Mitochondrial Reactive Oxygen Species Production Mediated by Romo1 Expression / 한양의대학술지

Release of reactive oxygen species (ROS) generated in the mitochondria to the cytosol is well controlled by various proteins in order to maintain and regulate redox homeostasis and cellular signaling pathways, however, the exact mechanisms by which the proteins located in the mitochondrial membrane control ROS release still remains to be identified. Although there are reports that several proteins play a role in mitochondrial ROS release to the cytosol, little is known about how it is released into the cytosol or its origin. Recently, several reports demonstrated that the ROS modulator 1 (Romo1) protein located on the mitochondrial membrane modulates ROS release into the cytosol and that these ROS are indispensible for survival in both normal cells and tumor cells. If these ROS are over-produced or dysregulated in pathological conditions, they may cause oxidative damages resulting in a variety of diseases. Therefore, understanding and identifying the mechanisms by which ROS are released to the cytosol may offer new strategies for pharmaceutical therapy of diseases related to oxidative stresses.

Manipulation of Human Telomerase Activity in Cancer and Stem Cells: Application of siRNA-induced Inhibition of Human Telomerase RNA (hTR)

BACKGROUND: We have determined the effects of human telomerase RNA inhibiton using siRNA in tumor cells and human embryonic and mesenchymal stem cells. METHODS: We selected the sequences against the predicted loop; these sequneces were comprised of nucleotides from 76 to 94 residues and from 143 to 163 residues as the target sequences, and we cloned these sequences into pU6sh75 and pU6sh143 cells. Three different kinds of cell lines were used: HeLa, SNUhES3, and human mesenchymal stem cells. The degree of inhibition of telomerase activity was assessed by TRAP assay and RT-PCR. RESULTS: The telomerase activity of the HeLa and SNUhES3 cells were 135.3+/-14.5 and 109.0+/-18.2; these cells showed higher activity than human mesenchymal stem cells and Wi38 cells (46.3+/-5.0 and 26.0+/-12.0), which were control cells. When each of the types of cells was treated with siRNA-hTR, the transfection efficiency of pU6sh75 for the HeLa, SNUhES3, and human mesenchymal stem cells was 91.0+/-8.4%, 83.3+/-16.0% and 81.9+/-12.3%, respectively. In the case of pU6sh143, its transfection efficiency was similar to pU6sh75; the HeLa, SNUhES3 and human mesenchymal stem cells tranfection efficiency was 90.1+/-9.0%, 79.9+/-18.2% and 79.4+/-15.1%, respectively. After two days of transfection, the level of telomerase activity in the pU6sh75 transfected cells decreased to 64.3+/-10.1% and 56.0+/-11.0% in the HeLa and SNUhES3 cells, respectively. When the cells were transfected with pU6sh143, the telomerase activity also decreased in the HeLa and SNUhES3 cells (71.3+/-9.1% and 61.6+/-8.3%, respectively). However, the difference of telomerase activity was not significant in the human mesenchymal stem cells: 43.0+/-7.2% with pU6sh75 and 46.0+/-9.0% with pU6sh143. CONCLUSION: Telomerase RNA inhibiton with siRNA may be a feasible way to inhibit the telomerase activity of human tumor and embryonic stem cells.

cDNA Microarray Analysis of Differential Gene Expression in Gastric Cancer Cells Sensitive and Resistant to 5-Fluorouracil and Cisplatin / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Gastric cancer is one of the most prevalent cancers worldwide. 5-fluorouracil (5-FU) and cisplatin are the most commonly used drugs for the treatment of gastric cancer. However, a significant number of tumors often fail to respond to chemotherapy. MATERIALS AND METHODS: To better understand the molecular mechanisms underlying drug resistance in gastric cancer the gene expression in gastric cancer cells, which were either sensitive or resistant to 5-FU and cisplatin, were examined using cDNA microarray analysis. To confirm the differential gene expression, as determined using the microarray, semiquantitative RT-PCR was performed on a subset of differentially expressed cDNAs. RESULTS: 69 and 45 genes, which were either up-regulated (9 and 22 genes) or down-regulated (60 and 25 genes), were identified in 5-FU- and cisplatin-resistant cells, respectively. Several genes, such as adaptor-related protein complex 1 and baculoviral IAP repeat-containing 3, were up-regulated in both drug-resistant cell types. Several genes, such as the ras homolog gene family, tropomyosin, tumor rejection antigen, protein disulfide isomerase-related protein, melanocortin 1 receptor, defensin, cyclophilin B, dual specificity phosphatase 8 and hepatocyte nuclear factor 3, were down-regulated in both drug-resistant cell types. CONCLUSION: These findings show that cDNA microarray analysis can be used to obtain gene expression profiles that reflect the effect of anticancer drugs on gastric cancer cells. Such data may lead to the assigning of signature expression profiles of drug-resistant tumors, which may help predict responses to drugs and assist in the design of tailored therapeutic regimens to overcome drug resistance.

Proteomic Profiling of Human Small Cell Lung Cancer Cell Line NCI-H211 / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Small cell lung cancer is one of the major causes of death from cancer worldwide. To explore the expressions of global protein in small cell lung cancer cells, a proteomic approach, to identify the proteins, was used and the establishment of a protein reference map attempted. MATERIALS AND METHODS: Two-dimensional gel electrophoresis (2-DE), with subsequent analysis by mass spectrometry (MS), was applied to the study of protein identification from a small cell lung cancer cell line, NCI- H211. The cells were lysed, and the extracts subjected to isoelectric focusing, with immobilized pH gradients, followed by second dimension SDS-PAGE. The polypeptides were identified by peptide mass fingerprinting, with MALDI-TOF MS, after in-gel protein digestion. RESULTS: From silver staining of the gel, around two thousands protein spots were separated by the 2-DE. Of these protein spots visualized in the gel, one hundred and ten were identified by peptide mass fingerprinting. Different proteins, such as enzymes, cytoskeletal proteins and proteins common to eukaryotic cells, were identified. CONCLUSION: The protein expressions of the small cell lung cancer cells were analyzed to establish a protein reference map. The reference map presented here may serve as a working tool for the further study of small cell lung cancer.

Correlation between Retinoic Acid Sensitivity and IGFBP-3, AFP Protein Expression in Hepatoma Cell Lines / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Retinoic acid (RA) has been known to inhibit the proliferation, and to induce apoptosis, of various cancer cell lines. We investigated the correlation between the protein levels of the RAR and RXR receptor families, IGFBP-3 and AFP, and the RA sensitivity in hepatoma cell lines. MATERIALS AND METHODS: The cell growth inhibition was examined by assaying various 1 to 10muM RA treated hepatoma cell lines. Western blot analysis for the RAR and RXR families, AFP and IGFBP-3 were performed after treatment with 10muM RA. RESULTS: The 1 to 10muM RA treatment induced growth inhibition in the SNU368, SNU354, SNU398 and HepG2 cells. The cell growth of SNU449 and Hep3B were not suppressed by 1muM, but were slightly suppressed by 10muM RA. An increased expression of IGFBP-3 in HepG2, SNU354, SNU398 and SNU368 cells, and a decreased expression of alpha-fetoprotein (AFP), was observed from the western blot analysis in all hepatoma cells tested, whereas no confirmed tendency of RAR expressions was seen. CONCLUSION: Our result showed that the growth inhibition of RA differed according to the sensitivity of the type of cells to RA. We supposed that RA-induced cell growth inhibition may be related to the expressions of IGFBP-3 and AFP, but no exact correlation exists between the growth inhibition and receptor expression status in hepatoma cell lines.

Microsatellite Alterations in Serum DNA of Lung Cancer Patients / Journal of the Korean Cancer Association, 대한암학회지

No abstract available.

Synergistic effect of peroxiredoxin II antisense on cisplatin-induced cell death

Peroxiredoxin II (Prx II) is known not only to protect cells from oxidative damage caused by hydrogen peroxide (H2O2), but also to endow cancer cells with resistance to both H2O2 and cisplatin and to grant them radioresistance. In this study, we examined whether Prx II antisense could enhance cisplatin-induced cell death. When gastric cancer cells were transfected with various concentrations of Prx II antisense plasmid, pPrxII/AS, and then treated with the same concentrations of cisplatin, Prx II antisense enhanced cisplatin-induced cell death. The combination index (CI) at all doses of the combination was below 1, indicating that Prx II antisense sensitized cisplatin-induced cell death. This synergism was also observed in the cells transfected with a Prx II antisense oligomer. Our present results, therefore, suggest that Prx II antisense would be a very good sensitizer for cisplatin, and that Prx II as a target for chemosensitizers constitutes a promising avenue for future research.

Synergistic effect of peroxiredoxin II antisense on cisplatin-induced cell death

Peroxiredoxin II (Prx II) is known not only to protect cells from oxidative damage caused by hydrogen peroxide (H2O2), but also to endow cancer cells with resistance to both H2O2 and cisplatin and to grant them radioresistance. In this study, we examined whether Prx II antisense could enhance cisplatin-induced cell death. When gastric cancer cells were transfected with various concentrations of Prx II antisense plasmid, pPrxII/AS, and then treated with the same concentrations of cisplatin, Prx II antisense enhanced cisplatin-induced cell death. The combination index (CI) at all doses of the combination was below 1, indicating that Prx II antisense sensitized cisplatin-induced cell death. This synergism was also observed in the cells transfected with a Prx II antisense oligomer. Our present results, therefore, suggest that Prx II antisense would be a very good sensitizer for cisplatin, and that Prx II as a target for chemosensitizers constitutes a promising avenue for future research.

Retinoic Acid Enhances Drug-Induced Cell Death in Anticancer Drug-Resistant Cell Lines / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Retinoids (RA), a group of vitamin A derivatives, is known to be important for regulation of normal cellular growth and differentiation. RA treatment of various cancers resulted in cell growth inhibition and apoptosis. Therefore, the chemotherapeutic and chemopreventative activities of various types of tumor have been examined. Biological actions of RA are mediated through nuclear receptors, including the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). In this study, we examined the effect of all-trans-retinoic acid (atRA) as an anticancer drug-sensitiser in cancer cell lines and in its drug- resistant cancer cell lines MATERIALS AND METGODS: Cells were maintained by RPMI 1640 medium containing 10% fetal bovine serum. Cells were treated with 1 micro M atRA for 48 h, then with the desired concentration of anticancer drug for 24 h. Cell viability was measured spectrophotometrically at 540 nm using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Western blot analyses were performed with the desired antibodies. RESULTS: We investigated if pre-treatment with atRA enhanced the drug-sensitivity of various cancer cell lines to either 5-fluorouracil, adriamycin, or cisplatin. 5-FU (SNU638-F2) and CDDP-resistant cell (SNU638-Cis) lines, from a Korean gastric cancer cell line (SNU638) and the ADR-resistant cells (AD600) was established from a colon cancer cell line (SW620). Treatment of each cell line, with 1 micro M atRA, prior to drug exposure resulted in enhanced cell death in these cell lines. Furthermore, the effect of atRA on growth inhibition, in each drug-resistant cell line, was more obvious than in their parent cell lines. Increased activity of Transglutaminase II (TgaseII) and cleavage of Poly (ADP-ribose) polymerase (PARP) were also observed (western blot analysis CONCLUSION: Based on our data, we suggest that atRA enhances anticancer drug-induced cell death and reverses the drug-sensitivity of the drug-resistant cancer cell lines.