Pei Yin Ho

In vitro and in vivo studies of the anticancer action of terbinafine in human cancer cell lines: G0/G1 p53‐associated cell cycle arrest

Terbinafine (TB) (Lamisil®), a promising oral antifungal agent used worldwide, has been used in the treatment of superficial mycosis. In our study, we demonstrated that TB dose‐dependently decreased cell number in various cultured human malignant cells. Flow cytometry analysis revealed that TB interrupts the cell cycle at the G0/G1 transition. The TB‐induced cell cycle arrest in colon cancer cell line (COLO 205) occurred when the cyclin‐dependent kinase (cdk) system was inhibited just as the levels of p53, p21/Cip1 and p27/Kip1 proteins were augmented. In the TB‐treated COLO 205, the binding between p53 protein and p53 consensus binding site in p21/Cip1 promoter DNA probe was increased. Pretreatment of COLO 205 with p53‐specific antisense oligodeoxynucleotide decreased the TB‐induced elevations of p53 and p21/Cip1 proteins, which in turn led to arrest in the cell cycle at the G0/G1 phase. Moreover, in the p53 null cells, HL60, TB treatment did not induce cell cycle arrest. Taken together, these results suggest an involvement of the p53‐associated signaling pathway in the TB‐induced antiproliferation in COLO 205. We further examined whether administration of TB could affect the growth of tumors derived from human colon cancer cells in an in vivo setting. COLO 205 cells implanted subcutaneously in nude mice formed solid tumor; subsequent intraperitoneal injections of TB (50 mg/kg) led to obvious decline in tumor size, up to 50–60%. In these tumors, increases in the p21/Cip1, p27/Kip1 and p53 proteins and the occurrence of apoptosis were observed. Combined treatment with TB and nocodazole (ND), a clinically used anticancer agent, potentiated the apoptotic effect in COLO 205. These findings demonstrate for the first time that TB can inhibit the proliferation of tumor cells in vitro and in vivo.

Inhibition of human vascular endothelial cells proliferation by terbinafine

We have demonstrated previously that terbinafine (TB), an oral antifungal agent used in the treatment of superficial mycosis, suppresses proliferation of various cultured human cancer cells in vitro and in vivo by inhibiting DNA synthesis and activating apoptosis. In our study, we further demonstrated that TB at a range of concentrations (0–120 μM) dose‐dependently decreased cell number in cultured human umbilical vascular endothelial cells (HUVEC). Terbinafine was not cytotoxic at a concentration of 120 μM, indicating that it may have an inhibitory effect on the cell proliferation in HUVEC. The TB‐induced inhibition of cell growth rate is reversible. [3H]thymidine incorporation revealed that TB reduced the [3H]thymidine incorporation into HUVEC during the S‐phase of the cell‐cycle. Western blot analysis demonstrated that the protein levels of cyclin A, but not cyclins B, D1, D3, E, CDK2 and CDK4, decreased after TB treatment. The TB‐induced cell‐cycle arrest in HUVEC occurred when the cyclin‐dependent kinase 2 (CDK2) activity was inhibited just as the protein level of p21 was increased and cyclin A was decreased. Pretreatment of HUVEC with a p21 specific antisense oligonucleotide reversed the TB‐induced inhibition of [3H]thymidine incorporation. Taken together, these results suggest an involvement of the p21‐associated signaling pathway in the TB‐induced antiproliferation in HUVEC. Capillary‐like tube formation and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti‐angiogenic effect of TB. These findings demonstrate for the first time that TB can inhibit the angiogenesis.

Lovastatin inhibits proliferation of anaplastic thyroid cancer cells through up-regulation of p27 by interfering with the Rho/ROCK-mediated pathway

Previously, we demonstrated that lovastatin, a HMG-CoA reductase inhibitor, induced apoptosis, differentiation, and inhibition of invasiveness of human anaplastic thyroid carcinoma cells (ATCs). Here, we further examined the effect of lovastatin on the growth of ARO cells. Lovastatin (0-20μM) concentration-dependently decreased cell number in cultured ATC and arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that lovastatin caused an increase of the protein level of p27 and cyclin-dependent kinase (CDK)4 and a decrease of the protein level of cyclin A2, cyclin D3, and phosphorylated Rb (pRb), but did not significantly change the protein levels of p21, cyclins D1 and E, and CDK2, in ARO cells. The formation of the CDK2-p27 complex was increased and the CDK2 activity was decreased in the lovastatin-treated ARO cells. Pretreatment of ARO cells with a p27, but not p21, antisense oligonucleotide prevented the lovastatin-induced G0/G1 arrest in ARO cells. The lovastatin-induced growth inhibition and translocation of RhoA and Rac1 in ARO cells were completely prevented by mevalonate and partially by geranylgeranyl pyrophosphate. Treatment of ARO cells with Y27632, an inhibitor of Rho-associated kinase, abolished the GGPP-mediated prevention of lovastatin-induced anti-proliferation and up-regulation and prolonged degradation of p27. Taken together, these data suggest that lovastatin treatment caused a reduction of Rho geranylgeranylation, which in turn increased the expression and stability of p27, and then inhibited ARO cell proliferation. These data suggest that lovastatin merits further investigation as multipotent therapy for treatment ATC.

Progesterone inhibits human endothelial cell proliferation through a p53-dependent pathway

Abstract.Previous studies have shown that progesterone inhibits endothelial cell proliferation through a nuclear receptor-mediated mechanism. Here, we further demonstrate that progesterone at physiologic levels (5 – 500 nM) dose- and time-dependently inhibited DNA synthesis of cultured human umbilical vein endothelial cells (HUVEC). The mRNA and protein levels of p21, p27, and p53 in HUVEC were increased by progesterone. The formation of CDK2-p21 and CDK2-p27 were increased and the CDK2 activity was decreased in the progesterone-treated HUVEC. The progesterone-inhibited [3H]thymidine incorporation was completely blocked when the expressions of p21 and p27 were knocked-down together. Transfection of HUVEC with dominant negative p53 cDNA prevented the progesterone-induced increases in p21 and p27 promoter activity and protein level, decreases in thymidine incorporation, and capillary-like tube formation. Matrigel angiogenesis assay in mice demonstrated the antiangiogenic effect of progesterone in vivo. These findings demonstrate for the first time that progesterone inhibited endothelial cell proliferation through a p53-dependent pathway.

Terbinafine inhibits endothelial cell migration through suppression of the Rho-mediated pathway

We showed previously that terbinafine, an allylamine with fungicidal activity, could inhibit angiogenesis by suppressing the endothelial cell proliferation. In the present study, we further showed that terbinafine (0–120 μmol/L) dose dependently inhibited the adhesion and migration of human umbilical vascular endothelial cells (HUVEC). Western blot analysis showed that terbinafine decreased the levels of Ras protein and membrane-bound RhoA protein. Moreover, the terbinafine-induced migration inhibition in HUVEC was prevented by pretreatment with farnesol or geranylgeraniol. Pretreatment of HUVEC with Ras inhibitor peptide or a ROCK (a kinase associated with RhoA for transducing RhoA signaling) inhibitor, Y27632, abolished the farnesol- or geranylgeraniol-induced prevention effect on the terbinafine-induced migration inhibition, respectively. These data suggest that the consuming or depletion of geranylgeranyl pyrophosphate and consequent suppression of protein geranylgeranylation and farnesylation, which is essential for activation of Rho GTPases and Ras, respectively, might account for the terbinafine-induced inhibition of HUVEC migration. The levels of phosphorylated focal adhesion kinase and paxillin protein and the mRNA levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 were also decreased by terbinafine treatment. Taken together, these results indicate that suppression of Rho-mediated pathway might be involved in the signal transduction leading to the inhibition of cell migration caused by terbinafine in HUVEC. [Mol Cancer Ther 2006;5(12):3130–8]

Anti-angiogenic action of 5,5-diphenyl-2-thiohydantoin-N10 (DPTH-N10).

Previously, we demonstrated that 5,5-diphenyl-2-thiohydantoin (DPTH) exerts an anti-proliferation effect on subcultured human umbilical vein endothelial cells (HUVEC). In the present study, we show that 2(naphthalen-2-ylmethylsulfanyl)-5,5-diphenyl-1,5-dihydro-imidazol-4-one (DPTH-N10), a derivative compound of DPTH, exerts a 5 times stronger inhibition of [3H]thymidine incorporation into HUVEC as compared with DPTH and at very low concentrations (0-20 microM) inhibited DNA synthesis and decreased cell number in cultured HUVEC in a concentration- and time-dependent manner, but not in human fibroblasts. [3H]thymidine incorporation analysis demonstrated that treatment of HUVEC with DPTH-N10 arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that the protein level of p21 in HUVEC increased after DPTH-N10 treatment. In contrast, the protein levels of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase (CDK)2, and CDK4 in HUVEC were not changed significantly after DPTH-N10 treatment. Immunoprecipitation showed that the formation of the CDK2-p21 complex, but not the CDK2-p27, CDK4-p21, and CDK4-p27 complex, was increased in the DPTH-N10-treated HUVEC. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in the DPTH-N10-treated HUVEC. Pretreatment of HUVEC with a p21, but not p27, antisense oligonucleotide reversed the DPTH-N10-induced inhibition of [3H]thymidine incorporation into HUVEC. Taken together, these data suggest that DPTH-N10 inhibits HUVEC proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally interrupts the cell cycle. Capillary-like tube formation, aortic ring culture, and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti-angiogenic effect of DPTH-N10.

Inhibition of the ERK phosphorylation plays a role in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells.

Previously, we showed that terbinafine (TB) induces cell-cycle arrest in cultured human umbilical vein endothelial cells (HUVEC) through an up-regulation of the p21 protein. The aim of this study is to delineate the molecular mechanisms underlying TB-induced increase of p21 protein. RT-PCR analysis demonstrated that the mRNA levels of p21 and p53 were increased in the TB-treated HUVEC. The p21 promoter activity was also increased by TB treatment. Transfection of HUVEC with p53 dominant negative (DN) abolished the TB-induced increases of p21 promoter activity and protein level, suggesting that the TB-induced increase of p21 is p53-dependent. Western blot analysis demonstrated that TB decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK). Over-expression of mitogen-activated protein kinase (MEK)-1, the immediate upstream activator kinase of ERK, abolished the TB-induced increases of p21 and p53 protein and decrease of thymidine incorporation. The ERK inhibitor (PD98059) enhanced the TB-induced inhibition of thymidine incorporation into HUVEC. Taken together, these data suggest that the decrease of ERK activity plays a role in the TB-induced up-regulation of p21 in HUVEC. On the other hand, pretreatment of the cells with geranylgeraniol (GGOH), farnesol (FOH), or Ras inhibitor peptide did not affect the TB-induced decrease of thymidine incorporation. Taken together, our results suggest that TB might cause a decrease of MEK, which in turn up-regulates p53 through the inhibition of ERK phosphorylation, and finally causes an increase of p21 expression and cell-cycle arrest.

Involvement of the JNK Activation in Terbinafine-Induced p21 Up-Regulation and DNA Synthesis Inhibition in Human Vascular Endothelial Cells

Previously, we demonstrated that the extracellular signal‐regulated kinase (ERK)‐mediated pathway contributes to the terbinafine (TB)‐induced increases of p21 and p53 protein level as well as decrease of DNA synthesis in human umbilical venous endothelial cells (HUVEC). The aim of this study is to examine the involvement of c‐Jun NH2‐terminal kinase (JNK) in the TB‐induced increase of p21 protein level and DNA synthesis inhibition. Western blot analysis and kinase assay demonstrated that TB treatment increased both the protein level and the kinase activity of JNK1/2 in HUVEC. Transfection of HUVEC with JNK1 dominant negative (DN‐JNK1) prevented the TB‐induced increases of p21 and p53 protein level and decrease of DNA synthesis, suggesting that JNK1/2 activation is involved in the TB‐induced cell cycle arrest in HUVEC. Moreover, over‐expression of mitogen‐activated protein kinase (MEK)‐1 prevented the TB‐induced increase of JNK1/2 protein levels, suggesting that MEK‐1 is an upstream inhibitor of JNK. Transfection of HUVEC with DN‐JNK1 prevented the TB‐induced inhibition of ERK phosphorylation, suggesting that JNK1/2 might serve as a negative regulator of ERK. Taken together, our results suggest that JNK activation is involved in the TB‐induced inhibition of ERK phosphorylation, p53 and p21 up‐regulation and DNA synthesis inhibition in HUVEC. J. Cell. Biochem. 108: 860–866, 2009.