Kim, Young June;Ahn, Kwang Sung;Kim, Minjeong;Kim, Min Ju;Ahn, Jin Seop;Ryu, Junghyun;Heo, Soon Young;Park, Sang-Min;Kang, Jee Hyun;Choi, You Jung;Shim, Hosup
Asian-Australasian Journal of Animal Sciences
/
v.30
no.3
/
pp.439-445
/
2017
Objective: Production of alpha-1,3-galactosyltransferase (${\alpha}GT$)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous ${\alpha}GT$ knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce ${\alpha}GT$-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods: Miniature pig fibroblasts were transfected with ${\alpha}GT$ gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous ${\alpha}GT$ gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-${\alpha}$-1,3-galactose, an epitope produced by ${\alpha}GT$. Using magnetic activated cell sorting, cells with monoallelic disruption of ${\alpha}GT$ were removed. Remaining cells with LOH carrying biallelic disruption of ${\alpha}GT$ were used for the second round NT to produce homozygous ${\alpha}GT$ gene-targeted piglets. Results: Monoallelic mutation of ${\alpha}GT$ gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous ${\alpha}GT$ gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous ${\alpha}GT$ knockout piglets. Conclusion: The present study demonstrates that the time required for the production of ${\alpha}GT$-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.
Lee, Su Yeon;Ju, Min Kyung;Jeon, Hyun Min;Kim, Cho Hee;Park, Hye Gyeong;Kang, Ho Sung
Journal of Life Science
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v.27
no.11
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pp.1245-1255
/
2017
Most cancer cells produce ATP predominantly through glycolysis instead of through mitochondrial oxidative phosphorylation, even in the presence of oxygen. The phenomenon is termed the Warburg effect, or the glycolytic switch, and it is thought to increase the availability of biosynthetic precursors for cell proliferation. EMTs have critical roles in the initiation of the invasion and metastasis of cancer cells. The glycolytic switch and EMT are important for tumor development and progression; however, their correlation with tumor progression is largely unknown. The Snail transcription factor is a major factor involved in EMT. The Snail expression is regulated by distal-less homeobox 2 (Dlx-2), a homeodomain transcription factor that is involved in embryonic and tumor development. The Dlx-2/Snail cascade is involved in Wnt-induced EMTs and the glycolytic switch. This study showed that in response to Wnt signaling, the Dlx-2/Snail cascade induces the expression of PFKFB2, which is a glycolytic enzyme that synthesizes and degrades fructose 2, 6-bisphosphate (F2,6BP). It also showed that PFKFB2 shRNA prevents Wnt-induced EMTs in the breast-tumor cell line MCF-7. The prevention indicated that glycolysis is linked to Wnt-induced EMT. Additionally, this study showed PFKFB2 shRNA suppresses in vivo tumor metastasis and growth. Finally, it showed the PFKFB2 expression is higher in breast, colon and ovarian cancer tissues than in matched normal tissues regardless of the cancers' stages. The results demonstrated that PFKFB2 is an important regulator of EMTs and metastases induced by the Wnt, Dlx-2 and Snail factors.
Purpose: The purpose of this review Is to provide an update on novel radiation treatments for head and neck cancer Recent Findings: Despite the remarkable advances In chemotherapy and radiotherapy techniques, the management of advanced head and neck cancer remains challenging. Epidermal growth factor receptor (EGFR) Is an appealing target for novel therapies In head and neck cancer because not only EGFR activation stimulates many important signaling pathways associated with cancer development and progression, and importantly, resistance to radiation. Furthermore, EGFR overexpression Is known to be portended for a worse outcome in patients with advanced head and neck cancer. Two categories of compounds designed to abrogate EGFR signaling, such as monoclonal antibodies (Cetuxlmab) and tyrosine kinase inhibitors (ZD1839 and 051-774) have been assessed and have been most extensively studied In preclinical models and clinical trials. Additional TKIs In clinical trials include a reversible agent, Cl-1033, which blocks activation of all erbB receptors. Encouraging preclinical data for head and neck cancers resulted In rapid translation Into the clinic. Results from Initial clinical trials show rather surprisingly that only minority of patients benefited from EGFR inhibition as monotherapy or In combination with chemotherapy. In this review, we begin with a brief summary of erbB- mediated signal transduction. Subsequently, we present data on prognostic-predictive value of erbB receptor expression in HNC followed by preclinlcal and clinical data on the role of EGFR antagonists alone or in combination with radiation In the treatment of HNC. Finally, we discuss the emerging thoughts on resistance to EGFR biockade and efforts In the development of multiple-targeted therapy for combination with chemotherapy or radiation. Current challenges for investigators are to determine (1 ) who will benefit from targeted agents and which agents are most appropriate to combine with radiation and/or chemotherapy, (2) how to sequence these agents with radiation and/or cytotoxlc compounds, (3) reliable markers for patient selection and verification of effective blockade of signaling in vivo, and (4) mechanisms behind intrinsic or acquired resistance to targeted agents to facilitate rational development of multi-targeted therapy, Other molecuiar-targeted approaches In head and neck cancer were briefly described, Including angloenesis Inhibitors, farnesyl transferase inhibitors, cell cycle regulators, and gene therapy Summary: Novel targeted theraples are highly appealing in advanced head and neck cancer, and the most premising strategy to use them Is a matter of intense Investigation.
Journal of the Korean Society of Food Science and Nutrition
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v.44
no.7
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pp.975-982
/
2015
Piceatannol (trans-3,4,3',5'-trihydroxystilbene), a natural stilbene, is an analogue of resveratrol. In the present study, possible mechanisms by which piceatannol exerts its pro-apoptotic action in cultured human oral cancer YD-15 cells were investigated. To investigate whether or not piceatannol has effects on cancer cell viability, human oral YD-15 cells were treated with piceatannol (0, 50, and $100{\mu}M$). Piceatannol treatment ($100{\mu}M$) showed the strongest inhibition of cell proliferation and reduced cell viability in a dose-dependent manner. Chromatin condensation detected by DAPI staining significantly increased in a concentration-dependent manner, indicating apoptosis. Piceatannol treatment activated initiator Bax (pro-apoptotic) and cPARP in a concentration-dependent manner. Further, piceatannol induced down-regulation of Bcl-2 (anti-apoptotic). We also evaluated the activity of piceatannol against oral cavity cancer tumors in mice. Piceatannol-treated nude mice bearing YD-15 xenograft tumors exhibited significantly reduced tumor volume and weight due to the potent effect of piceatannol on tumor cell apoptosis, as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Immunohistochemistry staining showed elevated expression of cleaved-caspase-3 as well as reduced expression of Ki-67 in the piceatannol-treated group. Therefore, piceatannol can be developed as a cancer preventive medicine due to its growth inhibitory effects and induction of apoptosis in human oral cancer cells.
Kim, Kwan-Chang;Kim, Yong-Jin;Kim, Soo-Hwan;Choi, Seung-Hwa
Journal of Chest Surgery
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v.42
no.6
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pp.685-695
/
2009
Background: Calcification is the most frequent cause of clinical failure of bioprosthetic tissues that are fabricated from Glutaraldehyde (GA)-fixed porcine valve or bovine pericardium. We recently used a multi-factorial approach of employing different mechanisms to investigate how to reduce the calcification of bioprosthetic tissues. The purpose of the present study was to evaluate the synchronized synergism using ethanol, L-lysine and $NaBH_4$ in glutaraldehyde treated porcine pericardium from the standpoint of calcification and tissue elasticity. Material and Method: Porcine pericardium was fixed with 0.625% GA (commercial fixation). An interim step of ethanol (80%; 1 day at room temperature) or L-lysine (0.1 M; 2 days at $37^{\circ}C$) or $NaBH_4$ (0.1 M; 2 days at room temperature) was followed by completion of the GA fixation (2 days at $4^{\circ}C$ and 7 days at room temperature). The tensile strength and thickness of the samples were measured. The treated pericardiums were implanted subcutaneously into three-week old Sprague-Dawley rats for 8 weeks. The calcium content was assessed by atomic absorption spectroscopy and the histology of the samples. Result: The amount of calcium in the pericardium pretreated with ethanol (13.6${\pm}$10.0 ug/mg, p=0.008), L-lysine (15.3${\pm}$1.0 ug/mg, p=0.002) and both (16.1${\pm}$11.1 ug/mg, p=0.012) was significantly reduced compared with the control (51.2${\pm}$8.5 ug/mg). However, $NaBH_4$ pretreatment (65.7${\pm}$61.8 ug/mg, p=0.653) and combined pretreatment that including ethanol, L-lysine and $NaBH_4$ (92.9${\pm}$58.3 ug/mg, p=0.288) were not significantly different from the controls(51.2${\pm}$8.5 ug/mg). Both the combined pretreatment using ethanol and L-lysine (7.60${\pm}$1.55, p=0.76) and the combined pretreatment that included ethanol, L-lysine and $NaBH_4$ (7.47${\pm}$1.85, p=0.33) increased the tensile strength/thickness ratio compared with that of the controls (4.75${\pm}$1.88). Conclusion: The combined pretreatment using ethanol and L-lysine seemed to decrease the calcification of porcine pericardium fixed with glutaraldehyde, as compared to single pretreatment, and it increase the tissue elasticity, but to the degree that showed synchronized synergism. $NaBH_4$ pretreatment seemed to increase the calcification of porcine pericardium, irrespective of whether single or combined pretreatment was used.
Hwang, Sung Il;Lee, Hak Jong;Kim, Kil Joong;Chung, Jin-haeng;Jung, Hyun Sook;Jeon, Jong June
Ultrasonography
/
v.32
no.2
/
pp.132-142
/
2013
Purpose: The purpose of this study is to investigate the correlations of various kinetic parameters derived from the time intensity curve in a xenograft mouse model injected with a prostate cancer model (PC-3 and LNCaP) using an ultrasound contrast agent with histopathologic parameters. Materials and Methods: Twenty nude mice were injected with human prostate cancer cells (15 PC-3 and five LNCaP) on their hind limbs. A bolus of $500{\mu}L$ ($1{\times}10^8$ microbubbles) of second-generation US contrast agent (SonoVue) was injected into the retroorbital vein. The region of interest was drawn over the entire tumor. The time intensity curve was acquired and then fitted to a gamma variate function. The maximal intensity (A), time to peak (Tp), maximal wash-in rate (washin), washout rate (washout), area under the curve up to 50 sec ($AUC_{50}$), area under the ascending slope ($AUC_{in}$), and area under the descending slope ($AUC_{out}$) were derived from the parameters of the gamma variate fit. Immunohistochemical staining for VEGF and CD31 was performed. Tumor volume, the area percentage of VEGF stained in a field, and the count of CD31 (microvessel density, MVD) positive vessels showed correlation with the parameters from the time intensity curve. Results: No significant differences were observed between the kinetic and histopathological parameters from each group. MVD showed positive correlation with A (r=0.625, p=0.003), washin (r=0.462, p=0.040), $AUC_{50}$ (r=0.604, p=0.005), and $AUC_{out}$ (r=0.587, p=0.007). Positive correlations were also observed between tumor volume and $AUC_{50}$ (r=0.481, p=0.032), washin (r=0.662, p=0.001), and $AUC_{out}$ (r=0.547, p=0.012). Washout showed negative correlations with MVD (r=-0.454, p=0.044) and tumor volume (r=-0.464, p=0.039). The area percentage of VEGF did not show any correlation with calculated data from the curve. Conclusion: MVD showed correlations with several of the kinetic parameters. CEUS has the potential for prediction of tumor vascularity in a prostate cancer animal model.
Background: Cellular remnants in the bioprosthetic heart valve are known to be related to a host's immunologic response and they can form the nidus for calcification. The extracellular matrix of the decellularized valve tissue can also be used as a biological scaffold for cell attachment, endothelialization and tissue reconstitution. Thus, decellularization is the most important part in making a bioprosthetic valve and biological caffold. Many protocols and agents have been suggested for decellularization, yet there ave been few reports about the effect of a treatment with hypotonic solution prior to chemical or enzymatic treatment. This study investigated the effect of a treatment with hypotonic solution and the appropriate environments such as temperature, the treatment duration and the concentration of sodium dodecylsulfate (SDS) for achieving proper decellularization. Material and Method: Porcine aortic valves were decellularized with odium dodecylsulfate at various concentrations (0.25%, 0.5%), time durations (6, 12, 24 hours) and temperatures ($4^{\circ}C$, $20^{\circ}C$)(Group B). Same the number of porcine aortic valves (group A) was treated with hypotonic solution prior to SDS treatment at the same conditions. The duration of exposure to the hypotonic solution was 4, 7 and 14 hours and he temperature was $4^{\circ}C$ and $20^{\circ}C$, respectively. The degree of decellularization was analyzed by performing hematoxylin and eosin staining. Result: There were no differences in the degree of decellularization between the two concentrations (0.25% 0.5%) of SDS. Twenty four hours treatment with SDS revealed the best decellularization effect for both roups A and B at the temperature of $4^{\circ}C$, but there was no differences between the roups at $20^{\circ}C$. Treatment with hypotonic solution (group A) showed a better ecellularization effect at all the matched conditions. Fourteen hours treatment at $4^{\circ}C$ ith ypotonic solution prior to 80S treatment revealed the best decellularization effect. The treatment with hypotonic solution at $20^{\circ}C$ revealed a good decellularization effect, but his showed significant extracellular matrix destruction. Conclusion: The exposure of porcine heart valves to hypotonic solution prior to SDS treatment is highly effective for achieving decellularization. Osmotic treatment with hypotonic solution should be considered or achieving decellularization of porcine aortic valves. Further study should be carried out to see whether the treatment with hypotonic solution could reduce the exposure duration and concentration of chemical detergents, and also to evaluate how the structure of the extracellular matrix of the porcine valve is affected by the exposure to hypotonic solution.
Purpose: Cancer specific killing can be achieved by therapeutic gene activated by cancer specific promotor. Expression of sodium iodide symporter (NIS) gene causes transportation and concentration of iodide into the cell, therefore radioiodine treatment after NIS gene transfer to cancer cell could be a form of radionuclide gene therapy. luciferase (Luc) gene transfected cancer cell can be monitored by in vivo optical imaging after D-luciferin injection. Aims of the study are to make vector with both therapeutic NIS gene driven by AFP promoter and reporter Luc gene driven by CMV promoter, to perform hepatocellular carcinoma specific radiodiodine gene therapy by the vector, and assessment of the therapy effect by optical imaging using luciferase expression. Materials and Methods: A Vector with AFP promoter driven NIS gene and CMV promoter driven Luc gene (AFP-NIS-CMV-Luc) was constructed. Liver cancer cell (HepG2, Huh-7) and non liver cancer cell (HCT-15) were transfected with the vector using liposome. Expression of the NIS gene at mRNA level was elucidated by RT-PCR. Radioiodide uptake, perchlorate blockade, and washout tests were performed and bioluminescence also measured by luminometer in these cells. In vitro clonogenic assay with 1-131 was performed. In vivo nuclear imaging was obtained with gamma camera after 1-131 intraperitoneal injection. Results: A Vector with AFP-NIS-CMV-Luc was constructed and successfully transfected into HepG2, Huh-7 and HCT-15 cells. HepG2 and Huh-7 cells with AFP-NIS-CMV-Luc gene showed higher iodide uptake than non transfected cells and the higher iodide uptake was totally blocked by addition of perchlorate. HCT-15 cell did not showed any change of iodide uptake by the gene transfection. Transfected cells had higher light output than control cells. In vitro clonogenic assay, transfected HepG2 and Huh-7 cells showed lower colony count than non transfected HepG2 and Huh-7 cells, but transfected HCT-15 cell did not showed any difference than non transfected HCT-15 cell. Number of Huh-7 cells with AFP-NIS-CMV-Luc gene transfection was positively correlated with radioidine accumulation and luciferase activity. In vivo nuclear imaging with 1-131 was successful in AFP-NIS-CMV-Luc gene transfected Huh-7 cell xenograft on nude mouse. Conclusion: A Vector with AFP promoter driven NIS and CMV promoter driven Luc gene was constructed. Transfection of the vector showed liver cancer cell specific enhancement of 1-131 cytotoxicity by AFP promoter, and the effect of the radioiodine therapy can be successfully assessed by non-invasive luminescence measurement.
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