Background: TRAIL is a cytokine that selectively induces apoptosis in various cancer cell lines. Gefitinib is new targeted drug applied in lung cancer that selectively inhibits EGFR tyrosine kinase. However, lung cancers have shown an initial or acquired resistance to these drugs. This study examined the effect of IGF-1R and its blockade on enhancing the sensitivity of lung cancer cell lines to TRAIL and gefitinib. Methods: Two lung cancer cell lines were used in this study. NCI H460 is very sensitive to TRAIL and gefitinib. On the other hand, A549 shows moderate resistance to TRAIL and gefitinib. The IGF-1R blockade was performed using adenoviruses expressing the dominant negative IGF-1R and shRNA to IGF-1R and AG1024 (IGF-1R tyrosine kinase inhibitor). Results: The adenovirus expressing dominant negative IGF-1R(950st) induced the increased expression of defective IGF-1R on the lung cancer cell surface, and the adenovirus-shIGF-1R effectively decreased the level of IGF-1R expression on cell surface. The genetic blockade of IGF-1R by the adenovirus-dnIGF-1R and AG1024 increased the sensitivity of A549 cells to TRAIL. The reduction of IGF-1R by transduction with ad-shIGF-1R also increased the sensitivity of the A549 cells to gefitinib. Conclusion: The blockade of IGF-1R through various mechanisms increased the sensitivity of the lung cancer cell line that was resistant to TRAIL and gefitinib. However, further studies using other cell lines showing acquired resistance as well as in vivo animal experiments will be needed.
Kim, Hyun;Cho, Sang-Rae;Kim, Dong Kyo;Choe, Changyong;Seong, Hwan-Hoo
Journal of Embryo Transfer
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v.30
no.3
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pp.195-200
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2015
The objective of this study was to evaluate the toxicities of permeable cryoprotectants and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Toxicities of permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), Glycerol, and 1,2-PROH were investigated using a murine embryo model. Female $F-{_1}$ mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to among DMSO, EG, Glycerol, and 1,2-PROH. Embryo development was evaluated up to the blastocyst stage. The total cell count of blastocysts that were treated with DMSO and Glycerol at the 2-cell stage was significantly lower than that were treated with EG ($81.1{\pm}15.1$), 1,2-PROH ($88.0{\pm}21.1$) or the control ($99.9{\pm}21.3$) (p<0.001). On comparison of four cryoprotectant treated groups, the DMSO and Glycerol treated group showed a decreased cell count compared with the EG and 1,2-PROH treated group (p<0.05). Both DMSO ($14.7{\pm}1.3$), EG ($12.1{\pm}1.1$), Glycerol ($15.2{\pm}1.8$), and 1,2-PROH ($11.5{\pm}1.3$) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control ($6.5{\pm}0.7$, p<0.0001). In addition, the DMSO or Glycerol treated group showed more apoptotic cells than the EG or 1,2-PROH treated group (p<0.001). The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to among DMSO, EG, Glycerol, and 1,2-PROH at room temperature. When comparing four permeable cryoprotective agents, EG and 1,2-PROH appeared to be less toxic than DMSO and Glycerol at least in a murine embryo model.
The aim of this study is to know how the rat submandibular gland changes under various emotional stress condition, using molecular biological methods. Restraint and chronic unpredictable mild stress (CUMS) experiment is conducted on fifty one 7-week old Sprague-Dawley rats (restraint stress experiment: 21, CUMS: 30). The rats were sacrificed, the submandibular glands were excised immediately at certain time, and examined by the use of immunohistochemistry and western blotting. In CUMS experiment, sucrose preference test, water intake change, weight change were implemented at 1 week interval for the experimental period The results are as follows: 1. The number of clusterin-secreting cells of restraint stress group compared to control group showed significantly decreasing tendency in all experimental groups except for the 1st hour group (p<0.001 in the 9th, 24th, 72nd, 120th, and 168th hour group). 2. The number of clusterin-secreting cells of CUMS group compared to control group showed significantly increasing tendency in the 2nd week group (p<0.01), and significantly decreasing tendency in the 4th and 5th week group (p<0.001). 3. Sucrose preference test in CUMS experiment showed significant difference between the 5th week experimental group and control group (p<0.01). 4. Weight change in CUMS experiment showed significant difference between the 5th week experimental group and control group (p<0.01), but water intake change didn't show significant difference compared to control group. 5. In western blot analysis, clusterin expression was decreased on a gradual basis in due time compared to the control group in the restraint stress group. As for CUMS group (chronic unpredictable mild stress group), it was increased till the 2nd week and decreased till the 5th week after that, which is similar to immunohistochemical analysis result and the decreasing tendency of sucrose preference and weigh changes. Through the test, it was proved that expression of clusterin in saliva glands decreases after receiving either acute or chronic stress, indicating relation with depression caused by chronic stress. Unlike other data, however, apoptotic tendency was hardly found in tissues. Diverse possibilities could be suggested on that: first, the stress was not enough to expedite apoptosis; second, apoptosis-related protein was already being secreted though not detected with microscope; third, clusterin, a major secretion molecule of saliva, decreased with saliva's malfunction due to stress. In the respect, it will be necessary to examine proteins expressed in case of cell death or other heat-shock proteins at the same time, in order to see whether any cellular change or death is caused by decreasing clusterin under high stress, and whether the original state is restored as time goes by under mild stress, through longer-term tests using even higher acute stress.
Backgroud: Sleep deprivation (SD) impairs learning and memory by inhibiting hippocampal functioning at molecular and cellular levels. Abnormal autophagy and apoptosis are closely associated with neurodegeneration in the central nervous system. This study is aimed to explore the alleviative effect and the underlying molecular mechanism of stem-leaf saponins of Panax notoginseng (SLSP) on the abnormal neuronal autophagy and apoptosis in hippocampus of mice with impaired learning and memory induced by SD. Methods: Mouse spatial learning and memory were assessed by Morris water maze test. Neuronal morphological changes were observed by Nissl staining. Autophagosome formation was examined by transmission electron microscopy, immunofluorescent staining, acridine orange staining, and transient transfection of the tf-LC3 plasmid. Apoptotic event was analyzed by flow cytometry after PI/annexin V staining. The expression or activation of autophagy and apoptosis-related proteins were detected by Western blotting assay. Results: SLSP was shown to improve the spatial learning and memory of mice after SD for 48 h, accomanied with restrained excessive autophage and apoptosis, whereas enhanced activation of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway in hippocampal neurons. Meanwhile, it improved the aberrant autophagy and apoptosis induced by rapamycin and re-activated phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling transduction in HT-22 cells, a hippocampal neuronal cell line. Conclusion: SLSP could alleviate cognitive impairment induced by SD, which was achieved probably through suppressing the abnormal autophagy and apoptosis of hippocampal neurons. The findings may contribute to the clinical application of SLSP in the prevention or therapy of neurological disorders associated with SD.
Purpose: The interaction of the Fas: Fas ligand has been recognized to play an important role in radiation induced apoptosis. The purpose of this study was to investigate the role of Fas and Fas ligand mutations, in radiation-induced apoptosis in vivo. Materials and Methods: Mice with a mutation in the Fas ($C57BL/6J-Fas^{lpr}$) and its normal control (C57BL/6J) and the Fas ligand ($C3H/HeJ-Fas^{gld}$) and its normal control (C3H/HeJ), were used in this study. Eight-week old male mice were given whole body radiation. After irradiation, the mice were killed at various time intervals, and their spleens collected. Tissue sample was stained with hematoxylin-eosin, and the numbers of apoptotic cells scored. The regulating molecules of apoptosis including the p53, Bcl-2, Bax, $Bcl-X_L\;and\;Bcl-X_s$ genes were also analyzed by Western blotting. Results: With 2.5 Gy and 10 Gy of irradiation, the levels of apoptosis were lower in the $C57BL/6J-Fas^{lpr}\;and\;C3H/HeJ-Fas^{gld}$ mice than in the control mice (p<0.05). With the expression of apoptosis regulating molecules, the Bax was increased in both the C57BL/6J and C3H/HeJ mice in response to radiation; the peak levels of Bax in the C57BL6J and C3H/HeJ were 3 and 3.3-fold higher after 8hr, respectively. However the Bax was not increased in either the $C57BL/6J-Fas^{lpr}\;or\;C3H/HeJ-Fas^{gld}$mice. The p53, Bcl-X_L,\;Bcl-X_S$and Bcl-2 showed no significant changes in the $C57BL/6J-Fas^{lpr},\;C3H/HeJ-Fas^{gld}$, C57BL/6J and C3H/HeJ mice. Conclusion: The levels of radiation-induced apoptosis were lower in the lpr and gld, than the control mice, which seemed to be related to the level of Bax activation due to the radiation in the lpr and gld mice. This result suggests that Fas/Fas L plays an important role in radiation-induced apoptosis in vivo.
Tributyltin (TBT) used world-wide in antifouling paints for ships is a widespread environmental pollutant and cause reproductive organs atrophy in rodents. At low doses, antiproliferative modes of action have been shown to be involved, whereas at higher doses apoptosis seems to be the mechanism of toxicity in reproductive organs by TBT. In this study, we investigated that the mechanisms underlying DNA fragmentation induced by TBT in the rat leyding cell line, R2C. Effects of TBT on intracellular Ca$\^$2+/ level and reactive oxygen species (ROS) were investigated in R2C cells by fluorescence detector. TBT significantly induced intracellular Ca$\^$2+/ level in a time-dependent manner. The rise in intracellular Ca$\^$2+/ level was followed by a time-dependent generation of reactive oxygen species (ROS) at the cytosol level. Simultaneously, TBT induced the release of cytochrome c from the mitochondrial membrane into the cytosol. Furthermore, ROS production and the release of cytochrome c were reduced by BAPTA, an intracellular Ca$\^$2+/ chelator, indicating the important role of Ca$\^$2+/ in R2C during these early intracellular events. In addition, Z-DEVD FMK, a caspase-3 inhibitor, decreased apoptosis by TBT. Taken together, the present results indicated that the apoptotic pathway by TBT might start with an increase in intracellular Ca$\^$2+/ level, continues with release of ROS and cytochrome c from mitochondria, activation of caspases,and finally results in DNA fragmentation.
Journal of the Korean Society of Food Science and Nutrition
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v.42
no.11
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pp.1717-1726
/
2013
Fermentation characteristics and health functionalities of kimchi by inoculating kimchi lactic acid bacteria (LAB) starters were studied. We manufactured single LAB starter kimchi (Lactobacillus plantarum pnuK, Lactobacillus plantarum 3099K, Leuconostoc mesenteroides pnuK), mixed LAB starter kimchi (Lb. plantarum pnu/Leu. mesenteroides pnuK, Lb. plantarum 3099/Leu. mesenteroides pnuK) with inoculum size of $10^6$ CFU/g, as well as naturally fermented kimchi (NK), and fermented them for 6 days at $15^{\circ}C$. The pH and acidity of the early phase of fermentation were not different, but kimchi with the starters showed rapid changes in the pH and acidity from 2 days of fermentation. As the fermentation progressed, the level of total aerobic bacteria and Lactobacillus sp. increased similarly with or without Lb. plantarum (LP) inoculation. However, the level of Leuconostoc sp. was high in kimchi inoculated with Leuconostoc sp. starter. In the sensory evaluation test, kimchi with starters received higher overall acceptability scores than those of NK; mixed starter added kimchi earned the highest score. In DPPH and hydroxyl radical scavenging activity, kimchi with the starters exhibited higher activity than that of NK. In the MTT assay of HCT-116 and HT-29 human colon cancer cells, NK showed inhibition rates of 63.4 and 51.9%, but LPpnuK achieved 77.1 and 68.8%, respectively. This study showed that inoculating starters in kimchi increased in vitro antioxidant and anticancer activities, and single starter (LP) added kimchi revealed higher functionality than the kimchi with mixed starter. Kimchis with the starters effectively up-regulated the gene expressions of the pro-apoptotic gene of Bax, but down-regulated Bcl-2. They promoted expressions of p53 and p21, and suppressed expressions of inflammation-related genes, iNOS and COX-2, compared with NK. Taken together, it is expected that using starters may help manufacture kimchi with improved sensory quality and health functionality.
The increased occurrence of hyperglycemia and oxidative stress in streptozotocin (STZ) induced type I diabetes has been implicated in the etiology and pathology of disease complication. STZ has known to be genotoxic in a variety of assays including tests for microbial mutagenesis and unscheduled DNA synthesis in rat kidney. Diabetes mellitus (DM) is a pathologic condition, resulting in severe metabolic imbalances and non-physiologic changes in many tissues. We examined the effect of gamma radiation and KWNP on preventing the development of insulin dependent diabetes mellitus using streptozotocin-induced Fisher 344 diabetic rats. The hematological values (red blood cell and white blood cell), serum biochemical constituents-alkaline phosphatase (ALP), total cholesterol, triglycerides and insulin-were checked and the organs (testis, spleen and kidney) were weighed. The gonad indices of the STZ treated groups were much lower than the value of the control group. But the gonad indices of the KWNP treated groups were higher than those of the treated groups. The ratio of the weight of kidney to the body weight of the STZ treated groups was higher than that of the control group. The value of the diabetic group treated with KWNP after irradiation (F group) was lower than the other STZ treated groups. The white blood cell and ALP values of the F group were lower than the other STZ groups, as well. The cholesterol and triglyceride values of all the KWNP treated groups were significantly lower than the other groups. A significant increase (about 10 times) of insulin was detected in the F group. The results of hematological assay showed the distinctive damage in the irradiated and STZ treated groups. The quantity of apoptotic cells in seminiferous tubule of testis confirmed a serious damage as assessed in the STZ treated groups. These experimental results have revealed that treatment of the products of KWNP after irradiation has the antidiabetic effect in the STZ-induced diabetic rats. But the F group showed higher recuperative power. These experimental results have revealed that treatment of the gamma irradiation and KWNP have the recovering effect in the STZ-induced diabetic rats.
Journal of the Korean Society of Food Science and Nutrition
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v.44
no.10
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pp.1422-1430
/
2015
This study was conducted to determine the effect of fermented water extracts from Ligularia fischeri (LAF) on reduction of hepatotoxicity induced by D-galactosamine (D-GalN) in rats. In this experiment, male Sprague-Dawley rats were used as experimental animals, which were divided into eight groups: normal group, D-GalN-treated group (control), D-GalN and non-fermented water extracts from Ligularia fischeri (LA)-treated groups [100, 200, and 400 mg/kg BW (body weight)], and D-GalN and LAF-treated groups (100, 200, and 400 mg/kg BW). ${\gamma}$-Glutamyl transferase, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase activities in serum of the D-GalN and LAF-treated groups decreased significantly compared to those of the control group (P<0.05). The high density lipoprotein-cholesterol levels of the D-GalN and LAF-treated groups increased significantly compared to those of the control group (P<0.05). The low density lipoprotein-cholesterol and triglyceride levels of the D-GalN and LAF-treated groups decreased significantly compared to those of the control group (P<0.05). The atherogenic index values of the D-GalN and LAF-treated groups decreased significantly compared to those of the control group (P<0.05), and their high density lipoprotein cholesterol by total cholesterol ratio increased significantly in these groups (P<0.05). Superoxide dismutase activity of liver tissues were enhanced significantly (P<0.05) in the D-GalN and LAF-treated groups compared to that of the control group (P<0.05), whereas their malondialdehyde content decreased significantly in these groups (P<0.05). The histopathological observations revealed apoptotic cells and mild portal inflammation in liver tissues of the D-GalN and LAF-treated groups. Taken together, these results demonstrate that LAF may improve plasma lipid profile and alleviate hepatic damage.
The addition of growth factors and cytokines to in vitro culture (IVC) media could affect embryo development and the quality of the resulting blastocysts. The present study was performed to investigate the effect of porcine induced pluripotent stem cell (piPSC)-culture conditioned medium (CM) on the in vitro maturation (IVM) and development of parthenogentic embryos (parthenotes) in pigs. Cumulus-oocyte complexes (COCs) or activated oocytes were cultured in IVM or IVC medium supplemented with 0 (control), 25, or 50% of stem cell medium (SM) or CM, respectively. The maturation rate of CM-25% group was significantly improved when compared with control group (p<0.05), but that was not different among SM or CM groups. Blastocyst formation rate was significantly higher in CM-25% group (29.2%) than that of control (20.7%), SM-50% (19.6%) and CM-50% (23.66%, p<0.05). Cell number and the apoptotic cell index in blastocysts was significantly lower in SM-25% than in CM-25% group (p<0.05). The embryo quality related genes, OCT4, KLF4, TERT and ZFP42, were significantly increased in CM-25% group compared with control (p<0.05). In conclusion, the addition of 25% of CM to IVM and IVC medium positively influences not only the developmental potential also quality of parthenotes in pig.
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