Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-Nacetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer.

Aberrant Promoter Hypomethylation of Sortilin 1: A Moyamoya Disease Biomarker / 대한뇌졸중학회지

BACKGROUND AND PURPOSE: The pathogenesis of moyamoya disease (MMD) remains poorly understood, and no reliable molecular biomarkers for MMD have been identified to date. The present study aimed to identify epigenetic biomarkers for use in the diagnosis of MMD. METHODS: We performed integrated analyses of gene expression profiles and DNA methylation profiles in endothelial colony forming cells (ECFCs) from three patients with MMD and two healthy individuals. Candidate gene mRNA expression and DNA methylation status were further validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and pyrosequencing analysis of an expanded ECFC sample set from nine patients with MMD and ten controls. We evaluated the diagnostic accuracy of the potential biomarkers identified here using receiver operating characteristic curve analyses and further measured major angiogenic factor expression levels using a tube formation assay and RT-qPCR. RESULTS: Five candidate genes were selected via integrated analysis; all five were upregulated by hypomethylation of specific promoter CpG sites. After further validation in an expanded sample set, we identified a candidate biomarker gene, sortilin 1 (SORT1). DNA methylation status at a specific SORT1 promoter CpG site in ECFCs readily distinguished patients with MMD from the normal controls with high accuracy (area under the curve 0.98, sensitivity 83.33%, specificity 100%). Furthermore, SORT1 overexpression suppressed endothelial cell tube formation and modulated major angiogenic factor and matrix metalloproteinase-9 expression, implying SORT1 involvement in MMD pathogenesis. CONCLUSIONS: Our findings suggest that DNA methylation status at the SORT1 promoter CpG site may be a potential biomarker for MMD.

Effect of Necrosis on the miRNA-mRNA Regulatory Network in CRT-MG Human Astroglioma Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Glioblastoma multiforme (GBM) is the most common adult primary intracranial tumor. The remarkable features of GBM include central necrosis. MicroRNAs (miRNAs) have been considered as diagnostic/prognostic biomarkers for many cancers, including glioblastoma. However, the effect of necrosis on the miRNA expression profile and predicted miRNA-mRNA regulatory information remain unclear. The purpose of this study is to examine the effect of necrotic cells on the modulation of miRNA and mRNA expression profiles and miRNA-mRNA network in CRT-MG cells. MATERIALS AND METHODS: We used human astroglioma cells, CRT-MG, treated with necrotic CRT-MG cells to examine the effect of necrosis on the modulation of miRNA and mRNA by next-generation sequencing. For preparation of necrotic cells, CRT-MG cells were frozen and thawed through cycle of liquid nitrogen–water bath. The putative miRNA-mRNA regulatory relationship was inferred through target information, using miRDB. RESULTS: The necrotic cells induced dysregulation of 106 miRNAs and 887 mRNAs. Among them, 11 miRNAs that had a negative correlation value of p < 0.05 by the hypergeometric test were screened, and their target mRNAs were analyzed by Gene Ontology enrichment analysis. Using the Kyoto Encyclopedia of Genes and Genomes database, we also found several necrotic cell treatment-activated pathways that were modulated by relevant gene targets of differentially expressed miRNAs. CONCLUSION: Our result demonstrated that dysregulation of miRNA and mRNA expression profiles occurs when GBM cells are exposed to necrotic cells, suggesting that several miRNAs may have the potential to be used as biomarkers for predicting GBM progression and pathogenesis.

Aberrant epigenetic regulation of GABRP associates with aggressive phenotype of ovarian cancer

Metastasis is a major cause of therapeutic failure in ovarian cancer. To elucidate molecular mechanisms of ovarian cancer metastasis, we previously established a metastatic xenograft mouse model using human ovarian carcinoma SK-OV-3 cells. Using gene expression profiling, we found that γ-aminobutyric acid (GABA)A receptor π subunit (GABRP) expression was upregulated (>4-fold) in metastatic tissues from our xenograft mice compared with SK-OV-3 cells. Importantly, GABRP knockdown diminished the migration and invasion of SK-OV-3 cells, and reduced extracellular signal-regulated kinase (ERK) activation while overexpression of GABRP exhibited significantly increased cell migration, invasion and ERK activation. Moreover, treatment with the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126 similarly suppressed the migration and invasion of SK-OV-3 cells, implying that GABRP promotes these cellular behaviors by activating the MAPK/ERK pathway. Using genome-wide DNA methylation profiling, we identified hypomethylated CpG sites in the GABRP promoter in metastatic tissues from the xenograft mice compared with SK-OV-3 cells. Treatment with a DNA methyltransferase inhibitor demonstrated that methylation at −963 bp from the GABRP transcription start site (−963 CpG site) was critical for the epigenetic regulation of GABRP. Finally, we analyzed human ovarian cancer patient samples and showed DNA hypomethylation at the GABRP −963 CpG site in advanced stage, but not early-stage, primary tumors compared with their paired normal tissues. These findings suggest that GABRP enhances the aggressive phenotype of ovarian cancer cells, and that the DNA methylation status of the GABRP −963 CpG site may be useful for predicting the metastatic potential in ovarian cancer patients.

Aberrant Hypomethylation of Solute Carrier Family 6 Member 12 Promoter Induces Metastasis of Ovarian Cancer

PURPOSE: Ovarian cancer (OC) is the most fatal of gynecological malignancies with a high rate of recurrence. We aimed to evaluate the expression of solute carrier family 6, member 12 (SLC6A12) and methylation of its promoter CpG sites in a xenograft mouse model of metastatic OC, and to investigate the regulatory mechanisms that promote aggressive properties during OC progression. MATERIALS AND METHODS: Expression of SLC6A12 mRNA was determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and DNA methylation status of its promoter CpGs was detected by quantitative methylation-specific PCR. The metastatic potential of SLC6A12 was evaluated by in vitro migration/invasion transwell assays. Gene expression and DNA methylation of SLC6A12 and clinical outcomes were further investigated from publicly available databases from curatedOvarianData and The Cancer Genome Atlas. RESULTS: SLC6A12 expression was 8.1–14.0-fold upregulated and its DNA methylation of promoter CpG sites was 41–62% decreased in tumor metastases. After treatment with DNA methyltransferase inhibitor and/or histone deacetylase inhibitor, the expression of SLC6A12 was profoundly enhanced (~8.0-fold), strongly supporting DNA methylation-dependent epigenetic regulation of SLC6A12. Overexpression of SLC6A12 led to increased migration and invasion of ovarian carcinoma cells in vitro, approximately 2.0-fold and 3.3-fold, respectively. The meta-analysis showed that high expression of SLC6A12 was significantly associated with poor overall survival [hazard ratio (HR)=1.07, p value=0.016] and that low DNA methylation levels of SLC6A12 at specific promoter CpG site negatively affected patient survival. CONCLUSION: Our findings provide novel evidence for the biological and clinical significance of SLC6A12 as a metastasis-promoting gene.

Overexpression of Mucin 13 due to Promoter Methylation Promotes Aggressive Behavior in Ovarian Cancer Cells

PURPOSE: Recent discoveries suggest that aberrant DNA methylation provides cancer cells with advanced metastatic properties. However, the precise regulatory mechanisms controlling metastasis genes and their role in metastatic transformation are largely unknown. To address epigenetically-regulated gene products involved in ovarian cancer metastasis, we examined the mechanisms regulating mucin 13 (MUC13) expression and its influence on aggressive behaviors of ovarian malignancies. MATERIALS AND METHODS: We injected SK-OV-3 ovarian cancer cells peritoneally into nude mice to mimic human ovarian tumor metastasis. Overexpression of MUC13 mRNA was detected in metastatic implants from the xenografts by expression microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The DNA methylation status within the MUC13 promoter region was determined using bisulfite sequencing PCR and quantitative methylation-specific PCR. We evaluated the effects of exogenous MUC13 on cell invasion and migration using in vitro transwell assays. RESULTS: MUC13 mRNA expression was up-regulated, and methylation of specific CpG sites within the promoter was reduced in the metastatic implants relative to those in wild-type SK-OV-3 cells. Addition of a DNA methyltransferase inhibitor to SK-OV-3 cells induced MUC13 expression, thereby implying epigenetic regulation of MUC13 by promoter methylation. MUC13 overexpression increased migration and invasiveness, compared to control cells, suggesting aberrant up-regulation of MUC13 is strongly associated with progression of aggressive behaviors in ovarian cancer. CONCLUSION: We provide novel evidence for epigenetic regulation of MUC13 in ovarian cancer. We suggest that the DNA methylation status within the MUC13 promoter region may be a potential biomarker of aggressive behavior in ovarian cancer.

DNA Hypomethylation-Mediated Overexpression of Carbonic Anhydrase 9 Induces an Aggressive Phenotype in Ovarian Cancer Cells

PURPOSE: Both genetic and epigenetic alterations can lead to abnormal expression of metastasis-regulating genes in tumor cells. Recent studies suggest that aberrant epigenetic alterations, followed by differential gene expression, leads to an aggressive cancer cell phenotype. We examined epigenetically regulated genes that are involved in ovarian cancer metastasis. MATERIALS AND METHODS: We developed SK-OV-3 human ovarian carcinoma cell xenografts in mice. We compared the mRNA expression and DNA methylation profiles of metastatic tissues to those of the original SK-OV-3 cell line. RESULTS: Metastatic implants showed increased mRNA expression of the carbonic anhydrase 9 (CA9) gene and hypomethylation at CpG sites in the CA9 promoter. Treatment of wild-type SK-OV-3 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reduced methylation of the CA9 promoter and increased CA9 mRNA expression. Eight CpGs, which were located at positions -197, -74, -19, -6, +4, +13, +40, and +86, relative to the transcription start site, were hypomethylated in metastatic tumor implants, compared to that of wild-type SK-OV-3. Overexpression of CA9 induced an aggressive phenotype, including increased invasiveness and migration, in SK-OV-3 cells. CONCLUSION: Alterations in the DNA methylation profile of the CA9 promoter were correlated with a more aggressive phenotype in ovarian cancer cells.

Regulatory B Subunits of Protein Phosphatase 2A Are Involved in Site-specific Regulation of Tau Protein Phosphorylation

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer's disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta (GSK3beta) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the GSK3beta kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.

Splenic Infarction Caused by Vivax Malaria

Splenic infarction caused by malaria is a rare complication and this is mostly caused by plasmodium falciparum. We report here on a 38 year-old female patient who developed symptomatic splenic infarction that was caused by vivax malaria. She presented with fever and left upper quadrant pain. Computed tomography showed multiple low density areas in the spleen, and the peripheral blood smear revealed plasmodium vivax infestation. We examined for other causes of splenic infarction, but all were negative. This is just the second report of symptomatic splenic infarction that was caused by vivax malaria only. Unlike the previous case, the levels of D-dimer and fibrinogen degradation factor were elevated. This may be related with the hypercoagulable state caused by malaria. Treatment was conservative and the further course was uneventful.

The Role of Abdomen-pelvis CT for the Diagnosis of Appendicitis

PURPOSE: We wanted to evaluate the value of intravenous contrast enhanced abdomen-pelvis computed tomography (CT) for diagnosing acute appendicitis and we wanted to determine which patients groups will benefit from preoperative CT. METHODS: Between January and June 2006, the medical records of 354 patients who had clinically suspected acute appendicitis were retrospectively reviewed. Appendectomy was performed in 260 patients and CT was conducted in 108 patients of the 260 patients. The 5mm slice CT scans were evaluated for the presence of appendicitis. The sensitivity, specificity and accuracy of CT were calculated. The negative appendectomy rate (NAR) was compared between the patients with CT scans and those without CT scans. Furthermore, the patients were classified into the children and adults groups and the male and female groups and the differences of the NARs were analyzed for each group. RESULTS: The sensitivity, specificity and accuracy were 95%, 93% and 94%, respectively. The NAR was lower for the patients with a CT scan (12%) compared to 27% for those patients without CT scans (P=0.002). The difference of the NAR between the preoperative CT group and the without CT group was statistically significant for the female (P=0.004) and adult groups (P=0.012) (14% vs 36%, 11% vs 26%, respectively). CONCLUSION: Preoperative intravenous contrast enhanced abdomen-pelvis CT was effective in reducing the negative appendectomy rate in patients who were suspected of having acute appendicitis. Especially, the adults and women benefit more from CT scanning and they had a significantly lower negative appendectomy rate than the children and men, respectively.

Detection of Neural Fates from Random Differentiation: Application of Support Vector MachineMin

Embryonic stem cells can be differentiated into various types of cells, requiring a tight regulation of transcription. Biomarkers related to each lineage of cells are used to guide the differentiation into neural or any other fates. In previous experiments, we reported the guided differentiation (GD)-specific genes by comparing profiles of random differentiation (RD). Interestingly 68% of differentially expressed genes in GD overlap with that of RD, which makes it difficult for us to separate the lineages by examining several markers. In this paper, we design a prediction model to identify the differentiation into neural fates from any other lineage. From the profiles of 11,376 genes, 203 differentially expressed genes between neural and random differentiation were selected by random variance T-test with 95% confidence and 5% false discovery rate. Based on support vector machine algorithm, we could select 79 marker genes from the 203 informative genes to construct the optimal prediction model. Here we propose a prediction model for the prediction of neural fates from random differentiation which is constructed with a perfect accuracy.

Clinical Significance of DAX-1 Expression using Immunihistochemical Staining in Breast Cancer / 한국유방암학회지

PURPOSE: There have been some reports that DAX-1 (Dosage-sensitive sex reversal, Adrenal hypoplasia critical region, on chromosome X, gene 1) can modify the estrogen receptor-beta and the progesterone and androgen receptors. Therefore, the aims of this work were to evaluate the expression pattern of DAX-1 in human breast cancer and its relationship to the steroid hormone receptors and other prognostic factors. METHODS: A retrospective analysis was performed using the clinical records of 161 patients diagnosed with invasive breast cancer, and who underwent surgical treatment and hormonal therapy between 1994 and 2004. We evaluated the presence and distribution of DAX-1 expressions in breast cancers using immunohistochemical staining. RESULTS: DAX-1 was expressed in 57 (35.4%) of the 161 cases. Also, the DAX-1 expression showed significant correlations with the size and nodal metastasis. In the androgen receptor positive cases (85 cases), the DAX-1 positive cases were statistically younger than the DAX-1 negative cases. In the progesterone receptor positive cases (81 cases), a statistical significance was noted between the DAX-1 expression and nodal metastasis. CONCLUSION: We conclude that DAX-1 can modulate the steroid hormone receptors including the progesterone and androgen receptors, in breast cancer. It could also be assumed that the influence of DAX-1 on the prognosis of breast cancer is different according to the kind of steroid hormonal receptor expressed.

Clinical Significance of DAX-1 Expression using Immunihistochemical Staining in Breast Cancer / 한국유방암학회지

PURPOSE: There have been some reports that DAX-1 (Dosage-sensitive sex reversal, Adrenal hypoplasia critical region, on chromosome X, gene 1) can modify the estrogen receptor-beta and the progesterone and androgen receptors. Therefore, the aims of this work were to evaluate the expression pattern of DAX-1 in human breast cancer and its relationship to the steroid hormone receptors and other prognostic factors. METHODS: A retrospective analysis was performed using the clinical records of 161 patients diagnosed with invasive breast cancer, and who underwent surgical treatment and hormonal therapy between 1994 and 2004. We evaluated the presence and distribution of DAX-1 expressions in breast cancers using immunohistochemical staining. RESULTS: DAX-1 was expressed in 57 (35.4%) of the 161 cases. Also, the DAX-1 expression showed significant correlations with the size and nodal metastasis. In the androgen receptor positive cases (85 cases), the DAX-1 positive cases were statistically younger than the DAX-1 negative cases. In the progesterone receptor positive cases (81 cases), a statistical significance was noted between the DAX-1 expression and nodal metastasis. CONCLUSION: We conclude that DAX-1 can modulate the steroid hormone receptors including the progesterone and androgen receptors, in breast cancer. It could also be assumed that the influence of DAX-1 on the prognosis of breast cancer is different according to the kind of steroid hormonal receptor expressed.

A Clinical Analysis of 100 Cases of Gasless Endoscopic Thyroidectomy

PURPOSE: With recent developments in endoscopic surgery for thyroid tumors, several approaches have been applied to endoscopic neck surgery. Gasless endoscopic thyroidectomy has some advantages over gas insufflating surgery. We evaluated the role of gasless endoscopic thyroidectomies on various thyroid tumors including malignant thyroid tumors. METHODS: We performed thyroidectomies for a total of 195 patients who were admitted to Uijongbu St. Mary's Hospital from November 1999 to February 2002. We compared the clinical data of 100 patients who received gasless endoscopic thyroidectomies with the data of 95 patients who underwent conventional thyroidectomies. Furthermore, we subclassified the 100 patients who received a gasless endoscopic thyroidectomy into two groups, before and after the start of 2001. RESULTS: The analysis of the clinical data showed that an endoscopic thyroidectomy gave superior results with respect to the time of postoperative recovery and cosmetic results. Also, the patients in the second half group showed significantly shorter operation times reduced from 153.4 min. to 128.6 min. and length of hospital stay similarly reduced(3.55 days vs. 5.35 days). CONCLUSION: A gasless endoscopic thyroidectomy is a safe and technically feasible alternative to a conventional thyroi-dectomy in benign and early malignant tumors, and provides good cosmetic results.

A Study on the Synthesis , Labeling and Its Biodistribution of Estradiol Derivatives / 대한핵의학회잡지

OBJECTIVES: Due to the heterogeneous receptor distribution and changes of receptor status over time, the biochemical measurement of estrogen receptor status of biopsy specimens is not sufficient to diagnose breast cancer. As a result, I-123 labeled estradiols have been applied for the diagnosis. The purpose of this study was to develop a suitable radioligand for imaging estrogen receptor-positive human breast tumors. METHODS: Among the various estradiol derivatives, 17alpha-[123I]iodovinyl estradiol ([123I]IVE) has been prepared from 17alpha-ethynyl estradiol. Labeling of E-17alpha-[123I]iodovinyl estradiol (E-[123I]IVE) was carried out using peracetic acid with [123I]NaI and Z-[123I]IVE labelling was archived using chloamine- T/HCl solution with [123I]NaI. Labeling yield was determined by silica thin-layer chromatography (TLC) and radiochemical purity was measured by high performance liquid chromatography (HPLC). The biodistribution of E-[123I]IVE was measured in immature female rats at 60 min, 120 min and 300 min after injection. RESULTS: The labeling yield of two isomers was 92% and 94% (E-[123I]IVE and Z-[123I]IVE, respectively). The radiochemical purity was more than 98% after purification. The highest uptake was observed at 120 min in uterus (3.11% ID/g for E-[123I]IVE). CONCLUSION: These results suggest the possibility of using E-[123I]IVE as an imaging agent for the evaluation of the presence of estrogen receptor in patients with breast cancer.

Study for the Synthesis of 123IIdoxifene and Its Uptake in the Breast Cancer Cell / 대한핵의학회잡지

PURPOSE: Idoxifene is currently entering phase II clinical trials for the treatment of advanced breast cancer. The radiolabeled idoxifene using 123I provides an opportunity for clinical pharmacology with single photon emission computed tomography (SPECT). The purpose of this study was to prepare radiolabeled idoxifene using 123I and to determine its cell uptake of breast cancer cell line. MATERIALS AND METHODS: With a view to evaluating new anticancer drugs, we are investigating the novel antiestrogen pyrrolidino- 4-iodotamoxifen (idoxifene). [123I]Idoxifene has been prepared in no-carrier-added form using a tributyl stannylated precursor which has been synthesized by means of (2-chloroethoxy)benzene with (+/-)-2- phenylbutanoic acid on the basis of previously reported standard methods. The biodistribution and dynamic behavior of the compound were investigated using the comparative breast cancer cell line, MCF-7 (estrogen receptor-positive) and MDA-MB-468 (non-estrogen receptor). RESULTS AND CONCLUSION: Acylation of (2-chloroethoxy)benzene with (+/-)-2-phenylbutanoic acid gave the versatile ketone (81%) which reacted with 1,4-diiodobenzene to give triphenylethylene as a mixture of E and Z geometric isomers, which were separated by the recrystallization in ethanol. The E-isomer was treated with pyrrolidine to give idoxifene (67%). In order to incorporate radioactive iodine into the 4-position, the 4-stannylated precursor was prepared (30%). The yield of radioiodination was 90-92% with a high radiochemical purity greater than 98%. The ratio of tumor uptake of the breast cancer cell line between MCF-7 and MDA-MB-468 was about 1.7.

Surgical experience of Takayasu'arteritis / 대한흉부외과학회지

No abstract available.