• Tuberculosis and Respiratory Diseases
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• v.54 no.4
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• pp.403-414
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• 2003

Background : Proteasome inhibitors can promote either cell survival or programmed cell death, depending on both the specific type and proliferative status of the cell. However, it is not well known whether inhibition of proteasome activity is related to apoptosis in lung cancer cells. In addition, the exact mechanisms responsible for apoptosis induced by proteasome inhibition are not well understood. In the present study, we have examined the effect of proteasome inhibition on lung cancer cells and tried to test the mechanisms that may be associated with the apoptosis of these cells. Methods : We examined the effect of proteasome inhibition with MG132 or PS-341 on cell survival in A549 and NCI-H157 lung cancer cells using MTT assay, and analyzed the cleavage of PARP by Western blot analysis to find evidence of apoptosis. Next, we evaluated the activation of caspase 3 by Western blot analysis and the activity of JNK by immunocomplex kinase assay. We also examined the changes in anti-apoptotic pathways like ERK and cIAP1 by Western blot analysis after inhibition of proteasome function. Results : We demonstrated that MG132 reduced cell survival by inducing apoptosis in A549 and NCI-H157 cells. Proteasome inhibition with MG132 or PS-341 was associated with activation of caspase 3 and JNK, reduced expression of activated ERK, and downregulation of cIAP1. Conclusion : Apoptosis induced by proteasome inhibition may be associated with the activation of pro-apoptotic pathways like caspase 3 and JNK and the inactivation of anti-apoptotic pathways in lung cancer cells.

• Biomedical Science Letters
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• v.21 no.2
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• pp.69-76
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• 2015

Cervical cancer is the third most common cancer in women worldwide and there is a significant association between human papillomavirus (HPV) infection and cervical cancer. Certain HPV groups, labeled high-risk (HR) HPV groups, are strongly associated with malignancies of the human cervix. HPV prevalence and genotype distribution were analyzed using the REBA $HPV-ID^{(R)}$ (YD Diagnostics, Yongin, Korea) assay based on the reverse blot hybridization assay (REBA) with a total of 324 liquid-based cytology samples from women in Western Shandong Province, East China and results were compared with cytological diagnosis. Most of the HPV genotypes that were detected in high-grade cervical lesions were HR-HPV genotypes such as HPV 16, 18, 33, 53, and 58. The prevalence of these HR-HPV genotypes increased in high-grade cervical lesions. However, from low- to high-grade cervical lesions, the ability to detect LR-HPV genotypes decreased. Additionally, in general, the single HPV genotype infection rate increases in proportion to the severity of the lesion. The study findings suggest that a currently available preventive vaccine against HPV 16 and 18 may have limited effectiveness for prevention of all HPV infection in this province. Finally, based on these findings, these data could guide national or regional vaccination programs in the Western Shandong Province of East China to substantially reduce the burden of cervical lesions.

• Archives of Plastic Surgery
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• v.41 no.6
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• pp.654-660
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• 2014

Background Reactive oxygen species (ROS) damages cell molecules, and modifies cell signaling. The nuclear factor E2-related factor (Nrf2) is a critical transcription regulator, which protects cells against oxidative damage. Nrf2 expression is increased in a large number of cancers. However, little information has been reported regarding the expression of Nrf2 in skin cancers. Hence, we explored the expression of Nrf2 protein in skin cancers. Methods The Nrf2 protein expression in 24 specimens, including 6 malignant melanomas (MM), 6 squamous cell carcinomas (SCC), 6 basal cell carcinomas (BCC), and 6 normal skin tissues, was evaluated by western blotting. Immunohistochemical staining was performed. The expression of Kelch-like ECH-associated protein 1 (Keap1), the key regulator of Nrf2, was also analyzed by western blotting. Results Small interfering RNA transfection to the melanoma cell line G361 confirmed that an approximately 66 kDa band was the true Nrf2 band. The western blot revealed that the Nrf2 protein was definitely expressed in normal skin tissues, but the Nrf2 expression was decreased in MM, SCC, and BCC. Immunohistochemical examination showed that expression of Nrf2 was decreased in all skin cancer tissues compared to the normal skin tissues. Keap1 was not expressed in all malignant skin tumors and normal skin tissues by western blot. Conclusions ROS was increased in various types of cancers which proteins were highly expressed or underexpressed. This study demonstrated that the expression of Nrf2 protein was down-regulated in human malignant skin tumors. We suggest that decreased expression of Nrf2 is related to skin cancers.

• Journal of the Korean Society of Radiology
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• v.8 no.6
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• pp.325-332
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• 2014

Metallic nanoparticles have attractive properties in biomedical applications such as diagnostics and therapeutics. Cross linked dextran coated iron oxide nanoparticles (SPIONs) and silica coated gadolinium oxide nanoparticles (SPGONs) have been synthesized as a radiosensitizer in the proton beam cancer therapy. The dextran and silicaused for the protective moieties on the SPIONs and SPGONs respectively. Size distributions of synthesized nanoparticles were confirmed 3~5 nm for SPIONs and 30~100 nm for SPGONs by transmission electron microscope (TEM). Cell survival fraction measurement and Western blot assay were performed to evaluate the radiosensitization effects of synthesized radiosensitizer. The calculated radiosensitization of SPIONs and SPGONs at 90 % cell death from the measured cell survival curves were 1.23 and 1.03 respectively. Western blotting results also show the same consistent results that the amount of released cytochrome c from mitochondria was considerably increased for the cancer cells taken up SPIONs.

• Clinical and Experimental Pediatrics
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• v.51 no.2
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• pp.170-180
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• 2008

Purpose : Some antibiotics were known to exert neuroprotective effects in the animal model of hypoxic-ischemic (H-I) brain injury, but the mechanism is still unclear. A recent study reported that geneticin (G418), an aminoglycoside antibiotic, increased survival of human breast cancer cells by suppressing apoptosis. We investigated the neuroprotective effects of systemically administrated geneticin via anti-apoptosis following the H-I brain injury Methods : Seven-day-old Sprague-Dawley rat pups were subjected to unilateral (left) common carotid artery occlusion followed by 2.5 hours of hypoxic exposure and the cortical cell culture of rat brain was done under a hypoxic incubator. Apoptosis was measured in the injured hemispheres 7 days after H-I insult and in the injured cells from hypoxic chamber using morphologic analysis by Terminal dUTP Nick-end Labeling(TUNEL) assay and immunohistochemistry for caspase-3, and cytologic analysis by western blot and real time PCR for bax, bcl-2, and caspase-3. Results : The gross appearance and hematoxylin and eosin stain revealed increased brain volume in the geneticin-treated animal model of perinatal H-I brain injury. The TUNEL assay revealed decreased apoptotic cells after administration of geneticin in the cell culture model of anoxia. Immunohistochemistry showed decreased caspase-3 expression in geneticin-treated cortical cell culture. Western blot and real-time PCR showed decreased caspase-3 expression and decreased ratio of Bax/Bcl-2 expression in geneticin-treated animal model. Conclusion : Geneticin appears to exert a neuroprotective effect against perinatal H-I brain injury at least via anti-apoptosis. However, more experiments are needed in order to demonstrate the usefulness of geneticin as a preventive and rescue treatment for H-I brain injuries of neonatal brain.

• Development and Reproduction
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• v.6 no.2
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• pp.131-139
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• 2002

Since GnRH and its receptor genes are expressed in the ovary, it has been suggested that ovarian GnRH might be involved in the regulation of ovarian function and the apoptosis of ovarian cells. However, it was not known well on the expression and function of GnRH and its receptor in the corpus luteum. The present study was undertaken to investigate whether GnRH and its receptor are expressed in luteal cells and GnRH has any effect on the apoptosis of luteal cells. Luteal cells obtained from the pregnant rats were cultured and stained for GnRH and its receptor proteins. Cultured luteal cells showed distinct immunoreactivity against both anti-GnRH and anti-GnRH receptor antibodies. In addition, the presence of GnRH receptor protein in cultured cells was confirmed by Western blot analysis. To investigate the effect of GnRH on the apoptosis of luteal cells, luteal cells were cultured in the presence of 10$^{-6}$ M GnRH-agonist(GnRH-Ag) for 3, 8, and 12h. TUNEL assay showed that the number of cells undergoing apoptosis increased 12h after culture(P<0.05). DNA fragmentation analysis confirmed the results such that the cells treated for 12h showed the greatest increase of fragmentation(p<0.05). Further, Western blot analysis of cytochrome c in the mitochondrial and cytoplasmic fractions of the luteal cells showed that GnRH-Ag treatment increased the content of cytochrome c in cytoplasm. These results demonstrate that the luteal cells express GnRH and its receptor and GnRH-Ag treatment induces apoptosis of the luteal cells via mitochondrial release of cytochrome c. The present study suggest that the releasing of cytochrome c from mitochondria might be involved in the luteal cell apoptosis induced by GnRH-Ag.

• The korean journal of orthodontics
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• v.31 no.6 s.89
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• pp.589-600
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• 2001

Insulin-like growth factor I (IGF-I) has the local tissue regulating actions. In bone, IGF-I increases the replication of osteoblastic lineage, probably preosteoblasts, and enhances osteoblastic collagen synthesis and matrix composition rates. The purpose of this study was to investigate the local regulatory effect of IGF-I on periodontium totally, both in an autocrine and paracrine manner. To examine the effect of IGF-I directly on osteoblast (OB) of test rats, and indirectlv on OB via periodontal ligament fibroblast (PDLF), and the effect of gingival fibroblast (GF) on OB via cellular paracrine manner for the understanding of humoral action of adjacent tissue, GF and PDLF were obtained from male Sprague-Dawley rats of six to eight weeks of age. OB was obtained iron frontal and parietal calvarial bone of Sprague-Dawley 21day-old-fetus. After each tell was Incubated 24 hours, for collecting conditioned medium, different concentrations of IGF-I (1,10,100 ng/ml,1ml/well) was adding in the GF, PDLF cells, and the supernatant from these cultures was put into the primary OB culture with $1{\times}10^4$cell/ml/well. The experimental group was divided into six groups control OB, IGF-I treated OB, OB culture with conditioned medium from PDLF, OB culture with conditioned medium from IGF-I treated PDLF, OB culture with conditioned medium from GF, OB culture with conditioned medium from IGF-I treated GF. After final IGF-I treatment, OB was Incubated for 24 hours, and alkaline phosphatase activity assay, BMP expression, cell proliferation measurement using MTT assay, total protein measurement, Collagen synthesis assay using western blot, and examination of bone nodule synthesis were done. Alkaline phosphatase expressions were increased in the group of PDLF-IGF-I supernatant treatment. Direct IGF-I treatment with concentrations of 100ng/m1 showed increased viable tell number measured by MTT assay. And IGF-I treatment did not increase total protein amount. The entire experimental group showed BMP2, 4 expression in western blot, and there was no significant difference between control and experimental groups. These results suggested that supernatant from PDLF effects on increasing cellular activities of OB regardless of IGF-I, and at high concentration, IGF-I increases OB tell proliferation.

• Microbiology and Biotechnology Letters
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• v.32 no.1
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• pp.72-77
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• 2004

Previously, we synthesized a novel Cyclin-dependent kinase inhibitor, MCS-5A. Also, we investigated the involvement of cell cycle regulatory events during MCS-5A-mediated apoptosis in HL-60(+p16/-p53) cells with up-regulation of p16 protein expression. In contrast, apoptosis was not observed in A549(-p16/+p53) cells. Therefore we propose that $p16^{INK4A}$ is a key enzyme for inducing apoptosis. In the present studies, we have explored the mechanism of $p16^{INK4A}$ -mediated cytotoxicity and the role of p16.sup INK4A/ overexpression in the induction of apoptosis in human tumor cells. The tumor suppressor gene $p16^{INK4A}$ is known as a cyclin-dependent kinase inhibitor (CKI) and cell cycle regulator. We expressed wild type $p16^{INK4A}$ in pcDNA3.1 vector and then transfected into non-small cell lung cancer (NSCLC) cell expressing different statue of p16$^{INK4A}$, p53 gene〔A549(-p16/+p53), H1299(-p16/-p53) and HeLa(+pl6/+p53) cell line〕. TUNEL assay (including propidium iodide staining following transfection of these cell line with pcDNA3.1-pl6) indicate that p16$^{INK4A}$-mediated cytotoxicity was associated with apoptosis. This is supported by studies demonstrating an induction of caspase 3 cleavage due to the transfection of A549, H1299 and HeLa cells with pcDNA3.1-pl6. These results suggest that p16$^{INK4A}$ has a new function of inducing apoptosis which is not related with the function of tumor suppressor gene p53.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.239-255
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• 1997

Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the pMal-pol clone (Lee et al. 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind T5 phage promoter and the $6{\times}His$ region of the pQE-3D expression vector, and it was called pQEVPl. Again, the $6{\times}$His-tagged VP1 DNA fragment in the pQEVP1 was cleaved with EcoRI and transferred into the VP1 site of the pBacVP1, resulting pBacHis-VP1 recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (LacZ-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay. The recombinant virus was named VP1-HcNPV-1. The $6{\times}$His-tagged VP1 protein produced by the pQEVP1 was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was $2.0{\times}10^5\;pfu/ml$ at 7 days postinfection.

• Nuclear Medicine and Molecular Imaging
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• v.42 no.5
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• pp.394-400
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• 2008

Purpose: Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Materials and Methods: Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was $19.1{\pm}4.7%$, $54.0{\pm}6.4%$, $85.7{\pm}8.7%$, and $98.4{\pm}1.3%$ at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and 1-125 uptake. Results: Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro 1-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC ($29,704{\pm}6,659\; picomole/10^6\;cells$) was greater than that in adeno-hNIS-rMSC at MOI 100 ($6,168{\pm}2,134\;picomole/10^6\;cells$). Conclusion: Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.