• Journal of Life Science
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• v.17 no.10
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• pp.1447-1451
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• 2007

Through the screening of marine natural compounds that inhibit cancer cell proliferation, we previously reported that pectenotoxin-2 (PTX-2) isolated from marine sponges exhibits selective cytotoxicity against several cell lines in p53-deficient tumor cells compared to those with functional p53. However, the molecular mechanisms of its anti-proliferative action on malignant cell growth are not completely known. To further explore the mechanisms of its anti-cancer activity and to test whether the status of p53 in liver cancer cells correlates with their chemo-sensitivities to PTX-2, we used two well-known hepatocarcinoma cell lines, p53-deficient Hep3B and p53-wild type HepG2. We have demonstrated that PTX-2 markedly inhibits Hep3B cell growth and induces apoptosis whereas HepG2 cells are much more resistant to PTX-2 suggesting that PTX-2 seems to act by p53-independent cytotoxic mechanism. The apoptosis induced by PTX-2 in Hep3B cells was associated with the modulation of DNA fragmentation factor (DFF) family proteins, up-regulation of pro-apoptotic Bcl-2 family members such as Bax and Bcl-xS and activation of caspases (caspase-3, -8 and -9). Blockade of the caspase-3 activity by caspase-3 inhibitor, z-DEVD-fmk, prevented the PTX-2-induced growth inhibition in Hep3B cells. Moreover, treatment with PTX-2 also induced phosphorylation of AKT and extracellular-signal regulating kinase (ERK), but not c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MARK). Specific inhibitors of PI3K inhibitor (LY294002) and ERK1/2 inhibitor (PD98059) significantly blocks PTX-2-induced-anti-proliferative effects, whereas a JNK inhibitor (SP600125) and a p38 MAPK inhibitor (SB203580) have no significant effects demonstrating that the pro-apoptotic effect of PTX-2 mediated through activation of AKT and ERK signal pathway in Hep3B cells.

• Tuberculosis and Respiratory Diseases
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• v.58 no.1
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• pp.43-53
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• 2005

Emphysema is characterized by air space enlargement and alveolar destruction. The mechanism responsible for the development of emphysema was thought to be protease/antiprotease imbalance and oxidative stress. A very recent study shows that alveolar cell apoptosis causes lung destruction and emphysematous changes. Thus, this study was performed to support the evidence for the role of apoptosis in the development of emphysema by characterizing cigarette smoke extract (CSE)-induced apoptosis in A549 (type II pneumocyte) lung epithelial cells. CSE induced apoptosis at low concentration (10% or less) and both apoptosis and necrosis at high concentration (20%). Apoptosis was demonstrated by DNA fragmentation using FACScan for subG1 fraction. Discrimination between apoptosis and necrosis was done by morphologic analysis using fluorescent microscopy with Hoecst 33342/propium iodide double staing and electron microscopy. Cytochrome c release was confirmed by using immunofluorescence with monoclonal anti-cytochrome c antibody. However, CSE-induced cell death did not show the activation of caspase 3 and was not blocked by caspase inhibitors. This suggests that CSE-induced apoptosis might be caspase-independent apoptosis. CSE-induced cell death was near completely blocked by N-acetylcystein and bcl-2 overexpression protected CSE-induced cell death. This results suggests that CSE might induce apoptosis through intracellular oxidative stress. CSE also activated p53 and functional knock-out of p53 using stable overexpression of HPV-E6 protein inhibited CSE-induced cell death. The characterization of CSE-induced cell death in lung epithelial cells could support the role of lung cell apoptosis in the pathogenesis of emphysema.

• Journal of Acupuncture Research
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• v.24 no.2
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• pp.1-14
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• 2007

목적 : 이 연구는 Vipera lebetina turanica의 사독약침파(蛇毒藥鍼波)(Snake venom toxin, SVT)이 in vitro에서 $NF-{\kappa}B$의 활성억제와 apoptosis 관련 단백질의 발현 조절을 통하여 세포자멸사(Apoptosis)를 유도하는지 in vivo에서 또한 전립선 암세포주인 PC-3 세포의 성장을 억제하는지 살펴보고자 하였다. 방법 : SVT를 처리한 후 PC-3의 성장억제를 관찰하기 위해 WST-1 assay, CCK-8 assay를 시행하였고，Apoptosis evaluation에는 DAPI, TUNEL staining assay를 시행하였으며，Apoptosis regulatory proteins의 변화 관찰에는 western blot analysis를 시행하였고，apoptosis와 연관된 $NF-{\kappa}B$의 활성 변화를 관찰하기 위해 EMSA시행하였으며，SVT의 핵내이동을 관찰하기 위해 Immunofluorescence Staining, Confocal immunocytochemistry를 시행하였으며，전립암세포의 종양형성에는 흉선을 제거한 쥐에 Tumorigenecity study를 시행하였다. 결과 : PC-3 세포에 SVT를 처리한 후，전립선 암세포의 성장，Apoptosis의 유발，Apoptosis관련 단백질의 발현，$NF-{\kappa}B$의 활성，SVT의 PC-3세포 핵내 이동여부 및 흉선제거 후 PC-3 세포를 이식한 쥐의 종양형성과정에 미치는 영향을 관찰하여 다음과 같은 결과를 얻었다. 1. PC-3 세포에서 SVT를 처리한 후 세포성장이 억제되고，세포자멸사가 유도되며，조절인자인 p53, caspase-3, -9는 증가되었고，Bcl-2는 감소되었다. 2. PC-3 세포에서 SVT를 처리한 후 $NF-{\kappa}B$의 활성이 유의하게 감소되었다. 3. DAPI로 염색된 상태에서 SVT가 PC-3 세포의 핵내로 이통되는 것이 관찰되었다. 4. 흉선제거 후 전립선 암세포주를 이식한 쥐에서 SVT를 피내로 주입한 결과 전립선암의 크기와 무게가 유의하게 감소하였다. 결론 : 이상의 결과는 SVT가 $NF-{\kappa}B$의 활성 억제를 통하여 인간 전립선암세포주인 PC-3의 세포자멸사를 유발함으로써 증식억제 효과가 있음을 입증한 것이며，이를 재확인한 생체 연구에서의 긍정적인 결과는 향후 SVT의 전립선암의 예방과 치료에 대한 효과적인 치료제 개발에 초석이 될 것으로 기대된다.

• Journal of Acupuncture Research
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• v.34 no.1
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• pp.23-30
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• 2017

Objectives : In this study, the roles of Interleukin (IL)-4 and Signal transducer and activator of transcription 6 (STAT6), which have been reported to play a role in the pathogenesis of inflammation and cancer, were evaluated in snake venom toxin (SVT)-induced apoptosis. Methods : Inflammation was induced in human HaCaT kerationocytes, by lipopolysaccharide (LPS; $1{\mu}g/mL$) or tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), followed by treatment with SVT (0, 1, or $2{\mu}g/mL$). Cell viability was assessed by MTT assays after 24 h, and the expression of levels of IL-4, STAT6, and the apoptosis-related proteins p53, Bax, and Bcl-2 were evaluated by western blotting. Electro mobility shift assays (EMSAs) were performed to evaluate the DNA binding capacity of STAT6. Results : MTT assays showed that inflammation-induced growth of HaCaT cells following LPS or TNF-${\alpha}$ stimulation was inhibited by SVT. Western blot analysis showed that p53 and Bax, which promote apoptosis, were increased, whereas that of Bcl-2, an anti-apoptotic protein, was decreased in a concentration-dependent manner in LPS- or TNF-${\alpha}$-induced HaCaT cells following treatment with SVT. Moreover, following treatment of HaCaT cells with LPS, IL-4 concentrations were increased, and treatment with SVT further increased IL-4 expression in a concentration-dependent manner. Western blotting and EMSAs showed that the phosphorylated form of STAT6 was increased in HaCaT cells in the context of LPS- or TNF-${\alpha}$-induced inflammation in a concentration-dependent manner, concomitant with an increase in the DNA binding activity of STAT6. Conclusion : SVT can effectively promote apoptosis in HaCaT cells in the presence of inflammation through a pathway involving IL-4 and STAT6.

• Microbiology and Biotechnology Letters
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• v.47 no.1
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• pp.43-53
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• 2019

The anticancer activity of guava (Psidium guajava L.) leaf extract (GLE) occurs via the induction of apoptosis in cancer cells. However, the mechanism behind GLE-induced apoptosis in the human hepatocellular carcinoma cell line HepG2 remains unclear. In the present study, we investigated the apoptotic effects and mechanism of action of GLE in cultured HepG2 cells. The results showed that GLE induced reactive oxygen species (ROS) synthesis and disrupted the mitochondrial membrane potential (${\Delta}{\Psi}m$). Moreover, GLE increased the expression of apoptotic pathway proteins, such as the cleaved forms of caspase-3, -8, and -9; the translocation of Bax and cytochrome c (cyt-c) from the mitochondria to the cytosol; and the downregulation of Bcl-2. In addition, p53 protein expression was increased upon GLE treatment. These observations indicate that the GLE-induced apoptosis in HepG2 cells is mediated by mitochondrial ROS generation, followed by caspase activation and cyt-c release, suggesting that GLE may be a promising candidate for the development of novel drugs for the treatment of liver cancers.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.1947-1959
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• 2016

Background: Colorectal cancer (CRC) is a major cause of morbidity and mortality, being the second most common type of cancer worldwide in both men and women. It accounts yearly for approximately 9% of all new cases of cancers. Furthermore, the current chemotherapeutic regimens seem unsatisfactory, so that exploration of novel therapeutic modalities is needed. The present study was undertaken to investigate the inhibitory effects of a crude alkaloid extract (CAERS) of a medicinal herb, Rhazya stricta, on proliferation of CRC HCT116 cells and to elucidate mechanisms of action. To achieve these aims, we utilized MTT, comet, DNA laddering and gene reporter assays, along with Western blot and RT-PCR analyses. Results: We found that CAERS inhibited cell proliferation and induced apoptotic cell death in HCT116 cells. Hallmarks of morphological and biochemical signs of apoptosis were clearly evident. CAERS down-regulated DNA-binding and transcriptional activities of NF-${\kappa}B$ and AP-1 proteins, while up-regulating expression of the Nrf-2 protein. It also down-regulated expression levels of the ERK MAPK, Bcl-2, cyclin D1, CDK-4, survivin and VEGF and up-regulated levels of Bax, caspase-3/7 and -9, p53, p21, Nrf-2. Markedly, it promoted mRNA expression levels of cytoprotective genes including the hemeoxygenase-1, NAD(P)H quinine oxidoreductase 1 and UDP-glucuronyltransferase. Conclusions: These findings indicate that CAERS exerts antiproliferative action on CRC cells through induction of apoptotic mechanisms, and suggest CAERS could be a promising agent for studying and developing novel chemotherapeutic agents aimed at novel molecular targets for the treatment of CRC.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.8
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• pp.4685-4688
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• 2013

We aimed to investigate the mechanism and effects of autophagy on cisplatin (DDP)-induced apoptosis in human gastric cancer cell line SGC7901. After SGC7901 cells were treated with DDP and/or chloroquine, cell proliferation was measured using MTT assay; cell apoptosis was determined by flow cytometry; autophagy and apotosis-related proteins expression were detected by Western blot; and quantitative analysis of autophagy after monodansylcadaverine (MDC) staining was performed using fluorescence microscopy. We found after treatment with 5 mg/L DDP for 24 h, the rates of cell apoptosis were ($21.07{\pm}2.12$)%. Autophagy, characterized by an increase in the number of autophagic vesicles and the level of LC3-II protein was observed in cells treated with DDP. After inhibition of autophagy by chloroquine, the rates of cell apoptosis were increased to ($30.16{\pm}3.54$)%, and the level of Caspase-3 and P53 protein were increased, and Bcl-2 protein was decreased. Therefore, autophagy protects human gastric cancer cell line SGC7901 against DDP-induced apoptosis, inhibition of autophagy can promote apoptosis, and combination therapy with DDP and chloroquine may be a promising therapeutic strategy for gastric cancer.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6605-6611
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• 2013

Background: WEE1 is a G2/M checkpoint regulator protein. Various studies have indicated that WEE1 could be a good target for cancer therapy. The main aim of this study was to asssess the tumor suppressive potential of WEE1 silencing in two different breast cancer cell lines, MCF7 which carries the wild-type p53 and MDA-MB468 which contains a mutant type. Materials and Methods: After WEE1 knockdown with specific shRNAs downstream effects on cell viability and cell cycle progression were determined using MTT and flow cytometry analyses, respectively. Real-time PCR and Western blotting were conducted to assess the effect of WEE1 inhibition on the expression of apoptotic (p53) and anti-apoptotic (Bcl2) factors and also a growth marker (VEGF). Results: The results showed that WEE1 inhibition could cause a significant decrease in the viability of both MCF7 and MDA-MB-468 breast cancer cell lines by more than 50%. Interestingly, DNA content assays showed a significant increase in apoptotic cells following WEE1 silencing. WEE1 inhibition also induced upregulation of the apoptotic marker, p53, in breast cancer cells. A significant decrease in the expression of VEGF and Bcl-2 was observed following WEE1 inhibition in both cell lines. Conclusions: In concordance with previous studies, our data showed that WEE1 inhibition could induce G2 arrest abrogation and consequent cell death in breast cancer cells. Moreover, in this study, the observed interactions between the pro- and anti-apoptotic proteins and decrease in the angiogenesis marker expression confirm the susceptibility to apoptosis and validate the tumor suppressive effect of WEE1 inhibition in breast cancer cells. Interestingly, the levels of the sensitivity to WEE1 silencing in breast cancer cells, MCF7 and MDA-MB468, seem to be in concordance with the level of p53 expression.

• Journal of Microbiology and Biotechnology
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• v.28 no.6
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• pp.1007-1021
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• 2018

Cancer represents one of the most significant threats to human health on a global scale. Hence, the development of effective cancer prevention strategies, as well as the discovery of novel therapeutic agents against cancer, is urgently required. In light of this challenge, this research aimed to evaluate the effects of several potent bioactive peptides and proteins contained in crocodile white blood cell extract (cWBC) against LU-1, LNCaP, PC-3, MCF-7, and CaCo-2 cancer cell lines. The results demonstrate that 25, 50, 100, and $200{\mu}g/ml$ cWBC exhibits a strong cytotoxic effect against all investigated cell lines ($IC_{50}$ $70.34-101.0{\mu}g/ml$), while showing no signs of cytotoxicity towards noncancerous Vero and HaCaT cells. Specifically, cWBC treatment caused a significant reduction in the cancerous cells' colony forming ability. A remarkable suppression of cancerous cell migration was observed after treatment with cWBC, indicating potent antimetastatic properties. The mechanism involved in the cancer cell cytotoxicity of cWBC may be related to apoptosis induction, as evidenced by typical apoptotic morphology features. Moreover, certain cWBC concentrations induced significant overproduction of ROS and significantly inhibited the $S-G_2/M$ transition in the cancer cell. The molecular mechanisms of cWBC in apoptosis induction were to decrease Bcl-2 and XIAP expression levels and increase the expression levels of caspase-3, caspase-8, and p53. These led to a decrease in the expression level of the cell cycle-associated gene cyclin-B1 and the arrest of cell population growth. Consequently, these findings demonstrate the prospect of the use of cWBC for cancer therapy.

• KSBB Journal
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• v.28 no.5
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• pp.303-309
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• 2013

The extract from Lysimachia foenum-graecum (LFE) has been known to possess various instructive characters including anti-oxidant, anti-obesity, fungicidal activities. However, the accurate mechanism of those effects of LFE is not well known. In that respect, we evaluated the apoptotic effect and anti-cancer efficacy of extracts of LFE in MCF-7 breast cancer cells. In this study, we hypothesized that LFE may exert cancer cell apoptosis through regulating p53 and mitochondria-mediated apoptotic proteins. And this substance can generate ROS to cause free radical-induced apoptosis. Accordingly, the generation of ROS by LFE triggers the activation of p53 which are accompanied by pro-apoptotic protein activation and suppression of pro-survival proteins. We determined with MTT assay, flow cytometry for detection of intracellular ROS and Annexin V-PI staining, Western blotting. Consequently, our researches demonstrated that the treatment of LFE to breast cancer cells resulted in an activation of p53, Puma, Bax, cleaved-PARP and an inhibition of Bcl-2 expressions.