We previously reported that 3-methylcholanthrene (3MC), an aryl-hydrocarbon receptor (AhR) agonist, inhibits the proliferation of human umbilical vascular endothelial cells (HUVECs; Juan et al., 2006, Eur J Pharmacol 530: 1-8). Herein, pretreatment of HUVECs with p21 or p27 small interfering (si)RNA reduced 3MC-induced elimination of [(3)H]thymidine incorporation, demonstrating their essential roles in the antiproliferation of HUVECs. The molecular mechanisms of p21 and p27 involved in the antiproliferative effects of 3MC were elucidated in this study. 3MC time- and concentration-dependently increased p21 and p27 levels, and decreased the protein level of CDK2 with no apparent alteration of p53. Interestingly, 3MC-mediated p21 and p27 inductions were eliminated by resveratrol, an AhR antagonist, suggesting their AhR dependency, further confirmed by AhR siRNA. Among the relevant pathways, p38MAPK activation sustained the levels of p21 and p27 induced by 3MC, which was eliminated by AhR antagonists and N-acetylcysteine (NAC), an antioxidant. 3MC concentration-dependently enhanced not only the consensus dioxin-responsive element (DRE)-driven luciferase activity, but also the binding activity of the AhR to the putative DRE derived from the p21 and p27 promoters. A deletion of the DRE (-285/-270) in p21 (-2,300/+8) only partially alleviated the 3MC-induced luciferase activity unless NAC was added, suggesting that there may be a DRE-independent mechanism associated with oxidative stress. However, a deletion of the DRE (-660/-645) in p27 (-1,358/-100) almost completely abrogated the activation. Our study demonstrated that both the functional DRE and the phosphorylation of p38MAPK are essential for the induction of p21 and p27, resulting in the antiproliferative action of 3MC in HUVECs.

Glucose transporters (GLUTs) are required for glucose uptake in malignant cells, and they can be used as molecular targets for cancer therapy. An RT-PCR analysis was performed to investigate the mRNA levels of 14 subtypes of GLUTs in human colorectal cancer (COLO 205 and HT-29) and normal (FHC) cells. RT-PCR (n = 27) was used to assess the differences in paired tissue samples (tumor vs normal) isolated from colorectal cancer patients. GLUT2 was detected in all tested cells. The average GLUT2 mRNA level in 12 of 27 (44.4%) cases was 2.4-fold higher in tumor compared to normal tissues (*, p = 0.027). Higher GLUT2 mRNA expression was preferentially detected in advanced-stage tumors (stage 0 vs 3 = 16.38-fold, 95% CI = 9.22-26.54-fold; *, p = 0.029). The apple polyphenol phloretin (Ph) and siRNA methods were used to inhibit GLUT2 protein expression. Ph (0-100 μM, for 24 h) induced COLO 205 cell growth cycle arrest in a p53-dependent manner, which was confirmed by pretreatment of the cells with a p53-specific dominant negative expression vector. Hepatocyte nuclear factor 6 (HNF6), which was previously reported to be a transcription factor that activates GLUT2 and p53, was also induced by Ph (0-100 μM, for 24 h). The antitumor effect of Ph (25 mg/kg or DMSO twice a week for 6 weeks) was demonstrated in vivo using BALB/c nude mice bearing COLO 205 tumor xenografts. In conclusion, targeting GLUT2 could potentially suppress colorectal tumor cell invasiveness.

Folate is important for normal cell division. Folate deficiency has been implicated in various diseases, including atherosclerosis, neural tube defects, and cancer. However, the effect of folate on angiogenesis was unclear. The aim of this study was to investigate the anti-angiogenic action of folic acid (FA). FA (0-10 μmol/L) concentration-dependently decreased DNA synthesis and proliferation in cultured human umbilical venous endothelial cells (HUVEC). Western blot analyses demonstrated that the levels of p21, p27 and p53 protein in HUVEC were increased by FA. The FA-inhibited [3H]thymidine incorporation was completely blocked when the expressions of p21 and p27 were knocked-down together. Knock-down of p53 prevented the FA-induced increases in p21 and p27 protein level. The levels of phosphorylated Src (p-Src) and p-Src-FA receptor (FR) complex in HUVEC were increased by FA. Knock-down of FR reduced the FA-induced increases of p-Src and p53. The FA-induced increases of p21, p27 and p53 protein levels were abolished when cSrc was knocked-down. FA also increased NF-κB nuclear translocation and binding onto the p53 promoter. The FA-induced up-regulation of the p53 promoter activity was prevented by knocked-down of ERK. Matrigel angiogenesis assay in mice demonstrate the anti-angiogenic effect of FA in vivo. In conclusion, our data indicate that FA bound to FR in HUVEC, subsequently activated the cSrc/ERK 2/NF-κB/p53 signaling pathway, which in turn up-regulated the expression of p21 and p27, and finally resulted in cell cycle arrest at the G0/G1 phase. In the present study, we uncover a completely novel role of FA for anti-angiogenesis.

We previously showed that progesterone (P4) inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) through a p53-dependent pathway. Now we investigated further the molecular mechanism underlying the hormone activity. In cultured HUVECs, P4 increased the protein levels of phosphorylated Src (p-Src), Raf-1, and ERK. The levels of p-Src and p-Src-progesterone receptor complex in HUVECs were increased by P4 treatment. These effects were blocked by pretreatment with a progesterone receptor antagonist, RU486. The P4-induced increase in p53 transactivity was abolished by pretreatment with Src kinase inhibitors. Moreover, administration with cSrc antisense oligonucleotide prevented the P4-induced increases of the levels of p53 mRNA and protein. These data suggest that P4-induced up-regulation of p53 might be mediated through activation of cSrc. Pretreatment with Src kinase inhibitors also prevented P4-induced membrane translocation of Kras and increases of the protein levels of phosphorylated Raf and phosphorylated ERK. Transfection with dominant-negative ERK2 prevented the P4-induced increases of protein level and promoter activity of p53 and a decrease of thymidine incorporation. P4 also increased nuclear factor-κB (NF-κB) nuclear translocation and NF-κB binding onto the p53 promoter. These effects were abolished by pretreatment with ERK inhibitors. The P4-induced up-regulation of the p53 promoter activity was prevented by preadministration with dominant-negative ERK2 or NF-κB inhibitors. Taken together, our data suggest that the cSrc/Kras/Raf-1/ERK2/NF-κB signaling pathway contributes to the P4-induced up-regulation of p53 in HUVECs. These findings highlight progesterone receptor activation of extranuclear signaling pathways in regulating p53 and cell cycle progression in HUVECs.

Previously, we showed that magnolol induces cell-cycle arrest in cultured colon and liver cancer cells through an upregulation of the p21 protein. The aim of this study was to delineate the molecular mechanism underlying this magnolol-induced increase of p21 protein. Thus our RT-PCR analysis demonstrated that the mRNA levels of p21 were increased at 1 h after magnolol treatment and sustained for at least 24 h. The p21 promoter activity was also increased by magnolol treatment. Western blot analysis demonstrated that treatment of COLO-205 cells with magnolol increased the levels of phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment of the cells with PD98059 abolished the magnolol-induced upregulation of p21 protein, suggesting the involvement of an ERK pathway in the magnolol-induced upregulation of p21 in COLO-205 cells. Ras inhibitor peptide abolished the magnolol-induced increase of phosphorylated ERK protein levels, increase of p21 protein, and decrease of thymidine incorporation. Moreover, treatment of COLO-205 with magnolol increased the phosphorylated Raf-1 protein (the Ras target molecule). Pretreatment of the cells with Raf-1 inhibitor reversed the magnolol-induced decrease in thymidine incorporation. Treatment of the cells with CaM kinase inhibitor, but not protein kinase A (PKA) inhibitor or phosphatidylinosital 3-kinase (PI3K) inhibitor, abolished the magnolol-induced activation of ERK and decrease of thymidine incorporation. Taken together, our results suggest that magnolol activates ERK phosphorylation through a Ras/Raf-1-mediated pathway. Subsequently, p21 expression is increased, and finally thymidine incorporation is decreased.