Human Adrenocortical Tumor Cell Research Articles

Angiotensin II Regulates Mitochondrial mTOR Pathway Activity Dependent on Acyl-CoA Synthetase 4 in Adrenocortical Cells.

Two well-known protein complexes in mammalian cells, mTOR type 1 and type 2 (mTORC1/2) are involved in several cellular processes such as protein synthesis, cell proliferation, and commonly dysregulated in cancer. An acyl-CoA synthetase type 4 (ACSL4) is one of the most recently mTORC1/2 regulators described, in breast cancer cells. The expression of ACSL4 is hormone-regulated in adrenocortical cells and required for steroid biosynthesis. mTORC1/2 have been reported to be crucial in the proliferation of human adrenocortical tumor cells H295R and interestingly reported at several subcellular locations, which has brought cell biology to the vanguard of the mTOR signaling field. In the present work, we study the regulation of mTORC1/2 activation by angiotensin II (Ang II)-the trophic hormone for adrenocortical cells-the subcellular localization of mTORC1/2 signaling proteins and the role of ACSL4 in the regulation of this pathway, in H295R cells. Ang II promotes activation by phosphorylation of mTORC1/2 pathway proteins in a time-dependent manner. Mitochondrial pools of ribosomal protein S6, protein kinase B (Akt) in threonine 308, and serine 473 and Rictor are phosphorylated and activated. Glycogen synthase kinase type 3 (GSK3) is phosphorylated and inactivated in mitochondria, favoring mTORC1 activation. Epidermal growth factor, a classic mTORC1/2 activator, promoted unique activation kinetics of mTORC1/2 pathway, except for Akt phosphorylation. Here, we demonstrate that ACSL4 is necessary for mTORC1/2 effectors phosphorylation and H295R proliferation, triggered by Ang II. Ang II promotes activation of mitochondrial mTORC1/2 signaling proteins, through ACSL4, with a direct effect on adrenocortical cellular proliferation.

MON-185 ACTH-Derived Peptide Antagonists as an Alternative Treatment Strategy for Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by different enzyme deficiencies in the steroid hormone synthesis leading to a disturbed cortisol biosynthesis. The medical treatment of CAH includes suboptimal ACTH-suppressing high glucocorticoid doses to reduce adrenal hyperplasia and overproduction of androgens. These inappropriate corticoid substitutions are often associated with undesirable side effects such as arterial hypertension, growth failure and obesity. Since the current therapy of patients with CAH is often unsatisfactory, innovative treatment options are required.The aim of our study was to specifically block the melanocortin 2 receptor (MC2R) signaling pathway as an alternative treatment strategy for CAH. We tested ACTH-derived selective peptides with incorporation of various synthetic non-natural amino acids in the activation motif of ACTH. To study the antagonistic activity of the peptides, cAMP production of MC2R/MRAP stably transfected human embryonic kidney (HEK) 293 cells were measured. All new synthesized peptide antagonists reduced ACTH-stimulated MC2R activity as competitive inhibitors indicated by a reduced in vitro cAMP response. Cells pre-incubated with peptide LNP009 showed the most efficient blockade of the MC2R and the highest shift of EC50 of ACTH (33.8 nM ± 0.08 vs. 7.3 nM ± 0.09). LNP009 was additionally tested for specificity concerning the other known melanocortin receptors and showed no antagonistic effect up to 1 μM on MC3, MC4 or MC5 receptor transiently transfected HEK 293 cells. To further investigate the inhibitory effect of our most potent antagonist peptide LNP009 on the steroid hormone response, we assessed steroidogenic enzyme expression of the human adrenocortical tumor cell line NCI-H295RA and performed mass spectrometry analyzes of steroids in the cell culture supernatant. Pre-incubation with LNP009 reduced the expression of the genes CYP21A2, CYP11B1 and HSD3B2 in NCI-H295RA cells and significantly reduced the synthesis of aldosterone (P=0.046; n=3), cortisol (P=0.020; n=3) and corticosterone (P=0.035; n=3).With the successful blocking of the ACTH binding and signal transduction by our antagonistic peptides, we anticipate an alternative approach for optimizing the treatment of CAH patients lacking the side effects of the currently used ACTH-suppressing corticoid therapy.

Downregulated ATF3 expression inhibited growth of adrenocortical carcinoma cells and investigation of its mechanisms

Objective To investigate the effect of downregulated activating transcription factor 3 (ATF3) expression on proliferation of adrenocortical carcinoma cells and its mechanisms. Methods Immunohistochemistry and Western blotting were used to detect the expression of ATF3 in human adrenocortical tumor tissues and cells. Adrenocortical carcinoma cells, Sw-13, and NCI-H259R cells, were transfected with siATF3 using lipidosome 2000, and expression of ATF3 mRNA was determined using RT-PCR; expression of ATF3, cleaved caspase 3, caspase 3, cleaved PARP, and PARP proteins were detected using Western blotting; cell growth inhibition rate and apoptosis rate were monitored using MTT and AnnexinV-FITC/PI, respectively. Sw-13 and NCI-H259R cells were treated with NVP-BEZ235, Perifosine, BKM120, IWP-2, PP2, KN93, Everolimus respectively followed by detected expression of ATF3 mRNA by realtime PCR. The effect of ATF3 on cell proliferation after inhibition of related signaling pathways were detected by MTT. Results The ATF3 in human adrenocortical gland tumor tissues and cells showed high expression. The levels of ATF3 mRNA and protein in Sw-13 and NCI-F259R cells transfected with siATF3 were significantly reduced. Compared with the negative control group (NC siRNA), siATF3 transfection significantly inhibited the proliferation of Sw-13 and NCI-F259R cells (P<0.05), and increased the apoptosis rate (P<0.05). Western blotting shown that the levels of cleaved caspase 3 and cleaved PARP protein in siATF3 transfected cells increased significantly; and realtime PCR results indicated that the expression of ATF3 mRNA was dramatically inhibited by PP2, KN93, and IWP-2 in NCI-F259R cells compared with control group (DMSO); but ATF3 significantly promoted the proliferation activity of NCI-F259R cells which treated by PP2, KN93, and IWP-2 signaling inhibitors. Conclusion High expression of ATF3 is existed in adrenocortical carcinoma cells. Downregulated ATF3 expression may inhibit cell proliferation and activate apoptosis pathway, resulting in apoptosis in Sw-13 and NCI-F259R cells, this mechanism of action is related to activating Wnt/β-catenin, CaMKI, and SRC pathway. (Chin J Endocrinol Metab, 2018, 34: 738-745) Key words: Adrenocortical carcinoma; Activating transcription factor 3; Signal pathway; Apoptosis; Mechanism

Cofilin is a cAMP effector in mediating actin cytoskeleton reorganization and steroidogenesis in mouse and human adrenocortical tumor cells

cAMP pathway plays a major role in the pathogenesis of cortisol-producing adrenocortical adenomas (CPA). cAMP-induced steroidogenesis is preceded by actin cytoskeleton reorganization, a process regulated by cofilin activity.In this study we investigated cofilin role in mediating cAMP effects on cell morphology and steroidogenesis in adrenocortical tumor cells.We demonstrated that forskolin induced cell rounding and strongly reduced phosphorylated (P)-cofilin/total cofilin ratio in Y1 (−52 ± 16%, p < 0.001) and human CPA cells (−53 ± 18%, p < 0.05). Cofilin silencing significantly reduced both forskolin-induced morphological changes and progesterone production (1.3-fold vs 1.8-fold in controls, p < 0.05), whereas transfection of wild-type or S3A (active), but not S3D (inactive) cofilin, potentiated forskolin effects on cell rounding and increased 3-fold progesterone synthesis with respect to control (p < 0.05). Furthermore, cofilin dephosphorylation by a ROCK inhibitor potentiated forskolin-induced cell rounding and steroidogenesis (2-fold increase vs forskolin alone).Finally, we found a reduced P-cofilin/total cofilin ratio and increased cofilin expression in CPA vs endocrine inactive adenomas by western blot and immunohistochemistry.Overall, these results identified cofilin as a mediator of cAMP effects on both morphological changes and steroidogenesis in mouse and human adrenocortical tumor cells.

Cofilin is a cAMP effector in mediating actin cytoskeleton reorganization and steroidogenesis in mouse and human adrenocortical tumor cells

Cofilin is a cAMP effector in mediating actin cytoskeleton reorganization and steroidogenesis in mouse and human adrenocortical tumor cells

Effects of Type 1 Insulin-Like Growth Factor Receptor Silencing in a Human Adrenocortical Cell Line.

Type 1 insulin-like growth factor receptor (IGF-1R) is overexpressed in a variety of human cancers, including adrenocortical tumors. The aim of the work was to investigate the effects of IGF-1R downregulation in a human adrenocortical cell line by small interfering RNA (siRNA). The human adrenocortical tumor cell line NCI H295R was transfected with 2 specific IGF1R siRNAs (# 1 and # 2) and compared with untreated cells and a negative control siRNA. IGF1R expression was determined by quantitative reverse-transcription PCR (qRTPCR) and Western blot. The effects of IGF-1R downregulation on cell proliferation and apoptosis were assessed. IGF-1R levels were significantly decreased in cells treated with IGF-1R siRNA # 1 or # 2. Relative expression of IGF1R mRNA decreased approximately 50% and Western blot analysis revealed a 30% of reduction in IGF-1R protein. Downregulation of this gene resulted in 40% reduction in cell growth in vitro and 45% increase in apoptosis using siRNA # 2. These findings demonstrate that decreasing IGF-1R mRNA and protein expression in NCI H295R cells can partially inhibit adrenal tumor cell growth in vitro. Targeting IGF1R is a promising therapy for pediatric malignant adrenocortical tumor and can still be an option for adult adrenocortical cancer based on personalized genomic tumor profiling.

Effects of everolimus, nilotinib, imatinib and ZOL in combination with mitotane in monolayer and spheroids human adrenocortical tumor cell line

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Deficiency of ALADIN impairs redox homeostasis in human adrenal cells and inhibits steroidogenesis.

Triple A syndrome is a rare, autosomal recessive cause of adrenal failure. Additional features include alacrima, achalasia of the esophageal cardia, and progressive neurodegenerative disease. The AAAS gene product is the nuclear pore complex protein alacrima-achalasia-adrenal insufficiency neurological disorder (ALADIN), of unknown function. Triple A syndrome patient dermal fibroblasts appear to be more sensitive to oxidative stress than wild-type fibroblasts. To provide an adrenal and neuronal-specific disease model, we established AAAS-gene knockdown in H295R human adrenocortical tumor cells and SH-SY5Y human neuroblastoma cells by lentiviral short hairpin RNA transduction. AAAS-knockdown significantly reduced cell viability in H295R cells. This effect was exacerbated by hydrogen peroxide treatment and improved by application of the antioxidant N-acetylcysteine. An imbalance in redox homeostasis after AAAS knockdown was further suggested in the H295R cells by a decrease in the ratio of reduced to oxidized glutathione. AAAS-knockdown SH-SY5Y cells were also hypersensitive to oxidative stress and responded to antioxidant treatment. A further impact on function was observed in the AAAS-knockdown H295R cells with reduced expression of key components of the steroidogenic pathway, including steroidogenic acute regulatory and P450c11β protein expression. Importantly a significant reduction in cortisol production was demonstrated with AAAS knockdown, which was partially reversed with N-acetylcysteine treatment. Conclusion: Our in vitro data in AAAS-knockdown adrenal and neuronal cells not only corroborates previous studies implicating oxidative stress in this disorder but also provides further insights into the pathogenic mechanisms in triple A syndrome.

Effect of a novel 17,20-lyase inhibitor, orteronel (TAK-700), on androgen synthesis in male rats

Endogenous androgens play a role in the development and progression of prostate cancer (PC), thus androgen suppression may offer an effective therapeutic strategy for this disease. Orteronel (TAK-700), 6-[(7S)-7-hydroxy-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-7-yl]-N-methyl-2-naphthamide, is a novel, non-steroidal, selective inhibitor of the 17,20-lyase activity of CYP17A – a key enzyme in the production of steroidal hormones – and is being developed as a therapy for PC. The purpose of this study was to elucidate the inhibitory activity of orteronel, in particular its specificity for androgen synthesis enzymes, in male rats – an androgen-synthesis model that largely reflects this pathway in humans.Orteronel inhibited 17,20-lyase activity in rats with an IC50 of 1200nM but did not inhibit 17α-hydroxylase or 11β-hydroxylase (CYP11B1) activity in rats at concentrations up to 10μM. In cellular steroidogenesis assays using rat testicular cells, orteronel suppressed testosterone and androstenedione production with an IC50 of 640nM and 210nM, respectively, but did not suppress either corticosterone or aldosterone production in rat adrenal cells at concentrations up to 30μM. In addition, serum testosterone and androstenedione levels in human chorionic gonadotropin-injected hypophysectomized rats were significantly reduced by single oral administration of orteronel at a dose of 30mg/kg (both p≤0.01); serum corticosterone and aldosterone levels in ACTH-injected hypophysectomized rats did not result in significant differences compared with controls, following orteronel administration at doses up to 300mg/kg. Serum testosterone levels in intact male rats were significantly reduced by orteronel 4h after dosing at 100mg/kg (p≤0.01); testosterone levels showed a tendency to recover afterward. In intact male rats, the weight of the prostate glands and seminal vesicles was decreased in a dose-dependent manner following multiple doses of orteronel at 37.5, 150, and 600mg/kg, TID for 4 days. The reversibility of orteronel was further confirmed using a human adrenocortical tumor cell line.In summary, orteronel is a selective and reversible 17,20-lyase inhibitor, and decreases the weight of androgen-dependent organs in male rats. Our data suggests that orteronel would therefore be effective for androgen-dependent disorders such as PC.

Abstract DD01-03: TAK-700, an inhibitor of 17,20-lyase

Abstract Background: Recent studies suggest that residual adrenal androgen after castration could be responsible for the progression of castration-resistant prostate cancer (CRPC). To develop an effective therapy for CRPC that inhibits the production of adrenal and testicular androgens, we searched for a novel, non-steroidal, selective, and potent inhibitor of 17,20-lyase, a key enzyme in androgen synthesis, and identified TAK-700 as a clinical candidate. Methods: Enzyme assay was conducted using radiolabeled substrates. The enzyme reaction products were separated by thin layer chromatography and the radioactivity was measured by a bio-image analyzer. We assessed the activity and specificity of TAK-700 on androgen production in vitro in monkey adrenal cells and human adrenocortical tumor cell line, using a radioimmunoassay. Using a single dose of [14C]TAK-700 1 mg/kg, we investigated the pharmacokinetics and bioavailability of TAK-700 in cynomolgus monkeys, and assessed the effects of TAK-700 7.5 and 15 mg/kg twice daily (BID) for 7 days on serum androgen levels in castrated and intact cynomolgus monkeys. Results: TAK-700 potently inhibited the activities of human and monkey 17, 20-lyase (CYP17A1) with IC50 values of 140 and 27nmol/L, respectively. The inhibitory activity of TAK-700 on 17-hydroxylase was lower than that on 17, 20-lyase in all the enzyme preparations. IC50 values of ketoconazole and abiraterone for human 17, 20-lyase were 110 and 27nmol/L, respectively. TAK-700 did not inhibit monkey 11-hydroxylase up to 10μmol/L. In addition, IC50 values of TAK-700 for all CYP-specific activities (drug-metabolizing enzymes) examined were higher than 10 μmol/L. TAK-700 suppressed the production of DHEA, androstenedione, cortisol, corticosterone and aldosterone by monkey adrenal cells with IC50 values of 110, 130, 310, &amp;gt;100,000 and 4,400nmol/L, respectively. The IC50 value for cortisol production was only 3-fold higher than that for DHEA production in monkeys, reflecting low specificity of inhibition between monkey 17, 20-lyase and 17-hydroxylase activity (IC50 values: 27 vs 38nmol/L). Ketoconazole suppressed the production of DHEA, androstenedione, cortisol, corticosterone and aldosterone by monkey adrenal cells with IC50 values of 340, 580, 340, 1,500 and 350nmol/L, respectively. TAK-700 inhibited the production of dehydroepiandrosterone (DHEA) and cortisol in human adrenocortical tumor line NCI-H295R cells with IC50 values of 37 and 990nmol/L, respectively, showing about 27-fold selectivity for DHEA production. Ketoconazole inhibited the production of DHEA and cortisol with IC50 values of 330 and 480nmol/L (selectivity 1.5-fold), respectively, and abiraterone inhibited the production of DHEA and cortisol with IC50 values of 2.7 and 23nmol/L (selectivity: 8.5-fold), respectively. TAK-700 showed the highest specificity of inhibition for DHEA production among these 3 compounds. After oral administration of [14C]TAK-700 1 mg/kg, maximum plasma concentration (Cmax) was 0.147 μg/mL, time to Cmax (Tmax) was 1.7 hours, elimination half-life (t1/2) was 3.8 hours, and exposure (area under the plasma concentration-time curve in 24 hours, AUC0-24h) was 0.727 μg·h/mL. Oral bioavailability of TAK-700 in monkeys was 70.5%. Oral dosing of TAK-700 7.5 and 15 mg/kg BID rapidly (within 1 day) suppressed serum levels of the androgens dehydroepiandrosterone (DHEA) and testosterone and of cortisol in both castrated and gonadally intact monkeys. While suppression of DHEA and cortisol levels persisted during the 7-day treatment period in castrated monkeys, suppression of testosterone was attenuated from the second day of treatment, possibly due to activation of the hypothalamus-pituitary-gonad axis. TAK-700 had a milder suppressive effect on cortisol levels than on DHEA levels. During the treatment period, mean DHEA levels decreased to 9.0% and 6.9% of baseline levels in castrated monkeys treated with TAK-700 7.5 and 15 mg/kg, respectively. The mean cortisol levels decreased to 26.0% and 17.3% of baseline levels with TAK-700 7.5 and 15 mg/kg, respectively. In vitro results have shown that the specificity of TAK-700 suppression of DHEA production over cortisol production is 10-fold greater in human adrenal-origin cells than in monkey adrenal cells. Therefore, the impact of TAK-700 on human serum cortisol levels may be smaller than that in monkeys. Conclusion: As TAK-700 potently and selectively inhibited 17, 20-lyase activity and DHEA production by H295R cells and suppressed serum androgen levels in castrated monkeys, TAK-700 may be effective for at least some types of CRPC, and clinical evaluation of TAK-700 is now underway in patients with CRPC. Citation Format: Masuo Yamaoka, Takahito Hara, Hiroshi Miki, Takenori Hitaka, Tomohiro Kaku, Naohiro Kawaguchi, Hitomi Yamasaki, Toshiyuki Takeuchi, Akihiro Tasaka, Masami Kusaka. TAK-700, an inhibitor of 17,20-lyase [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr DD01-03

Mechanisms of angiotensin II-mediated regulation of aldosterone synthase expression in H295R human adrenocortical and rat adrenal glomerulosa cells

In adrenal zona glomerulosa cells angiotensin II (Ang II) is a key regulator of steroidogenesis. Our purpose was to compare the mechanisms of Ang II-induced changes in the expression level of early transcription factors NR4A1 (NGFIB) and NR4A2 (Nurr1) genes, and the CYP11B2 gene encoding aldosterone synthase in H295R human adrenocortical tumor cells and in primary rat adrenal glomerulosa cells. Real-time PCR studies have demonstrated that Ang II increased the expression levels of NR4A1 and NR4A2 in H295R cells within 1 h after stimulation, which persisted up to 6 h; whereas in rat adrenal glomerulosa cells the kinetics of the expression of these genes were more rapid and transient. Ang II also induced prolonged nuclear translocation of Nurr1 and NGFIB proteins in both cell types. Studies using MEK inhibitor (PD98059, 20 μM), protein kinase C inhibitor (BIM1, 3 μM) and calmodulin kinase (CAMK) inhibitor (KN93, 10 μM) revealed that in rat adrenal glomerulosa cells CAMK-mediated mechanisms play a predominant role in the regulation of CYP11B2. In accordance with earlier findings, in H295R cells MEK inhibition increased the expression of NR4A1, NR4A2 and CYP11B2 genes, however, it decreased the Ang II-induced gene expression levels, suggesting that ERK activation has a role in control of expression of these genes. No such mechanism was detected in rat glomerulosa cells. Sar 1-Ile 4-Ile 8-AngII, which can cause G protein-independent ERK activation, also stimulated the expression of CYP11B2 in H295R cells. These data suggest that the previously reported CAMK-mediated stimulation of early transcription factors NGFIB and Nurr1 has a predominant role in Ang II-induced CYP11B2 activation in rat adrenal glomerulosa cells, whereas in H295R cells ERK activation and G protein-independent mechanisms also contribute to this process.

CBP/p300-Interacting Transactivator, with Glu/Asp-Rich C-Terminal Domain, 2, and Pre-B-Cell Leukemia Transcription Factor 1 in Human Adrenal Development and Disease

Disorders of adrenal development result in significant morbidity and mortality. However, the molecular basis of human adrenal development, and many forms of disease, is still poorly understood. We evaluated the role of two new candidate genes, CBP/p300-interacting transactivator, with Glu/Asp-rich C-terminal domain, 2 (CITED2), and pre-B-cell leukemia transcription factor 1 (PBX1), in human adrenal development and disease. CITED2 and PBX1 expression in early human fetal adrenal development was assessed using RT-PCR and in situ hybridization. The regulation of CITED2 and PBX1 by steroidogenic factor-1 (SF-1) and dosage-sensitive sex reversal, adrenal hypoplasia congenital, critical region on the X chromosome, gene-1 (DAX1) was evaluated in NCI-H295R human adrenocortical tumor cells by studying promoter regulation. Finally, mutational analysis of CITED2 and PBX1 was performed in patients with primary adrenal disorders. CITED2 and PBX1 are expressed in the human fetal adrenal gland during early development. Both genes are activated by SF-1 in a dose-dependent manner in NCI-H295R cells, and, surprisingly, PBX1 is synergistically activated by SF-1 and DAX1. Mutational analysis failed to reveal significant coding sequence changes in individuals with primary adrenal disorders. CITED2 and PBX1 are likely to be important mediators of adrenal development and function in humans, but mutations in these genes are not common causes of adrenal failure in patients in whom a molecular diagnosis remains unknown. The positive interaction between DAX1 and SF-1 in regulating PBX1 may be an important mechanism in this process.

Leukemia inhibitory factor protein and receptors are expressed in the bovine adrenal cortex and increase cortisol and decrease adrenal androgen release

The release of adrenal steroids during acute stress is primarily regulated by adrenocorticotropic hormone (ACTH). In contrast, during chronic inflammatory stress additional factors are involved in regulating adrenal function. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that increases ACTH release from the pituitary. In addition, LIF and LIF receptors (LIFR) are expressed in the human adrenal cortex and the human adrenocortical tumor cell line H295R. Furthermore, LIF increases basal and ACTH-stimulated cortisol release from H295R cells. However, the expression of LIF and LIFR in non-human adrenal glands and the effects of LIF on the release of cortisol from adrenal cells of non-human species have not been determined. Furthermore, the effects of LIF on adrenal androgen release from all species are unknown. In this study, immunohistochemistry, Western blots, RT-PCR, and nucleotide sequencing was utilized to demonstrate that LIF and its receptor are expressed throughout the bovine adrenal cortex. Although LIF did not modify basal cortisol release from dispersed cells isolated from the bovine adrenal zona fasciculate, this cytokine increased ACTH-stimulated release of cortisol from these cells in a manner dependent on the LIF concentration and exposure interval. In contrast, LIF in a concentration-dependent and time-dependent manner decreased basal and ACTH-stimulated adrenal androgen release from dispersed cells isolated from the bovine adrenal zona reticularis. Because LIF release increases during inflammatory stress and this cytokine stimulates adrenal cortisol release and inhibits adrenal androgen release, this cytokine may play an important role in regulating the release of adrenal steroids during inflammatory stress.

Side Population Does Not Define Stem Cell-Like Cancer Cells in the Adrenocortical Carcinoma Cell Line NCI h295R

Recent evidence suggests the existence of a stem cell-like subpopulation of cells in hematological and solid tumor entities, which determine the malignant phenotype of a given tumor through their proliferative potential and chemotherapy resistance. A recently used technique for the isolation of this cell population is through exclusion of the vital dye Hoechst 33342, which defines the so-called side population (SP). Herein we demonstrate the presence of SP cells in a variety of adrenal specimens, including primary cultures of human adrenocortical tumors and normal adrenal glands as well as established human and murine adrenocortical cancer cell lines by fluorescence-activated cell sorter analysis and confocal microscopy. On a functional level, SP cells from the human adrenocortical tumor cell line NCI h295R revealed an expression pattern consistent with a less differentiated phenotype, including lower expression of steroidogenic enzymes such as steroid acute regulatory protein (StAR) and side-chain cleavage enzyme (P450scc) in comparison with non-SP cells. However, proliferation between SP and non-SP cells did not differ (105.6 +/- 18.1 vs. 100.0 +/- 3.5%). Furthermore, re-sorting and tracing experiments revealed the capacity for both cell types to give rise to the original SP- and non-SP-containing cell population. Similarly to the baseline growth kinetics, no survival benefit was evident in SP cells after treatment with cytotoxic agents commonly used in adrenocortical carcinomas. Taken together, these findings provide evidence that Hoechst dye exclusion, in contrast to what has been reported for other tumor entities, is not a major tumor stem cell defining marker in adrenocortical NCI h295R tumor cells.

Angiotensin-II acute regulation of rapid response genes in human, bovine, and rat adrenocortical cells

Angiotensin-II (Ang-II) regulates adrenal steroid production and gene transcription through several signaling pathways. Changes in gene transcription occur within minutes after Ang-II stimulation, causing an increase in aldosterone production and subsequent increase in the overall capacity to produce aldosterone. Our goal was to compare the Ang-II regulation of early gene expression and confirm the up-regulation of selected genes using quantitative real-time RT-PCR (qPCR) across three species, such as, human, bovine, and rat. Microarray analyses were performed using samples from control and Ang-II (10 nM)-treated (1 h) cells from human adrenocortical tumor cell line H295R, and primary adrenal glomerulosa cells from bovine and rat, applied respectively to human, bovine, and rat chips. qPCR was performed to confirm up-regulation of selected genes using mRNA. The microarray comparison revealed 18% similarity among the top 50 up-regulated genes, with human/rat, 20%; human/bovine, 36%; and rat/bovine, 26% similarity. The gene list generated by this comparison included: activating transcription factor 3, B-cell translocation gene (BTG2), Nuclear receptor subfamily 4, group A, member 1 (NR4A1), NR4A2, NR4A3, early growth response 1, v-fos FBJ murine osteosarcoma viral oncogene homolog (c-FOS), FOSB, and Jun family member B (JUNB). Pretreatment of H295R cells with cycloheximide had no effect on Ang-II induction of these genes, suggesting that they are direct targets of Ang-II signaling. The Ang-II gene targets have been defined in three different adrenocortical model systems. Several of the listed genes have previously been described as being key regulators of adrenocortical function. The presence of adrenal cell common genes in such distinct cell models strengthens the hypothesis that these genes are regulators of aldosterone production.

Leptin Interferes with Adrenocorticotropin/3′,5′-Cyclic Adenosine Monophosphate (cAMP) Signaling, Possibly through a Janus Kinase 2-Phosphatidylinositol 3-Kinase/Akt-Phosphodiesterase 3-cAMP Pathway, to Down-Regulate Cholesterol Side-Chain Cleavage Cytochrome P450 Enzyme in Human Adrenocortical NCI-H295 Cell Line

Obesity has adverse effects on adrenocortical functions. Adipocyte-derived leptin, a biomarker molecule of obesity, may directly control adrenal steroidogenesis via an unclear mechanism. We studied the mechanism underlying leptin action on adrenal steroidogenesis in human adrenocortical NCI-H295 tumor cell line. Levels of progesterone, cortisol, and cAMP were determined by ELISA. Western blotting was used to detect protein amounts of P450 side-chain cleavage (P450scc), Janus kinase 2 (JAK2), Akt, and their phosphorylated forms. The mRNA expressions of P450scc and leptin receptors were measured by RT-PCR and real-time PCR. P450scc promoter activity was analyzed with a luciferase reporter system. Cholera toxin mimicked ACTH action by increasing adrenal cAMP levels and steroid secretion. Leptin did not affect basal release but significantly inhibited ACTH/cholera toxin-induced steroid secretion. The concomitant inhibitions by leptin on cholera toxin-induced protein and ACTH/cholera toxin-induced mRNA expression of P450scc were confirmed. Leptin inhibited ACTH/cholera toxin-induced CYP11A1 promoter activity via a known cAMP-responsive region located between -1.7 and -1.5 kb. Leptin activated phosphorylations of JAK2 and Akt. Inhibitory effects of leptin on ACTH/cholera toxin-induced cAMP levels, CYP11A1 promoter activity, and steroid secretion were blunted by either inhibitor of JAK2 (AG490) or phosphatidylinositol 3-kinase/Akt (wortmannin) as well as inhibitors of cAMP-degrading phosphodiesterases (PDEs), including nonspecific 3-isobutyl-1-methylxanthine and PDE3-specific SKF94836. Leptin failed to affect the inductions of CYP11A1 promoter activity and steroid secretion by PDE-nonhydrolyzable N(6)-monobutyryl-cAMP. Leptin interferes with ACTH/cAMP signaling, possibly through a cAMP-degrading mechanism involving activation of JAK2, phosphatidylinositol 3-kinase, and PDE3, to down-regulate P450scc expression and consequent adrenal steroidogenesis.

Blocking T-Type Ca2+ Channels with Efonidipine Decreased Plasma Aldosterone Concentration in Healthy Volunteers

Efonidipine can block both L- and T- type Ca2+ channels. In a previous in vitro study, we clarified that efonidipine dramatically suppresses aldosterone secretion from human adrenocortical tumor cells during angiotensin II (Ang II)- and K+-stimulation, whereas nifedipine, a dominant L-type Ca2+ channel antagonist, does not. This study was conducted to assess the in vivo effects of efonidipine and nilvadipine on the plasma aldosterone concentration. Placebo, 40 mg of efonidipine, or 2 mg of nilvadipine was administered to five healthy male volunteers. Hemodynamic parameters (pulse rate [PR] and blood pressure [BP]), plasma concentrations of neurohormonal factors (plasma renin activity, Ang II, aldosterone, and adrenocorticotropic hormone [ACTH]), and serum concentrations of Na+ and K+ were measured before and 6 h after administration of the agents. All three agents had little effect on PR and BP. Efonidipine and nilvadipine significantly increased plasma renin activity and Ang II. Both had little effect on ACTH, Na+, and K+. The plasma aldosterone concentration was significantly decreased after efonidipine treatment (88.3 +/- 21.3 to 81.6 +/- 24.9 pg/ml, p = 0.0407), whereas it was significantly increased after nilvadipine treatment (66.5 +/- 12.2 to 82.17 +/- 16.6 pg/ml, p = 0.0049). Placebo had little effect on neurohormonal factors. Efonidipine decreased plasma aldosterone concentration despite the increase in plasma renin activity and Ang II, suggesting that T-type Ca2+ channels may also play an essential role in the secretion of aldosterone in healthy human volunteers.

Neuroendocrine properties of human adrenocortical cells: GABA production and functional GABAB receptors in the NCI-H295R cell line

The neurotransmitter GABA, well known for its functions in the central nervous system, is also present in peripheral endocrine tissues. GABAergic paracrine or autocrine signaling pathways have been found in the pancreatic islets, the adenohypophysis, and in steroid-producing organs like ovary and testis. In the present study, we describe elements of a GABAergic system in a human adrenocortical tumor cell line (NCI-H295R). Immunocytochemistry, Western blots and RT-PCR experiments showed that this cell line, a model system for the study of adrenocortical function, expresses glutamic acid decarboxylase (GAD) isoform 67, the biosynthetic enzyme for GABA. We furthermore demonstrated that these cells decarboxylate glutamate to GABA by an in-vitro assay. In addition, NCI-H295R cells express the GABAB(1a), GABAB(1e) and GABAB(2) subunits of the heterodimeric GABAB receptor as shown by RT-PCR and by immunostaining for the GABAB(2) subunit. According to our findings, GABAB(1e), a truncated splice variant of the GABAB(1) subunit which lacks the transmembrane and intracellular domains, is the predominant GABAB(1) transcript. The GABAB receptors we identified are functional since the GABAB-agonist baclofen decreased the low-voltage activated (T-type) calcium currents in whole-cell patch clamp experiments. In summary, our results show that GABA is produced by steroidogenic cells derived from the human adrenal cortex and may act on these cells in an autocrine manner. In addition, adrenocortical GABA may also reach the adrenal medulla via the vasculature and influence chromaffin cells which are responsive to GABA.

Transforming growth factor beta1 inhibits aldosterone and cortisol production in the human adrenocortical cell line NCI-H295R through inhibition of CYP11B1 and CYP11B2 expression.

Transforming growth factor beta1 (TGFbeta1) has been shown to exert strong inhibitory effects on adrenocortical cell steroidogenesis. However, the molecular targets of TGFbeta1 in adrenocortical cells appear to differ between species. Here, we report the first characterization of the regulatory effects of TGFbeta1 on the steroidogenic functions of the human adrenocortical tumor cell line NCI-H295R. After treatment with 2 ng/ml TGFbeta1 for 24 h, basal production of corticosterone, cortisol and androstenedione was dramatically decreased. When TGFbeta1 was added simultaneously with forskolin, the production of cortisol and 11-hydroxyandrostenedione was decreased by 85% whereas that of deoxycortisol was increased. When TGFbeta1 was added simultaneously with angiotensin II, aldosterone production was reduced by 80%. We observed that TGFbeta1 strongly inhibits forskolin-induced steroid 11beta-hydroxylase activity and CYP11B1 mRNA levels, as well as angiotensin II-induced aldosterone synthase activity and CYP11B2 mRNA levels. CYP11B1 and CYP11B2 gene products thus appear as the major steroidogenic enzymes down-regulated by TGFbeta1 in the human adrenocortical tumor cell line NCI-H295R.

High expression of cyclin E and G1 CDK and loss of function of p57KIP2 are involved in proliferation of malignant sporadic adrenocortical tumors.

Maternal loss of heterozygosity (LOH) of the 11p15 region and overexpression of the insulin-like growth factor (IGF)-II gene are associated with the malignant phenotype in sporadic adrenocortical tumors. In the imprinted 11p15 region, the p57KIP2 gene is maternally expressed and encodes a cyclin-dependent kinase (CDK) inhibitor involved in G1/S phase of the cell cycle. We hypothesized that maternal LOH in malignant adrenocortical tumors could be responsible for loss of p57KIP2 gene expression and, thus, could favor progression through the cell cycle. We investigated 3 normal adrenals, 31 adrenocortical tumors [11 tumors with normal expression of the IGF-II gene (mainly benign) and 20 with IGF-II gene overexpression (mainly malignant)], and the human adrenocortical tumor cell line NCI H295R for expression of the p57KIP2 gene, G1 cyclins (cyclin D2 and E) and G1 CDK (CDK2, CDK3 and CDK4) protein contents and for kinase activity of G1 cyclin-CDK complexes. The expression of p57KIP2, G1 cyclins, and G1 CDKs in benign tumors was similar to that in normal adrenal tissues, as were kinase activities of G1 cyclin-CDK complexes. By contrast, abrogation of the p57KIP2 gene expression and increased expression of G1 cyclins (cyclin E) and G1 CDKs (CDK2 and CDK4) were associated with high activity of G1 cyclin-CDK complexes in malignant tumors and in the H295R cell line. These data suggest that the p57KIP2 gene might act as a tumor suppressor gene in adrenocortical tumors. Maternal LOH with duplication of the paternal allele or pathological functional imprinting of the 11p15 region are responsible for loss of expression of the p57KIP2 gene and increased expression of the IGF-II gene. Consequently, both events favor cell proliferation in malignant adrenocortical tumors.