Jhang Ho Pak

Peroxiredoxin 6 Overexpression Attenuates Cisplatin-Induced Apoptosis in Human Ovarian Cancer Cells

We examined the involvement of peroxiredoxin 6 (Prdx 6) in providing chemoprotection against cisplatin cytotoxicity in SKOV-3 ovarian cancer cells. Treatment of SKOV-3 cells with cisplatin-induced cytotoxicity that was associated with increased accumulation of intracellular reactive oxygen species (ROS) and apoptosis mediated by proteolytically activated caspase 3 and 9. Overexpression of Prdx 6 protein or exposure to N-acetylcysteine (NAC) reversed the apoptotic effect of cisplatin by reducing ROS levels and suppressing the caspase signaling pathway. These results indicate that targeting Prdx 6 may sensitize cancer cells to ROS-producing therapeutic treatments, such as anticancer drugs and radiation.

Differential gene expression profiling in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products

Clonorchiasis is associated with bile duct malignancy and the subsequent development of cholangiocarcinoma. Although this is likely caused by adult Clonorchis sinensis and its excretory–secretory products (ESP), the precise molecular mechanisms remain obscure. To evaluate the effect of C. sinensis infection on differential gene expression in host hepatocytes, we examined the kinetics of changes in gene expression in the human cholangiocarcinoma cell line HuCCT1 treated with ESP at different times. Using complementary DNA microarrays containing 23,920 human genes of known function, we initially identified 435 genes altered by ESP treatment. Of these, 31 were up-regulated and 35 were down-regulated more than twofold in a time-dependent manner following ESP treatment. Clustering of these genes by function revealed that several were involved in the cell cycle, oncogenesis, protein modification, immunity, signal transduction, cell structure, and developmental processes. These findings should provide a fundamental basis for further analysis of the molecular pathways and mechanisms involved in C. sinensis infection of host cells.

Proteomic analysis of differentially expressed proteins in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products.

Severe Clonorchis sinensis infection is a significant risk factor for malignant changes in bile ducts and surrounding liver tissues occurring as a result of direct contact with C. sinensis worms and their excretory–secretory products (ESP). However, the intrinsic molecular mechanisms involved in these processes remain obscure. To determine the effects of C. sinensis infection on protein expression in host bile duct epithelium, we examined proteomic profile changes in the human cholangiocarcinoma cell line (HuCCT1) treated with ESP at 24 h. Using a combination of 2‐DE, quantitative image and MALDI‐TOF MS analysis, we identified 83 proteins that were translationally modulated in response to ESP, among which 49 were up‐regulated and 34 down‐regulated. These proteins were classified under various biological categories, including metabolism, cell structure and architecture, proteolysis, protein modification, transport, signal transduction, and reactive oxygen species (ROS) detoxification. In particular, ESP induced the expression of redox‐regulating proteins, including peroxiredoxins (Prdx 2, 3, and 6) and thioredoxin 1 (Trx 1), possibly via intracellular ROS generation. Application of the proteomic approach to identify ESP response proteins should be a prerequisite before further investigation to clarify the molecular pathways and mechanisms involved in C. sinensis infection of host cells. J. Cell. Biochem. 108: 1376–1388, 2009.

Free radicals enzymatically triggered by Clonorchis sinensis excretory–secretory products cause NF-κB-mediated inflammation in human cholangiocarcinoma cells

Chronic clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis worms and their excretory-secretory products, is associated with hepatobiliary damage, inflammation, periductal fibrosis and even development of cholangiocarcinoma. Our previous report revealed that intracellular reactive oxygen species were generated in C. sinensis excretory-secretory product-treated human cholangiocarcinoma cells; however, their endogenous sources and pathophysiological roles in host cells were not determined. In the present study, we found that treatment of human cholangiocarcinoma cells with excretory-secretory products triggered increases in free radicals via a time-dependent activation of NADPH oxidase, xanthine oxidase and inducible nitric oxide synthase. This increase in free radicals substantially promoted the degradation of cytosolic IκB-α, nuclear translocation of nuclear factor-κB subunits (RelA and p50), and increased κB consensus DNA-binding activity. Excretory-secretory product-induced nuclear factor-κB activation was markedly attenuated by preincubation with specific inhibitors of each free radical-producing enzyme or the antioxidant, N-acetylcysteine. Moreover, excretory-secretory products induced an increase in the mRNA and protein expression of the proinflammatory cytokines, IL-1β and IL-6, in an nuclear factor-κB-dependent manner, indicating that enzymatic production of free radicals in ESP-treated cells participates in nuclear factor-κB-mediated inflammation. These findings provide new insights into the pathophysiological role of C. sinensis excretory-secretory products in host chronic inflammatory processes, which are initial events in hepatobiliary diseases.

Proteomic analysis of apoptosis related proteins regulated by proto‐oncogene protein DEK

A nuclear phosphoprotein, DEK, is implicated in certain human diseases, such as leukemia and antoimmune disorders, and a major component of metazoan chromatin. Basically as a modulator of chromatin structure, it can involve in various DNA and RNA‐dependent processes and function as either an activator or repressor. Despite of numerous efforts to suggest the biological role of DEK, direct target proteins of DEK in different physiological status remains elusive. To investigate if DEK protein triggers the changes in certain protein networks, DEK was knocked down at both types of cell clones using siRNA expression. Here we provide a catalogue of proteome profiles in total cell lysates derived from normal HeLa and DEK knock‐down HeLa cells and a good in vitro model system for dissecting the protein networks due to this proto‐oncogenic DEK protein. In this biological context, we compared total proteome changes by the combined methods of two‐dimensional gel electrophoresis, quantitative image analysis and MALDI‐TOF MS analysis. There were a large number of targets for DEK, which were differentially expressed in DEK knock‐down cells and consisted of 58 proteins (41 up‐regulated and 17 down‐regulated) differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. In the identified 58 spots, 16% of proteins are known to be associated with apoptosis. Among others, we identified apoptosis related proteins such as Annexins, Enolase1, Lamin A, and Glutathione‐S‐transferase omega 1. These results are consistent with recent studies indicating the crucial role of DEK in apoptosis pathway. We further demonstrated by ChIP analysis that knock‐down of DEK caused hyperacetylation of histones around Prx VI promoter which is upregulated in our profile. Using immunoblotting analysis, we have demonstrated the modulation of other caspase‐dependent apoptosis related proteins by DEK knock‐down and further implicate its role in apoptosis pathway. J. Cell. Biochem. 106: 1048–1059, 2009.

Protective effects of Peroxiredoxin 6 overexpression on amyloid β-induced apoptosis in PC12 cells

Abstract Oxidative stress triggered by amyloid beta (Aβ) accumulation contributes substantially to the pathogenesis of Alzheimers disease (AD). In the present study, we examined the involvement of the antioxidant activity of peroxiredoxin 6 (Prdx 6) in protecting against Aβ25–35-induced neurotoxicity in rat PC12 cells. Treatment of PC12 cells with Aβ25-35 resulted in a dose- and time-dependent cytotoxicity that was associated with increased accumulation of intracellular reactive oxygen species (ROS) and mitochondria-mediated apoptotic cell death, including activation of Caspase 3 and 9, inactivation of poly ADP-ribosyl polymerse (PARP), and dysregulation of Bcl-2 and Bax. This apoptotic signaling machinery was markedly attenuated in PC12 cells that overexpress wild-type Prdx 6, but not in cells that overexpress the C47S catalytic mutant of Prdx 6. This indicates that the peroxidase activity of Prdx 6 protects PC12 cells from Aβ25-35-induced neurotoxicity. The neuroprotective role of the antioxidant Prdx 6 suggests its therapeutic and/or prophylactic potential to slow the progression of AD and limit the extent of neuronal cell death caused by AD.

Transcriptional induction of minichromosome maintenance protein 7 (Mcm7) in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products.

Clonorchiasis is an infection associated with bile duct malignancy and subsequent development of cholangiocarcinoma. This disease is mainly caused by Clonorchis sinensis worms and their excretory-secretory products (ESP). However, the precise molecular mechanisms of carcinogenesis remain to be determined. Previously, we established differential gene expression profiles from microarrays containing 23,920 human genes of known function in a human cholangiocarcinoma cell line, HuCCT1, treated with ESP. Among the upregulated genes, we focused on minichromosome maintenance protein 7 (Mcm7), which is implicated in various cancer types, and analyzed transcriptional regulation mediated by ESP to further elucidate its role in cholangiocarcinoma development. Global histone acetylation levels were increased in ESP-treated cells, along with histone acetyltransferase (HAT) protein expression. Detailed promoter analysis using reporter and chromatin immunoprecipitation assays revealed that transcriptional activation of Mcm7 is mediated by HAT recruitment to the promoter region upon C. sinensis ESP treatment. These findings contribute to clarification of the intrinsic mechanism underlying the cellular carcinogenesis process stimulated by Mcm7 in C. sinensis-treated host cells.

The relationship between cisplatin resistance and histone deacetylase isoform overexpression in epithelial ovarian cancer cell lines.

Objective To investigate the relationship between cisplatin resistance and histone deacetylase (HDAC) isoform overexpression in ovarian cancer cell lines. Methods Expression of four HDAC isoforms (HDAC 1, 2, 3, and 4) in two ovarian cancer cell lines, SKOV3 and OVCAR3, exposed to various concentrations of cisplatin was examined by western blot analyses. Cells were transfected with plasmid DNA of each HDAC. The overexpression of protein and mRNA of each HDAC was confirmed by western blot and reverse transcriptase-polymerase chain reaction analyses, respectively. The cell viability of the SKOV3 and OVCAR3 cells transfected with HDAC plasmid DNA was measured using the cell counting kit-8 assay after treatment with cisplatin. Results The 50% inhibitory concentration of the SKOV3 and OVCAR3 cells can be determined 15-24 hours after treatment with 15 µg/mL cisplatin. The expression level of acetylated histone 3 protein in SKOV3 cells increased after exposure to cisplatin. Compared with control cells at 24 hours after cisplatin exposure, the viability of SKOV3 cells overexpressing HDAC 1 and 3 increased by 15% and 13% (p<0.05), respectively. On the other hand, OVCAR3 cells that overexpressed HDAC 2 and 4 exhibited increased cell viability by 23% and 20% (p<0.05), respectively, compared with control cells 24 hours after exposure to cisplatin. Conclusion In SKOV3 and OVCAR3 epithelial ovarian cancer cell lines, the correlation between HDAC overexpression and cisplatin resistance was confirmed. However, the specific HDAC isoform associated with resistance to cisplatin varied depending on the ovarian cancer cell line. These results may suggest that each HDAC isoform conveys cisplatin resistance via different mechanisms.

Identification and characterization of the second cysteine protease inhibitor of Clonorchis sinensis (CsStefin-2)

CsStefin-2, the second cysteine protease inhibitor of Clonorchis sinensis, was identified and characterized. CsStefin-2 is a cysteine protease inhibitor that belongs to family 1 stefins based on its phylogenetic and structural properties. However, CsStefin-2 had a QIVSG cystatin motif distinct from the common QVVAG cystatin motif that is well conserved in family 1 stefins. Mutagenesis analysis revealed that the two amino acid substitutions in the QIVSG cystatin motif of CsStefin-2 did not affect its inhibitory activity. Molecular modeling also indicated that no critical change was induced in the interaction between CsStefin-2 and its target enzyme. CsStefin-2 showed broad inhibitory activities against several cysteine proteases, including human cathepsins B and L, papain, and cathepsin Fs of C. sinensis (CsCFs), and effectively inhibited the autocatalytic maturation of CsCF-6. Native CsStefin-2 was assembled into a homo-tetramer, in which intermolecular disulfide bonds are not involved in the assembly of the tetramer. CsStefin-2 was expressed throughout the various developmental stages of the parasite and was localized in the intestinal epithelium, where CsCFs are actively synthesized. These results suggest that CsStefin-2 is the second active cysteine protease inhibitor of C. sinensis that shares functional redundancy with CsStefin-1 to modulate the activity and processing of CsCFs.

Induction of cancer-related microRNA expression profiling using excretory-secretory products of Clonorchis sinensis

Clonorchis sinensis is a carcinogenic human liver fluke by which chronic infection is strongly associated with the development of cholangiocarcinoma. Although this cholangiocarcinoma is caused by both physical and chemical irritation from direct contact with adult worms and their excretory–secretory products (ESPs), the precise molecular events of the host–pathogen interactions remain to be elucidated. To better understand the effect of C. sinensis infection on cholangiocarcinogenesis, we profiled the kinetics of changes in cancer-related microRNAs (miRNAs) in human cholangiocarcinoma cells (HuCCT1) treated with C. sinensis ESPs for different periods. Using miRNA microarray chips containing 135 cancer-related miRNAs, we identified 16 miRNAs showing differentially altered expression following ESP exposure. Of these miRNAs, 13 were upregulated and 3 were downregulated in a time-dependent manner compared with untreated controls. Functional clustering of these dysregulated miRNAs revealed involvement in cell proliferation, inflammation, oncogene activation/suppression, migration/invasion/metastasis, and DNA methylation. In particular, decreased expression of let-7i, a tumor suppressor miRNA, was found to be associated with the ESP-induced upregulation of TLR4 mRNA and protein, which contribute to host immune responses against liver fluke infection. Further real-time quantitative PCR analysis using ESP-treated normal cholangiocytes (H69) revealed that the expressions of nine miRNAs (miR-16-2, miR-93, miR-95, miR-153, miR-195, miR-199-3P, let7a, let7i, and miR-124a) were similarly regulated, indicating that the cell proliferation and inhibition of tumor suppression mediated by these miRNAs is common to both cancerous and non-cancerous cells. These findings constitute further our understanding of the multiple cholangiocarcinogenic pathways triggered by liver fluke infection.