The A549 human lung adenocarcinoma cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Culturing was maintained according to their recommendations. The A549 cell line was cultured in F-12K medium (Invitrogen Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) (both from Gibco, Grand Island, NY, USA) antibiotic solution. All cell cultures and subcultures were incubated at 37°C with 5% CO₂.

Crocodile blood powder was purchased from a local vendor and dissolved in phosphate-buffered saline (PBS). After sonification, the mixture was centrifuged at 10,000 × g for 20 min. The supernatant was collected. After precipitation by ammonium sulfate overnight at 4°C, the mixture was centrifuged at 10,000 × g for 20 min. The supernatant and residues were collected and desalted by Sephadex G25.

An ÄKTA FPLC system (Amersham Pharmacia Biotech, Sweden) and a Sephadex G25 column (50×4.6 mm; GE Healthcare, USA) were used. The samples were separated at 5 ml/min. The mobile phase was distilled water with a constant elution component. The column temperature was approximately 25°C and detected at 280 nm. The detector with conductance was recruited.

An Agilent 1100 system (Agilent Technologies, Palo Alto, CA, USA) and a Prevail C18 column (250×4.6 mm I.D., 5 µm; Alltech, USA) were used. The samples were separated by HPLC at a flow rate of 0.8 ml/min. The mobile phase consisted of 0.1% trifluoroacetic acid in water (A) and acetonitrile (B) with a gradient elution of 5% B at 0–5 min; 20% B at 6–15 min; 30% B at 16–25 min; 60% B at 26–35 min; 90% B at 36–45 min; 5% B at 46–50 min. The column temperature was set at 25°C with injection volume of 10 µl. Detection was set at 280 nm.

A549 cells were seeded into a 96-well plate at a density of 1×10⁴ cells/well. Cells were treated with different concentrations of sample solution for 24, 48 and 72 h, respectively, and those without treatment as control. A total of 50 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) solution was added to each well and incubated for 1 h. The purple formazan formed was dissolved in 200 µl of DMSO and quantified by measuring the absorbance at 540 nm with a microplate reader (Tecan Infinite M200; Tecan Group, Switzerland). The relative cell viability was compared to the control. The half maximal inhibitory concentration (IC₅₀) was determined according to the relative cell viability curve.

A549 cells were seeded into a 96-well plate at a density of 1×10⁴ cells/well. Cells were treated with different concentrations of sample solution for 24 h. Measurement was carried out according to the manufacturer's instructions (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, G1780; Promega, Madison, WI, USA). In brief, after centrifugation at 250 × g for 4 min, 50 µl of the supernatant was transferred into a new 96-well plate. A total of 50 µl of substrate was added and the mixture was incubated for 30 min at room temperature. Measurement was carried out using a microplate reader at 490 nm.

A549 cells were seeded in 100-mm dishes at a confluency of 5×10⁶. The cells were treated with 200 µg/ml of S2 for 24, 48 and 72 h, respectively and harvested with trypsin (Invitrogen Life Technologies) and PBS. The cell pellets were then re-suspended in 500 µl of DNA digestion buffer with Proteinase K (BioVision, USA) added to a 0.5 mg/ml final concentration overnight at 55°C with gentle shaking. A total of 700 µl of neutralized phenol/chloroform/isoamyl alcohol (25:24:1) was added and mixed fairly vigorously. The samples were spinned at 10,000 × g for 5 min and 500 µl of the upper phase was transferred to a new microfuge tube. DNA was precipitated with 1 ml 100% ethanol at room temperature and centrifuged at 10,000 × g. The sample was then washed with 70% ethanol twice and air dried. The DNA precipitate was dissolved in 50 µl of TE buffer and electrophoresed on a 1.5% agarose gel with GelRed Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA) and detected under UV light.

After A549 cells grew to 80% confluency on 100-mm culture dishes, different concentrations of S2 were added after 24 h. The cells were harvested with trypsin and washed with cold PBS twice. After centrifugation at 250 × g for 5 min, the cell pellets were re-suspended in cold 70% ethanol and fixed at −20°C overnight. The supernatant was discarded after centrifugation at 250 × g for 5 min. The cell pellets were washed with cold PBS twice and re-suspended with 0.5 ml propidium iodide (PI) solution. After incubation at 37°C in the dark, the cells were transferred into a 5 ml polystyrene round-bottom tube and sorted with 10,000 events/sample in triplicate using a flow cytometer (BD FACSCanto; BD Biosciences, USA). The results were analyzed with ModFit 3.0 software.

A549 cells were cultured into 100-mm dishes and treated with different concentrations of S2 for 24 h. After harvesting with trypsin and washing twice with cold PBS, detection was carried out according to the manufacturer's instructions (FITC Annexin V Apoptosis Detection kit; BD Biosciences). In brief, the cell pellets were re-suspended in 0.5 ml of 1X binding buffer. A total of 5 µl Annexin V-FITC solution and 5 µl 1X PI solution were added to 100 µl of the cell suspension. The mixture was incubated for 15 min in the dark at room temperature. Before injecting into the flow cytometer, 0.4 ml 1X binding buffer was added to each sample mixture. Analysis was achieved with 10,000 events/sample in triplicate using a flow cytometer (BD FACSCanto; BD Biosciences). The results were analyzed with WinMDI 2.9 software.

A549 cells were cultured into 6-well plates and treated with different concentrations of S2 for 3 h. After harvesting with trypsin and washing twice with cold PBS, the cell pellets were re-suspended in 0.5 ml of JC-1 solution (MitoScreen kit; BD Biosciences). After incubation for 15 min at 37°C in the dark, the cell mixture was centrifuged at 250 × g for 5 min and the supernatant was discarded. Before re-suspending with 0.5 ml of 1X assay buffer solution, the cell pellets were washed twice with 1 ml of 1X binding buffer at room temperature. Analysis was achieved with 10,000 events/sample in triplicate using a flow cytometer. The results were analyzed with WinMDI 2.9 software.

A549 cells were cultured in 6-well plates with coverslips inside at a density of 1×10⁵ cells/well and treated with different concentrations of S2 for 24 h. After washing with warm Hank's Balanced Salt Solution (HBSS; Gibco, Gaithersburg, MD, USA), a sufficient amount of 25 µM carboxy-H₂DCFDA (Image-iT™ LIVE Green Reactive Oxygen Species Detection kit; BD Biosciences) working solution was applied to the coverslips followed by a 30-min incubation at 37°C in the dark. To stain the nucleus, a final concentration of 1.0 µM of Hoechst was added to the cells at the last 5 min of incubation. The coverslips were washed with warm HBSS (Thermo Fisher Scientific, USA) prior to reversely cover the slides with mounting medium. The slides were detected under a fluorescence microscope under a excitation/emission wavelength of 350/461 nm for Hoechst and 495/529 nm for carboxy-H₂DCFDA.

A549 cells were seeded onto 96-well plates at the concentration of 1×10⁴ cells/well. The cells were treated with different concentrations of S2 for 24 h. A total of 100 µl of 1X caspase substrate Z-DEVD solution (Apo-ONE Homogeneous Caspase-3/7 Assay kit; Promega) was added to each well (1:1). The mixture was gently mixed using a plate shaker at 250 × g and incubated at room temperature for 3 h. The fluorescence of each well was measured at 485/520 nm using a microplate reader.

A549 cells were cultured into 6-well plates with coverslips inside at a density of 1×10⁵cells/well and treated with different concentrations of S2 for 24 h. An optimal concentration of mitochondrial dye MitoTracker Red (M7512; Invitrogen Life Technologies) was added and incubated for 30 min at 37°C. After washing with warm HBSS, 1 ml of 4% paraformaldehyde was added for 30 min, followed by 0.5% of Triton-X for 15 min. After washing with PBS, 3% bovine serum albumin (BSA) was added. An optimal concentration of purified mouse anti-cytochrome c (BD Sciences) and goat anti-mouse antibody conjugating FITC (Thermo Fisher Scientific) were added to the coverslips. At the last 5 min of incubation, the nuclear staining dye Hoechst (33342; Molecular Probes) was added. All coverslips were mounted to the slides conversely. Scanning and detection were processed under confocal laser scanning microscopy (FV1000; Olympus).

A549 cells were seeded in 100-mm dishes followed by 24 h of treatment with different concentrations of S2. After harvesting and washing, the cell pellets were re-suspended in whole cell lysis buffer on ice. The cell lysate was collected by centrifugation at 10,000 × g at 4°C for 30 min after incubation overnight at −20°C. The protein concentration of each sample supernatant was quantified by using the DC Protein Assay kit (Bio-Rad, Hercules, CA, USA). After normalization, 80 µg of proteins were loaded to 4–12% SDS-PAGE to separate under a voltage of 80–120 V. After transferring to the polyvinylidene fluoride (PVDF) membrane (Millipore Corp., USA), the membrane was blocked with non-fat dry milk (5% w/v) or BSA at room temperature for 1 h. Dilution of the primary antibodies and secondary antibodies were adjusted according to the manufacturer's instructions (Cell Signaling Technologies, Danvers, MA, USA; Santa Cruz Biotechnology). Blots were developed using the ECL Chemiluminescence Detection Reagent (GE Healthcare) according to the manufacturer's instructions. Densitometric analysis was performed with ImageJ software.

Statistical analysis of the raw data was carried out by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The data are expressed as means ± SD as indicated. Significance was accepted at p<0.05.

The fractions collected from the precipitation and supernatant were separated according to molecular size (Figs. 1 and 2). The chromatogram profile showed a higher concentration of less polarity in whole blood (Fig. 3A). After precipitation, the less polar parts could not be found but the most polar portion were eluted (Fig. 3B).

Cell viability showed no significant change after treatment with residues including P1 and P2, but decreased after treatment with S1 and S2. S2 displayed more significant efficacy (Fig. 4). The whole blood extract displayed inhibitory effects on cell viability (Fig. 5). Cell growth was prohibited in a dose- and time-dependent manner after treatment with S2 (Fig. 6A) with a low IC₅₀ value (Fig. 6B). Treatment with S2 revealed no cytotoxicity to cells concurrent with increasing concentrations (Fig. 7). DNA ladder was found in the early 24 h of treatment while fragments were not detected in the control under the same exposure time (Fig. 8). DNA condensations were observed after treatment with S2 after 24 h (Fig. 9). There was a significant increase in the percentage of cells in the G0/G1 phase compared to that noted in the control but a decreasing trend in the S phase, as well as the slight changes in the G2/M phase were not obvious (Fig. 10). The percentage of FITC-positive and double-positive cells showed an increasing trend with S2 concentrations (Fig. 11). The appearance of ROS in the control group was not detected but the amount and intensities of ROS increased (Fig. 12). After treatment with various concentrations of S2, the cell numbers decreased with the increase in S2 concentrations with a signal of higher intensity of FITC and lower intensity of PE (Fig. 13B). Moreover, at the higher concentration of S2, a more obvious change was observed after 24 h (Fig. 13A). Aggregations were found in mitochondria followed by treatment with S2 (Fig. 14). The co-localization of green vs. red signal shifted after treatment with S2 (Fig. 15). The fluorescent intensity of caspase-3/7 showed a significant increase (Fig. 16).

Following treatment with various concentrations of S2, the expression level of Bcl-2 decreased and the trend became significant when the S2 concentrations increased whereas expression of Bax showed a total opposite trend. With increasing concentrations, an upregulated expression level of Bax was noted (Fig. 17). Expression levels of another protein in the Bcl-2 family, Bid, displayed an increasing trend with a reverse change in caspase-8 (Fig. 18). The expression of caspase family proteins showed similar changes, with downregulation of wild types and upregulation of the cleaved forms. The expression of PARP decreased together with the decreasing expression of cleaved PARP (Fig. 19). The expression level of cell cycle-related proteins, MDM2 and PCNA, showed a decrease when p21 and p53 were upregulated (Fig. 20). The expression of proteins associated with the PI3K/AKT survival pathway, wild-type Akt, PDK1 and GSK-3β showed no significant changes compared to the control. In contrast, the phosphorylated forms of the above proteins and c-Raf were decreased. Furthermore, wild-type PTEN showed an increasing trend while phosphorylated PTEN exhibited a significant decrease (Fig. 21).