In-Wook Kim

Differential DNA copy number aberrations in the progression of cervical lesions to invasive cervical carcinoma

Host genomic alterations in addition to human papillomavirus (HPV) are needed for cervical precursor lesions to progress to invasive cancer because only a small percentage of women infected by the virus develop disease. However, the genomic alterations during the progression of cervical lesions have not been systematically examined. The aim of this study was to identify differential genomic alterations among cervical intraepithelial neoplasia CIN1, CIN2, CIN3 and cervical squamous cell carcinoma (SCC). Genomic alterations were examined for 15 cases each of CIN1, CIN2, CIN3 and SCC by array-based comparative genomic hybridization (array CGH). The chromosomal regions showing significant differential in DNA copy number aberrations (DCNAs) among CIN1, CIN2, CIN3 and SCC were successfully identified by resampling-based t-test. The chromosomal regions of 5q35.3 and 2q14.3 showed significant DCNAs between CIN1 and CIN2, and between CIN2 and CIN3, respectively, while a significant difference in DCNAs between CIN3 and SCC was observed at 1q24.3, 3p14.1, 3p14.2, 5q13.2, 7p15.3, 7q22.1 and 13q32.3. In addition, the status of DCNAs in 1q43, 2p11.2, 6p11.2, 7p21.1, 7p14.3, 10q24.1, 13q22.3, 13q34 and 16p13.3 was conserved throughout the progression of CIN to SCC. The presence of differential and common DCNAs among CIN1, CIN2, CIN3 and SCC supports that the CIN progression may include continual clonal selection and evolution. This approach also identified 34 probe sets consistently overexpressed when amplified, suggesting an unbiased identification of candidate genes in SCC during cervical cancer progression.

Selenium enhances the efficacy of Radachlorin mediated-photodynamic therapy in TC-1 tumor development

Selenium, an essential trace element possessing anti-carcinogenic properties, can induce apoptosis in cancer cells. Our goal was to investigate the enhanced antitumor effects of photodynamic therapy (PDT) plus selenium in TC-1 tumor cells and implanted mice. Cell viability was evaluated at various time intervals after PDT treatment and/or selenium by methyl thiazolyl tetrazolium (MTT) assay. When only PDT treatment was administered to TC-1 tumor cells, TC-1 cell growth recovered over time. On the other hand, co-treatment of PDT and selenium extended the inhibition time of tumor cell growth. Co-treatment of PDT and selenium showed serious morphological changes in TC-1 cells and induced a more apoptotic population by FACS analysis. By signal transduction pathway SuperArray analysis, genes closely involved in the NFκB, p53 and phopholipase C pathways, such as VCAM1, MDM2 and FOS, were significantly downregulated at least 10-fold in TC-1 cells following PDT and selenium cotreatment. In an in vivo study, tumor-bearing mice were intravenously injected with Radachlorin 3 h before irradiation with 300 J/cm2 of light. Selenium was administered daily for 20 days. Combination therapy against the mouse tumors generated by TC-1 cells was more effective than PDT or selenium alone. These data suggest that selenium plus PDT can induce a significant tumor suppression response compared with PDT alone. Additionally, it can be an effective anticancer therapy strategy.

Target-based molecular signature characteristics of cervical adenocarcinoma and squamous cell carcinoma.

There is an urgent need for molecular marker studies of adenocarcinoma (AC) and squamous cell carcinoma (SCC) of the uterine cervix. This study utilized oligomicroarray and pathway analyses to characterize a transcriptomic signature with molecular networks associated with AC and SCC. A 10K oligomicroarray was used to identify potential transcripts that were differentially expressed in cervical cancers from 28 patients and common reference RNAs from 17 different normal cervixes. Molecular networks were correlated using genomics tools to globally explore cellular pathways. Gene expression levels of 46 transcripts separated cancer samples into AC and SCC groups. Genes including: KRT17, IGFBP2, CALCA and VIPR1 were differentially expressed in AC and SCC. In addition, we identified a transcriptomic signature that predicted tumor classification and progression based upon its cellular processes. The downregulated signatures for SCC were cell death of pheochromocytoma cells (P=0.0037), apoptosis of neurons (P=0.009) and damage to DNA (P=0.0038). By contrast, the upregulated molecular signatures in AC were immunological disorder (P=0.006), splenomegaly (P=0.0053) and hepatic system disorder (P=0.006). The G2/M DNA damage checkpoint regulation pathway (P=0.05) was found to be significantly linked to IGF1R as a new regulatory component of a putative cytoplasmic signaling cascade in SCC. By contrast, the antigen presenting canonical pathway (P=0.038) appeared to be linked to PPARγ in AC. Taken together, these experiments provide important new information regarding the role of molecular networks in mediating SCC and AC, possibly through two independent pathways, and contribute to provide new targets for the prevention and treatment of cervical cancer.

Synthesis of chlorin-based unsaturated fatty acid conjugates: Their in vitro phototoxicity on TC-1 cancer cell line

Chlorin-based photosensitizers in photodynamic therapy are the promising anticancer agents, but some of their properties such as specific-targeting to tumor need to be improved. The aim of this study was to synthesize chlorin-based unsaturated fatty acid conjugates to obtain an optimal photosensitizers. Thus four chlorin-based fatty acid conjugates were successfully synthesized through an esterification reaction using carbodiimide coupling reagents in enough yields. Then, structures of these conjugates were confirmed by (1)H NMR, MALDI-MS, and UV-vis spectroscopy. Furthermore, their in vitro phototoxicity and cellular uptake were evaluated on TC-1 lung cancer cell line and HeLa cell line.

Multiplexed bead-based immunoassay of four serum biomarkers for diagnosis of ovarian cancer.

The purpose of the present study was to evaluate multiplex liquid assay-based measurement of multiple ovarian cancer-associated biomarkers such as hemoglobin, haptoglobin and apolipoprotein E, together with CA125, which has been widely used in the diagnosis of ovarian cancer, in order to provide a higher diagnostic power. We measured the serum levels of CA125, hemoglobin, haptoglobin and apolipoprotein E from the serum of 76 healthy individuals and 69 ovarian cancer patients using a multiplex liquid assay system, Luminex 100. The results were analyzed according to normal versus ovarian cancer, tumor stages and tumor histology. In addition, to validate the use of these biomarkers for the diagnosis of ovarian cancer, the sensitivity and specificity of each biomarker was analyzed by its receiver operating characteristics (ROC) curve. The serum levels of all four biomarkers in ovarian cancer patients were significantly higher than those of healthy individuals. When CA125 was combined with the biomarkers, the overall sensitivity and specificity were significantly improved in the ROC curve, which showed 95 and 75% sensitivity and specificity, respectively. At 95% specificity for all stages the sensitivity increased to 75% compared to 41% for CA125 alone. For stage I+II increased the sensitivity to 68% from 36% for CA125 alone. For stage III+IV the corresponding values were 100 and 95%, respectively. Taken together, the new combination of hemoglobin, haptoglobin and apolipoprotein E with CA125 significantly improved both the sensitivity and the specificity of ovarian cancer diagnosis compared with those of individual biomarkers. These findings suggest the benefit of the combination of these markers for the diagnosis of ovarian cancer.

Identification of candidate biomarkers using the Experion™ automated electrophoresis system in serum samples from ovarian cancer patients

Ovarian cancer is the most common cause of disease-related death in women globally. Detection of ovarian cancer using new biomarkers is necessary for early diagnosis. To date, there have been no obvious biomarkers for ovarian cancer detection in the incipient stage. In this study, we discovered potential diagnostic serological biomarkers for ovarian cancer using the Experion™ automated electrophoresis system. Sera from 14 healthy women and 84 ovarian cancer patients at stages I- IV were applied to the Experion to compare the protein expression levels. To examine the protein expression pattern of Experion data, proteins in the samples were resolved using 10 and 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. The candidate biomarkers elevated in ovarian cancer were purified and determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. α-2-macroglobulin (173.7 kDa), ceruloplasmin (147 kDa), inter-α-trypsin inhibitor family heavy chain-related protein (126 kDa), C-1 inhibitor (115.2 kDa) and hemoglobin α/β (14.4 kDa were overexpressed in the ovarian cancer sera. This study documents a novel way to measure ovarian cancer or cancer-related proteins for biomarkers using the Experion assay system, which should be easily adaptable for high-throughput diagnosis to establish databases of ovarian cancer for clinical applications.

Synthesis of di-pyropheophorbide-a-platinum(II) complex and the in vitro cytotoxicity against TC-1 tumor cells

This report focuses on the conjugation of a chemotherapeutic drug and photodynamic therapy (PDT) agent with the intention of obtaining an optimal anticancer agent for use in combination cancer treatment. We have used pyropheophorbide-a (PPa) as a PDT agent, which was linked with ethylenediamine using carbodimide/N-hydroxysuccinicimide coupling reagents to obtain a resulting reactive amine group. Next, the free amine-bearing PPa and potassium tetrachloroplatinate (K2PtCl4) were reacted in aqueous solution to obtain PPa-cisplatin complex. Subsequently, the PPa-based platinum complex was characterized through several methods including NMR spectroscopy, mass spectrometer and UV absorption spectroscopy and its in vitro cytotoxicity and cellular uptake were evaluated on the TC-1 cancer cell line. The results indicated that the conjugate has a greater cytotoxic effect in the dark than cisplatin, even though its phototoxic effect was slightly lower than that of free photosensitizer; in addition, the cellular uptake of complex at 0.125 and 0.25 µM was higher than that of free photosensitizer.

Direct measurement of singlet oxygen by using a photomultiplier tube-based detection system

The effective dosimetry for photodynamic therapy (PDT) can be specified by direct measurement of singlet oxygen ((1)O2) production. The purpose of this study was to investigate the feasibility of a newly developed photomultiplier tube (PMT)-based singlet oxygen detection (SOD) system. The lowest and highest (1)O2 concentrations detectable by the PMT-SOD system were 15nM and 10μM, respectively. Dose-dependent quenching, by NaN3, of the fluorogenic reaction was observed, which was negatively correlated with the (1)O2 level measured by the PMT-SOD system. The lifetime of (1)O2, as measured by the PMT-SOD system, was found to be lengthened when H2O was replaced with deuterium oxide. (1)O2 photon counts were significantly and dose-dependently correlated with intracellular fluorescence intensity after photosensitizer treatments. In vitro cell viability test and in vivo xenografted-tumor mass shrinkage showed a positive association between PDT-induced cytotoxicity and (1)O2 production concomitantly measured by the PMT-SOD system. It was concluded that the PMT-SOD system is capable of measuring (1)O2 production directly and accurately, demonstrating that this system can be useful in the determination of dosimetry for PDT.

RETRACTED ARTICLE: Antimicrobial and antifungal effects of green tea extracts against microorganisms causing vaginitis

Green tea extracts (GTE) including catechins and caffeine possess strong antimicrobial activity against a variety of microorganisms. In this study, we examined their possible antimicrobial and antifungal effects on bacteria (Proteus mirabilis and Streptococcus pyogenes) and yeast species (Candida albicans) that cause vaginitis. The GTE and ethyl acetate fractions exhibited strong growth inhibitory activity against P. mirabilis and S. pyogenes, whereas the aqueous fraction possessed poor antimicrobial activity. Among the catechins, epigallocatechin gallate showed strong antibacterial activity. C. albicans was potently inhibited by the methylene chloride fraction and caffeine, while antifungal activity of GTE was contributed by caffeine. Furthermore, we evaluated whether in vitro activity of GTE included purified solvent fractions that were stable to heat and pH. Antimicrobial activity was potent in alkaline environment for P. mirabilis and acid environment for S. pyogenes and C. albicans. Herein GTE can be a valuable therapeutic agent for the treatment of vaginal-related pathogens.

In vitro phototoxicity and dark-toxicity of a novel synthesized pyropheophorbide-a-paclitaxel conjugate against cancer cell lines

Synthesis of pyropheophorbide-a-paclitaxel (PPa-PTX) conjugate was performed in high yield with the aim of searching for an optimal agent for cancer treatment. After synthesis, the conjugate was confirmed to be linked through an ester bond at the 2′ position of the paclitaxel moiety using multi-nuclear magnetic resonance spectroscopy. Phototoxicity of PPa and PPa-PTX conjugate, as well as PTX, was evaluated with three human cancer cell lines (HeLa, CaSki and TC-1). The new conjugate at 0.01–0.06 μM displayed 20–40% higher phototoxicity in HeLa and CaSki cell lines than free PPa and PTX. Furthermore, cellular uptake of these bio-molecules was examined by confocal laser scanning microscopy. Although PPa-PTX showed a delayed uptake compared to PPa, it penetrated completely into cells within 24 h incubation.