TNF-alpha has been shown to induce matrix metalloproteinase-9 (MMP-9) expression, which, in turn, degrades extracellular matrix in the inflammatory responses. However, the inductive mechanisms of the MMP-9 by TNF-alpha remain unclear. In human tracheal smooth muscle cells, TNF-alpha induced MMP-9 expression and Akt phosphorylation in a time-dependent manner, which was attenuated by the inhibitors of Src (PP1), epidermal growth factor receptor (AG1478), PDGFR (AG1296), and PI3K (LY294002), respectively, revealed by reporter gene assay, RT-PCR, zymographic, and Western blot analyses. Transfection with the dominant negative mutants of c-Src (KM, K295M [kinase inactive mutant]), p85, and Akt (KA, K179A) also reduced MMP-9 expression. These findings indicated that MMP-9 expression was regulated by PI3K/Akt via the transactivation of growth factor receptors. Furthermore, LY294002 or wortmannin inhibited Akt phosphorylation but had no effect on NF-kappaB translocation, which was blocked by helenalin. Mutated NF-kappaB DNA binding element in the MMP-9 promoter and helenalin also attenuated MMP-9 expression, suggesting that PI3K/Akt and NF-kappaB independently regulated MMP-9 expression. To support this notion, immunofluorescence staining and immunoprecipitation were applied to characterize the transcription factors involved in these responses. The results showed that LY294002 and curcumin blocked Akt translocation into nucleus. In contrast, p300, acetyl-histone (H3), and NF-kappaB p65 were found to be coimmunoprecipitated with the phosphorylated Akt, indicating that these components associated with the MMP-9 promoter are revealed by chromatin immunoprecipitation assay. Thus, our study provides a new insight into the molecular mechanisms that TNF-alpha-stimulated Akt phosphorylation mediated through transactivation of Src and growth factor receptors may stimulate the recruitment of p300, assemble transcription factor (p65), and then lead to MMP-9 expression.

The effects of swimming and lactate on the release of testosterone were examined in male rats. During in vivo experiments, male rats were catheterized via the right jugular vein and blood was collected at 0, 10, 15, 30, and 60 min following the exercise, or they were catheterized via the right jugular vein and the left femoral vein and blood was collected at 0, 2, 5, 10, 15, 30, 60, and 120 min after a 10-min infusion at lactate (13 mg.kg-1.min-1). Trunk blood and blood from the testicular vein were also collected after 10 min of swimming or water immersion. In an in vitro experiment, testicular fragments were challenged with lactate (0.01-10 mM) and/or human chorionic gonadotropin (hCG; 0.5 IU.mL-1), and the mediobasal hypothalamus (MBH) was challenged with lactate (8 mM). The post-exercise levels of plasma lactate and testosterone at 10, 15, and 30 min were higher than resting levels. Plasma luteinizing hormone (LH) was increased following 30 min of swimming. Administration of lactate or hCG increased in a dose dependent manner testicular cyclic adenosine 3':5' monophosphate (cAMP) and testosterone release. Plasma testosterone increased after swimming and lactate infusion. Incubation of MBH with lactate increased the gonadotropin-releasing hormone (GnRH) level in the medium. These results suggest that the increased plasma testosterone levels in male rats during exercise is at least partially a result of a direct and LH-independent stimulatory effect of lactate on the secretion of testosterone by increasing testicular cAMP production. Swim-elevated plasma LH may be a result of a rise of GnRH caused by lactate.

The incidence of thyroid cancer increases with age, and it is twice in women as common as in men. The undifferentiated thyroid cancer (UTC) is the most aggressive of all thyroid cancers. Unfortunately, there are almost no efficacious therapeutic modalities. It is important to develop some new effective therapies. Evodiamine is a chemical extracted from a kind of Chinese herb named Wu-Chu-Yu and has been demonstrated to be effective in preventing the growth of a variety of cancer cells. In the present study, the mechanism by which evodiamine inhibited the undifferentiated thyroid cancer cell line ARO was examined. Based on 3-(4,5-dimethylthiazol -2-yle)2,5-diphenyltetrazolium bromide (MTT) assay, cell proliferation rate was reduced dose-dependently by evodiamine, but not by rutaecarpine. According to the flow cytometric analysis, evodiamine treatment resulted in G2/M arrest and DNA fragmentation in ARO cells. The G2/M arrest was accompanied with an increase of the expression of cdc25C, cyclin B1, and cdc2-p161 protein, and it was also with a decrease of the expression of cdc2-p15. Furthermore, by using the TUNEL assay, evodiamine-induced apoptosis was observed at 48 h and extended to 72 h. Western blotting demonstrated that evodiamine treatment induced the activation of caspase-8, caspase-9, caspase-3, and the cleavage of poly ADP-ribose polymerase (PARP). These results suggested that evodiamine inhibited the growth of the ARO cells, arrested them at M phase, and induced apoptosis through caspases signaling.

Previously we found that the increased plasma testosterone levels in male rats during exercise partially resulted from a direct and luteinizing hormone (LH)-independent stimulatory effect of lactate on the secretion of testosterone. In the present study, the acute and direct effects of lactate on testosterone production by rat Leydig cells were investigated. Leydig cells from rats were purified by Percoll density gradient centrifugation subsequent to enzymatic isolation of testicular interstitial cells. Purified rat Leydig cells (1 x 10(5) cells/ml) were in vitro incubated with human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (an adenylyl cyclase activator, 10(-5) M), or 8-bromo-adenosine-3':5'-cyclic monophosphate (8-Br-cAMP, 10(-4) M), SQ22536 (an adenylyl cyclase inhibitor, 10(-6)-10(-5) M), steroidogenic precursors (25-hydroxy-cholesterol, pregnenolone, progesterone, and androstenedione, 10(-5) M each), nifedipine (a L-type Ca(2+) channel blocker, 10(-5)-10(-4) M), or nimodipine (a potent L-type Ca(2+) channel antagonist, 10(-5)-10(-4) M) in the presence or absence of lactate at 34 degrees C for 1 h. The concentration of medium testosterone was measured by radioimmunoassay. Administration of lactate at 5-20 mM dose-dependently increased the basal testosterone production by 63-187% but did not alter forskolin- and 8-Br-cAMP-stimulated testosterone release in rat Leydig cells. Lactate at 10 mM enhanced the stimulation of testosterone production induced by 25-hydroxy-cholesterol in rat Leydig cells but not other steroidogenic precursors. Lactate (10 mM) affected neither 30- nor 60-min expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein. The lactate-stimulated testosterone production was decreased by administration of nifedipine or nimodipine. These results suggested that the physiological level of lactate stimulated testosterone production in rat Leydig cells through a mechanism involving the increased activities of adenylyl cyclase, cytochrome P450scc, and L-type Ca(2+) channel.

Hypoxia has been shown to stimulate the expression of vascular endothelial growth factor (VEGF), which is a major mediator for angiogenesis and vasculogenesis. During hypoxia, VEGF promotes angiogenesis in the testis. However, the effect of VEGF on the steroidogenesis of testosterone and the cell proliferation in Leydig cells is unclear. To assess the effects and the action mechanisms of hypoxia, a mouse TM3 Leydig cell line was employed in the present study. The Leydig cells were incubated in an incubator chamber (95% N2-5% CO2) for 1–24 h. The cultured media were collected and assayed by testosterone RIA and VEGF enzyme immunoassay. 3-(4,50-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide assay was used to detect the proliferation of Leydig cells. The present results showed that the proliferation of Leydig cells was enhanced significantly by hypoxia. The basal VEGF release was increased, and the response of VEGF production to human chorionic gonadotropin (hCG) was also enhanced in hypoxic condition. During hypoxia, administration of hCG or VEGF stimulated proliferation of Leydig cells, but the stimulatory effect was abolished by the administration of anti-VEGF antibody. Higher doses of VEGF stimulated testosterone release in a dose-dependent manner. Administration of anti-VEGF antibody abolished the stimulatory effect of VEGF on testosterone release. These data suggest that hypoxia stimulates cell proliferation and testosterone release in Leydig cells via an increase of VEGF production.

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1 Digoxin (10 77 ± 10 75 M) or digitoxin (10 77 ± 10 75 M) decreased the basal and human chorionic gonadotropin (hCG)-stimulated release of progesterone from rat granulosa cells. 2 Digoxin (10 75 M) or digitoxin (10 75 M) attenuated the stimulatory eects of forskolin and 8-bromo-cyclic 3' : 5'-adenosine monophosphate (8-Br-cAMP) on progesterone release from rat granulosa cells. 3 Digoxin (10 75 M) or digitoxin (10 75 M) inhibited cytochrome P450 side chain cleavage enzyme (cytochrome P450 scc ) activity (conversion of 25-hydroxyl cholesterol to pregnenolone) in rat granulosa cells but did not in¯uence the activity of 3b-hydroxysteroid dehydrogenase (3b-HSD). 4 Neither progesterone production nor P450scc activity in rat granulosa cells was altered by the administration of ouabain. 5 Digoxin (10 75 M) or digitoxin (10 75 M), but not ouabain, decreased the expression of P450scc and steroidogenic acute regulatory (StAR) protein in rat granulosa cells. 6 The present results suggest that digoxin and digitoxin decrease the progesterone release by granulosa cells via a Na + ,K + -ATPase-independent mechanism involving the inhibition of post-cyclic AMP pathway, cytochrome P450scc and StAR protein functions.

Hypoxia induces metabolic alteration in cancer cells by stabilizing hypoxia-inducible factor 1a (HIF-1a (HIF1A)), which regulates the bioenergetic genes of glycolysis and lipid metabolic pathways. However, the target genes of hypoxia-induced metabolic alterations in the prostate remain uncertain. Mitochondrial aconitase (mACON) (ACONM) is an enzyme that is central to carbohydrate and energy metabolism and is responsible for the interconversion of citrate to isocitrate as part of the citric acid cycle in the human prostate. We evaluated the effects of the molecular mechanisms of hypoxia on mACON gene expression in PC-3 and LNCaP human prostate carcinoma cells. Immunoblotting assays revealed that hypoxia modulated mACON and lactate dehydrogenase A (LDHA) protein expression, while these effects were attenuated when HIF-1a was knocked down. Hypoxia induced fatty acid synthase (FASN) in PC-3 cells while hypoxia blocked FASN gene expression in LNCaP cells after 24-h incubation. Results of real-time RT-qPCR, immunoblotting, and transient gene expression assays revealed that hypoxia treatment or co-transfection with HIF-1a expression vector enhanced gene expression of mACON, implying that hypoxia modulated mACON at the transcriptional level. Hypoxia-induced mACON promoter activity is dependent on the DNA fragment located at K1013 to K842 upstream of the translation initiation site. L-mimosine, an iron chelator, stabilized HIF-1a but downregulated mACON gene expression, suggesting that iron chelation blocked the hypoxia-induced mACON gene expression. These results suggest that hypoxia dysregulates the expressions of LDHA, FASN, and mACON genes, and the hypoxia-induced mACON gene expression is via the HIF-1a-dependent and iron-dependent pathways in prostate carcinoma cells.