• Development and Reproduction
• /
• v.2 no.1
• /
• pp.1-8
• /
• 1998

In mammalian ovary, major portion(>99%) of ovarian follicles undergo atresia. Recent studies have shown that this phenomenon is mediated via GC apoptosis. Ceramide, a product of sphingomyelin hydrolysis, has been proposed as a novel lipid second messenger with specific roles in mediating antiproliferative responses including apoptosis and cell cycle arrest. In the present study, we have examined the effect of ceramide on apoptotic cell death of GC in vitro. GCs were harvested by squeezing the antral follicles from the immature mice (3-4 weeks) and cultured in MEM medium with 10% fetal bovine serum. The cells were treated with various concentrations of ceramide (0 to 50 \mu M)and cultured up to 24 h.Cell death was determined by MTT cell viability assay and apoptosis was examined by acridine orange staining, in situ 3'-end labeling(TUNEL), and flow cytometry. Ceramid treatment induced apoptotic cell death of GC in a time- and a dose-dependent manner. Results of flow cytometric analysis showed that creamide-induced cell death was mostly confined to the $G_{0}$/$G_{1}$ cells. these results provide an evidence for ceramide as a lipid second messenger of apoptosis in mouse GC.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2002.11a
• /
• pp.117-117
• /
• 2002

The oocytes from most of animal species accumulate genetic information and other necessary materials during oogenesis for the later use in the early development. Over the years oocyte maturation has been studied extensively both in vitro and in vivo. Particularly, maturation of follicular oocyte in vitro becomes one of the important tools for the studies of basic cell biology, the in vitro technology of animal production, and in particular, the somatic cell cloning by nuclear transfer. We examined meiotic maturation and cumulus expansion in the presence of translation or transcription inhibitors for varying periods of in viかo maturation (IVM) of pig oocyte. In Experiment 1, the results revealed that translation and transcription inhibitors inhibited cumulus expansion and meiotic maturation during 35h of IVM. However, 50 to 60% of the oocytes underwent nuclear maturation without cumulus expansion during 75h of IVM. The rest of the oocytes were arrested at metaphase I (40-50%) in the presence of the inhibitors. In Experiment II, the OCCs were exposed to the drugs only for 15h to examine translation and transcription inhibitors on cumulus expansion and meiotic maturation. Transcription inhibitors for 15h did not arrest meiotic maturation when the oocytes were cultured for subsequent, necessary period of IVM, whereas cumulus expansion was completely inhibited, suggesting that initial 15h is critical transcription activity far cumulus expansion. Translation inhibitors for 15h exposure did not alter cumulus expansion and meiotic maturation during subsequent culture in the absence of the drugs. In Experiment III, the OCCs were exposed to the drugs only for later 30h to examine the influence of transcription and translation inhibitors on oocyte maturation. Interestingly, all meiotic maturation underwent normally with full expansion of cumulus. Similar results were obtained from Experiment IV where 5h of exposure from 15 to 20h of IVM culture to the drugs was performed and subsequently cultured for same period in fresh medium. Taken there results together, both transcription and translation are necessary for nuclear maturation and cumulus expansion, and first 15h IVM for cumulus expansion is critical. The arrested oocytes by the drugs were still capable of undergoing nuclear maturation, although cumulus expansion was affected.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2002.11a
• /
• pp.98-98
• /
• 2002

Recruitment of primordial follicles(PMF) is crucial for female fertility. however, factors and mechanisms that regulate this process is poorly understood. The present study was conducted to obtain an inclusive view of the gene expression and to identify novel factors and their pathways of regulating PMF arrest and/or growth initiation. Ovaries from one-day neonatal(consists of oocyte and PMF) and five-day old(consists of PMF and primary follicles, PRIF) mice were collected, either total RNA or mRNA was isolated, and suppression subtractive hybridization(SSH) was used to isolate and clone genes that differentially expressed in day 1 and day 5 ovaries. Confirmation that some of these genes are differentially expressed in PMF and/or in PRIF was accomplished by using laser captured microdissection(LCM), RT-PCR. in situ hybridization(ISH) and/or immunohistochemistry(IHC). In toto, 357 clones were sequenced and analyzed by BLAST and RIKEN program. Sequences of 330 clones significantly matched database entries while 27 clones were novel. Forty-two and 47 different genes were identified as differentially expressed in day 1 and day 5 ovaries, respectively, while 7 genes were expressed in both stages of ovaries. Day 5-subtracted library included several genes known as markers far growing follicles, such as ZP2, MATER, and fetuin. Among the genes with assigned functions, 23.8% was associated with cell cycle/apoptosis regulation, 7.1% with cellular structure, 11.9% with metabolism, 26.2% with signal transduction, and 31.0% with gene/protein expression in day 1; while 10.6%, 17.0%, 23.5%, 25.5%, and 23.4% in day 5, respectively. Genes such as GDF-8, Lats2, Septin2, and Weel were the highly expressed genes in PMF, while HSP84, Laminin2, MATER, MTi7, PTP, and Wrn were highly expressed genes in PRIF. We have successfully discovered list of genes expressed in day 1 and day 5 ovaries and confirmed that some of them are differentially expressed in PMF and/or PRIF. Gene expression profile from the present study would provide insight for the future study on the mechanism(s) involved in primordial-primary follicular transition. This work was Supported by Korean Health 21 RND Project, Ministry of Health and Welfare, Korea (01-PJ10-PG6-01GN13-0002).

• The Korean Journal of Emergency Medical Services
• /
• v.9 no.2
• /
• pp.55-78
• /
• 2005

The purpose of this study was to provide basic data for the improvement of the guidelines and training programs regarding the cardiopulmonary resuscitation performance of bystanders who can respond to the incidents in earlier times as the first responder of the cardiac arrest incident, by reviewing the performance of basic CPR and the influencing factors after providing 70 students of Department of Emergency Medical Technology with the CPR training. For the purpose of the study, the collected data were computerized and analyzed by SPSS-WIN program(ver. 10.1). The results for this study were as follows The duration of session between the groups in the BLS CPR were 3 minutes and 36 seconds, 2 minutes and 32 seconds respectively. The average compression number per minute were 24.3 times and 33,2 times respectively(p=.000), and the average compression rate per minute were 112 times and 122 times respectively(p=.000). The average ventilation number per minute were 3.54 times and 5.1 times respectively(p=.000). The errors in compression "Too shallow" were 20.73 times(34.6%) and 23,23 times(38,7%) out of 60 times in 4 cycles with the standard of 38 nun. In CPR performance results according to gender in the first episode, males showed better results in compression depth as 41.5 mm comparing to females average 38.2 mm(p=.015). When ventilation results were compared according to the use of FS, the average ventilation number per minute, total ventilation per minute and the average volume per episode were significantly higher when FS was not used(<.040), There was no significant difference in ventilation accuracy between two groups. According to the results, we need to improve and distribute portable barrier devices, and to be familiar with those devices. We need to enforce ventilations as well as to include compressions so that faster and more accurate CPR can be performed. Additionally, we need to exclude ventilation only cases, minimize the interference time of chest compression due to inaccurate ventilation, simplify or minimize the complicatedness of CPR performance and responding time related to breathing, provide first responders with various training programs such as initial assessment and ventilations only, or initial assessment and chest compression-only CPR and than provide advanced training with AHA BLS education including CPR for more than two people according to CPR skills and target characteristics.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.1
• /
• pp.125-131
• /
• 2015

Currently, taxol is mainly extracted from the bark of yews; however, this method can not meet its increasing demand on the market because yews grow very slowly and are a rare and endangered species belonging to first-level conservation plants. Recently, increasing efforts have been made to develop alternative means of taxol production; microbe fermentation would be a very promising method to increase the production scale of taxol. To determine the activities of the taxol extracted from endophytic fungus N. sylviforme HDFS4-26 in inhibiting the growth and causing the apoptosis of cancer cells, on comparison with the taxol extracted from the bark of yew, we used cellular morphology, cell counting kit (CCK-8) assay, staining (HO33258/PI and Giemsa), DNA agarose gel electrophoresis and flow cytometry (FCM) analyses to determine the apoptosis status of breast cancer MCF-7 cells, cervical cancer HeLa cells and ovarian cancer HO8910 cells. Our results showed that the fungal taxol inhibited the growth of MCF-7, HeLa and HO8910 cells in a dose-and time-dependent manner. IC50 values of fungal taxol for HeLa, MCF-7 and HO8910 cells were $0.1-1.0{\mu}g/ml$, $0.001-0.01{\mu}g/ml$ and $0.01-0.1{\mu}g/ml$, respectively. The fungal taxol induced these tumor cells to undergo apoptosis with typical apoptotic characteristics, including morphological changes for chromatin condensation, chromatin crescent formation, nucleus fragmentation, apoptotic body formation and G2/M cell cycle arrest. The fungal taxol at the $0.01-1.0{\mu}g/ml$ had significant effects of inducing apoptosis between 24-48 h, which was the same as that of taxol extracted from yews. This study offers important information and a new resource for the production of an important anticancer drug by endofungus fermentation.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.7
• /
• pp.3519-3528
• /
• 2012

Inhibitory effects of Maclura amboinenesis Bl, one plant used traditionally for the treatment of cancers, on metastatic potential of highly metastatic B16F10 melanoma cells were investigated in vitro. Cell proliferation was assessed using the MTT colorimetric assay. Details of metastatic capabilities including invasion, migration and adhesion of B16F10 melanoma cells were examined by Boyden Chamber invasion and migration, scratch motility and cell attachment assays, respectively. The results demonstrated that n-hexane and chloroform extracts exhibited potent anti-proliferative effects (p<0.01), whereas the methanol and aqueous extracts had less pronounced effects after 24 h exposure. Bioactivity-guided chromatographic fractionation of both active n-hexane and chloroform extracts led to the isolation of two main prenylated xanthones and characterization as macluraxanthone and gerontoxanthone-I, respectively, their structures being identified by comparison with the spectral data. Interestingly, both exhibited potent effective effects. At non-toxic effective doses, n-hexane and chloroform extracts (10 and $30{\mu}g/ml$) as well as macluraxanthone and gerontoxanthone-I (3 and $10{\mu}M$) significantly inhibited B16F10 cell invasion, to a greater extent than $10{\mu}m$ doxorubicin, while reducing migration of cancer cells without cellular cytotoxicity. Moreover, exposure of B16F10 melanoma cells to high concentrations of chloroform ($30{\mu}g/ml$) and geratoxanthone-I ($20{\mu}M$) for 24 h resulted in delayed adhesion and retarded colonization. As insights into mechanisms of action, typical morphological changes of apoptotic cells e.g. membrane blebbing, chromatin condensation, nuclear fragmentation, apoptotic bodies and loss of adhesion as well as cell cycle arrest in the G1 phase with increase of sub-G1 cell proportions, detected by Hoechst 33342 staining and flow cytometry were observed, suggesting DNA damage and subsequent apoptotic cell death. Taken together, our findings indicate for the first time that active n-hexane and chloroform extracts as well as macluraxanthone and gerontoxanthone-I isolated from Maclura amboinensis Bl. roots affect multistep of cancer metastasis processes including proliferation, adhesion, invasion and migration, possibly through induction of apoptosis of highly metastatic B16F10 melanoma cells. Based on these data, M. amboinensis Bl. represents a potential candidate novel chemopreventive and/or chemotherapeutic agent. Additionally, they also support its ethno-medicinal usage for cancer prevention and/or chemotherapy.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.8
• /
• pp.3581-3586
• /
• 2014

Aim: To investigate the effects of tetramethypyrazine (TMP) on proliferation and apoptosis of the human gastric carcinoma cell line 7901 and its possible mechanism of action. Methods: The viability of TMP-treated 7901 cells was measured with a 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and cell apoptosis was analyzed by flow cytometry. The distribution of cells in different phases of cell cycle after exposure of TMPs was analyzed with flow cytometry. To investigate the molecular mechanisms of TMP-mediated apoptosis, the expression of NF-${\kappa}Bp65$, cyclinD1 and p16 in SGC-7901 cells was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Results: TMP inhibited the proliferation of human gastric carcinoma cell line 7901 in dose and time dependent manners. Cell growth was suppressed by TMP at different concentrations (0.25, 0.5, 1.0, 2.0 mg/ml), the inhibition rate is 0.46%, 4.36%, 14.8%, 76.1% (48h) and 15.5%, 18.5%, 41.2%, 89.8% (72h) respectively. When the concentration of TMPs was 2.0mg/ml, G1-phase arrest in the SGC-7901 cells was significant based on the data for cell cycle distribution. RT-PCR demonstrated that NF-${\kappa}Bp65$ and cyclin D1 mRNA expression was significantly down-regulated in 7901 cells treated with 2.0 mg/ml TMP for 72h (p<0.05), while the p16 mRNA level was up-regulated (p<0.05). The protein expression of NF-${\kappa}Bp65$ and cyclin D1 decreased gradually with the increase in TMP concentration, compared with control cells (p<0.05), while expression of protein p16 was up-regulated (p<0.01). Conclusion: TMP exhibits significant anti-proliferative and pro-apoptotic effects on the human gastric carcinoma cell line SGC-7901. NF-${\kappa}Bp65$, cyclinD1 and p16 may also play important roles in the regulation mechanisms.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.5
• /
• pp.2169-2177
• /
• 2014

Matrine, a main active component extracted from dry roots of Sophora flavecens, has been reported to exert antitumor effects on A549 human non-small lung cancer cells, but its mechanisms of action remain unclear. To determine effects of matrine on proliferation of A549 cells and assess possible mechanisms, MTT assays were employed to detect cytotoxicity, along with o flow cytometric analysis of DNA content of nuclei of cells following staining with propidium iodide to analyze cell cycle distribution. Western blotting was performed to determined expression levels of Bax, Bcl-2, VEGF and HDAC1, while a microarray was used to assessed changes of miRNA profiles. In the MTT assay, matrine suppressed growth of human lung cancer cell A549 in a dose- and timedependent manner at doses of 0.25-2.5 mg/ml for 24h, 48h or 72h. Matrine induced cell cycle arrest in G0/G1 phase and decreased the G2/M phase, while down-regulating the expression of Bcl2 protein, leading to a reduction in the Bcl-2/Bax ratio. In addition, matrine down regulated the expression level of VEGF and HDAC1 of A549 cells. Microarray analysis demonstrated that matrine altered the expression level of miRNAs compared with untreated control A549 cells. In conclusion, matrine could inhibit proliferation of A549 cells, providing useful information for understanding anticancer mechanisms.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.5
• /
• pp.2291-2295
• /
• 2014

Although nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis and expression has been observed in some types of human cancer and stem cells, the molecular mechanisms involved in mediation of cell proliferation and cell cycling remains largely elusive. The aim of the present study was to evaluate NS as a potential target for gene therapy of human breast carcinoma by investigating NS gene expression and its effects on SKBR-3 cell proliferation and apoptosis. NS mRNA and protein were both found to be highly expressed in all detected cancer cell lines. The apoptotic rate of the pcDNA3.1-NS-Silencer group ($12.1-15.4{\pm}3.8%$) was significantly higher than those of pcDNA3.1-NS ($7.2-12.0{\pm}1.7%$) and non-transfection groups ($4.1-6.5{\pm}1.8%$, P<0.01). MTT assays showed the knockdown of NS expression reduced the proliferation rate of SKBR-3 cells significantly. Matrigel invasion and wound healing assays indicated that the number of invading cells was significantly decreased in the pcDNA3.1-NS-siRNA group (P<0.01), but there were no significant difference between non-transfected and over-expression groups (P>0.05). Moreover, RNAi-mediated NS down-regulation induced SKBR-3 cell G1 phase arrest, inhibited cell proliferation, and promoted p53 pathway-mediated cell apoptosis in SKBR-3 cells. NS might thus be an important regulator in the G2/M check point of cell cycle, blocking SKBR-3 cell progression through the G1/S phase. On the whole, these results suggest NS might be a tumor suppressor and important therapeutic target in human cancers.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.10
• /
• pp.6007-6012
• /
• 2013

ANXA2, a member of the annexin family, is overexpressed and plays important roles in tumor development. However, the significance of ANXA2 expression in gastric carcinoma has not been clarified.To elucidate its roles in growth of gastric cancer, ANXA2 expression in SGC-7901 cells was inhibited with a designated siRNA, then cell proliferation, cell cycling, apoptosis and motility were determined by MTT assay, flow cytometry, Hoechst 33342 staining and wound healing assay, respectively. To further assess the behavior of ANXA2 deleted SGC-7901 cells, changes of microstructures were observed under fluorescence microscopy, laser scanning confocal microscopy and electron microscopy. We found that inhibition of ANXA2 expression caused cell proliferation to decrease significantly with G1 arrest, motility to be reduced with changes in pseudopodia/filopodia structure and F-actin and ${\beta}$-tubulin expression, and apoptosis to be enhanced albeit without significance. At the same time, ANXA2 deletion resulted in fewer pseudopodia/filopodia, non-stained areas were increased, contact inhibition among cells reappeared, and expression of F-actin and ${\beta}$-tubulin was decreased, with induction of polymerized disassembled forms. Taken together, these data suggest that ANXA2 overexpression is important to maintain the malignancy of cancer cells, and this member of the annexin family has potential to be considered as a target for the gene therapy of gastric carcinoma.