Chemicals and reagents
Baccatin III, Dimethyl sulfoxide (DMSO), Hoechst 33258, Paclitaxel (Taxol), propidium Iodide (PI), Proteinase K and RNase A were purchased from Sigma-Aldrich. Pancaspase inhibitor, caspase-2 inhibitor, caspase-3 inhibitor, caspase -9 inhibitor and caspase-10 inhibitor were obtained from R&D systems Inc. (Minneapolis, MN) and Calbiochem. Dulbecco’s modified Eagle medium (DMEM), RPMI-1640 medium and fetal bovine serum (FBS) were purchased from GIBCO. 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 compounds were of analytical grade.
Isolation of taxol and baccatin III from F. solani
As described earlier , taxol and baccatin III were isolated from F. solani. Briefly, the fungus was grown in 500 ml of potato dextrose liquid medium in 2 l Erlenmeyer flasks at 25°C in the dark in stationary cultures. After 21 days, 1 l of culture (medium plus mycelium) was extracted twice with an equal volume of methylene chloride . The obtained crude extract was subjected to thin-layer chromatographic analysis using solvent system, chloroform:methanol (9.2:0.8; v/v). After chromatography, the area with silica gel on the plates containing putative taxol and baccatin III was scraped at the appropriate relative front (Rf) and exhaustively eluted with methanol and further separated by high performance liquid chromatography (HPLC) using Kromasil C18-column (250 × 4.6 mm) at 227 nm. Authentic taxol and baccatin III standards were used as reference.
Cell lines and culture conditions
HeLa (human cervical carcinoma cell line), HepG2 (human liver carcinoma cell line), Jurkat-JR4 (human leukemia T cell line), Ovcar-3 (human ovarian carcinoma cell line), T47D (human ductal breast epithelial tumor cell line), Jurkat-JR16 (Jurkat cells overexpressing Bcl2) and caspase-8 deficient Jurkat cells were used for the experiments. The Jurkat cell lines were grown in RPMI-1640 medium. HepG2, HeLa, Ovcar-3 and T47D cells were cultured in DMEM. All culture media were supplemented with 10% fetal bovine serum (FBS), 100 iu ml-1 penicillin and 100 μg ml-1 streptomycin. Cell lines were grown in a humidified 5% CO2 environment at 37°C and were passaged every 3–4 days. Stock solutions of paclitaxel, baccatin III, fungal taxol and fungal baccatin III dissolved in DMSO were stored at -80°C. Stocks were diluted in culture medium at the required concentration at the time of treatment.
Analysis and quantification of apoptosis
Analysis of hypodiploid cells were performed using Propidium Iodide (PI) staining . Flow cytometric analysis (FACScan) of the cell lines was performed after treatment with taxol and baccatin III. Cells (0.5 × 106) treated with different concentrations of standards or fungal taxol and baccatin III in 500 μl of medium for various time intervals were harvested and washed once with 0.2% BSA containing PBS (50 mM phosphate buffer, pH 7.2 containing 0.85% NaCl) and fixed in 70% ethanol for 1 h at -20°C. The cells were then centrifuged at 1000 × g and suspended in staining solution containing 50 μg/ml PI, 50 μg/ml RNase A and 100 μM EDTA in PBS for 1 h at 42°C. Analysis was carried out using a flow cytometer. Cell cycle distribution is presented as the number of cells versus the amount of DNA, and the extent of apoptosis was determined by counting cells of DNA content within the subG1 peak.
Effect of caspases on fungal taxol and baccatin III induced apoptosis
In order to find out the involvement of caspases in the fungal taxol and baccatin III induced apoptotic pathway, caspase inhibitors were employed. Jurkat cells (0.25 × 106) in 250 μl of RPMI supplemented with 10% FBS were first pretreated with 25, 50 and 100 μM of cell permeable Z-VAD-FMK (inhibitor of all caspases) or Z-LEHD-FMK (caspase 9 inhibitor) or Z-DEVD-FMK (caspase 3 inhibitor) or Z-AEVD-FMK (caspase-10 inhibitor) or Z-VDVAD-FMK (caspase-2 inhibitor) for 1 h. The cells were then cultured for 24 and 48 h with 6 nM of fungal taxol (TFUNG) or 3.5 μM of fungal baccatin III (BFUNG). The cells were processed for PI staining and subjected to FACScan analysis as described above.
Determination of the mitochondrial membrane potential (JC-1 Assay)
The change in mitochondrial membrane potential or MMP (ΔΨm) was measured using the potentiometric dye JC-1 as described earlier . The assay was carried out in 24-well plates. Cells were treated with fungal taxol (6 nM) or fungal baccatin III (3.5 μM) for 6, 12, 24 and 36 h. The cells were then incubated with 2.5 μg ml-1 of JC-1 dye for 15 min at 37°C, washed once with ice-cold PBS containing 2% (v/v) FBS, resuspended in the same and analyzed immediately by flow cytometry. JC-1 monomers emit at 530 nm (FL-1 channel- green fluorescence) and J-aggregates emit at 590 nm (FL-2 channel- red fluorescence). 2, 4-Dinitrophenol (2,4-DNP) is used as the positive control to set the gates along with the untreated cells as the negative control. The percentage of MMP (MFI590nm/MFI525nm) was plotted against time upon fungal taxol or baccatin III treatment. Data analysis was carried out using CellQuest Pro software.
Determination of nuclear morphology
The changes in chromatin organization upon treatment with fungal taxol or baccatin III was determined microscopically by staining either with Hoechst 33258 or acridine orange-ethidium bromide (AO-EB) dual stain . After overnight adherence on cover slips (in case of HeLa cells), the cells were incubated with fungal taxol (0.1 μM for both JR4-Jurkat and HeLa cells) or baccatin III (3.5 μM for JR4-Jurkat and 3 μM for HeLa cells) for 12 h. The cells were then fixed with 3.7% (v/v) paraformaldehyde, permeabilized with 0.1% Triton X-100 and stained with Hoechst 33258 (1 mg ml-1 in PBS) . After washing twice with PBS, cells were examined by fluorescence microscopy (360/40 nm excitation and 460/50 nm emission filters). The apoptotic cells were identified by the presence of highly condensed chromatin or fragmented nuclei. For AO/EB staining , after treatment with indicated concentrations of taxol or baccatin III for 12 h, the cells were incubated with 3 μl of RNase A (10 mg ml-1) at 37°C for 30 min. After washing twice with PBS, the cells were fixed with 3.7% (v/v) paraformaldehyde for 10 min at room temperature. Then the cells were stained with an AO/EB mixture for 15 min and washed with PBS, the cells were observed under fluorescence microscope at 10× magnification using 485 nm excitation and 535 nm emission filter sets.
DNA fragmentation analysis
DNA fragmentation was studied as described earlier . Jurkat cells (3 × 106) were treated with fungal taxol (6 nM) or baccatin III (3.5 μM), whereas HeLa cells, after overnight adherence were treated with fungal taxol (6 nM) or baccatin III (3 μM), for 36 h. After treatment, the cells were harvested and washed with 1 ml of PBS, resuspended in 100 μl of PBS and fixed in 70% chilled ethanol overnight. The cells were spun down at 1000 × g and resuspended in 40 μl of phosphate-citrate (PC) buffer consisting of 192 parts of 0.2 M Na2HPO4 and 8 parts of 0.1 M of citric acid (pH 7.8), at RT for 30 min. After centrifugation at 1000 × g at RT for 5 min, the supernatant was transferred to fresh tubes and concentrated by vacuum in SpeedVac concentrator (Savant Instruments Inc., Farmingdale, NY). 3 μl of 0.25% Nonidet-40 (Sigma Chemicals Co., USA) in distilled water was then added to the tubes, followed by 3 μl of a solution of RNase A (Sigma Aldrich Co., USA). After incubation for 30 min at 37°C, 3 μl of a proteinase K (Sigma Aldrich Co.,USA) was added and incubated for additional 30 min at 37°C. Gel loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol) was the added and the entire content of the tube was transferred to 1.2% agarose gel and electrophoresed at 2 V/cm for 16 h. The DNA present in the gels was visualized under UV light after staining with ethidium bromide (5 μg ml-1).
Statistical analysis was performed using GraphPad Prism software 5.0 (GraphPad Software, La Jolla, CA, USA). Student’s t-test was used to analyze the data. Values of p <0.05 or less were considered statistically significant.