Pseudolaric acid B inhibits PAX2 expression through Wnt signaling and induces BAX expression, therefore promoting apoptosis in HeLa cervical cancer cells / 부인종양

OBJECTIVES: Pseudolaric acid B (PAB) has been shown to inhibit the growth of various tumor cells, but the molecular details of its function are still unknown. This study investigated the molecular mechanisms by which PAB induces apoptosis in HeLa cells. METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to investigate the effect of PAB treatment in various cervical cancer cell lines. Annexin V/propidium iodide staining combined with flow cytometry and Hoechst 33258 staining were used to assess PAB-induced apoptosis. Additionally, we performed bioinformatics analyses and identified a paired box 2 (PAX2) binding site on the BAX promoter. We then validated the binding using luciferase and chromatin immunoprecipitation assays. Finally, western blotting assays were used to investigate PAB effect on the Wnt signaling and the involved signaling molecules. RESULTS: PAB promotes apoptosis and downregulates PAX2 expression in HeLa cells in a time- and concentration-dependent manner. PAX2 binds to the promoter of BAX and inhibits its expression; therefore, PAX2 inhibition is associated with increased levels of BAX, which induces apoptosis of HeLa cells via the mitochondrial pathway. Additionally, PAB inhibits classical Wnt signaling. CONCLUSION: PAB effectively inhibits Wnt signaling and PAX2 expression, and increases BAX levels, which induce apoptosis in HeLa cells. Therefore, PAB is a promising natural molecule for the treatment of cervical cancer.

Bleomycin Inhibits Proliferation via Schlafen-Mediated Cell Cycle Arrest in Mouse Alveolar Epithelial Cells / 결핵

BACKGROUND: Idiopathic pulmonary fibrosis involves irreversible alveolar destruction. Although alveolar epithelial type II cells are key functional participants within the lung parenchyma, how epithelial cells are affected upon bleomycin (BLM) exposure remains unknown. In this study, we determined whether BLM could induce cell cycle arrest via regulation of Schlafen (SLFN) family genes, a group of cell cycle regulators known to mediate growth-inhibitory responses and apoptosis in alveolar epithelial type II cells. METHODS: Mouse AE II cell line MLE-12 were exposed to 1–10 µg/mL BLM and 0.01–100 µM baicalein (Bai), a G1/G2 cell cycle inhibitor, for 24 hours. Cell viability and levels of pro-inflammatory cytokines were analyzed by MTT and enzyme-linked immunosorbent assay, respectively. Apoptosis-related gene expression was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Cellular morphology was determined after DAPI and Hoechst 33258 staining. To verify cell cycle arrest, propidium iodide (PI) staining was performed for MLE-12 after exposure to BLM. RESULTS: BLM decreased the proliferation of MLE-12 cells. However, it significantly increased expression levels of interleukin 6, tumor necrosis factor α, and transforming growth factor β1. Based on Hoechst 33258 staining, BLM induced condensation of nuclear and fragmentation. Based on DAPI and PI staining, BLM significantly increased the size of nuclei and induced G2/M phase cell cycle arrest. Results of qRT-PCR analysis revealed that BLM increased mRNA levels of BAX but decreased those of Bcl2. In addition, BLM/Bai increased mRNA levels of p53, p21, SLFN1, 2, 4 of Schlafen family. CONCLUSION: BLM exposure affects pulmonary epithelial type II cells, resulting in decreased proliferation possibly through apoptotic and cell cycle arrest associated signaling.

TGF-β1 upregulates the expression of hyaluronan synthase 2 and hyaluronan synthesis in culture models of equine articular chondrocytes

We investigated the effect of transforming growth factor beta 1 (TGF-β1) on equine hyaluronan synthase 2 (HAS2) gene expression and hyaluronan (HA) synthesis in culture models of articular chondrocytes. Equine chondrocytes were treated with TGF-β1 at different concentrations and times in monolayer cultures. In three-dimensional cultures, chondrocyte-seeded gelatin scaffolds were cultured in chondrogenic media containing 10 ng/mL of TGF-β1. The amounts of HA in conditioned media and in scaffolds were determined by enzyme-linked immunosorbent assays. HAS2 mRNA expression was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The uronic acid content and DNA content of the scaffolds were measured by using colorimetric and Hoechst 33258 assays, respectively. Cell proliferation was evaluated by using the alamarBlue assay. Scanning electron microscopy (SEM), histology, and immunohistochemistry were used for microscopic analysis of the samples. The upregulation of HAS2 mRNA levels by TGF-β1 stimulation was dose and time dependent. TGF-β1 was shown to enhance HA and uronic acid content in the scaffolds. Cell proliferation and DNA content were significantly lower in TGF-β1 treatments. SEM and histological results revealed the formation of a cartilaginous-like extracellular matrix in the TGF-β1-treated scaffolds. Together, our results suggest that TGF-β1 has a stimulatory effect on equine chondrocytes, enhancing HA synthesis and promoting cartilage matrix generation.

Cytotoxic activity of Androctonus australis hector venom and its toxic fractions on human lung cancer cell line

Background: Several studies have showed that animal venoms are a source of bioactive compounds that may inhibit the growth of cancer cells, which makes them useful agents for therapeutic applications. Recently, it was established that venom toxins from scorpions induced cytotoxic, antiproliferative and apoptogenic effects on cancer cells. Therefore, the present study aims to investigate the cytotoxic activity of Androctonus australis hector (Aah) scorpion venom and its toxic fractions (FtoxG-50 and F3) on NCI-H358 human lung cancer cells. Methods: The cytotoxic and antiproliferative activities were estimated using MTT assay, lactate dehydrogenase release and clonogenic assays. Apoptosis was evaluated by Hoechst 33258 staining, DNA fragmentation assay and caspase-3 activity. Oxidative stress was analyzed by reactive oxygen species, nitric oxide, malondialdehyde and protein carbonyl levels along with assessment of antioxidant status. In addition, alteration of mitochondrial membrane potential was analyzed by JC1 fluorescent dye. Results: The present findings showed that F3 fraction was more cytotoxic towards NCI-H358 lung cancer cells with an IC50 of 27.05 ± 0.70 μg/mL than venom alone (396.60 ± 1.33 μg/mL) and its toxic fraction FtoxG-50 (45.86 ± 0.91 μg/mL). Nevertheless, F3 fraction was not cytotoxic at these concentrations on normal human lung fibroblast MRC-5 cells. Inhibition of NCI-H358 cell proliferation after F3 fraction exposure occurred mainly by apoptosis as evidenced by damaged nuclei, significant DNA fragmentation level and caspase-3 activation in a dose dependent manner. Moreover, F3 fraction enhanced oxidative and nitrosative stress biomarkers and dissipated mitochondrial membrane potential in lung cancer cells along with significant depletion in cellular enzymatic and non-enzymatic antioxidants. Further, the apoptosis induced by F3 fraction was markedly prevented by the antioxidant N-acetylcysteine (NAC) suggesting the potential mechanism of oxidative stress. Conclusion: These findings suggest that F3 fraction could induce apoptosis in lung cancer cells through involvement of oxidative stress and mitochondrial dysfunction. Hence, these properties make F3 fraction a promising candidate for development of new anticancer agents.(AU)

Anti-carcinogenic actions of glycoprotein conjugated with isoflavones from submerged-liquid culture of Agaricus blazei mycelia through reciprocal expression of Bcl-2 and Bax proteins / 동물의과학연구지

Glycoproteins isolated from fruit bodies and mycelial cultures of mushrooms exhibit anti-carcinogenic actions in human cancer cells and animal tumor cells by induction of apoptosis. Here, we report that isoflavone-conjugated glycoproteins (designate Gluvone), exhibit strong anti-carcinogenic effects on human breast cancer MCF-7 cells by induction of apoptosis. Gluvone with 9.4 kDa of molecular weight was isolated from submerged-liquid culture of Agaricus blazei mycelia (ABM) in soy flake-containing liquid medium. MCF-7 cells were incubated with various amounts of Gluvone (0~250 microM) for a period of 6 days. Gluvone exhibited anti-proliferative actions in a dose-dependent manner and 62% growth inhibition at 200 microM for 4 days relative to control. Hoechst 33258 staining analysis revealed that Gluvone induced formation of apoptotic bodies. Gluvone was associated with down-regulation of anti-apoptotic Bcl-2 protein expression as well as up-regulation of pro-apoptotic Bax protein expression. Gluvone treatment induced proteolytic activation of caspase-9 and caspase-3 through cytochrome c release from mitochondria to cytosol as well as concomitant degradation of poly (ADP-ribose) polymerase (PARP). In addition, Gluvone induced activation of caspase-8. Taken all together, these results indicate that the anti-proliferative effect of Gluvone is associated with induction of apoptotic cell death through the mitochondrial dysfunction pathway mediated by enhancement of Bax protein expression and suppression of Bcl-2 protein expression.

Protective Effects of Verapamil against H2O2-Induced Apoptosis in Human Lens Epithelial Cells

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.

Parthenolide Sensitizes Human Colorectal Cancer Cells to Tumor Necrosis Factor-related Apoptosis-inducing Ligand through Mitochondrial and Caspase Dependent Pathway

BACKGROUND/AIMS: Combination therapy utilizing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in conjunction with other anticancer agents, is a promising strategy to overcome TRAIL resistance in malignant cells. Recently, parthenolide (PT) has proved to be a promising anticancer agent, and several studies have explored its use in combination therapy. Here, we investigated the molecular mechanisms by which PT sensitizes colorectal cancer (CRC) cells to TRAIL-induced apoptosis. METHODS: HT-29 cells (TRAIL-resistant) were treated with PT and/or TRAIL for 24 hours. The inhibitory effect on proliferation was detected using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Annexin V staining, cell cycle analysis, and Hoechst 33258 staining were used to assess apoptotic cell death. Activation of an apoptotic pathway was confirmed by Western blot. RESULTS: Treatment with TRAIL alone inhibited the proliferation of HCT 116 cells in a dose-dependent manner, whereas proliferation was not affected in HT-29 cells. Combination PT and TRAIL treatment significantly inhibited cell growth and induced apoptosis of HT-29 cells. We observed that the synergistic effect was associated with misregulation of B-cell lymphoma 2 (Bcl-2) family members, release of cytochrome C to the cytosol, activation of caspases, and increased levels of p53. CONCLUSION: Combination therapy using PT and TRAIL might offer an effetive strategy to overcome TRAIL resistance in certain CRC cells.

Synergistic Effect of Parthenolide in Combination with 5-Fluorouracil in SW480 Cells

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.

Effects of diallyl disulfide on apoptosis of human leukemia K562 cells and expression of Fas, FasL and caspase-8 / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effects of diallyl disulfide (DADS) on apoptosis of human leukemia K562 cells and possible mechanisms.</p><p><b>METHODS</b>The morphologic changes of leukemia K562 cells after DADS treatment were observed by Hoechst 33258 staining. Cell apoptosis rates after different concentrations and different durations of DADS treatment were determined by flow cytometry. Fas, FasL and caspase-8 mRNA expression was estimated by reverse transcription-polymerase chain reaction (RT-PCR) 48 hrs after DADS treatment.</p><p><b>RESULTS</b>The characteristics of apoptosis in K562 cells induced by DADS were observed. After 24 hrs of DADS treatment, the apoptosis rate of K562 cells increased from (11.60 ± 0.83)% at the concentration of 10 mg/L to (37.94 ± 0.87)% at the concentration of 40 mg/L. The apoptosis rate of K562 cells increased after 40 mg/L DADS with the increasing time from (37.94 ± 0.87)% (24 hrs) to (47.02 ± 0.66)% (72 hrs). Expression of Fas and caspase-8 mRNA increased, while FasL mRNA expression decreased significantly 48 hrs after DADS treatment compared with the control group (P<0.05).</p><p><b>CONCLUSIONS</b>DADS can induce apoptosis of human leukemia K562 cells in a time- and concentration-dependent manner, possibly through increasing Fas and caspase-8 expression and decreasing FasL expression.</p>

Chromosomal variation in three human-mouse hybridoma cell lines after various passaging intervals as assessed with two different staining methods

The main objective of this study was to investigate the status of chromosome stability in 3 human-mouse hybridoma cell lines over a period of time in various passages. Metaphase spreads from 3 human-mouse cell lines [HF2X653, SPMO-4 and F3B6] that had been cultured in 4 successive passages, from 1 to 4 weeks, were prepared and analyzed. Metaphase chromosomes stained in Giemsa and a fluorescent dye, Hoechst 33258, for differential staining. This staining was performed for differentiating human and mouse chromosomes. Numerical chromosome analysis showed that although in successive passages the total number of chromosomes in hybridoma cells remained unchanged, some changes occurred in the number of human and mouse metacentric and acrocentric chromosomes during different passages. These changes were detectable, using fluorescence staining method. Since one of the main uses of human-mouse hybridoma cells is producing monoclonal antibody, chromosomal instability in these cells causes the loss of human chromosomes coding the antibody of interest occasionally. Therefore, cytogenetical analysis and characterization of these cells, especially by using the appropriate ways of chromosomal identification, is essential prior to use

Clusterin Expression and Apoptosis in Transitional Cell Carcinoma of the Bladder / 대한비뇨기과학회지

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.

Neuroprotection of chloride channel blockers against NMDA-induced apoptosis of cultured rat hippocampal neurons / 南方医科大学学报

Activation of N-methyl-d-aspartic acid (NMDA) receptor plays an important role in neuronal apoptosis induced by cerebral ischemia but the underlying mechanisms are still unclear. The present study examined the neuroprotection of three chloride blockers in an in vitro cell model of cerebral ischemia established by treatment of cultured rat hippocampal neurons with NMDA. Hoechst 33258 staining and MTT assay were used to detect neuronal apoptosis and cell viability, respectively. The neuroprotective effects of chloride channel blockers on the cell viability and neuronal apoptosis were only observed when the blockers were applied before NMDA exposure. In comparison with DIDS, SITS showed more potent protective effect in a dose-dependent manner, whereas NPPB showed no significant neuroprotective effect. The results demonstrate that pretreatment with both SITS and DIDS have protective effect against neuronal apoptosis, which is achieved by blocking both NMDA receptor and chloride channel.

Study on the Mitochondrial Dysfunction by p53 Regulation in Ceramide-induced Neuronal Cell Death / 체질인류학회지

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.

Cytotoxicity of COX-2 Inhibitor (Nimesulide) in Non-small Cell Lung Cancer Cell Line / 대한흉부외과학회지

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.

Role of p-38 MAP Kinase in apoptosis of hypoxia-induced osteoblasts / 대한치과교정학회지

Tooth movement by orthodontic force effects great tissue changes within the periodontium, especially by shifting the blood flow in the pressure side and resulting in a hypoxic state of low oxygen tension. The aim of this study is to elucidate the possible mechanism of apoptosis in response to hypoxia in MC3T3E1 osteoblasts, the main cells in bone remodeling during orthodontic tooth movement. MC3T3E1 osteoblasts under hypoxic conditions (2% oxygen) resulted in apoptosis in a time-dependent manner as estimated by DNA fragmentation assay and nuclear morphology stained with fluorescent dye, Hoechst 33258. Pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, or Z-DEVD-CHO, a specific caspase-3 inhibitor, completely suppressed the DNA ladder in response to hypoxia. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-1 activity (YVADase) was detected. To confirm what caspases are involved in apoptosis, Western blot analysis was performed using anti-caspase-3 or -6 antibodies. The 10-kDa protein, corresponding to the active products of caspase-3, and the 10-kDa protein of the active protein of caspase-6 were generated in hypoxia-challenged cells in which the processing of the full length form of caspase-3 and -6 was evident. While a time course similar to this caspase-3 and -6 activation was evident, hypoxic stress caused the cleavage of lamin A, which was typical of caspase-6 activity. In addition, the stress elicited the release of cytochrome c into the cytosol during apoptosis. Furthermore, we observed that pre-treatment with SB203580, a selective p38 mitogen activated protein kinase inhibitor, attenuated the hypoxia-induced apoptosis. The addition of SB203580 suppressed caspase-3 and -6-like protease activity by hypoxia up to 50%. In contrast, PD98059 had no effect on the hypoxia-induced apoptosis. To confirm the involvement of MAP kinase, JNK/SAPK, ERK, or p38 kinase assay was performed. Although p38 MAPK was activated in response to hypoxic treatment, the other MAPK -JNK/SAPK or ERK- was either only modestly activated or not at all. These results suggest that p38 MAPK is involved in hypoxia-induced apoptosis in MC3T3E1 osteoblasts.

ATP and Adenosine Induce Growth Inhibition and Apoptosis in Human Trophoblast-like (TL) Cell Line / 대한산부인과학회잡지

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.

Effects of Exposure of Propidium Iodide and Bisbenzimide on Differential Staining of Mouse Blastocysts / 대한불임학회지

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:

Parthenogenetic Mouse Embryonic Stem Cells have Similar Characteristics to In Vitro Fertilization mES Cells / 대한불임학회지

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.

Induction of Apoptosis and Inhibition of Cellular Proliferation in Aspirin-treated SNU-668 Human Gastric Adenocarcinoma Cell Lines / 대한암학회지

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.

Radioprotective effects of DNA ligands Hoechst-33342 and 33258 in whole body irradiated mice.

Radioprotective effects of bisbenzimidole derived DNA ligands Hoechst-33342 (H-342) and Hoechst-33258 (H-258) have been investigated in whole body irradiated stain-A and Balb/c mice (Co-60 Gamma-ray, absorbed doses of 2.5 to 10 Gy delivered at dose rates of 0.01 to 0.50 Gy/min). Biodistribution of Hoechst dyes (2 or 5 mg/kg, body wt., i.v.) and their effects on cell cycle kinetics in bone marrow were studied by flow cytometry. Protection against radiation-induced chromosomal aberrations, micronuclei formation, alterations in DNA content dispersion, inhibition of erythropoiesis and animal lethality were investigated. Significant amount of DNA bound Hoechst could be observed in liver, intestine, kidney and brain for more than 14 days after its administration, while in the bone marrow cells, a reduction in the bound Hoechst was noticed after 7 days. H-342 significantly reduced the radiation-induced chromosome aberrations mainly due to a decrease in the frequency of acentrics (nearly 30%), while a marginal decrease (10%) in the dicentrics was observed at all the dose rates studied. Both H-342 and H-258 reduced the radiation-induced micronuclei formation in a dose dependent manner (2-10 mg/kg body wt.) and this protective effect was observed up to 6 days after the administration. Neither of the two compounds induced any cytogenetic damage in the bone marrow cells of unirradiated animals nor induced tumours at the doses used here (< 5 mg/kg, body wt. i.v.). Reduction in cytogenetic damage of bone marrow cells led to a faster recovery of erythropoesis as observed by increased PCE/NCE ratio in the peripheral blood erythrocytes of the animals which received Hoechst before irradiation. H-258 (5 mg/kg body wt.) given 18 hr before irradiation reduced radiation-induced animal death (5-9 Gy), while no significant effect was observed at higher doses (10 Gy). However, H-342, which has a higher cell permeability, even at a lower dose (2 mg/kg body wt.) showed significant protection at 10 Gy. The protective effects could be enhanced further, by combining these DNA binding agents with the glucose analogue, 2-deoxy-D-glucose (2-DG) which has been shown earlier to protect bone marrow cells against radiation damage.