Journal: Nature Communications
Article Title: Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance
Figure Lengend Snippet: Genetical inhibition of Wnt/β-catenin signalling augments the cytotoxic effects of temozolomide. ( a , b ) Inhibition of β-catenin expression by shRNA or siRNA sensitizes LS174T ( a ) and SW480 ( b ) colon carcinoma cells to temozolomide. LS174T cells were grown in medium containing ±1 mg ml −1 doxycycline (doxy) and treated with increasing concentrations of temozolomide (TMZ) twice (at 0 and 48 h) for totally 96 h. Doxy-induced shRNA knockdown of β-catenin expression significantly augment the cytotoxic effect of TMZ ( t -test on log IC 50 , P =0.0003). Overexpression of MGMT reversed the cytotoxic effect of TMZ caused by β-catenin knockdown ( t -test on log IC 50 , P =0.0491). Each concentration was tested in triplicate and the experiments were repeated twice. Values are mean±s.e.m. and presented as the percentage of matched control cells. Only depletion of β-catenin inhibited cell growth to 46±5.8% (mean±s.e.m.) of untreated control. ( b ) SW480 cells were transiently transfected with siRNA against β-catenin and subsequently treated with 100 μM TMZ for 48 h. Significant growth inhibition was observed in cells treated with a combination of siRNA against β-catenin and TMZ compared with only TMZ ( t -test, P =0.002). The experiment was repeated twice. Values are mean±s.e.m. ( c ) Clonogenic capacity of LS174T cells ±1 μg ml −1 doxy to induce shRNA against β-catenin and treated with 50 μM TMZ, as a single treatment or repeated treatment. The TMZ treatment was significantly more efficient in the shRNA-induced LS174T cells ( t -test, P =0.032). Each experimental point was performed in triplicate. The experiment was repeated with similar results. Mean±s.d. are displayed. ( d ) β-catenin knockdown significantly increases TMZ-induced apoptosis in LS174T cells. LS174T cells were incubated with ±1 μg ml −1 doxy to induce shRNA against β-catenin and treated with 50 μM TMZ for 48 h ( t -test, P =0.036). Apoptosis was analysed by flow cytometry measurement of cells in sub-G0 phase. The experiment was repeated three times. Values are mean±s.d.
Article Snippet: Chemicals Celecoxib (Pfizer, Täby, Sweden), dimethyl-PGE2 (dmPGE2 ), cyclophosphamide (given as the active metabolite 4-hydroxycyclophosphamide), temozolomide, doxycycline, XAV-939, salinomycin (all from Sigma-Aldrich, Solna, Sweden), O6-BG, Wnt-C59 and LGK974 (Cayman Chemical, Ann Arbor, MI, USA) were dissolved in dimethyl sulfoxide (Sigma-Aldrich).
Techniques: Inhibition, Expressing, shRNA, Over Expression, Concentration Assay, Transfection, Incubation, Flow Cytometry, Cytometry