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.

Terbinafine (TB), an oral antifungal agent used in the treatment of superficial mycosis, has been reported to exert an anti-tumor effect in various cancer cells. However, the effect of TB on oral cancer has not been evaluated. Herein we demonstrate that TB (0-60 µM) concentration-dependently decreased cell number in cultured human oral squamous cell carcinoma (OSCC), KB cells. The anti-proliferation effect of TB was also observed in two other OSCC cell lines, SAS and SCC 15. TB (60 µM) was not cytotoxic and its inhibition on KB cell growth was reversible. [(3) H]thymidine incorporation and flow cytometric analyses revealed that TB-inhibited DNA synthesis and induced the G0/G1 cell-cycle arrest. The TB-induced cell-cycle arrest occurred when the cyclin-dependent kinase 2 activity was inhibited just as the protein levels of p21(cip1) and p27(kip1) were increased. The TB-induced G0/G1 cell-cycle arrest was completely blocked when the expressions of p21(cip1) and p27(kip1) were knocked-down together. Taken together, these results suggest that the p21(cip1) - and p27(kip1) -associated signaling pathways might be involved in the TB-induced anti-proliferation in KB cells. In vivo, TB (50 mg/kg, i.p.) significantly inhibited the KB tumor size. In these TB-treated tumors, increases in the levels of p21(cip1) and p27(kip1) protein and decreases in the number of proliferating cell nuclear antigen-positive cells and the microvessel density were observed. These findings demonstrate for the first time that TB might have potential to serve as a therapeutic tool in the treatment of oral cancer.

No abstract

We previously showed that progesterone (P4) could inhibit the proliferation of human umbilical venous endothelial cells (HUVECs) through the p53-dependent pathway. In the present study, we further demonstrated that P4 at physiologic levels (5-500 nM) concentration-dependently inhibited migration of HUVECs. This effect was blocked by pre-treatment with the P4 receptor (PR) agonist-antagonist, RU486, suggesting that the P4-induced migration inhibition in HUVECs was through the PR-mediated signaling pathway. Western blot analyses demonstrated that the levels of RhoA and Rac-1 protein were reduced in the P4-treated HUVECs. P4 also inhibited the membrane translocation of RhoA and Rac-1 protein. Moreover, the P4-induced migration inhibition in HUVECs was prevented by over-expression of the constitutively active RhoA construct (RhoA V14). However, pre-treatment with the ROCK (a kinase associated with RhoA for transducing RhoA signaling) inhibitor, Y27632, abolished the over-expression of RhoA-induced prevention effect on the P4-induced migration inhibition in HUVECs. These data suggest that the inhibition of Rho GTPases might account for the P4-induced migration inhibition of HUVECs. Pre-treatment with the cSrc inhibitor, PP2, prevented the P4-induced migration inhibition in HUVEC. The levels of phosphorylated focal adhesion kinase (FAK) and paxillin protein were also decreased by P4 treatment. Taken together, these results suggest that suppression of the Rho-mediated pathway might be involved in the signal transduction leading to the inhibition of cell migration caused by P4 in HUVECs.