Chien Ho Chen

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.

Molecular mechanisms of G0/G1 cell-cycle arrest and apoptosis induced by terfenadine in human cancer cells.

Terfenadine (TF), a highly potent histamine H1 receptor antagonist, has been shown to exert no significant central nervous system side effects in clinically effective doses. In this study, we demonstrated that TF induced significant growth inhibition of human cancer cells, including Hep G2, HT 29, and COLO 205 cells, through induction of G0/G1 phase cell‐cycle arrest. The minimal dose of TF induced significant G0/G1 arrest in these cells was 1–3 μM. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated, whereas the kinase activities of cyclin‐dependent kinase 2 (CDK2) and CDK4 were inhibited simultaneously in the TF‐treated cells. On the other hand, significant apoptosis, but not G0/G1 arrest, was induced in the HL 60 (p53‐null) or Hep 3B (with deleted p53) cells when treated with TF (3–5 μM). To clarify the roles of p21/Cip1 and p27/Kip1 protein expression, which was involved in G0/G1 arrest and apoptosis induced by TF in human cancer cells, antisense oligodeoxynucleotides (ODNs) specific to p21/Cip1 and p27/Kip1 were used, and the expression of the p21/Cip1 and p27/Kip1 were monitored by immunoblotting analysis. Our data demonstrated that the percentage of the apoptotic cells detected by annexin V/PI analysis in the TF‐treated group was clearly attenuated by pretreatment with p27/Kip1–specific ODNs. These results indicated that p27/Kip1 (but not p21/Cip1) protein indeed played a critical role in the TF‐induced apoptosis. We also demonstrated that the TF‐induced G0/G1 cell‐cycle arrest effect was not reversed by TF removal, and this growth inhibition lasted for at least 7 d. Importantly, the occurrence of apoptosis and cell growth arrest was not observed in the TF‐treated normal human fibroblast, even at a dose as high as 25 μM. Our study showed the molecular mechanisms for TF‐induced cell growth inhibition and the occurrence of apoptosis in human cancer cells.

Production of the chemokine eotaxin-1 in osteoarthritis and its role in cartilage degradation†

The expression of the chemokine, eotaxin‐1, and its receptors in normal and osteoarthritic human chondrocytes was examined, and its role in cartilage degradation was elucidated in this study. Results indicated that plasma concentrations of eotaxin‐1 as well as the chemokines, RANTES, and MCP‐1α, were higher in patients with osteoarthritis (OA) than those in normal humans. Stimulation of chondrocytes with IL‐1β or TNF‐α significantly induced eotaxin‐1 expression. The production of eotaxin‐1 induced expression of its own receptor of CCR3 and CCR5 on the cell surface of chondrosarcomas, suggesting that an autocrine/paracrine pathway is involved in eotaxin‐1s action. In addition, eotaxin‐1 markedly increased the expressions of MMP‐3 and MMP‐13 mRNA, but had no effect on TIMP‐1 expression in chondrocytes. However, pretreatment of anti‐eotaxin‐1 antibody significantly decreased the MMP‐3 expression induced by IL‐1β. These results first demonstrate that human chondrocytes express the chemokine, eotaxin‐1, and that its expression is induced by treatment with IL‐1β and TNF‐α. The cytokine‐triggered induction of eotaxin‐1 further results in enhanced expressions of its own receptor of CCR3, CCR5, and MMPs, suggesting that eotaxin‐1 plays an important role in cartilage degradation in OA.

Hyaluronan regulates PPARγ and inflammatory responses in IL-1β-stimulated human chondrosarcoma cells, a model for osteoarthritis

The carbohydrate polymer, hyaluronan, is a major component of the extracellular matrix in animal tissues. Exogenous hyaluronan has been used to treat osteoarthritis (OA), a degenerative joint disease involving inflammatory changes. The underlying mechanisms of hyaluronan in OA are not fully understood. Pro-inflammatory interleukin (IL)-1β downregulates peroxisome proliferator-activated receptor gamma (PPARγ), and increases expression of matrix metalloproteinases (MMPs) which are responsible for the degeneration of articular cartilage. The effects of low- and high-molecular-weight hyaluronan (oligo-HA and HMW-HA) on the inflammatory genes were determined in human SW-1353 chondrosarcoma cells. HMW-HA antagonized the effects of IL-1β by increasing PPARγ and decreasing cyclooxygenase (COX)-2, MMP-1, and MMP-13 levels. It promoted Akt, but suppressed mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NFκB) signaling, indicating anti-inflammatory effects. In contrast, the cells had overall opposite responses to oligo-HA. In conclusion, HMW-HA and oligo-HA exerted differential inflammatory responses via PPARγ in IL-1β-treated chondrosarcoma cells.

Chondroprotective effects of glucosamine involving the p38 MAPK and Akt signaling pathways.

The purpose of the present study was to elucidate the possible signal transduction pathway involved in the underlying mechanism of glucosamine (GLN)’s influence on the gene expression of matrix metalloproteinases (MMPs) in chondrocytes stimulated with IL-1β. Using chondrosarcoma cells stimulated with IL-1β, the effects of GLN on the mRNA and protein levels of MMP-3, the activation of JNK, ERK, p38, NF-κB, and AP-1, the nuclear translocation of NF-κB/Rel family members, and PI3-kinase/Akt activation were studied. GLN inhibited the expression and the synthesis of MMP-3 induced by IL-1β, and that inhibition was mediated at the level of transcription involving both the NF-κB and AP-1 transcription factors. Translocation of NF-κB was reduced by GLN as a result of the inhibition of IκB degradation. A slightly synergistic effect on the activation of AP-1 induced by IL-1β was shown in the presence of GLN. Among MAPK pathways involved in the transcriptional regulation of AP-1, phosphorylation of JNK and ERK was found to increase with the presence of GLN under IL-1β treatment, while that for p38 decreased. It was also found that GLN alone, but also synergistically with IL-1β, was able to activate the Akt pathway. The requirements of NF-κB translocation and p38 activity are indispensably involved in the induction of MMP-3 expression in chondrosarcoma cells stimulated by IL-1β. Inhibition of the p38 pathway in the presence of GLN substantially explains the chondroprotective effect of GLN on chondrocytes that regulate COX-2 expression, PGE2 synthesis, and NO expression and synthesis. The chondroprotective effect of GLN through the decrease in MMP-3 production and stimulation of proteoglycan synthesis may follow another potential signaling pathway of Akt.

Ketoconazole potentiates terfenadine-induced apoptosis in human Hep G2 cells through inhibition of cytochrome p450 3A4 activity

Terfenadine (TF) is a highly potent histamine H1 receptor antagonist that in clinically effective doses is free of significant central nervous system side effects. Ketoconazole (KT) is a worldwide used oral antifungal agent with a broad spectrum of activity against both superficial and systemic mycosis. Simultaneously administration of KT and TF has been reported to induce several potent symptoms including cardiotoxicity, excitotoxicity, inhibition of blood mononuclear cells proliferation, and cardiovascular toxicity. However, the intracellular molecular mechanisms of TF–KT interactions in cells were still uncertain. In this study, we first demonstrated that TF (5–30 μM) induced apoptosis in several types of human cancer cell lines including human hepatoma (Hep G2), colorectal cancer (COLO 205), and fibroblast (CCD 922SK) cells for 24 h. The cellular responses to TF‐induced apoptosis were demonstrated to be associated with the p53‐signaling pathway, including induction of p53, p21/Cip1, p27/Kip1, bax protein expression and inhibition of bcl‐2 protein expression. To realized the role of H1 receptor involved in TF‐induced apoptosis, different H1 receptor antagonists including promethazine, mequitazine, and chlorpheniramin (50–100 μM) were administered and demonstrated that these chemicals cannot induced apoptosis through the H1 receptor signaling pathway. Interestingly, we found that the apoptotic effect of TF (2.5 μM) was significantly potentiated by KT (1 μM) treatment in Hep G2 cells through inhibition of the cytochrome p450 3A4 (CYP 3A4) activity. Such results were demonstrated by decreased of the TF activity with recombinant CYP 3A4, which prepared from baculovirus‐infected insect cells. Our results provide the molecular basis of TF–KT interaction and this information should allow more rational forecasting of the risk for TF therapy during co‐administration of KT. J. Cell. Biochem. 87: 147–159, 2002.

Ketoconazole potentiates the antitumor effects of nocodazole: In vivo therapy for human tumor xenografts in nude mice

Our previous studies demonstrated that the oral antifungal agent ketoconazole (KT) induces apoptosis and G0/G1 phase cell cycle arrest in human cancer cell lines. In this study, we first demonstrated that KT (1 μM) potentiated the apoptotic effects of nocodazole (ND, 1 nM) in COLO 205 cancer cells. We further demonstrated the therapeutic efficacy of a combined treatment of KT (50 mg/kg/three times per week) and ND (5 mg/kg/three times per week) in vivo by treating athymic mice bearing COLO 205 tumor xenografts. The antitumor effects of ND were significantly potentiated by KT in mice after 6 wk of treatment. No gross signs of toxicity were observed in mice receiving these treatment regimens. The apoptotic cells were detected in a microscopic view of the terminal deoxynucleotidyl transferase–mediated dUTP nick‐end labeling staining and by observation of DNA fragmentation in KT + ND–treated tumor tissues. The levels of cell cycle regulatory proteins were determined by Western blot analysis. Treatment with KT inhibits tumor growth through elevation of p53, p21/CIP1, and p27/KIP1 as well as inhibition of cyclin D3 and cyclin‐dependent kinase 4 protein expression. Immunohistochemical staining analysis showed that p53, p21/CIP1, and p27/KIP1 immunoreactivity were induced in the tumor tissues. To clarify the roles of the p21/CIP1 and p27/KIP1 protein expression involved in G0/G1 arrest and/or apoptosis induced by a combined treatment with KT and ND, antisense oligodeoxynucleotides (ODNs) specific to p21/CIP1 and p27/KIP1 were used. Our results demonstrated that apoptotic phenomena, including BAX induction and cytochrome C released from mitochondria induced by KT + ND, were significantly attenuated by pretreatment the cells with the p27/KIP1–specific antisense ODNs. These results indicate that p27/KIP1 protein does indeed play a critical role in the KT + ND–induced apoptosis. Our study revealed the molecular mechanism of KT + ND in regression of the tumor growth. The apoptotic effects of KT in a great variety of cancer cells make it a very attractive agent for cancer chemotherapy.

Disease-modifying effects of Glucosamine HCl involving regulation of metalloproteinases and chemokines activated by interleukin-1β in human primary synovial fibroblasts†

The purpose of this study was to investigate the possible involvement of synovium in cartilage destruction in osteoarthritis (OA) patients. Using human primary synovial fibroblasts (HPSFs), we examined the effects of glucosamine (GLN) on the regulation of the expression of matrix metalloproteinases (MMP‐1, ‐2, and ‐13) and chemokines (IL‐8, MCP‐1, and RANTES) as well as the involvement of MAPK signal pathways (JNK, ERK, and p‐38) and the transcription factor of NF‐κB on the present or absence of interleukin (IL)‐1β. Our experiments showed that protein production and mRNA expressions of MMP‐1, MMP‐3, MMP‐13, IL‐8, MCP‐1, and RANTES were downregulated by treatment with glucosamine in HPSFs. The results further showed that GLN could inhibit IκBα phosphorylation and IκBα degradation leading to inhibition of the translocation of NF‐κB to nuclei. However, GLN upregulated MAPKs pathways in HPSFs cells with or without IL‐1β. The results suggest that the inhibition of MMP‐1, ‐3, and ‐13 expressions as well as IL‐8, MCP‐1, and RANTES productions by GLN might mediate suppression of NF‐κB signal pathways, and HPSFs seem to have a potential functions as an alternative source of MMPs and chemokines for inducing the degradation of cartilage in OA. J. Cell. Biochem. 104: 38–50, 2008.

15-Deoxy-Δ12,14-prostaglandin J2 inhibits fibrogenic response in human hepatoma cells

Liver fibrosis can be induced by environmental chemicals or toxicants, and finally stimulates fibrogenic cytokines expression, such as transforming growth factor-beta (TGF-beta) and its downstream mediator connective tissue growth factor (CTGF). 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a metabolite of arachidonic acid, can act as a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, and function as either anti-inflammatory or inflammatory agents in different cell types. In this study, CTGF was detected in three human hepatoma cell lines, Hep3B, HepG2, and Huh-7, and it was up-regulated by TGF-beta. 15d-PGJ(2) significantly inhibited TGF-beta-induced CTGF protein and mRNA expressions, and promoter activity in hepatoma cells. 15d-PGJ(2) suppressed TGF-beta-induced Smad2 phosphorylation, however enhancing the phosphorylation of ERK, c-Jun N-terminal kinase (JNK), and p38 in TGF-beta-treated Hep3B cells. Other PPAR ligands like the PPARgamma agonist, troglitazone; the PPARalpha agonist, Wy-14643, and bezafibrate were also able to inhibit TGF-beta-induced CTGF. The results suggest that 15d-PGJ(2) inhibits TGF-beta-induced CTGF expression by inhibiting the phosphorylation of Smad2, which is independent of PPAR, and 15d-PGJ(2) might also act through a PPAR-dependent mechanism in human hepatoma cells. 15d-PGJ(2) might have a beneficent effect on prevention of liver fibrosis induced by environmental toxicants.

Targeting annexin A2 reduces tumorigenesis and therapeutic resistance of nasopharyngeal carcinoma

The expression of annexin A2 (ANXA2) in nasopharyngeal carcinoma (NPC) cells induces the immunosuppressive response in dendritic cells; however, the oncogenic effect and clinical significance of ANXA2 have not been fully investigated in NPC cells. Immunohistochemical staining for ANXA2 was performed in 61 patients and the association with clinicopathological status was determined. Short hairpin (sh)RNA knockdown of ANXA2 was used to examine cellular effects of ANXA2, by investigating alterations in cell proliferation, migration, invasion, adhesion, tube-formation assay, and chemo- and radiosensitivity assays were performed. RT-qPCR, Western blotting, and immunofluorescence were applied to determine molecular expression levels. Clinical association studies showed that the expression of ANXA2 was significantly correlated with metastasis (p = 0.0326) and poor survival (p = 0.0256). Silencing of ANXA2 suppressed the abilities of cell proliferation, adhesion, migration, invasion, and vascular formation in NPC cell. ANXA2 up-regulated epithelial-mesenchymal transition associated signal proteins. Moreover, ANXA2 reduced sensitivities to irradiation and chemotherapeutic drugs. These results define ANXA2 as a novel prognostic factor for malignant processes, and it can serve as a molecular target of therapeutic interventions for NPC.