Figure 1.
SecinH3 reduces proliferation of lung cancer cells expressing wild-type EGFR.
A, structures of the inhibitors used in this study. B – C, proliferation of A549 (B) and H460 (C) cells was determined by MTT assay in the presence of the indicated inhibitors. ***, p<0.001 relative to DMSO-treated cells.
Figure 2.
SecinH3 and gefitinib are synergistic.
A – B, proliferation of H460 cells in the presence of gefitinib or SecinH3 alone or in their combination was determined by MTT assay (A) or by colony formation assay (B). In A the numbers give the relative value of proliferation with untreated cells set as 1. The proliferation assuming an additive effect of SecinH3 and gefitinib (expected) is compared to the value found in the experiments (observed). Observed values below the expected values indicate synergism.
Figure 3.
Survivin and p27/Kip expression.
A – C, H460 cells were treated overnight with 1 µM gefitinib, 15 µM SecinH3 or their combination. A, the expression of p27Kip and survivin was analyzed by western blot. B, C, Statistical evaluation of the western blot data shown in A. ** in B indicates p<0.01 relative to DMSO-treated controls as well as to gefitinib-treated cells. *** in C indicates p<0.001 relative to DMSO-treated controls. n = 3.
Figure 4.
Effect of gefitinib and SecinH3 on EGFR signaling.
A – D, serum-starved cells were stimulated with EGF for 5 min and probed by western blot for the activation of the indicated proteins. A, representative Western blot. B – D, Statistical evaluation of the phosphorylation of EGFR (B), IRS1 (C), and Akt (D) in EGF-stimulated H460 cells in presence of gefitinib, SecinH3, or a combination of both inhbitors. p indicates the phosphorylated, i.e. activated, form of the protein. The signals were normalized for the loading control Hsc70. The error bars give the standard error. *, p<0.05, **, p<0.01, ***, p<0.001 relative to EGF-treated cells except for those comparisons which are specified by brackets.
Figure 5.
SecinH3 reduces IGF1R-dependent signaling and proliferation.
A, H460 cells were serum-starved and treated overnight with 0.5 µM AG1024, 15 µM SecinH3 or their combination. After stimulation with IGF1 for 5 min the phosphorylation state of the indicated proteins was analyzed by western blot. The graphs summarize 3 experiments, the error bars give the standard error. *, p<0.05, **, p<0.01, ***, p<0.001 relative to IGF1-treated cells except for those comparisons which are specified by brackets. B, proliferation of H460 cells in the presence of the indicated inhibitors was determined by MTT assay. The numbers give the relative value of proliferation with untreated cells set as 1. The proliferation if an additive effect of SecinH3 and gefitinib is assumed (expected) is compared to the value found in the experiments (observed). Observed values below the expected values indicate synergism. ***, p<0.001.
Figure 6.
SecinH3 retards growth of H460 xenografts in vivo.
Mice bearing subcutaneous H460 xenografts were treated with SecinH3 by daily intraperitoneal injections. A, tumor volume was determined each day. On day 9 the experiment had to be stopped because the tumor volume in the control animals exceeded 1 cm3. B, distribution of tumor volumes on day 9. C – D, the degree of apoptosis was analyzed in sections of tumors on day 9. C, the number of fragmented nuclei was determined by TUNEL staining. D, cleaved, i.e. activated, caspase-3 was detected by imunohistochemistry. Representative tumor sections are shown. 5× and 40× indicate the magnification of the objective. For all graphs, means and standard errors are shown (n = 14). *, p<0.05, **, p<0.01, ***, p<0.001.