Su Mi Bae

Antiproliferative effects of quercetin through cell cycle arrest and apoptosis in human breast cancer MDA-MB-453 cells

To explore the anticancer effects of the flavonoid quercetin on human breast cancer MDA-MB-453 cells via cell cycle regulation and the induction of apoptosis, the antiproliferative effect of quercetin was first examined by MTT assay. When MDA-MB-453 cells were treated with quercetin for various periods of time (3–24 hrs) and at various doses (1–100 μM), cell growth decreased significantly in a time-and dose-dependent manner. To elucidate the mechanism underlying the antiproliferative effect of quercetin, cell cycle progression and the induction of apoptosis in MDA-MB-453 cells exposed to 100 μM quercetin for 24 hrs were investigated. Quercetin caused a remarkable increase in the number of sub-G1 phase cells, and an Annexin-V assay revealed that exposure to quercetin affected apoptosis. Moreover, treatment with quercetin increased Bax expression but decreased Bcl-2 expression. Cleaved caspase-3 and PARP expression was also increased by quercetin. Thus, quercetin has probable anticancer activity. Our results suggest the existence of multiple pathways for the induction of cell cycle arrest and apoptosis by quercetin.

Targeted cellular process profiling approach for uterine leiomyoma using cDNA microarray, proteomics and gene ontology analysis

Summary.  This study utilized both cDNA microarray and two‐dimensional protein gel electrophoresis technology to investigate the multiple interactions of genes and proteins involved in uterine leiomyoma pathophysiology. Also, the gene ontology analysis was used to systematically characterize the global expression profiles at cellular process levels. We profiled differentially expressed transcriptome and proteome in six‐paired leiomyoma and normal myometrium. Screening up to 17 000 genes identified 21 upregulated and 50 downregulated genes. The gene‐expression profiles were classified into mutually dependent 420 functional sets, resulting in 611 cellular processes according to the gene ontology. Also, protein analysis using two‐dimensional gel electrophoresis identified 33 proteins (17 upregulated and 16 downregulated) of more than 500 total spots, which was classified into 302 cellular processes. Of these functional profilings, downregulations of transcriptomes and proteoms were shown in cell adhesion, cell motility, organogenesis, enzyme regulator, structural molecule activity and response to external stimulus functional activities that are supposed to play important roles in pathophysiology. In contrast, the upregulation was only shown in nucleic acid‐binding activity. Taken together, potentially significant pathogenetic cellular processes were identified and showed that the downregulated functional profiling has a significant impact on the discovery of pathogenic pathway in leiomyoma. Also, the gene ontology analysis can overcome the complexity of expression profiles of cDNA microarray and two‐dimensional protein analysis via its cellular process‐level approach. Therefore, a valuable prognostic candidate gene with relevance to disease‐specific pathogenesis can be found at cellular process levels.

CpG-ODN-stimulated dendritic cells act as a potent adjuvant for E7 protein delivery to induce antigen-specific antitumour immunity in a HPV 16 E7-associated animal tumour model.

We previously reported that both E7 and CpG‐oligodeoxynucleotide (ODN) are required for protecting animals from human papillomavirus (HPV) 16 E7‐associated tumour challenge. Here we investigate dendritic cells (DC)‐based approach in this protection. In the study, we isolated bone marrow‐derived DC and stimulated DC with E7 and ODN. In vitro stimulation of DC with E7 plus ODN resulted in more production of interleukin‐12, as compared to that with E7 or ODN alone. Further injection with E7+ODN‐stimulated DC resulted in more significant tumour protection, as compared to stimulation with E7 or ODN alone. We further evaluated the levels of immune responses induced by DC stimulated with E7+ODN. We observed little enhancement of E7‐specific antibody and T helper cell proliferative responses by E7+ODN stimulation, as compared to E7 stimulation. However, there was some enhancement of interferon‐γ (IFN‐γ) production from CD4+ T cells and a more significant production of IFN‐γ from CD8+ T cells by E7+ODN stimulation, as compared to E7 stimulation alone. This was consistent with intracellular IFN‐γ staining levels of CD8+ T cells. Tumour protection further appeared to be mediated by CD8+ T cells, as determined by in vivo T‐cell depletion. Thus, these data suggest that upon ODN stimulation DC might function as a potent adjuvant for E7 protein delivery for induction of protective cellular immunity against HPV E7‐associated tumour challenge.

Polymerase chain reaction-based fluorescent Luminex assay to detect the presence of human papillomavirus types.

Becuase 40% of human papillomavirus (HPV) infections are mixed infections, the accurate identification of high‐risk HPV genotypes in mixed infections is important for defining a womans risk for progression to cervical cancer. Thus, advanced Luminex‐based HPV genotyping has been developed to simultaneously detect the presence of multiple HPV types. Here, we describe the development of a Luminex‐based HPV genotyping that combines polymerase chain reaction amplification with hybridization to fluorescence‐labeled polystyrene bead microarrays (Luminex suspension array technology). New HPV type‐specific oligonucleotide probes and YBT L1/GP6‐1 primers were used to detect the HPV types in 132 clinical samples. We simultaneously evaluated the usefulness of this technique on clinical samples. We detected 15 specific HPV types (6, 16, 18, 31, 35, 42, 51, 52, 55, 56, 58, 59, 66, 67 and 68) examined with specificity without known cross‐reaction to other HPV types. The detection limit for the different HPV types was above 500 plasmids. We compared the performance of the Luminex‐based assay to the established HPV DNA microarray chip for polymerase chain reaction products derived from 53 clinical samples. The evaluation showed excellent agreement. The Luminex‐based HPV genotyping was a sensitive, reproducible technique for the simultaneous genotyping of all clinically relevant genital HPV types. This assay system may be used to provide critical clinical information for early detection of HPV, especially in cases where the HPV copy numbers are low and the latency period of HPV infection is prolonged. (Cancer Sci 2007; 98: 549–554)

Development of Multiplexed Bead-Based Immunoassays for the Detection of Early Stage Ovarian Cancer Using a Combination of Serum Biomarkers

CA125 as a biomarker of ovarian cancer is ineffective for the general population. The aim of this study was to evaluate multiplexed bead-based immunoassay of multiple ovarian cancer-associated biomarkers such as transthyretin and apolipoprotein A1, together with CA125, to improve the identification and evaluation of prognosis of ovarian cancer. We measured the serum levels of CA125, transthyretin, and apolipoprotein A1 from the serum of 61 healthy individuals, 84 patients with benign ovarian disease, and 118 patients with ovarian cancer using a multiplex liquid assay system, Luminex 100. The results were then analyzed according to healthy and/or benign versus ovarian cancer subjects. When CA125 was combined with the other biomarkers, the overall sensitivity and specificity were significantly improved in the ROC curve, which showed 95% and 97% sensitivity and specificity, respectively. At 95% specificity for all stages the sensitivity increased to 95.5% compared to 67% for CA125 alone. For stage I+II, the sensitivity increased from 30% for CA125 alone to 93.9%. For stage III+IV, the corresponding values were 96.5% and 91.6%, respectively. Also, the three biomarkers were sufficient for maximum separation between noncancer (healthy plus benign group) and stage I+II or all stages (I−IV) of disease. The new combination of transthyretin, and apolipoprotein A1 with CA125 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.

Equol induces apoptosis through cytochrome c-mediated caspases cascade in human breast cancer MDA-MB-453 cells

This study investigated the role of the caspase activation cascade in extrinsic and intrinsic apoptosis induced by equol in human breast cancer MDA-MB cells. First, the antiproliferative effect of equol was determined in cells treated with 1-100 microM equol for 24, 48, and 72h. Equol significantly inhibited cell proliferation in a dose-dependent manner (p<0.05). Exposure to 50 or 100 microM equol for 72h strongly promoted apoptosis. Under the same conditions, remarkable cytochrome c release was observed. Subsequently, caspase-9, which acts in mitochondria-mediated apoptosis, was cleaved by equol at high concentrations, but caspase-8 activation of receptor-mediated apoptosis was not observed. At both equol concentrations, the caspase-8 and -9 activity assays showed similar patterns. In addition, equol treatment activated caspase-3, which is downstream from caspase-9, and this was accompanied by the cleavage of capase-6 and -7. Activation of these caspases leads to increased activation of PARP, lamin, and ICAD. This study suggests that equol induces the intrinsic pathway of apoptosis via caspase-9 and cytochrome c, independent of caspase-8, in human breast cancer MDA-MB-453 cells.

Self-assembled, covalently linked, hollow phthalocyanine nanospheres

A rational design and synthesis of covalently linked Pc nanospheres with a very thin shell and hollow interior, composed of approximately 12 000 Pc units on average, was demonstrated through thiol–ene “click” chemistry without using any templates or emulsifiers. The ZnPc nanospheres allow post-synthetic modification to improve their dispersibility in aqueous solution without altering the morphology of the nanospheres or the properties of ZnPc cores. More importantly, the ZnPc nanospheres showed higher singlet oxygen generation efficiency and in vitro phototoxicity than monomeric Pc molecules, suggesting that ZnPc nanospheres are potentially useful as a PS for PDT. We anticipate that the ZnPc nanospheres would allow other post-synthetic modifications such as the introduction of targeting ligands to deliver the nanospheres to specific target sites and perform a dual chemo- and photodynamic therapy by the encapsulation of therapeutic agents. The easy synthesis of a hollow spherical framework with a high Pc content, coupled with facile post-synthetic modification may allow Pc nanospheres to be a versatile platform for a diverse range of medical and non-medical applications.

Synergistic Anti-Tumor Effects of Combination of Photodynamic Therapy and Arsenic Compound in Cervical Cancer Cells: In Vivo and In Vitro Studies

The effects of As4O6 as adjuvant on photodynamic therapy (PDT) were studied. As4O6 is considered to have anticancer activity via several biological actions, such as free radical production and inhibition of VEGF expression. PDT or As4O6 significantly inhibited TC-1 cell proliferation in a dose-dependent manner (P<0.05) by MTT assay. The anti-proliferative effect of the combination treatment was significantly higher than in TC-1 cells treated with either photodynamic therapy or As4O6 alone (62.4 and 52.5% decrease compared to vehicle-only treated TC-1 cells, respectively, P<0.05). In addition, cell proliferation in combination of photodynamic therapy and As4O6 treatment significantly decreased by 77.4% (P<0.05). Cell survival pathway (Naip1, Tert and Aip1) and p53-dependent pathway (Bax, p21Cip1, Fas, Gadd45, IGFBP-3 and Mdm-2) were markedly increased by combination treatment of photodynamic therapy and As4O6. In addition, the immune response in the NEAT pathway (Ly-12, CD178 and IL-2) was also modulated after combination treatment, suggesting improved antitumor effects by controlling unwanted growth-stimulatory pathways. The combination effect apparently reflected concordance with in vitro data, in restricting tumor growth in vivo and in relation to some common signaling pathways to those observed in vitro. These findings suggest the benefit of combinatory treatment with photodynamic therapy and As4O6 for inhibition of cervical cancer cell growth.

cDNA Microarray Analysis of Gene Expression Profiles Associated with Cervical Cancer.

PURPOSE The molecular pathology of cervical cancers associated with human papillomavirus infection is presently unclear. In an effort to clarify this issue, the gene expression profiles and pathogenic cellular processes of cervical cancer lesions were investigated. MATERIALS AND METHODS Cervical cancer biopsies were obtained from patients at the Department of Obstetrics and Gynecology, The Catholic University of Korea. The disease status was assigned according to the International Federation of Gynecology and Obstetrics. The tissue samples of 11 patients (invasive cancer stage Ib- IIIa) were investigated by a cDNA microarray of 4, 700 genes, hierarchical clustering and the Gene Ontology (GO). Total RNA from cervical cancer and non-lesional tissues were labeled with Cy5 and Cy3. The HaCaT human epithelial keratinocyte cell line was used as a negative control cell. The stages of invasive cancer were Ib to IIIb. All specimens were obtained by punch-biopsies and frozen in liquid nitrogen until required. RESULTS 74 genes, showing more than a 2 fold difference in their expressions, were identified in at least 8 of the 11 patients. Of these genes, 33 were up-regulated and 41 were down-regulated. The gene expression profiles were classified into 345 mutually dependent function sets, resulting in 611 cellular processes according to their GO. The GO analysis showed that cervical carcinogenesis underwent complete down-regulation of cell death, protein biosynthesis and nucleic acid metabolism. The genes related to nucleic acid binding and structural molecule activity were also significantly down-regulated. In contrast, significant up-regulation was shown in the skeletal development, immune response and extracellular activity. CONCLUSION These data are suggestive of potentially significant pathogenetic cellular processes, and showed that the down-regulated functional profiling has an important impact on the discovery of pathogenic pathways in cervical carcinogenesis. GO analysis can also overcome the complexity of the expression profiles of the cDNA microarray via a cellular process level approach. Thereby, a valuable prognostic candidate gene, with real relevance to disease-specific pathogenesis, can be found at the cellular process levels.

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.