Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, United States.
J Steroid Biochem Mol Biol. 2010 Oct;122(4):219-31. doi: 10.1016/j.jsbmb.2010.06.006. Epub 2010 Jun 15.
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.
cRNA 微阵列和实时 PCR（qPCR）研究表明，五种细胞周期途径（CCP）基因（CCNA2、CCNE2、CDC25A、CDKN1B 和 PLK-1）是黄体素在 PC-3 前列腺癌细胞中的靶点[Shoulars 等人，J. Steroid Biochem. Mol. Biol. 118（2010）41-50]。在本文中，微阵列数据的 Ingenuity 通路分析鉴定了黄体素调节的 7 个基因（EGFR、c-Fos、SOS、GRB2、JNK1、MKK4 和 RasGAP）在表皮生长因子信号通路（EGFSP）中，这些基因可能参与黄体素调节 CCP 基因和细胞增殖。为了研究这些可能性，我们通过实时 PCR（qPCR）和 Western blot 分析比较了黄体素和吉非替尼对这些 EGFSP 和 CCP 基因的反应谱（RNA 和蛋白质）。黄体素和吉非替尼是 EGFR 相关酪氨酸蛋白激酶的已知拮抗剂。因此，如果两条通路中的基因都通过这种共同的作用机制来控制，那么 EGFR 调节的 EGFSP 或 CCP 基因的反应谱应该非常相似。用黄体素处理 PC-3 细胞 24 小时导致 c-Fos 基因表达增加 4 倍，CCP 基因显著抑制（p<0.001）和 G2/M 期阻滞。用吉非替尼处理 PC-3 细胞也以类似于黄体素的方式抑制大多数 CCP 基因的表达，然而，EGFR 拮抗剂抑制 c-Fos 基因表达，刺激 CDKN1B（p27）并将细胞阻滞在 G0/G1 期。因此，尽管大多数 CCP 基因对黄体素或吉非替尼的反应模式相似，但两种化合物对 EGFSP 基因表达和细胞周期阻滞的影响显然不同。联合研究表明，黄体素对 EGFSP 基因的反应不受吉非替尼的影响，尽管这两种化合物在抑制 CCNA2、CCNE2、CDC25A 和 PCNA 方面具有相加作用。这些发现表明，黄体素和吉非替尼通过涉及 EGFR 相关酪氨酸激酶的共同机制调节 CCP 基因表达。相反，黄体素通过一种不依赖于 EGFR 酪氨酸激酶的机制调节 PC-3 细胞增殖，可能涉及通过组蛋白 H4 结合相互作用对 EGFSP 基因表达的表观遗传调控，导致 c-Fos 和 p21 基因表达上调。
