Sung Eun Namkoong

A Major Constituent of Green Tea, EGCG, Inhibits the Growth of a Human Cervical Cancer Cell Line, CaSki Cells, through Apoptosis, G1 Arrest, and Regulation of Gene Expression

A constituent of green tea, (-)-epigallocatechin-3-gallate (EGCG) has been known to possess antiproliferative properties. In this study, we investigated the anticancer effects of EGCG in human papillomavirus (HPV)-16 associated cervical cancer cell line, CaSki cells. The growth inhibitory mechanism(s) and regulation of gene expression by EGCG were also evaluated. EGCG showed growth inhibitory effects in CaSki cells in a dose-dependent fashion, with an inhibitory dose (ID)(50) of approximately 35 microM. When CaSki cells were further tested for EGCG-induced apoptosis, apoptotic cells were significantly observed after 24 h at 100 microM EGCG. In contrast, an insignificant induction of apoptotic cells was observed at 35 microM EGCG. However, cell cycles at the G1 phase were arrested at 35 microM EGCG, suggesting that cell cycle arrests might precede apoptosis. When CaSki cells were tested for their gene expression using 384 cDNA microarray, an alteration in the gene expression was observed by EGCG treatment. EGCG downregulated the expression of 16 genes over time more than twofold. In contrast, EGCG upregulated the expression of four genes more than twofold, suggesting a possible gene regulatory role of EGCG. This data supports that EGCG can inhibit cervical cancer cell growth through induction of apoptosis and cell cycle arrest as well as regulation of gene expression in vitro. Furthermore, in vivo antitumor effects of EGCG were also observed. Thus, EGCG likely provides an additional option for a new and potential drug approach for cervical cancer patients.

A Therapy Modality Using Recombinant IL-12 Adenovirus plus E7 Protein in a Human Papillomavirus 16 E6/E7-Associated Cervical Cancer Animal Model

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.

Protein kinase C modulates telomerase activity in human cervical cancer cells

Telomerase, a ribonucleoprotein reverse transcriptase that extends telomeres of eukaryotic chromosomes is repressed in normal somatic cells but is activated during development and neoplasia. The regulation mechanism of telomerase activity in cancer cells is not clearly known. In this report, a possible affect of PKC on telomerase activity was examined using HeLa and CUMC-6 cervical cancer cell lines. Exposure of cells to PKC inhibitor, bisindolylmaleimide I and Go6976, and high levels of PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA) resulted in the inhibition of PKC activity in both cells. Telomerase activities were also inhibited by bisindolyl-maleimide I and Go6976, respectively, in a time-dependent manner. As PKC activity changes in TPA-treated cervical cancer cells, telomerase activities were increased at low dose of TPA and decreased at high dose. The expression levels of human telomerase subunits, human telomerase RNA (hTR) were not influenced by PKC modulating drugs. In contrast, the expression of full-length human telomerase reverse transcriptase (hTERT) was decreased after exposure to bisindolylmaleimide I and Go6976 in a time-dependent manner. hTERT expression was not affected by low dose of TPA. In contrast, high dose of TPA inhibited hTERT expression level. But the expression patterns of β-deletion transcript of hTERT after 72 h of treatment with PKC inhibitors or high dose of TPA exposure were not discernable as compared with those of full-length hTERT transcripts to PKC modulating drugs. These results suggest that PKC-modulating drugs altered telomerase activities by affecting full-length hTERT expression profile in human cervical cancers.

AAC-11 overexpression induces invasion and protects cervical cancer cells from apoptosis.

To identify the genes involved in cervical carcinogenesis, we applied the mRNA differential display (DD) method to analyze normal cervical tissue, cervical cancer, metastatic lymph node, and cervical cancer cell line. We cloned a 491-bp cDNA fragment, CC231, which was present in metastatic tissue and cervical cancer cell line, but absent in normal cervical and cervical cancer tissues. The 491 bp cDNA fragment has 98% homology to the previously published sequence, AAC-11 (antiapoptosis clone 11). The levels of AAC-11 mRNA expressions in nine normal cervical and nine primary cervical cancer tissues were low. Its expression was higher in three metastatic tissues and five cervical cancer cell lines (HeLa, CaSki, SiHa, CUMC-3, and CUMC-6). Invasion of matrigel and adhesion to laminin by AAC-11 transfected CUMC-6 cells were increased by approximately 2-fold and 4-fold, respectively. Northern blot analysis showed that matrix metalloproteinase (MMP)-2 and membrane type 1 MMP (MT1-MMP) genes were found to be expressed in high levels in AAC-11-transfected cancer cells. But MMP-2 and MT1-MMP were not expressed in cells transfected with vector alone or wild-type cells. AAC-11-transfected cells expressed an elevated level of MMP-2 protein as assessed by immunoblotting. On the contrary, tissue inhibitor of MMP (TIMP-2) expression was detectable in cells transfected with vector alone or wild-type cells, respectively. Its expression was undetectable in AAC-11 transfected cells. In cervical cancer cells transfected with AAC-11, the expression of β-catenin was up-regulated. These suggest that overexpressions of MMP-2 and MT1-MMP, loss of TIMP-2 expression, and up-regulation of β-catenin by AAC-11 transfection may contribute to the development of cervical cancer invasion. AAC-11 gene transfection increased cervical cancer cell colonization. The effect of AAC-11 on cultured cervical cancer cells was associated with antiapoptotic process. Approximately 50% of the AAC-11 transfected cells in serum-free medium died after 2 weeks, compared to 1 week for vector alone or wild-type cells. These results suggest that AAC-11 may serve as a candidate metastasis-related and apoptosis-inhibiting gene in human cervical cancer.

Human papillomavirus genotyping by the DNA chip in the cervical neoplasia

Human papillomavirus (HPV) is implicated as an etiologic agent in neoplasitc lesions of the cervix. In this study, we used an HPV DNA chip to detect the type-specific sequence of HPV from cervical swabs in women with biopsy- proven neoplastic lesions of the cervix. Four hundred seventy-one patients were involved and classified into four groups based on the cytopathologic diagnosis: group I (normal, n = 290), group II (low-grade squamous intraepithelial lesions (SIL), n = 68), group III (high-grade SIL, n = 51), and group IV (invasive cervical cancer, n = 55). HPV detection rates were 17.6% (51 of 290), 73.5% (50 of 68), 92.2% (47 of 51), and 95.2% (59 of 62) in patients of group I to group IV, respectively. HPV-16 was the most frequent type (21.8%) in all specimens tested, and significantly increased the prevalence by advancing the grade of the cervical lesions (P < 0.01). The next frequent virus types were HPV-18 and HPV-58. The prevalence of multiple HPV infections was 37.3, 43.7, 27.7, and 28.8%, and no significant difference was detected between each group (P > 0.05). This suggests that the HPV DNA chip is a sensitive diagnostic tool for the detection of HPV in cervical specimens, and that it would provide more useful information on viral genotype and multiple HPV infections. Taken together, molecular biological data on HPV might be beneficial for the prevention and management of cervical neoplastic lesions.

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

PURPOSEnThe 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.nnnMATERIALS AND METHODSnCervical 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.nnnRESULTSn74 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.nnnCONCLUSIONnThese 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.

The Role of Radiation Therapy for the Extramammary Paget's Disease of the Vulva ; Experience of 3 Cases

We have experienced three cases of extramammary Pagets disease (EMPD) of the vulva that received radiation therapy (RT). Here, we analyze the efficacy of RT and include a literature survey.Three patients with EMPD of the vulva were treated with curative RT between 1993 and 1998. One of the patients had associated underlying adenocarcinoma of the vulva. The total doses of radiation administered were 54~78 Gy/6~8 weeks. Radiation fields encompassed 2 to 3 cm outer margins free from all visible disease including or not including the inguinal area using a 9 MeV electron or a 6 MV photon beam. Follow-up durations after radiotherapy were 0.6~11 years. Complete response was obtained in all three patients. Marginal failure occurred in one patient, and another patient with underlying adenocarcinoma treated by vulvectomy with bilateral inguinal lymph node dissection followed by external RT showed no relapse. Radiation induced side effects were transient acute confluent wet desquamation in the treated area resulting in mild late atrophic skin changes.Although surgery is currently considered the preferred primary treatment for EMPD, it has a high relapse rate due to the multifocal nature of the disease. We conclude that RT is of benefit in some selected cases of EMPD.

The increasing frequency of cervical cancer in Korean women under 35.

PURPOSEnThe goal of this study was to determine the clinical and epidemiological trends of cervical cancer in young Korean women. Social behavior including sexual habits has changed in Korean women, with sexual activity commencing at a younger age. These changes are likely to influence certain risk factors of cervical cancer, resulting in changing trends in the occurrence of the disease.nnnMATERIALS AND METHODSnThe incidence of cervical cancer in women less than 35 years-old between January 1990 and December 2006 was analyzed, and available medical records from January 1996 to December 2006 were reviewed. The clinical, pathological and epidemiologic characteristics and changing trends among these young patients were analyzed.nnnRESULTSnOver the last two decades, the incidence of young (< 35 years) cervical cancer patients increased, more patients had an aggressive form of the disease, and there was a higher rate of women with more advanced education. Human papillomavirus (HPV) infection was detected in 94.0% of the women (63/67) tested. HPV 16 (82.5%) and HPV 18 (12.7%) were the two most common viral infections detected throughout the study period.nnnCONCLUSIONSnThe changing trends and risk factors identified suggest a need for more active education of young women about cervical cancer prevention strategies. In addition, young women are strongly recommended to undergo a regular screening test and HPV vaccination.

A new cell line from human undifferentiated carcinoma of the ovary: establishment and characterization

A cell line designated CUMO-2 has been established from an undifferentiated ovarian carcinoma. The s.c. injection of cells into nude mice gave rise to fast-growing tumors, while the i.p. route induced a peritoneal carcinomatosis with ascites. Histopathologically, the transplanted s.c. tumors closely resembled the original tumor, but tumors developed in the peritoneal cavity were highly anaplastic. The epithelial nature of the cells was confirmed by ultrastructural analysis. Sequential cytogenetic analyses on early and late passages revealed highly aneuploid tumor cells with consistent structural aberrations of chromosomes 1, 3, 8 and 11. CUMO-2 cells were found to produce CA 125 in vitro and in vivo. Cytosol estrogen receptor (ER) was found but progesterone receptor (PR) was not measured. HLA typing indicated the presence of DR8 and DQw4. A gonadotropin-releasing hormone (Gn-RH) analog inhibited cell growth and Gn-RH receptor mRNA was detected by reverse transcription/polymerase chain reaction in this cell line. Administration of transforming growth factor β1 inhibited both cell growth and c-myc mRNA expression. This cell line demonstrated a conformational band shift in exon 7 of thep53 gene. It was a frameshift mutation.

Expression profiling of the cellular processes in uterine leiomyomas: omic approaches and IGF-2 association with leiomyosarcomas.

PURPOSEnThis study utilized both cDNA microarray and 2D protein gel electrophoresis technology to investigate the multiple interactions of the genes and proteins involved in the pathophysiology of uterine leiomyomas. Also, Gene Ontology (GO) analysis was used to systematically characterize the global expression profiles, which were found to correlate with the leiomyosarcomas.nnnMATERIALS AND METHODSnThe uterine leiomyoma biopsies were obtained from patients in the Department of Obstetrics and Gynecology, The Catholic University of Korea. Differentially expressed transcriptome and proteome, in 6 paired leiomyoma and normal myometrium, were profiled. The total RNAs from the leiomyoma and normal myometrium were labeled with Cy5 and Cy3. All specimens were punch-biopsy-obtained, and frozen in liquid nitrogen.nnnRESULTSnScreening of up to 17,000 genes identified 71 that were either up-regulated or down-regulated (21 and 50, respectively). The gene expression profiles were classified into 420 mutually dependent functional sets, resulting in 611 cellular processes, according to the gene ontology. Also, the protein analysis, using 2D gel electrophoresis, identified 33 proteins (17 up-regulated and 16 down-regulated) with more than 500 total spots, which were classified into 302 cellular processes. O f these functional profilings, transcriptomes and proteoms down-regulations were shown in the cell adhesion, cell motility, organogenesis, enzyme regulator, structural molecule activity and responses to external stimulus functional activities, which are supposed to play important roles in the pathophysiology. In contrast, up-regulation was only shown in the nucleic acid binding activity. The CDKN2A, ADH1A, DCX, IGF2, CRABP2 and KIF5C were found to increase the reliability of this study, and correlate with the leiomyosarcomas.nnnCONCLUSIONnPotentially significant pathogenetic cellular processes showed that down-regulated functional profiling has an important impact on the discovery of the pathogenic pathways in leiomyomas and leiomyosarcomas. GO analysis can also overcome the complexity of the expression profiles of cDNA microarrays and 2D protein analyses, via a cellular process level approach. Thereby, a valuable prognostic candidate gene, with real relevance to disease-specific pathogenesis, can be found at cellular process levels.