Thirty-three patients with complaints of idiopathic oligo-asthenozoospermia were treated with traditional Chinese medicine. Their symptom complexes were diagnosed from the states of qi and blood, the conditions of body fluid, and pathological changes of zan-fu organs by means of four traditional methods in Chinese medicine, i. e., inspection, auscultation and olfaction, inquiry, and pulse-feeling and palpation. The patients were classified into Stagnation of the Liver-Qi (Kankiutsu-sho, 11 cases), Deficiency of Kidney (Jinkyosho, 5 cases), Deficiency of the Spleen-Qi (Hikikyo-sho, 9 cases), Phlegm-Dampness (Tanshitsu-sho, 5 cases), Damp-Heat (Shitsunetsu-sho, 3 cases).
The changes in target symptoms and signs, semen qualities, and serum hormones were compared before and after six months of treatment with the corresponding prescriptions. Seven successful pregnancies have been accomplished, with a pregnancy rate of 21.2%. Although no significant increases in sperm concentration and motility were recorded, most target symptoms and signs were improved, and the serum testosterone and estradiol were significantly decreased after treatment.

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

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

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

AIMTo study the mechanism of dracorhodin perchlorate-induced Hela cell apoptosis.

METHODSCell viability was measured by MTT method. Morphological changes were observed by phase contrast microscopy and Hoechst 33258 staining. DNA fragmentation was assayed by agarose gel electrophoresis. Protein expression was detected by Western blot analysis.

RESULTSDracorhodin perchlorate induced Hela cell apoptosis. The apoptosis was partially reversed by caspase-1, -3, -8, -9 and caspase family inhibitors. Treatment of Hela cells with dracorhodin perchlorate for 12 h increased the protein expression ratio of Bax/Bcl-XL; procaspase-3, -8, ICAD and PARP were cleaved to smaller molecules.

CONCLUSIONDracorhodin perchlorate induced Hela cell death via alteration of Bax/Bcl-XL ratio and activation of caspases.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; Arecaceae ; chemistry ; Benzopyrans ; isolation & purification ; pharmacology ; Caspase Inhibitors ; Caspases ; metabolism ; Cell Line, Tumor ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; HeLa Cells ; Humans ; Plants, Medicinal ; chemistry ; Proteins ; metabolism

Pseudolaric acid B was isolated from Pseudolarix kaempferi Gordon (Pinaceae) and was evaluated for the anti-cancer effect in HeLa cells. We observed that pseudolaric acid B inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. HeLa cells treated with pseudolaric acid B showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. JNK inhibitor, SP600125, markedly inhibited pseudolaric acid B-induced cell death. In addition, Bcl-2 expression was down-regulated while Bax protein level was up-regulated. Caspase-3 inhibitor, z-DEVD-fmk, partially blocked pseudolaric acid B-induced cell death, and the expression of two classical caspase substrates, PARP and ICAD, were both decreased in a time- dependent manner, indicative of downstream caspase activation.
Anthracenes/pharmacology ; *Apoptosis ; Caspases/antagonists & inhibitors/*metabolism ; Cell Proliferation/drug effects ; Cysteine Proteinase Inhibitors/pharmacology ; Diterpenes/*pharmacology ; Down-Regulation ; Enzyme Activation ; Hela Cells ; Humans ; JNK Mitogen-Activated Protein Kinases/drug effects/*metabolism ; Oligopeptides/pharmacology ; Protein Kinase Inhibitors/pharmacology ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Signal Transduction/*drug effects ; Up-Regulation

Interleukin-1beta (IL-1beta) is a pivotal proinflammatory cytokine. To investigate the mechanism of IL-1beta-induced cell death in human malignant melanoma A375-S2 cells, MTT assay, photomicroscopical observation, DNA agarose gel electrophoresis, radioimmunoassay and Western blot analysis were carried out. IL-1beta did not only induce nuclear condensation and DNA fragmentation, but also increased degradation of two substrates of caspase-3, poly ADP-ribose polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD). Simultaneously, release of precursor of IL-1beta (pro-IL-1beta) and endogenous IL-1beta production were involved in the apoptotic process. IL-1beta enhanced the ratio of Bax/Bcl-2 and Bax/Bcl-xL expression and up-regulated apoptosis inducing factor (AIF) expression, which required the activation of downstream caspases. These results suggest that IL-1beta induces endogenous IL-1beta production, enhances cleavage of caspase downstream substrates and promotes mitochondria mediated apoptosis in A375-S2 cells.
Apoptosis/*drug effects ; Blotting, Western ; Caspase 1/metabolism ; Caspases/metabolism ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Comparative Study ; DNA Fragmentation/drug effects ; Deoxyribonucleases/metabolism ; Dose-Response Relationship, Drug ; Enzyme Activation/drug effects ; Humans ; Interleukin-1/biosynthesis/*pharmacology/physiology ; Interleukin-6/pharmacology ; Lymphotoxin/pharmacology ; Melanoma/metabolism/pathology/physiopathology ; Mitochondria/*physiology ; Poly(ADP-ribose) Polymerases/metabolism ; Proto-Oncogene Proteins c-bcl-2/biosynthesis ; Time Factors

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