Fig. 2

Combination of curcumin and gefitinib downregulates EGFR through inhibiting SP1 and HDAC1 binding induced-EGFR transcription activity. a H157 and H1299 cells were treated with gefitinib, or curcumin alone, or the two combination at indicated concentrations for 48 and evaluated for the protein expression as indicated by immunoblot analysis. b Curcumin-induced suppression of HDCA1 and HDCA2 proteins in H157 and H1299 cells. Protein expression was determined as outlined above in A. c Expressions of Sp1 and other indicated proteins in control and Sp1 or Sp3 knocked down cells were determined by immunoblotting in H157 and H1299 cells. d H157 cells were transfected with Sp1 plasmids (PCMV6-Sp1) and control plasmid DNA (PCMV6). After 8 h, cells were treated with 5 μM gefitinib, or 10 μM curcumin alone, or two drug combination for an additional 48 h. And then the expressions of Sp1, HDAC1, EGFR, Survivin and c-Met proteins were determined by immunoblotting. Similar results were obtained from three independent experiments. Typical immunoblots were presented in the Figure. e H157 cells were pre-incubated with curcumin and nuclear extracts were prepared. The immunoprecipitation was performed using anti-Sp1 and anti-HDAC1 antibodies. The immunoprecipitated pellets were analyzed by immunoblotting with anti-acetyl-lysine and anti-Sp1 antibodies. f H157 cells treated with DMSO or curcumin at indicated concentrations were stained for HDAC1 (green) and Sp1 (red) using immunofluorescence assay. g Effect of Sp1 on EGFR promoter activity. Cells were treated with curcumin (10 μM) plus gefitinib (5 μM), or transfected with siSp1 or plasmids containing Sp1 (PCMV6-Sp1), and luciferase activity was determined as described in Materials and Methods. (**P<0.01 compared with gefitinib plus curcumin, or siSp1, or gefitinib plus curcumin in combination with pCMV6-Sp1, respectively)