The latex of F. asafoetida was collected by incision of the root collar from plants growing in Jandagh (Isfahan, Iran) at an altitude of 1500 m above sea level. The plant material was identified by Dr. Mohammad-Reza Kanani, Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran, where a voucher specimen is kept (School of Pharmacy, Isfahan, No. 5729). The latex (100 g) was dried and extracted with acetone (2 × 1 L) for 2 days with continuous shaking to afford a gummy residue (30 g), that was fractionated by gravity column chromatography on silica gel and further purified by HPLC to obtain pure compounds as previously described (Shokoohinia et al., 2013).

Normal human epidermal melanocytes (NHEM) were purchased from Lonza (Walkersville, MD, USA) and were grown in Melanocyte growth medium 2 (Lonza). The melanoma cells lines B16/F10, Sk-Mel-5, and Sk-Mel-28 were purchased from IRCCS AOU San Martino—IST (Genova, Italy), A375 from Sigma-Aldrich (Milan, Italy), and WM3060 and WM983A were purchased from Rockland (Limerick, Ireland). B16/F10, Sk-Mel-5, Sk-Mel-28, and A375 were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 μmol/L non-essential amino acids, penicillin (100 U/mL), streptomycin (100 μg/mL), and 1 mmol/L sodium pyruvate (all from Sigma-Aldrich, Milan, Italy). WM3060 and WM983A were cultured in Tumor Specialized Media (1:5 Leibovitz's—MCDB153), containing 2% Inactivated FBS and 1.68 mM CaCl₂. Cells were grown at 37°C in a humidified incubator under 5% CO₂. The cell line PES 43 was isolated from a lung metastases of a patient from the National Cancer Institute, G. Pascale Foundation (Scala et al., 2006) and cultured in Iscove's modified Dulbecco's medium (Cambrex Bioscience, Verviers, Belgium) supplemented with heat-inactivated 10% fetal bovine serum, penicillin, and streptomycin (100 units/mL each). ADA was diluted in DMSO to produce a stock solution of 10 mM for in vitro experiments. All cell lines used in this study were characterized by the cell bank were they were purchased.

Cell proliferation was measured by the 3-[4,5-dimethyltiazol-2yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The melanoma cells (A375, SK-Mel-5, SK-Mel-28, PES43, B16/F10, WM3060, and WM983A) and the NHEM cells were seeded on 96-well plates (1 × 10⁴ cells/well) and treated with ADA or with the other compounds: Propionyl deacylasadisulfide (PDA); methoxylatifolone (MEF); foetisulfide A (FSA); arachyl deacylasadisulfide (ARDA); deacylasadisulfide (DA)]; (10-30-100 μM) for 24–48–72 h before adding 25 μl of MTT (Sigma, Milan, Italy) (5 mg/ml in saline). Cells were incubated for additional 3 h at 37°C. Thereafter, cells were lysed and dark blue crystals were solubilized with a solution containing 50% (vol/vol) N,N-dimethyl formamide, 20% (wt/vol) sodium dodecylsulfate with an adjusted pH of 4.5. The OD of each well was measured with a microplate spectrophotometer (TitertekMultiskan MCC/340) equipped with a 620-nm filter.

The characterization of the potential H₂S-generating properties of the tested compound ADA has been carried out by an amperometric approach, through the Apollo-4000 free radical analyzer (WPI) detector and H₂S-selective mini-electrodes. The experiments have been carried out at room temperature (20°C). Following the manufacturer's instructions, a “PBS buffer 10x” was prepared (NaH₂PO₄·H₂O 1.28 g, Na₂HPO₄.12H₂O 5.97 g, NaCl 43.88 g in 500 ml H₂O) and stocked at 4°C. Immediately before the experiments, the “PBS buffer 10x” was diluted using distilled water (1:10) to obtain the assay buffer and the pH adjusted to 7.4. The H₂S-selective mini-electrode was equilibrated in 10 ml of the assay buffer, until the recovery of a stable baseline. Then, 100 μl of a DMSO solution of ADA was added (the final concentration of the tested compound was 100 μM; the final concentration of DMSO in the assay buffer was 1%). The eventual generation of H₂S was observed for 20 min. Preliminary experiments demonstrated that DMSO 1% did not produce any interference on the amperometric recording. When required by the experimental protocol, the nucleophil L-cysteine (4 mM) was added 10 min before the addition of ADA. L-Cysteine did not produce any amperometric response. The correct relationship between the amperometric currents (recorded in pA) and the corresponding concentrations of H₂S was previously determined by suitable calibration curves, which were obtained by the use of increasing concentrations of NaHS (1, 3, 5, and 10 μM) at pH 4.0.

Apoptosis was detected with an Annexin V-FITC kit purchased from BD Pharmingen (San Diego, CA, USA) according to the manufacturer's instructions. PES 43 cells were seeded in 35 mm culture dishes and allowed to attach overnight. The cells were treated with ADA (100 μM) for 24–48–72 h, collected, and washed twice with PBS. Samples were then taken to determine baseline and drug-induced apoptosis by Annexin V-FITC/Propidium Iodide (PI) (Beckman Coulter; Brea, CA) double staining or PI staining and flow cytometry analysis using a FACSCanto II 6-color flow cytometer (Becton Biosciences, San Jose, CA), as described previously (Ianaro et al., 2009). To detect early and late apoptosis, both adherent and floating cells were harvested together and resuspended in annexin V binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) at a concentration of 10⁶ cells/mL. Subsequently, 5 μL of FITC-conjugated Annexin V and 5 μL of PI were added to 100 μL of the cell suspension (10⁵ cells). The cells were incubated for 15 min at room temperature in the dark. Finally, 400 μL of annexin V binding buffer was added to each tube. A minimum of 50000 events for each sample were collected and data were analyzed using FacsDiva software (Becton Biosciences).

PES 43 and A375 cells were treated with ADA 100 μM for 15–30–60'or for 3–6–24–48 h. Whole-cell or nuclear extracts were prepared as previously described (Ialenti et al., 2005; Panza et al., 2016). The protein concentration was measured by the Bradford method (Bio-Rad, Milan, Italy). Equal amounts of protein (40 μg/sample) from whole or nuclear cell extracts were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membranes (Trans-Blot Turbo Transfer Starter System, Biorad). The membranes were blocked for 2 h in 5% low-fat milk in PBS with 0.1% Tween 20 (PBST) at room temperature. Then the filters were incubated with the following primary antibodies: IκBα (sc-1643 Cruz Biotechnology, Santa Cruz, CA; diluted 1:200); Bcl-2 (2876, Cell Signaling, USA; diluted 1:1000), caspase 3 (9662, Cell Signaling, USA; diluted 1:1000), PARP (9542, Cell Signaling, USA; diluted 1:1000), p44/42 MAPK (Erk1/2) (9102, Cell Signaling, USA; diluted 1:1000), Phospho-p44/42 Erk MAPK (Erk1/2, Thr202/Tyr204) XP (4370, Cell Signaling, USA; diluted 1:2000), AKT (9272, Cell Signaling, USA; diluted 1:1000), Phospho-AKT (Ser473) XP (4060, Cell Signaling, USA; diluted 1:2000), c-FLIP (06-864, Millipore; diluted 1 μg/ml); XIAP (R&D System, Minneapolis; 1 μg/ml); NF-κB p65 (F-6): (sc-8008 Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1:200); β-actin (Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1:1000), GAPDH (2118, Cell Signaling, USA; diluted 1:1000) overnight at 4°C. The membranes were washed 3 times with PBST and then incubated with horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1:2000) for 2 h at room temperature. The immune complexes were visualized by the ECL chemiluminescence method and acquired by the Image Quant 400 system (GE Healthcare).

The assay was performed using chambers with polycarbonate filters with 8-μm nominal pore size (Millipore, USA) coated on the upper side with Matrigel (Becton Dickinson Labware, USA) as previously described (Ivanov et al., 2003). Briefly, the chambers were placed into a 24-well plate and melanoma cells (2.5 × 10⁵/mL) were plated in the upper chamber, with or without ADA (10–30 μM), in serum-free DMEM. After the incubation period (16 h), the filter was removed, and non-migrant cells on the upper side of the filter were detached with the use of a cotton swab. Filters were fixed with 4% formaldehyde for 15 min, and cells located in the lower filter were stained with 0.1% crystal violet for 20 min and then washed with PBS. The filters were examined microscopically and cellular invasion was determined by counting the number of stained cells on each filter in at least 4–5 randomly selected fields. Resultant data are presented as a mean of invaded cells ± SEM /microscopic field of three independent experiments.

The experimental procedures, according to Italian (DL 26/2014) and European (n. 63/2010/UE) regulations on the protection of animals used for experimental and other scientific purposes, were approved by the Italian Ministry. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). Mice were observed daily and humanely euthanized by CO₂ inhalation if a solitary subcutaneous tumor exceeded 1.5 cm in diameter or mice showed signs referable to metastatic cancer. All efforts were made to minimize suffering. Female C57BL/6 mice (18−20 g) were purchased from Charles River Laboratories, Inc. Mice were housed at the Animal Research Facility of the Department of Pharmacy of the University of Naples Federico II.

B16/F10 (5 × 10⁵) murine melanoma cells were collected in PBS and injected via tail vein into syngeneic C57BL/6J mice. The mice were equally randomized into three groups (8 mice/group): 0.9% normal saline control group, ADA 5 mg·kg⁻¹ group, ADA 50 mg·kg⁻¹ group. Starting on the 1st day after tumor cell inoculation, test compounds were given orally every day for 14 days. Then the mice were weighed and sacrificed. The lungs were removed and washed with PBS. The percentage of metastatic area was calculated using ImageJ software (ImageJ).

Data are expressed as mean ± SEM of n experiments. Data were analyzed and presented using GRAPHPAD PRISM software (GraphPad). Significance was determined using Student's 2-tailed t-test. Results were considered significant at P-values ≤ 0.05 and are labeled with a single asterisk. In addition, P-values ≤ 0.01 and 0.001 are designated with double and triple asterisks, respectively.

In preliminary experiments, the disulfide compounds obtained from asafoetida were evaluated for their anti-proliferative activity on human melanoma cells A375 (Figure 1). After an initial screening, we found that all the sulfur-containing compounds ADA, PDA, FSA, ARDA, and DA inhibited the proliferation of melanoma cells although with different potency (Figure 1). On the other hand, the structurally unrelated MEF was practically inactive. Therefore, the disulfide group is essential for the anti-proliferative effect. Among the active compounds ADA and PDA showed similar activity on the human melanoma cell line A375 (IC₅₀ 51.4 and 54.6 μM, respectively). The selection of ADA for further investigation was based on the relative abundance of this metabolite in the plant, the higher availability for pharmacological evaluation as well as the presence of a clear concentration-related effect as compared to PDA.

ADA also inhibited the proliferation of all the other human melanoma cell lines employed in this study, namely SK-Mel-5, SK-Mel-28, WM983A, and PES43 cell lines, all carrying the BRAF^v600E mutation and the WM3060 cell line wild type for BRAF, without affecting NHEM proliferation (Table 1). A marked inhibition of cell proliferation was observed also in the murine cell line B16/F10 after incubation with increasing concentration of ADA (Table 1). Among the human cell lines employed the high metastatic PES43 and A375 were the most sensitive to the anti-proliferative activity of ADA. In fact, as shown in Table 1 increasing concentration of ADA (10-30-100 μM) inhibited PES43 proliferation at 72 h by 38, 61, and 71%, respectively (P < 0.001, n = 5) and A375 cell proliferation by 28, 54, and 76% respectively (P < 0.001, n = 5). Thus, these cell lines were selected for the molecular studies.

To verify if the anti-proliferative effect of ADA was related to its ability to induce apoptosis of cancer cells, the cytofluorimetric assay, with Annexin V/PI dual staining, was carried out on melanoma cells PES43 and A375. This dual staining distinguishes between unaffected (unlabeled; quadrant 3, Q3), early apoptotic (Annexin V positive; quadrant 4, Q4), late apoptotic (Annexin V positive, PI positive; quadrant 2, Q2), and necrotic (PI positive; quadrant 1, Q1) cells. Treatment of PES 43 and A375 cells for 24–48–72 h with ADA (100 μM) resulted in a significant and time–dependent induction of apoptosis (Figures 2A,B,D,E). The pro-apoptotic effect of ADA was confirmed by a time-dependent cleavage of caspase 3, the main effector caspase, and of its substrate poly (adenosine diphosphate-ribose) polymerase (PARP) (Figures 2C,F).

It has been reported that in melanoma constitutive activation of NF-κB confers tumor survival capacity and apoptosis avoidance (Ueda and Richmond, 2006). In order to analyze if the inhibition of apoptosis induced by ADA was involving the NF-κB pathway, PES 43 and A375 cells were treated with ADA 100 μM for 15, 30, and 60 min and western blot analysis was carried out on cellular extracts. As shown in Figures 3A,E an inhibition of IκBα degradation at the earliest time points following ADA incubation was observed. This effect was abided by the inhibition of NF-κB nuclear translocation as demonstrated by a reduction in band intensity of the subunit p65 (Figures 3B,F). Finally, western blot analysis showed that ADA markedly decreased the expression of three anti-apoptotic proteins whose expression is modulated by the transcriptional activity of NF-κB, (XIAP, c-FLIP, and Bcl-2) confirming NF-κB involvement (Figures 3C,G).

The Mitogen-Activated Protein Kinase (MAPK)/ERK and the Phosphoinositide 3-Kinase (PI3K)/AKT pathways are two of the most frequently deregulated pathways in melanoma (Hodis et al., 2012). They play an important role in melanoma development and progression and are involved in the mechanism of resistance to targeted therapy (Flaherty et al., 2012). As shown in Figures 3D,H, both phospho-AKT (p-AKT) and phospho-ERK (p-ERK) band intensity was time-dependently reduced following treatment with ADA (100 μM).

To determine whether ADA affected the invasive ability of the metastatic melanoma cells PES 43 and A375, we performed cell invasion assays using a transwell system. As shown in Figures 4A,B, ADA at 10 and 30 μM significantly inhibited the invasiveness of PES43 cell line by 33 and 63%, respectively (P < 0.01; P < 0.001 vs. control; n = 5). ADA (10–30 μM) produced a comparable effect on A375 cell line (Figures 4C,D) inhibiting the invasiveness by 45 and 67% respectively (P < 0.001 vs. control; n = 5).

To properly investigate on the role of ADA on melanoma progression, we exploited a murine model of metastatic melanoma induced following the injection of the murine melanoma cells B16/F-10 into the tail vein of C57BL/6J mice. Groups of mice were treated with different doses of ADA (5 and 50 mg·kg⁻¹, p.o.) while control group received the vehicle only. Fourteen days after tumor implant, lungs were removed and the percentage of metastatic affected area was calculated by using the software Image J. As shown in Figures 5A,B, ADA (5–50 mg·kg⁻¹) caused a dose-dependent reduction of lung metastases. The higher dose tested e.g., 50 mg·kg⁻¹ reduced the total area of about 96% (2.74% vs. control 68.1%).

The incubation of ADA 100 μM in aqueous solution in presence of the nucleophil L-cysteine led to the generation of a time-related increase in H₂S concentration. The rise of the H₂S production reached a steady-state after about 10 min and the highest concentration of H₂S, recorded after 20 min was 5.05 ± 0.04 μM (Figure 6).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.