• Korean Journal of Clinical Laboratory Science
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• v.39 no.2
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• pp.68-75
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• 2007

After reports on regression of cancer in humans and animals infected with microbial pathogens date back more than 100 years, much effort has been spent over the years in developing a wild type or attenuated bacterial and purified bacterial proteins for the treatment of cancer. Pseudomonas aeruginosa exotoxin A (ETA) is known to inhibit cell growth and trigger significant cell death in various cancer cells. Although ETA induces apoptosis of cancer cells, its exact mechanism of action is not known yet. Four different assays were performed in this study: morphological assessment of apoptotic cells, cell cytotoxity, annexin-V binding assay, and cell cycle analysis. The proliferation and survival of the K-562 cells treated with ETA were decreased in a dose dependent manner. In addition, the apoptotic body of K-562 cells was induced by ETA treatment in a dose dependent manner. The ETA-induced apoptosis was confirmed by annexin-V binding assay. Flow cytometric analysis was examined to ascertain whether ETA could arrest the cell cycle at the sub-G1 phase. Our results suggest that P. aeruginosa ETA inhibits cell growth and induces apoptosis in human leukemia K-562 cells.

• Journal of Life Science
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• v.33 no.10
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• pp.767-775
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• 2023

Sulfasalazine is a disease-modifying antirheumatic abiotic agent. It is a derivative of aminosalicylic acid and has been used for the treatment of various inflammatory diseases, such as rheumatoid arthritis, ulcerative colitis, and Crohn's disease, since it was first synthesized in 1941 and approved as a medicine in the United States in 1950. However, its mechanism of action has not yet been clearly identified. In this study, the effects of sulfasalazine on cell survival, apoptosis, and cell cycle progression in macrophages, which are major immune cells that regulate inflammatory responses, were investigated using mouse macrophage RAW 264.7 cells. Sulfasalazine inhibited the viability of RAW 264.7 cells in a dose-dependent manner, starting at a concentration of 0.25 mM. Annexin-V staining was used to confirm that the decrease in cell viability was due to apoptosis, and the number of Annexin-V-positive cells increased significantly at a concentration of 0.25 mM or higher. The effect of sulfasalazine on the expression of key proteins that regulate the G0/G1 phase of the cell cycle was also investigated. Sulfasalazine treatment significantly increased the expression of the cyclin-dependent kinase inhibitors p21 and p27 in RAW 264.7 cells. Although sulfasalazine is frequently used as a control drug in studies on inflammatory diseases, such as inflammatory colitis and rheumatoid arthritis, studies on its effect on macrophages are very limited. Therefore, the results of this study are expected to provide vital information on the use of sulfasalazine as a disease treatment.

• Biomedical Science Letters
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• v.26 no.4
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• pp.249-255
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• 2020

Radiation therapy is one of the primary options for the treatment of malignant tumors. Even though it is an effective anti-cancer treatment, it can cause serious complications owing to radiation-induced damage to the normal tissue around the tumor. It was recently reported that normal stem cell response to the genotoxic stress of ionizing radiation can boost the therapeutic effectiveness of radiation by repairing damaged cells. Therefore, we focused on annexin A-1 (ANXA1), one of the genes induced by low-dose irradiation, and assessed whether it can protect adipose-derived stem cells (ADSCs) against oxidative stress-induced damage caused by low-dose irradiation and improve effectively cell survival. After confirming ANXA1 expression in ADSCs transfected with an ANXA1 expression vector, exposure to hydrogen peroxide (H2O2) was used to mimic cellular damage induced by a chronic oxidative environment to assess cell survival under oxidative conditions. ANXA1-transfected ADSCs demonstrated that increased viability compared with un-transfected cells and exhibited enhanced anti-oxidative properties. Taken together, these results suggest that ANXA1 could be used as a potential therapeutic target to improve the survival of stem cells after low-dose radiation treatment.

• International Journal of Oral Biology
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• v.32 no.2
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• pp.45-49
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• 2007

In this study, the cytotoxicity of commonly used local anesthetics was evaluated on odontoblasts which are essential for pulpal homeostasis in vitro. Local anesthetics, such as articaine, bupivacaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, and procaine, were tested on the odontoblast cell line, MDPC-23. The concentration-and time-dependent cytotoxic effects of local anesthetics on odontoblasts were measured by MTT assay. Among local anesthetics treated for 18 h, only bupivacaine significantly showed cell death in a concentration-($LC_{50}=1.2mM$) and time-dependent manner. To confirm cell death induced by bupivacaine, the observation of cell morphology and FACS using Annexin V and propidium iodide (PI) staining were performed. As a result of Annexin V and PI staining, as well as the morphological change, only bupivacaine induced apoptotic cell death on odontoblasts when compared with levobupivacaine and lidocaine. These results suggest that bupivacaine might affect normal pulpal integrity even after uneventful local anesthesia.

• Journal of Veterinary Science
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• v.21 no.2
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• pp.33.1-33.15
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• 2020

Bovine ephemeral fever virus (BEFV) causes bovine ephemeral fever, which can produce considerable economic damage to the cattle industry. However, there is limited experimental evidence regarding the underlying mechanisms of BEFV. Annexin A2 (AnxA2) is a calcium and lipid-conjugated protein that binds phospholipids and the cytoskeleton in a Ca²⁺-dependent manner, and it participates in various cellular functions, including vesicular trafficking, organization of membrane domains, and virus proliferation. The role of the AnxA2 gene during virus infection has not yet been reported. In this study, we observed that AnxA2 gene expression was up-regulated in BHK-21 cells infected with the virus. Additionally, overexpression of the AnxA2 gene promoted the release of mature virus particles, whereas BEFV replication was remarkably inhibited after reducing AnxA2 gene expression by using the small interfering RNA (siRNA). For viral proteins, overexpression of the Matrix (M) gene promotes the release of mature virus particles. Moreover, the AnxA2 protein interaction with the M protein of BEFV was confirmed by GST pull-down and co-immunoprecipitation assays. Experimental results indicate that the C-terminal domain (268-334 aa) of AxnA2 contributes to this interaction. An additional mechanistic study showed that AnxA2 protein interacts with M protein and mediates the localization of the M protein at the plasma membrane. Furthermore, the absence of the AnxA2-V domain could attenuate the effect of AnxA2 on BEFV replication. These findings can contribute to elucidating the regulation of BEFV replication and may have implications for antiviral strategy development.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.4
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• pp.1663-1666
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• 2012

Background and Objective: Protein expression in colon and rectal cancer (CRC) and paired normal tissues was examined by two-dimensional gel electrophoresis (2-DE) to identify differentially expressed proteins. Materials and Methods: Five fresh colorectal cancer and paired adjacent normal tissues were obtained and differentially expressed protein spots were determined using PDQuest software, with identification on the basis of MALDI-TOF mass spectra. Results: Compared with normal colorectal mucosa, protein abnormal expression of 65 spots varying more than 1.5 times were found in 2-DE gels from colorectal cancer samples (P<0.05); forty-two proteins were up-regulated and 23 were down-regulated; twelve protein spots were identified using mass spectrometry, of which 8 were up-regulated, includimng HSPB1and Annexin A4, while 4 were down-regulated, the results being consistent with Western blot findings. Conclusions: Two-dimensional electrophoresis reference maps for CRC tissues and adjacent normal mucosa (NMC) were established and 12 differentially expressed proteins were identified. Up-regulated HSPB1 and Annexin A4 may play many important roles in the pathogenesis of colorectal cancer.

• Nutrition Research and Practice
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• v.10 no.4
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• pp.393-397
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• 2016

BACKGROUND/OBJECFTIVES: Artemisinin, a natural product isolated from Gaeddongssuk (artemisia annua L.) and its main active derivative, dihydroartemisinin (DHA), have long been used as antimalarial drugs. Recent studies reported that artemisinin is efficacious for curing diseases, including cancers, and for improving the immune system. Many researchers have shown the therapeutic effects of artemisinin on tumors such as breast cancer, liver cancer and kidney cancer, but there is still insufficient data regarding glioblastoma (GBM). Glioblastoma accounts for 12-15% of brain cancer, and the median survival is less than a year, despite medical treatments such as surgery, radiation therapy, and chemotherapy. In this study, we investigated the anti-cancer effects of DHA and transferrin against glioblastoma (glioblastoma multiforme, GBM). MATERIALS/METHODS: This study was performed through in vitro experiments using C6 cells. The toxicity dependence of DHA and transferrin (TF) on time and concentration was analyzed by MTT assay and cell cycle assay. Observations of cellular morphology were recorded with an optical microscope and color digital camera. The anti-cancer mechanism of DHA and TF against GBM were studied by flow cytometry with Annexin V and caspase 3/7. RESULTS: MTT assay revealed that TF enhanced the cytotoxicity of DHA against C6 cells. An Annexin V immune-precipitation assay showed that the percentages of apoptosis of cells treated with TF, DHA alone, DHA in combination with TF, and the control group were $7.15{\pm}4.15%$, $34.3{\pm}5.15%$, $66.42{\pm}5.98%$, and $1.2{\pm}0.15%$, respectively. The results of the Annexin V assay were consistent with those of the MTT assay. DHA induced apoptosis in C6 cells through DNA damage, and TF enhanced the effects of DHA. CONCLUSION: The results of this study demonstrated that DHA, the derivative of the active ingredient in Gaeddongssuk, is effective against GBM, apparently via inhibition of cancer cell proliferation by a pharmacological effect. The role of transferrin as an allosteric activator in the GBM therapeutic efficacy of DHA was also confirmed.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.4
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• pp.467-472
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• 2018

Objective: Molecular cloning and bioinformatics analysis of annexin A2 (ANXA2) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. Methods: The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer (Cervus Nippon hortulorum) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). Results: The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus. Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period). Conclusion: ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

• Asia-pacific Journal of Multimedia Services Convergent with Art, Humanities, and Sociology
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• v.7 no.1
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• pp.395-405
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• 2017

To confirm potential anti-cancer activities of ethylacetate (EtOAc) fraction from Orostachys japonicus on the A549 human lung cancer cells, this study examined. As a result of conducting MTS assay for measuring cell viability, the EtOAc fraction inhibited the proliferation of A549 cells in a dose-dependent manner. To investigate whether the inhibiting A549 cell viability was caused by apoptosis, this study analyzed chromatin condensation in A549 cells using DAPI staining. The morphological changes such as the formation of nuclear condensation were formed in a dose-dependent manner. Also, this study performed Annexin V-FITC staining for detecting phosphatidylinositol (PS). As a result of Annexin V-FITC staining to investigate level of early and late apoptosis, the apoptosis level treated with EtOAc fraction was higher than that of control. RT-PCR was performed to study the correlation between G2/M cell cycle arrest and cell cycle control genes. The anti-cancer activity of EtOAc fraction was accompanied by inhibition of CDK1, 4, cyclin B1 and D1 mRNA. This study also examined the expression of various marker proteins: p53, Bax, Bcl-2 and pro-caspase 3. Western blotting revealed that p53 and Bax proteins were up-regulated, and Bcl-2 and pro-caspase 3 proteins down-regulated in a time and dose-dependent manner.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.7
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• pp.2719-2724
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• 2015

Objective: The aim of this study was to investigate the clinical significance of annexin a1 (ANXA1) and provide molecular evidence to support that decreased ANXA1 expression could enhance cancer migration and invasion in pancreatic ductal adenocarcinoma (PDAC). Materials and Methods: Immunohistochemistry of a tissue microarray with 162 surgically resected PDAC specimens was performed to examine the expression of ANXA1. We also investigated the relationship between ANXA1 expression and clinicopathological factors and prognosis of PDAC patients. We further studied the role of ANXA1 in PDAC cell proliferation, migration and invasion by cell proliferation assay, migration assay and matrigel invasion assay with reduced ANXA1 expression by RNAi. Western blotting was used to detect matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression. We also detected MMP-9 enzyme activity by gelatin zymography. Results: Decreased expression of ANXA1 was significantly associated with poor differentiation, lymph node metastasis and advanced TNM stage of PDAC patients (p<0.05). Moreover, decreased expression of ANXA1 was correlated with poor survival (p<0.05). Furthermore, we found that ANXA1 knockdown inhibited cell proliferation, induced G1 phase cell cycle arrest, increased PDAC cell migration and invasion capacity compared with controls. In addition, Western blotting showed that ANXA1 knockdown increased the MMP-9 protein level and decreased TIMP-1 expression. Gelatin zymography showed that MMP-9 enzyme activity was also elevated. Conclusions: Negative ANXA1 expression is a most unfavorable prognostic factor for PDAC patients. ANXA1 knockdown inhibits cell proliferation by inducing G1 phase cell cycle arrest and increases migration and invasion of PDAC cells through up-regulating MMP-9 expression and activity, implying that ANXA1 may serve as a promising prognostic biomarker and therapeutic target for PDAC.