• Asian Pacific Journal of Cancer Prevention
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• 제16권14호
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• pp.6039-6046
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• 2015

Aims: To investigate effect of metallic nanoparticles, silver (AgNPs) and gold nanoparticles (AuNPs) as antitumor treatment in vitro against human breast cancer cells (MCF-7) and their associated mechanisms. This could provide new class of engineered nanoparticles with desired physicochemical properties and may present newer approaches for therapeutic modalities to breast cancer in women. Materials and Methods: A human breast cancer cell line (MCF-7) was used as a model of cells. Metallic nanoparticles were characterized using UV-visible spectra and transmission electron microscopy (TEM). Cytotoxic effects of metallic nanoparticles on MCF-7 cells were followed by colorimetric SRB cell viability assays, microscopy, and cellular uptake. Nature of cell death was further investigated by DNA analysis and flow cytometry. Results: Treatment of MCF-7 with different concentrations of 5-10nm diameter of AgNPs inhibited cell viability in a dose-dependent manner, with IC50 value of $6.28{\mu}M$, whereas treatment of MCF-7 with different concentrations of 13-15nm diameter of AuNPs inhibited cell viability in a dose-dependent manner, with IC50 value of $14.48{\mu}M$. Treatment of cells with a IC50 concentration of AgNPs generated progressive accumulation of cells in the S phase of the cell cycle and prevented entry into the M phase. The treatment of cells with IC50 concentrations of AuNPs similarly generated progressive accumulation of cells in sub-G1 and S phase, and inhibited the entrance of cells into the M phase of the cell cycle. DNA fragmentation, as demonstrated by electrophoresis, indicated induction of apoptosis. Conclusions: Our engineered silver nanoparticles effectively inhibit the proliferation of human breast carcinoma cell line MCF-7 in vitro at high concentration ($1000{\mu}M$) through apoptotic mechanisms, and may be a beneficial agent against human carcinoma but further detailed study is still needed.

• 미생물학회지
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• 제42권3호
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• pp.195-198
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• 2006

Anthrax lethal toxin은 탄저병의 치사원인이 되는 독소이며, Lethal toxin은 두 종류의 단백질 PA (Protective antigen)과 LF (lethal factor)로 구성되어 진다. PA는 세포표면의 수용체와 결합하여 LF를 세포질 안으로 이동시켜 주는 역할을 한다. LF는 금속 이온$(Zn^{2+})$ 의존적 단백질 가수분해 효소로써 MKKs[MAPK (mitogen-activated protein kinase) kinases] 집단 단백질의 아미노 말단 부분을 절단하여 대상 세포를 죽음으로 유도하는 것으로 알려져 있다. 본 연구에서는 LF에 대한 특성 분석 및 억제제 개발에 과한 연구를 위해 cell-based high-throughtput screens 개발에 선행되어야 하는 기초 자료를 마련하는데 그 목적이 있다. 이를 위하여 LF의 절단 대상이 되는 기질이 MEK1을 yeast내에서 동시 발현시켜 LF의 활성을 검증하였다. 먼저 효모(Saccharomyces cerevisiae)를 숙주로 하여 LF의 기질인 MEK1 발현 vector를 구축하였고, 구축된 발현 system을 기본을 LF 활성을 검증하고자 yeast에 형질전환하여 plasmid의 안전성 및 MEK1 유전자의 발현 및 LF에 의한 MEK1 아미노말단의 절단 부위를 확인하였다. 본 연구는 세포내 검증 system 도입의 기초적 자료를 제공하였으며, yeast내의 MEK1 발현은 탄저병의 저해제 선별 및 활성 측정 검증을 생체에서 고효율적이며, 안정적으로 할 수 있다는 가능성을 나타냈다.

• 대한수의학회지
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• 제42권1호
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• pp.35-43
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• 2002

Cryopreservation is commonly used for an efficient utilization of semen, oocytes and embryos but has disadvantage in the survival, development of the post-thawed eggs. The high risk in the survival, development of eggs after thawing is thought to be caused by inappropriate internal regulation of $Ca^{2+}$ and/or formation of intracellular ice crystals. In this experiment, we tested whether the $Ca^{2+}$ current (iCa), a decisive factor to $Ca^{2+}$ entry, was altered in post-thawed oocytes by using whole cell voltage clamp technique. The quality and survival rates of the oocytes derived from both fresh and frozen groups were examined by morphology and FDA-test. Vitrified oocytes (VOs) were incubated for 4 hr after thawing and then donated to this experiment. Ethyleneglycol-ficoll-galactose (EFG) was used as a cryoprotectant for vitrification. The membrane potential was held at -80 mV and step depolarizations of 250 ms were applied from -50 mV to 50 mV in 10 mV increments. The survival rates showed a higher in VOs vitrified with EFG containing $Ca^{2+}$ than in VOs vitrified with EFG under the $Ca^{2+}$-free condition (82.0% vs 14%). In group with/without $Ca^{2+}$, the survival rates were significantly (P<0.01) difference. In the fresh metaphase II oocytes (FOs), current-voltage (I-V) relationship showed that iCa began to activate at -40 mV and reached its maximum at -10 mV. With same voltage pulses, inward currents were elicited in VOs. I-V relationships observed in VOs were similar to those in FOs. Time constants of activation and inactivation of the inward current shown in VOs were not different to those in FOs. This accordance in I-V relations and time constants in FOs with those in VOs indicates that the inward currents in FOs are unaltered by vitrification and thawing. Therefore, vitrification with EFG does not play as a factor to deteriorate $Ca^{2+}$ entry across the membrane of the oocytes.

• 대한미생물학회지
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• 제34권5호
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• pp.435-443
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• 1999

Orientia tsutsugamushi is obligate intracellular bacterium that grows within the cytoplasm of the eukaryotic host cells. Therefore capability of the attachment, entry into the host cell and intracellular survival should be critical process for oriential infection. In this study we investigated the cellular invasion mechanism of Orientia tsutsugamushi and the role of transmembrane heparan sulfate proteoglycan, which binds diverse components at the cellular microenvironment and is implicated as host cell receptors for a variety of microbial pathogens. First of all Orientia tsutsugamushi can invade a wide range of nonprofessional phagocytic cells including fibroblast, epithelial cells and endothelial cells of various host species, including Band T lymphocytes. Thus, it was postulated that the attachment of O. tsutsugamushi requires the recognition of ubiquitous surface structures of many kinds of host cells. Treatments with heparan sulfate and heparin inhibited the infection of Orientia tsutsugamushi in dose-dependent manner for L cell, mouse fibroblast, whereas other glycosaminoglycans such as chondroitin sulfate had no effect. Collectively, these findings provide strong evidence that initial interaction with heparan sulfate proteoglycan is required for the oriential invasion into host cells.

• BMB Reports
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• 제34권2호
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• pp.176-181
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• 2001

Using a retrovirus, foreign genes can be introduced into mammalian cells. The purpose of this study is to produce a retrovirus that can make the infected cells express two genes; the human multidrug resistance gene (MDR1) and the HLA-B7 gene, which is one of the major human histocompatibility complex (MHC) class I genes. For the expression of these genes, the internal ribosome entry site (IRES) was used, which was derived from the encephalomyocarditis (EMC) virus. In order to produce retroviruses, a retroviral vector was transfected into a packaging cell line and the transfected cells were treated with vincristine, which is an anti-cancer drug and a substrate for the MDRI gene product. This study revealed that two genes were incorporated into chromosomes of selected cells and expressed in the same cells. The production of the retrovirus was confirmed by the reverse transcription (RT)-PCR of the viral RNA. The retrovirus that was produced infected mouse fibroblast cells as well as the human U937. This study showed that packaging cells produced the retroviruses, which can infect the target cells. Once the conditions for the high infectivity of retrovirus into human cells are optimized, thus virus will be used to infect hematopoietic stem cells to co-express MDRl and HLA-B7 genes, and develop the lymphocytes that can be used for the immnogene therapy.

• Nutrition Research and Practice
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• 제14권2호
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• pp.95-101
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• 2020

BACKGROUND/OBJECTIVES: Folate plays a critical role in DNA synthesis and methylation. Intracellular folate homeostasis is maintained by the enzymes folylpolyglutamate synthase (FPGS) and γ-glutamyl hydrolase (GGH). FPGS adds glutamate residues to folate upon its entry into the cell through a process known as polyglutamylation to enhance folate retention in the cell and to maintain a steady supply of utilizable folate derivatives for folate-dependent enzyme reactions. Thereafter, GGH catalyzes the hydrolysis of polyglutamylated folate into monoglutamylated folate, which can subsequently be exported from the cell. The objective of this review is to summarize the scientific evidence available on the effects of intracellular folate homeostasis-associated enzymes on cancer chemotherapy. METHODS: This review discusses the effects of FPGS and GGH on chemosensitivity to cancer chemotherapeutic agents such as antifolates, such as methotrexate, and 5-fluorouracil. RESULTS AND DISCUSSION: Polyglutamylated (anti)folates are better substrates for intracellular folate-dependent enzymes and retained for longer within cells. In addition to polyglutamylation of (anti)folates, FPGS and GGH modulate intracellular folate concentrations, which are an important determinant of chemosensitivity of cancer cells toward chemotherapeutic agents. Therefore, FPGS and GGH affect chemosensitivity to antifolates and 5-fluorouracil by altering intracellular retention status of antifolates and folate cofactors such as 5,10-methylenetetrahydrofolate, subsequently influencing the cytotoxic effects of 5-fluorouracil, respectively. Generally, high FPGS and/or low GGH activity is associated with increased chemosensitivity of cancer cells to methotrexate and 5-fluorouracil, while low FPGS and/or high GGH activity seems to correspond to resistance to these drugs. Further preclinical and clinical studies elucidating the pharmocogenetic ramifications of these enzyme-induced changes are warranted to provide a framework for developing rational, effective, safe, and customized chemotherapeutic practices.

• Molecules and Cells
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• 제40권9호
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• pp.621-631
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• 2017

Vaccinia-related kinase 1 (VRK1) and VRK3 are members of the VRK family of serine/threonine kinases and are principally localized in the nucleus. Despite the crucial roles of VRK1/VRK3 in physiology and disease, the molecular and functional interactions of VRK1/VRK3 are poorly understood. Here, we identified over 200 unreported VRK1/VRK3-interacting candidate proteins by affinity purification and LC-MS/MS. The networks of VRK1 and VRK3 interactomes were found to be associated with important biological processes such as the cell cycle, DNA repair, chromatin assembly, and RNA processing. Interactions of interacting proteins with VRK1/VRK3 were confirmed by biochemical assays. We also found that phosphorylations of XRCC5 were regulated by both VRK1/VRK3, and that of CCNB1 was regulated by VRK3. In liver cancer cells and tissues, VRK1/VRK3 were highly upregulated and its depletion affected cell cycle progression in the different phases. VRK3 seemed to affect S phase progression and G2 or M phase entry and exit, whereas VRK1 affects G1/S transition in the liver cancer, which could be explained by different interacting candidate proteins. Thus, this study not only provides a resource for investigating the unidentified functions of VRK1/VRK3, but also an insight into the regulatory roles of VRK1/VRK3 in biological processes.

• 대한바이러스학회지
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• 제30권1호
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• pp.83-99
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• 2000

A defective HIV-1 helper virus DNA, pHyPC, was assembled by deleting the RNA packaging signal, env, nef and the 3'LTR sequences. HIV-1 like virus particles that carry the HIV-1 receptor, CD4 were generated by co expression of pHyPC and plasmid DNAs encoding different chimeric CD4 proteins. The CD4 particles, sharing the CD4 ectodomain, precisely fused to different membrane anchors. CD4(+) particles specifically bound to HIV-1 Env expressing cells, but any signs of infection into these cells were not detected. Binding was only partially blocked by either polyclonal anti-CD4 antibodies or by high concentrations of soluble CD4. Surprisingly, CD4(+) particles also adsorbed to HeLa, CHO, NIH3T3 and COS-7 cells in the absence of HIV-1 Env expression. Adsorption was comparable in strength and speed to the highly specific CD4-Env interaction. CD4(-) particles exhibited only background levels of binding. Cell binding was CD4. dependent, but it was independent of the cell type from which the CD4(+) particles originated. Interestingly, CD4-dependent/Env-independent binding was only found when CD4 was present on virus particles. This suggests that the micro-environment of CD4 on virus particles uniquely expose this new cell binding activity. Its high affinity could explain in part why infection of Env(+) cells by CD4(+) particles was not detected. Further experiments will be required to evaluate whether this strong membrane interaction could represent one step in the multiple-step viral entry process.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.170-175
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• 2012

Clostridium difficile toxin A glucosylates Rho family proteins, resulting in actin filament disaggregation and cell rounding in cultured colonocytes. Given that the cellular toxicity of toxin A is dependent on its receptor binding and subsequent entry into the cell, we herein sought to identify additional colonocyte proteins that might bind to toxin A following its internalization. Our results revealed that toxin A interacted with ERK1 and ERK2 in two human colonocyte cell lines (NCM460 and HT29). A GST-pulldown assay also showed that toxin A can directly bind to ERK1 and ERK2. In NCM460 cells exposed to PMA (an ERK1/2 activator), the phosphorylation of ERK1/2 did not affect the interaction between toxin A and ERK1/2. However, an in vitro kinase assay showed that the direct binding of toxin A to ERK1 or ERK2 inhibited their kinase activities. These results suggest a new molecular mechanism for the cellular toxicity seen in cells exposed to toxin A.

• 한방비만학회지
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• 제18권2호
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• pp.57-63
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• 2018

Objectives: Metabolic syndrome is defined by a cluster of major cardiovascular risk factors: obesity, insulin resistance, dyslipidemia, and arterial hypertension. Several members of a large family of nonselective cation entry channels, e.g., transient receptor potential vanilloid 4 (TRPV4) channels have been associated with the development of dyslipidemia and hypertension. The purpose of this study was to investigate the effects of Leejung-tang (Lizhong-tang), Rikkunshito, and Bojungikgi-tang (Buzhongyiqi-tang) on TRPV4 channel. Methods: Human embryonic kidney 293 cells stably transfected with the TRPV4 expression vectors were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, $5{\mu}g/mL$ blasticidin, and 0.4 mg/mL zeocin in a humidified 20% $O_2/10%$ $CO_2$ atmosphere at $37^{\circ}C$. Whole-cell patch clamp recordings were obtained using an Axopatch 700B amplifier and pClamp v.10.4 software (Molecular Devices, San Jose, CA, USA), and signals were digitalized at 5 kHz using Digidata 1422A. Results: Leejung-tang and Rikkunshito (10, 30 and 50 mg/mL) had no effects on the TRPV4 whole-cell currents at dose dependent manner. However, Bojungikgi-tang (10, 30, and 50 mg/mL) inhibited the TRPV4 whole-cell currents in a dose dependent manner and the half maximal inhibitory concentration (IC50) of Bojungikgi-tang was 18.2 mg/mL. Conclusions: These results suggest that Bojungikgi-tang plays an important roles to inhibit the TRPV4 channel, suggesting that Bojungikgi-tang is considered one of the candidate agents for the treatment of metabolic syndrome such as dyslipidemia and hypertension.