Chemicals and reagents
Vincristine sulphate and Dragendorff’s reagent were procured from Sigma Aldrich (USA). Dimethyl sulfoxide (DMSO), Dulbecco’s modified eagle medium (DMEM), Sodium dodecyl sulfate (SDS), Tris–HCl, Ethylenediaminetetraacetic acid (EDTA), DAPI, Ethidium bromide, Fetal bovine serum, Propidium iodide, RNase A, Proteinase K, 2,4-DNP were obtained from Sigma Chemicals Company (San Diego, USA). DCFH-DA and Annexin-V stains were obtained from Life Technologies (USA). JC-1 (5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolyl carbocyanine iodide) dye was purchased from Molecular probes (Eugene, OR, USA). All other reagents and chemicals were of analytical grade.
Eutypella fungal culture
The fungus used in this study was isolated from stem cuttings of C. roseus plant collected from the nursery of the Indian Institute of Science, Bangalore, India. This fungus was identified as Eutypella species. (Order: Xylariales, Family: Diatrypaseae) on the basis of sequence data which included the 5.8 s rDNA gene and ITS genes. The partial ITS sequence was deposited in NCBI GenBank with accession no. KC920840. The fungus was maintained on potato dextrose agar at 25 °C ± 2 °C with regular sub-culturing.
Cultivation of Eutypella spp – CrP14 and extraction of vincristine
The endophytic fungus was grown on PDA plates for about 10 days. Three 5 mm agar plugs containing mycelium of Eutypella spp were transferred to 400 ml of potato dextrose broth, in a 1 L conical flask and incubated at 25 °C ± 2 °C under stationary condition for 21 days. After completion of incubation, the culture was passed through two layers of muslin cloth, in order to get a clear filtrate devoid of mycelia. For alkaloid extraction, a modified protocol was used, based on the methods described elsewhere [10–12]. The pH of the filtrate was adjusted to 2.0 with 1 N H2SO4 and extracted with equal volume of ethyl acetate. The aqueous phase obtained was retained and the pH was adjusted to 10.0 with 1 N NaOH, and again extracted with equal volume of ethyl acetate. The organic phase was evaporated to dryness at 35 °C using rotary vacuum evaporator and the extract was subsequently used in the experiments.
Analysis of fungal vincristine (FVCR) from Eutypella spp-CrP14
Thin layer chromatography (TLC) analysis was carried out on Merck 0.25 mm silica gel plates. The fungal filtrate culture extract as well as standard vincristine were chromatographed concurrently on a TLC plate and developed in solvent chloroform: methanol (7:3). Upon completion, the fungal vincristine (FVCR) and also the reference standard on TLC plate were visualized by spraying with alkaloid-specific Dragendorff’s reagent.
Mass spectroscopy was done on the fungal filtrate culture extract using the electrospray ionization technique. Fungal extract and also a standard vincristine (SVCR) solution were infused separately into the mass spectrometer through a reverse-phase C18 column (BDS Hypersil), 250 mm × 4.6 mm. The mobile phase used was acetonitrile: 1 % acetic acid (20: 80) at a flow rate of 0.5 ml / min and the data was acquired over a m/z range of 200–100 in positive ion mode.
Quantification of FVCR was performed by HPLC analysis. Fungal filtrate culture extract was subjected to HPLC separation using Agilent C18 column (4.6 × 150 mm, 3 μm pore size and particle size of 120 A) in a Thermofischer PDA detector with a wave length of 252 nm. A gradient elution of 10 % - 100 % was performed using 0.1 % formic acid in 5 mM ammonium acetate (solution A) and methanol (solution B) at a flow rate of 0.8 ml/min. A sample of 20 μl was injected and analyzed. The identification of FVCR was accomplished by comparison of retention times with authentic standard vincristine.
Apoptotic activity of FVCR
Preparation of FVCR
The fungal extract prepared from Eutypella spp-CrP14 was first separated on TLC. The spot corresponding to vincristine in crude was scraped and eluted twice with methanol. The resulting partially purified material was used as ‘fungal vincristine’ in apoptotic studies.
Cell lines and maintenance
The cell lines A431, HeLa, A549 and HEK 293 were procured from National Centre for Cell Sciences (NCCS) Pune, India. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and penicillin + streptomycin (100 μg/ml each) mixture in a 5 % CO2 incubator at 37 °C. The cell lines were maintained with regular passaging.
Antiproliferative activity of FVCR on different cancer cell lines
Cells at a density of 1 × 104 cells per well were seeded in 100 μl of Dulbecco’s modified Eagle’s medium (DMEM) with 10 % fetal bovine serum (FBS) in 96-well plates and grown for 24 h at 37 °C in a 5 % CO2 incubator. The cells were then treated with FVCR at various concentrations, ranging from 5 to 100 μg/ml. The cells were then treated with FVCR at various concentrations, ranging from 5 to 100 μg/ml. After 24 h, MTT solution (10 μl of a 5 mg/ml stock) in PBS was added to each well and incubated for 2 h; the supernatant was then removed, and DMSO (100 μl) was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader. The most sensitive cell line was further tested with authentic vincristine, which served as positive control. Standard vincristine is known to inhibit cell proliferation in this test system in our laboratory .
Cytotoxicity of FVCR – Live dead cell assay using PI staining
The assay was performed using propidium iodide (PI), a membrane impermeable dye that is generally excluded from viable cells. It binds to double stranded DNA by intercalating between base pairs. Briefly, A431 cells (1 × 106) were cultured with different concentrations of FVCR (5, 10 and 20 μg/ml) for 24 h and re-suspended in 100 μl phosphate buffer saline (PBS). Propidium iodide (PI) stain (5 μl) was added to each sample just prior to FACScan analysis and data were acquired for unstained and PI stained cells. Dot-plot of forward scatter versus PI was recorded to check the percentage of cell death.
Cell cycle analysis to determine cell cycle distribution
A431 cells (2.5 × 105) were seeded in 24-well plates containing a 500 μl culture medium and incubated for 24 h in a 5 % CO2 incubator at 37 °C. Cells were then treated with different concentrations (5, 10 and 20 μg/ml) of FVCR and incubated for 24 h. Cells were trypsinized, re-suspended in phosphate buffer saline (PBS), centrifuged for10 min and fixed in ice-cold ethanol (70 % v/v) for 30 min. The cells were then stained with PI and analyzed by flow cytometer as described previously . The data were analyzed using CellQuest Pro software.
Detection of apoptosis and cell death using annexin-V-FITC and PI dual staining
The apoptotic cell death induced by FVCR was measured by flow cytometry using the annexin V-FITC/PI staining method . Cells (2.5 × 105) were seeded in 24-well plates and treated with different concentrations (5, 10 and 20 μg/ml) of FVCR for 24 h in the DMEM-FBS medium. The cells were then stained with recommended concentrations of annexin V-FITC with and without PI and quantified through flow cytometry using the FACScan Calibur (Becton Dickinson, USA). Cells were labeled in the four quadrants as follows: Q3-Annexin V negative and PI negative were considered to be viable cells; Q4- Annexin V positive and PI negative as early apoptotic cells; Q2- Annexin V positive and PI positive as late apoptotic cells or necrotic cells; and Q1-Annexin V negative and PI positive as necrotic cells/ dead cells. The data obtained with an excitation (λex)/emission (λem) wavelengths of 488/520 nm for annexin V-FITC and 540/630 nm for PI were analyzed using CellQuest Pro software.
Determination of DNA fragmentation
The DNA fragmentation analysis was carried out by agarose gel electrophoresis. FVCR-treated and untreated A431 cells were harvested and re-suspended in phosphate-citrate buffer (40 μl containing 192 parts of 0.2 M Na2HPO4 and 8 parts of 0.1 M of citric acid, pH 7.8) and left for 30 min at room temperature. The cells were then centrifuged for 5 min followed by addition of Nonidet P-40 (3 μl of 0.25 %), RNase A (3 μg) and incubated at 37 °C. After 30 min, proteinase K (3 μg) was added and fragmented DNA was precipitated with the addition of ethanol overnight at −20 °C followed by centrifugation. The DNA pellet obtained was dissolved in TE buffer, and analyzed by 0.8 % agarose gel electrophoresis. The fragmented DNA ladder formation in A431 cells was visualized on a UV transilluminator after staining with 5 μg ethidium bromide.
Determination of mitochondrial membrane potential (ΔΨm)
The mitochondrial membrane potential was assessed by using JC-1 dye, a lipophilic cationic fluorescent dye with dual (red and green) emission wavelengths. It exists as a monomer (green) in cytosol and accumulates as red aggregates on selective entry into mitochondria in normal cells . In apoptotic and necrotic cells, JC-1 exists in monomeric form and stains green. A431cells (2.5 × 105) were cultured in the presence or absence of different concentrations (5, 10 and 20 μg/ml) of FVCR for 24 h. The cells were then stained with 2.5 μg/ml of JC-1 dye and incubated for 15 min at 37 °C in a CO2 incubator. At the end of incubation, the cells were washed with ice-cold PBS containing FBS (2 % v/v), and both red and green fluoresence were analysed with a flow cytometry using FACS Calibur (Becton Dickinson, USA). JC-1 monomers emit at 530 nm (channel-green fluorescence) and JC-1-aggregates emit at 590 nm (channel-red fluorescence). Valinomycin-treated (10 μM) cells served as the positive control, and untreated cells were taken as the negative control. Data were analyzed using CellQuest Pro software.
Measurement of ROS production
The involvement of ROS in FVCR induced apoptosis pathway was studied using the dye 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) . Briefly, A431 cells (2.5 × 105) were seeded in 24-well plates, incubated for 24 h and treated with different concentrations (5, 10 and 20 μg/ml) of FVCR for 24 h in DMEM-FBS medium. In order to detect the production of ROS, the cells were harvested, washed with PBS, re-suspended in PBS containing DCFH-DA (10 mM) and incubated at 37 °C for 30 min. Hydrogen peroxide (800 μM) was used as a positive control. Fluorescence generated due to the hydrolysis of DCFH-DA to dichlorodihydrofluorescein (DCFH) by non-specific cellular esterases and the subsequent oxidation of DCFH by peroxides was measured by flow cytometry for green fluorescence at 530 nm.
All data were expressed as a mean ± standard deviation of three independent experiments.