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 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.

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 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.

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. [

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

AIMTo study the mechanism of evodiamine-induced cell death of A375-S2.

METHODSThe changes in cell morphology were observed by invert microscopy and Hoechst 33258 staining. DNA fragmentation was assayed by agarose gel electrophoresis. The effects of evodiamine on apoptosis and cell cycle were studied by flow cytometric analysis.

RESULTSEvodiamine was shown to markedly inhibit the growth of A375-S2 cells in dose- and time-dependent manners. At the early stage, evodiamine activated caspase cascades, which unexpectedly did not induce typical DNA fragmentation. At later stage, caspase inhibitors failed to block A375-S2 cell death induced by evodiamine. Evodiamine-induced cell death was shown to be not directly associated with cell cycle arrest.

CONCLUSIONAt the early stage, evodiamine initiates caspase-dependent and a typical apoptosis pathway in A375-S2 cells, but later it induces cell death through caspase-independent pathway which might be necrosis.

Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; Apoptosis ; Caspase Inhibitors ; Caspases ; metabolism ; Cell Cycle ; Cell Division ; drug effects ; DNA Fragmentation ; physiology ; Dose-Response Relationship, Drug ; Evodia ; chemistry ; Humans ; Melanoma ; enzymology ; pathology ; Plant Extracts ; administration & dosage ; isolation & purification ; pharmacology ; Quinazolines ; administration & dosage ; isolation & purification ; pharmacology ; Time Factors ; Tumor Cells, Cultured

Silibinin is a polyphenolic flavanoid derived from fruits and seeds of milk thistle (Silybum marianum). To investigate the effect and mechanism of silibinin on beta-isoproterenol-induced rat neonatal cardiac myocytes injury, the viability, the activation of lactate dehydrogenase (LDH) and the content of maleic dialdehyde (MDA) were chosen for measuring the degree of cardiac myocytes injury. Superoxide dismutase (SOD) activity, mitochondrial membrane potential (deltapsi) detected by flow cytometric analysis, and Western blotting analysis were applied to determine the related proteins. Silibinin protected isoproterenol-treated rat cardiac myocytes from death and significantly decreased LDH release and MDA production. Silibinin increased superoxide dismutase (SOD) activity, and increased mitochondrial membrane potential (deltapsi). Furthermore, the release of pro-apoptotic cytochrome c from mitochondria was reduced by silibinin. Silibinin increased the expression of anti-apoptotic Bcl-2 family protein Bcl-2, and up-regulation of SIRT1 inhibited the translocation of Bax from cytoplasm to mitochondria, which caused mitochondrial dysfunction and cell injury. Silibinin protects cardiac myocytes against isoproterenol-induced injury through resuming mitochondrial function and regulating the expression of SIRT1 and Bcl-2 family members.
Animals ; Animals, Newborn ; Blotting, Western ; Cardiotonic Agents ; isolation & purification ; pharmacology ; Cell Survival ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Isoproterenol ; toxicity ; L-Lactate Dehydrogenase ; metabolism ; Malondialdehyde ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; Milk Thistle ; chemistry ; Mitochondria, Heart ; drug effects ; metabolism ; physiology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Silymarin ; isolation & purification ; pharmacology ; Sirtuin 1 ; Sirtuins ; metabolism ; Superoxide Dismutase ; metabolism ; Up-Regulation ; bcl-2-Associated X Protein ; metabolism

To study the mechanism of downregulation of apoptosis by autophagy induced by oridonin in HeLa cells, the cell viability was measured by MTT method. DNA fragmentation was assayed by agarose gel electrophoresis. Autophagic and apoptotic ratio was determined by flowcytometric analysis. Protein expression was detected by Western blotting analysis. Oridonin induced both apoptosis and autophagy in HeLa cells. Apoptosis was upregulated by introduction of the inhibitor of autophagy, 3-methyladenine (3-MA). Addition of oridonin increased Bax/Bcl-2 expression ratio and cytochrome c, whereas the expression of SIRT-1 was decreased, and 3-MA pre-application enhanced these changes. Oridonin-induced autophagy antagonized apoptosis in HeLa cells through mitochondrial pathway.
Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Apoptosis ; drug effects ; Autophagy ; drug effects ; Blotting, Western ; Cytochromes c ; metabolism ; Diterpenes ; isolation & purification ; pharmacology ; Diterpenes, Kaurane ; isolation & purification ; pharmacology ; Flow Cytometry ; HeLa Cells ; Humans ; Isodon ; chemistry ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Sirtuin 1 ; Sirtuins ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo study the mechanisms of oridonin-induced U937 cell apoptosis, and to examine the role of ERK MAPK.

METHODMTT, Hoechst 33258 staining, DNA agarose gel electrophoresis and Western blot analysis were used.

RESULTOridonin inhibited U937 cell growth in a time- and dose-dependent manner. Apoptotic bodies were found with Hoechst 33258 staining after treatment with 27 micromol x L(-1) oridonin. Simultaneously, ERK phosphorylation was significant. ERK inhibitor PD98059 partially blocked the growth-inhibitory effect as well as DNA fragmentation. The expression of antiapoptotic mitochondrial protein Bcl-XL decreased time-dependently, and that of proapoptotic protein Bax increased. However, PD98059 reversed the effect of oridonin on Bcl-XL and Bax.

CONCLUSIONOridonin induces U937 cell apoptosis through activation of ERK and alteration of the ratio of Bax/Bcl-XL.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; DNA Fragmentation ; drug effects ; Diterpenes ; administration & dosage ; isolation & purification ; pharmacology ; Diterpenes, Kaurane ; administration & dosage ; isolation & purification ; pharmacology ; Dose-Response Relationship, Drug ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Flavonoids ; pharmacology ; Humans ; Isodon ; chemistry ; Phosphorylation ; Plants, Medicinal ; chemistry ; U937 Cells ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism