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Hsinchu, Taiwan

Wu H.-Y.,National Chiao Tung University | Wu H.-Y.,National Pingtung University of Science and Technology | Lin C.-Y.,National Chiao Tung University | Chen T.-C.,Hsinchu Hospital | And 2 more authors.
International Journal of Biochemistry and Cell Biology | Year: 2011

Mammalian Ste20-like protein kinase 3 (Mst3) is a key player in inducing apoptosis in a variety of cell types and has recently been shown to participate in the signaling pathway of hypoxia-induced apoptosis of human trophoblast cell line 3A-sub-E (3A). It is believed that oxidative stress may occur during hypoxia and induce the expression of Mst3 in 3A cells via the activation of c-Jun N-terminal protein kinase 1 (JNK1). This hypothesis was demonstrated by the suppressive effect of dl-α-lipoic acid, a reactive oxygen species scavenger, in hypoxia-induced responses of 3A cells such as Mst3 expression, nitrotyrosine formation, JNK1 activation and apoptosis. Similar results were also observed in trophoblasts of human placental explants in both immunohistochemical studies and immunoblot analyses. These suggested that the activation of Mst3 might trigger the apoptotic process in trophoblasts by activating caspase 3 and possibly other apoptotic pathways. The role of nitric oxide synthase (NOS) and NADPH oxidase (NOX) in hypoxia-induced Mst3 up-regulation was also demonstrated by the inhibitory effect of N G-nitro-l-arginine and apocynin, which inhibits NOS and NOX, respectively. Oxidative stress was postulated to be induced by NOS and NOX in 3A cells during hypoxia. In conclusion, hypoxia induces oxidative stress in human trophoblasts by activating NOS and NOX. Subsequently, Mst3 is up-regulated and plays an important role in hypoxia-induced apoptosis of human trophoblasts. © 2011 Elsevier Ltd.

Ko J.-C.,Hsinchu Hospital | Ko J.-C.,Yuanpei University | Ko J.-C.,National Chiao Tung University | Tsai M.-S.,National Chiayi University | And 5 more authors.
Biochemical Pharmacology | Year: 2011

Thymidine phosphorylase (TP) is the rate-limiting enzyme for the activation of capecitabine (pro-drug of fluorouracil), and as a useful predictor of tumor response to capecitabine-based chemotherapy. Overexpression of Rad51 and ERCC1 induce resistance to chemotherapeutic agents. Emodin, one of the main bioactive anthraquinone derivatives in the roots and rhizomes of numerous plants, possesses potent antitumor effects. Accordingly, we aimed to explore the molecular mechanism of emodin enhances the capecitabine-induced cytotoxicity through controlling Rad51, ERCC1, and TP expression in human non-small cell lung cancer (NSCLC). The results show that capecitabine increases the phosphorylation of MKK1/2-ERK1/2 and protein levels of Rad51 and ERCC1 through enhancing the protein stability. Depletion of endogenous Rad51 or ERCC1 expression by specific small interfering RNA transfection significantly increases capecitabine-induced cell death and growth inhibition. Emodin enhances the capecitabine-induced cytotoxic effects through ERK1/2 inactivation and decreasing the Rad51 and ERCC1 protein levels induced by capecitabine. Enhancement of ERK1/2 signaling by constitutively active MKK1/2 (MKK1/2-CA) results in increasing Rad51 and ERCC1 protein levels and cell viability in NSCLC cell lines treated with emodin and capecitabine. Interestingly, emodin enhances TP mRNA and protein expression in capecitabine treated NSCLC cell lines, and depletion of the TP expression decreases the cytotoxic effects induced by capecitabine and emodin. We conclude that enhancing the cytotoxicity to capecitabine by emodin is mediated by down-regulation the expression of Rad51 and ERCC1 and up-regulation TP expression. © 2010 Elsevier Inc. All rights reserved.

Ko J.-C.,Hsinchu Hospital | Tsai M.-S.,National Chiayi University | Chiu Y.-F.,National Chiayi University | Weng S.-H.,National Chiayi University | And 2 more authors.
Journal of Pharmacology and Experimental Therapeutics | Year: 2011

Chemotherapy for advanced human non-small-cell lung cancer (NSCLC) includes platinum-containing compound such as cisplatin in combination with a second- or third-generation cytotoxic agent. 5-Fluorouracil (5-FU) belongs to antimetabolite chemotherapeutics, and its mechanism of cytotoxicity is involved in the inhibition of thymidylate synthase (TS). TS and thymidine phosphorylase (TP) are key enzymes of the pyrimidine salvage pathway. In this study, we have examined the molecular mechanism of TS and TP in regulating drug sensitivity to cisplatin in NSCLC cell lines. Cisplatin could increase the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MKK1/2)-extracellular signal-regulated kinase 1/2 (ERK1/2) and the protein levels of TS and TP through enhancing the protein stability in A549 and H1975 cells. Blocking ERK1/2 activation by MKK1/2 inhibitor [U0126; 1,4-diamino-2,3-dicyano-1,4-bis(2- aminophenylthio)butadiene)] decreased TS and TP protein levels in both cell lines treated with cisplatin. Depletion of endogenous TS or TP expression by specific small interfering RNA transfection significantly increased cisplatin-induced cell death and growth inhibition. Combined treatment with 5-FU could decrease cisplatin-induced ERK1/2 activation and the induction of TS and TP, which subsequently resulted in synergistic cytotoxic effects. Enforced expression of constitutive active MKK1/2 vectors rescued the protein levels of phospho-ERK1/2, TS, and TP, and the cell viability that were decreased by cisplatin and 5-FU combination. In contrast, U0126 enhanced drug sensitivity to cisplatin and/or 5-FU in lung cancer cells. In conclusion, the up-regulation of ERK1/2-dependent TS and TP can protect human lung cancer cells from cisplatin-induced cytotoxicity. Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics.

Ko J.-C.,Hsinchu Hospital | Su Y.-J.,National Chiayi University | Lin S.-T.,National Chiayi University | Jhan J.-Y.,National Chiayi University | And 3 more authors.
Biochemical Pharmacology | Year: 2010

Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. Emodin exhibits anticancer effects against a variety of cancer cells, including lung cancer cells. ERCC1 and Rad51 proteins are essential for nucleotide excision repair and homologous recombination, respectively. Furthermore, ERCC1 and Rad51 overexpression induces resistance to DNA-damaging agents that promote DNA double-strand breaks. Accordingly, the aim of this study was to determine the role of ERCC1 and Rad51 in emodin-mediated cytotoxicity in human non-small cell lung cancer (NSCLC) cells. Both ERCC1 and Rad51 protein levels as well as mRNA levels were decreased in four different NSCLC cell lines after exposure to emodin. These decreases correlated with the inactivation of the MKK1/2-ERK1/2 pathway. Moreover, cellular ERCC1 and Rad51 protein and mRNA levels were specifically inhibited by U0126, a MKK1/2 inhibitor. We found that transient transfection of human NSCLC cells with si-ERCC1 or si-Rad51 RNA and cotreatment with U0126 could enhance emodin-induced cytotoxicity. In contrast, overexpression of constitutively active MKK1/2 vectors (MKK1/2-CA) was shown to significantly recover reduced phospho-ERK1/2, ERCC1, and Rad51 protein levels and to rescue cell viability upon emodin treatment. These results demonstrate that activation of the MKK1/2-ERK1/2 pathway is the upstream signal regulating the expressions of ERCC1 and Rad51, which are suppressed by emodin to induce cytotoxicity in NSCLC cells. Crown Copyright © 2009.

Ko J.-C.,Hsinchu Hospital | Su Y.-J.,National Chiayi University | Lin S.-T.,National Chiayi University | Jhan J.-Y.,National Chiayi University | And 6 more authors.
Lung Cancer | Year: 2010

Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants; it exhibits an anticancer effect on many malignancies. The most important chemotherapeutic agent for patients with advanced non-small cell lung cancer (NSCLC) is a platinum-containing compound such as cisplatin or carboplatin. The molecular mechanism underlying decreased NSCLC cell viability after treatment with emodin and cisplatin is unclear. Therefore, the aim of this study was to assess the cytotoxic effect of combined emodin and cisplatin on NSCLC cell lines and to clarify underlying molecular mechanisms. Exposure of human NSCLC cells to emodin decreased cisplatin-elicited ERK1/2 activation and ERCC1 protein induction by increasing instability of ERCC1 protein. Cisplatin alone did not affect expression of ERCC1 mRNA. However, emodin alone or combined with cisplatin significantly decreased expression of ERCC1 mRNA levels. Enhancement of ERK1/2 activation by transfection with constitutively active MKK1/2 (MKK1/2-CA) vector increased ERCC1 protein levels and protein stability, as well as increasing viability of NSCLC cells treated with emodin and cisplatin. In contrast, blocking ERK1/2 activation by U0126 (an MKK1/2 inhibitor) decreased cisplatin-elicited ERCC1 expression and enhanced cisplatin-induced cytotoxicity. Depletion of endogenous ERCC1 expression by si-ERCC1 RNA transfection significantly enhanced cisplatin's cytotoxic effect. In conclusion, ERCC1 protein protects NSCLC cells from synergistic cytotoxicity induced by emodin and platinum agents. Further investigation of combined emodin and cisplatin may lead to novel therapy in the future for NSCLC through down-regulating expression of ERCC1. © 2009 Elsevier Ireland Ltd.

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