The aerial parts of A. ebracteatus in powder form were purchased from a Thai traditional medicine shop (Chao-krom-poe, Bangkok, Thailand).

A. ebracteatus powder (250 g) was extracted with 0.5% sodium dodecyl sulfate (SDS) (Ajax finechem) for 24 h at 37°C in a shaker (Gallenkamp). The extracts were then centrifuged at 9,100 × g for 30 min at 4°C and protein was precipitated with 80% cold acetone (Avantor) in a ratio of 1:2 (v/v) at −20°C for 24 h. The supernatant was removed via centrifugation at 5,800 × g for 30 min at 4°C. Then, the protein precipitate was dissolved in 0.2 M sodium acetate buffer (Ajax finechem), pH 4.5 and digested with pepsin (Sigma-Aldrich; Merck KGaA) in a ratio of 1:25 (w/w) for 24 h at 37°C followed by heat inactivation for 10 min at 60–70°C. Following this, this protein hydrolysate was centrifuged at 5,800 × g for 30 min at 4°C and pH was adjusted from 6.8 to 7.0 followed by protein filtration through a membrane with a molecular weight cut-off of 3 kDa (GE Healthcare). Finally, the protein content was determined using Bradford assay (Bio-Rad Technologies) according to the manufacturer's protocol.

Vero (normal kidney cells, ATCC^® CCL-81™) and A431 (skin cancer cells; ATCC^® CRL-1555™) cells were cultured in DMEM (Gibco; Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) heat inactivated fetal bovine serum (Gibco; Invitrogen; Thermo Fisher Scientific, Inc.) and 1% (v/v) antimycotic-antibiotic solution (Gibco; Invitrogen; Thermo Fisher Scientific, Inc.). Both cell lines were cultured in 75 cm² sterile flasks (SPL) at 37°C under 5% CO₂ atmosphere (Heal force).

All cell lines were cultures in 75 cm² sterile tissue culture flasks at 37°C with 5% CO₂ in DMEM supplemented with 10% heat inactivated FBS (10% v/v). Cells were regularly passaged to maintain exponential growth. Basic cell culture techniques were performed as previously described (17). All cells were checked for mycoplasma contamination using PCR and staining with Hoechst 33258. All cell lines were confirmed to be mycoplasma-free cultures.

Cell viability was evaluated using MTT assay (Invitrogen; Thermo Fisher Scientific, Inc.) as previously described (18). Briefly, 1.5×10³ cells were seeded in 96 well-microplate (SPL) and incubated under 5% CO₂ at 37°C for 24 h. Cells were them treated with various final concentrations of A. ebracteatus protein hydrolysate from 0.00 (water only), 3.33, 33.33, 333.33 and 1,666.67 ng protein/ml. For analyzing the cytotoxicity of the partially purified protein hydrolysate, cells were treated with 425.9 ng protein/ml [the half inhibitory concentration (IC₅₀) value of the A. ebracteatus protein hydrolysate against A431] of each fraction. For evaluating the cytotoxicity of the purified peptides, cells were treated with different concentrations of single or combined peptides, ranging from 100–500 µM. Following the addition of protein hydrolysates or peptides, cells were incubated for 5 days without changing the media nor substituting the protein hydrolysates or peptides. Treated cells were then subjected to MTT assay according to the manufacturer's protocol.

HPLC analysis with a UV detector (RP-HPLC) was performed to partially purified peptides from A. ebracteatus protein hydrolysate. The column employed was an ODS-2 HYPERSIL C18; 250×4.6 mm, 5 µm particle size (Thermo Fisher Scientific, Inc.). A gradient mobile phase of 0–50 min was applied, with HPLC-grade 0.1% trifluoroacetic acid (TFA) (Thermo Fisher Scientific, Inc.), acetonitrile (Thermo Fisher Scientific, Inc.) and water (90:10 v/v). The injected sample volume was 100 µl (17.05 µg protein/ml) and the flow rate was 1.00 ml/min. Detection was performed at 220 nm. Each fraction was collected using a fraction collector (Amersham Pharmacia Biotech). Finally, fractions were lyzed in 50 mM Tris/HCl buffer (Thermo Fisher Scientific, Inc.) and then lyophilized using a freeze dryer (LaboGene).

The peptide sequences were identified using LC-MS/MS. Briefly, the peptides were acidified with 0.1% (v/v) formic acid and subjected to an Ultimate 3000 capillary LC system (Dionex). Peptide separation was performed using an Acclaim PepMap RSLC 75 µm ×15 cm nanoviper C18 with a 2 µm particle sizes and a 100 Å pore size (Thermo Fisher Scientific, Inc.) at flow rate of 300 nl/min. Mobile phase A consisted of 2% acetonitrile and 0.1% formic acid in HPLC grade water and mobile phase B consisted of 0.1% formic acid in HPLC grade acetonitrile. The instrument was controlled by Hystar software. DataAnalysis™ and Biotools software version 3.2 were used for the data interpretation and de novo sequencing.

Four peptide sequences (Table I) were commercially synthesized by GenScript^® for >95% purity. The pI and molecular weight (MW) of each peptide were calculated using a peptide property calculator (19).

Cells (1×10⁵ cells) were seeded in 24 well-microplates (SPL) and incubated in the presence of 5% CO₂ at 37°C for 24 h. Cells were then treated with 425.9 ng protein/ml of the partially purified peptide. Cells were incubated in the presence of 5% CO₂ at 37°C for 3 days. Following incubation, cells were washed twice with cold phosphate buffer saline (PBS) and then harvested. For the positive control, cells were induced with 50 µg/ml dihydrochloride hydrate (Sigma-Aldrich; Merck KgaA) for 30 min. Cells were then stained using Annexin V and dead cells assay kit (Merck Millipore) in the dark for 15 min. Finally, cells were analyzed using a flow cytometer (Merck Millipore).

Cells (1×10⁵ cells) were seeded in 24-well microplates and incubated at 5% CO₂, 37°C for 24 h. Cells were then treated with 425.9 ng protein/ml of the partially purified peptides and incubated in the presence of 5% CO₂, 37°C for 5 days. Protein extraction was performed using passive lysis buffer (PLB) (Promega), and the protein concentration was determined using a Bradford assay. For the immunoblotting step, the protein was firstly separated using NuPAGE^® Bis-Tris gel (Invitrogen; Thermo Fisher Scientific, Inc.) and transferred on to PVDF membrane (Merck Millipore). Then, the membrane was blocked with 5% skimmed milk in TBS-T for 1 h. Next, the membrane was washed with TBS-T buffer and then incubated overnight using primary antibodies that were diluted in 0.01% BSA in TBS-T. The dilution ratios for anti-p53 (Invitrogen; Thermo Fisher Scientific, Inc.), anti-RelA (p65), anti-Cyclin D1 (Santa Cruz Biotechnology) were equal to 1:1,000 (v/v); for anti-alpha-tubulin (Invitrogen; Thermo Fisher Scientific, Inc.) was equal to 1:1,500 (v/v); and anti-Ser15P was equal to 1:300 (Abcam). The membrane was then washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Abcam) for 1 h. Finally, the ECL substrate (GE Healthcare) was added onto a membrane and analyzed using a molecular imager (Bio-Rad Technologies).

An unpaired Student's t-test was performed using GraphPad QuickCalcs (GraphPad software). Unless any statistically significant differences between the mean of three or more independent groups were compared, one-way ANOVA with Dunnett's T3 analysis was performed using IBM SPSS software. P<0.05 was considered to indicate a statistically significant result.

Natural bioactive peptides are primarily released from their precursor proteins by digestive enzymes during gastrointestinal digestion (14). Therefore, the production of bioactive peptides in the present study mimicked the human digestive system in the body by digesting the extracted lysates with the enzyme pepsin. The MTT assay was then performed to evaluate the anti-non-melanoma skin cancer activity of A. ebracteatus protein hydrolysate. The non-melanoma skin cancer cells (A431) were treated with various concentrations of protein hydrolysate ranging from 0–1,666.7 ng protein/ml. Notably, Vero cells, an epithelial cell line from African green monkeys were used to represent normal cells in the present study. As a result, A. ebracteatus protein hydrolysate significantly inhibited the cell viability of A431 in a dose dependent manner (Fig. 1A). By treating cells with 333.3 ng protein/ml, the cell viability of A431 was only 47.5±10.8% (P<0.001) without affecting the Vero cells (Fig. 1A). The IC₅₀ of A. ebracteatus protein hydrolysate against A431 cells calculated by GraphPad was equal to 425.9 ng protein/ml (Fig. 1B). Furthermore, A. ebracteatus protein hydrolysate was revealed to induce anticancer activities against a board range of cancer cell lines (Fig. S1).

Subsequently, partially purified peptides of the A. ebracteatus protein hydrolysate were then investigated in order to identify bioactive fractions.

To partially purify protein hydrolysate and identify fractions that possess anti-epidermoid cancer activities, a reverse phase (RP) HPLC with a UV detector was performed. The chromatogram indicated several major and minor peaks, which were sorted into 19 fractions (Fig. 2A). According to the IC₅₀ value of A. ebracteatus protein hydrolysate against A431 cells (Fig. 1B), the concentration at 425.9 ng protein/ml of each fraction was then administered to the A431 cells. After 5 days of incubation, the cells were subjected to an MTT assay. The results demonstrated that the majority of the fractions had no inhibitory effects on the cells (Fig. 2B). Nevertheless, the fraction number 1 (FR1) demonstrated approximately 53±11% inhibition against A431 cell viability (P<0.0001) without affecting the Vero normal cells (Fig. 2B). Therefore, the FR1 was selected for further analysis to identify their potential peptide sequences using LC-MS/MS and de novo analyses.

The LC-MS/MS and bioinformatic analyses on the FR1 identified four peptide sequences; these were named AE1-AE4, as presented in Table I. Excluding peptide AE1, peptides AE2-AE3 had a molecular weight of ~1,300 g/mol and pI values <7. On the other hand, AE1 was the smallest size about 929 g/mol and had a pI of 9.9.

These peptides were synthesized and their anti-epidermoid cancer activities were evaluated using an MTT assay with A431 cells. Subsequently, A431 cells were treated with single or combined peptides ranging from 100–500 µM. After 5 days of incubation, the cells were subjected to the MTT assay. The results revealed that each single peptide had no cytotoxicity effects on either A431 or Vero cells (Fig. 3A-D). Nevertheless, 500 µM of the combined treatment with the 4 peptides (in the same ratio, 125 µM of each peptide) revealed ~35 and 50% inhibition against Vero and A431 cells, respectively. (Fig. 3E). These results implied that the mixture of the 4 peptides provided synergistic cytotoxic effects against the cultured cells. Further MTT assays were then performed and the IC₅₀ of the combined peptides against A431 cells was equal to 482.8 µM (Fig. 3F). Despite this IC₅₀ demonstrating no cytotoxic effect against the normal cells (Fig. 3F), the value was considerably high. Subsequently, the partially purified FR1 (Fig. 2B) was considered and investigated to identify its effect on the apoptosis pathway.

To investigate the effects of partially purified peptides on the apoptosis pathway, A431 cells were treated with 425.9 ng protein/ml of the FR1 and incubated for 3 days rather than 5 days to ensure that enough cell population were presented for testing the apoptotic assay. After 3 days of incubation, cells defined as positive control were treated with 50 µg/ml dihydrochloride hydrate, and apoptosis inducer. All cells were then stained using the Muse^® Annexin V and Dead Cell kit that uses a single reagent and two stains to reliably stain and differentiate live, dead and apoptotic cells. The assay relies on the binding of fluorescently labeled Annexin V to phosphatidylserine (PS) molecules, which translocate to the outer surface of the cell membrane upon the onset of apoptosis (20). 7-Amino-Actinomycin D (7-AAD) is a fluorescence indicator that is used as a dead cell marker. It also binds selectively to GC regions of DNA (21). Following staining, apoptotic cells were analyzed using a flow cytometer.

The result from the mock analysis revealed that the majority of the A431 cells were alive (~90%) with 5% of naturally occurring late apoptotic cells and 1% of naturally occurring early apoptotic cells and 1% of cell dead (Fig. 4A and D). The addition of the apoptotic inducer increased the number of late apoptotic cells by 3 fold. However, the apoptotic inducer only significantly increased numbers of late apoptotic cells (Fig. 4B and D) whereas the effects of FR1 increased numbers of early apoptotic cells by 2 fold, and moderately increased late apoptotic cells by ~1.5 fold when compared with mock (Fig. 4C and D). Therefore, these results suggested that the FR1 contains bioactive compounds that may inhibit the viability of cancer cells by promoting the apoptosis pathway in A431 cells. Subsequently, the effects FR1 on the protein expression of p53, Ser15P, NF-κB (RelA/p65) and cyclin D1 were further investigated.

A431 cells are reported to carry a p53 mutation at codon 273 (p53-R273H) (22,23). This R273H mutation affected the DNA-binding ability of p53, thus, p53-R273 could not act as a transcription factor (24). Furthermore, p53-R273H has been reported to induce drug resistance (25). Therefore, whether the FR1 could restore p53 transcriptional activity in A431 cells or not was further investigated via assessing the protein expression levels of p53 and Ser15P. Furthermore, the effects of partially purified peptide FR1 were also determined via the protein expression levels of another important transcription factor, RelA (p65), which is involved in the regulation of cell apoptosis, the cell cycle and carcinogen transformation (26). Finally, the effects of FR on the protein expression of cyclin D1, an important regulator of cell cycle progression (27), was also investigated.

The results from the western blot analysis indicated that partially purified peptide FR1 from A. ebracteatus may increase the expression of p53. On the other hand, FR1 decreased the protein expression of RelA (p65) and cyclin D1 (Fig. 5A and B). Ser15P was barely detected in mock as its expression is often very low in carrying p53 mutants such as cancer cells (28–30); however, FR1 had no effect on the expression levels of Ser15P (Fig. 5A and B).