Programmed cell death or apoptosis is associated with changes in K+ concentration in many cell types. Recent studies have demonstrated that two-pore domain K+ (K2P) channels are involved in mouse embryonic development and apoptotic volume decrease of mammalian cells. In cerebellar granule neurons that normally undergo apoptosis during the early developmental stage, TASK-1 and TASK-3, members of K2P channels, were found to be critical for cell death. This study was performed to identify the role of K+ channels in the H2O2-induced or cryo-induced cell death of mouse and bovine embryos. Mouse and bovine two-cell stage embryos (2-cells) exposed to H2O2 for 4 h suffered from apoptosis. The 2-cells showed positive TUNEL staining. Treatment with high concentration of KCl (25mM) inhibited H2O2-induced apoptosis of 2-cells by 19%. Cryo-induced death in bovine blastocysts showed positive TUNEL staining only in the cells near the plasma membrane. Cryoprotectant supplemented with 25 mM KCl reduced apoptosis slightly compared to cryoprotectant supplemented with 5 mM KCl. However, the combination of antioxidants (β-mercaptoethanol) with 25 mM KCl significantly decreased the rate of H2O2-induced and cryo-induced apoptosis compared to treatments with only antioxidants or 25 mM KCl. These results show that blockage of K+ channel efflux for a short-time reduces H2O2- and cryo-induced apoptosis in mouse and bovine embryos. Our findings suggest that apoptosis in mouse and bovine embryos might be controlled by modulation of K+ channels which are highly expressed in a given cell type.

Epimedium koreanum Nakai has been used in traditional medicine as an aphrodisiac, hypotensive, and neurasthenia;however, the cellular mechanism of E. koreanum Nakai cultivated in North Korea on HCT116 colon cancer cellactivity has not been investigated. This study is to investigate the effect of E. koreanum Nakai on apoptosis of thehuman colon cancer cell line HCT116. The anti-proliferative activity of E. koreanum Nakai on HCT116 was iden-tified through MTT, Western Blot, and RT-PCR analyses. The results show that 70% ethyl alcohol extracts of E.koreanum Nakai inhibited the growth of HCT116 colon cancer cells (IC50: 1.2mg/mL on 24 hr). Concomitant acti-vation of the mitochondria-dependent apoptotic pathway occurred via modulation of Bcl-2 and Bax expressions,resulting in activation of cleaved caspase-3 and cleaved caspase-9.

Several studies have shown that curcumin, which is derived from the rhizomes of turmeric, possesses antimicrobial, antioxidant and anti-inflammatory properties. The antitumor properties of curcumin have also now been demonstrated more recently in different cancers. This study was undertaken to investigate the modulation of cell cycle-related proteins and the mechanisms underlying apoptosis induction by curcumin in the SCC25 human tongue squamous cell carcinoma cell line. Curcumin treatment of the SCC25 cells resulted in a time- and dose-dependent reduction in cell viability and cell growth, and onset of apoptotic cell death. The curcumin-treated SCC25 cells showed several types of apoptotic manifestations, such as nuclear condensation, DNA fragmentation, reduced MMP and proteasome activity, and a decreased DNA content. In addition, the treated SCC25 cells showed a release of cytochrome c into the cytosol, translocation of AIF and DFF40/CAD into the nuclei, a significant shift in the Bax/Bcl-2 ratio, and the activation of caspase-9, caspase-7, caspase-6, caspase-3, PARP, lamin A/C, and DFF45/ICAD. Furthermore, curcumin exposure resulted in a downregulation of G1 cell cycle-related proteins and upregulation of p27KIP1. Taken together, our findings demonstrate that curcumin strongly inhibits cell proliferation by modulating the expression of G1 cell cycle-related proteins and inducing apoptosis via proteasomal, mitochondrial, and caspase cascades in SCC25 cells.

Dibenzylideneacetone (DBA), an analogue of curcumin has been shown to have anti-cancer activity in a variety of tumor cell lines. However, the anti-cancer activity of DBA and its molecular mechanism in HN22 oral cancer cell line have not been fully explored. The effects of DBA on anti-proliferative and apoptotic activity were evaluated by the trypan blue exclusion assay, 4’-6-diamidino-2-phenylindole (DAPI) staining, Western blot analysis, and reverse transcriptase-polymerase chain Reaction (RT-PCR). Our data showed that the treatment of DBA to HN22 cells exerted anti-proliferative and apoptotic activities and the activity was accompanied by a decrease in Sp1 protein, Sp1 mRNA and its promoter activity. DBA also reduced the expression level of Sp1 protein and caused apoptotic cell death in HN22 cells simultaneouly. Phosphorylation of ERK and JNK were regulated by DBA whereas phosphorylation of p38 was not altered. Overall, our results suggest that the regulation of Sp1 activities and ERK/JNK are involved in DBA-induced apoptosis and DBA can be a promising anticancer drug candidate for the treatment of oral cancer.

Cathepsin B is abundantly expressed peptidase of the papain family in the lysosomes, and closely related to the cell degradation system such as apoptosis, necrosis and autophagy. Abnormal degradation of organelles often occurs due to release of cathepsin B into the cytoplasm. Many studies have been reported that relationship between cathepsin B and intracellular mechanisms in various cell types, but porcine embryos has not yet been reported. Therefore, this study evaluated the effect of cathepsin B inhibitor (E-64) on preimplantation developmental competence and quality of porcine embryos focusing on apoptosis and oxidative stress. The expression of cathepsin B mRNA in porcine em-bryos was gradually decreased in inverse proportion to E-64 concentration by using real-time RT-PCR. When putative zygotes were cultured with E-64 for 24 h, the rates of early cleavage and blastocyst development were decreased by increasing E-64 concentration. However, the rate of blastocyst development in 5 μM treated group was similar to the control. On the other hand, both the index of apoptotic and reactive oxygen species (ROS) of blastocysts were sig-nificantly decreased in the 5 μM E-64 treated group compared with control. We also examined the mRNA expression levels of apoptosis related genes in the blastocysts derived from 5 μM E-64 treated and non-treated groups. Expre-ssion of the pro-apoptotic Bax gene was shown to be decreased in the E-64 treated blastocyst group, whereas expre-ssion of the anti-apoptotic Bcl-xL gene was increased. Taken together, these results suggest that proper inhibition of cathepsin B at early development stage embryos improves the quality of blastocysts, which may be related to not only the apoptosis reduction but also the oxidative stress reduction in porcine embryos.

Prostate cancer is a leading cause of death among the aging men. Surgical or radio therapy is effective when the cancer is confined to the prostate gland but once the cancer spreads beyond the pelvis, even chemotherapy and hormonal ablation therapy fails in curing this disease. Our previous studies have shown that β-glucan induces apoptosis in prostate cancer cells. Cellular viability of these cells treated with β-glucan was measured by MTT assay. β-glucan induced dose- and time-dependent inhibition of cell viability in LNCaP cancer cells. In flow cytometry analysis, β-glucan induced dose- and timedependent apoptotic activities in LNCaP cancer cells. In addition, increased of expression caspase-3, caspase-9, Bax, and cytochrome C but decreased of expression Bcl-2 was observed in LNCaP cells treated with β-glucan. These results suggest that β-glucan induces apoptosis in LNCaP human prostate cancer cells mediated mainly through the increased of expression caspase-3, -9, Bax, cytochrome C and decreased of expression Bcl-2.

The present study was performed to investigate the effect of Hh-Ag1.5, a small-molecule chemical agonist of SMOothened receptor, on the in vitro maturation and development of in vitro fertilized (IVF) embryos in pigs. Oocytes or fertilized embryos were cultured in a maturation or embryo culture medium supplemented with 0 (control), 25, 50 or 100 nM of Hh-Ag1.5, respectively. Although the maturation rate were not different among treatment groups, the blastocyst formation rate in the group treated with 25 nM Hh-Ag1.5 was significantly increased compared to other groups (P<0.05). While the highest dose of Hh-Ag1.5 (100 nM) did negatively affect to the embryo development and cell number in blastocysts compared to other groups (P<0.05), the apoptotic cell index in blastocysts was significantly lower in 25 and 50 nM groups than in control and 100 nM groups (P<0.05). The mRNA expression of the proapoptotic gene Bax and the ratio of Bax/Bcl-XL decreased in among treatment groups compared to control (P<0.05). The embryo quality related genes, Tert and Zfp42, were significantly decreased in 50 and 100 nM groups compared with control and 25 nM groups (P<0.05). In conclusion, the addition of 25 nM Hh-Ag1.5 to in vitro maturation and culture medium can enhance the developmental potential as well as quality of IVF embryos in pig.

The objective of this study was to examine the effects of high concentrations of glucose on porcine parthenotes developing in vitro. Addition of 55 mM glucose to the culture medium of embryos at the four-cell-stage significantly inhibited blastocyst formation, resulting in fewer cells in blastocyst-stage embryos and increased levels of apoptosis and autophagy compared to control. Quantitative reverse transcriptase (RT) PCR analysis revealed that the expression of pro-apoptotic genes (Caspase 3, Bax and Bak) and autophagy genes (Atg6 and Atg8/Lc3) were increased significantly by the addition of 55 mM glucose to the culture medium compared to control. MitoTracker Green fluorescence revealed a decrease in the overall mitochondrial mass compared to control. However, the addition of 55 mM glucose had no effect on mRNA expression of the nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1. These results suggest that hyperglycemia reduced the mitochondrial content of porcine embryos developing in vitro and that this may hinder embryonic development to the blastocyst stage and embryo quality by increasing apoptosis and autophagy in these embryos.

The key regulators of apoptosis are the interacting protein of the Bcl-2 family. Bcl-2, an important member of this family, blocks cytochrome C release by sequestering pro-apoptotic BH3-only proteins such as Bid, Bad, Bax and Bim. The pro-survival family members (Bcl-2, Bcl-XL, Bcl-W) are critical for cell survival, since loss of any of them causes cell death in certain cell type. However, its role during early porcine embryonic development is not sufficient. In this study, we traced the effects of Bcl-2 inhibitor, ABT-737, on early porcine embryonic development. We also investigated several indicators of developmental potential, including gene expression (apoptosis-related genes) and apoptosis, which are affected by ABT-737. Porcine embryos were cultured in the PZM-3 medium with or without ABT-737 for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without ABT-737 (14.7±3.0 vs 30.3±4.8%, p<0.05). TUNEL assay showed that the number of containing fragmented DNA at the blastocyst stage increased in the ABT-737 treated group compared with control (4.7 vs 3.7, p<0.05). The mRNA expression of the pro-apoptotic gene Bax increased in ABT-737 treated group (p<0.05), whereas expressions of the anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL, Bcl-W) decreased (p<0.05). Also, expressions of the ER stress indicator genes (GRP78, XBP-1 and sXBP-1) increased in ABT-737 treated group (p<0.05). In conclusion, Bcl-2 is closely associated with of apoptosis- and ER stress-related genes expressions and developmental potential in pig embryos.

Calcineurin-binding protein 1 (Cabin1) regulates calcineurin phosphatase activity as well as the activation, apoptosis, and inflammatory responses of fibroblast-like synoviocytes (FLSs), which actively participate in the chronic inflammatory responses in rheumatoid arthritis (RA). However, the mechanism of action of Cabin1 in FLS apoptosis is not clear. The aim of this study was to define the regulatory role of Cabin1 in FLSs of mice with collagen-induced arthritis (CIA). Transgenic mice overexpressing human Cabin1 in joint tissues, under the control of a type II collagen promoter, were generated. hCabin1 expression in joints and FLSs was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of cytokines, matrix metalloproteinases (MMPs), and apoptosis-related genes in FLSs was determined by enzyme- linked immunosorbent assay, gelatin zymography, and RT-PCR, respectively. Joints were histologically examined after H&E and TRAP staining. hCabin1-transgenic CIA mice had less severe arthritis than wild-type CIA mice, based on hind paw thickness and histology. This was accompanied by significantly enhanced apoptosis in transgenic mice, evidenced by significantly more TUNEL-positive cells in synovial tissues. The expression of inflammatory cytokines and MMPs was reduced, and the transgenic CIA mice exhibited decreased Akt activation and increased expression of p53, caspase-3, caspase-9, and Bax. hCabin1 plays a critical role in promoting apoptosis of FLSs and in attenuating inflammation and the destruction of cartilage and bone in RA. These findings help elucidate the pathogenic mechanisms of RA and suggest that Cabin1 is a potential target for RA treatment.

Doxorubicin, a widely used chemotherapeutic agent, were found rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis of oocyte, and a little known is actions of this drug in early embryos. Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, also plays the important role during the apoptosis of cell. The cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Cleaved PARP (cPARP) can be a marker of apoptosis.Doxorubicin inhibited the early embryo development, but the treatment could still reach the BL (blastocyst) stagethat suggested that involved in DNA synthesis and repaired progress. Herein, the higher expression of PARP family shown especially in 2, 4 cell stagy. There was evidence of expression of Caspase3 and Bcl2l1 during embryogenesis (2 cell, 4 cell, morula and BL stage), suggesting that modulationsof apoptosis-related genes and PARP were cause by DXR. Furthermore, the effect of doxorubicin on early embryo development was assessed different stage rates, and apoptosis index also conformed doxorubicin modulate embryo development by regulating apoptosis- related genes and PARP family genes. In conclusion, Doxorubicin blocked pre- implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by Parp.

Although there are a number of reports regarding the toxicity evaluation of inorganic nanoparticles, knowledge on biodegradable nanomaterials, which have always been considered safe, is still limited. For example, the toxicity of chitosan nanoparticles, one of the most widely used drug/gene delivery vehicles, is largely unknown. In this report, we examined the cytotoxic effects of chitosan nanoparticles on mouse embryos at the blastocyst stage and in vivo implantation by embryo transfer. Blastocysts treated with 250 nm chitosan nanoparticles exhibited significantly increased apoptosis and a corresponding decrease in total cell number, which was concentration‐dependent. Moreover, the TUNEL positive signal in the embryos exposed to chitosan nanoparticles showed an increased of the ICM and the implantation success rate was lower than that of their control counterparts. Our results collectively indicate that in vitro exposure to chitosan nanoparticles induces apoptosis and retards implantation development after transfer to host mice. The results collectively show that chitosan nanoparticles have the potential to induce embryo cytotoxicity. Further studies are required to establish effective protection strategies against the cytotoxic effects of these nanoparticles.

Doxorubicin, a widely used chemotherapeutic agent, were found rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis of oocyte. In this report, we elucidated the molecular requirements for actions of this drug in early embryos. Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues have recently been shown in female oocyte cells. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Cleaved PARP (cPARP) may be considered a marker of apoptosis. Doxorubicin inhibited the early embryo development, but the treatment could still reach the BL (blastocyst) stage that suggested that involved in DNA synthesis and repaired progress. Herein, the higher expression of PARP family shown especially in 2, 4 cell stagy. There was evidence of expression of Caspase3 and Bcl2l1 during embryogenesis (2 cell, 4 cell, morula and BL stage), suggesting that modulations of apoptosis-related genes and PARP were cause by DXR. Furthermore, the effect of doxorubicin on early embryo development was assessed different stage rates, and apoptosis index also conformed doxorubicin modulate embryo development by regulating apoptosis-related genes and PARP family genes. In conclusion, Doxorubicin blocked pre-implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by Parp.

The purpose of this thesis is to examine the effect of hormone treatment in blastocyst development of in vitro cultured porcine oocyte. Oocytes used in the study was matured in vitro in the presence of 10% FBS or 10% pFF, and treated with FSH, LH or FSH+ LH, and the rate of blastocyst development was assessed based on the expression of autophagic genes. There was no significant differences in blastocyst development between oocytes maturaed in 10% FBS or 10% pFF. In vitro matured oocytes treated with FSH+LH showed blastocyst development rate as high as that of untreated oocytes, while groups treated with LH only showed a decrease in blastocyst development. About the expression of cell death assosiated factors, mRNA levels of autophagy and apoptosis genes were increased in oocytes matured in 10% FBS and treated with LH. Oocytes that did not receive hormone treatment showed low expression of most cell death genes except ATG5. When oocytes were matured in 10% pFF, ATG5 expression was the highest in FSH treated group, while LC3 showed strong expression in all hormone treated groups. On the other hand, the expression level of mTOR and caspase-3 did not show significant differences between groups. We also examined the protein level of apoptotic genes in the blastocyst. The amount of caspase-3 protein was similar between groups matured in 10% FBS and 10% pFF, but was the highest when treated with LH. Blastocysts treated with FSH and FSH+LH showed similar level of caspase-3 protein, while the level was the lowest when hormone treatment was not given. Within the blastocyst, caspase-3 was mostly expressed in trophoblast cells when matured in 10% FBS, while maturation in 10% pFF caused expression of this protein in the inner cell mass (ICM). Expression of MAP1LC3A was higher in groups matured in 10% pFF than groups matured in 10% FBS in all types of hormone treatment. Among the blastocysts matured in 10% pFF, MAP1LC3A level increased in the order of untreated < FSH < FSH+ LH. Expression of MAP1LC3A within the FBS-matured blastocyst was concentrated to the trophoblast, while pFF-matured blastocyst showed expression in both trophoblast and ICM. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in 10% FBS group without hormone treatment. In both FBS and pFF group, and in all three combinations of hormone treatment, mTOR expression was ovserved mostly in ICM. Together, these results indicated that hormone treatments tend to induce expression of genes associated with programmed cell death. We suggest that proper induction of programmed cell death by FSH and LH treatment would increase the rate of blastocyst development. * This work was supported by BioGreen 21 Program (No. PJ008029). Rural Development Administation, Republic of Korea.