br Cell proliferation assays br The effects
2.2. Cell proliferation assays
The effects of MET, SIM, and combination MET+SIM treatments on proliferation were examined using MTS assays. Ishikawa and HEC-1B POM 1 were plated at 3000 cells/well and RL95-2 cells were plated at 5000 cells/well in 96-well plates in their corresponding media for 24 h then treated with 1–12 μM SIM (Sigma-Aldrich, St. Louis, MO), 2–8 mM MET (Medisca, Plattsburgh, NY), or combination MET+SIM for 72 h. Viability was quantified using the formazan dye-based MTS assay (Promega, Madison, WI) according to the manufacturer's specifi-cations. The plates were incubated at 37 °C until the untreated wells ex-hibited an A490 of 0.7–0.9. Wells containing medium alone were used as blanks. Viability was expressed as a percentage of untreated controls on the same 96-well plate. Each experiment was performed in quadrupli-cate and repeated at least twice to assure consistency.
Data were analyzed isobolographically using CompuSyn software (Biosoft) to characterize the inhibitory effect of treatment agents on
the cell lines. Raw data for each agent or combination was entered sin-gly to generate a median effect plot. From this plot, the combination index (CI) equation was applied to determine whether the drug effects were additive, synergistic or antagonistic. CI values of b1, =1 or N1 indi-cated synergy, additivity or antagonism, respectively.
Apoptosis was assessed by deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) in RL95-2, Ishikawa, and HEC-1B cells seeded into 60 mm dishes and incubated overnight as follows: RL95-2 cells were seeded at 1.5 × 106 cells/dish (24 h) and 1.0 × 106 cells/dish (48 h). Ishikawa and HEC-1B were seeded at 4.0 × 105 cells/dish (24 h) and 3.5 × 105 cells/dish (48 h). The culture medium was then re-placed with fresh medium containing 4–8 mM MET with and without 1–8 μM SIM, and incubated for 24 and 48 h. All cells, floating and adher-ent, were collected, washed, fixed, and stained by TUNEL using the APO-Direct kit (BD Pharmingen, San Jose, CA), according to the manufac-turers' protocols. Ten thousand cells were measured by FACS Calibur (BD Biosciences) and the data were analyzed using CellQuest Pro 5.2.1 software. Each sample was gated to include only a singlet population using dual parameters of DNA width (x-axis) and DNA area (y-axis). In assessing apoptosis, the resultant gates were used to generate dual parametric graphs of DNA area (x-axis) and FITC-dUTP (y-axis). Gates were based on increased dUTP-FITC labeling compared to the untreated controls. Results are expressed as fold-increase over controls. Experi-ments were repeated at least three times and/or run in triplicate.
2.4. Annexin V apoptosis assay
Apoptosis was independently assessed using Annexin V in RL95-2 cells seeded and treated for 24 h as described above for TUNEL assay. All cells, floating and adherent, were collected, washed and stained by FITC Annexin V Apoptosis Detection Kit 1 (BD BioSciences), according to the manufacturer's protocols. Ten thousand cells were measured by FACS Calibur and the data were analyzed using CellQuest Pro 5.2.1 soft-ware. Gates were based on increased Annexin-FITC-only labeling com-pared to the untreated controls. Results are expressed as fold-increase over controls. Experiments were repeated at least three times and/or run in triplicate.
2.5. Caspase-3 apoptosis assay
Apoptosis induction was verified following SIM, MET, and MET+SIM treatments using a fluorometric caspase-3 assay kit (BioVision Technol-ogies, Milipitas, CA) or the Caspase-Glo 3/7 assay (Promega Corpora-tion) as per manufacturer's instructions. These kits detect activity of caspases that recognize the sequence DEVD. For the fluorometric assay, cells were plated in 60 mm dishes at concentrations as described above. Following 24 h of incubation, fresh medium was used to treat cells with SIM (1–8 μM), MET (4 mM), or combination MET+SIM for 24–48 h. Cells were then lysed and protein concentrations measured and loaded equally onto a 96-well plate. Reagents were added as di-rected and samples were read using a 400 nm excitation filter and