OBJECTIVE: These experiments were conducted to investigate the optimal expose length of propidium iodide (PI) and bisbenzimide on differential staining of mouse blastocysts. MATERIALS AND METHODS: A total 964 blastocysts (early~hatched) was exposed to PI (n=831) (group I: Animals ; Bisbenzimidazole* ; Blastocyst* ; Mice* ; Propidium*

BACKGROUND: Although nucleotides -like Adenosine Triphosphate (ATP) and its derivatives Adenosine, were known to induce growth inhibition and apoptosis in diverse cell lines, little is known about their effects on trophoblast. OBJECTIVE: To elucidate the effects of extracellular ATP and adenosine on trophoblast cell growth and to delineate if apoptosis is involved in this mechanism. MATERIALS AND METHODS: We used TL cell line, derived from human term placenta. The cells were cultured for 24, 48, and 72 hours after being treated with ATP and adenosine, each. Also, cell growth according to different concentrations of ATP and adenosine was evaluated. To test whether apoptosis was induced by each nucleotide, DNA fragmentation and nuclear condensation by Hoechst 33258 stain and P53 protein expression were evaluated. RESULTS: Cell growth was inhibited by ATP and adenosine in time and dose-dependent manner. Furthermore, the growth inhibitory effect of adenosine was stronger than ATP, whereas signs of DNA fragmentation and nuclear condensation were observed in ATP treated cells, but not in adenosine treated ones. CONCLUSION: Our results shows that ATP and adenosine exert inhibitory effect on growth in TL cell line. These findings suggest that pathological production of ATP or its metabolites, adenosine, may lead to a pathologic status such as preeclampsia or intrauterine growth restriction.
Adenosine Triphosphate* ; Adenosine* ; Apoptosis* ; Bisbenzimidazole ; Cell Line* ; DNA Fragmentation ; Humans* ; Nucleotides ; Placenta ; Pre-Eclampsia ; Trophoblasts

Ceramide induces cell death in a dose- and time-dependent manner in neuroblastoma SK-N-SH cells. To investigate the mechanism of SK-N-SH cell death by C2-ceramide, morphological features and Hoechst 33258 staining were analyzed. In these morphlogic study the cell death by ceramide showed typical apoptotic features, nuclear condensation, fragmentation, and membrane blebbing. Ceramide-induced apoptosis was accompanied by nuclear accumulation of p53. Inhibition of p53 expression with p53 antisense oligonucleotides inhibited apoptosis evoked by ceramide. Also, ceramide induced mitochondrial event, collapse of mitochondrial membrane potential (delta psi m) and interestingly, inhibition of p53 attenuated collapse of mitochondrial membrane potential, suggests that ceramide induces mitochondrial dysfunction through upregulation of p53 expression. These results suggest that ceramide-induced apoptosis is dependent upon increase in cellular p53 levels which play a critical role in the regulation of apoptotic cell death and p53 modulates mitochondrial function such as mitochondrial membrane potential level.
Apoptosis ; Bisbenzimidazole ; Blister ; Cell Death* ; Membrane Potential, Mitochondrial ; Membranes ; Neuroblastoma ; Neurons* ; Oligonucleotides, Antisense ; Up-Regulation

PURPOSE: The aims of this study was to examine the anti-proliferative effect and apoptosis induction by aspirin using SNU-668 human gastric adenocarcinoma cell lines. MATERIALS AND METHODS: After treating SNU-668 cell lines with various concentrations of aspirin, cell growth was quantified by MTT assay. Apoptosis was determined by comparing aspirin treated cell lines by immunofluores cence with control cells after Hoechst 33258 staining. Cell lines were further examined using ELISA. Cell cycle was evaluated by FACS. RESULTS: Inhibition of cellular proliferation occurred when cells were treated with aspirin at concetrations of 1 mM or more. Aspirin also induced apoptosis =in these cell lines. Percentages of induction were 3.0+/- 0.6% , 4.8, +/- 0.6%, 17.5+/-0.8%, and 19.2+/-0.7% at 0, 0.5, 1 and 2 mM concentration of aspirin, respectively. ELISA confirmed apoptosis in these cells. However, cell cycle was not affected. CONCLUSION: These results indicate that induction of apoptotic cell death contribute to the anti-proliferative effect of aspirin on SNU-668 human gastric adenocarcinoma cell lines without affecting cell cycle. These findings suggest aspirin may play an important role in cancer prevention and tumor regression in humans.
Adenocarcinoma* ; Apoptosis* ; Aspirin ; Bisbenzimidazole ; Cell Cycle ; Cell Death ; Cell Line* ; Cell Proliferation* ; Enzyme-Linked Immunosorbent Assay ; Humans* ; Stomach Neoplasms

OBJECTIVE: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. MATERIAL AND METHODS: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and 5microgram/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal perm of hybrid F1 male mice(1x106/ml). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, blastocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identity ES cells, the surface markers alkaline phosphatase, SSEA-1, 3, 4 and Oct4 staining were examined in replated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined. RESULTS: Although the cleavage rate (> or =2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic blastocysts (9.6+/-3.1, 35.1+/-5.2) were significantly lower than those of IVF blastocysts (19.5+/-4.7, 63.2+/-13.0) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-1 and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac differentiation derived from mES or P-mES cells was confirmed. CONCLUSION: This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.
Alkaline Phosphatase ; Animals ; Antigens, CD15 ; Bisbenzimidazole ; Blastocyst ; Cell Count ; Cell Line ; Embryonic Stem Cells* ; Embryonic Structures ; Ethanol ; Female ; Fertilization in Vitro* ; Humans ; Male ; Mice* ; Oocytes ; Propidium

A large number of bacterial pathogens have been identified as mediators of apoptosis in vitro and the induction of apoptosis might be an important step in the pathogenesis of these bacteria. In this study, we analyzed the interactions of Orientia tsutsugamuchi with J774 murine macrophage-like cells. The J774 cells were infected with Boryong strain of O. tsutsugamushi and the DNA was analyzed with agarose gel electrophoresis. We observed the typical laddering pattern of DNA fragmentation indicative of apoptosis in infected cells but not cells infected with heat-killed O. tsutsugamushi. We performed terminal deoxynucleotidyl transferase (TdT) assay to label the 3'-hydroxy ends of DNA breaks and observed intense brown staining of nuclei of infected macrophages. With Hoechst 33258 for staining nucleus, strong chromatin condensation was observed only in infected J774 cells. We also examined the cytokine secretion pattern of J774 cells during the rickettsial infection. The large amount of TNF-alpha and IL-10 were secreted after 24 hrs of infection, but the secretion of IL-1beta was increased in small amount. These results showed that O. tsutsugamushi induce apoptosis in murine macrophage-like cells by different mechanism from that of shigella which cause secretion of large amount of IL-1beta.
Apoptosis* ; Bacteria ; Bisbenzimidazole ; Chromatin ; Chungcheongnam-do ; DNA ; DNA Breaks ; DNA Fragmentation ; DNA Nucleotidylexotransferase ; Electrophoresis, Agar Gel ; Interleukin-10 ; Macrophages ; Orientia tsutsugamushi* ; Shigella ; Tumor Necrosis Factor-alpha

Verapamil is used in the treatment of hypertension, angina pectoris, and atrial fibrillation. Recently, several studies have demonstrated that verapamil increased the optic nerve head blood flow and improved the retrobulbar circulation. All these show that verapamil is potentially useful for ophthalmic treatment. Thus, the aim of this study is to investigate whether verapamil could protect human lens epithelial cell (HLEC) from oxidative stress induced by H2O2 and the cellular mechanism underlying this protective function. The viability of HLEC was determined by the MTT assay and apoptotic cell death was analyzed by Hoechst 33258 staining. Moreover, Caspase-3 expression was detected by immunocytochemistry and flow cytometry analysis. We also detected Caspase-3 mRNA expression by reverse-transcription-polymerase chain reaction and the GSH content in cell culture. The results showed that oxidative stress produced significant cell apoptotic death and it was reduced by previous treatment with the verapamil. Verapamil was effective in reducing HLEC death mainly through reducing the expression level of apoptosis-related proteins, caspase-3, and increasing glutathione content. Therefore, it was suggested that verapamil was effective in reducing HLEC apoptosis induced by H2O2.
Angina Pectoris ; Apoptosis* ; Atrial Fibrillation ; Bisbenzimidazole ; Caspase 3 ; Cell Culture Techniques ; Cell Death ; Epithelial Cells* ; Flow Cytometry ; Glutathione ; Humans ; Hypertension ; Immunohistochemistry ; Optic Disk ; Oxidative Stress ; RNA, Messenger ; Verapamil*

BACKGROUND/AIMS: Parthenolide (PT) is responsible for the bioactivities of Feverfew. Besides its potent anti-inflammatory effect, this compound has recently been reported to induce apoptosis in cancer cells. Unfortunately, many of the therapies that use 5-fluorouracil (5-FU) alone or in combination with other agents are likely to become ineffective due to drug resistance. In the present study, we investigate the antitumor effect of PT combined with 5-FU on colorectal cancer cells. METHODS: SW480 cell was employed as a representative of human colorectal carcinoma (CRC) cells. We performed MTT, annexin-V assay, and Hoechst 33258 staining to measure the synergistic effect. Western blotting was used to demonstrate apoptotic pathway. RESULTS: Our result demonstrated that PT inhibited the viability of colorectal cancer cells and had synergistic anti-proliferation in combination with 5-FU. After combined treatment of 5-FU and PT, enhanced apoptotic cell death is observed using annexin-V FITC assay and it was revealed by the condensed chromatin and fragmented DNA. Compared with 5-FU or PT alone, the apoptosis of colorectal cancer cells treated with PT and 5-FU enhanced the activation of caspase-8, caspase-3. CONCLUSIONS: Combined treatment with PT may offer an efficacious strategy to overcome 5-FU resistance in certain CRC cells.
Apoptosis ; Bisbenzimidazole ; Blotting, Western ; Caspase 8 ; Cell Death ; Chromatin ; Colorectal Neoplasms ; DNA ; Drug Resistance ; Fluorescein-5-isothiocyanate ; Fluorouracil ; Humans ; Sesquiterpenes ; Tanacetum parthenium

PURPOSE: The clusterin expression has been associated with tumorigenesis of various malignancies, including tumors of the prostate, colon and breast. Furthermore, the expression of clusterin is modulated by many factors that are believed to regulate tumor growth and apoptosis. We studied the clusterin expression in transitional cell carcinoma (TCC) of the bladder and we investigated its correlation with apoptosis. MATERIALS AND METHODS: Eighty five bladder tumor specimens from radical cystectomy or transurethral resection were subjected to immunohistochemical clusterin staining with Ig G clusterin Ab. We examined the immunohistochemical localization of clusterin, and this was followed by TUNEL staining to detect the apoptotic cells. After double-staining with Hoechst 33258, we detected the apoptotic cells under a fluorescence microscope and we calculated the apoptotic index. RESULTS: Invasive TCC showed a stronger positive expression of clusterin as compared with superficial TCC, but the positivity of the clusterin expression was not in proportion to the tumor grade. The apoptotic indices of cancer were 0.52+/-0.28%, 0.30+/-0.16% and 0.17+/-0.11% in Grade I, Grade II and Grade III superficial TCC, respectively, and it was 0.23+/-0.13% in Grade III invasive TCC. Apoptotic cells were not detected in the cancer cells stained with clusterin. Conversely, clusterin was not expressed in the cells showing apoptosis. CONCLUSIONS: These results suggest that clusterin could be used as a marker to provide prognostic information for the TCC patients. The apoptotic index revealed that apoptosis and the clusterin expression have correlation with transitional cell cancer. Further study will be needed to clarify the role of clusterin as a therapeutic target for cancer treatment.
Apoptosis* ; Bisbenzimidazole ; Breast ; Carcinogenesis ; Carcinoma, Transitional Cell* ; Clusterin* ; Colon ; Cystectomy ; Fluorescence ; Humans ; In Situ Nick-End Labeling ; Prostate ; Urinary Bladder Neoplasms ; Urinary Bladder*

BACKGROUND: In recent years, a combination of two demographic phenomena, an increased number of older people in the population and an increase in the incidence of lung cancer with age, has made it mandatory to develop therapeutic modalities with less toxicity for the treatment of inoperable elderly patients with lung cancer. Therefore, we investigated the correlation between COX-2 expression and cytotoxicity of Nimesulide, a specific COX-2 inhibitor. MATERIAL AND METHOD: Immunohistochemical staining of COX-2 was performed. After exposure of Nimesulide, XTT analysis, FACS analysis and Hoechst staining were carried out. RESULT: COX-2 protein was expressed in non- treated A549 cells strongly, but not in H1299. Cytotoxicity of Nimesulide against A549 cell and H1299 cell were similar and IC50 of Nimesulide in both cell lines were 70.9 microM in A549 cell line and 56.5 microM in H1299 cell line respectively. FACS analysis showed G0/G1 arrest in both cell lines and the S phase cell fraction was decreased. Morphologic assessment of apoptosis by Hoechst 33258 staining, many apoptotic cells were detected in both cell lines. CONCLUSION: Selective COX-2 inhibitor, Nimesulide, can inhibit the proliferation of non-small cell lung cancer cell lines in vitro. Inhibitory effect of Nimesulide are induction of apoptosis and G0/G1 arrest. There is no correlation between COX-2 expression and cytotoxicity of Nimesulide, a specific COX-2 inhibitor. Therefore, highly selective COX-2 inhibitors such as Nimesulide can be expected to lead to even greater efficacy of their use as adjuncts to various anticancer angents and radiation therapy for the treatment of high-risk patients.
Aged ; Apoptosis ; Bisbenzimidazole ; Carcinoma, Non-Small-Cell Lung* ; Cell Line* ; Cyclooxygenase 2 Inhibitors ; Humans ; Incidence ; Inhibitory Concentration 50 ; Lung Neoplasms ; S Phase