Paulus S. Wang

INHIBITORY EFFECTS OF DIGITALIS ON THE PROLIFERATION OF ANDROGEN DEPENDENT AND INDEPENDENT PROSTATE CANCER CELLS

PURPOSE Digitalis or cardiac glycosides have been noted to induce tumor static or oncolytic effects in various types of cancer. We evaluated the effects and underlying mechanisms of cardiac glycosides, including digoxin, digitoxin and ouabain, on the proliferation of hormone dependent and independent prostate cancer cell lines. MATERIALS AND METHODS Cell proliferation of the 3 human prostate cancer cell lines LNCaP, DU145 and PC3 was measured by 3-(4,5-dimethylthiazol-2-yle)2,5-diphenyltetralozium bromide (Sigma Chemical Co., St. Louis, Missouri) colorimetric assay. The cytotoxic effects of digitalis on prostate cancer cells were determined by lactate dehydrogenase measurements of the culture medium. Intracellular Ca2+ was measured by a dual wavelength spectrometer system. The percent of apoptotic cells after digitalis treatment was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and flow cytometry. RESULTS Digoxin, digitoxin and ouabain significantly inhibited the proliferation of LNCaP, DU145 and PC3 cells at a dose of 1 or 10 microM. after 1 to 4 days of culture. Cytotoxicity of digitalis on the DU145 and LNCaP cells was dose dependent but cytotoxicity was not obvious in PC3. Digitalis (1 microM.) significantly increased intracellular Ca2+ in LNCaP and DU145 after 12 hours of culture but PC3 cells needed a 24-hour treatment to show any effect. In the apoptosis measurement digitalis at a dose of 1 and 10 microM. also significantly increased the percent of apoptotic cells in the LNCaP, DU145 and PC3 cell lines. Normal control human glomerular epithelial cells showed no response to digitalis treatment at all tested doses. CONCLUSIONS Digitalis may inhibit the proliferation of prostate cancer cell lines, although the 3 cell lines showed varied sensitivity to digitalis. These effects are possibly the result of a mechanism involving sustained elevation of the concentration of intracellular Ca2+ and of apoptosis.

Inhibitory effects of evodiamine on the growth of human prostate cancer cell line LNCaP

Evodiamine, isolated from a Chinese herbal drug named Wu‐Chu‐Yu, possesses many biological functions. Recently, it has been reported that Wu‐Chu‐Yu exerts an antiproliferative effect on several cancers. Prostate carcinoma initially occurs as an androgen‐dependent tumor and is the second leading cause of cancer death in American males. In the present study, the effect of evodiamine on the growth of androgen‐dependent prostate cancer cell line LNCaP in vitro was examined. Based on [3‐(4,5‐dimethylthiazol‐2‐yle)2,5‐diphenyltetrazolium bromide] (MTT) assay, evodiamine significantly inhibited the growth of LNCaP cells in a concentration‐dependent manner. A significant and concentration‐dependent inhibitory effect of evodiamine on LNCaP cell growth was observed at 24 hr and persisted for 96 hr. The examination of lactate dehydrogenase (LDH) assay showed that the cytotoxic effects of evodiamine on LNCaP cells were concentration dependent. Furthermore, we examined the influences of evodiamine on cell death and cell cycle. The flow cytometric analysis of evodiamine‐treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest reached a maximum at 24 hr after evodiamine treatment. The G2/M arrest was accompanied by an elevated p34cdc2 kinase activity and an increase in the protein expression of cyclin B1 and phosphorylated form of p34cdc2 (Thr 161). Examination of TUNEL showed that evodiamine‐induced apoptosis was observed at 24 hr and extended for 72 hr. Evodiamine elevated caspase‐3, and caspase‐9 activities and the processing of caspase‐3 and caspase‐9. These results suggested that evodiamine inhibits the growth of prostate cancer cell line, LNCaP, through an accumulation of cell cycle at G2/M phase and an induction of apoptosis.

Effects of polybrominated diphenyl ethers on steroidogenesis in rat Leydig cells

BACKGROUND Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that have been defined as major environmental pollutants. While previous studies have found that PBDEs may enhance the levels of sex-steroid hormones, their effects on testosterone secretion from rat Leydig cells are unclear. This study investigated the effects and mechanisms of PBDE-710, a mixture of tetra- and penta-PBDEs, on testosterone biosynthesis in rat Leydig cells. METHODS Leydig cells from adult male rats were challenged with different concentrations of PBDE-710 (0.5-15 ng/ml) to evaluate the effects on testosterone steroidogenesis. Concentrations of testosterone and of cAMP and pregnenolone in medium were measured by radioimmunoassay (RIA) and by enzyme-linked immunosorbent assay, respectively. Nuclear translocation of protein kinase A α (PKAα) was determined by immunofluorence assay and western blot assay, and the mRNA expression of steroidogenic acute regulatory protein (StAR) was analyzed by quantitative real-time polymerase chain reaction. RESULTS In this in vitro study, PBDE-710 (5 or 15 ng/ml) increased basal testosterone secretion and cAMP production by 3- and 2-fold, respectively. The stimulatory effect was abolished by adenylyl cyclase inhibitor. Enzyme activity of CYP11A1, as determined by the pregnenolone concentration, was stimulated by PBDE-710 treatment. Furthermore, nuclear translocation of PKAα was increased by 20% and StAR gene expression was elevated by 4-fold after PBDE-710 treatment. CONCLUSIONS These results suggest that low concentrations of PBDE-710 could stimulate testosterone secretion by acting directly on Leydig cells to activate the cAMP pathway and increase expression of StAR.

Vasoactive intestinal polypeptide appears to be one of the mediators in misoprostol‐enhanced small intestinal transit in rats

Abstract Background and Aims: Prostaglandin analogs have the pharmacologic effect of speeding up small intestinal transit (SIT). It remains unknown whether some gut peptides also mediate this enhancement. We studied the effect of misoprostol on rat SIT and looked at the role of vasoactive intestinal polypeptide (VIP) release during its action.

Direct effect of propylthiouracil on progesterone release in rat granulosa cells

The present study was to investigate the direct effect and action mechanism of propylthiouracil (PTU), an antithyroid drug, on the production of progesterone in rat granulosa cells. PTU (3–12 mM) decreased the basal and human chorionic gonadotropin (hCG)‐stimulated release of progesterone from rat granulosa cells. PTU (3–12 mM) attenuated the stimulatory effects of forskolin and 8‐bromo‐cyclic 3′:5′‐adenosine monophosphate on progesterone release from rat granulosa cells. PTU (12 mM) inhibited the activities of both the cytochrome P450 side‐chain cleavage enzyme (P450scc, conversion of 25‐hydroxyl cholesterol to pregnenolone) and the 3β‐hydroxysteroid dehydrogenase (conversion of pregnenolone to progesterone) in rat granulosa cells. PTU decreased the Vmax but increased the Km of P450scc. PTU (12 mM) decreased the hCG‐increased amount of steroidogenic acute regulatory (StAR) protein in rat granulosa cells. The present results suggest that PTU decreases the progesterone release by granulosa cells via a thyroid‐independent mechanism involving the inhibition of post‐cAMP pathway, and the activities of intracellular calcium, steroidogenic enzyme, and StAR protein functions.

Effects of nonylphenol on aldosterone release from rat zona glomerulosa cells

Alkylphenol ethoxylate, which consists of approximately 80% nonylphenol ethoxylate (NPE), is a major nonionic surfactant. Nonylphenol (NP), the primary degradation product of NPE, has been reported to interfere with reproduction in fish, reptiles, and mammals by inducing cell death in the gonads and by affecting other reproductive parameters. However, the effects of NP on rat adrenal zona glomerulosa cells (ZG) and the underlying mechanisms remain unclear. In this study, we explored the effects of NP on aldosterone release. ZG cells were incubated with NP in the presence or absence of the secretagogues angiotensin II (ANG II), potassium, 8-Br-cAMP, 25-OH-cholesterol, corticosterone or cyclopiazonic acid (CPA). After performing radioimmunoassay (RIA) and Western blot analysis, we found that (1) NP stimulated aldosterone release in cells induced by ANG II, KCl, 8-Br-cAMP, 25-OH-cholesterol, corticosterone, and CPA; (2) NP triggered the release of higher amounts of pregnenolone in cells treated with vehicle and 25-OH-cholesterol+trilostane than in cells treated with other compounds; and (3) the stimulatory effect of NP seemed to be mediated through steroidogenic acute regulatory protein (StAR) and aldosterone synthase activity. These observations suggest that the effects of NP are mediated via increased free Ca(2+) in the cytoplasm.

Effects of dehydroepiandrosterone on aldosterone release in rat zona glomerulosa cells

The present study was to investigate the effects and action mechanisms of dehydroepiandrosterone (DHEA) on steroidogenesis in rat adrenal zona glomerulosa cells (ZG). ZG cells were incubated with DHEA in the presence or absence of angiotensin II (AngII), a high concentration of potassium, 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone, deoxycorticosterone, corticosterone, A23187, or cyclopiazonic acid (CPA) at 37 degrees C for 1 h. The concentration of aldosterone or pregnenolone in the culture medium was then measured by radioimmunoassay (RIA). The cells were used to determine the cellular cAMP content. The data demonstrated that: (1) DHEA inhibited AngII-, high concentration of KCl-, forskolin-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone-, deoxycorticosterone-, corticosterone-, A23187-, or CPA-stimulated aldosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA noncompetitively inhibited aldosterone synthase but showed uncompetitive inhibition of P450scc. These results suggest that DHEA acts directly on rat ZG cells to diminish aldosterone secretion by inhibition of a post-cAMP pathway or by acting on intracellular Ca2+ mobilization. In addition it affects the function of post-P450scc steroidogenic enzymes.

Role of serum- and glucocorticoid-inducible kinase-1 in regulating torsion-induced apoptosis in rats

Serum- and glucocorticoid-inducible kinase-1 (SGK1) is a serine/threonine protein kinase that responds to various stimuli and mediates cell survival. Although it is known that testicular torsion leads to testicular damage and male infertility, the role of SGK1 in torsion remains unclear. This study investigated whether torsion-induced apoptosis is associated with changes in phosphoinositide-dependent protein kinase-1 (PDK1), SGK1 and forkhead transcription factor FOXO3a expression and/or phosphorylation in rats. Sprague-Dawley rats were divided into four groups: sham (control), 1, 2 and 4 h of unilateral torsion. Bilateral testes, testicular interstitial fluid (TIF) and blood samples were collected immediately after torsion. Our results revealed that SGK1 protein and mRNA were abundantly present in testes and were induced by 2 h of torsion, but that phosphorylation of SGK1, PDK1 and FOXO3a decreased simultaneously. After 2 h of torsion, the testosterone secretion capacity of the primary Leydig cells and testicular interstitial cells (TICs) was impaired and apoptotic spermatogonia and TICs were observed; in addition, the mean seminiferous tubular diameter was decreased. Torsion increased plasma corticosterone levels, but decreased plasma luteinizing hormone and testosterone levels. However, the testosterone levels of the TIF in the ipsilateral testes were significantly enhanced after 2 h of torsion, but suppressed in the contralateral testes. This animal study suggests that PDK1, SGK1 and FOXO3a are involved in torsion-induced apoptosis and that medical therapy should be performed as early as 2 h after the occurrence of torsion to prevent further damage.

Stimulatory Effects of Propylthiouracil on Pregnenolone Production through Upregulation of Steroidogenic Acute Regulatory Protein Expression in Rat Granulosa Cells

Propylthiouracil (PTU) is a common and effective clinical medicine for the treatment of hyperthyroidism. Our previous study demonstrated that short-term treatment with PTU inhibits progesterone production in rat granulosa cells. However, our present results indicate that a 16-h treatment with PTU was able to stimulate pregnenolone production in rat granulosa cells, although progesterone production was diminished by PTU through inhibition of 3β-hydroxysteroid dehydrogenase. Notably, we found that PTU treatment enhanced the conversion of cholesterol into pregnenolone, whereas the protein level of the cytochrome P450 side-chain cleavage enzyme (P450scc, which is the enzyme responding to this conversion) was not affected. Interestingly, the levels of steroidogenic acute regulatory protein (StAR) in both total cell lysate and the mitochondrial fraction were significantly increased by PTU treatment. Furthermore, the binding of steroidogenic factor-1 (SF-1) to the StAR promoter region was also enhanced by PTU treatment, which suggests that PTU could upregulate StAR gene expression. In addition to SF-1 regulation, we found that mitogen-activated protein (MAP) kinase kinase activation is an important regulator of PTU-stimulated StAR protein expression, based on the effects of the MEK inhibitor PD98059. In conclusion, these results indicate that PTU plays opposite roles in the production of progesterone and its precursor, pregnenolone. The regulation of negative feedback on speeding the cholesterol transportation and pregnenolone conversion after a 16-h PTU treatment may be the mechanism explaining PTUs inhibition of progesterone production in rat granulosa cells.

Radioimmunoassay for Ghrelin: Evaluation of Method and the Effect of Fasting in Rats

A double antibody radioimmunoassay (RIA) for ghrelin has been developed and characterized. Antisera against the human ghrelin conjugated to bovine serum albumin (BSA) were produced in the rabbit. Since the ghrelin of human and rat are close homologies, this antiserum (No. YJC 7-36) is cross-reacted (100%) to rat ghrelin. Therefore, it can be applied to both human and rat RIA of ghrelin for measurement of ghrelin in serum. No cross-reactivities with human insulin, rat growth hormone, somatostatin, and glucagon were detected in this RIA system. Very weak cross-reactivity with human growth hormone was found (0.65%). Sensitivity of the RIA was 300 pg per assay tube. The within-assay precision of serum samples were 4.8% in the human being and 8.7% in the rat. The between-assay precision was 9.4% in human being and 9.0% in rats. The serum ghrelin levels of 12 rats were 1.87±0.29 ng/ml in non-fasting status, but 4.28±0.58 ng/ml in fasted rats.