Our previous study demonstrated that Coprisin, a peptide from Copris tripartitus infected with bacterial pathogens, has an antibacterial activity. We assessed in this study whether Coprisin caused cellular toxicity in various mammalian cell lines. Coprisin selectively caused a marked drop of cell viability in Jurkat T cells, U937 cells and AML-2 cells belonging to the human leukemia cells but not in Caki cells and Hela cells. Fragmentation of DNA, a maker of apoptosis, was also confirmed in theleukemia cell lines but not in other cells. The Coprisin-induced apoptosis in leukemia cells was mediated by AIF (apoptosis inducing factor), a caspase -independent pathway.

Oral squamous cell carcinoma (SCC) is one of the leading causes of cancer mortality worldwide. It is generally thought that adjuvant chemotherapy provides modest prolongation of survival in various carcinoma. Docetaxel (Taxotere, TXT) play a significant role in the treatment of various solid tumors of epithelial origin. CsA (immunosuppressive drug) was widely used as adjunct for the treatment of cancer. Thus, it is important to pursue the apoptosis of IHOK and oral SCC induced by TXT combined with CsA related to the pathogenesis of oral SCC. But TXT combined CsA effect on IHOK and oral SCC remains unclear. After cultured IHOK and HN 22 oral squamous cell carcinoma cell line treated by 10 nM TXT and 1 μM, and caspase inhobitor, respectively, apoptosis index, cytochrome c and caspase-3 -8, -9 mRNA expression by RT-PCR, and procaspase-3 protein amount by immunoslot blotting was prepared. The purpose of this study were to examine the TXT-induced apoptosis pathway via caspase activation by CsA enhancement, and to apply these results to an effective therapeutic treatment plan for oral SCC by TXT combined CsA . 10 nM TXT showed about 60%, 55% celluar apoptosis of IHOK and HN 22, cell line, respectively, while CsA alone did not induce apoptosis in IHOK and HN 22 cell line. 1 μM CsA combined with 10 nM TXT increased apoptosis in IHOK and HN 22 cell line through caspase-3 and cytochrome c mRNA expression, while could not effect on caspase-8 and -9. Caspase inhibitor suppressed apoptosis of IHOK and HN 22 cell line induced by a combination of 1 μM CsA and 10 nM TXT. Immnoslot blotting showed procaspase-3 activation by a combination 1 μM CsA and 10 nM TXT, while caspase inhibitor inhibited activation. It suggested that a combination of CsA and TXT might induce increased apoptosis of IHOK and HN 22 oral squamous cell carcinoma cell line through caspase-3 activation. This treatment with a combination of TXT and CsA may be an effective therapeutic strategy for oral squamous cell carcinoma

Cordycepin (3’-deoxyadenosin), a polyadenylation specific inhibitor, is the main functional component in Cordyceps militaris which is one of the top three famous traditional Chinese medicine. It has been shown to possess many pharmacological activities including immunologically stimulating, anti-cancer, anti-bacterial, and anti-virus, anti-infection effects. However, its anti-cancer molecular mechanisms are poorly understood. In this study, the apoptotic effects by cordycepin were investigates in human leukemia cells. Treatment of cordycepin significantly inhibited cells growth in a concentrationdependent manner by inducing apoptosis, as evidenced by morphological change and apoptotic cell death such as formation of apoptotic bodies, DNA fragmentation and increased populations of sub-G1. Induction of apoptosis by cordycepin was associated with modulation of Bcl-2 and inhibitor of apoptosis proteins (IAP) family expression. Cordycepin also increased reactive oxygen species (ROS) generation, activation of casepase-3, caspase-8, caspase-9, cleavage of poly(ADP-ribose) polymerase (PARP), β-catenin and phospholipase C (PLC)-γ1 protein. The quenching of ROS generation by N-acetyl-L-cysteine administration, a scavenger of ROS, reversed the cordycepin-induced apoptosis effects. Theresults suggested that cordycepin may be a potential chemotherapeutic agent for the treatment of leukemia patients [This work was supported by Blue-Bio Industry RIC at Dong-Eui University as a RIC (08-06-07) program of ITEP under Ministry of Knowledge Economy].

COPRISIN is an antibiotic substance extracted from Copris tripartitus. This study is intended to identify various cell biological stimuli that COPRISIN, widely known as an antibacterial substance, has on human cells and to identify its molecule mechanism. A variety of human cell lines were divided into epithelial cells including kidney cells or womb cells, and immunocyte including T cells or macrophages and, after their being cultivated and maintained, cell biological changes of the respective cells according to COPRISIN treatment were compared. As a result, it was confirmed that, different from other experiment cells, COPRISIN specifically caused cell kill in T cells and macrophages. That is, fragmentation of DNA, typical characteristics observed in the process of apoptosis, was confirmed in the nucleus of cells dying owing to COPRISIN treatment. An Apoptosis process is one dependent upon activity of caspase family protein, it was proved that COPRISIN medium cell kill process was one through a caspase-independent route such as AIF. Though it was found out that transcription of TNF-α and extracellular TNF-α secretion increased in blood cells stimulated by COPRISIN, it was also confirmed that TNF-α was a major medium factor in a COPRISIN induced cell kill process from the fact that a cell kill process by COPRISIN was not inhibited at all with TNF-α inhibiting antibody treatment. Above results revealed that COPRISIN, different from other tissue origin cells including kidney cells, can specifically induce apoptosis in immunocyte, which is caused by a caspase-independent cell signal transmission route.

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at in 5% , 5% for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

Cordycepin (3’-deoxyadenosine) is a polyadenylation specific inhibitor, one of the components of Cordyceps militaris. It has been shown to possess many pharmacological activities including immunologically stimulating, anti-tumor, anti-virus, and anti-infection effects. However, its molecular mechanisms are poorly understood. In this study, the apoptotic effects by cordycepin were investigated in human leukemia cells. Cordycepin treatment inhibited leukemia cells growth a concentration-dependent manner by inducing apoptosis,as evidenced by morphological change and apoptotic cell death such as formation of apoptotic bodies, DNA fragmentation and increased populations of apoptoticsub G1 phase. Induction of apoptosis by cordycepin in leukemia cells were associated with modulation of Bcl-2 member and inhibitor of apoptosis (IAP) proteins expression. Cordycepin also increased ROS generation, activation of caspase-3, caspase-8, caspase-9, cleavage of poly(ADP-ribose) polymerase (PARP), -catenin and phospholipase (PLC)-1 protein. Both the this effect by cordycepin treatment were significantly inhibited by NAC, a ROS scavenger, demonstrating the important role of ROS in the observed cytotoxic effect. This results suggested that cordycepin may be a potential chemotherapeutic agent for the treatment of leukemia patients.

Chios gum mastic (CGM) is a resin produced from the stem and leaves of Pistiacia lentiscus L var chia, a plant which grows only on Chios Island in Greece. CGM has been used for many centuries as a dietary supplement and folk medicine for stomach and duodenal ulcers in many Mediterranean countries and is known also to induce cell cycle arrest and apoptosis in some cancer cells. In this study, we further investigated the induction and mechanisms underlying the apoptotic response to CGM treatment in the SCC25 human tongue squamous cell carcinoma cell line. The viability of SCC25 cells, human normal keratinocytes (HaCaT cells) and human gingival fibroblasts (HGF-1 cells), and the growth inhibition of SCC25 cells were assessed by MTT assay and clonogenic assay, respectively. Staining with Hoechst and hemacolor dyes and TUNEL assays were employed to detect SCC25 cells undergoing apoptosis. SCC25 cells were treated with CGM, and this was followed by western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, MMP activity and proteasome activity analyses. CGM treatment of SCC25 cells was found to result in a time- and dosedependent decrease in cell viability, a dose-dependent inhibition of cell growth, and apoptotic cell death. Interestingly, CGM showed a remarkable level of cytotoxicity in SCC25 cells but not in normal cells. Tested SCC25 cells also showed several lines of apoptotic manifestation. Taken together, our present findings demonstrate that CGM strongly inhibits cell proliferation by modulating the expression of G1 cell cycle-related proteins and induces apoptosis via the proteasome, mitochondria and caspase cascades in SCC25 cells.

Artemisia scoparia (A. scoparia), perennial herb is indigenous to Korea and has been traditionally used in liver damage. We investigated the effect of the essential oil obtained from A. scoparia on apoptosis of KB cells. Cytotoxicity and cellular DNA content were analyzed by MTT assay and flow cytometry, agarose gel electrophoresis, and Hoechst 33258 staining. The caspase-3 and poly (ADP-ribose) polymerase (PARP) proteins were estimated by Western blotting method. We found that the essential oil induced the apoptosis of the KB cells by concentrations of 0.4 to 0.2 mg/ml which was verified by DNA fragmentation, apoptotic bodies, and the sub-G0/G1 ratio. The essential oil also transient caspase-9 and caspase-3 activity and cleavage of PARP in KB cells for 24 h. The essential oil-induced apoptotic cell death was accompanied by up-regulation of Bax and down-regulation of Bcl-2. In conclusion, we demonstrated that the essential oil of A. scoparia induces apoptosis in KB cells

Nitric oxide (NO) acts as an intracellular messenger at the physiological level but can be cytotoxic at high concentrations. The cells within periodontal tissues, such as gingival and periodontal fibroblasts, contain nitric oxide syntheses and produce high concentrations of NO when exposed to bacterial lipopolysaccharides and cytokines. However, the cellular mechanisms underlying NO-induced cytotoxicity in periodontal tissues are unclear at present. In our current study, we examined the NO-induced cytotoxic mechanisms in human gingival fibroblasts (HGF). Cell viability and the levels of reactive oxygen species (ROS) were determined using a MTT assay and a fluorescent spectrometer, respectively. The morphological changes in the cells were examined by Diff-Quick staining. Expression of the Bcl-2 family and Fas was determined by RT-PCR or western blotting. The activity of caspase-3, -8 and -9 was assessed using a spectrophotometer. Sodium nitroprusside (SNP), a NO donor, decreased the cell viability of the HGF cells in a dose- and time-dependent manner. SNP enhanced the production of ROS, which was ameliorated by NAC, a free radical scavenger. ODQ, a soluble guanylate cyclase inhibitor, did not block the SNP-induced decrease in cell viability. SNP also caused apoptotic morphological changes, including cell shrinkage, chromatin condensation, and DNA fragmentation. The expression of Bax, a member of the proapoptotic Bcl-2 family, was upregulated in the SNP-treated HGF cells, whereas the expression of Bcl-2, a member of the anti-apoptotic Bcl-2 family, was downregulated. SNP augmented the release of cytochrome c from the mitochondria into the cytosol and enhanced the activity of caspase-8, -9, and -3. SNP also upregulated Fas, a component of the death receptor assembly. These results suggest that NO induces apoptosis in human gingival fibroblast via ROS and the Bcl-2 family through both mitochondrial- and death receptor-mediated pathways. Our data also indicate that the cyclic GMP pathway is not involved in NO-induced apoptosis.

당뇨망막병증은 서구에서 성인들의 실명을 일으키는 원인이다. 당뇨병이 있을 때 고혈당증은 여러 세포 형태에서 세포자연사를 유도하지만 그 기작은 명확하게 밝혀지지 않았다. 본 연구의 목적은 인간망막 내피세포에서 고혈당 포도당이 세포자연사에 미치는 영향에 대하여 알아보았다. 망막 내피세포는 5, 25, 50 mM 포도당이 포함된 IMDM배지에서 37℃, 5% CO2조정된 항온기에서 24, 36, 48시간 동안 배양하였다. 여러 농도의

본 연구는 한우의 난포낭종 및 황체낭종에서 세포자멸사 관련 유전자의 발현 변화를 조사함으로써 난소낭종과 세포자 멸사간의 관계를 정리하고자 한다. 마이크로어레이 분석 결과, 난포낭종에서 PIK3R2와 AKT1가 유의하게 증가하였고, 황체낭종에서는 증가되는 세포자멸사 관련 DEGs, TNF-RAF2, PRLR, FOXL2, STK4 및 COL4A3와 감소하는 DEGs, INHA, CIDEB, BCL10 및 FASLG가 포함되었다. 정량적 역전사 중합 효소