Chen W.-S.,Tzu Chi University |
Yu Y.-C.,Tzu Chi University |
Lee Y.-J.,National Yang Ming University |
Chen J.-H.,Tzu Chi University |
And 3 more authors.
International Journal of Radiation Oncology Biology Physics | Year: 2010
Purpose: Radiotherapy is one of the best choices for cancer treatment. However, various tumor cells exhibit resistance to irradiation-induced apoptosis. The development of new strategies to trigger cancer cell death besides apoptosis is necessary. This study investigated the role of securin in radiation-induced apoptosis and senescence in human cancer cells. Methods and Materials: Cell survival was determined using clonogenic assays. Western blot analysis was used to analyze levels of securin, caspase-3, PARP, p53, p21, Rb, γ-H2AX, and phospho-Chk2. Senescent cells were analyzed using a β-galactosidase staining assay. A securin-expressed vector (pcDNA-securin) was stably transfected into securin-null HCT116 cells. Securin gene knockdown was performed by small interfering RNA and small hairpin RNA in HCT116 and MDA-MB-231 cells, respectively. Results: Radiation was found to induce apoptosis in securin wild type HCT116 cells but induced senescence in securin-null cells. Restoration of securin reduced senescence and increased cell survival in securin-null HCT116 cells after irradiation. Radiation-induced γ-H2AX and Chk2 phosphorylation were induced transiently in securin-wild-type cells but exhibited sustained activation in securin-null cells. Securin gene knockdown switches irradiation-induced apoptosis to senescence in both HCT116 p53-null and MDA-MB-231 cells. Conclusions: Our results demonstrated that the level of securin expression plays a determining role in the radiosensitivity and fate of cells. Depletion of securin impairs DNA repair after irradiation, increasing DNA damage and promoting senescence in the residual surviving cells regardless of functional p53 expression. The knockdown of securin may contribute to a novel radiotherapy protocol for the treatment of human cancer cells that are resistant to irradiation. © 2010 Elsevier Inc. All rights reserved. Source
Leu J.-D.,Taipei City Hospital Renai Branch |
Chiu Y.-W.,National Yang Ming University |
Lo C.-C.,National Yang Ming University |
Chiang P.-H.,National Yang Ming University |
And 11 more authors.
International Journal of Radiation Biology | Year: 2013
Purpose: A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity. Materials and methods: Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by Comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632. Results: When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation-induced double-strand break (DSB) repair systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1. Conclusion: CFL-1 over-expression enhances radiosensitivity and this is associated with reduced DNA repair capacity. Although phosphorylated CFL-1 seems to be involved in radiosensitivity, further studies are required to address the importance of CFL-1 activity to the regulation of radiosensitivity. © 2013 Informa UK, Ltd. Source
Huang T.-H.,National Yang Ming University |
Chiu S.-J.,Tzu Chi University |
Chiu S.-J.,Institute of Radiation science |
Chiang P.-H.,National Yang Ming University |
And 7 more authors.
Anticancer Research | Year: 2011
Background: N-Heterocyclic indolyl glyoxylamide compounds are derived from the antimicrotubule agent D-24851, which exhibits anticancer activity after oral administration. The actions of these compounds on lung cancer cells are still unknown. Here, we investigated the effects of two N-heterocyclic indolyl glyoxylamides, BPR0C259 and BPR0C123, on non-small human lung cancer cells. Materials and Methods: 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the half maximal inhibitory concentration (IC 50), cell viability and radiation response of A549 cells and H1299 cells. Apoptosis was determined by sub-G 1 ratio, colony formation assay and caspase-3 activation. Cell cycle distribution was detected using flow cytometry. Results: Both compounds were able to inhibit the viability of human lung cancer cells, although the IC 50 of BPR0C123 was lower than that of BPR0C259. Both compounds induced significant sub-G1 and caspase-3 activation as low as 0.1 μM in both cell lines. These effects were independent of p53 activation because the level of serine-15 phosphorylated p53 was not affected after drug treatment. Furthermore, both compounds induced similar levels of G 2/M phase arrest and radiosensitivity in these lung cancer cells. Conclusion: Current data suggest that N-heterocyclic indolyl glyoxylamides can suppress the proliferation of and potentially increase radiosensitivity of human lung cancer cells. Source
Lin L.-T.,National Yang Ming University |
Wang B.-S.,National Yang Ming University |
Chen J.-C.,National Yang Ming University |
Liu C.-H.,Chang Gung University |
And 9 more authors.
Journal of Biomedical Optics | Year: 2013
Bacteriophytochrome infrared fluorescent protein (IFP) has a long emission wavelength that is appropriate for detecting pathophysiological effects via near-infrared (NIR) based imaging. However, the brightness and photostability of IFP are suboptimal, although an exogenous supply of biliverdin (BV) IXα is able to enhance these properties. In this study, we fused a far red mPlum fluorescent protein to IFP 1.4 via a linker deoxyribonucleic acid (DNA) sequence encoding eight amino acids. The brightness of mPlum-IFP 1.4 fusion protein at the IFP emission channel was comparable to that of native IFP 1.4 protein when fusion protein and IFP 1.4 were excited by 543 and 633 nm using confocal microscopy, respectively. Visualization of IFP 1.4 fluorescence by excitation of mPlum in mPlum-IFP 1.4 fusion protein is likely to be associated with Forster resonance energy transfer (FRET). The FRET phenomenon was also predicted by acceptor photobleaching using confocal microscopy. Furthermore, the expression of mPlum-IFP 1.4 fusion protein could be detected in cell culture and in xenograft tumors in the absence of BV using in vivo imaging system, although the BV was still essential for detecting native IFP 1.4. Therefore, this innovative fluorescent fusion protein would be useful for NIR-based imaging in vitro and in vivo. ©2013 Society of Photo-Optical Instrumentation Engineers. Source
Chen W.-S.,National Taiwan University |
Chen W.-S.,China Medical University at Taichung |
Yen C.-J.,National Cheng Kung University |
Chen Y.-J.,E DA Hospital |
And 15 more authors.
Oncotarget | Year: 2015
Maspin suppresses tumor progression by promoting cell adhesion and apoptosis and by inhibiting cell motility. However, its role in tumorigenesis of hepatocellular carcinoma (HCC) remains unclear. The gene regulation of maspin and its relationship with HCC patient prognosis were investigated in this study. Maspin expression was specifically reduced in HBV-associated patients and correlated with their poor prognosis. Maspin downregulation in HCC cells was induced by HBx to promote their motility and resistance to anoikis and chemotherapy. HBx-dependent induction of microRNA-7, -107, and -21 was further demonstrated to directly target maspin mRNA, leading to its protein downregulation. Higher expressions of these microRNAs also correlated with maspin downregulation in HBV-associated patients, and were associated with their poor overall survival. These data not only provided new insights into the molecular mechanisms of maspin deficiency by HBx, but also indicated that downregulation of maspin by microRNAs confers HBx-mediated aggressiveness and chemoresistance in HCC. Source