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

Taxol(paclitaxel) is used in chemotherapy against several cancer. Treatment of tumor cell lines with taxol induces apoptosis, but exact mechanism is not sufficiently understood. The purpose of this study was to investigate apoptosis by the inhibitory effect of paclitaxel on the motility properties of human salivary gland adenocarcinoma cell lines. Paclitaxel inhibited cell motility induced by soluble and immobilized attractant. It suggested that paclitaxel would be a potent inhibitor of salivary gland adenocarcinoma cell motility independent of its cytotoxic and apoptotic activity.

This study investigated the developmental ability and gene expression of somatic cell nuclear transfer embryos using ear skin fibroblast cells derived from miniature pig. When miniature pig (m) and landrace pig (p) were used as donor cells, there were no differences in cleavage (79.2 vs. 78.2%) and blastocyst rates (27.4 vs. 29.7%). However, mNT blastocysts showed significantly higher apoptosis rate than that of pNT blastocysts (6.1 vs. 1.7%) (p<0.05). The number of nuclei in pNT blastosysts was significantly higher than that of mNT (35.8 vs. 29.3) (p<0.05). Blastocysts were analyzed using Realtime RT-PCR to determine the expression of Bax-, Bcl-xl, H19, IGF2, IGF2r and Xist. Bax- was higher in mNT blastocyst than pNT blastocyst (p<0.05). There was no difference in Bcl-xl between two NT groups. Bax-/Bcl-xl was, however, significantly higher in mNT blastocyst compared to pNT. The expression of imprinting genes were aberrant in blastocysts derived from NT compared to in vivo blastocysts. H19 and IGF2r were significantly lower in mNT blastocysts (p<0.05). The expression of IGF2 and Xist was similar in two NT groups. However, imprinting genes were expressed aberrantly in mNT compared to pNT blastocysts. The present results suggest that the NT between donor cells derived from miniature pig and recipient oocytes derived from crossbred pig might affect reprogramming of donor cell, resulting in high apoptosis and aberrant expression patterns of imprinting genes.

Cordyceps militarisis well known as a traditional herbal ingredient, which has been used for patients suffering from cancer in oriental medicine. In this study we have investigated the biochemical mechanisms of anti-proliferative effects by C. militarisextract(CME) in human breast cancer MDA-MB-231 cells. It was found that CME treatment induced chromatin condensation, mitochondrial energization, annexin V staining and sub-G1 phase DNAcontent. These indicators of apoptosis correlate with the mitochondrial dependent pathway, which results in the activation of caspase-3 activity. Both the cytotoxic effect by CME treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor,demonstrating the important role of caspase-3 in the observed cytotoxic effect. Cotreatment of CME and LY294002, resulted in significantly induction of apoptosis. These results indicate that caspase-3 is a key regulator of apoptosis in response to CME in human breast cancer MDAMB- 231 through downregulation of Akt, and that the C. militaris extract may therefore have therapeutic potential against human breast cancer.

Agaricus blazei is well known as a traditional medicinal mushroom and it has been shown to exhibit immunostimulatory and anti-cancer activity. However, the cellular and molecular mechanism of apoptosis of cancer cells is poorly understood. In this study, we have investigated whether A. blazei extract (ABE) exerts anti-proliferative and apoptotic effects on human leukemia THP-1 cells. It was found that ABE induced a time- and dose-dependent increase in leukemia cells apoptosis through caspase-3 activation and PARP cleavage. Activation of caspase- 9 induced by ABE suggested that ABE-induced signaling was mediated through a mitochondrial death pathway. In addition, we observed an elevation of ROS and a consequent loss of mitochondrial membrane potential, further suggesting that ABE-induced death signaling was mediated through a mitochondrial oxygen stress pathway. The antioxidant Nacetylcysteine, however, opposed ABE-mediated mitochondrial dysfunction, caspase activation, and apoptosis, supporting the role of ROS in the apoptotic process. We conclude that ABE induces apoptosisin human leukemia cells through a reactive oxygen species and caspase-dependent mitochondrial pathway.

In this study, the apoptotic effects of the actin disruption agent, latrunculin B(LB) have been investigated on p53 deficient chronic myeloid leukemia cell line K562. A dose-dependent decrease in K562 cell proliferation was observed after LB treatment with maximum decrease in cell proliferation being at 1.5μM where the percent inhibition was 66.53%. F-actin stained with TRITC-phalloidin was shown as a peripheral ring or appeared diffusely distributed throughout the cytoplasm in untreated cells, this actin ring was decreased following LB treatment, and even large focal actin aggregates were formed. Treatment of K562 with LB(1.5μM) generated ROS substantially. LB activated expression in a dose-dependent manner. Therefore it can be concluded that LB, depolymerising agent of actin, induces apoptosis by producing ROS and up-regulating NF-kB and COX-2 activation.