Seung Joon Kim

Nitric oxide induces MUC5AC mucin in respiratory epithelial cells through PKC and ERK dependent pathways

BackgroundNitric oxide (NO) is generally increased during inflammatory airway diseases. This increased NO stimulates the secretion of mucin from the goblet cell and submucosal glands but the mechanism is still unknown precisely. In this study, we investigated potential signaling pathways involving protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the NO-induced MUC5AC mucin gene and protein expression in A549 cells.MethodsNitric oxide was donated to the A549 cells by NOR-1. MUC5AC mucin levels were assayed by enzyme-linked immunosorbent assay (ELISA). MUC5AC promoter activity was determined by measuring luciferase activity after the lysing the transfected cells. Activation of PKC isoforms were measured by assessing the distribution of the enzyme between cytosolic and membrane fractions using immunoblotting. Immunoblotting experiments using a monoclonal antibody specific to PKC isoforms were performed in the cytosol and membrane fractions from A549 cells. Western blot analysis for pERK and p38 were performed using the corresponding antibodies from the cell lysates after donating NO to the A549 cells by NOR-1.ResultsThe transcriptional activity of MUC5AC promoter was maximal at the concentration of 0.1 mM NOR-1 for 1 hour incubation in transfected A549 cells. (±)-(E)-methyl-2-((E)-hydroxyimino)-5-nitro-6-methoxy-3-hexenamide (NOR-1) markedly displaced the protein kinase C (PKC)α and PKCδ from the cytosol to the membrane. Furthermore, the PKC-α,βinhibitors, GÖ6976 (10 nM) and PKCδ inhibitors, rottlerin (4 μM) inhibited the NOR-1 induced migration of PKCα and PKCδ respectively. NOR-1 also markedly increased the MUC5AC promoter activity and mRNA expression, mucin synthesis and ERK1/2 phosphorylation. The PKC inhibitors also inhibited the NOR-1 induced MUC5AC mRNA and MUC5AC protein synthesis by inhibiting the activation of PKCα and PKCδ with ERK1/2 pathways.ConclusionExogenous NO induced the MUC5AC mucin gene and protein through the PKCα and PKCδ – ERK pathways in A549 cells. Inhibition of PKC attenuated NO-mediated MUC5AC mucin synthesis. In view of this findings, PKC inhibitors might be useful in the treatment of bronchial asthma and chronic bronchitis patients where NO and mucus are increased in the bronchial airways.

Distribution and cytokine production of CD4 and CD8 T-lymphocyte subsets in patients with acute asthma attacks

BACKGROUNDnThe activation of T cells and the elevation of Th2-type cytokines have been observed in asthmatic patients, but the relative role of CD4 and CD8 T cell is still unclear.nnnOBJECTIVEnTo investigate the role of T cell subset in patients with acute asthma attacks, we analyzed the distribution, activation status, and cytokine production of CD4 and CD8 cells.nnnMETHODSnThe percentages of the CD4 and CD8 cell in peripheral blood (PB) and bronchoalveolar lavage (BAL) fluid were analyzed by flow cytometry. The cytokines (IL-4, IL-5, and IFN-gamma) and soluble IL-2 receptor (sIL-2R) were measured by ELISA in culture supernatants of CD4 and CD8 cells purified from PB.nnnRESULTSnThe CD4/CD8 ratio in PB of asthmatic patients was significantly higher than that of controls, which was significantly reduced after treatment. In contrast, there was a tendency to high percentage of CD8 cells in asthmatic patients as compared with controls in BAL, which resulted in a decreased CD4/CD8 ratio. Comparing the T cell subsets in BAL with paired PB in asthma, the CD4 cells were higher in PB, but CD8 cells were higher in BAL. The IL-4, IL-5, and sIL-2R produced by CD4 cells were significantly higher than those produced by CD8 cells in asthmatic patients.nnnCONCLUSIONSnOur results provide evidence that activated CD4 T cells increase and produce type 2 cytokines in PB, but CD8 T cell are more sequestrated than CD4 T cells in the airway during an acute asthma attack.

Detection of c-K-ras point mutation in ovarian cancer.

Point mutations of c-K-ras in ovarian cancer were detected by replacement of GGT of codon 12 by GAT, AGT, TGT and GTT, polymerase chain reaction, agarose gel electrophoresis and Southern blot hybridization with a digoxigenin detection system. The incidence of four-typed point mutations of c-K-ras oncogene in 37 ovarian cancers was 35.1% (13/37) and the distributions were 32.4% (12/37), 2.7% (1/37), 0% and 0% of GGT to GAT, GGT to AGT, GGT to TGT, and GGT to GTT, respectively. The incidence of c-K-ras point mutations on codon 12 among 37 patients with ovarian cancer was 35.5% (8/22) in those with serous cystadenocarcinomas and 28.6% (2/7) in those with mucinous cystadenocarcinomas. c-K-ras point mutations on codon 12 were detected in 14.3% (1/7) of patients with stage I disease, 28.6% (2/7) with stage II disease, and in 43.5% (10/23) with stage III/IV disease, and there was a statistically significant increase in point mutations of c-K-ras oncogene with advancing clinical stage. The incidence of c-K-ras point mutations on codon 12 among 33 patients who had a pelvic lymph node dissection was 52.4% (11/21) in those with pelvic lymph node metastases and 16.7% (2/12) in those without pelvic lymph node metastases, a statistically significant difference. Furthermore, point mutation of c-K-ras gene was found most frequently in patients with advanced stage disease, and in those with pelvic lymph node metastases. Activation of c-K-ras oncogene seems to be a major factor in ovarian carcinogenesis and tumor progression.

Abstract 4671: Dual mutational changes of ALK gene fusion combined with EGFR or K-ras oncogenes in lung adenocarcinomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnPurposennThe mutations of the EGFR, K-ras or ALK oncogenes are closely associated with tumorigenesis and progression of lung adenocarcinoma. Their mutational changes are known to be mutually exclusive, but double oncogenic mutations of ALK combined with EGFR or K-ras gene are rarely found. We analyzed the clinical impacts of double oncogenic mutations on clinical characteristics and treatment outcome, according to EGFR, K-ras, ALK mutation or triple negative (EGFR, K-ras, ALK wild) in lung adenocarcinomas.nnPatients and MethodsnnFor one hundred ninety six lung cancer patients conformed histologically as adenocarcinoma, we examined activating EGFR (exons 18 and 21) and K-ras (exon 2 and 3) mutations by direct nucleotide sequencing and. ALK gene rearrangements by FISH using break-apart probe. Among 196 patients, 86 patients (43.9%) were triple negative adenocarcinoma, 73 patients (37.2%) presented with activating EGFR mutation, 17 patients (8.7%) with ALK and 13 patients (6.6%) with K-ras mutation. Median age of 17 patients harboring ALK rearrangement was 54 years (range 25∼79), approximately 7 years younger than the patient population harboring EGFR or K-ras mutation (62 and 61 years, respectively). Fifteen patients (88.2%) had stage III or IV and 9 patients (52.9%) were never smoker. Platinum doublet chemotherapy or EGFR TKIs was administered for 14 patients initially presented as stage IV disease or recurred as metastatic disease after local treatment. The median PFS of the patients receiving platinum doublet chemotherapy and EGFR TKIs as first-line treatment was 5.67 months (range 2.97-20.9) and 3.58 months (range 3.53-3.63), respectively. Of 17 ALK-positive patients, dual oncogenic mutations of ALK together with either EGFR or K-ras were identified in 7 patients (3.6%). Six patients (85.7%) receiving platinum-doublet or EGFR TKI as initial treatment. showed partial response, and 1 patient (14.3%) showed stable disease. These seven patients are all alive at present.nnConclusionnnTaken together, our data suggest the existence of dual oncogenic mutations in lung adenocarcinomas is not infrequent. We are undergoing the experiment for detection for circulating tumor cells harboring ALK rearrangements, EGFR or K-ras mutation in these patients peripheral blood.nnCitation Format: Jieun Lee, Suk Hee Hong, Eun Kyung Jeon, Jung Oh Kim, Seung Joon Kim, Young Kyoon Kim, Jae Gil Park, Sook-Whan Sung, Tae-Jung Kim, Jin Hyoung Kang. Dual mutational changes of ALK gene fusion combined with EGFR or K-ras oncogenes in lung adenocarcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4671. doi:10.1158/1538-7445.AM2014-4671

Abstract B41: Different clinical outcomes of systemic chemotherapy in non-small cell lung cancer without EGFR and K-ras mutation according to smoking status.

Background: The current treatment strategy in non-small cell lung cancer (NSCLC) adenocarcinoma without epidermal growth factor receptor (EGFR) activating mutation or anaplastic large cell kinase gene (ALK) rearrangement does not differentiate between genotype or smoking status even though resent advances in molecular study in NSCLC. In terms of second line treatment, pemetrexed is considered as a standard treatment in non-squamous NSCLC without EGFR activating mutation or ALK rearrangement. We retrospectively analyzed clinical outcomes of systemic chemotherapy according to molecular characteristics.nnMethods: Between 2006 and 2012, 131 patients with stage 4 NSCLC adenocarcinoma patients who were enrolled in a Seoul St. Marys hospital lung cancer multidisciplinary center registry were available for molecular pathology regarding EGFR and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation status. Mutations in EGFR (exons 18 to 21) and KRAS (codons 12 and 13) were determined by direct sequencing.nnResults: Among 131 patients, the frequency of EGFR mutations, KRAS mutations, and no mutations in both genes (double negative [DN]) was 28.2%, 5.3% and 66.4% respectively. Among the DN genotype, 47.1%were never smoker and 37.9% were female. The median progression free survival (PFS) with 1st line platinum based combination chemotherapy was 5.4 months (95% CI: 3.7-7.02) for DN genotype and 7.4 months (95% CI: 5.73-9.06) for EGFR mutation group (p=0.047). According to smoking status, the median PFS with 1st line platinum based combination chemotherapy for never smokers in EGFR mutation group was significantly longer than the DN group (DN-N) (8.4 vs 6.7 months: p=0.018). However among the smokers, no significant difference in median PFS with 1st line chemotherapy between EGFR mutation and DN group (DN-S) were found (6.7 vs 4.6 months: p=0.515). In terms of second line treatment except EGFR tyrosine kinase inhibitors (EGFR-TKI), 64% of DN group and 70.3% of EGFR mutation group received pemetrexed, 21% of DN group and 29.7% of EGFR mutation group received docetaxel. 15.1% of DN group received EGFR-TKIs. The median PFS with second line treatment in DN-N group was 7.7 months (95% CI: 2.1-18.9), 3.1 months (95% CI: 1.4-4.8) and 4.4 months (95% CI: 2.2-6.5) for pemetrexed, docetaxel and EGFR-TKI respectively (p=0.009). However, no significant differences in median PFS between second line treatments was found in DN-S group [3.0 vs 2.8 vs 1.7 months for pemetrexed, docetaxel and EGFR-TKI respectively (p=0.100)]. In EGFR mutation group, no significant difference between pemetrexed and docetaxel was found according to the smoking status.nnConclusions: NSCLC without EGFR and K-ras mutation (DN group) had different clinical outcomes according to smoking history. Smoking history could be a useful clinical marker to choose a second-line treatment. However, further genomic study to find a druggable target in this group should be conducted.nnCitation Information: Mol Cancer Ther 2013;12(11 Suppl):B41.nnCitation Format: Sook Hee Hong, JinHyoung Kang, Eun kyung Jeon, Seung Joon Kim, Kyo-Young Lee, Sang young Roh. Different clinical outcomes of systemic chemotherapy in non-small cell lung cancer without EGFR and K-ras mutation according to smoking status. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B41.

Abstract 2372: Predictive markers in patients with non-small cell lung cancer with pemetrexed treatment.

Background: This study is desiged to evaluate biomarkers that are correlated with the efficacy of pemetrexed in patients with non-small-cell lung cancer (NSCLC). Methods: Patients with stage III or IV NSCLC who received pemetrexed monotherapy after failure of one or more chemotherapy regimen between January 2003 and August 2011 were included. Polymorphism of reduced folate carrier gene (SLC19A, rs1051298), 5,10-methylenetetrahydroflate reductase (MTHFR, rs1801133) and thymidylate synthase (TS, rs45445694, rs16430) gene were analyzed by PCR and direct sequencing method using peripheral blood. Results: Total 123 patients were included. The median age was 61 years (range 35-82). Fifty eight (47.2%) patients were female. Predominant histology was adenocarcinoma (92.7%). Metastatic diseases were 114 (80.5%) patients. Patients received one or more lines of systemic therapy before pemetrexed therapy, one line in 53 (43.1%) patients, two in 51(41.5%), three in 14 (11.02%), and four in 4 (3.3%). Median 4 cycles of chemotherapy were given (range 1-44). Response rate was 22.7% (CR, n=2, PR, n=26) and stable disease was detected in 54 patients (43.9%). Median follow up duration was 28.7months (range 4.07-84.33months). Median progression free survival (PFS) was 4.2 months (95% CI: 2.9-5.6) and median overall survival (OS) was 18.9 months (95% CI: 13.3-24.6). Clinical factors like, sex, histology of tumor (adenocarcinoma vs, nonadenocarcinoma), differentiation, smoking status and EGFR mutation status (active mutation vs. other mutation vs. wild type) did not have any significant difference on response and PFS. Only polymorphisms of TS promoter enhancer region (TSER) were related with PFS. 2R/2R repeat in the TSER (2R/2R vs 2R/3R, 3R/3R 1.0m vs 4.3m, p Conclusions: This study suggests that polymorphisms of TSER associated PFS in pemetrexed treatment. Further prospective studies will be needed to validate these polymorphism as a useful biomarker of pemetrexed treatment in NSCLC patients. Citation Format: Hiun S. Chae, Yoon Ho Ko, Eun Kyoung Jeon, Sook Hee Hong, Jeong-Oh Kim, Seung Joon Kim, Kyo Young Lee, Young Kyoon Kim, HoonKyo Kim, Seok young Park, In Sook Woo, Jin Hyoung Kang. Predictive markers in patients with non-small cell lung cancer with pemetrexed treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2372. doi:10.1158/1538-7445.AM2013-2372