Hyemi Kim

Role of reactive oxygen species-mediated mitochondrial dysregulation in 3-bromopyruvate induced cell death in hepatoma cells : ROS-mediated cell death by 3-BrPA

Hexokinase type II (HK II) is the key enzyme for maintaining increased glycolysis in cancer cells where it is overexpressed. 3-bromopyruvate (3-BrPA), an inhibitor of HK II, induces cell death in cancer cells. To elucidate the molecular mechanism of 3-BrPA-induced cell death, we used the hepatoma cell lines SNU449 (low expression of HKII) and Hep3B (high expression of HKII). 3-BrPA induced ATP depletion-dependent necrosis and apoptosis in both cell lines. 3-BrPA increased intracellular reactive oxygen species (ROS) leading to mitochondrial dysregulation. NAC (N-acetyl-l-cysteine), an antioxidant, blocked 3-BrPA-induced ROS production, loss of mitochondrial membrane potential and cell death. 3-BrPA-mediated oxidative stress not only activated poly-ADP-ribose (PAR) but also translocated AIF from the mitochondria to the nucleus. Taken together, 3-BrPA induced ATP depletion-dependent necrosis and apoptosis and mitochondrial dysregulation due to ROS production are involved in 3-BrPA-induced cell death in hepatoma cells.

Double-stranded RNA mediates interferon regulatory factor 3 activation and interleukin-6 production by engaging Toll-like receptor 3 in human brain astrocytes

Toll‐like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. In this study, human brain astrocytes were found to constitutively express TLR3, and this expression was increased by interferon‐γ (IFN‐γ) or double‐stranded RNA (dsRNA). Treatment employing dsRNA in astrocytes induced IFN regulatory factor 3 (IRF3) phosphorylation, dimer formation and nuclear translocation followed by STAT1 activation. This treatment also activated nuclear factor‐κB, p38 and c‐Jun N‐terminal kinase significantly, while activating extracellular signal‐regulated kinase to a lesser extent. Treatment with anti‐TLR3 antibody inhibited dsRNA‐mediated interleukin‐6 (IL‐6) production. In the presence of mitogen‐activated protein kinase inhibitors, astrocytes failed to secrete IL‐6 in response to dsRNA treatment. Therefore, dsRNA‐induced IL‐6 production is dependent on mitogen‐activated protein kinases and type I IFN production is dependent on IRF3 in brain astrocytes. These results suggest that brain inflammation, which produces inflammatory cytokines and type I IFNs, may enhance TLR3 expression in astrocytes. Additionally, upregulated TLR3 might modulate inflammatory processes by producing proinflammatory cytokines.

Rhodamine cyclic hydrazide as a fluorescent probe for the detection of hydroxyl radicals.

A new rhodamine fluorescent probe for monitoring ˙OH has been developed based on the oxidative C-H abstraction reaction of rhodamine cyclic hydrazide. The probe exhibits excellent selectivity for ˙OH with virtually no interference by other ROS/RNS species. Fluorescent imaging of A549 and RAW264.7 cells is also successfully demonstrated to detect intracellular ˙OH in live cells.

Differentiation of benign and malignant ampullary obstructions on MR imaging

OBJECTIVE To define diagnostic criteria for differentiating malignant ampullary carcinoma from benign ampullary obstruction on MR imaging. MATERIALS AND METHODS Nineteen patients with ampullary carcinoma and 22 patients with benign ampullary obstruction were enrolled. At the first session, two radiologists independently evaluated specific imaging findings, and then reached consensus decisions. At the second session, another two radiologists, who were informed about useful differentiation criteria based on the results from the first session, reviewed images and determined the causes of ampullary obstruction. Sensitivity and specificity were calculated for each interpretation session, and the Cohen κ statistic was used to evaluate interobserver agreement. RESULTS Findings of the presence of an ampullary mass (P<0.001), papillary bulging (P<0.001), irregular (P=0.021) and asymmetric (P<0.001) common bile duct (CBD) narrowing, and proportional biliary dilatation (P<0.001) were more commonly seen in patients with an ampullary carcinoma. The sensitivity and specificity of the first session were 84.2% and 86.4% after consensus. The sensitivity increased to 100% for both the readers at the second session, while the specificity decreased to 63.6% and 59.1%, respectively. CONCLUSIONS Identification of an ampullary mass, papillary bulging, irregular and asymmetric narrowing of the CBD, or proportional biliary dilatation may improve the diagnosis of ampullary carcinoma in patients with ampullary obstruction.

The prognostic effects of tumor infiltrating regulatory T cells and myeloid derived suppressor cells assessed by multicolor flow cytometry in gastric cancer patients

The prognostic effects of tumor infiltrating lymphocytes (TILs), especially regulatory T cells (Tregs) and myeloid derived suppressing cells (MDSCs) are inconclusive in gastric cancers. We investigated the frequencies of TILs including CD8+ T cells, CD45+CD4+CD25± FOXP3+ Tregs, CD45+CD11b+ CD14+ HLA−DR− MDSCs in 28 gastric cancer tissues by using multicolor flow cytometry. In gastric cancer tissue, the percentage of Tregs among the CD4+ T cell subset was substantially increased compared to that of Tregs among peripheral blood CD4+ T cells from the controls. High frequency of CD8+ T cells among CD3+ T cells correlated with increased overall survival (OS) (p = 0.005). High frequency of Tregs among CD4+ T cells correlated with increased OS (p < 0.001), and disease-free survival (DFS) (p = 0.039) and was an independent prognostic factor in OS (Hazard ratio: 0.047; 95% confidence interval, 0.006-0.372; p = 0.004). High frequency of MDSCs among total examined cells correlated with decreased OS (p = 0.027) and was an independent prognostic factor in OS (Hazard ratio 8.601; 95% confidence interval, 1.240-59.678; p = 0.029). We have demonstrated that high levels of Tregs among tumor-infiltrating CD4+ T cells were favorable, but an increased proportion of MDSCs was an adverse independent prognostic factor in gastric cancer. Our results may provide important insights for future immunotherapy in gastric cancer.

Usefulness of impulse oscillometry and fractional exhaled nitric oxide in children with Eosinophilic bronchitis.

Eosinophilic bronchitis (EB) is a common cause of chronic cough. Although EB shares many immunopathologic features with asthma, it does not show airway hyperresponsiveness or reversible airway obstruction by spirometry.

Targeted therapy for Epstein-Barr virus-associated gastric carcinoma using low-dose gemcitabine-induced lytic activation

The constant presence of the viral genome in Epstein-Barr virus (EBV)-associated gastric cancers (EBVaGCs) suggests the applicability of novel EBV-targeted therapies. The antiviral nucleoside drug, ganciclovir (GCV), is effective only in the context of the viral lytic cycle in the presence of EBV-encoded thymidine kinase (TK)/protein kinase (PK) expression. In this study, screening of the Johns Hopkins Drug Library identified gemcitabine as a candidate for combination treatment with GCV. Pharmacological induction of EBV-TK or PK in EBVaGC-originated tumor cells were used to study combination treatment with GCV in vitro and in vivo. Gemcitabine was found to be a lytic inducer via activation of the ataxia telangiectasia-mutated (ATM)/p53 genotoxic stress pathway in EBVaGC. Using an EBVaGC mouse model and a [125I] fialuridine (FIAU)-based lytic activation imaging system, we evaluated gemcitabine-induced lytic activation in an in vivo system and confirmed the efficacy of gemcitabine-GCV combination treatment. This viral enzyme-targeted anti-tumor strategy may provide a new therapeutic approach for EBVaGCs.

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus.

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.

Regulation of HK2 expression through alterations in CpG methylation of the HK2 promoter during progression of hepatocellular carcinoma.

Hexokinase 2 (HK2) is a rate-determining enzyme in aerobic glycolysis, a process upregulated in tumor cells. HK2 expression is controlled by various transcription factors and epigenetic alterations and is heterogeneous in hepatocellular carcinomas (HCCs), though the cause of this heterogeneity is not known. DNA methylation in the HK2 promoter CpG island (HK2-CGI) and its surrounding regions (shore and shelf) has not previously been evaluated, but may provide clues about the regulation of HK2 expression. Here, we compared HK2 promoter methylation in HCCs and adjacent non-cancerous liver tissues using a HumanMethylation450 BeadChip array. We found that, while the HK2-CGI N-shore was hypomethylated, thereby enhancing HK2 expression, the HK2-CGI was itself hypermethylated in some HCCs. This hypermethylation suppressed HK2 expression by inhibiting interactions between HIF-1α and a hypoxia response element (HRE) located at −234/−230. HCCs that were HK2negative and had distinct promoter CGI methylation were denoted as having a HK2-CGI methylation phenotype (HK2-CIMP), which was associated with poor clinical outcome. These findings indicate that HK2-CGI N-shore hypomethylation and HK2-CGI hypermethylation affect HK2 expression by influencing the interaction between HIF 1α and HRE. HK2-CGI hypermethylation induces HK2-CIMP and could represent a prognostic biomarker for HCC.

Serum Dickkopf-1 as a Biomarker for the Diagnosis of Hepatocellular Carcinoma

Purpose Dickkopf-1 (DKK-1) is a Wnt/β-catenin signaling pathway inhibitor. We investigated whether DKK-1 is related to progression in hepatocellular carcinoma (HCC) cells and HCC patients. Materials and Methods In vitro reverse-transcription polymerase chain reaction (RT-PCR), wound healing assays, invasion assays, and ELISAs of patient serum samples were employed. The diagnostic accuracy of the serum DKK-1 ELISA was assessed using receiver operating characteristic (ROC) curves and area under ROC (AUC) analyses. Results RT-PCR showed high DKK-1 expression in Hep3B and low in 293 cells. Similarly, the secreted DKK-1 concentration in the culture media was high in Hep3B and low in 293 cells. Wound healing and invasion assays using 293, Huh7, and Hep3B cells showed that DKK-1 overexpression promoted cell migration and invasion, whereas DKK-1 knock-down inhibited them. When serum DKK-1 levels were assessed in 370 participants (217 with HCC and 153 without), it was significantly higher in HCC patients than in control groups (median 1.48 ng/mL vs. 0.90 ng/mL, p<0.001). The optimum DKK-1 cutoff level was 1.01 ng/mL (AUC=0.829; sensitivity 90.7%; specificity 62.0%). Although DKK-1 had a higher AUC than alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) (AUC=0.829 vs. 0.794 and 0.815, respectively), they were statistically similar (all p>0.05). When three biomarkers were combined (DKK-1 plus AFP plus DCP), they showed significantly higher AUC (AUC=0.952) than single marker, DKK-1 plus AFP, or DKK-1 plus DCP (all p<0.001). Conclusion DKK-1 might be a key regulator in HCC progression and a potential therapeutic target in HCC. Serum DKK-1 could complement the diagnostic accuracy of AFP and DCP.