• Journal of Life Science
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• v.26 no.3
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• pp.376-386
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• 2016

Apoptosis induction has been proposed as an efficient mechanism by which malignant tumor cells can be removed following chemotherapy. The intrinsic mitochondria-dependent apoptotic pathway is frequently implicated in chemotherapy-induced tumor cell apoptosis. Since DNA-damaging agent (DDA)-induced apoptosis is mainly regulated by the tumor suppressor protein p53, and since more than half of clinical cancers possess inactive p53 mutants, microtubule-damaging agents (MDAs), of which apoptotic effect is mainly exerted via p53-independent routes, can be promising choice for cancer chemotherapy. Recently, we found that the apoptotic signaling pathway induced by MDAs (nocodazole, 17α-estradiol, or 2-methoxyestradiol) commonly proceeded through mitotic spindle defect-mediated prometaphase arrest, prolonged Cdk1 activation, and subsequent phosphorylation of Bcl-2, Mcl-1, and Bim in human acute leukemia Jurkat T cells. These microtubule damage-mediated alterations could render the cellular context susceptible to the onset of mitochondria-dependent apoptosis by triggering Bak activation, Δψm loss, and resultant caspase cascade activation. In contrast, when the MDA-induced Bak activation was inhibited by overexpression of anti-apoptotic Bcl-2 family proteins (Bcl-2 or Bcl-xL), the cells in prometaphase arrest failed to induce apoptosis, and instead underwent mitotic slippage and endoreduplication cycle, leading to formation of populations with 8N and 16N DNA content. These data indicate that cellular apoptogenic mechanism is critical for preventing polyploid formation following MDA treatment. Since the formation of polyploid cells, which are genetically unstable, may cause acquisition of therapy resistance and disease relapse, there is a growing interest in developing new combination chemotherapies to prevent polyploidization in tumors after MDA treatment.

• Tuberculosis and Respiratory Diseases
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• v.60 no.3
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• pp.304-313
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• 2006

Background : Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes the oxidative degradation of heme to form biliverdin, carbon monoxide (CO), and free iron. The current evidence has indicated a critical role of HO-1 in cytoprotection and also in other, more diverse biological functions. It is known that the high expression of HO-1 occurs in various tumors, and that HO-1 has an important role in rapid tumor growth because of its antioxidative and antiapoptotic effects. Therefore, the role of HO-1 was analyzed in human lung cancer cell lines, and especially in the A549 cell line. Material and Methods : Human lung cancer cell lines, i.e., A549, NCI-H23, NCI-H157 and NCI-H460, were used for this study. The expression of HO-1 in the untreated state was defined by Western blotting. ZnPP, which is the specific HO inhibitor we used, and the viability of cells were tested for by conducting MTT assaysy. The HO enzymatic activity, as determined via the bilirubin level, was also indirectly measured. Moreover, the generation of intracellular hydrogen peroxide (H2O2) was monitored fluorimetrically with using a scopoletin-horse radish peroxidase (HRP) assay and 2',7'-dichlorofluorescein diacetate (DCFH-DA). We have also transfected small HO-1 interfering RNA (siRNA) into A549 cells, and the apoptotic effects were evaluated by flow cytometric analysis and Western blotting. Results : The A549 cells had a greater expression of HO-1 than the other cell lines, whereas ZnPP significantly decreased the viability of the A549 cells more than the viability of the other lung cancer cells in a dose-dependant fashion. Consistent with the viability, the HO enzymatic activity also was decreased. Moreover, intracellular H2O2 generation via ZnPP was induced in a dose-dependent manner. Apoptotic events were, then induced in the HO-1 siRNA transfected A549 cells. Conclusion : HO-1 provides new important insights into the possible molecular mechanism of the antitumor therapy in lung cancer.

• Tuberculosis and Respiratory Diseases
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• v.65 no.6
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• pp.476-486
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• 2008

Background: TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates $NF-{\kappa}B$ in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate $NF-{\kappa}B$ in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of $NF-{\kappa}B$ activation using proteasome inhibitor MG132 which blocks $I{\kappa}B{\alpha}$ degradation can sensitize lung cancer cells to TRAIL-induced apoptosis. Methods: A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells were used and cell viability test was done by MTT assay. Apoptosis was confirmed with Annexin V assay followed by FACS analysis. To study $NF-{\kappa}B$-dependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgG ${\kappa}-NF-{\kappa}B$ luciferase construct. To investigate DNA binding of $NF-{\kappa}B$ activated by TRAIL, electromobility shift assay was used and supershift assay was done using anti-p65 antibody. Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation. Results: A549 and NCI-H1299 cells were relatively resistant to TRAIL-induced apoptosis showing only 20~30% cell death even at the concentration 100 ng/ml, but MG132 ($3{\mu}M$) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced $NF-{\kappa}B$ transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of $NF-{\kappa}B$ activated by TRAIL and supershift with p65 antibody. $I{\kappa}B{\alpha}$ degradation was proven by western blot. MG132 completely blocked both TRAIL-induced $NF-{\kappa}B$ dependent luciferase activity and DNA binding of $NF-{\kappa}B$. Conclusion: This results suggest that inhibition of $NF-{\kappa}B$ can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer.

• Clinical and Experimental Pediatrics
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• v.46 no.1
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• pp.56-66
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• 2003

Purpose : Human umbilical vein endothelial cells(HUVECs) play an important role in regulating blood flow by releasing vasoactive substances. It has been reported that endothelial impairment and dysfunction might be a primary cause of placental vascular disease, which is manifested clinically as preeclampsia in mother and intrauterine growth restriction in fetus. Furthermore, the frequency of apoptotic changes is increased in umbilical and placental tissues from growth-restricted pregnancies. However, the various mechanisms of umbilical endothelial cell apoptosis have not been broadly proposed. We investigate the effects of amiloride derivatives on apoptotic death of HUVECs and identify their ionic mechanism. Methods : HUVECs were purchased from Clonetics, and cultured on endothelial cell growth medium. MTT assay and flow cytometry were used for assessing cytotoxic effect and confirming the apoptosis. Changes in intracellular ion concentrations were measured with specific fluorescent dyes and fluorescence imaging analysis system. Results : Amiloride derivatives elicited cytotoxic effects on HUVECs with dose-dependent manners and the rank order of potency is HMA($IC_{50}\;11.2{\mu}M$), MIA>EIPA>>amiloride. HMA-induced cytotoxicity is dependent on extra- and intracellular pH, that is, increase extra- and intracellular pH augmented the cytotoxic effects of HMA. HMA dose-dependently reduced intracellular major ions, such as $K^+$ and $Cl^-$. Interestingly, the depletion of intracellular ions induced by HMA was also significantly enhanced at alkaline extracellular pH. Conclusion : Amiloride derivatives induce apoptosis of HUVECs with dose and pH dependent manners. They reduce intracellular $K^+$ and $Cl^-$ concentration, which is also extracellular pH dependent.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.80-80
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• 2003

${\beta}$-lapachone(${\beta}$-Lap), a natural o-naphthoquinone, presents in the bark of the Lapacho tree. ${\beta}$-Lap is cytotoxic against a variety of human cancer cells and it potentiates the anti-tumor effect of Taxol. In addition, ${\beta}$-Lap has been reported to radiosensitize cancer cells by inhibiting the repair of radiation-induced DNA damage.In the present study, we investigated the cytotoxicity of ${\beta}$-Lap against RKO human colorectal cancer cells as well as the combined effect of ${\beta}$-LaP and ionizing radiation. An incubation of RKO cells with 5 ${\mu}$M of ${\beta}$-Lap for 4 h killed almost 90％ of the clonogenic cells. An incubation of RKO cells with 5 ${\mu}$M of ${\beta}$-Lap for 4 h or longer also caused massive apoptosis. Unlike other cytotoxic agents, ${\beta}$-Lap did not increase the expression of p53 and p21 and it suppressed the NFkB expression. The expression of Caspase 9 and 3 was minimally altered by ${\beta}$-Lap. Radiation and ${\beta}$-Lap acted synergistically in inducing clonogenic cell death and apoptosis in RKO cells when ${\beta}$-Lap treatment was applied after but not before the radiation exposure of the cells. Interestingly, a 4 h treatment with 5 ${\mu}$M of ${\beta}$-Lap starting 5 h after irradiation was as effective as that starting immediately after irradiation. The mechanisms of ${\beta}$-Lap-induced cell killing is controversial but a recent hypothesis is that ${\beta}$-Lap is activated by NAD(P)H: quinone-onidoreductase (NQO1) in the cells followed by an elevation of cytosolic Ca$\^$2+/ level and activation of proteases leading to apoptosis. It has been reported that NQO1 level in cells is markedly up-regulated for longer than 10 h after irradiation. Indeed, using immunological staining of NQO1, we observed a significant elevation of NQO1 expression in RKO cells 5h after 2-4 Gy irradiation. Such a prolonged elevation of NQO1 level after irradiation may be the reasons why the ${\beta}$-Lap treatment applied S h after irradiation was as effective as that applied immediately after irradiation in killing the cells. In view of the fact that the repair of radiation-induced damage is usually completed within 1-2 h after irradiation, it is highly likely that the ${\beta}$-Lap treahment applied 5 h after irradiation could not inhibit the repair of radiation-induced damage. For in vivo study, RKO cells were injected S.C. into the hind-leg of Nu/Nu mice, and allowed to grow to 130 mm3 tumor. The mice were i.p. injected with ${\beta}$-lapachone or saline 2 h after irradiation of tumors with 10 Gy of X-rays. The radiation induced growth delay was increased by 2.4 $\mu\textrm{g}$/g of ${\beta}$-lapachone. Taken together, we may conclude that the synergistic interaction of radiation and ${\beta}$-Lap in killing cancer cells is not due to radiosensitization by ${\beta}$-Lap but to an enhancement of ${\beta}$-Lap cytotoxicity by radiation through an upregulation of NQO1. The fact that NQO1 is elevated in tumors and that radiation causes prolonged increase of the NQO1 expression may be exploited to preferentially kill tumor cells using ${\beta}$-Lap in combination with radiotherapy.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.3
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• pp.217-229
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• 2010

Objective: Apoptosis plays an important role for the maintenance of the normal pregnancy. Expression of X-linked inhibitor of apoptosis (XIAP) is able to effectively prevent apoptosis and controls trophoblast cells death throughout placental development, but it is still unknown in the function of XIAP in trophoblast cells exposed to hypoxic condition, which is one of the factors causing preeclampsia. Therefore, we conducted to compare XIAP expression in normal and pre-eclamptic placenta tissues and analyzed the function of XIAP in HTR-8/SVneo trophoblast cell line exposed to hypoxic condition. Methods: The expression of XIAP was analyzed in placental tissues from the following groups of patients (none underwent labor): 1) term normal placenta (n=15); 2) term with pre-eclamptic placeneta (n=15); and 3) pre-term with pre-eclamptic placenta (n=11) using semi-quantitative RT-PCR, immunohistochemistry, and Western blot. In order to evaluate the function of XIAP in HTR-8/SVneo trophoblast cells under hypoxic condition, HIF-$1{\alpha}$ plasmids, and hypoxic condtion were transfected and treated into HTR-8/SVneo trophoblast cells for 24 hours, respectively. Results: We observed that XIAP are expressed in the syncytiotrophoblasts and syncytial knot of placental villi. The expression of XIAP was significantly decreased in preeclamptic placenta tissues than in normal placenta tissues without labor (p<0.05). Furthermore, we confirmed the XIAP expression in HTR-8/SVneo trophbolast cells exposed to hypoxia was translocated from cytoplasm into nucleus and decreased XIAP by hypoxic condition induced apoptosis in HTR-8/SVneo trophoblast cells through up-regulation of pro-apoptotic proteins. Conclusion: These results suggest that the expression of XIAP is involved in placental development as well as decreased expression of XIAP by hypoxia is associated with pre-eclampsia through inducing trophoblast cells apoptosis.

• Korean Journal of Food Science and Technology
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• v.53 no.4
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• pp.423-433
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• 2021

This study was performed to examine the neuroprotective effect of the ethyl acetate fraction from Eucommia ulmoides oliver leaf (EFEL) on PM_2.5-induced cytotoxicity. EFEL had higher total phenolic and flavonoid contents than the other fractions. In ABTS and DPPH radical scavenging activities, the IC₅₀ of EFEL was measured as 212.80 and 359.13 ㎍/mL, respectively. To investigate the neuroprotective effect of EFEL, MTT and DCF-DA assays were performed on HT22, MC-IXC, and BV-2 cells. EFEL effectively decreased PM_2.5-induced intercellular reactive oxygen species (ROS) content and inhibited PM_2.5-induced cell death. In the results of protein expression related to cellular cytotoxicity on microglial cells (BV-2), EFEL had an improvement effect on cell apoptosis and inflammatory pathways. Rutin and chlorogenic acid were identified as the main physiological compounds. Moreover, it was expected that EFEL, including rutin and chlorogenic acid, could be functional food substances with neuroprotective effects against PM_2.5-induced oxidative stress.

• Korean Journal of Food Science and Technology
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• v.51 no.4
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• pp.379-392
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• 2019

The current study investigated the effect of Gabjubaekmok (Diospyros kaki) ethanolic extract (GEE) on $H_2O_2$-induced human neuroblastoma MC-IXC cells and amyloid beta $(A{\beta})_{1-42}$-induced ICR (Institute of Cancer Research) mice. GEE showed significant antioxidant activity that was evaluated based on ABTS, DPPH scavenging activity, and inhibition of malondialdehyde (MDA) and acetylcholinesterase activity. Further, GEE inhibited ROS production and increased cell viability in $H_2O_2$-induced MC-IXC cells. Administration of GEE ameliorated the cognitive dysfunction on $A{\beta}$-induced ICR mice as evaluated using Y-maze, passive avoidance, and Morris water maze tests. Results of ex vivo test using brain tissues showed that, GEE protected the cholinergic system and mitochondrial functions by increasing the levels of antioxidants such as ROS, mitochondrial membrane potential (MMP), and adenosine triphosphate (ATP) against $A{\beta}$-induced cognitive dysfunction. Moreover, GEE decreasd the expression levels of apoptosis-related proteins such as $TNF-{\alpha}$, p-JNK, p-tau, BAX and caspase 3. While, expression levels of p-Akt and $p-GSK3{\beta}$ increased than $A{\beta}$ group. Finally, gallic acid was identified as the main compound of GEE using high performance liquid chromatography.

• Korean Journal of Food Science and Technology
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• v.44 no.6
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• pp.750-758
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• 2012

This study investigated flavonoid, total phenol, total flavonoid content, antioxidant and antiproliferative activity on human breast cancer cells (MCF-7, MDA-MB-231). Four varieties of Korean green peppers (KP: kkuri pepper, PP: phut pepper, CP: cheongyang pepper, OP: ohi pepper) and one foreign green pepper (JP: jalapeno) were used. The contents of luteolin, quercetin and apigenin, which are abundant flavonoids in green pepper, were the highest in KP. Also, the contents of total phenol, and total flavonoids were the highest in KP, followed by CP, JP, PP, and OP (KP: total phenol $13.29{\pm}0.45$ mg GAE/g D.W., total flavonoid $7.02{\pm}0.13$ mg QE/g D.W. In DPPH ABTS radical-scavenging activity, KP showed the most potent antioxidant activity. In the result of viability in human breast cancer cells, KP had the highest antiproliferative effect. These results suggest that green peppers have significant antioxidant activity and can be a possible candidate for treatment of breast cancer.