Hsiao-Fung Pu

Apoptotic signaling in bufalin‐ and cinobufagin‐treated androgen‐dependent and ‐independent human prostate cancer cells

Prostate cancer has its highest incidence in the USA and is becoming a major concern in Asian countries. Bufadienolides are extracts of toxic glands from toads and are used as anticancer agents, mainly on leukemia cells. In the present study, the antiproliferative and apoptotic mechanisms of bufalin and cinobufagin on prostate cancer cells were investigated. Proliferation of LNCaP, DU145, and PC3 cells was measured by 3‐(4,5‐dimethylthiazol‐2‐yle)‐2,5‐diphenyltetrazolium bromide assay and the doubling time (tD) was calculated. Bufalin and cinobufagin caused changes in the tD of three prostate cancer cell lines, which were more significant than that of human mesangial cells. In addition, bufadienolides induced prostate cancer cell apoptosis more significantly than that in breast epithelial cell lines. After treatment, the caspase‐3 activity and protein expression of caspase‐3, ‐8, and ‐9 were elevated. The expression of other apoptotic modulators, including mitochondrial Bax and cytosolic cytochrome c, were also increased. However, expression of p53 was only enhanced in LNCaP cells. Downregulation of p53 by antisense TP53 restored the cell viability suppressed by bufalienolides. Furthermore, the increased expression of Fas was more significant in DU145 and PC3 cells with mutant p53 than in LNCaP cells. Transfection of Fas small interfering RNA restored cell viability in the bufadienolide‐treated cells. These results suggest that bufalin and cinobufagin suppress cell proliferation and cause apoptosis in prostate cancer cells via a sequence of apoptotic modulators, including Bax, cytochrome c, and caspases. The upstream mediators might be p53 and Fas in androgen‐dependent LNCaP cells and Fas in androgen‐independent DU145 and PC3 cells. (Cancer Sci 2008; 99: 2467–2476)

Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3

Prostate carcinoma is one of the most common malignant tumors and has become a more common cancer in men. Previous studies demonstrated that evodiamine (EVO) exhibited anti‐tumor activities on several cancers, but its effects on androgen‐independent prostate cancer are unclear. In the present study, the action mechanisms of EVO on the growth of androgen‐independent prostate cancer cells (DU145 and PC3 cells) were explored. EVO dramatically inhibited the growth and elevated cytotocixity of DU145 and PC3 cells. The flow cytometric analysis of EVO‐treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest was accompanied by elevated Cdc2 kinase activity, an increase in expression of cyclin B1 and phosphorylated Cdc2 (Thr 161), and a decrease in expression of phosphorylated Cdc2 (Tyr 15), Myt‐1, and interphase Cdc25C. TUNEL examination showed that EVO‐induced apoptosis was observed at 72 h. EVO elevated the activities of caspase 3, 8, and 9 in DU145 cells, while in PC3 cells only the activities of caspase 3 and 9 were elevated. EVO also triggered the processing of caspase 3 and 9 in both DU145 and PC3 cells. We demonstrate that roscovitine treatment result in the reversion of G2/M arrest in response to EVO in both DU145 and PC3. However, inhibitory effect of roscovitine on EVO‐induced apoptosis could only be observed in DU145 rather than PC3. In DU145, G2/M arrest might be a signal for initiation of EVO‐triggered apoptosis. Whereas EVO‐triggered PC3 apoptosis might be independent of G2/M arrest. These results suggested that EVO inhibited the growth of prostate cancer cell lines, DU145 and PC3, through an accumulation at G2/M phase and an induction of apoptosis. J. Cell. Biochem. 101: 44–56, 2007.

Anti-Proliferative Effects of Evodiamine on Human Thyroid Cancer Cell Line ARO

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. J. Cell. Biochem. 110: 1495–1503, 2010.

Ginkgo biloba extract enhances male copulatory behavior and reduces serum prolactin levels in rats

The aim of this study was to investigate the effects of Ginkgo biloba extract (EGb 761) on male copulatory behavior in rats. EGb 761 (1 mg/ml) induced significant production of testosterone (T) in rat Leydig cells in vitro. Its effects on sexual behavior were then tested in Long-Evans male rats after 7, 14, 21, or 28 days of oral gavage of vehicle (distilled water) or EGb 761 at doses of 10, 50, or 100 mg/kg. Administration of 50 mg/kg of EGb 761 for 28 days and of 100 mg/kg for 14 or 21 days significantly increased intromission frequency compared to controls on the same day. An increase in ejaculation frequency was seen after treatment with 50 mg/kg of EGb 761 for 14, 21, or 28 days when compared to either the control group on the same day or the same group on day 0. A reduction in ejaculation latency was only seen after administration of 50 mg/kg of EGb 761 for 14 days compared to the vehicle-treated group. After treatment for 28 days, no significant difference was seen in mount latency, intromission latency, serum T levels, reproductive organ weight, sperm number, or levels of the metabolite of dopamine, 3,4-dihydroxyphenylacetic acid in the brain with any dose of EGb 761, but significantly reduced serum prolactin levels and increased dopamine levels in the medial preoptic area and arcuate nucleus were seen at the dose of 50 mg/kg. These findings show that EGb 761 (especially at the dose of 50 mg/kg) enhances the copulatory behavior of male rats and suggest that the dopaminergic system, which regulates prolactin secretion, may be involved in the facilitatory effect of EGb 761.

Increased concentrations of atrial and plasma atrial natriuretic peptide in castrated male rats

The effects of orchiectomy and testosterone replacement on the plasma concentration and the atrial stores of atrial natriuretic peptide (ANP) were studied in the rats. Male rats were orchiectomized (Orc) three weeks before replacement with testosterone propionate (TP, 20 mg/ml/kg body weight) or sesame oil for five days. Immunoreactive ANP (IR-ANP) in the extracted right atria and plasma of experimental rats was measured. Plasma ANP concentrations were 206 +/- 22, 927 +/- 151, and 264 +/- 61 pg/ml in normal control, Orc, and Orc + TP rats, respectively. ANP contents in right atria were higher in Orc (108 +/- 9 ng/mg tissue) and TP-treated Orc rats (123 +/- 9 ng/mg tissue) than in normal animals (32 +/- 7 ng/mg tissue). These results indicate an increased plasma concentration and atrial stores in the castrated male rats. Replacement of testosterone in the castrated male rats does not decrease the atrial ANP stores, but decreases the plasma ANP concentration.

The non‐genomic effects on Na+/H+‐exchange 1 by progesterone and 20α‐hydroxyprogesterone in human T cells

Progesterone is an endogenous immunomodulator and can suppress T‐cell activation during pregnancy. We have previously shown that the non‐genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress cellular genomic responses to mitogens. This study aimed to show that acidification is due to a non‐genomic inhibition of Na+/H+‐exchange 1 (NHE1) by progesterone and correlate this with immunosuppressive phytohemagglutinin (PHA)‐induced T‐cell proliferation. The presence of amiloride‐sensitive NHE 1 was identified in T cells. The activity of NHE1 was inhibited by progesterone but not by 20α‐hydroxyprogesterone (20α‐OHP). Furthermore, 20α‐OHP was able to compete with progesterone and release the inhibitory effect on the NHE1. The inhibition of NHE1 activity by progesterone‐BSA demonstrated non‐genomic action via plasma membrane sites. Finally, co‐stimulation with PHA and progesterone or amiloride, (5‐(N, N‐dimethyl)‐amiloride, DMA), inhibited PHA‐induced T‐cell proliferation, but this inhibition did not occur with 20α‐OHP and PHA co‐stimulation. However, when DMA was applied 72 h after PHA stimulation, it was able to suppress PHA‐induced T‐cell proliferation. This is the first study to show that progesterone causes a rapid non‐genomic inhibition of plasma membrane NHE1 activity in T cells within minutes which is released by 20α‐OHP. The inhibition of NHE1 leads to immunosuppressive T‐cell proliferation and suggests that progesterone might exert a major rapid non‐genomic suppressive effect on NHE1 activity at the maternal–fetal interface in vivo and that 20α‐OHP may possibly be able to quickly release the suppression when T cells circulated away from the interface. J. Cell. Physiol. 211: 544–550, 2007.

Effects of prolactin on aldosterone secretion in rat zona glomerulosa cells

Acute effects and action mechanisms of prolactin (PRL) on aldosterone secretion in zona glomerulosa (ZG) cells were investigated in ovariectomized rats. Administration of ovine PRL (oPRL) increased aldosterone secretion in a dose‐dependent manner. Incubation of [3H]‐pregnenolone combined with oPRL increased the production of [3H]‐aldosterone and [3H]‐deoxycorticosterone but decreased the accumulation of [3H]‐corticosterone. Administration of oPRL produced a marked increase of adenosine 3′,5′‐cyclic monophosphate (cAMP) accumulation in ZG cells. The stimulatory effect of oPRL on aldosterone secretion was attenuated by the administration of angiotensin II (Ang II) and high potassium. The Ca2+ chelator, ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid (EGTA, 10−2 M), inhibited the basal release of aldosterone and completely suppressed the stimulatory effects of oPRL on aldosterone secretion. The stimulatory effects of oPRL on aldosterone secretion were attenuated by the administration of nifedipine (L‐type Ca2+ channel blocker) and tetrandrine (T‐type Ca2+ channel blocker). These data suggest that the increase of aldosterone secretion by oPRL is in part due to (1) the increase of cAMP production, (2) the activation of both L‐ and T‐type Ca2+ channels, and (3) the activation of 21‐hydroxylase and aldosterone synthase in rat ZG cells. J. Cell. Biochem. 72:286–293, 1999.

Mitotane exhibits dual effects on steroidogenic enzymes gene transcription under basal and cAMP-stimulating microenvironments in NCI-H295 cells

Adrenocortical carcinoma (ACC) is an extremely rare and aggressive endocrine malignancy with a poor prognosis. The most common symptom of ACC is hypercortisolism (Cushings syndrome), which has the highest mortality. Mitotane is used as a steroidogenesis inhibitor for Cushings syndrome or as a chemical adrenalectomy drug for ACC. Mitotane induces adrenal cortex necrosis, mitochondrial membrane impairment, and irreversible binding to CYP proteins. In this study, we explored the molecular effect of mitotane on steroidogenesis in human adrenocortical cancer NCI-H295 cells. Mitotane (10-40μM) inhibited basal and cAMP-induced cortisol secretion but did not cause cell death. Mitotane exhibited an inhibitory effect on the basal expression of StAR and P450scc protein. Furthermore, 40μM of mitotane significantly diminished StAR, CYP11A1 and CYP21 mRNA expression. HSD3B2 and CYP17 seem to be insensitive to mitotane. The stimulatory effects of mitotane on CYP11B1 were more remarkable than its inhibitory effects. In contrast, the activation of cAMP signaling strongly elevated the expression of all these genes. Mitotane (40μM) almost completely neutralized this positive effect and returned 8-Br-cAMP-induced StAR, CYP11A1, CYP17 and CYP21 mRNA to control levels. After cAMP activation, mitotane did not change the levels of CYP11B1 mRNA. The present study demonstrates that mitotane can inhibit cortisol biosynthesis due to a non-specific interference with the gene transcription of steroidogenic enzymes under both basal and 8-Br-cAMP-activated conditions in NCI-H295 cells. We also identified that StAR and CYP11A1 key enzymes that participate in the rate-limiting step of steroidogenesis, were more sensitive to mitotane. In addition, the biphasic effect of mitotane on CYP11B1 was also elucidated.

Aldosterone and Mortality in Hemodialysis Patients: Role of Volume Overload

Background Elevated aldosterone is associated with increased mortality in the general population. In patients on dialysis, however, the association is reversed. This paradox may be explained by volume overload, which is associated with lower aldosterone and higher mortality. Methods We evaluated the relationship between aldosterone and outcomes in a prospective cohort of 328 hemodialysis patients stratified by the presence or absence of volume overload (defined as extracellular water/total body water >48%, as measured with bioimpedance). Baseline plasma aldosterone was measured before dialysis and categorized as low (<140 pg/mL), middle (140 to 280 pg/mL) and high (>280 pg/mL). Results Overall, 36% (n = 119) of the hemodialysis patients had evidence of volume overload. Baseline aldosterone was significantly lower in the presence of volume overload than in its absence. During a median follow-up of 54 months, 83 deaths and 70 cardiovascular events occurred. Cox multivariate analysis showed that by using the low aldosterone as the reference, high aldosterone was inversely associated with decreased hazard ratios for mortality (0.49; 95% confidence interval, 0.25–0.76) and first cardiovascular event (0.70; 95% confidence interval, 0.33−0.78) in the presence of volume overload. In contrast, high aldosterone was associated with an increased risk for mortality (1.97; 95% confidence interval, 1.69–3.75) and first cardiovascular event (2.01; 95% confidence interval, 1.28−4.15) in the absence of volume overload. Conclusions The inverse association of aldosterone with adverse outcomes in hemodialysis patients is due to the confounding effect of volume overload. These findings support treatment of hyperaldosteronemia in hemodialysis patients who have achieved strict volume control.

Differential Effect of Age on Transforming Growth Factor-β1 Inhibition of Prolactin Gene Expression Versus Secretion in Rat Anterior Pituitary Cells1

Transforming growth factor-β1 (TGF-β1) synthesized in the pituitary may act as an autocrine/paracrine regulator of lactotrope function. We examined the effects of TGF-β1 on PRL messenger RNA (mRNA), PRL synthesis, and PRL secretion in cultured anterior pituitary (AP) cells from rats at different ages. APs excised from ovariectomized female Sprague-Dawley rats, either young (2–3 months old; average serum PRL: 9 ng/ml), middle-aged (11–12 months old; average serum PRL: 133 ng/ml), or old (24 months old; average serum PRL: 159 ng/ml), were dispersed and cultured for 5 days. Then, cells were washed and challenged with increasing doses of TGF-β1 (0–100 ng/ml) for 1–48 h in serum-free medium. Northern blot analysis showed an increase in basal PRL mRNA levels, and a decrease in responsiveness to TGF-β1 with age. TGF-β1 suppressed PRL mRNA in a dose- and time-dependent manner in cells from young rats. Maximum inhibition was observed at 0.5–1 ng/ml of TGF-β1. At 0.5 ng/ml TGF-β1, significant reduction in PRL mRNA ...