2.1 Material
3-(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), 5 – fluroracil (5 – FU), 4’,6-diamidino-2-phenylinole (DAPI), Propidium Iodide (PI), Ethidium bromide (EB), RNase A (Ribonuclease A), EDTA, Phosphate-buffered Saline (PBS) pH 7.4, Dulbecco’s modified eagle medium (DMEM), Antibiotic solution, Fetal bovine serum (FBS), Trypsin 0.25% solution 1X, Dimethyl sulphoxide (DMSO), were purchased from Himedia Laboratories, Mumbai, India.
2.2. Assay of inhibitory activity of PFL-L on hyphal growth
Human pathogenic test fungus, Aspergillus flavus (AF017), Mucor sp (MC02). and Aspergillus niger (AN019) were obtained from Fungal Culture Collection Facility, CAS in Botany, University of Madras, Chennai, Tamil Nadu. Strains mature spore suspensions were grown on Potato Dextrose Broth (PDB) liquid medium at room temperature in a rotary shaker (200 rpm). Growth stage maintaining with proper subculture at fungal culture collection laboratory. After 7 days the strains were used as a further study. The spore’s suspension concentrations at 105 spores/ml of (80 µL) were added to a 96 well Microtiter plate [33].
The activities of PFL-L were determined against three different fungal strains using micro-dilution assays [34, 35]. PFL- L concentrations viz., 30 µg/ml, 40 µg/ml, 50 µg/ml, and 60 µg/ml of PFL-L were transferred to the Microtiter plate (n = 3). The media (PDB) were added to the all well and it gives final concentration at 120 µL. Standard drugs (70 µg/ml) were then added to the wells containing PDB suspension. The microtiter plates were maintained at an aseptic condition with tight tinfoil, sealed with parafilm, and incubated at 23–25ºC for 24 hours. Finally, the microtiter plates were measured at 570 nm by using a spectrophotometer (SpectraMaxM2e, Multimode Plate Readers, California). To evaluate the IC50 value for the antifungal activity ten doses (10–100 µg/ml) of PFL-L and standard drug as per our previous report [36].
All measurements were conducted in triplicate. The percentage (%) of cell viability is calculated following formula,
$$\text{C}\text{e}\text{l}\text{l} \text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y} \%=\frac{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e} \text{o}\text{f} \text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d} \text{c}\text{e}\text{l}\text{l}\text{s} }{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e} \text{o}\text{f} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{c}\text{e}\text{l}\text{l}\text{s} }\times 100$$
2.3. In-vitro anticancer activity
The HT29, A549 cancer cells, and fibroblast cell lines (3T3), were obtained from National Centre for Cell Science (NCCS), Pune, India. Cell lines were grown DMEM medium supplemented with FBS 10% (v/v) and Streptomycin with penicillin (250 U/mL) incubated 5% CO2 incubator at 37˚C. The cells were allowed to grow for 24–48 hrs. The cell cultures were seeded in a 96 well plate, at a cell density of 1 × 105 cells/well and incubated for 24 h at 37˚C and 5% CO2 [36, 37]. The cell viability and proliferation were evaluated after the cells get attached were treated against PFL-L (µg/ml) of different concentrations ranging from 10 to 100 µg/ml. After 24hr, the treated cells were washed with PBS (pH 7.4). Twenty microliters of MTT solution (5 mg/mL) were added to each well and allowed to stand for 4 h at 37˚C. Then, the medium with MTT was discarded and 100 µL of DMSO was added to each well to neutralize the dark blue formazan crystals. The microtiter plate was read a SpectraMaxM2e, Multimode Plate Readers, California at 595nm. All the experiments were conducted in triplicates. The PFL-L inhibited at 50% of cell proliferation was observed as IC50 value. Morphology of all cell lines was observed using by bright field microscope.
The percentage (%) of cell viability are calculated following formula,
$$\text{C}\text{e}\text{l}\text{l} \text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y} \%=\frac{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e} \text{o}\text{f} \text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d} \text{c}\text{e}\text{l}\text{l}\text{s} }{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e} \text{o}\text{f} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{c}\text{e}\text{l}\text{l}\text{s} } \times 100$$
2.4. Identification of apoptotic cells
To identify apoptotic cells, in presence of PFL-L treated cancer cells (HT29, A549) was examined by using a double staining method. The tested cells were washed with phosphate-buffered saline at pH 7.2 and fixed cells using, methanol: acetic acid 3:1 (v/v) for 10 min. After incubation, the tested cells were added in propidium iodide (PI), 50 µg/mL, and kept for 20 min to neutralize the reagents. Morphological analysis of the apoptotic cells was performed after staining with PI under a fluorescence microscope.
2.5. Determination of DNA damage by alkaline comet assay
The damage of the DNA was evaluated under alkaline conditions [38, 39]. The tested PFL-L and control cells were run the alkaline-based agarose gel with lysis buffer the following components, Tris – HCl (10 mM), pH 10, EDTA (100mM), sodium chloride (2.5M), DMSO (5%) and Triton X-100. HT29 and A549 cancer cells were embedded in low-melting-point agarose were lysed overnight at 4°C in lysis buffer. The unwinding step was performed by NaOH (300mM) and EDTA (1mM) for 40 min at 4°C (pH > 13). After running the gel, the slides were neutralized with Tris – HCl (0.4mM) pH at 7.5, and the cells were stained with ethidium bromide (2µg/ml). The DNA tail moment length was measured. The tail formation was scored by using a fluorescence microscope, Nikon, Japan.
2.6. Cell cycle analysis using flow cytometry method
To measure DNA ploidy as well as to measure alternations of cell cycle profiles characteristic of DNA fragmentation (necrosis) compared to patterned DNA cleavage (apoptosis). To determine the cell cycle analysis, 2.5 × 105 cells were incubated in 24 well plates with PFL-L in 0.5 ml of the medium at 5% CO2 at 37˚C in the incubator [40]. After treatment, floating cells in the medium were pooled with attached cells collected by trypsinization. Cells were collected by centrifugation at 600g for 10 min at 4 ºC. Cells were washed with ice-cold PBS and fixed in 80% ethanol in PBS at − 20ºC. The fixed cells were pelleted and stained with PI (50 µg/mL) in the presence of RNase A (20 µg/mL) for 30 min at 37ºC. Apoptosis was quantitatively determined by flow cytometry after incubation at 4 ºC in the dark for at least 24 h cells containing nuclei with sub diploid DNA content. The number of cells at each stage of the cell was estimated by fluorescence-activated cell sorting (FACS) and monitored by flow cytometry. The data were analyzed to determine the percentage of cells at each phase of the cell cycle (G0/G1, S, and G2/M) or in the aneuploid peak.
2.7. Western blot analysis
To study the apoptosis nature of proteins on HT29 and A549 tested cells after 24 hours with PFL-L [41]. The equal amount of PFL-L was mixed with the sample buffer (2 X) and boiled for 5 minutes. The tested cell's protein was separated on SDS-PAGE (10%) and electro-transferred onto a PVDF membrane (Bio-Rad, USA). The blots were blocked with a blocking buffer (5%) for 4 hrs. After blocking, the membrane was incubated with respective rabbit monoclonal antibodies. Goat monoclonal antibody Bcl-Xl, Bcl-2, Procaspase-3, procaspase-9, MMP-3, MMP-9, B6, N-Cadherin, and E-Cadherin, in 1:5000 dilutions overnight at 4˚C. Then, the membranes were washed thrice with T-TBS for 10 minutes each, followed by horseradish peroxidase-conjugated secondary antibody (1:10,000 dilutions) and was incubated for 45 min at room temperature. Finally, signals were visualized using an enhanced chemiluminescent system (Pierce Biotechnology Inc, USA).
2.8. Statistical analysis
All experiments on both antifungal and anticancer activity tests were the average of triplicate analyses. The data were recorded as mean ± standard deviation.