Cytotoxicity

The effects of metformin, GaM, and Didox on cell proliferation in vitro were measured by MTT assay as previously described [46]. Cells were plated in culture medium in 96-well plates and incubated for 24 h at 37° C in a CO₂ incubator. GaM or metformin alone or in combination were added to wells and the incubation continued for an additional 3–7 days. At specified times, 10 μL MTT (5 mg/ml stock solution) was added to each well and cells were incubated at 37° C for an additional 4 h. Cells were solubilized by the addition of 200 μl of 0.04 N HCl in isopropyl alcohol. Similar experiments were run with combination metformin and Didox. The absorbance of each well was determined spectrophotometrically at 570 nm using EL-X808 ultra-microplate auto reader (Biotech Instruments, Winooski, VT) and the absorbance of the wells containing additives was compared with that of the wells without additives (control). Drug interactions were evaluated for synergy as described by Chou and Talalay [23] using a computer program (Dose-effect analysis with microcomputers by J. Chou and T-C Chou, Biosoft, Cambridge, UK).