• Biomolecules & Therapeutics
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• 제23권6호
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• pp.582-589
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• 2015

Several theories emphasize that aging is closely related to oxidative stress and disease. The formation of excess ROS can lead to DNA damage and the acceleration of aging. Vigna angularis is one of the important medicinal plants in Korea. We isolated vitexin from V. angularis and elucidated the lifespan-extending effect of vitexin using the Caenorhabditis elegans model system. Vitexin showed potent lifespan extensive activity and it elevated the survival rates of nematodes against the stressful environments including heat and oxidative conditions. In addition, our results showed that vitexin was able to elevate antioxidant enzyme activities of worms and reduce intracellular ROS accumulation in a dose-dependent manner. These studies demonstrated that the increased stress tolerance of vitexin-mediated nematode could be attributed to increased expressions of stress resistance proteins such as superoxide dismutase (SOD-3) and heat shock protein (HSP-16.2). In this work, we also studied whether vitexin-mediated longevity activity was associated with aging-related factors such as progeny, food intake, growth and movement. The data revealed that these factors were not affected by vitexin treatment except movement. Vitexin treatment improved the body movement of aged nematode, suggesting vitexin affects healthspan as well as lifespan of nematode. These results suggest that vitexin might be a probable candidate which could extend the human lifespan.

• Biomolecules & Therapeutics
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• 제23권6호
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• pp.517-524
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• 2015

Human mesenchymal stem cells (MSCs) have been used in cell-based therapy to promote revascularization after peripheral or myocardial ischemia. High levels of reactive oxygen species (ROS) are involved in the senescence and apoptosis of MSCs, causing defective neovascularization. Here, we examined the effect of the natural antioxidant lycopene on oxidative stress-induced apoptosis in MSCs. Although $H_2O_2$ ($200{\mu}M$) increased intracellular ROS levels in human MSCs, lycopene ($10{\mu}M$) pretreatment suppressed $H_2O_2$-induced ROS generation and increased survival. $H_2O_2$-induced ROS increased the levels of phosphorylated p38 mitogen activated protein kinase (MAPK), Jun-N-terminal kinase (JNK), ataxia telangiectasia mutated (ATM), and p53, which were inhibited by lycopene pretreatment. Furthermore, lycopene pretreatment decreased the expression of cleaved poly (ADP ribose) polymerase-1 (PARP-1) and caspase-3 and increased the expression of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax), which were induced by $H_2O_2$ treatment. Moreover, lycopene significantly increased manganese superoxide dismutase (MnSOD) expression and decreased cellular ROS levels via the PI3K-Akt pathway. Our findings show that lycopene pretreatment prevents ischemic injury by suppressing apoptosis-associated signal pathway and enhancing anti-oxidant protein, suggesting that lycopene could be developed as a beneficial broad-spectrum agent for the successful MSC transplantation in ischemic diseases.

• 대한약침학회지
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• 제19권2호
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• pp.129-136
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• 2016

Objectives: The crude extracts of Scutellaria barbata D. Don (SB) have traditionally demonstrated inhibitory effects on numerous human cancers both in vitro and in vivo. Gastric cancer is one of the most common types of cancer on world. The authors investigated the effects of an ethanol extract of Scutellaria barbata D. Don (ESB) on the growth and survival of MKN-45 cells (a human gastric adenocarcinoma cell line). Methods: The MKN-45 cells were treated with different concentrations of ESB, and cell death was examined using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Analyses of sub-G1 peaks, caspase-3 and -9 activities, and mitochondrial membrane depolarizations were conducted to determine the anti-cancer effects of SB on MKN-45 cells. Also, intracellular reactive oxygen species (ROS) generation was investigated. Results: ESB inhibited the growth of MKN-45 cells, caused cell cycle arrest, and increased the sub-G1 population. In addition, ESB markedly increased mitochondrial membrane depolarization and the activities of caspase-3 and -9. ESB exerted anti-proliferative effects on MKN-45 cells by modulating the mitogen-activated protein kinase (MAPK) signaling pathway and by increasing the generation of ROS. Furthermore, combinations of anti-cancer drugs plus ESB suppressed cell growth more than treatments with an agent or ESB, and this was especially true for cisplatin, etoposide, and doxorubicin. Conclusion: ESB has a dose-dependent cytotoxic effect on MKN-45 cells and this is closely associated with the induction of apoptosis. ESB-induced apoptosis is mediated by mitochondria-, caspase- and MAPK dependent pathways. In addition, ESB enhances ROS generation and increases the chemosensitivity of MKN-45 cells. These results suggest that treatment with ESB can inhibit the proliferation and promote the apoptosis of human gastric adenocarcinoma cells by modulating the caspase-, MAPK- and ROS-dependent pathway.

• Journal of Korean Neurosurgical Society
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• 제42권5호
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• pp.392-399
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• 2007

Objective : Arsenic trioxide ($As_2O_3$) has been used as an anticancer agent in traditional Chinese medicine for thousand years and berberine is an isoquinoline alkaloid present that has indicated significant antimicrobial activity. We have examined the combined anticancer effects of $As_2O_3$ and berberine against the human neuroblastoma (HNB) SH-SY5Y cells in vitro, and to elucidate underlying molecular mechanism. Methods : HNB SH-SY5Y cells were treated with $2\;{\mu}M\;As_2O_3$ and $75\;{\mu}g/ml$ berberine, and their survival, cell death mechanism as well as synergistic cytotoxic effects were estimated by using MTT assay, DAPI staining, agarose gel electrophoresis, flow cytometric analysis, and western blot analysis. Results : The combined treatment of two drugs also markedly decreased cell viability. The cytotoxic effects of two drugs were revealed as apoptosis characterized by chromatin condensation, DNA fragmentation, and the loss of mitochondrial membrane potential. The apoptotic cytotoxicity was accompanied by activation of caspase-3 protease as well as decreased the expression of Bcl-2, Bid, and Bcl-x/L. In addition, the cells treated with combination of two drugs also showed significantly increased intracellular reactive oxygen species levels and lipid peroxidation compared to cells $As_2O_3$or berberine only. Conclusion : Combined treatment of $As_2O_3$ with berberine induced activation of apoptotic signaling pathways in HNB SH-SY5Y cells. These results suggest that the possibility of the combined treatment of two chemotherapeutic agents with low concentration improving cytotoxic effect for cancer cells with minimal side effects.

• Asian Pacific Journal of Cancer Prevention
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• 제17권2호
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• pp.581-590
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• 2016

Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.

• Asian Pacific Journal of Cancer Prevention
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• 제15권1호
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• pp.161-166
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• 2014

Lung cancer is the most common causes of cancer-related deaths worldwide, and a lack of effective methods for early diagnosis has greatly impacted the prognosis and survival rates of the affected patients. Tumor-initiating cells (TICs) are considered to be largely responsible for tumor genesis, resistance to tumor therapy, metastasis, and recurrence. In addition to representing a good potential treatment target, TICs can provide clues for the early diagnosis of cancer. MicroRNA (miRNA) alterations are known to be involved in the initiation and progression of human cancer, and the detection of related miRNAs in TICs is an important strategy for lung cancer early diagnosis. As Hsa-miR-155 (miR-155) can be used as a diagnostic marker for non-small cell lung cancer (NSCLC), a smart molecular beacon of miR-155 was designed to image the expression of miR-155 in NSCLC cases. TICs expressing CD133 and CD338 were obtained from A549 cells by applying an immune magnetic bead isolation system, and miR-155 was detected using laser-scanning confocal microscopy. We found that intracellular miR-155 could be successfully detected using smart miR-155 molecular beacons. Expression was higher in TICs than in A549 cells, indicating that miR-155 may play an important role in regulating bio-behavior of TICs. As a non-invasive approach, molecular beacons could be implemented with molecular imaging to diagnose lung cancer at early stages.

• The Korean Journal of Physiology and Pharmacology
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• 제17권1호
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• pp.43-50
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• 2013

Palmitic acid (PAM), one of the most common saturated fatty acid (SFA) in animals and plants, has been shown to induce apoptosis in exocrine pancreatic AR42J cells. In this study, we investigated cellular mechanisms underlying protective effects of oleic acid (OLA) against the lipotoxic actions of PAM in AR42J cells. Exposure of cells to long-chain SFA induced apoptotic cell death determined by MTT cell viability assay and Hoechst staining. Co-treatment of OLA with PAM markedly protected cells against PAM-induced apoptosis. OLA significantly attenuated the PAM-induced increase in the levels of pro-apoptotic Bak protein, cleaved forms of apoptotic proteins (caspase-3, PARP). On the contrary, OLA restored the decreased levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1) in PAM-treated cells. OLA also induced up-regulation of the mRNA expression of Dgat2 and Cpt1 genes which are involved in triacylglycerol (TAG) synthesis and mitochondrial ${\beta}$-oxidation, respectively. Intracellular TAG accumulation was increased by OLA supplementation in accordance with enhanced expression of Dgat2 gene. These results indicate that restoration of anti-apoptotic/pro-apop-totic protein balance from apoptosis toward cell survival is involved in the cytoprotective effects of OLA against PAM-induced apoptosis in pancreatic AR42J cells. In addition, OLA-induced increase in TAG accumulation and up-regulation of Dgat2 and Cpt1 gene expressions may be possibly associated in part with the ability of OLA to protect cells from deleterious actions of PAM.

• International Journal of Oral Biology
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• 제33권2호
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• pp.71-76
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• 2008

The neurotrophin plays an important role in the development, differentiation and survival of the nervous system in vertebrates. It exerts its cellular effects through two different receptors, the Trk receptor tyrosine kinase neurotrophin receptor and the p75 neurotrophin receptor, a member of the tumor necrosis factor receptor superfamily. Trk and p75 neurotrophin receptors utilize specific target proteins to transmit signals into the cell. An ankyrin-rich membrane spanning protein (ARMS) was identified as a new p75 interacting protein and serves as a novel downstream target of p75 neurotrophin receptor. We sought to delineate the interaction between p75 and ARMS by deletion constructs of p75 and green fluorescent protein (GFP)-tagged ARMS. We examined the interaction between these two proteins after overexpressing them in HEK-293 cells. Using both Western blot analysis and immunocytochemistry followed by confocal laser scanning microscopy, we found out that the intracellular domain of the p75 neurotrophin receptor was important for the interaction with ARMS. The results from this study suggest that ARMS may play an important role for mediating the signals from p75 neurotrophin receptor into the cell.

• KSBB Journal
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• 제28권5호
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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.

• BMB Reports
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• 제40권6호
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• pp.928-935
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• 2007

Three Angelica sinensis polysaccharide fractions (APFs), named APF1, APF2 and APF3, were isolated and purified from Radix A. sinensis and their antioxidant activities were evaluated in isolated mouse peritoneal macrophages by pretreatment with APFs before exposure to 0.2 mM tertbutylhydroperoxide (t-BHP). The results showed that pretreatment of the macrophages with APFs as low as $10{\mu}g$/ml could significantly enhance t-BHP-decreased cell survival, intracellular glutathione (GSH) content and superoxide dismutase (SOD) activity, and also inhibited t-BHP-increased lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) formation (p < 0.05), and APF3 was the most active fraction, followed by APF2 and APF1 in decreasing order. Furthermore, we found for the first time that the bound-protein in APF3 was associated closely with the protective effects and the polysaccharide inhibited the excess NO release from t-BHP-activated macrophages to protect host cells.