Ho Lin

Cdk5 regulates STAT3 activation and cell proliferation in medullary thyroid carcinoma cells

The biological behaviors of thyroid cancer are varied, and the pathological mechanisms remain unclear. Some reports indicated an apparent aggregation of amyloid accompanying medullary thyroid carcinoma (MTC). Amyloid aggregation in neurodegeneration leads to hyperactivation of Cdk5 and subsequent neuronal death. Based on the connection with amyloid, the role of Cdk5 in MTC is worthy of investigation. Initially, the expression of Cdk5 and its activator, p35, in MTC cell lines was identified. Cdk5 inhibition by specific inhibitors or short interfering RNA decreased the proliferation of MTC cell lines, which reveals the importance of Cdk5 in MTC cell growth. Although p35 cleavage has been considered as an important element in neurodegeneration, it seems that p35 cleavage was not a major cause in Cdk5 activity-dependent MTC cell proliferation because neither Cdk5 activity nor cell growth was affected by the inhibition of p35 cleavage. Clearance of amyloid by antibody neutralization indicated that MTC cell proliferation was supported by calcitonin-derived extracellular amyloid and subsequent Her2 and Cdk5 activation. Significantly, the STAT3 pathway was involved in Cdk5-dependent proliferation of MTC cells through Ser-727 phosphorylation. In addition, Cdk5 inhibition reduced nuclear distributions of both the Cdk5-p35 complex and phospho-STAT3 in MTC cells. Finally, Cdk5 inhibition retarded tumor formation in vivo accompanying the reduction of phospho-STAT3. Our findings suggest the first demonstration of a novel and specific role for Cdk5 kinase in supporting the proliferation of the medullary thyroid carcinoma cells and could shed light on a new field for diagnosis and therapy of thyroid cancer.

Regulation of Androgen Receptor and Prostate Cancer Growth by Cyclin-dependent Kinase 5

Prostate cancer is the most frequently diagnosed male malignancy. The normal prostate development and prostate cancer progression are mediated by androgen receptor (AR). Recently, the roles of cyclin-dependent kinase 5 (Cdk5) and its activator, p35, in cancer biology are explored one after another. We have previously demonstrated that Cdk5 may regulate proliferation of thyroid cancer cells. In addition, we also identify that Cdk5 overactivation can be triggered by drug treatments and leads to apoptosis of prostate cancer cells. The aim of this study is to investigate how Cdk5 regulates AR activation and growth of prostate cancer cells. At first, the data show that Cdk5 enables phosphorylation of AR at Ser-81 site through direct biochemical interaction and, therefore, results in the stabilization of AR proteins. The Cdk5-dependent AR stabilization causes accumulation of AR proteins and subsequent activation. Besides, the positive regulations of Cdk5-AR on cell growth are also determined in vitro and in vivo. S81A mutant of AR diminishes its interaction with Cdk5, reduces its nuclear localization, fails to stabilize its protein level, and therefore, decreases prostate cancer cell proliferation. Prostate carcinoma specimens collected from 177 AR-positive patients indicate the significant correlations between the protein levels of AR and Cdk5 or p35. These findings demonstrate that Cdk5 is an important modulator of AR and contributes to prostate cancer growth. Therefore, Cdk5-p35 may be suggested as diagnostic and therapeutic targets for prostate cancer in the near future.

Effects of estradiol on aldosterone secretion in ovariectomized rats.

The effects of estradiol benzoate (EB) on steroidogenesis in rat zona fasciculata‐reticularis (ZFR) cells were studied. Female rats were ovariectomized (Ovx) for 2 weeks and then injected subcutaneously with oil or EB for 3 days before decapitation. ZFR cells were isolated and incubated with adrenocorticotropin (ACTH) or prolactin (PRL) for 1 h. Corticosterone concentrations in plasma and cell media, and adenosine 3′,5′‐cyclic monophosphate (cAMP) production in ZFR cells were determined by radioimmunoassay. The effects of EB replacement in vivo on the activities of steroidogenic enzymes in ZFR cells were measured by the amounts of intermediate steroidal products separated by thin‐layer chromatography. Replacement of EB in vivo resulted in a dose‐dependent increase of plasma PRL and corticosterone in Ovx rats. The basal, ACTH‐, and PRL‐stimulated release of corticosterone by ZFR cells was greater in EB‐ than in oil‐treated animals. Forskolin‐induced production of cAMP was greater in the EB‐replaced rats than in oil‐treated animals, which correlated with the increase of corticosterone production. The 3‐isobutyl‐l‐methylxanthine (IBMX) plus ACTH‐, IBMX plus PRL‐, and forskolin plus PRL‐stimulated productions of cAMP were higher in EB‐ than in oil‐treated rats. The enzyme activities of postpregnenolone were not affected by EB replacement in Ovx rats. These results suggest that the EB‐related increase of corticosterone production in Ovx rats is associated with an increase of cAMP generation and the stimulatory effect of PRL on ZFR cells. J. Cell. Biochem. 77:560‐568, 2000.

5-Fluorouracil Induced Intestinal Mucositis via Nuclear Factor-κB Activation by Transcriptomic Analysis and In Vivo Bioluminescence Imaging

5-Fluorouracil (5-FU) is a commonly used drug for the treatment of malignant cancers. However, approximately 80% of patients undergoing 5-FU treatment suffer from gastrointestinal mucositis. The aim of this report was to identify the drug target for the 5-FU-induced intestinal mucositis. 5-FU-induced intestinal mucositis was established by intraperitoneally administering mice with 100 mg/kg 5-FU. Network analysis of gene expression profile and bioluminescent imaging were applied to identify the critical molecule associated with 5-FU-induced mucositis. Our data showed that 5-FU induced inflammation in the small intestine, characterized by the increased intestinal wall thickness and crypt length, the decreased villus height, and the increased myeloperoxidase activity in tissues and proinflammatory cytokine production in sera. Network analysis of 5-FU-affected genes by transcriptomic tool showed that the expression of genes was regulated by nuclear factor-κB (NF-κB), and NF-κB was the central molecule in the 5-FU-regulated biological network. NF-κB activity was activated by 5-FU in the intestine, which was judged by in vivo bioluminescence imaging and immunohistochemical staining. However, 5-aminosalicylic acid (5-ASA) inhibited 5-FU-induced NF-κB activation and proinflammatory cytokine production. Moreover, 5-FU-induced histological changes were improved by 5-ASA. In conclusion, our findings suggested that NF-κB was the critical molecule associated with the pathogenesis of 5-FU-induced mucositis, and inhibition of NF-κB activity ameliorated the mucosal damage caused by 5-FU.

Stimulatory effect of lactate on testosterone production by rat Leydig cells

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 × 105 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 Ca2+ channel blocker, 10−5–10−4 M), or nimodipine (a potent L‐type Ca2+ channel antagonist, 10−5–10−4 M) in the presence or absence of lactate at 34°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 Ca2+ channel. J. Cell. Biochem. 83: 147–154, 2001.

Antineuroinflammatory effects of lycopene via activation of adenosine monophosphate-activated protein kinase-α1/heme oxygenase-1 pathways.

Microglia play an important role in the immune defense in the central nervous system. Activation of microglia leads to the production of excessive inflammatory molecules and deleterious consequences, including neuronal death. Lycopene, 1 of the major carotenoids present in tomatoes, has been shown to exert antioxidant properties and to inhibit cancer cell proliferation. However, the effects of lycopene on neuroinflammatory responses in microglia remain unknown. In this study, we investigated the signaling pathways involved in lycopene-inhibited expression of cyclooxygenase (COX)-2 and inflammation mediators in BV-2 microglia, mouse primary cultured microglia, and rat primary cultured microglia. Lycopene inhibited the enhancement of lipopolysaccharide (LPS)-induced nuclear factor-kappaB (NF-κB) and activator protein 1 (AP-1) DNA binding activity. In the present study, we demonstrated that lycopene inhibits LPS-induced COX-2 expression through heme oxygenase-1 (HO-1) activation. Our results also demonstrate that stimulation with lycopene increases the phosphorylation of liver kinase B1 (LKB1), calmodulin-dependent protein kinase II (CaMKII), and adenosine monophosphate-activated protein kinase (AMPK)-α1. Treatment with AMPK inhibitors effectively antagonized lycopene-stimulated HO-1 expression. Interestingly, we also found that lycopene increased phospho-AMPKα1 accumulation in the nucleus in microglia. Preincubation of cells with HO-1 and AMPK selective pharmacological inhibitors dramatically reversed the inhibitory effect of lycopene on LPS-induced COX-2 and prostaglandin E2 production. Transfection of microglia with HO-1 and AMPKα small interfering RNA (siRNA) also effectively reversed the inhibitory effect of lycopene on LPS-induced COX-2 expression. In a mouse model, lycopene showed significant antineuroinflammatory effects on microglial activation and motor behavior deficits. These findings suggest that lycopene-inhibited LPS-induced COX-2 expression is mediated by HO-1 activation through the AMPK pathway. Therefore, lycopene might be useful as a therapeutic agent for the treatment of neuroinflammation-associated disorders.

Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration.

Although Abl functions in mature neurons, work to date has not addressed Abls role on Cdk5 in neurodegeneration. We found that β-amyloid (Aβ42) initiated Abl kinase activity and that blockade of Abl kinase rescued both Drosophila and mammalian neuronal cells from cell death. We also found activated Abl kinase to be necessary for the binding, activation, and translocalization of Cdk5 in Drosophila neuronal cells. Conversion of p35 into p25 was not observed in Aβ42-triggered Drosophila neurodegeneration, suggesting that Cdk5 activation and protein translocalization can be p25-independent. Our genetic studies also showed that abl mutations repressed Aβ42-induced Cdk5 activity and neurodegeneration in Drosophila eyes. Although Aβ42 induced conversion of p35 to p25 in mammalian cells, it did not sufficiently induce Cdk5 activation when c-Abl kinase activity was suppressed. Therefore, we propose that Abl and p35/p25 cooperate in promoting Cdk5-pY15, which deregulates Cdk5 activity and subcellular localization in Aβ42-triggered neurodegeneration.

Desipramine Protects Neuronal Cell Death and Induces Heme Oxygenase-1 Expression in Mes23.5 Dopaminergic Neurons

Background Desipramine is known principally as a tricyclic antidepressant drug used to promote recovery of depressed patients. It has also been used in a number of other psychiatric and medical conditions. The present study is the first to investigate the neuroprotective effect of desipramine. Methodology/Principal Findings Mes23.5 dopaminergic cells were used to examine neuroprotective effect of desipramine. Western blot, reverse transcription-PCR, MTT assay, siRNA transfection and electrophoretic mobility shift assay (EMSA) were carried out to assess the effects of desipramine. Desipramine induces endogenous anti-oxidative enzyme, heme oxygenase-1 (HO-1) protein and mRNA expression in concentration- and time-dependent manners. A different type of antidepressant SSRI (selective serotonin reuptake inhibitor), fluoxetine also shows similar effects of desipramine on HO-1 expression. Moreover, desipramine induces HO-1 expression through activation of ERK and JNK signaling pathways. Desipramine also increases NF-E2-related factor-2 (Nrf2) accumulation in the nucleus and enhances Nrf2-DNA binding activity. Moreover, desipramine-mediated increase of HO-1 expression is reduced by transfection with siRNA against Nrf2. On the other hand, pretreatment of desipramine protects neuronal cells against rotenone- and 6-hydroxydopamine (6-OHDA)-induced neuronal death. Furthermore, inhibition of HO-1 activity by a HO-1 pharmacological inhibitor, ZnPP IX, attenuates the neuroprotective effect of desipramine. Otherwise, activation of HO-1 activity by HO-1 activator and inducer protect 6-OHDA-induced neuronal death. Conclusions/Significance These findings suggest that desipramine-increased HO-1 expression is mediated by Nrf2 activation through the ERK and JNK signaling pathways. Our results also suggest that desipramine provides a novel effect of neuroprotection, and neurodegenerative process might play an important role in depression disorder.

The role of cyclic AMP production, calcium channel activation and enzyme activities in the inhibition of testosterone secretion by amphetamine

1 The aim of this study was to investigate the mechanism by which amphetamine exerts its inhibitory effect on testicular interstitial cells of male rats. 2 Administration of amphetamine (10−12–10−6 M) in vitro resulted in a dose‐dependent inhibition of both basal and human chorionic gonadotropin (hCG, 0.05 iu ml−1)‐stimulated release of testosterone. 3 Amphetamine (10−9 M) enhanced the basal and hCG‐increased levels of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) accumulation in vitro (P<0.05) in rat testicular interstitial cells. 4 Administration of SQ22536, an adenylyl cyclase inhibitor, decreased the basal release (P<0.05) of testosterone in vitro and abolished the inhibitory effect of amphetamine. 5 Nifedipine (10−6 M) alone decreased the secretion of testosterone (P<0.01) but it failed to modify the inhibitory action of amphetamine (10−10–10−6 M). 6 Amphetamine (10−10–10−6 M) significantly (P<0.05 or P<0.01) decreased the activities of 3β‐hydroxysteroid dehydrogenase (3β‐HSD), P450c17, and 17‐ketosteroid reductase (17‐KSR) as indicated by thin‐layer chromatography (t.l.c.). 7 These results suggest that increased cyclic AMP production, decreased Ca2+ channel activity and decreased activities of 3β‐HSD, P450c17, and 17‐KSR are involved in the inhibition of testosterone production induced by the administration of amphetamine.

Inhibitory effect of digoxin on testosterone secretion through mechanisms involving decreases of cyclic AMP production and cytochrome P450scc activity in rat testicular interstitial cells

In vivo and in vitro experiments were performed to examine inhibitory effects of digoxin on testosterone secretion and to determine possible underlying mechanisms. A single intravenous injection of digoxin (1 μg kg−1) decreased the basal and human chorionic gonadotropin (hCG)‐stimulated plasma testosterone concentrations in adult male rats. Digoxin (10−7–10−4 M) decreased the basal and hCG‐stimulated release of testosterone from rat testicular interstitial cells in vitro. Digoxin (10−7–10−4 M) also diminished the basal and hCG‐stimulated production of cyclic 3′ : 5′‐adenosine monophosphate (AMP) and attenuated the stimulatory effects of forskolin and 8‐Br‐cyclic AMP on testosterone production by rat testicular interstitial cells. Digoxin (10−4 M) inhibited cytochrome P450 side chain cleavage enzyme (cytochrome P450scc) activity (conversion of 25‐hydroxy cholesterol to pregnenolone) in the testicular interstitial cells but did not influence the activity of other steroidogenic enzymes. These results suggest that digoxin inhibits the production of testosterone in rat testicular interstitial cells, at least in part, via attenuation of the activities of adenylyl cyclase and cytochrome P450scc.