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The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.

Simple Summary It is generally accepted that fibroblasts represent a heterogeneous population of cells with different functions depending on the cell type. Although numerous reports have stated that cancer-associated fibroblast (CAF) promotes cancer progression, few studies have shown that they inhibit cancer progression. We propose that CAFs derived from some HNSCC patients is less effective in promoting cancer progression than CAFs from other patients and that specific collagen proteins may be involved in this process. Abstract Background: The critical effect of the tumor microenvironment on cancer progression is well recognized. Recent research suggests that the cancer-promoting properties of the tumor stroma may be attributed to fibroblasts. However, the effect of cancer-associated fibroblast (CAF) on the progression of head and neck squamous cell carcinoma (HNSCC) is not well known. Methods: From the immunohistochemical analysis of head and neck squamous cell carcinoma (HNSCC) tissues, we divided CAF into two groups depending on the presence or absence of a well-demarcated boundary between epithelial cancer cells and the surrounding extracellular matrix (ECM). Primary culture of CAF was performed, followed by co-transplantation with HNSCC cells into mice oral mucosa, and the tumorigenesis was compared. The mRNA expression patterns between these two CAF groups were compared using DNA microarray analysis. Results: CAFs from cancer tissues that showed no demarcation between ECM and epithelial cancer cells (CAF-Promote) tended to stimulate Matrigel invasion of HNSCC cells. Conversely, CAFs from cancer tissues that showed a boundary with epithelial cancer cells (CAF-Delay) caused no remarkable increase in Matrigel invasion. Compared with CAF-P, CAF-D is less effective in promoting FaDu tumorigenicity in the mouse model. In DNA microarray analysis, COL3A1 and COL6A6 showed particularly high expression in the CAF-D group. Conclusions: These cancer stroma-derived collagen proteins might delay the HNSCC progression. These findings are expected to provide vital information for predicting HNSCC prognosis and developing drug targets in the future.

We evaluated potential biomarkers in human whole saliva for the early diagnosis of oral squamous cell carcinoma (OSCC). We selected 30 candidate genes with relevance to cancer from recent reports in PubMed. Saliva samples were obtained from 34 non-tumor control and 33 OSCC patients. Real-time PCR was performed, and mRNA levels were compared. Normalized mRNA levels of six genes (NGFI-A binding protein 2 (NAB2), cytochrome P450, family 27, subfamily A, polypeptide 1 (CYP27A1), nuclear pore complex interacting protein family, member B4 (NPIPB4), monoamine oxidase B (MAOB), sialic acid acetyltransferase (SIAE), and collagen, type III, alpha 1 (COL3A1)) were significantly lower in saliva of OSCC patients. Receiver operating characteristics (ROC) analysis was used to individually evaluate the predictive power of the potential biomarkers for OSCC diagnosis. The area under the curve (AUC) values were evaluated for the OSCC vs. non-tumor groups via univariate ROC analyses, as well as multivariate ROC analyses of combinations of multiple potential biomarkers. The combination of CYP27A1 + SIAE showed a favorable AUC value of 0.84. When we divided saliva samples into two groups according to age using a 60-year cut-off, with OSCC patients and controls evaluated together, the AUC of MAOB&ndash, NAB2 was more predictive of OSCC in the under-60 group (AUC, 0.91, sensitivity, 0.92, and specificity, 0.86) than any other gene combination. These results are expected to aid the early diagnosis of OSCC, especially in patients under 60 years of age. While more studies with larger numbers of patients are necessary, our result suggest that salivary mRNA would be a potent biomarker for early OSCC diagnosis.

Background/aim Few studies have examined the effect of 2'-hydroxycinnamaldehyde (HCA) on head and neck squamous cell carcinoma (HNSCC) cell invasion. This study examined the role of BMP7 on the anti-migration and anti-invasion activity of HCA using HNSCC cells. Materials and methods Matrigel invasion and wound healing assays were conducted to investigate cell migration or invasion. BMP7 overexpression vector or siRNA mixture was used for transient regulation of gene expression. Results HCA attenuated HNSCC cell migration and spheroids Matrigel invasion without cytotoxicity. mRNA and protein expression of BMP7 increased with HCA treatment. Exogenous BMP7 overexpression without HCA treatment attenuated Matrigel invasion of cells. Furthermore, suppression of BMP7 by siRNA alleviated the inhibitory effect of HCA on the invasion of Matrigel by the cell, indicating that BMP7 is responsible for the anti-migration effect of HCA in HNSCC cells. Conclusion HCA treatment led to a remarkable up-regulation of BMP7, which resulted in the attenuation of HNSCC cell invasion.

Background: Anemia is a major risk factor that contributes to mortality in patients with chronic kidney disease. There is controversy over the optimal hemoglobin (Hb) target in these patients. This study investigated the association between Hb level and mortality in a cohort of hemodialysis (HD) patients in Korea. Methods: This study was a multicenter prospective observational study of maintenance HD patients that was performed for 5 years in western Seoul, Korea. Three hundred and sixty-two participants were enrolled. Laboratory values and mortality were accessed every 6 months. Repeated measures of laboratory values in each interval were averaged to obtain one semiannual mean value. The Hb values were divided into six groups: (1) Hb

Background The TNM classification system is used widely for tumour staging, and directs the treatment and prognosis of patients with cancer. The aim of this study was to assess the prognostic value of extranodal extension (ENE) in patients with early gastric cancer. Methods All patients who underwent gastrectomy with lymphadenectomy for primary gastric cancer with lymph node metastases between January 2003 and June 2006 were reviewed. Histological slides of metastatic nodes were reviewed by two gastrointestinal pathologists. The association of ENE with clinicopathological characteristics was assessed. The disease-specific survival rate was calculated by the Kaplan–Meier method, and a multivariable Cox regression model was used to identify independent prognostic factors. Results Some 1143 patients were included. ENE was associated with advanced pT and pN category, larger tumour size and lymphovascular/perineural invasion. In multivariable analysis, pT category, pN category, ENE, lymphovascular invasion and perineural invasion were found to be independent prognostic factors in node-positive gastric carcinoma. The 5-year survival rate of patients with ENE was 48·1 per cent, compared with 78·2 per cent for patients without ENE (P < 0·001). In the subgroup of patients with early gastric cancer, ENE was associated with a worse 5-year survival rate in patients with early (T1) gastric cancer: 75 per cent in patients with ENEversus 96·9 per cent in those without (P < 0·001). Conclusion ENE is an independent prognostic factor in patients with early and advanced gastric cancer.

MsrA and MsrB catalyze the reduction of methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, to methionine in different cellular compartments of mammalian cells. One of the three MsrBs, MsrB3, is an endoplasmic reticulum (ER)-type enzyme critical for stress resistance including oxidative and ER stresses. However, there is no evidence for the presence of an ER-type MsrA or the ER localization of MsrA. In this work, we developed an ER-targeted recombinant MsrA construct and investigated the potential effects of methionine-S-sulfoxide reduction in the ER on stress resistance. The ER-targeted MsrA construct contained the N-terminal ER-targeting signal peptide of human MsrB3A (MSPRRSLPRPLSLCLSLCLCLCLAAALGSAQ) and the C-terminal ER-retention signal sequence (KAEL). The over-expression of ER-targeted MsrA significantly increased cellular resistance to H2O2-induced oxidative stress. The ER-targeted MsrA over-expression also significantly enhanced resistance to dithiothreitol-induced ER stress; however, it had no positive effects on the resistance to ER stresses induced by tunicamycin and thapsigargin. Collectively, our data suggest that methionine-S-sulfoxide reduction in the ER compartment plays a protective role against oxidative and ER stresses.

Cancer-associated fibroblast (CAF)-specific proteins serve as both prognostic biomarkers and targets for anticancer drugs. In this study, we investigated the role of NGFI-A-binding protein (NAB)2 derived from CAFs in the progression of head and neck squamous cell carcinoma (HNSCC). Patient-derived HNSCC and paired metastatic lymph node tissues were examined for NAB2 expression by immunohistochemistry. Primary CAF cultures were established from HNSCC patient tissue, with paired non-tumor fibroblasts (NTFs) serving as a control. CAF or NTF was used to evaluate the effect of NAB2 on HNSCC progression using FaDu cell spheroids and an in vivo mouse xenograft model. NAB2 was detected in interstitial CAFs in primary and metastatic lymph node tissues of HNSCC patients. NAB2 mRNA and protein levels were higher in CAFs as compared to paired NTFs. Conditioned medium (CM) of NAB2-overexpressing CAFs increased the invasion of FaDu spheroids in the Matrigel invasion assay as compared to CM of NTF. Co-injection of NAB2-overexpressing CAFs with FaDu spheroids into mice enhanced the growth of tumors. These data suggest that NAB2-overexpressing CAFs promotes HNSCC progression and is a potential therapeutic target for preventing HNSCC metastasis.

NGFI-A binding protein 2 (NAB2) represses the transcriptional activation of early growth response protein-1 (EGR1), a tumor-suppressor. However, Epidermal Growth Factor (EGF) promotes tumor progression even with significant EGR1 upregulation. The molecular mechanism through which NAB2 is involved in cancer is largely unknown. Therefore, we evaluated how the NAB2-mediated suppression of EGR1 facilitates head and neck squamous cell carcinoma (HNSCC) cancer progression, in association with Sp1, which competes with EGR1 as a transcriptional regulator. The effect of NAB2 on EGR1/SP1 binding to the consensus promoter sequences of MMP2 and MMP9 was evaluated by chromatin immunoprecipitation (ChIP) and promoter luciferase assay. The correlation between EGR1-NAB2 expression and metastatic status was investigated using The Cancer Genome Atlas (TCGA) for HNSCC patients. Our data showed that NAB2 knockdown in FaDu and YD-10B HNSCC cells alleviated EGF-dependent increase of Matrigel invasion. In addition, NAB2 upregulation in EGF-treated FaDu cell diminishes EGR1 transcriptional activity, resulting in the upregulation of Sp1-dependent tumor-promoting genes. TCGA data analysis of 483 HNSCC tumors showed that higher levels of both EGR1 and NAB2 mRNA were significantly associated with metastasis, corresponding to in vitro results. Our data suggest that NAB2 upregulation facilitates EGF-mediated cancer cell invasion through the transactivation of Sp1-dependent tumor-promoting genes. These results provide insight into the paradoxical roles of EGF-EGR1 in cancer progression.

Background Endoscopic submucosal dissection (ESD) is not considered appropriate for all submucosal cancers owing to the risk of lymph node metastasis and difficulty estimating the deep margin status. This study aimed to determine predictive factors for lymph node metastases in submucosal cancer and to explore in which patients ESD might be feasible. Methods Details of patients who had curative gastrectomy for submucosal gastric cancer at Asan Medical Centre from 2007 to 2011 were reviewed retrospectively to determine the relationship between lymph node metastasis and clinicopathological characteristics, including age, sex, tumour location, size, gross appearance, depth of invasion, histological type/differentiation, presence of lymphovascular/perineural invasion, and immunohistochemical staining results for p53, human epidermal growth factor receptor (HER) 1 and HER2. Results A total of 1773 patients were analysed. The presence of lymphovascular invasion was related most strongly to lymph node metastasis. Multivariable analysis revealed that depth of invasion, tumour size, differentiation, gross appearance and perineural invasion were also related. Metastatic lymph nodes were found in four of 105 patients who met the classical criteria for ESD; all showed a moderately differentiated histological appearance. No lymph node metastases were observed in well differentiated SM1 tumours of any size (infiltration into upper third of submucosa), or in well differentiated SM2 (infiltration into middle third of submucosa) tumours of 2 cm or less without lymphovascular invasion. Conclusion Patients with well differentiated SM1 cancer of any size and those with well differentiated SM2 cancer of 2 cm or less without lymphovascular invasion may be suitable candidates for ESD.

MiRNAs are non-coding RNAs that play a role in the regulation of major processes. The inhibition of miRNAs using antisense oligonucleotides (ASOs) is a unique and effective technique for the characterization and subsequent therapeutic targeting of miRNA function. Recent advances in ASO chemistry have been used to increase both the resistance to nucleases and the target affinity and specificity of these ASOs.Peptide nucleic acids (PNAs) are artificial oligonucleotides constructed on a peptide-like backbone. PNAs have a stronger affinity and greater specificity to DNA or RNA than natural nucleic acids and are resistant to nucleases, which is an essential characteristic for a miRNA inhibitor that will be exposed to serum and cellular nucleases.For increasing cell penetration, PNAs were conjugated with cell penetrating peptides (CPPs) at N-terminal. Among the tested CPPs, Tat-modified peptide-conjugated PNAs have most effective function for miRNA inhibition. PNA-based ASO was more effective miRNA inhibitor than other DNA-based ASOs and did not show cytotoxicity at concentration up to 1,000 nM. The effects of PNA-based ASOs were shown to persist for 9 days. Also, PNA-based ASOs showed considerable stability at storage temperature. These results suggest that PNA-based ASOs are more effective ASOs of miRNA than DNA-based ASOs and PNA-based ASO technology, compared with other technologies used to inhibit miRNA activity can be an effective tool for investigating miRNA functions.

In this paper, we investigated the (002) preferred orientation and morphology characteristics of AlN thin film by using reactive rf sputtering. Additionally, AlN thin films grown in the range from 150 to 300 W were studied under room temperature without substrate heating and post annealing. Sputtered AlN thin films were well grown on Si substrates and the (002) main peak in XRD patterns showed the highest intensity at 300 W with degree of full width at half-maximum (FWHM). As increased RF power, the surface roughness was increased from 1.0 to 3.4 nm. In Fourier transformation infrared spectroscopy (FTIR), (TO) and (TO) mode closed to AlN thin film confirmed the changes with increasing the intensity rate. From these results, we could confirm a chance of the growth of AlN thin film by only low temperature.

With development of electronic products the demands for miniaturization and weight-lightening have increased until a recent date. Accordingly, The effort to substitute glass substrates was widely made. However, polymer substrates have weak point that substrates were damaged at high temperature. In this paper, we deposited transparent conductive film at low temperature. And we inserted Au thin film between oxide to compensate for deteriorated electrical characteristics. Ga-doped ZnO(GZO) multilayer coatings were deposited on glass substrate by DC sputtering. The optimization of deposition conditions of both AZO and Au layers were performed to obtain better electrical and optical characteristics in advance. We presumed that the properties of multilayer were affected by the deposition process of both GZO and Au layers. The best multilayer coating exhibited the resistivity of and transmittance of 77 %. From these results, we can confirm a possibility of the application as transparent conductive electrodes.

In this study we investigated the variation of the substrate temperatures using RF sputtering to identify the effect on the structure and electrical properties by c-axis orientation of ZnO thin film. ZnO thin films were prepared on Al/Si substrate. In our experimental results, ZnO thin film at was well grown with (002) peak of ZnO thin film, the thin film showed the high resistivity with the value of and the roughness with 27.06 nm. As increased the substrate temperatures, the grain size of ZnO thin films was increased. From these results, we could confirm the suitable substrate temperature of ZnO thin films for FBAR(film bulk acoustic resonator).

In this study we present the effect of annealing temperatures on the structural, electrical and optical characteristics of Ga-doped ZnO (GZO) films. GZO target is deposited on coming 7059 glass substrates by DC sputtering. and then GZO films are annealed at temperatures of 400, 500, in air ambient for 20 min. in this case of as-grown film, it shows the resistivity of and transmittance under 85%, whereas the electrical and optical properties of film annealed at are enhanced up to and 90%, respectively.

Facilities of electric railway should be maintained only at night and it is necessary to maintain by human power because facilities are installed at a high place. This paper presents the development and application of a real-time data acquisition system for the characteristics measurement of overhead contact wires in electric railway. The system is designed to perform in the telemetry environments that developed to store data by wireless in a live wire state of 25 kV power source. The field test results show that the proposed technique and the developed system can be practically applied to measure characteristics of temperature, displacement, and strain on overhead contact lines.

In this study, we fabricated FBAR(film bulk acoustic resonator) by using as a function of annealing temperature and concentrated on effect of frequency characteristic of FBAR. The results show that the annealing affects resistivity and crystallity. The optimum properties were observed for film annealed at . The resistivity was and the roughness was 21.10 nm. And the return loss is improved from -24.9 at to -29.8 at without the resonant frequency change. We finally confirmed the improvement on the frequency characteristics of FBAR device by annealing process at the optimized condition.

Hypoxia-inducible factor (HIF) is a key regulator of tumor growth and angiogenesis. Recent studies have shown that, BIX01294, a G9a histone methyltransferase (HMT)-specific inhibitor, induces apoptosis and inhibits the proliferation, migration, and invasion of cancer cells. However, not many studies have investigated whether inhibition of G9a HMT can modulate HIF-1α stability and angiogenesis. Here, we show that BIX01294 dose-dependently decreases levels of HIF-1α in HepG2 human hepatocellular carcinoma cells. The half-life of HIF-1α, expression of proline hydroxylase 2 (PHD2), hydroxylated HIF-1α and von Hippel-Lindau protein (pVHL) under hypoxic conditions were decreased by BIX01294. The mRNA expression and secretion of vascular endothelial growth factor (VEGF) were also significantly reduced by BIX01294 under hypoxic conditions in HepG2 cells. BIX01294 remarkably decreased angiogenic activity induced by VEGF in vitro, ex vivo, and in vivo, as demonstrated by assays using human umbilical vein endothelial cells (HUVECs), mouse aortic rings, and chick chorioallantoic membranes (CAMs), respectively. Furthermore, BIX01294 suppressed VEGF-induced matrix metalloproteinase 2 (MMP2) activity and inhibited VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR-2), focal adhesion kinase (FAK), and paxillin in HUVECs. In addition, BIX01294 inhibited VEGF-induced formation of actin cytoskeletal stress fibers. In conclusion, we demonstrated that BIX01294 inhibits HIF-1α stability and VEGF-induced angiogenesis through the VEGFR-2 signaling pathway and actin cytoskeletal remodeling, indicating a promising approach for developing novel therapeutics to stop tumor progression.

Since its discovery in 1962 by Ritossa, the heat shock response has been extensively studied by a number of investigators to understand the molecular mechanism underlying the cellular response to heat stress. The most well characterized heat shock response is induction of the heat shock proteins that function as molecular chaperones and exert cell cycle regulatory and anti-apoptotic activities. While most investigators have focused their studies on the toxic effects of heat stress in organisms such as severe heat stress-induced cell cycle arrest and apoptosis, the cellular response to fever-ranged mild heat stress has been rather underestimated. However, the cellular response to mild heat stress is likely to be more important in a physiological sense than that to severe heat stress because the body temperature of homeothermic animals increases by only 1-2 degrees C during febrile diseases. Here we provide information that mild heat stress does have some beneficial role in organisms via positively regulating cell proliferation and differentiation, and immune response in mammalian cells.

Background: The study was to examine Perceived Health Status, Climacteric Symptoms, and Health-Promoting Lifestyle among Middle-Aged Women residing in Jeju Island, and to identify the relationship among variables, ultimately to provide the basic data in developing health-promoting program for them in future.Methods: The data were collected from Sep. 1st to Sep. 20th, 2004, and the subjects consisted of 112 women aged between 40 years old and 60 years old, residing at Jejudo. Data analyses were conducted by using Pearson correlation coefficients, t-test, and ANOVA.Results: In Health-promoting lifestyle, the more educated subjects and those who have experienced diseases were found doing more health-promoting lifestyle. In perceived health status, the subjects educated over college, the subjects satisfied with marriage, and the subjects who have not experienced diseases were indicating higher. In climacteric symptoms, subjects aged 50 to 59 years old, subjects educated less than primary school, subjects not satisfied with marriage, and subjects with monthly family income over 2 million Won were higher. Subjects with experience of diseases were also higher than subjects with no experience of diseases. Health-promoting lifestyle was positively correlated with perceived health status and showed no correlation with climacteric symptoms.Conclusion: It was suggested that similar studies should be carried out for more subjects from various provinces, as the study was limited to only 112 middle-aged women residing at Jejudo, and thereby the development of health-promoting program for middle-aged women should be conducted.

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as sodium salicylate, aspirin and indomethacin have been reported to activate the heat shock transcription factor (HSF) without enhancing the expression of the heat shock proteins (HSPs). We investigated the effects of NSAIDs on the heat shock-induced HSP expression in fish cell line CHSE-214. Here we reveal that in CHSE-214 cells, co-exposure to sodium salicylate/aspirin and heat shock (24°C) enhances and prolongs the heat shock-induced HSP70 expression presumably through activation of the HSF. In contrast, the heat shock-induced HSP90 expression was inhibited by these NSAIDs. Thus, sodium salicylate and aspirin are likely to exert different effects on the heat shock-induced HSP70 and HSP90 expression. Indomethacin, another cyclooxygenase inhibitor, had no stimulatory or inhibitory effects on the heat shock-induced HSP70 and 90 expression, thereby indicating that sodium salicylate and aspirin may modulate the heat shock response via pathways not...

The expression of p21WAF1/ClP1/SDI1, one of the cyclin‐dependent kinase inhibitors, is regulated by a variety of transcription factors including p53 and STAT. Heat shock induces the expression of p21 in a temperature‐ and time‐dependent manner. Although the p21 induction by heat shock has been reported to be controlled by p53, a p53‐independent mechanism is also involved. To understand the p53‐independent regulation of heat shock‐induced p21 expression, we searched the promoter region of p21 gene and found one or two heat shock element (HSE)‐like sequences in human, rat, and mouse. Electromobility shift assay (EMSA) showed that heat shock factor (HSF) could bind to these HSE‐like sequences in response to heat shock, even though to a lesser extent than to HSE. In addition, p21 promoter deletion analysis revealed that heat shock activated a p21 deletion promoter construct containing the HSE‐like sequences but lacking p53‐binding sites, but not a promoter construct containing neither HSE‐like sequences nor t...

Background: This phase 3 study evaluated the efficacy of new adjuvant chemotherapy (MFP), which intensified the mitomycin-C (MMC) plus short-term doxifluridine (Mf) for gastric cancer. Patients and methods: A total of 855 patients (424 in Mf, 431 in MFP) with pathological stage II–IV (M0) gastric cancer after D2 gastrectomy were randomly assigned to receive either Mf (MMC 20 mg m−2, followed by oral doxifluridine 460–600 mg m−2 per day for 3 months) or MFP (MMC 20 mg m−2, followed by oral doxifluridine 460–600 mg m−2 per day for 12 months with 6 monthly infusions of 60 mg m−2 of cisplatin) chemotherapy. Results: With a median follow-up of 6.6 years, there was no difference between the two groups in recurrence-free survival (RFS) (5-year RFS 61.1% in Mf and 57.9% in MFP; hazard ratio 1.10 (95% CI 0.89–1.35); P=0.39) and overall survival (OS) (5-year OS 66.5% in Mf and 65.0% in MFP; hazard ratio 1.11 (95% CI 0.89–1.39); P=0.33). Conclusion: Intensification of Mf adjuvant chemotherapy by prolonging the duration of oral fluoropyrimidine and adding cisplatin was safe but not effective to improve the survivals in curatively resected gastric cancer patients.

Cancer stem cells (CSCs), a group of cancer cells, are associated with resistance to radiation and chemotherapy and are implicated in recurrent of cancer. Exostosin 1 (EXT1) gene is widely reported as tumor suppressor and its indispensable role in elongation of heparan sulfate (HS) can speculate probable role as tumor promotor. In recent years, a number of tumors are reported to over express EXT1. Here, we investigated the role of EXT1 in the maintenance of cancer cell stemness. MCF7/ADR cells developed by exposing MCF7, breast cancer cells, to doxorubicin for several months in culture, showed high resistance to doxorubicin compared to MCF7, parental cell line. Doxorubicin resistant MCF7/ADR cells have enhanced P-glycoprotein (P-gp) expression. In FACs analysis, MCF7/ADR cells were accounted for high number of CSC population; they possessed large number of ALDH+ and CD44+/CD24- population compared to MCF7. CSC marker, CD44, was found overexpressed in MCF7/ADR compared to MCF7. Besides that, in mammosphere culture, MCF7/ADR cells formed large number of mammospheres in contrast to MCF7. In microarray data analysis, several genes were found upregulated in MCF7/ADR cells and EXT1 was selected as a candidate gene. Overexpressed EXT1 in MCF7/ADR cells was confirmed by real time PCR. The critical role of EXT1 in maintaining cancer cell stemness was confirmed by siRNA mediated knockdown of EXT1. Knockdown of EXT1 repressed CSC markers, reduced population of ALDH+ and CD44+/CD24-, and suppressed expression of CD44. As a molecular mechanism, we detected suppressed expression of heparan sulfate, syndecan1(SDC1) with siEXT1. With these results, we can suggest that EXT1 level might be a determining factor for doxorubicin sensitive breast cancer cells and enhanced expression of EXT1 can take part in development of CSC phenotype on exposure to anthracycline based therapy through upregulation of SDC1. Citation Format: Sarala Manandhar, Chang-gu Kim, Su Young Oh, Sun-Hee Lee, Jiyoon Seok, Yuk-Dong Jung, Hye-Eun Lee, Young-Sun Choi, You Mie Lee*. Exostosin 1 regulates cancer cell stemness in breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B21.

Endothelial progenitor cells (EPCs) are bone marrow (BM) -derived stem cells to be committed and differentiated into mature endothelial cells. Runt-related transcription factor 3 (RUNX3) is a tumor suppressor involved in TGF-β signaling pathway and participate in cell cycle arrest and anti-angiogenesis. RUNX3 is frequently deleted or transcriptionally silenced in a variety of cancers by epigenetic mechanisms. The aim of study was to determine the role of RUNX3 in EPC function and vasculogenesis. Here we found that RUNX3 homozygous knockout (Rx3-/-) embryos had vascular defects in embryonic day (ED) 10 and developmental abnormalities in ED 16.5 organs. The number of EPC colonies and circulating EPCs (CD34+/VEGFR2+ cells), migration and tube formation capacity of EPCs derived from RUNX3 heterozygous (Rx3+/-) mice were increased in compared to those wild type (WT) mice, suggesting that deletion of RUNX3 can enhance vasculogenesis. Expression level of VEGF, VEGFR2, SDF-1, CXCR4 and endothelial nitric oxide synthase (eNOS) mRNA was significantly up-regulated in Rx3+/- EPCs. Protein expression of VEGF, p-VEGFR2, p-Akt, p-ERK, p-SAPK/JNK and p-eNOS was also augmented in Rx3+/- EPCs. Finally, we found that recovery of blood flow was highly stimulated in Rx3+/- mice using hindlimb ischemia mouse models. Taken together, our study revealed that RUNX3 might inhibit vasculogenic processes in physiological ischemia by inhibition of EPC functions. Citation Format: Su Young Oh, Se-Hyung Lee, Sun Hee Lee, You Mie Lee. Heterozygous deletion of RUNX3 promotes vasculogenesis by enhancing endothelial progenitor cells function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2485.

Salicylate and jasmonates are two different types of plant hormone that play critical roles in plant defense responses against insect herbivores and microbial pathogens, through activating defense genes. These two naturalproducts have been shown to have similar activities in animal cells: the compounds are able to induce cell cycle arrest or apoptosis in a variety of human cancer cells including those of colon, prostate, breast, and leukemia, suggesting the chemicals may potentially be a novel class of anti-cancer drugs. Since sodium salicylate can induce the heat shock response in animals, we examined the effects of jasmonates on the heat shock response in C6 glioma cells. Here, we show that brief exposure to methyl jasmonate (MeJA), but not to jasmonic acid, induces heat shock protein 72 (HSP72), but not HSP73 and HSP90, via heat shock factor I (HSF1) activation in C6 glioma cells without affecting cell viability. Intracellular H 2 O 2 and O 2 - , and mitochondrial ROS were prominently increased in response to 5 mM MeJA in C6 cells. MeJA-induced HSP72 expression, HSF1 DNA binding, and human HSP70 promoter-driven CAT activity were prevented by N-acetyl-L-cysteine (a general antioxidant), catalase (a specific antioxidant for H 2 O 2 ), and sodium formate (an inhibitor of OH-), but not by Rac1 dominant negative mutant Rac1N17 and diphenylene-iodonium (a NADPH oxidase inhibitor), indicating that MeJA induces HSP72 expression though HSF1 that is activated via Racl-NADPH oxidase-independent ROS production pathway. These results suggest that the plant stress hormones share the ability to induce heat shock response in animal cells.

Sodium salicylate, one of anti-inflammatory agents, is known to partially induce the heat shock response: it stimulates the DNA-binding of heat shock factor 1 (HSF1) without inducing heat shock gene expression. Here we show that when C6 glioma cells are recovered from sodium salicylate treatment, they highly induce heat shock protein 72 (HSP72), but not HSP73 and HSP90, demonstrating that salicylate-induced inert HSF1 can be fully activated into a transcriptionally competent form by sodium salicylate recovery (SR)-specific mechanism. Fluorescent analysis using 2',7'-dichlorodihydrofluorescein diacetate revealed that sodium salicylate enhanced reactive oxygen species (ROS) production. N-acetyl-L-cysteine (NAC, a ROS scavenger) completely suppressed SR-induced HSP72 synthesis and HSP72 promoter-driven CAT reporter gene transcription as well as salicylate-induced HSF1-DNA binding, indicating a critical role(s) of ROS in the SR-induced HSP72 gene regulation. We also show that treatment of C6 cells with sodium salicylate activated p38MAPK and inactivated ERK1/2 in a ROS-independent manner and activities of these protein kinases returned during recovery period to the control level. Inhibiting p38MAPK and ERK1/2 with the p38MAPK inhibitors (SB203580 and SB202190) and the MEK1/2 inhibitor (PD98059 and U0126) or with expression of dominant negative p38MAPK and ERK1/2 abolished SR-induced HSP72 synthesis and HSP70 promoter-driven CAT activity. However, sodium salicylate-induced HSF1-DNA binding was not affected by the p38MAPK inhibitor or the MEK1/2 inhibitor. These findings suggest that sodium salicylate partially activates HSF1 via ROS production and p38MAPK activation and the salicylate-induced inert HSF1 can be fully activated into a transcriptionally competent form by the ERK1/2 signaling pathways that are activated independently of ROS during SR.

Heat shock induces c-Jun N-terminal kinase (JNK) activation as well as heat shock protein (HSP) expression through activation of the heat shock factor (HSF), but its signal pathway is not clearly understood. Since a small GTPase Rac1 has been suggested to participate in the cellular response to stresses, we examined whether Rac1 is involved in the heat shock response. Here we show that moderate heat shock (39-41 degrees C) induces membrane translocation of Rac1 and membrane ruffling in a Rac1-dependent manner. In addition, Rac1N17, a dominant negative mutant of Rac1, significantly inhibited JNK activation by heat shock. Since Rac1V12 was able to activate JNK, it is suggested that heat shock may activate JNK via Rac1. Similar inhibition by Rac1N17 of HSF activation in response to heat shock was observed. However, inhibitory effects of Rac1N17 on heat shock-induced JNK and HSF activation were reduced as the heat shock temperature increased. Rac1N17 also inhibited HSF activation by l-azetidine-2-carboxylic acid, a proline analog, and heavy metals (CdCl)), suggesting that Rac1 may be linked to HSF activation by denaturation of polypeptides in response to various proteotoxic stresses. However, Rac1N17 did not prevent phosphorylation of HSF1 in response to these proteotoxic stresses. Interestingly, a constitutively active mutant Rac1V12 did not activate the HSF. Therefore, Rac1 activation may be necessary, but not sufficient, for heat shock-inducible HSF activation and HSP expression, or otherwise a signal pathway(s) involving Rac1 may be indirectly involved in the HSF activation. In sum, we suggest that Rac1 may play a critical role(s) in several aspects of the heat shock response.

The miRNAs are approximately ∼23 nucleotides non-coding RNAs. They regulate major processes such as development, apoptosis, cell proliferation, hematopoiesis, and patterning of the nervous system. MiRNA inhibition using antisense oligonucleotide is an effective tool for understanding miRNA function. The peptide nucleic acid (PNA) is an artificial oligonucleotide with a peptide backbone. PNAs have a stronger affinity and greater specificity than DNA or RNA and are resistant to nuclease, which is essential characteristic for a miRNA inhibitor that will be exposed to abundant serum and cellular nucleases. In here, we evaluated PNAs™ miRNA inhibitors in cultured cells including stem cell using luciferase assay and qPCR. FACS analysis was showed that PNAs™ miRNA inhibitors can penetrate to intra cells without transfection reagent. Also, miRNA inhibition effects are sustained until 15 days after treatment. PNAs™ miRNA inhibitor has a lower cytotoxicity than other nucleic acid-based miRNA inhibitors. PNAs™ miRNA inhibitors can be stored at room temperature for 20 weeks without losing the activity. These results demonstrate that PNAs are a powerful and effective tool to studies of miRNA mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4073.

Hyperthermia such as that occurring during fever may improve cell survival during infection, although its mechanism of action is largely unknown. Here we show that acute exposure to mild, but not severe, heat shock induces the synthesis of cyclin D1 that plays a critical role(s) in G1 progression of the cell cycle. This induction seemed to be regulated through multiple Ras signal pathways involving extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Rac1/NADPH oxidase, all of which have well been documented to be responsible for growth factor-induced cyclin D1 expression. In a physiological sense, mild heat shock may regulate cell proliferation through inducing cyclin D1 along with growth factors.