Figure 1.
Identification of small molecules that simultaneously activate p53 and inhibit NF-κB.
C6 cells derived from rat glioma carrying wild type p53 were used to screen the library. (A) The dose-dependent response of the NF-κB reporter gene with increasing concentrations of parthenolide. (B) Screening of compounds that inhibit the NF-κB reporter gene using the library of 200,000 compounds. Compounds that inhibited 100 ng/ml LPS-induced reporter activity more than 80% were selected as primary hits (n = 797). (C) Screening of the primary hit compounds for activation of the p53 reporter gene. Compounds that activated the p53 reporter more than 3-fold were selected as the final hits (n = 9). (D) Summary of HTS procedures. The total hit ratio was approximately 0.0045%.
Figure 2.
Classification of the bivalent molecules identified by HTS.
Compounds that induced the p53 reporter more than 3-fold and simultaneously inhibited the NF-κB reporter by more than 80% were included in the analyses (n = 9). In addition, 8 less active derivatives of similar structure were included for structure: activity relationship evaluation. Values in parentheses refer to maximum p53-responsive reporter induction in cells treated for 8 h with each compound (0.4–25 µM). Relative NF-κB inhibition is presented as the percentage inhibition of the NF-κB reporter in the presence of 10 µM of each compound. The 17 compounds were grouped according to structural similarity (Groups A–D).
Figure 3.
Effect of N-2 on LPS-stimulated gene expression and serine phosphorylation of NF-κB.
(A) The dose dependence of NF-κB reporter gene luciferase activity was determined in C6 cells with increasing concentrations of 9AA, QC, and N-2 (mean ± SD of three independent experiments). (B) The dose-dependent inhibition of nitric oxide (NO) production was determined with increasing concentrations of 9AA, QC, and N-2 in RAW 264.7 cells. The results shown are the mean ± SD of three experiments. (C) Levels of IL-6 and MCP-1 in RAW 264.7 cells following treatment with parthenolide (Par) or N-2 in the presence of LPS. Each data point represents the mean ± SD of four assays. * p<0.01 by paired Student's t-test. (D) Analysis of Ser536 phosphorylation of the p65 subunit of NF-κB in RAW 264.7 cells following treatment for 1 h with 20 µM of QC or 1 µM of N-2 in the presence of LPS. β-actin was used as a loading control.
Figure 4.
Effect of N-2 on p53 and expression of its target gene p21.
(A) The levels of endogenous p53 and p21 proteins were evaluated following treatment of A549 and B16F10 cells with 1 µM of N-2. (B) Dose dependency of N-2 on endogenous p53 and p21 proteins. (C) Dose dependency of N-2 and QC on endogenous p53 and p21 proteins in HCT116 cells. (D) The p53-responsive reporter activity in HCT116 cells treated for 12 h with 9AA, QC, doxorubicin (Dox) or N-2 over a range of concentrations (0.4–25.0 µM). (E) Activation kinetics of the p53-responsive reporter activity in HCT116 cells treated for 2–40 h with 3 µM of N-2, 3 µM of taxol, 1 µM of Dox or 10 µM of QC. (F) The p53 reporter activity in C6, HCT116, A549, or B16F10 cells treated for 12 h with 9AA, QC, or N-2 over a range of concentrations (0.4–25 µM). The data are shown as the relative fold induction of the p53 reporter gene at the most effective concentration of each compound. The results shown are the average of three experiments; the bars indicate standard deviation (D–F). (G) Phosphorylation of p53 at various serine residues and phosphorylated histone γ-H2AX in A549 cells treated for 12 h with QC (10 µM), 9AA (5 µM), Dox (1 µM) or N-2 (1 µM).
Figure 5.
Effect of N-2 on diverse tumor cell line growth in vitro and in vivo.
(A) Comparison of the IC50 concentrations of 9AA, QC, and N-2 in different wild-type and mutant p53 cell lines. Each point represents the IC50 of a particular type of cell, which are grouped as follows: (i) circles, p53 wild-type cell lines (A549, H460, HCT116, C6, SH-SY5Y, and B16F19); and (ii) triangles, p53 mutant cell lines (H2009, SW480, Jurkat, U937, and LLC). (B) Time course of body weight changes following treatment with N2. Error bars represent the SD. (C) The anti-tumor activity of N-2 and QC on B16F10 allografts. Tumor volume and a representative excised melanoma at day 15 are shown. (D) The anti-tumor activity of N-2 on LLC allografts. The tumor volume, appearance of allografts, and excised LLC tumors at day 14 are shown. Error bars represent the SD (C–D). * indicates significance compared with the control, p<0.05, # p<0.01 with ANOVA. (E) Effect of N-2 anchorage-independent growth in A549 cells. Each data point represents the mean ± SD of three assays. *p<0.05, # p<0.01 by paired Student's t-test.
Figure 6.
Global expression profiling of N-2-responsive genes in A549 cells.
(A) Interaction map of DEGs. Overall network reveals many interesting interactions that may be connected to p53 or NF-κB. The node color represents fold change (green, downregulation; red, upregulation). (B) N-2 up-regulates the mRNA levels of stress response genes (DDIT3, DDIT4, SESN2, and GADD45a) and down-regulates the mRNA level of genes associated with proliferation (PCNA and CCND3). The transcription level of selected genes was confirmed with RT-PCR. * p<0.01, ** p<0.001.