• Tuberculosis and Respiratory Diseases
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• v.70 no.1
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• pp.21-27
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• 2011

Background: Although the gold standard method for research trials on epidermal growth factor receptor (EGFR) mutations has been direct sequencing, this approach has the limitations of low sensitivity and of being time-consuming. Peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping is known to be a more sensitive detection tool. The aim of this study was to compare the detection rate of $EGFR$ mutation and EGFR-tyrosine kinase inhibitor (TKI) responsiveness according to $EGFR$ mutation status using both methodologies. Methods: Clinical specimens from 112 NSCLC patients were analyzed for $EGFR$ mutations in exons 18, 19, 20, and 21. All clinical data and tumor specimens were obtained from 3 university hospitals in Korea. After genomic DNA was extracted from paraffin-embedded tissue specimens, both PNA-mediated PCR clamping and direct-sequencing were performed. The results and clinical response to $EGFR$-TKIs were compared. Results: Sequencing revealed a total of 35 (22.9%) mutations: 8 missense mutations in exon 21 and 26 deletion mutations in exon 19. PNA-mediated PCR clamping showed the presence of genomic alterations in 45 (28.3%) samples, including the 32 identified by sequencing plus 13 additional samples (6 in exon 19 and 7 in exon 21). Conclusion: PNA-mediated PCR clamping is simple and rapid, as well as a more sensitive method for screening of genomic alterations in $EGFR$ gene compared to direct sequencing. This data suggests that PNA-mediated PCR clamping should be implemented as a useful screening tool for detection of $EGFR$ mutations in clinical setting.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.3
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• pp.167-174
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• 2012

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-$kit^+$ bone marrow cells (c-$kit^+$ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-$kit^+$ BM cells that give rise to $CD2^+CD8^+$ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-$kit^+$ BM cells than those of controls. And, we compared the ability of the cytotoxicity of $CD2^+CD8^+$ NK cells differentiated by cytokines from c-$kit^+$ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100 : 1 for 4 h once a week. In results, $CD2^+CD8^+$ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-$kit^+$ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-$kit^+$ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.

• Journal of Periodontal and Implant Science
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• v.49 no.3
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• pp.138-147
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• 2019

Purpose: Several studies have shown that the oral cavity is a secondary location for Helicobacter pylori colonization and that H. pylori is associated with the severity of periodontitis. This study investigated whether H. pylori had an effect on the periodontium. We established an invasion model of a standard strain of H. pylori in human periodontal ligament fibroblasts (hPDLFs), and evaluated the effects of H. pylori on cell proliferation and cell cycle progression. Methods: Different concentrations of H. pylori were used to infect hPDLFs, with 6 hours of co-culture. The multiplicity of infection in the low- and high-concentration groups was 10:1 and 100:1, respectively. The Cell Counting Kit-8 method and Ki-67 immunofluorescence were used to detect cell proliferation. Flow cytometry, quantitative real-time polymerase chain reaction, and western blots were used to detect cell cycle progression. In the high-concentration group, the invasion of H. pylori was observed by transmission electron microscopy. Results: It was found that H. pylori invaded the fibroblasts, with cytoplasmic localization. Analyses of cell proliferation and flow cytometry showed that H. pylori inhibited the proliferation of periodontal fibroblasts by causing G2 phase arrest. The inhibition of proliferation and G2 phase arrest were more obvious in the high-concentration group. In the low-concentration group, the G2 phase regulatory factors cyclin dependent kinase 1 (CDK1) and cell division cycle 25C (Cdc25C) were upregulated, while cyclin B1 was inhibited. However, in the high-concentration group, cyclin B1 was upregulated and CDK1 was inhibited. Furthermore, the deactivated states of tyrosine phosphorylation of CDK1 (CDK1-Y15) and serine phosphorylation of Cdc25C (Cdc25C-S216) were upregulated after H. pylori infection. Conclusions: In our model, H. pylori inhibited the proliferation of hPDLFs and exerted an invasive effect, causing G2 phase arrest via the Cdc25C/CDK1/cyclin B1 signaling cascade. Its inhibitory effect on proliferation was stronger in the high-concentration group.

• Tuberculosis and Respiratory Diseases
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• v.85 no.2
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• pp.155-164
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• 2022

Background: The remarkable efficacy of osimertinib in non-small cell lung cancer (NSCLC) with acquired T790M mutation has been widely documented in clinical trials and real-world practice. However, some patients show primary resistance to this drug. Even patients who initially show a favorable response have inconsistent clinical outcomes later. Therefore, the aim of this study was to identify additional clinical predictive factors for osimertinib efficacy. Methods: A prospective cohort of patients with acquired T790M positive stage IV lung adenocarcinoma treated with osimertinib salvage therapy in Hallym University Medical Center were analyzed. Results: Sixty-one eligible patients were analyzed, including 38 (62%) women and 39 (64%) who never smoked. Their mean age was 63.3 years. The median follow-up after treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) was 36.0 months (interquartile range, 24.7-50.2 months). The majority (n=45, 74%) of patients were deceased. Based on univariate analysis, low baseline neutrophil-to-lymphocyte ratios (NLR), age ≥50 years, never-smoking history, stage IVA at osimertinib initiation, and prolonged response to previous TKIs (≥10 months) were associated with a significantly longer progression-free survival (PFS). Multivariate analysis showed that never-smoking status (hazard ratio [HR], 0.54; 95% confidence interval [CI], 0.30-0.98; p=0.041) and a baseline NLR less than or equal to 3.5 (HR, 0.23; 95% CI, 0.12-0.45; p<0.001) were independently associated with a prolonged PFS with osimertinib. Conclusion: Smoking history and high NLR were independent negative predictors of osimertinib PFS in patients with advanced NSCLC developing EGFR T790M resistance after the initial EGFR-TKI treatment.

• Animal Bioscience
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• v.35 no.8
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• pp.1235-1249
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• 2022

Objective: The purpose of this study was to evaluate the protection of glutamate (GLU) against the impairment in intestinal barrier function induced by lipopolysaccharide (LPS) stress in weaned pigs. Methods: Twenty-four weaned pigs were divided into four treatments containing: i) non-challenged control, ii) LPS-challenged control, iii) LPS+1.0% GLU, and iv) LPS+2.0% GLU. On day 28, pigs were treated with LPS or saline. Blood samples were collected at 0, 2, and 4 h post-injection. After blood samples collection at 4 h, all pigs were slaughtered, and spleen, mesenteric lymph nodes, liver and intestinal samples were obtained. Results: Dietary GLU supplementation inhibited the LPS-induced oxidative stress in pigs, as demonstrated by reduced malondialdehyde level and increased glutathione level in jejunum. Diets supplemented with GLU enhanced villus height, villus height/crypt depth and claudin-1 expression, attenuated intestinal histology and ultrastructure impairment induced by LPS. Moreover, GLU supplementation reversed intestinal intraepithelial lymphocyte number decrease and mast cell number increase induced by LPS stress. GLU reduced serum cortisol concentration at 4 h after LPS stress and downregulated the mRNA expression of intestinal corticotropin-releasing factor signal (corticotrophin-releasing factor [CRF], CRF receptor 1 [CRFR1], glucocorticoid receptor, tryptase, nerve growth factor, tyrosine kinase receptor A), and prevented mast cell activation. GLU upregulated the mRNA expression of intestinal transforming growth factor β. Conclusion: These findings indicate that GLU attenuates LPS-induced intestinal mucosal barrier injury, which is associated with modulating CRF signaling pathway.

• Journal of Life Science
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• v.33 no.2
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• pp.191-198
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• 2023

As a member of the focal adhesion complex of the plasma membrane, Src is a nonreceptor tyrosine kinase that controls cell adhesion and motility. However, how Src activity is regulated in the plasma membrane microdomain in response to components of the extracellular matrix (ECM) remains unclear. This study compared and investigated the activity of Src in response to three representative ECM proteins: collagen type 1, fibronectin, and laminin. Genetically encoded FRET-based Src biosensors for plasma membrane subdomains were used. FRET-based biosensors allow the real-time analysis of protein activity in living cells based on their high spatiotemporal resolution. The results showed that Src activity was maintained at a high level under all ECM conditions of the lipid raft, and there was no significant difference between the ECM conditions. In contrast, Src activity was maintained at a low level in the non-lipid raft membrane. In addition, the Src activity of lipid rafts remained significantly higher than that of non-lipid raft regions under the same ECM conditions. In conclusion, this study demonstrates that Src activity can be controlled differently by lipid rafts and non-lipid raft microdomains.

• Journal of Life Science
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• v.33 no.5
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• pp.397-405
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• 2023

Although glutathione (GSH) has been shown to play an important role in the prevention of oxidative damage as an antioxidant, studies on immune regulation by it have not been properly conducted. In this study, we investigated whether luthione^®, a reduced GSH, has an immune enhancing effect in murine macrophage RAW 264.7 cells. The results of flow cytometry and immunofluorescence experiments indicated that luthione increased phagocytic activity, a representative function of macrophages, compared to the control cells. According to the results of the cytokine array, the expression of interleukin (IL)-5, IL-1β, and IL-27 was significantly increased in the luthione-treated cells. Luthione also enhanced the production of tumor necrosis factor-α and IL-1β through increased expression of their proteins, and increased release of the immune mediators such as nitric oxide (NO) and prostaglandin E₂ was associated with increased expression of inducible NO synthase and cyclooxygenase-2. In addition, the expression of cluster of differentiation 86, an M1 macrophage marker, was dramatically enhanced in RAW 264.7 cells treated with luthione. Furthermore, as a result of heat map analysis, we found that cytokine signaling 1/3-mediated signal transducer and activator of transcription/Janus tyrosine kinase signaling pathway was involved in the immunomodulatory effect by luthione. In conclusion, our data suggested that luthione could act as a molecular regulator in M1 macrophage polarization and enhance immune capacity by promoting macrophage phagocytic function.

• Korean Journal of Clinical Laboratory Science
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• v.55 no.4
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• pp.298-305
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• 2023

Burkitt's lymphoma is a distinct subtype of non-Hodgkin's lymphoma originating from B-cells that is notorious for its aggressive growth and association with immune system impairments, potentially resulting in rapid and fatal outcomes if not addressed promptly. Optimizing the use of Food and Drug Administration-approved medications, such as combining known safe drugs, can lead to time and cost savings. This method holds promise in accelerating the progress of novel treatments, ultimately facilitating swifter access for patients. This study explores the potential of a dual-targeted therapeutic strategy, combining the bruton tyrosine kinase-targeting drug Ibrutinib and the epidermal growth factor receptor/human epidermal growth factor receptor-2-targeting drug Lapatinib. Ramos and Daudi cell lines, well-established models of Burkitt's lymphoma, were used to examine the impact of this combination therapy. The combination of Ibrutinib and Lapatinib inhibited cell proliferation more than using each drug individually. A combination treatment induced apoptosis and caused cell cycle arrest at the S and G2/M phases. This approach is multifaceted in its benefits. It enhances the efficiency of the drug development timeline and maximizes the utility of currently available resources, ensuring a more streamlined and resource-effective research process.

• Tuberculosis and Respiratory Diseases
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• v.58 no.5
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• pp.473-479
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• 2005

Background : Gefitinib targets the epidermal growth factor receptor r(EGFR), and Gefitinib has antitumor activity in patient with non-small cell lung cancer (NSCLC). However, only 10 to 20 percent of patients show a clinical response to this drug, and the molecular mechanisms underlying patient sensitivity to gefitinib are unknown. PTEN (Phosphatase and tensin homolog deleted on chromosome Ten) plays a role for the modulation of the phosphatidylinositol 3-kinase pathway (PI3K), which is involved in cell proliferation and survival, so that it can inhibit cell cycle progression and induce G1 arrest. Therefore, we analyzed the relationship between PTEN expression and gefitinib's responsiveness in patients having advanced non small cell lung cancer that had progressed after previous chemotherapy. Methods : The expression of PTEN was studied by immunohistochemistry in paraffin-embedded tumor blocks that were obtained from 22 patients who had been treated with gefitinib from JAN, 2001 to AUG. 2004. For the evaluation of the relationships between the PTEN expression, the clinical stage and the basal characteristics, those cases that showed the respective antigen expression in >50% of the tumor cells were considered positive. Results : The positive rate of PTEN staining was 55% of the total of 22 patients. There was a significant relationship between the increased expression of PTEN and the response group (p=0.039). However, there was no significant relationship between the expression of PTEN and other clinicopathologic characteristics. Conclusion: The expression of PTEN in patients with advanced non small cell lung cancer that has progressed after previous chemotherapy may play a role in gefitinib's responsiveness.

• Radiation Oncology Journal
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• v.22 no.2
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• pp.77-90
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• 2004

Purpose: The purpose of this review Is to provide an update on novel radiation treatments for head and neck cancer Recent Findings: Despite the remarkable advances In chemotherapy and radiotherapy techniques, the management of advanced head and neck cancer remains challenging. Epidermal growth factor receptor (EGFR) Is an appealing target for novel therapies In head and neck cancer because not only EGFR activation stimulates many important signaling pathways associated with cancer development and progression, and importantly, resistance to radiation. Furthermore, EGFR overexpression Is known to be portended for a worse outcome in patients with advanced head and neck cancer. Two categories of compounds designed to abrogate EGFR signaling, such as monoclonal antibodies (Cetuxlmab) and tyrosine kinase inhibitors (ZD1839 and 051-774) have been assessed and have been most extensively studied In preclinical models and clinical trials. Additional TKIs In clinical trials include a reversible agent, Cl-1033, which blocks activation of all erbB receptors. Encouraging preclinical data for head and neck cancers resulted In rapid translation Into the clinic. Results from Initial clinical trials show rather surprisingly that only minority of patients benefited from EGFR inhibition as monotherapy or In combination with chemotherapy. In this review, we begin with a brief summary of erbB- mediated signal transduction. Subsequently, we present data on prognostic-predictive value of erbB receptor expression in HNC followed by preclinlcal and clinical data on the role of EGFR antagonists alone or in combination with radiation In the treatment of HNC. Finally, we discuss the emerging thoughts on resistance to EGFR biockade and efforts In the development of multiple-targeted therapy for combination with chemotherapy or radiation. Current challenges for investigators are to determine (1 ) who will benefit from targeted agents and which agents are most appropriate to combine with radiation and/or chemotherapy, (2) how to sequence these agents with radiation and/or cytotoxlc compounds, (3) reliable markers for patient selection and verification of effective blockade of signaling in vivo, and (4) mechanisms behind intrinsic or acquired resistance to targeted agents to facilitate rational development of multi-targeted therapy, Other molecuiar-targeted approaches In head and neck cancer were briefly described, Including angloenesis Inhibitors, farnesyl transferase inhibitors, cell cycle regulators, and gene therapy Summary: Novel targeted theraples are highly appealing in advanced head and neck cancer, and the most premising strategy to use them Is a matter of intense Investigation.