Figure 1. Simultaneous inhibition of the PI3K/mTOR and MEK/ERK pathways fails to synergistically reduce NHL cell proliferation in vitro.

(a) Proliferation of NHL cell lines treated with drug(s) or vehicle were evaluated by MTS Assay using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer’s instructions. Cells (1 X 10⁵/mL) were incubated with drug(s) or vehicle for 72 hours in a 12-well plate. Twenty microliters of MTS reagent was added to 100µL of cell suspension in a 96-well plate and incubated at 37°C for 1 hour. Percent of viable cells after 72 hours treatment is shown on the y-axis. The percent of viable cells for untreated cells was set as 100% for each sample. Elevated absorbance values are indicative of metabolically active cells. The COLO-205 cells (5 X 10³ cells/100µL) were added to each well of a 96-well plate. Increasing concentrations of drug(s) or vehicle were added the following day. The MTS reagent was added after 72 hours following the addition of the drug(s) or vehicle, and incubated with the cells for 4 hours at 37°C. Absorbance was measured at 490 nm using a FLUOstar OPTIMA spectrophotometer. Percent viable cells after 72 hours treatment is shown on the y-axis. The percent of viable cells for vehicle treated was set as 100% for each sample. Elevated absorbance values are indicative of metabolically active cells.(b) Phosphorylation of ERK 1/2 and S6 kinase were determined by Western blot of NHL cell lines treated with AZD6244 (1µM) and/or NVP-BEZ235 (30 nM) for 24 hours. Equal volume of DMSO (0.1%) was added for vehicle control. (c) Proliferation of cells with 25 µM CAL-101 and increasing concentrations of AZD6244 was determined by MTS assay after 48 hours following the addition of the drug(s) or vehicle as described in panel a. (d) Phosphorylation of Akt, ERK 1/2 and S6 kinase were determined by Western blot of NHL cell lines treated with AZD6244 (1µM) and/or CAL-101 (25µM) for 24 hours.