Figure 1

Effect of PHA665752 treatment on cell proliferation and apoptosis in PTC cell lines. (A) PHA665752 inhibits the proliferation of PTC cells. B-CPAP and TPC-1 cells were incubated with 0, 1, 2.5, 5, 10 and 25 µmol/L PHA665752 for 24 h. Cell proliferation assays were performed by using MTT as described in Materials and Methods. Columns, mean of three independent experiments with replicates of six wells for all the doses and vehicle control for each experiment; bars, SD. *0.001 > P < 0.05; **P < 0.001, statistically significant. (B) PHA665752 causes cell-cycle arrest in B-CPAP cells. B-CPAP cells were treated with 5 and 10 µmol/L PHA665752 for 24 h. Thereafter, the cells were washed and stained with propidium iodide and analyzed for DNA content by flow cytometry. (C) PHA665752-induced apoptosis detected by Annexin V/propidium iodide dual staining. B-CPAP and TPC-1 cells were treated with various doses of PHA665752 (as indicated) for 24 h and cells were subsequently stained with fluorescein-conjugated Annexin V and propidium iodide and analyzed by flow cytometry. Columns, mean of three independent experiments; bars, SD. *0.05 < P < 0.01; **P < 0.001. (D) Caspase 9 and 3 activation and PARP cleavage following PHA665752 treatment. B-CPAP and PTC-1 cells were treated with and without 5 and 10 µmol/L PHA665752 for 24 h. Cytoplasmic extracts were prepared. Then 10 µg protein from each sample was separated on SDS-PAGE and transferred to PVDF membrane and immunoblotted with antibodies against caspase 9, caspase 3, cleaved caspase 3 and PARP. The blots were probed with an antibody against β-actin for equal loading. (E) Effect of zVAD/fmk on PHA665752-induced apoptosis detected by Annexin V/propidium iodide dual staining. B-CPAP cells were pretreated with 80 µmol/L zVAD/fmk for 2 h and subsequently treated with 10 µmol/L PHA665752 for 24 h, and then cells were stained with fluorescein-conjugated Annexin V and propidium iodide and analyzed by flow cytometry. Columns, mean of three independent experiments; bars, SD. *P < 0.01. (F) Effect of zVAD/fmk on PHA665752-induced activation of caspase 3 and cleavage of PARP.B-CPAP cells were pretreated with 80 µmol/L zVAD/fmk for 2 h and subsequently treated with 10 µmol/L PHA665752 for 24 h. Then cells were lysed and equal amounts of proteins were separated on SDS-PAGE and transferred to PVDF membrane, and immunoblotted with antibodies against caspase 3, PARP and β-actin.