• Journal of Life Science
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• v.26 no.12
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• pp.1383-1391
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• 2016

Anti-estrogen drugs such as tamoxifen have been used for treating patients with ER-positive, early breast cancer. However, resistance to anti-estrogen treatment is inevitable in most patients. Breast cancer anti-estrogen resistance-3 (BCAR3) has been identified as the protein responsible for the induction of tamoxifen resistance in estrogen-dependent human breast cancer. We have previously reported that BCAR3 regulates the cell cycle progression and the signaling pathway of EGF and insulin leading to DNA synthesis. In this study, we investigated the functional role of BCAR3 in regulating c-Jun transcription in non-tumorigenic human breast epithelial MCF-12A cells. A transient transfection of BCAR3 increased both the mRNA and protein of c-Jun expression, and stable expression of BCAR3 increased c-Jun protein expression. The overexpression of BCAR3 directly activated the promoter of c-jun, AP-1, and SRE but not that of $NF-{\kappa}B$. Furthermore, single-cell microinjection of BCAR3 expression plasmid in the cell cycle-arrested MCF-12A cells induced c-Jun protein expression, and co-injection of dominant negative mutants of Ras, Rac, and Rho suppressed the transcriptional activity of c-Jun in the presence of BCAR3. Furthermore, stable expression of BCAR3 increased the proliferation of MCF-12A cells. The microinjection of inhibitory materials such as anti-BCAR3 antibody and siRNA BCAR3 inhibited EGF-induced c-Jun expression but did not affect IGF-1 induced upregulation of c-Jun. Taken together, we propose that BCAR3 plays a crucial role in c-Jun protein expression and cell proliferation and that small GTPases (e.g., Ras, Rac, and Rho) are required for the BCAR3-mediated activation of c-Jun expression.

• Journal of Life Science
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• v.19 no.7
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• pp.949-955
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• 2009

Mucosal epithelia sense external stress signals and transmit them to the intracellular cascade responses. Ribotoxic stress-producing chemicals such as deoxynivalenol (DON) or other trichothecene mycotoxins have been linked with gastrointestinal inflammatory diseases by Fusarium-contamination. The purpose of this study was to test the hypothesis that DON evokes the epithelial sentinel signals of RNA-dependent protein kinase (PKR) and early growth response gene 1 (EGR-1), which together contribute to the pro-inflammatory cytokine interleukin 8 (IL-8) in human intestinal epithelial cells. PKR suppression by the dominant negative PKR expression attenuated DON-stimulated interleukin-8 production. Moreover, 1L-8 transcriptional activation by DON was also reduced by PKR inhibition in the human intestinal epithelial cells. Treatment with the PKR inhibitor also suppressed EGR-1 promoter activity, mRNA and protein induction, although mitogen-activated protein (MAP) kinases such as extracellular signal-regulated protein kinases (ERK) 1/2, p38, c-Jun N-terminal Kinase (INK) were little affected or even enhanced in presence of a PKR inhibitor. These patterns were also compared in the EGR-1-suppressed cells, which showed much more suppressed production of 1L-8. All things taken into consideration, DON-activated sentinel signals of EGR-1 via PKR mediated interleukin-8 production in human intestinal epithelial cells, which provide insight into the possible general mechanism associated with mucosal inflammation as an intestinal toxic insult by ribotoxic trichothecene mycotoxins.

• Proceedings of the Korean Society of Toxicology Conference
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• 1998.10a
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• pp.36-36
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• 1998

Tumorigenicity of benzo(a)pyrene (BP) and benzo(a)pyrene diol epoxides ((+)BPDE-1, (-)BPDE-1) was investigated in transgenic TG-AC mice carrying v-Ha-ras oncogene fused to the promoter of the mouse embryonic a-like, z-globin gene. Animals were topically treated twice per week for 25weeks with BPDE (10$\mu$g/mouse) and BP (10, 20, 40$\mu$g/mouse). In addition, animals were treated with BPDE or BP (initiated) followed by TPA (2$\times$2.5$\mu$g/week, for 4 weeks) for promotion study. In the continuous treatment of BPDE or BP, animals treated with 40$\mu$g BP showed $100\%$ tumor response after 20 weeks, $40\%$ of mice for 20$\mu$g BP, and $20\%$ for (+)BPDE-1, but (-)BPDE-1 and 10$\mu$g BP did not show any tumor response. After 25 weeks, most tumors turned out to be carcinomas in animals treated with 40$\mu$g BP. In BPDE or BP/TPA Initiation-promotion study, papilloma response occurred earlier (6 weeks after TPA treatment) than in continuously treated animals with BPDE or BP. RT-PCR assay for transgene expression showed that BP or BPOE was not transgene dependent in its tumorigenicity, but TPA was. Several Cytokine genes(TGF-a, TNF-a) and c-myc gene expressions were monitored in skin tissues during BP carcinogenesis. In early stage of BP treatment, the gene expressions were elevated(c-myc,TGF-a) or unchanged(TNF-a) compared to control, but the levels were gradually decreased during both middle and late stages of cacinogenesis, Gene expression levels of skin papillomas in acetone initiated-TPA promoted animals were close to those of middle stage or between middle and late stages. i-NOS was also highly expressed in carcinoma and papilloma, These data suggest that transgene expressions of TG-AC mice were not dependent on BP carcinogenesis and that TG-AC mice were more sensitive to TPA regardless of types of initiators. In addition, genes(TGF-a, c-myc, TNF-a, i-NOS) were modulated in the skin during BP cacinogenesis or TPA promotion.

• KSBB Journal
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• v.24 no.6
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• pp.591-597
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• 2009

B-type natriuretic peptide is a neurohormone secreted in the cardiac ventricles. BNP levels are elevated in patients with ventricular dysfunction. Therefore, the concentration of BNP is important factor to reflect diagnosis and prognosis for cardiovascular disease. In this respect, anti-BNP scFv is an urgent requirement for early diagnosis in the field of biosensor. Herein, the genetic codes of anti-BNP scFv were chemically synthesized and cloned into both pET22b (+) and pColdⅣ vector, respectively. The recombinant scFv was successfully expressed as a functional form in cytoplasm of E. coli and detected through Western blot and ELISA. The highest level of functional expression of anti-BNP scFv was achieved using pET22b (+) vector at $15^{\circ}C$ by addition of 0.1 mM IPTG. Additionally, being exposed to both BNP and ANP, anti-BNP scFv specifically captured only BNP. Therefore, anti-BNP scFv expressed in this study will be applied to measure the concentration of BNP as a diagnostic recognition molecule.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.6
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• pp.2753-2758
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• 2014

Background: GC-binding factor 2 (GCF2) is a transcriptional regulator that represses transcriptional activity of the epidermal growth factor receptor (EGFR) by binding to a specific GC-rich sequence in the EGFR gene promoter. In addition to this function, GCF2 has also been identified as a tumor-associated antigen and regarded as a potentially valuable serum biomarker for early human hepatocellular carcinoma (HCC) diagnosis. GCF2 is high expressed in most HCC tissues and cell lines including HepG2. This study focused on the influence of GCF2 on cell proliferation and apoptosis in HepG2 cells. Materials and Methods: GCF2 expression at both mRNA and protein levels in HepG2 cells was detected with reverse transcription (RT) PCR and Western blotting, respectively. RNA interference (RNAi) technology was used to knock down GCF2 mRNA and protein expression. Afterwards, cell viability was analyzed with a Cell Counting Kit-8 (CCK-8), and cell apoptosis and caspase 3 activity by flow cytometry and with a Caspase 3 Activity Kit, respectively. Results: Specific down-regulation of GCF2 expression caused cell growth inhibition, and increased apoptosis and caspase 3 activity in HepG2 cells. Conclusions: These primary results suggest that GCF2 may influence cell proliferation and apoptosis in HepG2 cells, and also provides a molecular basis for further investigation into the possible mechanism at proliferation and apoptosis in HCC.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.10
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• pp.4281-4287
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• 2014

Background: Methylation of tumor suppressor genes has been investigated in all kinds of cancer. Tumor specific epigenetic alterations can be used as a molecular markers of malignancy, which can lead to better diagnosis, prognosis and therapy. Therefore, the aim of this study was to evaluate the association between gene hypermethylation and expression of fragile histidine triad (FHIT), glutathione S-transferase P1 (GSTP1) and p16 genes and various clinicopathologic characteristics in primary non-small cell lung carcinomas (NSCLC). Materials and Methods: The study included 28 primary non-small cell lung carcinomas, where an additional 28 tissue samples taken from apparently normal safety margin surrounding the tumors served as controls. Methylation-specific polymerase chain reaction (MSP) was performed to analyze the methylation status of FHIT, GSTP1 and p16 while their mRNA expression levels were measured using a real-time PCR assay with SYBR Green I. Results: The methylation frequencies of the genes tested in NSCLC specimens were 53.6% for FHIT, 25% for GSTP1, and 0% for p16, and the risk of FHIT hypermethylation increased among patients with NSCLC by 2.88, while the risk of GSTP1 hypermethylation increased by 2.33. Hypermethylation of FHIT gene showed a highly significant correlation with pathologic stage (p<0.01) and a significant correlation with smoking habit and FHIT mRNA expression level (p<0.05). In contrast, no correlation was observed between the methylation of GSTP1 or p16 and smoking habit or any other parameter investigated (p>0.05). Conclusions: Results of the present study suggest that methylation of FHIT is a useful biomarker of biologically aggressive disease in patients with NSCLC. FHIT methylation may play a role in lung cancer later metastatic stages while GSTP1 methylation may rather play a role in the early pathogenesis.

• Korean journal of applied entomology
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• v.56 no.1
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• pp.19-28
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• 2017

Polydnaviruses (PDVs) are a group of double-stranded DNA viruses symbiotic to some endoparasitoid wasps. Cotesia plutellae bracovirus (CpBV) is a PDV symbiotic to an endoparasitoid wasp, C. plutellae, parasitizing young larvae of Plutella xylostella. An early expressed gene, CpBV-ELP1, plays an important role in the parasitism by suppressing host cellular immunity by its cytotoxic activity against hemocytes. This study aimed to test its oral toxicity against insect pest by expressing it in a recombinant tobacco plant. A recombinant CpBV-ELP1 protein was produced using a baculovirus expression system and secreted to cell culture medium. The cell cultured media were used to purify CpBV-ELP1 by a sequential array of purification steps: ammonium sulfate fractionation, size exclusion chromatography, and ion exchange chromatography. Purified rCpBV-ELP1 exhibited a significant cytotoxicity against Spodoptera exigua hemocytes. CpBV-ELP1 was highly toxic to the fifth instar larvae of S. exigua by injection to hemocoel. It also showed a significant oral toxicity to fifth instar larvae of S. exigua by a leaf-dipping assay. CpBV-ELP1 was cloned into pBI121 vector under CaMV 35S promoter with opaline synthase terminator. Resulting recombinant vector (pBI121-ELP1) was used to transform Agrobacterium tumefaciens LBA4404. The recombinant bacteria were then used to induce callus of a tobacco (Nicotiana tabacum Xanthi) leaves and subsequent generation (T1) plants were selected. T1 generation tobacco plants expressing CpBV-ELP1 gave significant insecticidal activities against S. exigua larvae. These results suggest that CpBV-ELP1 gene can be used to control insect pests by constructing transgenic crops.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.4031-4036
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• 2012

Background: The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. Material and Methods: An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Results: Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. Conclusion: This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.2
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• pp.231-236
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• 2001

This study was conducted to investigate the effect of supplemented enzyme complex on growth performance and nutrient digestibility in pigs weaned at 14 days of age. Eighty pigs ($4.02{\pm}0.11kg$ of average body weight) were allotted in a completely randomized block design. Treatments were as follows: 1) control (negative), 2) control (positive, $Kemzyme^{(R)}$), 3) 0.1%, 4) 0.2% and 5) 0.3% of newly developed enzyme complex. Each treatment has 4 replicates with 4 pigs per replicate. During phase I period (d 0 to 14), ADG and ADFI were numerically higher in pigs fed diets supplemented enzyme complex regardless of their inclusion levels compared to pigs fed control (negative) diet. Feed/gain (F/G) was also better in pigs fed enzyme complex diet than that of pigs fed control (negative) diet. In addition, with increasing the inclusion level of enzyme complex, ADG and ADFI were improved. However, there was no significant difference between treatment in all growth parameters. During phase II period (d 15 to 28), ADG, ADFI and F/G showed the same tendency as in phase I period. For overall period (d 0 to 28) ADG was highest in pigs fed diet included 0.2% enzyme complex in all treatments but not significantly different. During phase I period, the digestibilities of all nutrients did not showed any significant difference between treatments. However, pigs fed diet contained enzyme complex and positive control diet (Kemzyme) showed numerically higher nutrient digestibilities in all nutrients than pigs fed negative control diet. During phase II period, data were consistent with those observed in phase I period. Especially, the digestibility of phosphorus was significantly higher in pigs fed diets contained enzyme complex including phytase than pigs fed control (negative and positive) diets (p<0.05). For overall experimental period, fecal or ileal amino acid digestibility were not affected by dietary treatment. Enzyme complex newly developed and used in this study can be possibly recommended as a growth promoter when supplemented in diet for early weaned piglets.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.2185-2193
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• 2016

Background: The pathogenesis of sporadic colorectal cancer (CRC) is influenced by the patient genetic background and environmental factors. Based on prior understanding, these are classified in two major pathways of genetic instability. Microsatellite instability (MSI) and CPG island methylator phenotype (CIMP) are categorized as features of the hypermethylated prototype, and chromosomal instability (CIN) is known to be indicative of the non-hypermethylated category. Secreted frizzled related protein 2 (SFRP2), APC1A in WNT signaling pathway and the DNA repair gene, O6-methylguanine-DNA methyltransferase (MGMT), are frequently hypermethylated in colorectal cancer. Detection of methylated DNA as a biomarker by easy and inexpensive methods might improve the quality of life of patients with CRC via early detection of cancer or a precancerous condition. Aim: To evaluate the rate of SFRP2 and MGMT hypermethylation in both polyp tissue and serum of patients in south Iran as compared with matched control normal population corresponding samples. Materials and Methods: Methylation-specific PCR was used to detect hypermethylation in DNA extracted from 48 polypoid tissue samples and 25 healthy individuals. Results: Of total polyp samples, 89.5% had at least one promoter gene hypermethylation. The most frequent methylated locus was SFRP2 followed by MGMT-B (81.2 and 66.6 percent respectively). Serologic detection of hypermethylation was 95% sensitive as compared with polyp tissue. No hypermethylation was detected in normal tissue and serum and its detection in patients with polyps, especially of serrated type, was specific. Conclusions: Serologic investigation for detection of MGMT-B, SFRP2 hypermethylation could facilitate prioritization of high risk patients for colonoscopic polyp detection and excision.