• Journal of Life Science
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• v.28 no.4
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• pp.454-464
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• 2018

Black soybeans are used as food sources as well as for traditional medicines because they contain an abundance of natural phenolic compounds. In this study, total phenolic contents (TPCs) of Korean black seed coat soybean varieties Socheongja (SCJ), Socheong 2 (SC2) and Cheongja 2 (CJ2) as well as their antioxidant capacities were investigated. Among them, TPCs were abundantly present in the order of CJ2$H_2O_2$-stimulated HaCaT human keratinocytes. Our results revealed that treatment with SCJ and SC2 prior to $H_2O_2$ exposure significantly increases the viability of HaCaT cells, indicating that the exposure of HaCaT cells to SCJ and SC2 conferred a protective effect against oxidative stress. SCJ and SC2 also effectively inhibited $H_2O_2$-induced apoptotic cell death through the blocking of mitochondrial dysfunction. SCJ and SC2 also attenuated the phosphorylation of Histone H2AX. Furthermore, they effectively induced the levels of thioredoxin reductase (TrxR) 1, a potent antioxidant enzyme, which is associated with the induction of nuclear transcription factor erythroid-2-like factor 2 (Nrf2); however, the protective effects of SCJ and SC2 were significantly reversed by Auranofin, a TrxR inhibitor. These results indicate that they have protective activity through the blocking of cellular damage related to oxidative stress via the Nrf2 signaling pathway. In conclusion, our study indicated that SCJ and SC2 might potentially serve as novel agents for the treatment and prevention of skin disorders caused by oxidative stress.

• Applied Microscopy
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• v.38 no.4
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• pp.293-306
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• 2008

The essential factor of acute respiratory distress syndrome (ARDS), an acute lung injury accompanied commonly by sepsis syndrome is accumulation of neutrophils in lung tissue. The study attempted to confirm whether a lung injury would be decreased with the anti-inflammatory effect of germanium by the treated germanium prior to the development of ARDS and whether nitric oxide influence in suppressing a lung injury. Test groups were divided in the following structure for experiment; CON that has been administered with sodium chloride to airway, LPS administered with endotoxin for 5 hours in the same amount and 5 hours of endotoxin administered Ge+LPS following 1 hours of pre-treated germanium. The result of a test using experimental animals, infilteration of neutrophils (p<0.001) in bronchoalveolar lavage fluid (BALF) was significantly decreased, the structure of lung tissue was preserved relatively well, and much neutrophils with distinct positive were observed on tunel staining which showed increase of apoptotic neutrophils in the pre-treated germanium group compare to the endotoxin administrated group. In observation of ultrastructural changes of cell in BALF, phagocytic alveolar macrophage was increased in alveolar space, the nucleus of most engulfed neutrophils were condensed, and some apoptosis neutrophils appears to be DNA fragmentation and effacement of cellular organelles were found in intercellular matrix in the pre-treated germanium group. However, the nitric oxide showed increase in all the groups excluding CON, and the nitric oxide effect such as degranulation diminishing of mast cells and apoptosis increase of neutrophils in the pre-treated group only. The situation appears that there was change in internal environment of the experimental animal by the pre-treated germanium before the nitric oxide is produced and the anti-inflammatory effect activated the pre-processed germanium by nitric oxide which activated following the change. Therefore, the nitric oxide created from macrophage in accordance with the pre-treated germanium appears to influence in alleviating a lung injury. Accordingly, acute lung injury is alleviated by the anti-inflammatory effect of germanium such as inhibition of neutrophils migration, induction of neutrophil apoptosis and increase of phagocytic function of phagocyte, and the nitric oxide produced from activated macrophage by germanium would influence in suppressing a lung injury.

• Journal of Oral Medicine and Pain
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• v.34 no.3
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• pp.261-276
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• 2009

Lactacystin, a microbial natural product synthesized by Streptomyces, has been commonly used as a selective proteasome inhibitor in many studies. Proteasome inhibitors is known to be preventing the proliferation of cancer cells in vivo as well as in vitro. Furthermore, proteasome inhibitors, as single or combined with other anticancer agents, are suggested as a new class of potential anticancer agents. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying induction of apoptosis in SCC25 human tongue sqaumous cell carcinoma cell line treated with lactacystin. The viability of SCC25 cells, human normal keratinocytes (HaCaT cells) and human gingiva fibroblasts (HGF-1 cells), and the growth inhibition of SCC25 cells were assessed by MTT assay and clonogenic assay respectively. The hoechst staining, hemacolor staining and TUNEL staining were conducted to observe SCC25 cells undergoing apoptosis. SCC25 cells were treated with lactacystin, and Western blotting, immunocytochemistry, confocal microscopy, FAScan flow cytometry, MMP activity, and proteasome activity were performed. Lactacystin treatment of SCC25 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. Interestingly, lactacytin remarkably revealed cytotoxicity in SCC25 cells but not normal cells. And tested SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, the up-regulation of Bax, and the activation of caspase-7, caspase-3, PARP, lamin A/C and DFF45 (ICAD). Flow cytometric analysis revealed that lactacystin resulted in G1 arrest in cell cycle progression which was associated with up-regulation in the protein expression of CDK inhibitors, $p21^{WAF1/CIP1}$ and $p27^{KIP1}$. We presented data indicating that lactacystin induces G1 cell cycle arrest and apoptois via proteasome, mitochondria and caspase pathway in SCC25 cells. Therefore our data provide the possibility that lactacystin could be as a novel therapeutic strategy for human tongue squamous cell carcinoma.

• Journal of Life Science
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• v.18 no.1
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• pp.120-128
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• 2008

The death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/ Apo1L is a cytokine that activates apoptosis through cell surface death receptors. TRAIL has sparked growing interest in oncology due to its reported ability to selectively trigger cancer cell death. Euphorbiae humifusae Wind has been used in traditional Oriental medicine as a folk remedy used for the treatment of cancer. However, the mechanism responsible for the anticancer effects of E. humifusae not clearly understood. Here, we show that treatment with subtoxic doses of water extract of E. humifusae (WEEH) in combination with TRAIL induces apoptosis in TRAIL-resistant human gastric carcinoma AGS cells. Combined treatment with WEEH and TRAIL induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis were correlated with the induction of caspase activity that resulted in the cleavage of poly (ADP-ribose) polymerase. Combined treatment also triggered the loss of mitochondrial membrane potential. Furthermore, co-treatment with WEEH and TRAIL down-regulated the protein levels of the anti-apoptotic proteins such as Bcl-2, Bcl-xL, XIAP and cIAP-1. Although more study will be needed to examine the detailed mechanisms, this combined treatment may offer an attractive strategy for safely treating gastric adenocarcinomas and the results provide important new insights into the possible molecular mechanisms of the anticancer activity of E. humifusae.

• Tuberculosis and Respiratory Diseases
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• v.54 no.5
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• pp.495-509
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• 2003

Background : Neutrophil-mediated inflammation is usually self-limiting, because neutrophils have a remarkably short life span. Prolonged neutrophil survival, which is caused by decreased spontaneous apoptosis, leads to persistent inflammation in sepsis. Because many inflammatory cytokines, which generate signals that delay apoptosis, are regulated by nuclear factor-${\kappa}B$ transcription factor, we hypothesized that nuclear factor-${\kappa}B$ might be related to the reduced neutrophil apoptosis observed in sepsis. Methods : Neutrophils of healthy volunteers and sepsis patients were freshly isolated from venous blood. Neutrophil apoptosis was assayed with two approaches : by counting apoptotic cells under a microscope and by flow cytometry using Annexin V. The activity of nuclear factor-${\kappa}B$ was assessed by immunofluorescent staining or electrophoretic mobility shift assay. Expression of X-linked inhibitor of apoptosis was measured by western blot assay. Results : We confirmed reduced spontaneous neutrophil apoptosis in patients with sepsis. The number of apoptotic neutrophils in patients with sepsis increased to the level of that in healthy controls after cycloheximide treatment, suggesting that decreased spontaneous neutrophil apoptosis is dependent on de novo protein synthesis. In patients with sepsis, basal neutrophil nuclear factor-${\kappa}B$ was activated compared to the level in healthy controls. Moreover, a blockade of nuclear factor-${\kappa}B$ activity reversed the decreased spontaneous neutrophil apoptosis in sepsis patients. Meanwhile, X-linked inhibition of apoptosis expression, which is regulated by nuclear factor-${\kappa}B$, decreased 24 hours after incubation in healthy persons, but persisted for 24 hours in patients with sepsis. Conclusion : These observations suggest that the reduced spontaneous neutrophil apoptosis observed in patients with sepsis may be related to the induction of survival protein by nuclear factor-${\kappa}B$.

• Radiation Oncology Journal
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• v.16 no.4
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• pp.377-388
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• 1998

Purpose : To evaluate changes in expression of cell cycle related genes during apoptosis induced in HL60 cells by X-irradiation to understand molecular biologic aspects in mechanism of radiation therapy. Material and Methods : HL-60 cell line (promyelocytic leukemia cell line) was grown in culture media and irradiated with 8 Gr by linear accelerator (6 MV X-ray). At various times after irradiation, ranging from 3 to 48 hours were analyzed apoptotic DNA fragmentation assay for apoptosis and by western blot analysis and semi-quantitative RT-PCR for expression of cell cycle related genes (cyclin A, cyclin B, cyclin C, cyclin Dl, cyclin E, cdc2, CDK2, CDK4, $p16^{INK4a}$, $p21^{WAF1}$, $p27^{KIP1}$, E2F, PCNA and Rb). Results : X-irradiation (8 Gy) induced apoptosis in HL-60 cell line. Cycline A protein increased after reaching its peak 48 h after radiation delivery and cyclin E, E2F, CDK2 and RB protein increased then decreased after radiation. Radiation induced up-regulation of the expression of E2F is due to mostly increase of Phosphorylated retinoblastoma proteins (ppRb). Cyclin Dl, PCNA, CDC2, CDK4 and $p16^{INK4a}$ protein underwent no significant change at any times after irradiation. There was not detected $p21^{WAF1}$ and $p27^{KIP1}$ protein. Cyclin A, B, C mRNA decreased immediately after radiation and then increased at 12 h after radiation. Cyclin Dl mRNA increased immediately and then decreased at 48 h after radiation. After radiation, cyclin E mRNA decreased with the lapse of time. CDK2 mRNA decreased at 3h and increased at eh after radiation. CDK4 mRNA rapidly increased at 6 to 12 h after radiation. There was no change of expression of $p16^{INK4a}$ and not detected in expressin of $p21^{WAF1}$ and $p27^{KIP1}$ mRNA. Conclusion : We suggest that entry into S phase may contribute to apoptosis of HL60 cells induced by irradiation. Increase of ppRb and decrease of pRb protein are related with radiation induced auoptosis of HL60 cells and tosis of HL60 cells induced by irradiation. Increase of ppRb and decrease of PRb protein are related with radiation induced apoptosis of HL60 cells and this may be associated with induction of E2F and cyclinE/CDK2. These results support that $p21^{WAF1}$ and $p27^{KIP1}$ are not related with radiation induced-apoptosis.

• Journal of Life Science
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• v.31 no.9
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• pp.827-833
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• 2021

Dendropanax morbiferus leaves (DPL) has been used as a medicine since ancient times in various diseases such as inflammation, diabetes, and cancer. In particular, it has been found to have anticancer effects on several types of cancer cells, but the anticancer effect on breast cancer cells SK-BR-3 has not yet been revealed. Therefore, in this study, DPL caused proliferation inhibition in breast cancer cells SK-BR-3 and the anticancer effect by inducing apoptosis was confirmed, through an in vitro experiment. In order to examine the effect of DPL on cell viability, MTT assay was performed to confirm a significant decrease in the concentration of cell viability. DAPI staining was performed to examine the effect of DPL on cellular morphological changes and increase of apoptotic bodies. To supplement this, an increase in the apoptosis rate was also confirmed through flow cytometry after staining with annexin V/PI. Western blot was performed to confirm apoptosis-related proteins. DPL increased the expression of Cleaved-PARP, Bax whereas decreased the expression of Bcl-2. Changes in the expression levels of MAPK pathway proteins p-ERK1/2, p-JNK, and p-p38 were also confirmed, and a significant increase in p-p38 was observed. These results indicated that DPL induced apoptosis, through p-p38 MAPK signal pathway in SK-BR-3 breast cancer cells.

• Journal of Food Hygiene and Safety
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• v.34 no.2
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• pp.183-190
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• 2019

Ursolic acid is recognized for various effects such as anti-cancer, antioxidant, and anti-inflammatory activity. In this study, we confirmed the anti-cancer effect of ursolic acid on human melanoma cancer cells, A375SM and A375P. Survival rate of the melanoma cells was confirmed by MTT assay and the proliferation rate was confirmed by wound healing assay. The rate of apoptotic bodies was confirmed by DAPI staining, and apoptosis rate was confirmed by flow cytometry. The induction of apoptosis protein was examined by western blotting according to the concentration of ursolic acid in melanoma cells. The survival and proliferation rates of melanoma cells were decreased according to the treatment concentrations of ursolic acid. DAPI staining showed that chromosomal condensation of melanoma cells was increased with increasing concentrations of ursolic acid, and increased apoptosis rate of melanoma cells by ursolic acid was confirmed by flow cytometry. We also confirmed by western blotting that cleaved-PARP and Bax were increased and Bcl-2 was decreased at $12{\mu}M$ concentration of uricolic acid in melanoma cells. This study was carried out at low concentrations of ursolic acid, 0 to $20{\mu}M$, and analyzed 24 h after treatment. As a result of this study, it is thought that ursolic acid has the anti-cancer effect through the regulation of apoptosis-related proteins in melanoma cells A375SM and A375P.

• Journal of Life Science
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• v.19 no.5
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• pp.620-624
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• 2009

D-Ala2-Leu5-enkephalin (DADLE), a hibernation inducer, can induce hibernation-like state in vivo and in vitro. We treated U937 human leukemia cancer cells with DADLE and investigated its possible effect on transcription and proliferation. Treatment of U937 cells with DADLE resulted in growth inhibition and induction of apoptotic cell death on high-dose as measured by MTT assay and DNA flow cytometer analysis. Bcl-XL, c-IAP-2 and survivin genes especially showed decreases in mRNA levels. DADLE treatment also inhibited the levels of cyclooxygenase (COX)-2 mRNA without alteration of COX-1 expression. DNA flow cytometer analysis revealed that DADLE caused arrest of the cell cycle on low-dose, which was associated with a down-regulation of cyclin E at the transcriptional level. DADLE treatment induced a marked down-regulation of cyclin-dependent kinase (Cdk)-2, -4 and -6. In addition, treatment with DADLE decreased telomere associated genes such as, c-myc and TERT, and increased TEP-1 in U937 cells. These results suggest that DADLE can be an inhibition agent in the cell cycle of the human leukemia cancer U937 cell.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4597-4606
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• 2012

Cholangiocarcinoma (CCA) is an uncommon adenocarcinoma which arises from the epithelial cells of the bile ducts. The aim of the study was to investigate the cytotoxicity, toxicity, and anticancer activity of a crude ethanolic extract of ginger (Zingiber officinale Roscoe) against CCA. Cytotoxic activity against a CCA cell line (CL-6) was assessed by calcein-AM and Hoechst 33342 assays and anti-oxidant activity was evaluated using the DPPH assay. Investigation of apoptotic activity was performed by DNA fragmentation assay and induction of genes that may be involved in the resistance of CCA to anticancer drugs (MDR1, MRP1, MRP2, and MRP3) was examined by real-time PCR. To investigate anti-CCA activity in vivo, a total of 80 OV and nitrosamine (OV/DMN)-induced CCA hamsters were fed with the ginger extract at doses of 1000, 3000, and 5000 mg/kg body weight daily or every alternate day for 30 days. Control groups consisting of 10 hamsters for each group were fed with 5-fluorouracil (positive control) or distilled water (untreated control). Median $IC_{50}$ (concentration that inhibits cell growth by 50%) values for cytotoxicity and anti-oxidant activities of the crude ethanolic extract of ginger were 10.95, 53.15, and $27.86{\mu}g/ml$, respectively. More than ten DNA fragments were visualized and up to 7-9 fold up-regulation of MDR1 and MRP3 genes was observed following exposure to the ethanolic extract of ginger. Acute and subacute toxicity tests indicated absence of any significant toxicity at the maximum dose of 5,000 mg/kg body weight given by intragastric gavage. The survival time and survival rate of the CCA-bearing hamsters were significantly prolonged compared to the control group (median of 54 vs 17 weeks). Results from these in vitro and in vivo studies thus indicate promising anticancer activity of the crude ethanolic extract of ginger against CCA with the absence of any significant toxicity. Moreover, MDR1 and MRP3 may be involved in conferring resistance of CCA to the ginger extract.