Aim To study the mechanisms of Pseudolaric acid B(PAB)-induced MCF-7 cell apoptosis and mitotic arrest.Methods MTT assay was performed to assess the cell growth inhibition,contrast phase microscope was used to observe cellular morphologic alteration,and the change of DNA was detected by fluorescent microscopy.The distribution of cell cycle was determined by flow cytometric analysis of propidium iodide staining,and the protein expression was examined by Western blot analysis.Results PAB inhibited MCF-7 cell growth in a dose-and time-dependent manner.4 ?mol?L-1 PAB induced DNA condensation at 24 h.PAB cleaved PARP in a time-dependent manner.At 36 h,PAB up-regulated the expression of cdc 2 and nuclear cyclin B1.Fas antagonistic antibody UB2 had no effect on apoptosis,but agonistic antibody CH11 enhanced the apoptosis induced by PAB.UB2 exerted no effect on cell cycle arrest,and CH11 had the same action as UB2 except for reducing the mitotic arrest through enhancing apoptotic subdiploid peak.Conclusion PAB inhibited MCF-7 cell growth through mitotic arrest and apoptosis.Apoptosis and mitotic arrest were independent of Fas pathway.

BACKGROUND: Uterine cervix cancer is one of the diseases to damage female health and degrade quality of life. It has been a hot spot to decrease the side effects of radiotherapy and chemotherapy and anti-tumor medicine by using Chinese medicine in medical domain. In Europe, silymarin had been used to treat hepatitis. It had little toxic action and large contents in Chinese herb. Therefore, many researchers are studying its anti-tumor effects now.OBJECTIVE: To study the pharmacological mechanism of silymarin on human cervical cancer cell.DESIGN: The cells cultured in 10% serum medium served as negative control group in vitro.MATERIALS: The experiment was finished in the China-Japan Research Institute of Medical and Pharmaceutical Sciences in Shenyang Pharmaceutical University; Department of Pathological Science of Showa Medical UniversityMATERIASLS: Human cervical cancer cell (HeLa) is the preservative cell line of American ATCC in the Lab. The experiment was completed in the Cell Lab of China-Japan Research Institute of Medical and Pharmaceutical Sciences in Shenyang Pharmaceutical University from Feburary 2003 to July 2003.METHODS: HeLa cell death ratio was measured by methyl thiazol tetrazolium(MTT) assay. Cellular morphologic changes were observed by phase-contrast and fluorescence microscopy. Cell cycle distribution was analyzed by flow cytometry.MAIN OUTCOME MEASURES: The changes of cell death ratio and cell cycle distribution.RESULTS: Silymarin-induced HeLa cell death was relevant to the content of serum in medium. At the does of 80 μmol /L silymarin, the cell death ratio in serum-free group reached 85.27%. The cell death ratio in 5% and 10%serum groups was 35.53% and 7.71% respectively. Morphological changes of HeLa cells demonstrated that silymarin induced HeLa cell death through apoptotic pathway at lower concentration in serum-free condition. Flow cytometry analysis showed that silymarin induced 70. 04% cells death, most of which were in G0/G1 phase.CONCLUSION: Silymarin mainly induce G0/G1 phase HeLa cell death through apoptotic pathway in serum-free medium.

AIM: To study the molecular biological mechanism and signal transduction pathway of interleukin-1? (IL-1?)-induced apoptosis in A375-S2 melanoma cells. METHODS: Photomicrocropy showed typical apoptotic changes. The cytotoxic effect of IL-1? in vitro and influences of caspases in this effect were measured by MTT assay. The cytotoxicity of cells was assessed by LDH-based assay. Degradation of DNA was detected by agarose gel electrophoresis. RESULTS: The inhibitory effect of IL-1? on A375-S2 cell growth was in a dose and time-dependent manner, and cell death rate reached more than 90% at 72 h after treatment with 10~(-9)mol/L IL-1?. The inhibitors of caspase-family, -1, -3, -8, -9, and -10, partially blocked cell death at early stage. LDH assay showed that major IL-1?-induced cell death was apoptosis, and in a dose and time-dependent manner. Typical apoptotic DNA ladder was observed in agarose gel electrophoresis. CONCLUSION: IL-1? induced apoptosis in melanoma A375-S2 cells by activating caspase pathway. [

AIM: To compare the cytotoxic effect of evodiamine with chemotherapy drugs on A375-S2 cells, and to examine the relationship between the effects of PKC and ERK on evodiamine-induced cell death. METHODS: MTT assay and Western blot analysis were applied. RESULTS: Compared to actinomycin D, cisplatin and 5-FU, evodiamine showed less cytotoxic effects on A375-S2 cells, but it induced more significant inhibition of proliferation in A375-S2 cells incubated with evodiamine for 24 h, followed by continuous culture in drug-free medium. The activation of PKC induced by 10 ?g?L -1 PMA partially blocked evodiamine-induced cell death, which was reversed by PKC and ERK inhibitors. Moreover, evodiamine down-regulated the expressions of ERK and phosphorylated ERK. CONCLUSION: Evodiamine has a strong inhibitory influence on proliferation of A375-S2 cells, even after removal of evodiamine. Evodiamine blocks the protective role of ERK to A375-S2 cells through the downregulation of ERK and phosphorylated ERK expression. [

AIM: To study the effect of oridonin on the phagocytosis of apoptotic U937 cells by macrophage-like cells. METHODS: DNA agarose gel electrophoresis, Giemsa staining, Hoechst 33258 staining and photomicroscopical observation were used. RESULTS: UV irradiation (2.4 J/cm~2, 4 min) induced U937 cell apoptosis. Marked DNA fragmentation in agarose gel electrophoresis was observed. Oridonin augmented phagocytosis of apoptotic U937 cells by U937 cell-derived macrophages in a time- and dose-dependent manner. However, less effect on synthetic fluoresbrite micropheres was observed. The oridonin-augmented phagocytosis was attenuated by anti-human TNF? or anti-human IL-1? antiserum. In addition, the similar effect of phagocytosis was observed in oridonin-treated human monocyte-derived macrophages at 4 day maturation. CONCLUSION: Oridonin enhances phagocytosis of apoptotic U937 cells by macrophage-like cells. The releases of TNF? and IL-1? are involved in this mechanism.

AIMTo study the role of PKC in evodiamine-induced A375-S2 cell death.

METHODSRatio of apoptosis induced by evodiamine was determined by TUNEL assay. MTT assay was carried out to assess cytotoxic effect of evodiamine. The influence on expression of ERK, phospho-ERK and Bcl-2 was detected by Western blotting analysis.

RESULTSTUNEL assay indicated that apoptosis was the type of A375-S2 cell death induced by evodiamine treatment for 24 h. Both staurosporine (inhibitor of PKC) and PD98059 (inhibitor of ERK) cooperated with evodiamine to further induce A375-S2 cell death. Evodiamine inhibited PKC activity, down-regulated the expression of ERK, phospho-ERK and Bcl-2, and staurosporine was capable of augmenting these effects induced by evodiamine.

CONCLUSIONPKC lies upstream and exhibits regulatory effect on ERK and Bcl-2 in evodiamine-induced cell death.

Apoptosis ; drug effects ; Cell Line, Tumor ; Evodia ; chemistry ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Flavonoids ; pharmacology ; Humans ; Melanoma ; pathology ; Plant Extracts ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Protein Kinase C ; antagonists & inhibitors ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Quinazolines ; isolation & purification ; pharmacology ; Staurosporine ; pharmacology

Norcantharidin (NCTD) is the demethylated form of cantharidin, which is the active substance of mylabris. To examine the pathway of NCTD-induced A375-S2 cell death, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-dipheyltetrazolium bromide (MTT) assay, photomicroscopical observation, DNA agarose gel electrophoresis, caspase activity assay and Western blot analysis were carried out. A375-S2 cells treated with NCTD exhibited several typical characteristics of apoptosis. The inhibitory effect of NCTD on human melanoma, A375-S2 cells, was partially reversed by the inhibitors of pan-caspase, caspase-3 and caspase-9. The activities of caspase-3 and -9 were significantly increased after treatment with NCTD at different time. The expression of inhibitor of caspase-activated DNase was decreased in a time-dependent manner, simultaneously, the ratio of Bcl-2/Bax or Bcl-xL/Bax was decreased and the expression ratio of proteins could be reversed by caspase-3 inhibitor. The expression of cytochrome c in cytosol was increased after NCTD treatment and caspase- 3 inhibitor had no significant effect on the up-regulation of cytochrom c. These results suggest that NCTD induced A375-S2 cell apoptosis and the activation of caspase and mitochondrial pathway were involved in the process of NCTD-induced A375-S2 cell apoptosis.
Animals ; Apoptosis/*physiology ; Bicyclo Compounds, Heterocyclic/chemistry/metabolism/*pharmacology ; Caspases/antagonists & inhibitors/*metabolism ; Cell Line, Tumor/*drug effects ; Cell Shape ; DNA Fragmentation ; Enzyme Activation ; Humans ; Mitochondria/*metabolism ; Molecular Structure ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Signal Transduction/*physiology

A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was used to determine that apoptosis causes HeLa cell death induced by pseudolaric acid B. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 decreased p53 protein expression during exposure to pseudolaric acid B. SP600125 decreased the phosphorylation of p53 during pseudolaric acid B exposure, indicating that JNK mediates phosphorylation of p53 during the response to pseudolaric acid B. SP600125 reversed pseudolaric acid B-induced down-regulation of phosphorylated extracellular signal-regulated protein kinase (ERK), and protein kinase C (PKC) was activated by pseudolaric acid B, whereas staurosporine, calphostin C, and H7 partly blocked this effect. These results indicate that p53 is partially regulated by JNK in pseudolaric acid B-induced HeLa cell death and that PKC participates in pseudolaric acid B-induced HeLa cell death.
Tumor Suppressor Protein p53/metabolism/*physiology ; Protein Kinase C/metabolism ; Phosphorylation ; JNK Mitogen-Activated Protein Kinases/*physiology ; Humans ; Hela Cells ; Diterpenes/*pharmacology ; DNA Fragmentation/drug effects ; Cell Death/*drug effects ; Anthracenes/pharmacology

Eight major policies were implemented by Japanese Government since Oct. 2001, to deal with bovine spongiform encephalopathy (BSE). These are; 1) Surveillance in farm by veterinarian, 2) Prion test at healthy 1.3mi cows/yr, by veterinarian, 3) Elimination of specified risk material (SRM), 4) Ban of MBM for production, sale use, 5) Prion test for fallen stocks, 6) Transparent information and traceability, 7) New Measures such as Food Safety Basic Law, and 8) Establish of Food Safety Commission in the Cabinet Office. At this moment, the extent of SRM risk has only been indicated by several reports employing tests with a limited sensitivity. There is still a possibility that the items in the SRM list will increase in the future, and this indiscriminately applies to Japanese cattle as well. Although current practices of SRM elimination partially guarantee total food safety, additional latent problems and imminent issues remain as potential headaches to be addressed. If the index of SRM elimination cannot guarantee reliable food safety, we have but to resort to total elimination of tissues from high risk-bearing and BSE-infected animals. However, current BSE tests have their limitations and can not yet completely detect highrisk and/or infected animals. Under such circumstances, tissues/wastes and remains of diseased, affected fallen stocks and cohort animals have to be eliminated to prevent BSE invading the human food chain systems. The failure to detect any cohort should never be allowed to occur, and with regular and persistent updating of available stringent records, we are at least adopting the correct and useful approach as a reawakening strategy to securing food safety. In this perspective, traceability based on a National Identification System is required.
Animal Feed/*standards ; Animals ; Cattle ; Consumer Product Safety/*legislation&jurisprudence ; Encephalopathy, Bovine Spongiform/epidemiology/*prevention&control ; Female ; Food Chain ; Japan/epidemiology ; Meat/*standards ; Risk Assessment ; Risk Management

BACKGROUNDWe have reported that norcantharidin (NCTD) induces human melanoma A375-S2 cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in the apoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD.

METHODSWe assessed the effects of NCTD on cell growth inhibition using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay, DNA fragmentation (DNA agarose gel electrophoresis), and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were also collected.

RESULTSThe NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKC inhibitors. The expression of phosphorylated JNK and p38 also increased after the treatment with NCTD, and inhibitors of c-Jun NH2-terminal kinase (JNK) and p38 (SP600125 and SB203580, respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38 expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation of phosphorylated JNK and phosphorylated p38, but had little effect on extracellular signal-regulated kinase (ERK) expression.

CONCLUSIONThese results suggest that the activation of JNK and p38 MAPK promotes the process of NCTD-induced A375-S2 cell apoptosis and that PKC plays an important regulation role in the activation of MAPKs.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line, Tumor ; DNA Fragmentation ; drug effects ; Enzyme Activation ; Humans ; Melanoma ; drug therapy ; pathology ; Mitogen-Activated Protein Kinases ; physiology ; Protein Kinase C ; antagonists & inhibitors ; physiology ; Staurosporine ; pharmacology