Shu-Fen Kan

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)

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.

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.

Inhibitory mechanisms of Agaricus blazei Murill on the growth of prostate cancer in vitro and in vivo

Agaricus blazei Murill (A. blazei) has been conventionally used as a health food for the prevention of cancer. However, little is known about the direct effects and action mechanisms of A. blazei on human prostate cancer. In the present study, the effects of A. blazei on the growth of human prostate cancer were examined in vitro and in vivo. A. blazei, especially the broth fraction, inhibited cell proliferation in both androgen-dependent and androgen-independent prostate cancer cell lines. The broth of A. blazei induced lactate dehydrogenase leakage in three cancer cell lines, whereas the activities of caspase 3 and the DNA fragmentation were enhanced the most in androgen-independent PC3 cells. The protein expressions of apoptosis-related molecules were elevated by the broth of A. blazei in PC3 cells. Oral supplementation with the broth of A. blazei (with the higher ratio of beta-glucan) significantly suppressed tumor growth without inducing adverse effects in severe combined immunodeficient mice with PC3 tumor xenograft. Tumor xenografts from A. blazei-fed mice showed decreased proliferating cell nuclear antigen-positive cells and reduced tumor microvessel density. Based on these results, we found that the broth of A. blazei may directly inhibit the growth of prostate cancer cell via an apoptotic pathway and suppress prostate tumor growth via antiproliferative and antiangiogenic mechanisms. We therefore suggest that A. blazei might have potential therapeutic use in the prevention and treatment of human prostate cancer.

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.

Effects of hyperprolactinemia on testosterone production in rat Leydig cells

The pathogenesis of hyperprolactinemia (hyperPRL) induced hypogonadism has been suggested to be related with a dysfunction of hypothalamus–pituitary–testis axis. While the direct inhibitory effects of prolactin (PRL) on testosterone (T) release have been demonstrated, the mechanism is still unclear. Our previous study demonstrated a diminished T release in the testicular interstitial cells (TICs) from the anterior pituitary (AP)‐grafted rats as compared with the control, and the pattern was in agreement with the in vivo model. However, TICs incubation cannot totally represent the response of the Leydig cells. Therefore, a Percoll gradient purified Leydig cell model was adopted to explore the response of T release under similar challenges in this study to investigate the effects of hyperPRL on the Leydig cells per se. HyperPRL in male rats was induced by grafting rat AP under the renal capsule. The control animals were grafted with rat brain cortex tissue (CX). Six weeks after grafting, the rats were sacrificed. Either TICs or Leydig cells were isolated, respectively, for in vitro incubation and challenge. Challenge drugs included human chorionic gonadotropin (hCG, 0.05 IU/ml), steroidogenic precursors (25‐OH‐cholesterol, 10−6 M; pregnenolone, 10−6 M), forskolin (an anenylyl cyclase activator, 10−4 M) and 8‐bromo‐3′:5′ cyclic adenosine monophosphate (cAMP) (8‐Br‐cAMP 10−4 M). T released by TICs or Leydig cells was determined by radioimmunoassay. The TICs from the AP‐grafted rats showed lower levels of T release than the control group while the purified Leydig cells demonstrated a reverse pattern in response to challenges of hCG, steroidogenic precursors, forskolin and 8‐Br‐cAMP. In hyperPRL rats, a paradoxical pattern of T release between TICs and purified Leydig cells is observed. The purified Leydig cells from AP‐grafted rats demonstrated a higher level amount of T release than the control after stimulation. The phenomenon can be attributed to the change of Leydig cell sensitivity to the stimulation after the effects of chronic hyperPRL. Moreover, another possibility is the role played by other interstitial cells to modulate steroidogenesis in Leydig cells. J. Cell. Biochem. 80:313–320, 2001.

Direct effects of prolactin on corticosterone release by zona fasciculata‐reticularis cells from male rats

The role of prolactin (PRL) in the male is not fully defined. The aim of this study was to investigate the function and mechanism of PRL on the production of corticosterone by zona fasciculata‐reticularis (ZFR) cells in vitro. The ZFR cells were obtained from male rats under normal, hyperprolactinemic, or hypoprolactinemic situation. PRL stimulated the corticosterone release in a dose‐dependent pattern in the ZFR cells from normal male rats. The cellular adenosine 3′‐5′‐cyclic monophosphate (cAMP) concentration positively correlated with PRL concentration in the presence of forskolin or 3‐isobutyl‐1‐methylxanthine (IBMX). PRL enhanced the stimulatory effects of cAMP mimetic reagents, i.e., forskolin, 8‐bromo‐adenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP), and IBMX on the release of corticosterone. The adenylate cyclase inhibitor (SQ22536) inhibited the corticosterone release in spite of presence of PRL. Nifedipine (L‐type calcium channel blocker) did not inhibit corticosterone release. The hyperprolactinemic condition was actualized by transplantation of donor rat anterior pituitary glands (APs) under kidney capsule. By comparison with the cerebral cortex (CX)‐grafted group, AP‐graft resulted in an increased release of corticosterone, 3β‐hydroxysteriod dehydrogenase (HSD) activity and cAMP production by ZFR cells. Acute hypoprolactinemic status was induced by bromocriptine for 2 days. The results showed the productions of corticosterone were lower in hypoprolactinemic group than in control group, which were persistent along with different ACTH concentrations. These results suggest that PRL increase the release of corticosterone by ZFR cells via cAMP cascades and 3β‐HSD activity. J. Cell. Biochem. 73:563–572, 1999.

Inhibitory effects of digoxin and ouabain on aldosterone synthesis in human adrenocortical NCI-H295 cells.

The present study was to investigate the effects and action mechanisms of digoxin and ouabain on steroidogenesis in human adrenocortical NCI‐H295 cells. Administration of digoxin or ouabain for 24 h decreased the basal and angiotensin II (Ang II)‐stimulated release of aldosterone by NCI‐H295 cells. The conversions of corticosterone (substrate of cytochrome P450 aldosterone synthase, P450c11AS) to aldosterone or deoxycortisol (substrate of cytochrome P450 11β‐hydroxylase, P450c11β) to cortisol were reduced by digoxin or ouabain. The basal and 22‐hydroxy‐cholesterol (a membrane‐permeable cholesterol, substrate of cytochrome P450 side‐chain cleavage enzyme, P450scc)‐stimulated pregnenolone release in mitochondria was inhibited by digoxin or ouabain. Digoxin or ouabain suppressed the basal and Ang II‐stimulated protein expression of steroidogenic acute regulatory (StAR) protein and P450scc. Incubation of digoxin or ouabain for 24 h reduced P450c11AS mRNA expression in NCI‐H295 cells. Digoxin or ouabain (10−6 M, 24 h)‐treated cells showed a lower resting intracellular Ca2+ concentration ([Ca2+]i) and an attenuated response of [Ca2+]i to Ang II. Since no significant cytotoxicity was observed at 10−6 M digoxin or ouabain, the digoxin‐ or ouabain‐induced decrease of aldosterone or cortisol release was not associated with cytotoxicity. These results demonstrate that digoxin or ouabain inhibits the aldosterone or cortisol release via reduction of P450c11AS or P450c11β and P450scc activities, inhibition of StAR and P450scc protein expression, suppression of P450c11AS mRNA expression, and attenuation of Ca2+ mobilization in NCI‐H295 cells.

Role of testicular interstitial macrophages in regulating testosterone release in hyperprolactinemia

Hyperprolactinemia‐induced hypogonadism has been linked to a dysfunction of the hypothalamus‐pituitary‐testis axis. The direct inhibitory effects of prolactin on the testicular release of testosterone have also been demonstrated, though their mechanisms remain unclear. Incubation of rat testicular interstitial cells (TICs) with prolactin stimulated the release of testosterone. TICs from rats with anterior pituitary‐grafting‐induced hyperprolactinemia release lower amounts of testosterone than controls. However, Leydig cells isolated from anterior pituitary‐grafted rats release a greater amount of testosterone. These paradoxical observations have remained unexplained. This study examined the roles of testicular interstitial macrophages and of their product, tumor necrosis factor‐α (TNF‐α), in regulating Leydig cells under condition of hyperprolactinemia. Hyperprolactinemia was induced by grafting two anterior pituitary glands of rats under the renal capsule. Control animals were grafted with rat cortex tissue. The rats were sacrificed 6 weeks later. TICs and macrophages, and Leydig cells were isolated for in vitro incubation and drugs challenge. Testosterone released by testicular interstitial or Leydig cells was measured by radioimmunoassay. TNF‐α concentration in the medium of TICs or macrophages was measured by enzyme‐linked immunosorbent assay (ELISA). A dose‐dependent stimulation of TNF‐α secretion in the medium of TICs or macrophages by the prolactin challenge was observed. Higher amounts of TNF‐α were released by TICs in the anterior pituitary‐grafted rats than in the control group. In contrast, the release of TNF‐α by testicular interstitial macrophages isolated from the anterior pituitary‐ and cortex‐grafted groups was quantitatively similar. Challenge with human chorionic gonadotropin did not modify the TNF‐α release by testicular interstitial macrophages in either group. Challenge of Leydig cells with TNF‐α inhibited their release of testosterone stimulated by human chorionic gonadotropin, but not their basal testosterone release. These different patterns of testosterone release in TICs versus Leydig cells cultures in anterior pituitary‐grafted rats may be due to the influence of testicular interstitial macrophages. These observations correlate with in vivo conditions, where prolactin increases the release of TNF‐α by testicular interstitial macrophages, which, in turn, decreases the human chorionic gonadotropin‐stimulated release of testosterone by Leydig cells. In summary, hyperprolactinemia‐induced hypogonadism involves a mechanism of prolactin‐originated, macrophage‐mediated inhibitory regulation of testosterone release by Leydig cells. TNF‐α, one of the cytokines secreted by macrophages, may play a key role in this mechanism.

Stimulatory effects of Hyperprolactinemia on aldosterone secretion in ovariectomized rats

Background To evaluate the effects of hyperprolactinemia on aldosterone secretion and its mechanisms of action in ovariectomized (OVX) rats. Methods Hyperprolactinemia was induced by the transplantation of rat anterior pituitary (AP) glands under the kidney capsule for 6 weeks in female rats. Control rats underwent cerebral cortex (CX) transplantation. Four weeks after transplantation, the rats were OVX 2 weeks before decapitation. After decapitation, the trunk blood was collected, and the adrenal glands of CX- and AP-grafted rats were prepared as zona glomerulosa (ZG) cells for in vitro study. Results Plasma prolactin and aldosterone in the rats were increased by AP gland transplantation. In the in vitro study, the basal aldosterone secretion by the adrenal ZG cells was higher in AP-grafted rats than in CX-grafted rats. The AP-grafted group showed increased responsiveness to angiotensin II (10-8 M), KCl (8x10-3 M), or 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-br-cAMP; 10-4 M, a membrane-permeable analogue of cAMP) with regard to aldosterone secretion as compared with the CX-grafted group. N-(2-[p-Bromocinnamylamine]ethyl)-5-isoquinolinesulfonamide (H89; 10-6, 10-5 M, a protein kinase A inhibitor) or tetrandrine (10-5 M, a blocker for both L-type and T-type Ca2+ channels) induced a greater suppression of aldosterone secretion in the AP-grafted group than in the CX-grafted group. No significant differences between the CX- and AP-grafted groups were observed, however, with regard to the adrenocorticotropic hormone (10-9 M)-, forskolin (10-5 M, an adenylyl cyclase activator)-, or nifedipine (10-5 M, an L-type Ca2+ channel blocker)-induced responsiveness of aldosterone secretion. In addition, there was no difference in the expression of desmolase (i.e., cytochrome P450 side-chain cleavage enzyme) in ZG cells between AP- and CX-grafted rats. The conversions of 25-OH-cholesterol into pregnenolone in the presence of trilostane (an inhibitor of 3β-hydroxysteroid dehydrogenase) and corticosterone into aldosterone, as well as the expression of the steroidogenic acute regulatory protein in ZG cells, were greater in AP-grafted rats than in CX-grafted rats. Conclusions These results suggest that hyperprolactinemia increases basal, angiotensin II- and KCl-stimulated aldosterone secretion by ZG cells in OVX rats through activation of T-type Ca2+ channels, the post-cAMP and protein kinase A pathway, cytochrome P450 side-chain cleavage enzyme, and aldosterone synthase, as well as by causing increased expression of steroidogenic acute regulatory protein in ZG cells.