Litsea populifolia, a plant species of the Lauraceae family, is widely distributed in the tropical and subtropical areas of Asia. The phylogenetic relationships and botanical characteristics of L. populifolia have been reported; however, its anti-oxidative and anti-cancer activities remain unclear. In this study, we evaluated the anti-oxidative and anti-cancer effects of ethanol extracts of L. populifolia (EELP) together with the molecular mechanism of its anti-cancer activity in human lung adenocarcinoma A549 cells. EELP showed significant anti-oxidative effects with a 50% inhibitory concentration at $11.71{\mu}g/ml$, which was measured by the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay. EELP exhibited cytotoxic activity and induced cell cycle arrest at the G1 phase in A549 cells in a dose-dependent manner, whereas EELP did not have the cytotoxic effect on the normal human lung cell line IMR90. Treatment with EELP also resulted in a decreased expression of G1/S transition-related molecules-including cyclin-dependent kinase (CDK) 2, CDK6, cyclin D1, and cyclin E-both for the transcription and translation levels. EELP-induced G1 arrest was associated with the phosphorylation of checkpoint kinase 2 (CHK2), p53, cell division cycle 25 homolog A (CDC25A), and the reduction of CDC25A expression in A549 cells. Collectively, these results suggest that EELP may exert an anti-cancer effect by cell cycle arrest at the G1 phase through both p53-dependent and p53-independent (ATM/CHK2/CDC25A/CDK2) pathways in A549 cells.
Lim, Han Hyuk;Song, Wung Joo;Kim, Gu-Hwan;Watkins, David;Rosenblatt, David S.;Kim, Yoo-Mi;Chang, Mea Young;Kil, Hong Ryang;Kim, Sook Za
Journal of The Korean Society of Inherited Metabolic disease
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v.19
no.1
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pp.12-19
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2019
Purpose: Isolated methylmalonic acidemia (MMA) is an autosomal recessive inherited disorder of propionate metabolism. There are two subtypes of MMUT gene defects. $Mut^0$ represents complete loss of methylmalonyl-CoA mutase (MCM) activity while mut- is associated with residual MCM activity, which can be stimulated by hydroxocobalamin (OHCbl) supplementation. The objective of this study is to investigate cobalamin responsiveness and mutations present in Korean MMA population. Methods: We evaluated 10 MMA patients using somatic cell complementation analysis on their fibroblasts to measure MCM activity and vitamin B12 responsiveness for the optimal treatment. MMUT gene was sequenced to identify the MMA mutations. Results: For all patients, the incorporation of $[^{14}C]-propionate$ was low, and there was no response to OHCbl. The incorporation of $[^{14}C]-methyltetrahydrofolate$ and $[^{57}Co]-CNCbl$ fell within the normal range. There was adequate synthesis of methylcobalamin while the synthesis of adenosylcobalamin was low. The complementation analysis showed all patients were $mut^0$. The sequence analysis identified 12 different MMUT mutations, including 2 novel mutations, p.Gln267Ter and p.Ile697Phe, were identified. All the patients in this study had neonatal onset of symptoms, belonged to $mut^0$ complementation class, and as a result, showed no cobalamin responsiveness. Conclusion: No Korean MMA patient showed cobalamin responsiveness.
Background : NF-${\kappa}B$ is a characteristic transcriptional factor which has been shown to regulate production of acute inflammatory mediators and to be involved in the pathogenesis of many inflammatory lung diseases. There has been some evidence that PI3K/Akt pathway could activate NF-${\kappa}B$ in human cell lines. However, the effect of PI3K/Akt pathway on the activation of NF-${\kappa}B$ varied depending on the cell lines used in the experiments. In this study we evaluated the effect of PI3K/Akt pathway on the activation of NF-${\kappa}B$ in human respiratory epithelial cell lines. Methods : BEAS-2B, A549 and NCI-H157 cell lines were used in this experiment. To evaluate the activation of Akt activation and I${\kappa}B$ degradation, cells were analysed by western blot assay using phospho-specific Akt Ab and $I{\kappa}B$ Ab. To block PI3K/Akt pathway, cells were pretreated with wortmannin or LY294002 and transfected with dominant negative Akt (DN-Akt). For IKK activity, immune complex kinase assay was performed. To evaluate the DNA binding affinity and transcriptional activity of NF-${\kappa}B$, electrophoretic mobility shift assay (EMSA) and luciferase assay were performed, respectively. Results : In BEAS-2B, A549 and NCI-H157 cell lines, Akt was activated by TNF-$\alpha$ and insulin. Activation of Akt by insulin did not induce $I{\kappa}B{\alpha}$ degradation. Blocking of PI3K/Akt pathway via wortmannin/LY294002 or DN-Akt did not inhibit TNF-$\alpha$-induced $I{\kappa}B{\alpha}$ degradation or IKK activation. Inhibition of PI3K/Akt did not affect TNF-$\alpha$-induced NF-${\kappa}B$ activation. Overexpression of DN-Akt did not block TNF-$\alpha$-induced transcriptional activation of NF-${\kappa}B$, but wortmannin enhanced TNF-$\alpha$-induced in NF-${\kappa}B$ transcriptional activity. Conclusion : PI3K/Akt was not involved in TNF-$\alpha$-induced $I{\kappa}B{\alpha}$ degradation or transcriptional activity of NF-${\kappa}B$ in human respiratory epithelial cell lines.
Vitamin D is known to exert its action by activating DNA and RBA within target cells to produce proteins and enzymes that can be used in bone resorption process. Particularly, the active form of vitmain D, 1,25-dihydroxycholecalciferol $[1,25-(OH)_2D_3]$, is considered to be one of the most potent stimulators of osteoclatic acitivity in vitro. The purpose of this study was to evaluate the effect of 1,25-Dihydroxyvitamin $D_3$ on the avtivity of periodotal ligament cells and, the experimental tooth movement. Human periodontal ligament cells were collected from the first premolar tooth extracted for the orthodontic treatment, and were incubated in the environment of $37^{\circ}C$, 5% $CO_2$ and 95% humidity. Microtitration(MIT) assay was done at 10, 25, 50 and 100ng/ml of 1,25-Dihydroxyvitamin $D_3$. 21 Sprague-Daft rats were divided into a control gmup(3), and experimental groups(18) where 100g of force from helical spring was applied across the maxillary incisors 1,25-Dihydroxyvitamin $D_3$ was injected into periodontal ligament at the mesial or distal surface of maxillary incisors so that we can compare the control side and the experimental side. Expreimental groups were sac rifled at 12, 24, 36, 48, 72hours and 7 days after force application, respectively. And the obtained tissues were evaluated histologically. The observed results were as follows. 1. The activity of periodontal ligament cells in l0ng/ml or 25ng/ml of 1,25-Dihydroxyvitamin $D_3$ 1,25-Dihydroxyvitamin $D_3$ was not significantly different to the control at the cultivation of 1, 2 and 3 days. 2. The activity of periodontal ligament cells was significantly increased at 3 days in 50 ng/ml of 1,25-Dihydroxyvitamin $D_3$ and 2, 3 days in 100g/ml of 1,25-Dihydroxyvitamin $D_3$. 3. Up to 7 days after force application, there was no difference in osteoblastic activity, tearing of periodontal ligament and proliferation of capillary at tension side between 1,25-Dihydroxyvitamin $D_3$ injection side and the control side. 4. The osteoclastic activity and the resorption of alveolar bone was greater in 1,25-Dihydroxyvitamin $D_3$ injection side than the control side at 36 hours after force application.
Yang Kwang Mo;Youn Seon-Min;Jeong Soo-Jin;Jang Ji-Yeon;Jo Wol-Soom;Do Chang-Ho;Yoo Y대-Jin;Shin Young-Cheol;Lee Hyung Sik;Hur Won Joo;Lim Young-Jin;Jeong Min-Ho
Radiation Oncology Journal
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v.21
no.3
/
pp.227-237
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2003
Purpose: The human chronic myelogenous leukemia cell line, K562, expresses the chimeric bcr-abl oncoprotein, whose deregulated protein tyrosine kinase activity antagonizes via DNA damaging agents. Previous experiments have shown that nanomolar concentrations of herbimycin A (HWA) coupled with X-irradiation have a synergistic effect in inducing apoptosis in the Ph-positive K562 leukemia cell line, but genistein, a PTK inhibitor, is non selective for the radiation-induced apoptosils on $p210^{bcr/abl}$ protected K562 cells. In these experiments, the cytoplasmic signal transduction pathways, the Induction on a number of transcription factors and the differential gene expression in this model were investigated. Materials and Methids: K562 cells in the exponential growth phase were used in this study. The cells were irradiated with 0.5-12 Gy, using a 6 Mev Linac (Clinac 1800, Varian, USA). Immediately after irradiation, the cells were treated with $0.25/muM$ of HMA and $25/muM$ of genistein, and the expressions and the activities of abl kinase, MAPK family, NF- kB, c-fos, c-myc, and thymidine kinase1 (TK1) were examined. The differential gene expressions induced by PTK inhibitors were also investigated. Results: The modulating effects of herbimycin A and genistein on the radiosensitivity of K562 cells were not related to the bcr-abl kinase activity. The signaling responses through the MAPK family of proteins, were not involved either in association with the radiation-induced apoptosis, which is accelerated by HMA, the expression of c-myc was increased. The combined treatment of genistein, with irradiation, enhanced NF- kB activity and the TK1 expression and activity. Conclusion: The effects of HMA and genistein on the radiosensitivity on the K562 cells were not related to the bcr-abl kinase activity in this study, another signaling pathway, besides the WAPK family responses to radiation to K562 cells, was found. Further evaluation using this model will provide valuable information for the optional radiosensitization or radioprotection.
A CpG island is a short stretch of DNA in which the frequency of the CG dinucleotide is higher than other regions. CpG islands are present in the promoters and exonic regions of approximately $30{\sim}60$% of mammalian genes so they are useful markers for genes in organisms containing 5-methylcytosine in their genomes. Recent evidence supports the notion that the hypermethylation of CpG island, by silencing tumor suppressor genes, plays a major causal role in cancer, which has been described in almost every tumor types. In this respect, CpG island search by computational methods is very helpful for cancer research and computational promoter and gene predictions. I therefore developed a window program (called CpGi) on the basis of CpG island criteria defined by D. Takai and P. A. Jones. The program 'CpGi' was implemented in Visual C++ 6.0 and can determine the locations of CpG islands using diverse parameters (%GC, Obs (CpG)/Exp (CpG), window size, step size, gap value, # of CpG, length) specified by user. The analysis result of CpGi provides a graphical map of CpG islands and G+C% plot, where more detailed information on CpG island can be obtained through pop-up window. Two human contigs, i.e. AP00524 (from chromosome 22) and NT_029490.3 (from chromosome 21), were used to compare the performance of CpGi and two other public programs for the accuracy of search results. The two other programs used in the performance comparison are Emboss-CpGPlot and CpG Island Searcher that are web-based public CpG island search programs. The comparison result showed that CpGi is on a level with or outperforms Emboss-CpGPlot and CpG Island Searcher. Having a simple and easy-to-use user interface, CpGi would be a very useful tool for genome analysis and CpG island research. To obtain a copy of CpGi for academic use only, contact corresponding author.
Journal of the Korean Association of Oral and Maxillofacial Surgeons
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v.30
no.6
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pp.465-473
/
2004
Purpose : The essential triad for nerve regeneration is nerve conduit, supporting cell and neurotrophic factor. In order to improve the peripheral nerve regeneration, we used polyglycolic acid(PGA) tube and brain-derived neurotrophic factor(BDNF) gene transfected Schwann cells in sciatic nerve defects of SD rat. Materials and methods : Nerve conduits were made with PGA sheet and outer surface was coated with poly(lactic-co-glycolic acid) for mechanical strength and control the resorption rate. The diameter of conduit was 1.8mm and the length was 17mm Schwann cells were harvested from dorsal root ganglion(DRG) of SD rat aged 1 day. Schwann cells were cultured on the PGA sheet to test the biocompatibility adhesion of Schwann cell. Human BDNF gene was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into E1 deleted region of adenovirus shuttle vector, pAACCMVpARS. BDNF-adenovirus was multiplied in 293 cells and purified. The BDNF-Adenovirus was then infected to the cultured Schwann cells. Left sciatic nerve of SD rat (250g weighing) was exposed and 14mm defects were made. After bridging the defect with PGA conduit, culture medium(MEM), Schwann cells or BDNF-Adenovirus infected Schwann cells were injected into the lumen of conduit, respectively. 12 weeks after operation, gait analysis for sciatic function index, electrophysiology and histomorphometry was performed. Results : Cultured Schwann cells were well adhered to PGA sheet. Sciatic index of BDNF transfected group was $-53.66{\pm}13.43$ which was the best among three groups. The threshold of compound action potential was between 800 to $1000{\mu}A$ in experimental groups which is about 10 times higher than normal sciatic nerve. Conduction velocity and peak voltage of action potential of BDNF group was the highest among experimental groups. The myelin thickness and axonal density of BDNF group was significantly greater than the other groups. Conclusion : BDNF gene transfected Schwann cells could regenerate the sciatic nerve gap(14mm) of rat successfully.
Park, Jae-Yong;Kim, Jeong-Ran;Chang, Hee-Jin;Kim, Chang-Ho;Park, Jae-Ho;Jung, Tae-Hoon
Tuberculosis and Respiratory Diseases
/
v.45
no.3
/
pp.587-595
/
1998
Background: Recombinant adenovirus hold promise as vectors to carry therapeutic genes for several reasons: 1) they can infect both dividing and non-dividing cells; 2) they have the ability to directly transduce tissues in vivo; 3) they can easily be produced in high titer; and 4) they have an established record of safety as vaccination material. However, one of the major limitation in the use of adenoviruses is that transgene expression is quite short because adenovirusees insert their DNA genome episomally rather than by chromosomal integration, and an immune response against the virus destroys cells expressing the therapeutic gene. Since sodium butyrate has been reported to induce adenovirus-mediated gene expression, we hypothesized that treatment of tumor cells, transduced with herpes simples virus thymidine kinase(HSVtk) gene using adenoviral vector, with butyrate could augment the effect of gene therapy. Methods: We transduced HSVtk gene, driven by the cytomegalovirus promoter, into REN cell line(human mesothelioma cell line). Before proceeding with the comparison of HSVtk/ganciclovir mediated bystander killing, we evaluated the effect of butyrate on the growth of tumor cells in order to rule out a potential antitumor effect of butyrate alone, and also on expression of HSVtk gene by Western blot analysis. Then we determined the effects of butyrate on bystander-mediated cell killing in vitro. Results: There was no inhibition of growth of cells exposed to butyrate for 24 hours at a concentration of 1.5mM/L. Toxic effects were seen when the concentration of butyrate was greater than 2.0mM/L. Gene expression was more stable and bystander effect was augmented by butyrate treatment of a concentration of 1.5mM/L. Conclusion: These results provide evidence that butyrate can augment the efficiency of cell killing with HSVtk/GCV system by inducing transgene expression and may thus by a promising new approach to improve responses in gene therapy using adenoviral vectors.
Objectives: Ethylene oxide (EtO) is classified as a human carcinogen, but EtO is still widely used to sterilize heat-sensitive materials in hospitals. Employees working around sterilizers are exposed to EtO after sterilization. The aim of the present study was to assess the exposure of EtO level, coupled with occupationally induced micronuclei from hospital workers. The influence of genetic polymorphisms of detoxifying genes (GSTT1 and GSTM1) and DNA repair genes (XRCC1 and XRCC3) on the frequencies of micronuclei in relation to exposure of EtO was also investigated. Methods: The study population was composed of 35 occupationally exposed workers to EtO, 18 student controls and 44 unexposed hospital controls in Korea. Exposure to EtO is measured by passive personal samplers. We analyzed the frequencies of micronuclei by performing cytokinesis-block micronucleus assay (CBMN assay) and GSTM1, GSTT1, XRCC1, and XRCC3 were also genotyped by performing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: The frequencies of micronuclei in EtO exposure group, student controls and hospital controls were $18.00{\pm}7.73$, $10.47{\pm}7.96$ and $13.86{\pm}6.35$ respectively and their differences were statistically significant, but no significant differences according to the level of EtO were observed. There was a dose-response relationship between the frequencies of micronuclei and cumulative dose of EtO, but no significantly differences were observed. We also investigated the influence of genetic polymorphisms (GSTM1, GSTT1, XRCC1, and XRCC3) on the frequencies of micronuclei, but there were no differences in the frequencies of micronuclei by genetic polymorphisms. Conclusions: The frequencies of micronuclei in EtO exposure group was significantly higher than control groups. A dose-response relationship was found between the level of EtO exposure and the frequencies of micronuclei, but no statistically differences were observed. We also found that the frequencies of micronuclei were increased according to cumulative EtO level. There was no association of the genetic GSTM1, GSTT1, XRCC1, and XRCC3 state with the frequency of micronuclei induced by EtO exposure.
Cho Jae-Shik;Baik Joon;Lim Sang-Chul;Cho Yeon;Yoon Je-Hwan;Seo Duk-Jung;Park Chang-Soo
Korean Journal of Head & Neck Oncology
/
v.17
no.2
/
pp.162-168
/
2001
Background: Inverted papilloma(IP) of the nasal cavity and paranasal sinuses is a benign neoplastic condition that can be associated with squamous cell carcinoma (SCC). Several studies have indicated an etiologic role for viruses in the development of inverted papilloma. And it is necessary to find out the significance of a biologic markers such as p53, c-erbB-2 to predict the malignant potential. The purposes of this study are to detect HPV in inverted papilloma of the nasal cavity and paranasal sinus, to examine role of HPV as an etiological agent, to examine the relationship between HPV subtype and malignant transformation of inverted papilloma, and to investigate the relation between expression rate of p53, c-erbB-2 and HPV in recurrent or malignant transformation cases. Material and Methods: Thirty two cases of inverted papilloma(IP) in the nasal cavity and paranasal sinuses were reviewed and classified into 3 groups; simple IP, IP with dysplasia group, IP with squamous cell carcinoma group. Paraffin embedded achival tissue was used in this study. The HPV was detected by in situ hybridzation (ISH) using HPV type 6/11, 16/18, 31/33/35 DNA probes. Expression of p53 and c-erbB-2 was examined by immunohistochemical staining. Results: 1) The HPV was detected in 6(19%) out of 32 cases. 2) The HPV 6/11 was dectected in 4 out of 21 cases of simple IP, HPV 16/18 in 1, HPV 31/33/35 in lout of 8 cases of IP with dysplasia respectively. 3) The positive expression of p53 was 13 cases out of 32 cases; 2 out of 21 cases of simple IP, all of 8 cases of IP with dysplasia and 3 cases of IP with squamous cell carcinoma 4) The positive expression of c-erbB-2 was in 24 out of 32 cases; 16 out of 21 cases of simple IP, 6 out of 8 cases of IP with dysplasia, 2 out of 3 cases of IP with squamous cell ca. 5) The recurrence of IP occurred in lout of 6 cases of positive for HPV, in 4 out of 26 cases negative for HPV. 6) The recurrence of IP occurred only in positive cases for p53. 7) The recurrence of IP occurred in 4(17%) out of 24 cases positive for c-erbB-2, in 1(13%) out of 8 cases negative for c-erbB-2. Conclusion: The p53 expression was associated with Inverted papillomas exhibiting evidence of malignant transformation. Also, there was a correlation between the p53 expression and recurrence.
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