possible that GLUTs are involved in the transportation of Ole into cancer cells. In this study we found that Ole was present in the cytoplasm of A375 cells after only 15 min of incubation (Figure S1b
), suggesting a fast uptake of Ole inside the cells, probably due to their higher level of GLUTs compared to the normal ones. This is a very important aspect that we can exploit in thinking of an Ole topical application directly on tumor cells. However, we did not exclude the possibility that Ole could enter into the cells using other routes, in particular, in areas of inflammatory reactions where several mediators are active.
Interestingly, in a very short time (48 h) Ole was able to induce a clear and significant induction of melanoma cell death, confirmed by an enhancement of markers of apoptosis. Ole affected the viability of melanoma cells, probably through the inhibition of phosphorylation of AKT and the S6 pathway. This finding is in accordance with Liu’s observation [52
], which indicates that inhibition of AKT is the mechanism underlying the pro-apoptotic and anti-invasive process promoted by Ole in glioma cells and with Yan’s indication [53
] about the induction of apoptosis by Ole through the PI3K/AKT pathway in HepG2 human hepatoma cell line.
Thus, we proceeded to investigate whether Ole blocking the AKT pathway may act in cooperation with the current chemotherapy treatments and allow for a decrease the doses that often lead to toxicity and severe side effects. We found that Ole potentiates the cytotoxic effect of DTIC, reducing the effective dose by 50%; this might be related to an enhanced reduction in AKT phosphorylation. In parallel experiments, Ole was also able to potentiate an mTOR inhibitor, such as RAD001, which is also in PLX4032-resistant melanoma cells. In the same way, Ole reduces the effective dose of RAD001 by 50% and this effect was linked to pAKT abrogation. Thus, Ole, by affecting the PI3K/AKT/mTOR pathway, might represent a new non-toxic agent of interest in the treatment of advanced melanoma. It is likely that Ole might also boost up the inhibition of mTOR through the AMPK/mTOR pathway, which in this study was not investigated, but that was suggested by our previous finding and by other authors [43
]. In addition, Ole may inhibit tumor angiogenesis and in vivo tumor growth, as recently found by the studies of Song et al. [55
] and Samara et al. [56
Although the in vitro studies are very promising, they do not consider Ole metabolism and bioavailability, such that the in vitro used concentrations, despite being in accordance with literature [25
], could seem far greater than those that could be realistically achieved in in vivo models.
Ole inevitably undergoes a metabolic process in in vivo models, and after ingestion, its metabolites are rapidly detected in plasma at different concentrations depending on gender [61
]; however, in the clinic, most agents are typically given via repeated administration that may lead to accumulation [62
], and this is quite close to the high doses used in in vitro experiments.
Furthermore our results suggest that an easily available product as an olive leaf extract enriched in Ole could be even more effective than Ole alone, probably because of the co-presence of other polyphenols among HT, which many authors have suggested to be the real active Ole metabolic product [23
]. Therefore, olive leaf extracts seem to have a powerful anticancer property as Samet I. et al. [63
] and Mijatovic SA et al. [64
] have also shown with regard to human chronic myelogenous leukemia K562 and melanoma cells, respectively, and their mixed phenolic composition with an enrichment in Ole could be useful to decrease the in vitro doses in order to obtain the same effect of Ole administration alone at higher concentration.
Nevertheless, polyphenol bioavailability is still a big drawback that many studies try to overcome through different approaches including the construction of granules containing probiotics and Olea europaea
extract in order to increase polyphenols bioavailability [65
], and the synthesis of more active Ole analogs with various chemical properties [56
In conclusion, Ole, and even more so olive leaf extracts, exhibited a promising potential as adjuvant in conventional anticancer therapies. Furthermore, it may reverse the drug resistance of cancer cells to chemotherapeutics and reduce adverse effects of conventional therapies on nontarget cells.
The limited in vivo animal studies, as recently summarized by two recent reviews [22
], and the paucity of in human studies, in particularly randomized controlled clinical trials, still represents the major drawback. Therefore, preclinical evidence needs to be substantiated by an evidence-based approach to determine the effective dose and best route of Ole administration on the basis of its bioavailability [67
] and any side effects related to chronic administration.