After 21 days, 1 l of culture was ex tracted twice with an equal volume selleck chem of methylene chloride. The obtained crude extract was subjected to thin layer chromatographic analysis using solvent system, chloroform methanol. After chromatography, the area with silica gel on the plates containing putative taxol and bacca tin III was scraped at the appropriate relative front and exhaustively eluted with methanol and further separated by high performance liquid chromatography using Kromasil C18 column at 227 nm. Authentic taxol and baccatin III standards were used as reference. Cell lines and culture conditions HeLa, HepG2, Jurkat JR4, Ovcar 3, T47D, Jurkat JR16 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 cul ture media were supplemented with 10% fetal bovine serum, 100 iu ml 1 penicillin and 100 ug 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 Propi dium Iodide staining. Flow cytometric analysis of the cell lines was performed after treatment with taxol and baccatin III. Cells treated with different concentrations of standards or fungal taxol and baccatin III in 500 ul of medium for various time intervals were harvested and washed once with 0.

2% BSA containing PBS and fixed in 70% ethanol for 1 h at ?20 C. The cells were then centrifuged at 1000 g and suspended in stain ing solution containing 50 ug/ml PI, 50 ug/ml RNase A and 100 uM 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 Entinostat 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 in hibitors were employed. Jurkat cells in 250 ul of RPMI supplemented with 10% FBS were first pretreated with 25, 50 and 100 uM of cell permeable Z VAD FMK or Z LEHD FMK or Z DEVD FMK or Z AEVD FMK or Z VDVAD FMK for 1 h. The cells were then cultured for 24 and 48 h with 6 nM of fungal taxol or 3. 5 uM of fungal baccatin III. The cells were processed for PI staining and sub jected to FACScan analysis as described above.