Table 1.
Cytotoxicity against NCI-H1975 cancer cell line and EGFR inhibitory activity of Gefitinib and its analogues.
Figure 1.
Effects of V1801 on NSCLC cells.
(a, b) The cells were treated with gefitinib or V1801 for 48 h, and analyzed by MTT assay. (c, d) The cells were treated with gefitinib or V1801 for indicated time points and assessed by trypan blue exclusion analysis. (e) Soft-agar colony formation assay for NCI-H1975 cells treated with or without V1801 or gefitinib. (f) Quantitation of foci counting. The mean+SD of three independent experiments is shown. (g, h) NCI-H1975 (g) and HCC827 (h) cells were treated with V1801 or gefitinib for 12 h, lysed, and Western blot assay was performed using indicated antibodies.
Figure 2.
V1801 induces apoptosis of NSCLC cells.
(a) NCI-H1975 cells were treated with gefitinib or V1801 for 24 h, and evaluated by Heochst33258 assay. (b) The cells were treated with gefitinib or V1801 for 24 h, and apoptotic cell death was analyzed by Annexin V/PI staining and flow cytometry. (c) NCI-H1975 cells were treated with V1801 or gefitinib (3 µM), lysed, and Western blot assay was performed using indicated antibodies. (d) A549 cells were treated with V1801 or gefitinib (3 µM) for 24 h, lysed, and Western blot assay was performed using indicated antibodies. (e) NCI-H1975 cells were pre-treated with z-VAD-fmk (20 µM) for 1 h followed by treatment with V1801 at 3 µM for 12 h, and the apoptotic cells were determined by Annexin V/PI staining and flow cytometry analysis. The mean+SD of three independent experiments is shown. **, P<0.01.
Figure 3.
Noxa plays a critical role in V1801-triggered apoptosis of gefitinib-resistant NSCLC cells.
(a) RT-PCR analysis of the expression of Noxa, Puma, Bim, Bcl-xl, and Mcl-1 at mRNA level in NCI-H1975 cells treated with gefitinib (3 µM) or V1801 (3 µM) for indicated time points. (b) NCI-H1975 cells were treated with V1801 at indicated concentration for 24 h, lysed, and analyzed by Western blot analysis using an anti-Noxa antibody. (c) NCI-H1975 cells were treated with V1801 at 3 µM for indicated time points, lysed, and analyzed by Western blot analysis using an anti-Noxa antibody. (d) Indicated cells were treated with V1801 or gefitinib for 24 h, lysed, and analyzed by Western blot analysis. (e) NCI-H1975 cells transfected with control or Noxa specific siRNA were treated with or without V1801 (3 µM) for 24 h, lysed, and Western blot analysis was performed. (f) Cells were transfected with siRNA and treated with V1801 as described in (e), then analyzed by Annexin V/PI staining and flow cytometry. (g) NCI-H1975 cells were treated with gefitinib or V1801, lysed, and immunoprecipitation/Western blot assays were performed using indicated antibodies. (h, i) NCI-H1975 cells were treated with V1801 or gefitinib for 24 h, lysed, cytosol and mitochondrial fractions were isolated by centrifugation, and Western blotting was conducted using indicated antibodies (h). The expression of cytochrome C was quantified by densitometry analysis and normalized against GAPDH or Hsp75 (i).
Figure 4.
c-Myc's role in V1801-induced Noxa up-regulation.
(a) NCI-H1975 cells were treated with gefitinib (3 µM) or V1801 (3 µM) for indicated time points, lysed, and Western blot assay was conducted using indicated antibodies. (b) Western blot analysis using lysates of NCI-H1975 cells transfected with control or c-Myc specific siRNA and anti-c-Myc antibody. (c) NCI-H1975 cells transfected with c-Myc specific siRNA were treated with or without V1801, lysed, and Western blotting was conducted using indicated antibodies. (d) NCI-H1975 cells transfected with c-Myc specific siRNA were treated with or without V1801, and evaluated by Anexin V/PI staining and flow cytometry. (e) NCI-H1975 cells were treated with indicated protocols for 24 h, lysed, and Western blot analysis was performed. (f) NCI-H1975 cells were treated with indicated protocols for 24 h, and assessed by MTT assay. *, p<0.05; **, p<0.01.
Figure 5.
V1801 interferes with Erk signal in NCI-H1975 cells.
(a) NCI-H1975 cells were treated with gefitinib (3 µM) or V1801 (3 µM) for indicated time points, lysed, and Western blot analysis was performed. (b) NCI-H1975 cells were treated with indicated protocols for 12 or 24 h, lysed, and Western blot analysis was performed. (c) NCI-H1975 cells were treated with indicated protocols for 24 h, and apoptotic cell death was evaluated by Annexin V/PI staining and flow cytometry. (d) NCI-H1975 cells were treated for 24 h with BOR and/or V1801, and then assessed by MTT assay. (e) NCI-H1975 cells were treated with V1801 (2 µM), gefitinib (2 µM), BOR (10 nM), or their combinations for indicated time points. The cells were then lysed and subjected to Western blotting using indicated antibodies.
Figure 6.
In vivo therapeutic efficacy of V1801 on mice bearing gefitinib-resistant NSCLC cells.
(a) Nude mice inoculated subcutaneously with NCI-H1975 cells were treated with gefitinib or V1801 for 3 weeks, and caliper measurements of the longest perpendicular tumor diameters were performed every two days to estimate the tumor volume. (b) Images of mice two weeks after initiation of the treatment. (c) Images of xenograft tumors obtained from mice. Eight representative tumors for each treatment group are shown. (d, e) Western blot analysis of lysates of tumor samples using indicated antibodies (d). Noxa expression was quantified by densitometry analysis and normalized against Actin expression (e).