The effect of aldosterone on connective tissue growth factor (CTGF) was examined in rat embryonic ventricular myocytes. Upon aldosterone treatment, CTGF expression was significantly increased in a dose and time-dependent manner. To explore the molecular mechanism for this upregulation, we examined the role of mineralocorticoid receptor. Pre-treatment of an antagonist (spironolactone) at 5-fold excess of aldosterone blocked the CTGF induction by aldosterone, suggesting that the upregulation was mediated by mineralocorticoid receptor. Aldosterone treatment resulted in activation of ERK1/2, p38 MAPK, and JNK pathways with a more transient pat-tern in p38 MAPK. Blocking studies using pre-treatment of the inhibitor of each path-way revealed that p38 MAPK cascade may be important for aldosterone-mediated CTGF upregulation as evidenced by the blocking of CTGF induction by SB203580 (p38 MAPK inhibitor), but not by PD098059 (ERK1/2 inhibitor) and JNK inhibitor I. Interestingly, JNK inhibitor I and PD098059 decreased the basal level of CTGF expression. On the other hand, pre-treatment of spironolactone abrogated the p38 MAPK activation, indicating that mineralocorticoid receptor mechanism is linked to p38 MAPK pathway. Taken together, our findings suggest that aldosterone induces CTGF expression via both p38 MAPK cascade and mineralocorticoid receptor and that cross-talk exists between the two pathways.
Aldosterone/*pharmacology ; Animals ; Cells, Cultured ; Dose-Response Relationship, Drug ; Gene Expression Regulation/drug effects/physiology ; Heart Ventricles/drug effects/embryology/metabolism ; Immediate-Early Proteins/*metabolism ; Intercellular Signaling Peptides and Proteins/*metabolism ; Myocytes, Cardiac/*drug effects/*metabolism ; Rats ; Receptors, Mineralocorticoid/*metabolism ; Research Support, Non-U.S. Gov't ; Signal Transduction/drug effects/physiology ; Spironolactone/pharmacology ; Up-Regulation/drug effects/physiology ; p38 Mitogen-Activated Protein Kinases/*metabolism

OBJECTIVETo study the effect of anticolchicine cytotoxicity of Dan Gua-Fang, a Chinesea Chinese), a Chinese herbal compound prescription on endothelial cells of vein (ECV304) cultivated in mediums of different glucose concentrations as well as the proliferation of those cells in the same conditions, in order to reveal the value of Dan Gua-Fang in preventing and treating endothelial damage caused by hyperglycemia in diabetes mellitus.

METHODSThe research was designed as three stages. The growing state and morphological changes were observed when ECV304 were cultivated in the culture mediums, which have different glucose concentrations with or without Dan Gua-Fang and at the same time with or without colchicine.

RESULTS(1) Dan Gua-Fang at all concentrations reduced the floating cell population of ECV304 cultivated in hyperglycemia mediums. (2) Dan Gua-Fang at all concentrations and hyperglycemia both had a function of promoting "pseudopod-like" structure formation in cultivated ECV304, but the function was not superimposed in mediums containing both hyperglycemia and Dan Gua-Fang. (3) Colchicine reduced and even vanished the "pseudopod-like" structure of the endotheliocyte apparently cultivated in mediums of hyperglycemia or with Dan Gua-Fang. The "pseudopod-like" structure of the endotheliocyte emerged quickly in Dan Gua-Fang groups after colchicine was removed, but it was not the case in hyperglycemia only without Dan Gua-Fang groups. (4) Dan Gua-Fang reduced the mortality of cells cultivated in mediums containing colchicine. The cell revived to its normal state fast after colchicine was removed.

CONCLUSIONDan Gua-Fang has the functions of promoting the formation of cytoskeleton and fighting against colchicine cytotoxicity.

Cell Culture Techniques ; Cell Line ; Cell Shape ; drug effects ; Colchicine ; adverse effects ; antagonists & inhibitors ; Culture Media ; adverse effects ; pharmacology ; Cytoprotection ; drug effects ; Cytotoxins ; adverse effects ; antagonists & inhibitors ; Drug Antagonism ; Drug Combinations ; Drug Evaluation, Preclinical ; Drug Synergism ; Drugs, Chinese Herbal ; adverse effects ; pharmacology ; Endothelial Cells ; drug effects ; physiology ; Glucose ; pharmacology ; Humans ; Umbilical Veins ; cytology ; drug effects ; Up-Regulation

OBJECTIVETo discuss the effect of protein kinase C (PKC) on regulation of ecto-5'-nucleotidase activity by lysophosphatidylcholine(LPC) in human umbilical endothelial cells (HUVEC).

METHODSExperiments were conducted in HUVEC grown on dishes which were divided into 4 groups (n=15): (1) Control group in which only eAMP (5 micromol/L) was added; (2) LPC group in which HUVEC were incubated with LPC (10 micromol/L) before eAMP was added; (3) Chelerythrine group in which cells were pre-incubated with the PKC inhibitor chelerythrine (100 micromol/L) before LPC and eAMP were added; (4) alpha, beta-Methyladenosine-5'-Diphosphate (AOPCP) group in which cells were incubated with AOPCP (10 micromol/L) before eAMP was added. Etheno-adenosine production was detected at 15th, 30th, 45th min with high performance liquid chromatography(HPLC) respectively.

RESULTSComparing to the control group LPC significantly increased etheno-adenosine production at three time points respectively (P < 0.05). Furthermore, PKC inhibitor chelerythrine abolished this effect of LPC and the ethenoadenosine production at three time points were at the same level of control group (P > 0.05). CD73 inhibitor AOPCP significantly decreased the etheno-adenosine production compared to the other three groups (P < 0.01).

CONCLUSIONEcto-5'-nucleotidase can be modulated within minutes following exposure of HUVEC to LPC and this response may be mediated by PKC in HUVEC.

5'-Nucleotidase ; metabolism ; Cells, Cultured ; GPI-Linked Proteins ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Lysophosphatidylcholines ; pharmacology ; Protein Kinase C ; physiology ; Up-Regulation ; drug effects

Macrophage colony-stimulating factor (M-CSF) is known as one of the factors essential for osteoclast development. In the present study, we examined effects of M-CSF on the apoptotic pathway of osteoclast precursors and their underlying molecular mechanisms. Osteoclast precursors underwent apoptosis in the absence of M-CSF, even in the presence of receptor activator of NF-kB ligand (RANKL). Active caspase-3 and -9 were detected in the osteoclast precursors and treatments of precursors with their specific inhibitors (Z- DEVD-FMK and Z-LEHD-FMK) decreased the apoptosis. M-CSF decreased apoptosis in a dose-dependent manner with decreasing in active caspases-3 and -9 levels and up-regulating Bcl-XL. Those effects of M-CSF on inhibiting apoptosis of osteoclasts precursor by regulating anti-apoptotic signals was more effective when combined with RANKL. These results demonstrate that M-CSF acts as a survival factor for the osteoclast precursors. Furthermore, it is believed that the apoptosis of osteoclast precursors may be involved in the activation of caspase-9 and that M-CSF may promote their survival through Bcl-XL-induced inhibition of caspase-9 activation.
Animals ; Apoptosis/drug effects/physiology ; Carrier Proteins/pharmacology ; Caspases/antagonists & inhibitors/drug effects/metabolism ; Cell Survival/drug effects ; Cells, Cultured ; Cysteine Proteinase Inhibitors/pharmacology ; Enzyme Activation/drug effects ; Female ; Macrophage Colony-Stimulating Factor/*pharmacology ; Membrane Glycoproteins/pharmacology ; Mice ; Mice, Inbred ICR ; Oligopeptides/pharmacology ; Osteoclasts/*cytology/drug effects ; Proto-Oncogene Proteins c-bcl-2/drug effects/*metabolism ; Stem Cells/cytology/*drug effects ; Up-Regulation

Hydroxyurea is commonly used to treat hematologic disorders and some type of solid tumors, but the mechanism for its therapeutic effect is not clearly known. In this study, we examined the effect of hydroxyurea on rat hepatoma McA-RH7777 cells, specifically, on the role of mitogen-activated protein (MAP) kinase signal transduction pathways and p21Waf1, p27Kip1 and p53. Rat hepatoma McA-RH7777 cells treated with hydroxyurea for 7 days, caused the inhibition of cell growth in a dose-dependent manner. But, this growth inhibition was not caused by necrosis or apoptosis but instead was associated with cell senescence-like change as evidenced by senescence associated-beta-galactosidase staining, and cells arrest at G1 phase of cell cycle. Phosphorylation of MAP kinases, such as ERK, JNK, and p38, was found to be decreased after treatment of cells with hydroxyurea. But, the expression of p21Waf1 was increased, while p27Kip1 and p53 were not detected in hydroxyurea treated rat hepatoma cells. Hydroxyurea treatment induced G1 arrest and a senescence-like changes in rat hepatoma McA-RH7777 cells may be the likely results of signal disruption of MAP kinases (ERK, JNK, and p38 MAP kinase) and p21Waf1 over-expression.
Animals ; Antineoplastic Agents/*pharmacology ; Cell Aging/drug effects ; Cell Cycle Proteins/analysis/metabolism/*physiology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; G1 Phase/drug effects/physiology ; Hydroxyurea/*pharmacology ; Liver Neoplasms, Experimental/enzymology/*metabolism ; Mitogen-Activated Protein Kinases/analysis/*physiology ; Phosphorylation/drug effects ; Protein p53/analysis/metabolism ; Rats ; Research Support, Non-U.S. Gov't ; Tumor Suppressor Proteins/analysis/metabolism ; Up-Regulation

OBJECTIVETo explore the effect of morphine on male reproductive ability and its mechanisms in the rat model of morphine tolerance.

METHODSTwenty male SD rats were equally randomized to groups I (control) and II (morphine tolerance). On the 1st day, the basic paw withdrawal thermal latency (PWTL) was obtained from all the rats followed by subcutaneous injection of morphine at 10 mg/kg and then calculation of the percentage of the maximal possible effect (MPE) at 30 min after administration. On the 2nd day, the rats of group I were injected subcutaneously with saline and those of group I with morphine at 10 mg/kg bid for 7 days. Then all the rats were killed after behavioral tests and their testes and epididymides harvested for sperm counting and determina- tion of the expressions of Bax and Caspase-3 by immunohistochemistry.

RESULTSOn the 1st day, no obvious differences were ob- served between the two groups in the basic PWTL or the percentage of MPE. On the 7th day, the percentage of MPE was significantly decreased in group II as compared with group I (P < 0.05), while the basic PWTL showed no marked difference between the two groups. Group II also exhibited a significantly reduced epididymal perm count (P < 0.05) and remarkably upregulated expressions of Bax and Caspase-3 in comparison with group I.

CONCLUSIONMorphine might increase testicular cell apoptosis and reduce sperm concentration by upregulating the expressions of Bax and Caspase-3 in the rat model of morphine tolerance.

Analgesics, Opioid ; pharmacology ; Animals ; Caspase 3 ; metabolism ; Drug Tolerance ; physiology ; Hot Temperature ; Male ; Morphine ; pharmacology ; Random Allocation ; Rats ; Reproduction ; drug effects ; Sperm Count ; Testis ; drug effects ; Time Factors ; Up-Regulation ; bcl-2-Associated X Protein ; metabolism

FTY720, a synthetic sphingoid base analog, was examined as a new sphingosine kinase inhibitor, which converts endogenous sphingosine into its phosphate form. With 20 micrometer of FTY720, sphingosine accumulated in the LLC-PK1 cells in a time- and dose-dependent manner. The FTY720 treated cells showed a high concentration of fragmented DNA, a high caspase-3 like activity and TUNEL staining cells. It was also found that the sphingosine and sphinganine level increased in a time- and dose-dependent manner within 12 h after the FTY720 treatment. The sphingosine kinase activity was reduced by FTY720 as much as other sphingosine kinase inhibitors, N, N-dimethylsphingosine (DMS), dl-threo-dihydrosphingosine (DHS). The fragmented DNA content as a result of the 20 micrometer of FTY720 treatment and by 5 micrometer of the exogenously added BSA-sphingosine complex indicated typical apoptosis. Under similar conditions, the accumulated sphingosine concentration in all the cells was almost identical even though the sphingosine distribution inside the cells was somewhat different. These results indicate that the FTY720 induced apoptosis is associated with the inhibition of the sphingosine kinase activity and is strongly associated with the successive accumulation of sphingosine.
Animals ; Apoptosis/*physiology ; Caspases/biosynthesis ; Cell Line ; DNA Fragmentation ; Endothelial Cells/drug effects ; Enzyme Inhibitors/*pharmacology ; Kidney/cytology ; Phosphotransferases (Alcohol Group Acceptor)/*antagonists & inhibitors/physiology ; Propylene Glycols/*pharmacology ; Research Support, Non-U.S. Gov't ; Sphingosine/pharmacology/*physiology ; Swine ; Up-Regulation

Proteasome inhibitors have shown remarkable success in the treatment of hematologic neoplasm. There has been a lot of attention to applying these drugs for solid tumor treatment. Recent preclinical study has signified the effectiveness on cell proliferation inhibition in lung adenocarcinoma treated by carfilzomib (CFZ), a second generation proteasome inhibitor. However, no insight has been gained regarding the mechanism. In this study, we have systematically investigated the CFZ functions in cell proliferation and growth, cell cycle arrest, and apoptosis in lung adenocarcinoma cells. Flow cytometry experiments showed that CFZ significantly induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma. MTS and colony formation assays revealed that CFZ substantially inhibited survival of lung adenocarcinoma cells. All results were consistently correlated to the upregulation expression of Gadd45a, which is an important gene in modulating cell cycle arrest and apoptosis in response to physiologic and environmental stresses. Here, upregulation of Gadd45a expression was observed after CFZ treatment. Knocking down Gadd45a expression suppressed G2/M arrest and apoptosis in CFZ-treated cells, and reduced cytotoxicity of this drug. The protein expression analysis has further identified that the AKT/FOXO3a pathway is involved in Gadd45a upregulation after CFZ treatment. These findings unveil a novel mechanism of proteasome inhibitor in anti-solid tumor activity, and shed light on novel preferable therapeutic strategy for lung adenocarcinoma. We believe that Gadd45a expression can be a highly promising candidate predictor in evaluating the efficacy of proteasome inhibitors in solid tumor therapy.
Adenocarcinoma of Lung/pathology* ; Apoptosis/drug effects* ; Cell Cycle Checkpoints/drug effects* ; Cell Cycle Proteins/genetics* ; Cell Line, Tumor ; Forkhead Box Protein O3/physiology* ; Gene Expression Regulation, Neoplastic/drug effects* ; Humans ; Lung Neoplasms/pathology* ; Oligopeptides/pharmacology* ; Proto-Oncogene Proteins c-akt/physiology* ; Up-Regulation

BACKGROUNDThe role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin.

METHODSThe inhibitory effect of shikonin on the growth of HeLa cells was measured by MTT assay. Fluorescent microscopic analysis of apoptotic cells stained with 4',6'-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis.

RESULTSShikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK), inhibitor PD 98059.

CONCLUSIONShikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.

Apoptosis ; drug effects ; Caspases ; physiology ; Cell Proliferation ; drug effects ; DNA Damage ; Flavonoids ; pharmacology ; HeLa Cells ; Humans ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Naphthoquinones ; pharmacology ; Phosphorylation ; Tumor Suppressor Protein p53 ; analysis ; Up-Regulation

This study was designed to investigate the effects of the prenylated flavonoid kurarinone on TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis and its underlying mechanism. A low dose of kurarinone had no significant effect on apoptosis, but this compound markedly promoted tumor cell death through elevation of Bid cleavage, cytochrome c release and caspase activation in HeLa cells treated with TRAIL. Caspase inhibitors inhibited kurarinone-mediated cell death, which indicates that the cytotoxic effect of this compound is mediated by caspase-dependent apoptosis. The cytotoxic effect of kurarinone was not associated with expression levels of Bcl-2 and IAP family proteins, such as Bcl-2, Bcl-xL, Bid, Bad, Bax, XIAP, cIAP-1 and cIAP-2. In addition, this compound did not regulate the death-inducing receptors DR4 and DR5. On the other hand, kurarinone significantly inhibited TRAIL-induced IKK activation, IkappaB degradation and nuclear translocation of NF-kappaB, as well as effectively suppressed cellular FLICE-inhibitory protein long form (cFLIPL) expression. The synergistic effects of kurarinone on TRAIL-induced apoptosis were mimicked when kurarinone was replaced by the NF-kappaB inhibitor withaferin A or following siRNA-mediated knockdown of cFLIPL. Moreover, cFLIP overexpression effectively antagonized kurarinone-mediated TRAIL sensitization. These data suggest that kurarinone sensitizes TRAIL-induced tumor cell apoptosis via suppression of NF-kappaB-dependent cFLIP expression, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.
Antineoplastic Agents/*pharmacology ; Apoptosis/*drug effects ; CASP8 and FADD-Like Apoptosis Regulating Protein/*genetics/metabolism ; Caspase 3/metabolism ; Caspase 8/metabolism ; Drug Synergism ; Enzyme Activation/drug effects ; Flavonoids/*pharmacology ; Gene Expression/drug effects ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; NF-kappa B/antagonists & inhibitors/*metabolism ; Protein Transport/drug effects ; RNA, Small Interfering/genetics ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand/*physiology ; Up-Regulation/drug effects