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Human hepatoma Hep3B cell line was purchased from American Type Culture Collection (ATCC) and maintained in our in-house National Cell Repository, National Centre for Cell Science (NCCS), Pune, India. Drug resistant clone was developed from and parental Hep3B cells by treated with gradually increasing concentrations of Paclitaxel in cell culture medium. All of these cells were cultured in DMEM (Invitrogen) and supplemented with 10% FBS and Penicillin/Streptomycin.
Development of resistant clones
Drug resistant clone of Hep3B (DRC) was developed by treating cells with increasing concentration of paclitaxel. Also resistant single cell clone of Hep3B (SCC) was developed by single cell suspension and then screening these for the sensitivity. Interestingly, both clones were less sensitive to paclitaxel in comparison with parental cells.
Cells were harvested and lysed in a buffer containing 50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 10mM NaF, 10mM sodium pyrophosphate, 2 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, 1% NP40 and Protease Inhibitor Cocktail (Roche Diagnostics, Germany) for 30 min on ice. Lysates were cleared by centrifugation at 14,000 rpm at 4°C for 30 min. Supernatants were collected and protein concentrations were determined by the Bradford assay (Bio-rad). The proteins were then separated with a SDS/polyacrylamide gel and transferred to a Nitrocellulose membrane (Millipore). After blocking in PBS with 5% non-fat dry milk for 1 hr, the membranes were incubated 90 min at 4-8°C with the primary antibodies in PBS with 2% non-fat dry milk. The following antibodies were utilized: anti-caveolin rabbit antibody (1:1000, Santacruz Biotechnology); anti-FASN rabbit antibody (1:1000 Santacruz Biotechnology), anti- cytochrome P450 Rabbit antibody (1:1000, Santacruz Biotechnology), anti-Bcl2 rabbit antibody (1:1000, Santacruz Biotechnology), anti-Hsp70 mouse monoclonal antibody (1:1000, Cell Signaling), and anti-β-tubulin monoclonal antibody (1:1000, Santacruz Biotechnology). Membranes were extensively washed with PBS and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody or anti-rabbit antibody (1:2,000, Santacruz Biotechnology). After additional washes with PBS, antigen-antibody complexes were visualized with the enhanced chemiluminescence kit (Cell signaling).
Cell survival MTT assay
Cell proliferation was determined by methylthiazole tetrazolium (MTT) assay. Cells were seeded at the density of 8,000 per well into 96 well plates and allowed to adhere for 24 h. Cells were treated with different concentration of paclitaxel for further 48 h. Fifty microliter of MTT (0.5 mg/ml) was added to each well and incubated for 4 h at 370C. Hundred microliter of 2-propanol was added and incubated in shaking condition at room temperature for 10 min. Absorbance was taken at 570 nm using 630 nm as reference filter. Absorbance given by untreated cells was considered as 100% cell survival.
Efflux of Rhodamine-123
To examine the transport activity of P-glycoprotein, efflux of Rhodamine-123 (Rh-123) was measured as described. Briefly, the cells were harvested and adjusted to 106 /ml cells, then incubated with 2 µM of Rh-123 for 30 min at 37°C. After 3 times of
washing to remove the extracellular free dye, the cells were incubated in dye-free media or media containing 10 µM verapamil (dissolved in DMSO) at 37°C. The efflux of Rh-123 was analyzed at successive time points by flow cytometry using a MOFLO BD Bioscience instrument.
Morphological observation of Taxol-resistant cells
The cells were seeded in 6-well plates at 3 x 105 cells per well in duplicate. After 12 hr incubation, cells were treated with or without 20 nM Taxol for 24 hrs, with untreated cells serving as controls. The cells were washed twice with PBS and then fixed with methanol/acetone (1:1), subsequently stained with 4',6-diamidino-2-phenylindole (DAPI) in order to visualize the morphology of cell nucleus. The morphology of cells was observed with the fluorescence microscope.
Cell apoptosis assay
The cancer cells were treated with 20 nM Taxol for 48 hrs. Two methods were used to detect apoptosis. 1) The early stage of apoptosis was detected by Annexin V/propidium iodide staining with the Apoptosis Detection Kit (BD PharMingen). Briefly, aliquots of 105 Taxol-treated cells were incubated with Annexin V/propidium iodide for 15 min at room temperature. The cells were then analyzed by flow cytometry (BD LSR _). 2) The late stage of apoptosis was detected by Cell Death Detection ELISA PLUS kit (Roche) according to the manufacturer's instruction.
siRNA oligonucleotides for LDH-A was purchased from Sigma, with a scrambled siRNA
(Sigma) used as a control. Transfection was performed using the Oligofectamine
Transfection reagent (Invitrogen) according to the manufacturer's protocol. Forty-eight
hours after transfection, whole-cell lysates were prepared for further analysis by Western
blot, LDH activity and Taxol cytotoxicity assay.
The unpaired Student's t-test was used for the data analysis. All data were shown as
mean ± standard error (SE). A statistical difference of P < 0.05 was considered