Eileen Jea Chien

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)

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.

Non‐genomic immunosuppressive actions of progesterone inhibits PHA‐induced alkalinization and activation in T cells

Progesterone is an endogenous immunomodulator, and can suppress T‐cell activation during pregnancy. When analyzed under a genome time scale, the classic steroid receptor pathway does not have any effect on ion fluxes. Therefore, the aim of this study was to investigate whether the non‐genomic effects on ion fluxes by progesterone could immunosuppress phytohemagglutinin (PHA)‐induced human peripheral T‐cell activation. The new findings indicated that, first, only progesterone stimulated both [Ca2+]i elevation and pHi decrease; in contrast, estradiol or testosterone stimulated [Ca2+]i elevation and hydrocortisone or dexamethasone stimulated pHi decrease. Secondly, the [Ca2+]i increase by progesterone was dependent on Ca2+ influx, and the acidification was blocked by Na+/H+ exchange (NHE) inhibitor, 3‐methylsulphonyl‐4‐piperidinobenzoyl, guanidine hydrochloride (HOE‐694) but not by 5‐(N,N‐dimethyl)‐amiloride (DMA). Thirdly, progesterone blocked phorbol 12‐myristate 13‐acetate (PMA) or PHA‐induced alkalinization, but PHA did not prevent progesterone‐induced acidification. Fourthly, progesterone did not induce T‐cell proliferation; however, co‐stimulation progesterone with PHA was able to suppress PHA‐induced IL‐2 or IL‐4 secretion and proliferation. When progesterone was applied 72 h after PHA stimulation, progesterone could suppress PHA‐induced T‐cell proliferation. Finally, immobilization of progesterone by conjugation to a large carrier molecule (BSA) also stimulated a rapid [Ca2+]i elevation, pHi decrease, and suppressed PHA‐induced proliferation. These results suggested that the non‐genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress the genomic responses to PHA. Progesterone might act directly through membrane specific nonclassical steroid receptors to cause immunomodulation and suppression of T‐cell activation during pregnancy. J. Cell. Biochem.

The non-genomic rapid acidification in peripheral T cells by progesterone depends on intracellular calcium increase and not on Na+/H+-exchange inhibition.

Progesterone is an endogenous immunomodulator that is able to suppress T cell activation during pregnancy. An increased intracellular free calcium concentration ([Ca(2+)](i)), acidification, and an inhibition of Na(+)/H(+)-exchange 1 (NHE1) are associated with this progesterone rapid non-genomic response that involves plasma membrane sites. Such acidification, when induced by phytohemagglutinin, is calcium dependent in PKC down-regulated T cells. We investigated the relationship between this rapid response involving the [Ca(2+)](i) increase and various membrane progesterone receptors (mPRs). In addition, we explored whether the induction of acidification in T cells by progesterone is a direct result of the [Ca(2+)](i) increase. The results show that the intracellular calcium elevation caused by progesterone is inhibited by SKF96365, U73122, and 2-APB, but not by pertussis toxin or U73343. The elevation is enhanced by the protein tyrosine kinase inhibitor staurosporine and the protein kinase C inhibitors Ro318220 and Go6983. These findings suggest that progesterone does not stimulate the [Ca(2+)](i) increase via the Gi coupled mPR(α). Furthermore, progesterone-induced acidification was found to be dependent on Ca(2+) entry and blocked by the inorganic channel blocker, Ni(2+). However, BAPTA, an intracellular calcium chelator, was found to prevent progesterone-induced acidification but not the inhibition of NHE1. This implies that acidification by progesterone is a direct result of the [Ca(2+)](i) increase and does not directly involve NHE1. Taken together, further investigations are needed to explore whether one or more mPRs or PGRMC1 are involved in bringing about the T cell rapid response that results in the [Ca(2+)](i) increase and inhibition of NHE1.

Non-genomic rapid inhibition of Na+/H+-exchange 1 and apoptotic immunosuppression in human T cells by glucocorticoids.

Glucocorticoids (GCs) have been employed as immunosuppressive agents for many years. However, it is still unclear how GCs instantly uncouple T cells from acute stressful inflammatory. In terms of time scale, the genomic activity of the classic GC receptor cannot fulfill this role under crisis; but a rapid non‐genomic response can. In a previous study, intracellular acidification was found to be due to a rapid non‐genomic inhibition of Na+/H+‐exchange 1 (NHE1) and this event led to the immunosuppression of T cell proliferation by progesterone. The aim of this study was to examine whether there is a rapid acidification response caused by an inhibition of NHE1 activity and to explore the differential non‐genomic effect on immunosuppression of hydrocortisone and dexamethasone. The IC50 values for NHE1‐dependent pHi recovery by hydrocortisone and dexamethasone are 250 and 1 nM, respectively. Co‐stimulation of GCs with phytohemagglutinin (PHA) is able to inhibit PHA‐induced IL‐2 secretion, IL‐4 secretion, and T‐cell proliferation. Furthermore, apoptosis in PHA‐activated T cells is not induced by hydrocortisone but by dexamethasone. The mechanism of immunosuppression on proliferation by dexamethasone was found to be different of hydrocortisone and seems to involve cytotoxicity against T cells. Moreover, apoptosis induced by dexamethasone and impermeable dexamethasone–bovine serum albumin suggests that the apoptotic immunosuppression occurs through both the plasma membrane and cytoplasmic sites. The rapid inhibitory responses triggered by GCs would seem to release T cells instantly when an acute stress‐related response is needed. Nonetheless, the apoptotic immunosuppression by dexamethasone is attributable to its severe cytotoxicity. J. Cell. Physiol. 223:679–686, 2010.

Interrelationship between Thyroxine and Estradiol on the Secretion of Thyrotropin-Releasing Hormone and Dopamine into Hypophysial Portal Blood in Ovariectomized-Thyroidectomized Rats

Effects of thyroxine (T4) on the secretion of thyrotropin-releasing hormone (TRH) and catecholamines into hypophysial portal blood and on the concentrations of arterial plasma thyroid-stimulating hormone (TSH) and prolactin (PRL) in ovariectomized and thyroidectomized (Ovx-Tx) rats were studied. Immediately after ovariectomy, rats were Tx or sham Tx. The Ovx-Tx rats were injected subcutaneously with estradiol benzoate (EB, 0.5 microgram/kg b.w.) or sesame oil, and T4 (20 micrograms/kg b.w.) or saline once daily for 2 weeks. The Ovx rats with intact thyroid gland were injected with saline and oil only. The hypophysial portal blood samples were collected and mixed with or without 2,3-dimercaptopropanol before extraction by methanol or perchloric acid, respectively. The femoral arterial blood was also collected. The concentrations of TRH in methanol-extracted portal plasma and that of TSH and PRL in arterial plasma were measured by radioimmunoassay. The concentrations of catecholamines in perchloric acid-extracted portal plasma samples were measured by radioenzymatic assay. Thyroidectomy in Ovx rats resulted in an increase in portal plasma TRH and arterial plasma TSH. Despite the presence or absence of estradiol, T4 replacement in Ovx-Tx rats decreased portal plasma TRH and arterial plasma TSH to euthyroid levels. Combination of the injection of T4 and EB in vivo caused significantly decreased levels of portal plasma dopamine and increased arterial plasma PRL compared with those in vehicle-injected Ovx-Tx animals. Concentrations of neither norepinephrine nor epinephrine in hypophysial portal plasma paralleled the altered concentrations of PRL or TSH in arterial plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone attenuates the inhibitory effects of cardiotonic digitalis on pregnenolone production in rat luteal cells.

Previous studies have shown that digoxin decreases testosterone secretion in testicular interstitial cells. However, the effect of digoxin on progesterone secretion in luteal cells is unclear. Progesterone is known as an endogenous digoxin‐like hormone (EDLH). This study investigates how digitalis affected progesterone production and whether progesterone antagonized the effects of digitalis. Digoxin or digitoxin, but not ouabain, decreased the basal and human chorionic gonadotropin (hCG)‐stimulated progesterone secretion as well as the activity of cytochrome P450 side chain cleavage enzyme (P450scc) in luteal cells. 8‐Br‐cAMP and forskolin did not affect the reduction. Neither the amount of P450scc, the amount of steroidogenic acute regulatory (StAR) protein, nor the activity of 3β‐hydroxysteroid dehydrogenase (3β‐HSD) was affected by digoxin or digitoxin. Moreover, in testicular interstitial and luteal cells, progesterone partially attenuated the reduction of pregnenolone by digoxin or digitoxin and the progesterone antagonist, RU486, blocked this attenuation. These new findings indicated that (1) digoxin or digitoxin inhibited pregnenolone production by decreasing the activity of P450scc enzyme, but not Na+–K+‐ATPase, resulting in a decrease on progesterone secretion in rat luteal cells, and (2) the inhibitory effect on pregnenolone production by digoxin or digitoxin was reversed partially by progesterone. In conclusion, digoxin or digitoxin decreased progesterone production via the inhibition of pregnenolone by decreasing P450scc activity. Progesterone, an EDLH, could antagonize the effects of digoxin or digitoxin in luteal cells. J. Cell. Biochem. 86: 107–117, 2002.

Response of alkalinization or acidification by phytohemagglutinin is dependent on the activity of protein kinase C in human peripheral T Cells

The increase of intracellular free calcium concentration ([Ca2+]i) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human T cells. However, a rapid change in intracellular pH (pHi), acidification or alkalinization during the activation, is also associated after these two signals. The aim of this study was to define whether the change in pHi is affected by calcium and protein kinase C (PKC), in phytohemagglutinin (PHA)‐stimulated T cells. T cells were isolated from human peripheral blood. The [Ca2+]i and the pHi were measured using, respectively, the fluorescent dyes, Fura‐2, and BCECF. In addition, down‐regulation of PKC activity by PMA (1 μM, 18 h) was confirmed in these cells using a protein kinase assay. The results indicated that, (1) alkalinization was induced by PHA or PMA in T cells; the results of alkalinization was PKC‐dependent and Ca2+‐independent, (2) in PKC down‐regulated T cells, PHA induced acidification; this effect was enhanced by pre‐treating the cells with the Na+/H+ exchange inhibitor, 5‐(N,N‐dimethyl)‐amiloride, (DMA, 10 μM, 20 min), (3) the acidification was dependent on the Ca2+ influx and blocked by removal of extracellular calcium or the addition of the inorganic channel blocker, Ni2+, and (4) Thapsigargin (TG), a Ca2+‐ATPase inhibitor, confirmed that acidification by the Ca2+ influx occurred in T cells in which PKC was not down‐regulated. These findings indicate two mechanisms, alkalinization by PKC and acidification by Ca2+ influx, exist in regulating pHi in T cells. This is the first report that PHA stimulates the acidification by Ca2+ influx but not alkalinization in T cells after down‐regulation of PKC. In conclusion, the activity of PKC in T cells determines the response in alkalinization or acidification by PHA. J. Cell. Biochem. 81: 604–612, 2001.

Bacterial lipopolysaccharide activates protein kinase C, but not intracellular calcium elevation, in human peripheral T cells

The increase of intracellular free calcium concentration ([Ca2+]i) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human peripheral T cells. Bacterial lipopolysaccharide (LPS) is nonmitogenic in human T cells. However, in the presence of monocytes, LPS becomes mitogenic to proliferate T cells. The aim of this study was to define the incompetency of LPS on two mitogenic signals in human peripheral T cells. T cells were isolated from human peripheral blood. [Ca2+]i and pHi were determined by loading the cells with the fluorescent dyes, Fura‐2 acetoxymethyl ester (Fura‐2/AM) and 2′,7′‐bis(2‐carboxyethyl)‐5‐(and 6)carboxyfluorescein acetoxymethyl ester (BCECF/AM). PKC activity was determined by protein kinase assay and cell proliferation was estimated from the incorporation of [3H]‐thymidine. The results indicated that (1) LPS (10 μg/ml) stimulated PKC activity significantly within 5 min, reached a plateau at 30 min, and maintained that level for at least 2 h; and (2) LPS stimulated cytoplasmic alkalinization but did not affect the levels of [Ca2+]i and [3H]‐thymidine incorporation into T cells. Moreover, the combination of calcium ionophore A23187 with LPS significantly stimulated [3H]‐thymidine incorporation into T cells. Thus, the results demonstrate that LPS failed to proliferate T cells, probably because of a lack of the machinery necessary to stimulate the mitogenic signal on [Ca2+]i elevation. J. Cell. Biochem. 76:404–410, 2000.