Sung Po Hsu

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.

Folic acid inhibits endothelial cell migration through inhibiting the RhoA activity mediated by activating the folic acid receptor/cSrc/p190RhoGAP- signaling pathway

Previously, our in vivo studies demonstrated that folic acid (FA) could inhibit angiogenesis and in vitro studies showed that FA reduced vascular endothelial cell proliferation through activating the cSrc/ERK-2/NFκB/p53 pathway mediated by FA receptor (FR). Here, we further examined the effect of FA on endothelial cell migration. Our results showed that FA (10 μM) inhibited the formation of lamellipodia, migration and capillary-like tube formation of human umbilical venous endothelial cells (HUVEC). These inhibition effects induced by FA treatment were not due to reduction of cell survival and cell adhesion on the collagen-coated plate. Treatment of HUVEC with FA (10 μM) increased the activity of cSrc and p190RhoGAP and decreased the activity of RhoA. Over-expression of the constitutively active RhoA construct (RhoA V14) prevented the FA-induced inhibition of migration and capillary-like tube formation in HUVEC. However, these preventive effects were abolished by pretreatment of HUVEC with a ROCK inhibitor, Y27632. Pretreatment with a cSrc inhibitor, PP2, prevented the FA-induced activation of p190GAP, reduction of the RhoA activity and migration inhibition in HUVEC. Moreover, pre-transfection with p190RhoGAP siRNA abolished the FA-induced reduction in the RhoA activity and migration inhibition in HUVEC. Taken together, our results suggest that FA might inhibit endothelial cell migration through inhibiting the RhoA activity mediated by activating the FR/cSrc/p190RhoGAP-signaling pathway. These findings further support the anti-angiogenic activity of FA.

Extra-nuclear activation of progesterone receptor in regulating arterial smooth muscle cell migration

We previously showed that progesterone (P4) inhibits the proliferation of rat aortic smooth muscle cells (RASMC). Here, we further demonstrate that P4 at physiologic levels (5-500 nM) concentration-dependently inhibited migration of cultured RASMC. The effect is blocked by pretreatment with progesterone receptor (PR) antagonist, RU486. The P4-induced RASMC migration inhibition was through RhoA inactivation induced by cSrc-enhanced RhoA degradation. The P4-induced increases of phosphorylated Src (pSrc) and PR-pSrc complex in RASMC were observed mainly in the membrane fraction. Pre-treatment with a cSrc inhibitor (PP2) or cSrc antisense oligonucleotides prevented the P4-induced decreases of the protein levels of RhoA, phosphorylated FAK (p-FAK) and paxillin phosphorylaton and migration inhibition in RASMC. These findings expend our knowledge of the basis of P4s effect on vascular smooth muscle cell migration and highlight novel pathways of signaling transduction of P4 through PR-mediated nongenomic mechanisms.

Involvement of cysteine-rich protein 61 in the epidermal growth factor-induced migration of human anaplastic thyroid cancer cells.

Anaplastic thyroid cancer (ATC) is among the most aggressive types of malignant cancer. Epidermal growth factor (EGF) plays a crucial role in the pathogenesis of ATC, and patients with thyroid carcinoma typically exhibit increased cysteine‐rich protein 61 (Cyr61). In this study, we found that EGF treatment induced cell migration, stress fiber formation, Cyr61 mRNA and protein expressions, and Cyr61 protein secretion in ATC cells. The recombinant Cyr61 protein significantly induced cell migration; however, inhibition of Cyr61 activity by a Cyr61‐specific antibody abrogated EGF‐induced cell migration. EGF treatment also affected epithelial‐to‐mesenchymal transition (EMT)‐related marker protein expression, as evidenced by an increase in vimentin and a decrease in E‐cadherin expression. Inhibition of Cyr61 expression by Cyr61 siRNA decreased cell migration and reversed the EMT‐related marker protein expression. EGF treatment increased the phosphorylation of the extracellular signal‐regulated kinase (ERK) and cAMP response element‐binding protein (CREB), and finally activated Cyr61 promoter plasmid activity. Our results suggest that Cyr61 is induced by EGF through the ERK/CREB signal pathway and that it plays a crucial role in the migration and invasion of ATC cells; moreover, Cyr61 might be a therapeutic target for metastatic ATC.

Combined Treatment with Troglitazone and Lovastatin Inhibited Epidermal Growth Factor-Induced Migration through the Downregulation of Cysteine-Rich Protein 61 in Human Anaplastic Thyroid Cancer Cells

Our previous studies have demonstrated that epidermal growth factor (EGF) can induce cell migration through the induction of cysteine-rich protein 61 (Cyr61) in human anaplastic thyroid cancer (ATC) cells. The aim of the present study was to determine the inhibitory effects of combined treatment with the peroxisome proliferator-activated receptor-γ (PPARγ) ligand troglitazone and the cholesterol-lowering drug lovastatin at clinically achievable concentrations on ATC cell migration. Combined treatment with 5 μM troglitazone and 1 μM lovastatin exhibited no cytotoxicity but significantly inhibited EGF-induced migration, as determined using wound healing and Boyden chamber assays. Cotreatment with troglitazone and lovastatin altered the epithelial-to-mesenchymal-transition (EMT) -related marker gene expression of the cells; specifically, E-cadherin expression increased and vimentin expression decreased. In addition, cotreatment reduced the number of filopodia, which are believed to be involved in migration, and significantly inhibited EGF-induced Cyr61 mRNA and protein expression as well as Cyr61 secretion. Moreover, the phosphorylation levels of 2 crucial signal molecules for EGF-induced Cyr61 expression, the cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK), were decreased in cells cotreated with troglitazone and lovastatin. Performing a transient transfection assay revealed that the combined treatment significantly suppressed Cyr61 promoter activity. These results suggest that combined treatment with low doses of troglitazone and lovastatin effectively inhibits ATC cell migration and may serve as a novel therapeutic strategy for metastatic ATC.

Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

Previously, we demonstrated that progesterone (P4) at physiologic levels (5-500 nM) inhibited proliferation in cultured rat aortic smooth muscle cells (RASMCs) through a P4 receptor (PR)-dependent pathway. We also showed that P4-induced cell cycle arrest in RASMCs occurs when the cyclin-CDK2 system is inhibited just as p21cip1 and p27kip1 protein levels are augmented. In the present study, we further investigated the molecular mechanism underlying P4-induced up-regulations of p21cip1 and p27kip1 in RASMCs. We used pharmacological inhibitors as well as dominant negative constructs and conducted Western blot analyses to delineate the signaling pathway involved. Our data suggest that P4 up-regulated the expression of p21cip1 and p27kip1 in RASMCs through increasing the level of p53 protein mediated by activating the cSrc/Kras/Raf-1/AKT/ERK/p38/IκBα/NFκB pathway. The findings of the present study highlight the molecular mechanism underlying P4-induced up-regulations in p21cip1 and p27kip1 in RASMCs.

Progesterone receptor-nfκb complex formation is required for progesterone-induced nfκb nuclear translocation and binding onto the p53 promoter

We previously demonstrated that progesterone (P4) up-regulates p53 expression in human umbilical venous endothelial cells (HUVECs) through P4 receptor (PR) activation of extranuclear signaling pathways. However, the involvement of nuclear PR in P4-increased p53 expression is still unclear. Here, the molecular mechanism underlying PR-regulated p53 expression in HUVECs was investigated. Treatment with P4 increased nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α phosphorylation (IκBα and nuclear factor-κB (NFκB) nuclear translocation. Interestingly, P4 also increased PR-A, but not PR-B, nuclear translocation in HUVECs. Immunoprecipitation assay illustrated that P4 increased the formation of PR-A-NFκB complex in both the cytosol and the nucleus of HUVEC. Chromatin immunoprecipitation assay showed an interaction between PR and the NFκB binding motif on the p53 promoter. Ablation of the NFκB binding motif in the p53 promoter completely abolished P4-increased p53 promoter activity. In the absence of P4, overexpression of NFκB did not increase NFκB nuclear translocation. In contrast, treatment of NFκB-overexpressing HUVECs with P4 for only 4 hours, which is much shorter than the time (21.5 h) required for P4-induced IκBα phosphorylation, increased NFκB nuclear translocation. Blockade of PR activity abolished this effect. Taken together, these results uncover a novel role of PR for P4-induced NFκB nuclear translocation and suggest that PR-A-NFκB complex formation is required for NFκB nuclear translocation and binding onto the p53 promoter in HUVECs. Our data indicate that both nuclear and extranuclear signaling pathways of PR are involved in P4-regulated p53 expression in HUVECs.

Simvastatin Inhibits Cell Proliferation and Migration in Human Anaplastic Thyroid Cancer

Malignant human anaplastic thyroid cancer (ATC) is pertinacious to conventional therapies. The present study investigated the anti-cancer activity of simvastatin and its underlying regulatory mechanism in cultured ATC cells. Simvastatin (0–20 μM) concentration-dependently reduced cell viability and relative colony formation. Depletions of mevalonate (MEV) and geranylgeranyl pyrophosphate (GGpp) by simvastatin induced G1 arrest and increased apoptotic cell populations at the sub-G1 phase. Adding MEV and GGpp prevented the simvastatin-inhibited cell proliferation. Immunoblotting analysis illustrated that simvastatin diminished the activation of RhoA and Rac1 protein, and this effect was prevented by pre-treatment with MEV and GGpp. Simvastatin increased the levels of p21cip and p27kip proteins and reduced the levels of hyperphosphorylated-Rb, E2F1 and CCND1 proteins. Adding GGpp abolished the simvastatin-increased levels of p27kip protein, and the GGpp-caused effect was abolished by Skp2 inhibition. Introduction of Cyr61 siRNA into ATC cells prevented the epidermal growth factor (EGF)-enhanced cell migration. The EGF-induced increases of Cyr61 protein expression and cell migration were prevented by simvastatin. Taken together, these results suggest that simvastatin induced ATC proliferation inhibition through the deactivation of RhoA/Rac1 protein and overexpression of p21cip and p27kip, and migration inhibition through the abrogation of Cyr61 protein expression.

A Synergistic Anti-Cancer Effect of Troglitazone and Lovastatin in a Human Anaplastic Thyroid Cancer Cell Line and in a Mouse Xenograft Model

Anaplastic thyroid cancer (ATC) is a malignant subtype of thyroid cancers and its mechanism of development remains inconclusive. Importantly, there is no effective strategy for treatment since ATC is not responsive to conventional therapies, including radioactive iodine therapy and thyroid-stimulating hormone suppression. Here, we report that a combinational approach consisting of drugs designed for targeting lipid metabolism, lovastatin (an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGCR) and troglitazone (an agonist of peroxisome proliferator-activated receptor gamma, PPARγ), exhibits anti-proliferation in cell culture systems and leads to tumor regression in a mouse xenograft model. The composition contains a sub-lethal concentration of both drugs and exhibits low toxicity to certain types of normal cells. Our results support a hypothesis that the inhibitory effect of the combination is partly through a cell cycle arrest at G0/G1 phase, as evidenced by the induction of cyclin-dependent kinase inhibitors, p21cip and p27kip, and the reduction of hyperphosphorylated retinoblastoma protein (pp-Rb)-E2F1 signaling. Therefore, targeting two pathways involved in lipid metabolism may provide a new direction for treating ATC.

Thy-1-induced migration inhibition in vascular endothelial cells through reducing the RhoA activity.

Our previous study indicated that Thy-1, which is expressed on blood vessel endothelium in settings of pathological and a specific of physiological, but not during embryonic, angiogenesis, may be used as a marker for angiogenesis. However, the function of Thy-1 during angiogenesis is still not clear. Here, we demonstrate that knock-down of the endogenous Thy-1 expression by Thy-1 siRNA transfection promoted the migration of human umbilical vein endothelial cells (HUVEC). In contrast, treatment with interleukin-1β (IL-1β) or phorbol-12-myristate-13-acetate (PMA) increased the level of Thy-1 protein and reduced the migration of HUVEC. These effects were abolished by pre-transfection of HUVEC with Thy-1 siRNA to knock-down the expression of Thy-1. Moreover, over-expression of Thy-1 by transfection of HUVEC with Thy-1 pcDNA3.1 decreased the activity of RhoA and Rac-1 and inhibited the adhesion, migration and capillary-like tube formation of these cells. These effects were prevented by co-transfection of the cell with constitutively active RhoA construct (RhoA V14). On the other hand, pre-treatment with a ROCK (a kinase associated with RhoA for transducing RhoA signaling) inhibitor, Y27632, abolished the RhoA V14-induced prevention effect on the Thy-1-induced inhibition of endothelial cell migration and tube formation. Taken together, these results indicate that suppression of the RhoA-mediated pathway might participate in the Thy-1-induced migration inhibition in HUVEC. In the present study, we uncover a completely novel role of Thy-1 in endothelial cell behaviors.