Androgen receptor W741C and T877A mutations in AIDL cells, an androgen-independent subline of prostate cancer LNCaP cells.

The androgen-independent LNCaP (AIDL) cell line was generated by maintaining prostate cancer LNCaP cells in a hormone-deprived medium. Notably, synthetic androgen R1881-related gene response is attenuated in AIDL cells as compared to the parental LNCaP cells. The aim of this study was to clarify the mechanisms underlying androgen sensitivity in AIDL cells. We first examined the expression of androgen receptor (AR) and its co-regulators. However, no significant difference in mRNA expression was found between LNCaP and AIDL cells. Remarkably, AR protein levels were induced by R1881 and DHT in LNCaP cells, but not in AIDL cells. We next performed the cDNA sequencing to detect mutations in the AR gene. The T877A mutation was detected both in LNCaP and AIDL cells. Furthermore, AIDL cells harbored a missense substitution (TGG → TGT) in the AR gene, which caused a point mutation at codon 741 (W741C). Double T877A and W741C AR mutants have been previously reported to exhibit reduced androgen sensitivity. Hence, the low-androgen-sensitive responses of AIDL cells may be explained, at least in part, by AR gene mutations.

AMP-activated protein kinase modulates the gene expression of aquaporin 9 via forkhead box a2.

Aquaporin 9 (AQP9) is permeable to glycerol, which is a source material in lipogenesis and gluconeogenesis in the liver. We investigated the transcriptional regulation of the AQP9 gene by AMP-activated protein kinase (AMPK), known as an energy sensor in cells since AMPK contributes to the metabolism of carbohydrate, lipid, and protein by regulating the expression of many enzymes and transcription factors in metabolic pathways. An AMPK activator, 5-aminoimidazole-4-carboxamide-1-ß-d-ribonucleoside (AICAR), was observed to suppress the expression of the AQP9 gene in HepG2 cells by promoting the phosphorylation of AMPK and AKT/PKB. Forkhead box a2 (Foxa2) was speculated to be one of the transcriptional regulators of AQP9 gene expression repressed by AICAR from the results of a reporter gene assay with a plasmid containing the promoter region of the AQP9 gene and knock-down of the Foxa2 gene by a specific siRNA. AICAR was determined to induce the phosphorylation and nuclear exclusion of Foxa2. Leptomycin B, inhibiting the binding of the nuclear exclusion signal sequence and chromosome region maintenance 1, prevented nuclear export of Foxa2 triggered by AICAR. These results suggest that the activated AMPK by AICAR causes suppression of the gene expression of AQP9 through transcriptional regulation by Foxa2.

Electrochemical and Mechanistic Study of Reactivities of α-, ß-, γ-, and δ-Tocopherol toward Electrogenerated Superoxide in N,N-Dimethylformamide through Proton-Coupled Electron Transfer.

Scavenging of superoxide radical anion (O2•-) by tocopherols (TOH) and related compounds was investigated on the basis of cyclic voltammetry and in situ electrolytic electron spin resonance spectrum in N,N-dimethylformamide (DMF) with the aid of density functional theory (DFT) calculations. Quasi-reversible dioxygen/O2•- redox was modified by the presence of TOH, suggesting that the electrogenerated O2•- was scavenged by α-, ß-, γ-TOH through proton-coupled electron transfer (PCET), but not by δ-TOH. The reactivities of α-, ß-, γ-, and δ-TOH toward O2•- characterized by the methyl group on the 6-chromanol ring was experimentally confirmed, where the methyl group promotes the PCET mechanism. Furthermore, comparative analyses using some related compounds suggested that the para-oxygen-atom in the 6-chromanol ring is required for a successful electron transfer (ET) to O2•- through the PCET. The electrochemical and DFT results in dehydrated DMF suggested that the PCET mechanism involves the preceding proton transfer (PT) forming a hydroperoxyl radical, followed by a PCET (intermolecular ET-PT). The O2•- scavenging by TOH proceeds efficiently along the PCET mechanism involving one ET and two PTs.

Involvement of interleukin 18 in indomethacin-induced lesions of the gastric mucosa in adjuvant-induced arthritis rat.

It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs) have significant side effects, such as gastroenteropathy, and rheumatoid arthritis patients taking NSAIDs are more susceptible to NSAIDs-induced gastric lesions in comparison with other patients. The pathogenic mechanism of these lesions is not fully understood. We demonstrate whether interleukin 18 (IL-18) expression relate the aggravation of gastric lesion in adjuvant-induced arthritis (AA) rats following the oral administration of indomethacin. Arthritis was induced by injecting 50 microl of a suspension of 10mg/ml heat-killed butyricum (Mycobacterium butyricum) in Bayol F oil into the plantar region of the right hind foot and tail of Dark Agouti rats resulting in an arthritis incidence of 100%. Two weeks after injection, the rats were administered indomethacin (40mg/kg) orally, and were killed under deep ether anesthesia 6h later. The gastric mucosa was then examined. Oral administration of indomethacin caused hemorrhagic lesions in the gastric mucosa of AA rats, and the lesion score for AA rats following indomethacin treatment was significantly higher than for normal rats administered indomethacin. The expression of the IL-18 mRNA and mature IL-18 protein in the gastric mucosa of AA rats administered indomethacin were also higher in comparison with normal rats receiving indomethacin. In addition, interferon-gamma and nitric oxide levels in the gastric mucosa of AA rats were increased by the oral administration of indomethacin. It is possible that IL-18 expression in AA rats is more sensitive to indomethacin, and the IL-18 may play a role in the aggravation of gastric lesions in AA rats treated with indomethacin.

Down-regulation of aquaporin 9 gene transcription by 10-hydroxy-2-decenoic acid: A major fatty acid in royal jelly.

10-Hydroxy-trans-2-decenoic acid (10H2DA) is a unique lipid component of royal jelly produced by worker honeybees that exerts insulin-like effects. We herein investigated the effects of 10H2DA on the gene expression of aquaporin 9 (AQP9), which functions as a glycerol transporter in the liver, to clarify whether 10H2DA modulates energy metabolism. 10H2DA suppressed AQP9 gene expression in HepG2 cells by promoting the phosphorylation of Akt and AMP-activated protein kinase (AMPK). This suppression was partially recovered by the treatment of cells with inhibitors for Akt and AMPK. Based on the result showing that leptomycin B partially recovered the suppression of AQP9 gene expression, 10H2DA inhibited the expression of Foxa2, a transcription factor for the AQP9 gene, and also induced its nuclear exclusion. Although 10H2DA up-regulated phosphoenolpyruvate carboxykinase and glucose-6-phosphatase gene expression, this was suppressed through the modulation of Foxa2 by insulin. These results suggest that 10H2DA suppresses AQP9 gene expression through the phosphorylation of Akt and AMPK and down-regulation of Foxa2 expression.

Determination of lamotrigine in human plasma using liquid chromatography-tandem mass spectrometry.

AIM: Lamotrigine (LTG) is a widely used anti-epileptic drug that is administered to avoid seizures and to maintain seizure-free status. However, several factors reportedly cause individual differences of plasma LTG levels, and the therapeutic target range of LTG varies between individuals. Thus, to optimize effective doses of LTG, we developed a rapid and simple method for determining plasma LTG concentrations. METHODS: Lamotrigine and the internal standard papaverine were extracted from human plasma using solid-phase extraction. After filtration, 5-µL aliquots of final samples were injected into the liquid chromatography-tandem mass spectrometry instrument and LTG and internal standard were separated using a Cadenza CD-C18 column (100 × 2 mm, 3 µm) with 0.1% formic acid in water/acetonitrile (2/1, v/v). RESULTS: The calibration curve was linear from 0.2 to 5.0 µg/mL, and assessments of recovery, intra- and inter-day precision and accuracy, matrix effects, freeze and thaw stability, and long-term stability demonstrated good reproducibility. Retention times of LTG and internal standard were 1.6 and 2.0 minutes, respectively, and the total run time was 3.5 minutes for each sample. CONCLUSION: We developed a rapid and simple method for determining plasma LTG concentrations. The present novel system could be used to inform LTG dose adjustments for individual patients.

Membrane trafficking of aquaporin 3 induced by epinephrine.

We investigated the membrane trafficking of AQP3 induced by epinephrine in Caco-2 cells to clarify the digestive absorption of glycerol permeated by AQP3. Epinephrine was found to promote within 60 min the translocation of AQP3 from the cytoplasmic fraction to the plasma membrane. This increased trafficking of AQP3 was suppressed by phospholipase C and protein kinase C (PKC) inhibitors and a phorbol ester accelerated the trafficking of AQP3 to the membrane fraction. In contrast, adenylyl cyclase (AC) and protein kinase A (PKA) inhibitors did not have any effect on the increased in trafficking of AQP3 by epinephrine and an AC activator did not affect the trafficking of AQP3. Phosphorylation of a threonine (514) residue in PKC was detected upon the treatment with epinephrine and the temporal transitional pattern of this phosphorylation paralleled that of the increased trafficking of AQP3. These results suggest that PKC modulates the trafficking of AQP3.

Regulation of aquaporin 3 expression by magnesium ion.

For understanding the actions of magnesium formulations, magnesium oxide and magnesium sulfate as a constituent of antacid, in the gastrointestinal tract, the effect of magnesium ion on the water channel aquaporin 3 (AQP3) known to be permeable mainly to water and glycerol was investigated in Caco-2 cells. The mRNA and protein of aquaporin 3 were detected by real-time RT-PCR and Western blotting, respectively, and found to increase significantly after treatment with magnesium acetate. Inhibitors for signal transducers, MDL-12330A, H-89, U0126, and Ro 31-8220, were shown to repress the increase in expression of the mRNA. A luciferase reporter vector containing bp -1382 to -12 of the 5'-flanking region of the aquaporin 3 gene was constructed for a reporter gene assay. The luciferase activity in transfectants increased on treatment with magnesium acetate. Serial deletion constructs revealed two regions responsible for the magnesium ion-mediated activation, one between bps -404 and -190, and the other between bps -190 and -82. siRNA for the cAMP response element-binding protein (CREB) sequence located between bp -404 and -190 counteracted the magnesium ion-mediated activation of aquaporin 3 transcription. These results suggest that signal transducers, adenylyl cyclase, protein kinase A (PKA), mitogen-activated protein kinase 1/2 (MEK1/2), and mitogen- and stress-activated protein kinase 1 (MSK1), were involved in the signaling pathway for regulating transcription of the aquaporin 3 gene and CREB is one of the transcriptional regulators for aquaporin 3 gene expression mediated by magnesium ion.

Downregulation of thymosin beta4 expression by androgen in prostate cancer LNCaP cells.

Androgen ablation therapy is an effective treatment for advanced prostate cancer, but the tumor often progresses toward a more aggressive phenotype. We determined the changes in genes associated with the malignant progression and found increased thymosin beta4, involved in tumor metastasis, in androgen-sensitive LNCaP cells grown in the medium with androgen-deficient, charcoal-stripped fetal calf serum. The mRNA expression of thymosin beta4 was determined by real-time polymerase chain reaction analysis. The transcriptional activity of thymosin beta4 was measured by luciferase assay using reporter plasmid containing 5'-flanking region of thymosin beta4. Thymosin beta4 mRNA expression was increased in LNCaP cells in the androgen-deficient condition and decreased by dihydrotestosterone treatment. Androgen receptor antagonist bicalutamide inhibited thymosin beta4 expression in a dose-dependent manner. In androgen receptor-negative PC-3 cells, no significant effects on thymosin beta4 gene expression were observed. The regulation of thymosin beta4 mRNA expression by androgen is due to the transcriptional activation. Deletion analysis revealed that the region between -83 bp and -46 bp of the thymosin beta4 gene is responsible for the regulation of the transcriptional activity by androgen. Thymosin beta4 expression is negatively controlled at the transcriptional level by androgen.

Isolation and characterization of LNCaP sublines differing in hormone sensitivity.

Prostate cancer is a heterogeneous disease with varying degrees of androgen sensitivity. In this study, we performed a limiting dilution of human prostate LNCaP cells, and isolated two sublines, LNCaP-E9 and LNCaP-G4, with differential hormone-sensitivity. Two LNCaP sublines were obtained by the limiting dilution method. The growth of E9 cells was decreased in the presence of androgens, while that of androgen-treated G4 cells was biphasic. Although the androgen receptor expression level in E9 cells was similar to that seen in G4 cells, the expression of PSA mRNA and protein was significantly lower in the E9 cells. Moreover, the androgen-based stimulation of PSA mRNA expression was less sensitive in E9 cells than G4 cells. Intracellular zinc level did not differ between E9 and G4 cells, but ZnT3 mRNA expression was significantly higher in the E9 cells. When the cells were grafted at the subrenal capsule, the number of CD31-positive vessels with a lumen was approximately 2.5 times higher than that in G4 tumors. LNCaP-E9 cells show lower androgen sensitivity than LNCaP-G4 cells. E9 and G4 cells would be helpful for understanding the biology of hormone-refractory prostate cancer.

Incadronate induces cell detachment and apoptosis in prostatic PC-3 cells.

BACKGROUND: Bisphosphonates are widely used for the treatment and prevention of osteoporosis and are also effective in the treatment of bone metastasis of prostate cancer. Several mechanisms underlying the antitumor effect of bisphosphonates have been proposed, including direct effects on tumor cells, such as induction of apoptosis and inhibition of invasion. MATERIALS AND METHODS: The detached and adherent cells after incadronate treatment were collected separately and stained with trypan blue solution. RESULTS: It was found that incadronate induced cell detachment with dephosphorylation of focal adhesion kinase (FAK). The induction of cell detachment by incadronate was prevented by coincubation with geranylgeraniol. The activation of caspase-3 was observed in incadronate-treated floating cells, but not in the adherent cells. A caspase inhibitor did not inhibit cell detachment by incadronate but it markedly prevented cell death. CONCLUSION: These results suggest that incadronate induces cell detachment, followed by caspase-dependent apoptosis.

Pamidronate down-regulates urokinase-type plasminogen activator expression in PC-3 prostate cancer cells.

BACKGROUND: Bisphosphonates are considered to be effective in preventing tumor metastasis to bone. Urokinase-type plasminogen activator (uPA) is thought to be critically involved in the metastatic phenotype of prostate cancer. In this study, we examined the effect of pamidronate on uPA expression in PC-3 prostate cancer cells. MATERIALS AND: The mRNA expression of uPA was assayed by real-time RT-PCR. The transcriptional activity of uPA was measured by a luciferase assay. RESULTS: Pamidronate inhibited uPA mRNA expression by about 90% at 24 h. The inhibition of uPA mRNA expression was prevented in part by cotreatment with geranylgeranyl diphosphate (GGPP). Moreover, GGTI-286, a selective inhibitor of geranylgeranyl transferase, also inhibited uPA mRNA expression. The luciferase activity of uPA reporter plasmid was significantly inhibited by pamidronate. CONCLUSION: These results indicate that the decrease in uPA expression brought about by pamidronate is dependent on the inhibition of geranylgeranylation of proteins and occurs at the transcriptional level.

Incadronate inhibits aminopeptidase N expression in prostatic PC-3 cells.

Bone metastasis is an important cause of morbidity in advanced prostate cancer. Bisphosphonates are widely used for the treatment and prevention of osteoporosis, but recently have been observed to be effective in controlling prostate cancer metastasis. Since aminopeptidase N (AP-N) is known to be involved in the metastasis of prostate cancer, we investigated the effect of bisphosphonate on AP-N expression. Incadronate induced inhibition of AP-N mRNA and protein expression in PC-3 cells. The inhibitory effect of AP-N mRNA expression was also observed in the cells treated with pravastatin and other nitrogen-containing bisphosphonates, which inhibit the key enzyme in the isoprenoid biosynthesis pathway. The decrease of AP-N mRNA expression induced by incadronate was inhibited by co-incubation with geranylgeranyl diphosphate (GGPP). Moreover, GGTI-286 treatment also resulted in reduced AP-N mRNA expression. The translocation of small G protein Rap1 from the cytosol to the membrane was inhibited by incadronate and pravastatin, respectively. These above results indicate that the decrease in AP-N expression elicited by bisphosphonate is related to the inhibition of the mevalonate pathway.

Inhibition of caveolin-1 expression by incadronate in PC-3 prostate cells.

BACKGROUND: Caveolin-1 is an essential component of caveolae and its expression is known to be increased in human prostate cancer. The reduction of caveolin-1 expression has been reported to decrease the tumorigenic and metastatic potential of prostate cancer. MATERIALS AND METHODS: Caveolin-1 expression was determined by real-time RT-PCR and Western blot analysis. RESULTS: Incadronate, a third-generation bisphosphonate, was found to inhibit the caveolin-1 mRNA and protein expression in PC-3 prostate cells. The decrease in caveolin-1 mRNA expression by incadronate was prevented by co-incubation with geranylgeranyol, but not with farnesol. Moreover, treatment of GGTI-286, a geranylgeranyl transferase inhibitor, but not FTI-277, a farnesyl transferase inhibitor, also resulted in the inhibition of caveolin-1 mRNA expression. CONCLUSION: These results indicate that the decrease in caveolin-1 expression elicited by incadronate is related to the inhibition of protein geranylgeranylation.

Correlation between ZIP2 messenger RNA expression and zinc level in rat lateral prostate.

Zinc content in rat lateral prostate (LP) is higher compared with the other tissues, but the zinc retention system in the prostate remains unclear. In the present study, we examined the expression of ZRT, a IRT-like protein (ZIP) family transporter in rat prostate. The zinc level in rat LP was higher compared with the ventral (VP) and dorsal prostate (DP). The predicted ZIP2 mRNA was really expressed in LP at a high level. The expression was decreased in LP from castrated rats, associated with a decrease in zinc level, and these changes were prevented by testosterone replacement. Moreover, ZIP2 expression levels in LP positively correlated with the zinc levels. These findings strongly suggest that ZIP2 is involved in zinc homeostasis of rat prostate.

Effect of buformin and metformin on formation of advanced glycation end products by methylglyoxal.

BACKGROUND: The formation and accumulation of advanced glycation end products (AGE) in various tissues are known to be involved in the aging process and complications of long-term diabetes. Aminoguanidine as AGE inhibitors was first studied, and metformin as biguanide compounds have been reported to react with reactive dicarbonyl precursors such as methylglyoxal. METHODS: We studied the effects of the biguanides of buformin and metformin on AGE formation by the methods of specific fluorescence, and enzyme-linked immunosorbent assay and a Western blot analysis using the anti-AGE antibody after incubating BSA or RNase with methylglyoxal. RESULTS: Buformin is a more potent inhibitor of AGE formation than metformin, and suggests that the amino group of buformin trap the carbonyl group of methylglyoxal to suppress formation of AGE. CONCLUSION: In addition to that of metformin, buformin may be clinically useful to prevent diabetic complications.

Anti-Androgenic Activity of Icarisid II from Epimedium Herb in Prostate Cancer LNCaP Cells.

Anti-androgens are regarded as potential therapeutic agents for the treatment of prostate cancer. We determined that an epimedium herb (EH) extract exhibited anti-androgenic activity in a luciferase assay using androgen receptor-positive prostate cancer LNCaP cells. Nine EH-derived flavonoids were examined. The results identified icarisid II as a very potent anti-androgenic EH-derived flavonoid. A quantitative RT-PCR analysis confirmed that the flavonol suppressed the expression of the androgen-responsive KLK3 gene.

Evidence that the prostate-specific antigen (PSA)/Zn2+ axis may play a role in human prostate cancer cell invasion.

Prostate-specific antigen (PSA), which is used as a marker for the diagnosis and monitoring of prostate cancer, is a kallikrein protease which could potentially play a role in human prostate cancer cell invasion. Zinc ions are effective inhibitors of a number of proteases. The enzymatic activity of purified PSA was strongly inhibited by Zn(2+). The ability of LNCaP cells which express and secrete PSA to invade Matrigel was strongly suppressed by Zn(2+) at a concentration similar to that inhibiting the activity of purified PSA. Zn(2+) effectively inhibited the degradation of Matrigel by purified PSA. These results suggest that Zn(2+) in human prostate may suppress the invasion and metastasis of prostate cancer cells through the regulation of the proteolytic activity of PSA. Loss of inhibition of the proteolytic activity of PSA by Zn(2+) in prostate tumors could contribute to invasion.

Zinc and metallothionein levels and expression of zinc transporters in androgen-independent subline of LNCaP cells.

Zinc levels in the prostate have been reported to be associated with the development and progression of malignant prostate cells. To investigate the reason why the zinc content decreases during the progression of prostate cancer to an androgen-independent state, we compared the expression levels of metallothionein and zinc transporters between androgen-responsive LNCaP cells and its androgen-independent subline, AIDL cells. AIDL cells showed lower zinc levels than LNCaP cells and comparable levels of androgen receptor expression to LNCaP cells, consistent with some clinical aspects of androgen-independent prostatic cancer. AIDL cells exhibited a lower expression of zinc transporter 1 (ZnT1) and higher expression of ZnT3 than LNCaP cells. The content of metallothionein, which is a major zinc-binding protein, was significantly lower in AIDL cells than in LNCaP cells. Furthermore, the expression of ZnT3 mRNA was decreased by incubating LNCaP cells in medium containing hormone-stripped fetal calf serum and increased by addition of synthetic androgen R1881 to the medium, whereas the intracellular zinc levels were not affected under these conditions. These findings suggest that factors such as ZnT1 and metallothioneins other than ZnT3 are associated with the low intracellular zinc content in AIDL cells.

High-level expression of zinc transporter-2 in the rat lateral and dorsal prostate.

Zinc is present at high concentrations in the prostate gland, however, the zinc-retention system in the prostate remains obscure. In this study, we investigated the expression of zinc transporters in the rat prostate and found that zinc transporter-2 (ZnT2), which sequesters zinc to the lysosome-like compartment, is expressed at high levels in the lateral prostate (LP) and dorsal prostate (DP), and that these areas contain higher levels of zinc than other tissues such as the ventral prostate (VP), liver, and kidney. Zinc levels in LP from castrated rats were lower than those in sham-operated rats. However, expression of ZnT2 in LP and DP was unaffected by castration. Expression of other zinc transporters (ZnT1, ZnT4, and divalent cation transporter 1) did not correlate with zinc levels. These results suggest that factors that regulate zinc homeostasis other than zinc transporters are involved in lowering zinc content after castration in rat prostate.