Abstract
cRNA microarray and real-time PCR (qPCR) studies from our lab identified five Cell Cycle Pathway (CCP) genes (CCNA2, CCNE2, CDC25A, CDKN1B, and PLK-1) as targets for luteolin in PC-3 prostate cancer cells [Shoulars et al., J. Steroid Biochem. Mol. Biol. 118 (2010) 41-50]. In this paper, Ingenuity Pathway Analysis of the microarray data identified 7 luteolin-regulated genes (EGFR, c-Fos, SOS, GRB2, JNK1, MKK4 and RasGAP) in the Epidermal Growth Factor Signaling Pathway (EGFSP) potentially involved in luteolin regulation of CCP genes and cell proliferation. To address these possibilities, we compared the response profiles (RNA and protein) of these EGFSP and CCP genes to luteolin and gefitinib by real-time PCR (qPCR) and Western blot analyses. Luteolin and gefitinib are known antagonists of EGFR-associated tyrosine protein kinase. Thus, the response profiles of EGFR regulated EGFSP or CCP genes should be very similar if genes in both pathways are controlled through this common mechanism of action. Treatment of PC-3 cell with luteolin for 24h caused a 4-fold stimulation of c-Fos gene expression, significant inhibition (p<0.001) of the CCP genes and G2/M arrest. Treatment of PC-3 cells with gefitinib also inhibited most of the CCP genes in a fashion similar to that of luteolin, however, the EGFR antagonist inhibited c-Fos gene expression, stimulated CDKN1B (p27) and arrested the cells in G0/G1. Thus, although the response patterns of most of the CCP genes to luteolin or gefitinib were similar, the effects of the two compounds on EGFSP gene expression and cell cycle arrest were clearly different. Combination studies revealed that the response of EGFSP genes to luteolin was not affected by gefitinib, even though the two compounds were additive with respect to their abilities to inhibit CCNA2, CCNE2, CDC25A and PCNA. These findings suggest that luteolin and gefitinib regulate CCP gene expression through a common mechanism involving EGFR-associated tyrosine kinase. Conversely, luteolin regulates PC-3 cell proliferation through an EGFR-tyrosine kinase independent mechanism(s), likely involving the epigenetic control of gene EGFSP gene expression through histone H4 binding interactions resulting in the upregulation of c-Fos and p21 gene expression.