Figure 1

Effects of MET and IR on proliferation, clonogenic survival and molecular signals in human LC cells. (A) Time course of MET effects on the pathways of AMPK. Cells were treated with 5 μ M MET doses for 1–72 h, lysed and probed with anti-total and -P-AMPK (T172) and -actin antibodies. Representative immunoblots from three experiments are shown. (B) A549, SK-MES1 and H1299 human LC cells were treated with increasing MET doses (5 μmol–5 mM) for 24 h before treatment with 0, 2 or 8 Gy IR. Cells were fixed 48 h later. Proliferation results (mean±s.e.) of three independent experiments (including six replicates per condition in each experiment) are shown. Statistically significant differences compared with corresponding control cells (not treated with MET) within the 0, 2 and 8 Gy IR treatment groups are shown (*P< 0.05, **P<0.001 for 0 Gy group; ^#P<0.05, ^##P<0.001 for 2 Gy group; ^xP<0.05 and ^xxP<0.001 for 8 Gy group respectively). (C) Metformin and IR reduced clonogenic survival of LC cells. A549 and H1299 LC cells were first treated with increasing doses of MET (0–50 μ M) and subsequently with combination of 0, 5 or 25 μ M MET and 0–8 Gy IR dose. Clonogenic survival was determined as outlined in Materials and Methods. Data were fitted into the linear quadratic model. Average results of three independent experiments are shown (* indicates statistical significance at P<0.05). (D) Comparison of MET and rapamycin effects in combination with IR on A549 human LC cell proliferation. Cells were treated with either 5 μ M, 100 μ M or 5 mM MET or 5, 25 and 500 nM rapamycin for 24 h, followed by treatment with either 0, 2 or 8 Gy of IR and incubation for an additional 24 h. Cells were fixed and proliferation rate was determined as outlined in (A). Results of 3–4 independent experiments (mean±s.e.) are shown. Statistically significant differences of MET or rapamycin treatments from no-drug treatment control in the same radiation dose group are shown: *P<0.05; **P<0.001 for 0 Gy group; ^#P<0.05; ^##P<0.001 for 2 Gy group; ^xP<0.05; ^xxP<0.001 for 8 Gy treatment group.