Journal articles: 'NCI-H295R'

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Samandari, Elika, Petra Kempná, Jean-Marc Nuoffer, Gaby Hofer, Primus E. Mullis, and Christa E. Flück. "Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3β-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities." Journal of Endocrinology 195, no. 3 (September 27, 2007): 459–72. http://dx.doi.org/10.1677/joe-07-0166.

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The human adrenal cortex produces mineralocorticoids, glucocorticoids, and androgens in a species-specific, hormonally regulated, zone-specific, and developmentally characteristic fashion. Most molecular studies of adrenal steroidogenesis use human adrenocortical NCI-H295A and NCI-H295R cells as a model because appropriate animal models do not exist. NCI-H295A and NCI-H295R cells originate from the same adrenocortical carcinoma which produced predominantly androgens but also smaller amounts of mineralocorticoids and glucocorticoids. Research data obtained from either NCI-H295A or NCI-H295R cells are generally compared, although for the same experiments no direct comparison between the two cell lines has been performed. Therefore, we compared the steroid profile and the expression pattern of important genes involved in steroidogenesis in both cell lines. We found that steroidogenesis differs profoundly. NCI-H295A cells produce more mineralocorticoids, whereas NCI-H295R cells produce more androgens. Expression of the 3β-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5, and sulfonyltransferase genes is higher in NCI-H295A cells, whereas expression of the cytochrome P450c17 (CYP17), 21-hydroxylase (CYP21), and P450 oxidoreductase genes does not differ between the cell lines. We found lower 3β-hydroxysteroid dehydrogenase type 2 but higher 17,20-lyase activity in NCI-H295R cells explaining the ‘androgenic’ steroid profile for these cells and resembling the zona reticularis of the human adrenal cortex. Both cell lines were found to express the ACTH receptor at low levels consistent with low stimulation by ACTH. By contrast, both cell lines were readily stimulated by 8Br-cAMP. The angiotensin type 1 receptor was highly expressed in NCI-H295R than NCI-H295A cells and angiotensin II stimulated steroidogenesis in NCI-H295R but not NCI-H295A cells. Our data suggest that comparative studies between NCI-H295A and NCI-H295R cells may help find important regulators of mineralocorticoid or androgen biosynthesis.

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Watanabe, Masatada, Mariko Noda, and Shizuo Nakajin. "Effect of epidermal growth factor and prostaglandin on the expression of aromatase (CYP19) in human adrenocortical carcinoma cell line NCI-H295R cells." Journal of Endocrinology 188, no. 1 (January 2006): 59–68. http://dx.doi.org/10.1677/joe.1.06214.

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We investigated the effects of epidermal growth factor (EGF) and prostaglandins (PG) on the expression of aromatase (CYP19) in human adrenocortical carcinoma cell line NCI-H295R cells. EGF significantly increased aromatase activity and CYP19 gene transcript in NCI-H295R cells. Exon PII was selected from among several tissue-specific exon I regions. Promoter II that abuts on exon PII was activated by EGF. PGE2 also significantly increased aromatase activity, CYP19 gene transcript, and promoter II activity. The results of experiments using protein kinase (PK) inhibitors suggest that the cAMP–PKA signaling pathway is involved in the up-regulation of aromatase expression by EGF. PGE2 activated promoter II activity in 4 h, while12 h was required for its activation by EGF. In addition, PGE2 was secreted from NCI-H295R cells in response to EGF. Selective agonists for prostaglandin receptors EP1 and EP2 significantly increased aromatase activity, which was decreased by the corresponding antagonists. Finally, antagonists for EP1 and EP2 inhibited the up-regulation of aromatase expression following EGF. These results suggest that PGE2 secondarily acts as an autocrine signal in the up-regulation of aromatase expression by EGF in NCI-H295R cells.

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Wenzel, Jan, Nicole Grabinski, Cordula A. Knopp, Andreas Dendorfer, Manjunath Ramanjaneya, Harpal S. Randeva, Monika Ehrhart-Bornstein, Peter Dominiak, and Olaf Jöhren. "Hypocretin/orexin increases the expression of steroidogenic enzymes in human adrenocortical NCI H295R cells." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 297, no. 5 (November 2009): R1601—R1609. http://dx.doi.org/10.1152/ajpregu.91034.2008.

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Hypocretins/orexins act through two receptor subtypes: OX1 and OX2. Outside the brain, orexin receptors are expressed in adrenal glands, where orexins stimulate the release of glucocorticoids. To further address the regulation of steroidogenesis, we analyzed the effect of orexins on the expression of steroidogenic enzymes in human adrenocortical National Cancer Institute (NCI) H295R cells by qPCR. In NCI H295R cells, OX2 receptors were highly expressed, as they were in human adrenal glands. After treatment of NCI H295R cells with orexin A for 12–24 h, the cortisol synthesis rate was significantly increased, whereas 30 min of treatment showed no effect. While CYP11B1 and CYP11B2 mRNA levels were increased already at earlier time points, the expression of HSD3B2 and CYP21 mRNA was significantly up-regulated after treatment with orexin A for 12 h. Likewise, orexin B increased CYP21 and HSD3B2 mRNA levels showing, however, a lower potency compared with orexin A. The mRNA levels of CYP11A and CYP17 were unaffected by orexin A. OX2 receptor mRNA levels were down-regulated after 12 and 24 h of orexin A treatment. Orexin A increased intracellular Ca2+ but not cAMP concentrations in NCI H295R cells. Furthermore, inhibition of PKC and MAPK kinase/ERK kinase (MEK1/2) prevented the increase of HSD3B2 expression by orexin A. Accordingly, orexin A treatment of NCI H295R cells markedly enhanced ERK1/2 phosphorylation that was prevented by PKC and, in part, PKA inhibition. In conclusion, orexins may influence adrenal steroidogenesis by differential regulation of the expression of steroidogenic enzymes involving Ca2+, as well as PKC-ERK1/2 signaling.

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Nicolson, Norman G., Reju Korah, and Tobias Carling. "Adrenocortical cancer cell line mutational profile reveals aggressive genetic background." Journal of Molecular Endocrinology 62, no. 4 (May 2019): 179–86. http://dx.doi.org/10.1530/jme-18-0262.

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Adrenocortical carcinomas are rare tumors with poor prognosis and limited treatment options. Although widely used as in vitro models to test novel therapeutic strategies, the adrenocortical carcinoma-derived cell lines NCI-H295R and SW-13 have only partially been described genetically. Our aim was to characterize the mutational landscape of these cells to improve their experimental utility and map them to clinical subtypes of adrenocortical carcinoma. Genomic DNA from NCI-H295R and SW-13 cells was subjected to whole-exome sequencing. Variants were filtered for non-synonymous mutations and curated for validated adrenocortical and pan-cancer driver gene mutations. Genes mutated in the cell lines were mapped using gene ontology and protein pathway tools to determine signaling effects and compared to mutational and clinical characteristics of 92 adrenocortical carcinoma cases from The Cancer Genome Atlas. NCI-H295R and SW-13 cells carried 1325 and 1836 non-synonymous variants, respectively. Of these, 61 and 76 were known cancer driver genes, of which 32 were shared between cell lines. Variant interaction analyses demonstrated dominant TP53 dysregulation in both cell lines complemented by distinct WNT (NCI-H295R) and chromatin remodeling (SW-13) pathway perturbations. Both cell lines genetically resemble more aggressive adrenocortical carcinomas with worse prognosis, for which development of targeted therapies is most critical. Careful incorporation of the genetic landscapes outlined in this study will further the in vitro utility of these cell lines in testing for novel therapeutic approaches for adrenocortical malignancy.

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Asif, Abdul R., Marija Ljubojevic, Ivan Sabolic, Vladimir Shnitsar, Maria Metten, Naohiko Anzai, Gerhard A. Müller, Gerhard Burckhardt, and Yohannes Hagos. "Regulation of steroid hormone biosynthesis enzymes and organic anion transporters by forskolin and DHEA-S treatment in adrenocortical cells." American Journal of Physiology-Endocrinology and Metabolism 291, no. 6 (December 2006): E1351—E1359. http://dx.doi.org/10.1152/ajpendo.00653.2005.

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Several important physiological functions are regulated by cortisol. Previously, we demonstrated the involvement of human organic anion transporter 3 (hOAT3) in cortisol release. In the present study, we investigated the influence of dehydroepiandrosterone sulfate (DHEA-S) and estrone sulfate on cortisol release in a human adrenocortical cell line (NCI-H295R) compared with forskolin stimulation. Additionally, we examined the impact of forskolin and DHEA-S on the expression of key enzymes in steroid biosynthesis and expression of hOAT3 and -4 in NCI-H295R cells. The cortisol release was increased 10-fold after 24-h incubation with DHEA-S, but incubation with estrone sulfate did not show any significant change in cortisol release. When cells were incubated with DHEA-S in the presence of forskolin, an additive influence of DHEA-S stimulation of cortisol was recorded over forskolin alone. The 24-h stimulation of NCI-H295R cells with forskolin increased the expression of steroidogenic acute regulatory protein (StAR), CYP17, CYP21A2, and CYP11A1, whereas only StAR mRNA expression was increased significantly by incubation with DHEA-S. Immunofluorescence analyses revealed strongly elevated expression of hOAT3 by forskolin as well as by DHEA-S stimulation. We conclude that the increased cortisol release of adrenocortical cells by DHEA-S and forskolin stimulation is probably due to high expression of the key enzymes of steroid biosynthesis and hOAT3.

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Belavgeni, Alexia, Stefan R. Bornstein, Anne von Mässenhausen, Wulf Tonnus, Julian Stumpf, Claudia Meyer, Evelyn Othmar, et al. "Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis." Proceedings of the National Academy of Sciences 116, no. 44 (October 14, 2019): 22269–74. http://dx.doi.org/10.1073/pnas.1912700116.

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Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis.

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Liu, J., X.-D. Li, A. Ora, P. Heikkilä, A. Vaheri, and R. Voutilainen. "cAMP-dependent protein kinase activation inhibits proliferation and enhances apoptotic effect of tumor necrosis factor-α in NCI-H295R adrenocortical cells." Journal of Molecular Endocrinology 33, no. 2 (October 2004): 511–22. http://dx.doi.org/10.1677/jme.1.01535.

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Adrenocorticotropin is the major regulator of adrenocortical development and function. It acts mainly through the cAMP-dependent protein kinase A (PKA) pathway. Our aim was to study the interaction of tumor necrosis factor-α (TNFα) and the PKA pathway in adrenocortical cell proliferation and apoptosis. The PKA activator Dibutyryl cAMP ((Bu)2cAMP) strongly induced differentiation and inhibited proliferation in the human adrenocortical cell line NCI-H295R (H295R). TNFα induced apoptosis of H295R cells. Interestingly, (Bu)2cAMP treatment clearly enhanced TNFα-induced apoptosis in H295R cells, but not in another human adrenocortical cell line SW-13, the mouse adrenocortical Y-1 cell line or the human HeLa cell line. This synergistic effect was not due to the (Bu)2cAMP-induced glucocorticoid secretion since dexamethasone had no significant effect on the TNFα-induced apoptosis. (Bu)2cAMP treatment rapidly increased the expression of the proto-oncogene c-myc in H295R cells, but not in SW-13, Y-1 or HeLa cells. In transient c-myc transfection assay, c-myc expression associated with decreased expression of the proliferation marker Ki-67 in H295R cells. In conclusion, cAMP-dependent protein kinase activation reduced proliferation and augmented TNFα-induced apoptosis in adrenocortical H295R cells, and these effects were associated with increased c-myc expression.

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Yu, Jin, Yuhuan Liu, Danying Zhang, Dongxia Zhai, Linyi Song, Zailong Cai, and Chaoqin Yu. "Baicalin inhibits recruitment of GATA1 to the HSD3B2 promoter and reverses hyperandrogenism of PCOS." Journal of Endocrinology 240, no. 3 (March 2019): 497–507. http://dx.doi.org/10.1530/joe-18-0678.

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High androgen levels in patients suffering from polycystic ovary syndrome (PCOS) can be effectively reversed if the herb Scutellaria baicalensis is included in traditional Chinese medicine prescriptions. To characterize the effects of baicalin, extracted from S. baicalensis, on androgen biosynthesis in NCI-H295R cells and on hyperandrogenism in PCOS model rats and to elucidate the underlying mechanisms. The optimum concentration and intervention time for baicalin treatment of NCI-H295R cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and ELISA. The functional genes affected by baicalin were studied by gene expression profiling (GEP), and the key genes were identified using a dual luciferase assay, RNA interference technique and genetic mutations. Besides, hyperandrogenic PCOS model rats were induced and confirmed before and after baicalin intervention. As a result, baicalin decreased the testosterone concentrations in a dose- and time-dependent manner in NCI-H295R cells. GEP revealed that 3β-hydroxysteroid dehydrogenase type II (HSD3B2) was the key enzyme of androgen biosynthesis, and baicalin inhibited the expression of HSD3B2 by regulating the binding of transcription factor GATA-binding factor 1 (GATA1) to the HSD3B2 promoter. Hyperandrogenic PCOS model rats treated with baicalin significantly reversed the high androgen levels of serum and the abnormal ovarian status, restored the estrous cyclicity and decreased the expression of HSD3B2 in ovarian. In summary, our data revealed that GATA1 is an important transcription factor activating the HSD3B2 promoter in steroidogenesis, and baicalin will potentially be an effective therapeutic agent for hyperandrogenism in PCOS by inhibiting the recruitment of GATA1 to the HSD3B2 promoter in ovarian tissue.

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Bollag, Wendy B., Patricia Kent, Stephanie White, Mariya V. Wilson, Carlos M. Isales, and Roberto A. Calle. "Phorbol ester increases mitochondrial cholesterol content in NCI H295R cells." Molecular and Cellular Endocrinology 296, no. 1-2 (December 2008): 53–57. http://dx.doi.org/10.1016/j.mce.2008.08.022.

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Jain, Meenu, Lisa Zhang, Mei He, Ya-Qin Zhang, Min Shen, and Electron Kebebew. "TOP2A is overexpressed and is a therapeutic target for adrenocortical carcinoma." Endocrine-Related Cancer 20, no. 3 (March 26, 2013): 361–70. http://dx.doi.org/10.1530/erc-12-0403.

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Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy with no effective therapy for patients with unresectable disease. The aim of the current study was i) to evaluate TOP2A expression and function in human adrenocortical neoplasm and ACC cells and ii) to determine the anticancer activity of agents that target TOP2A. TOP2A mRNA and protein expression levels were evaluated in 112 adrenocortical tissue samples (21 normal adrenal cortex, 80 benign adrenocortical tumors, and 11 ACCs). In vitro siRNA knockdown of TOP2A in ACC cell lines (NCI-H295R and SW13) was used to determine its effect on cellular proliferation, cell cycle, anchorage-independent growth, and cellular invasion. We screened 14 TOP2A inhibitors for their anticancer activity in ACC cells. TOP2A mRNA and protein expression was significantly higher in ACC than in benign and normal adrenocortical tissue samples (P<0.05). Knockdown of TOP2A gene expression in ACC cell lines significantly decreased cell proliferation, anchorage-independent growth, and invasion (P<0.05). A screening assay in NCI-H295R cells showed that 11 of 14 TOP2A inhibitors had antiproliferative activity, 5 of the 14 TOP2A inhibitors had a higher antiproliferative activity than mitotane, and aclarubicin was the agent with the highest activity. Aclarubicin was validated to significantly decrease proliferation and tumor spheroid size in both NCI-H295R and SW13 ACC cell lines (P<0.05). Our results suggest that TOP2A is overexpressed in ACC, regulates cellular proliferation and invasion in ACC cells, and is an attractive target for ACC therapy. Of the TOP2A inhibitors screened, aclarubicin is a good candidate agent to test in future clinical trials for patients with locally advanced and metastatic ACC.

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Logie, A., N. Boulle, V. Gaston, L. Perin, P. Boudou, Y. Le Bouc, and C. Gicquel. "Autocrine role of IGF-II in proliferation of human adrenocortical carcinoma NCI H295R cell line." Journal of Molecular Endocrinology 23, no. 1 (August 1, 1999): 23–32. http://dx.doi.org/10.1677/jme.0.0230023.

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In adrenocortical tumors, the malignant phenotype is associated with rearrangements (paternal isodisomy) at the 11p15 locus and IGF-II gene overexpression, strongly suggesting that the IGF system is a major determinant of adrenocortical tumor progression. The aim of this study was to validate an in vitro model for investigating the involvement of the IGF system in adrenocortical tumorigenesis. We analyzed the production of IGF mRNA and proteins, IGF-binding proteins (IGFBPs) and IGF receptors by the NCI H295R cell line, which is derived from a human adult adrenocortical carcinoma. H295R cells were shown to proliferate for a long period (26 days) in the absence of serum or any added growth factor. Northern blot analyses showed high IGF-II mRNA contents in H295R cells. The cells secreted large amounts of IGF-II protein (14 ng/10(6) cells per 48 h) although no IGF-I protein was detected. Western ligand blot analyses of conditioned media detected the presence of large amounts of a 34 kDa protein, which was identified as IGFBP-2 by immunoblotting. The presence of high-affinity binding sites for IGF-I and IGF-II on H295R cells was shown by binding experiments using radiolabeled IGFs and confirmed by reverse transcription PCR analyses showing type 1 and type 2 IGF receptors. Proliferation of H295R cells was inhibited by anti-IGF-II antibody (45%) and by anti-type 1 IGF receptor antibody (53%) indicating that IGF-II is an autocrine growth factor for these cells and that its effects are, at least in part, mediated by the type 1 IGF receptor. These findings confirm the involvement of the IGF system in adrenocortical tumors and suggest that the H295R cell line is a suitable in vitro model for studying the molecular mechanisms of adrenocortical tumor proliferation.

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Berber, Mesut, Sining Leng, Felix Beuschlein, David T. Breault, Johannes Loffing, and David Penton Ribas. "Calcineurin-NFATc4 Pathway Is Activated Upon K+-stimulation of Adrenal Aldosterone Production." Journal of the Endocrine Society 5, Supplement_1 (May 1, 2021): A805—A806. http://dx.doi.org/10.1210/jendso/bvab048.1638.

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Abstract The mineralocorticoid aldosterone secreted by the adrenal zona glomerulosa (ZG) cells promote renal K+ secretion and Na+ reabsorption; thereby it is critical for the control of ion homeostasis and blood pressure. While the Ca2+/calmodulin-dependent protein kinase (CAMK) pathway regulating K+ stimulated aldosterone production is well studied, little is known about the potentially involved phosphatases. Interestingly, immunosuppression therapy of transplanted patients with protein phosphatase 3 (calcineurin) inhibitors often results in rather low plasma aldosterone levels despite a concomitant hyperkalemia and hyperreninemia. Calcineurin (Cn) is a calcium and calmodulin-dependent protein phosphatase expressed in the adrenal cortex. We tested the hypothesis that Cn participates in the signal transduction pathway mediating the K+-dependent stimulation of aldosterone production. To address this question, we used the adrenocortical cell model NCI-H295R, mouse and human ex vivo adrenal preparations and a ZG-specific and inducible Cn knockout mouse model (ZG-CnB1-KO). Inhibition of Cn with tacrolimus abolished the K+-stimulated expression of CYP11B2 in NCI-H295R cell line as well as mouse and human adrenal pieces, ex vivo. Using a phosphoproteomics analysis, we identified nuclear factor of activated T-cells, cytoplasmic 4 (NFATc4) as a critical downstream factor mediating Cn function. In support of this result, genetic deletion of NFATc4 reduced the basal expression of CYP11B2 and impaired the K+-stimulated expression of this gene. Conversely, the expression of a constitutively active form of NFATc4 drastically increased the expression of CYP11B2 in NCI-H295R cells which remained unaltered upon treatment with K+ or tacrolimus. Finally, preliminary experiments using ZG-CnB1-KO mice suggest that Cn deletion in the ZG blunts the increase in aldosterone excretion triggered by high K+ diet. Altogether, our data indicate that Cn function is indispensable for the physiological regulation of aldosterone production. Moreover, Cn may represent a novel molecular target for the pharmacological treatment of primary aldosteronism.

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Liakos, P., D. Lenz, R. Bernhardt, JJ Feige, and G. Defaye. "Transforming growth factor beta1 inhibits aldosterone and cortisol production in the human adrenocortical cell line NCI-H295R through inhibition of CYP11B1 and CYP11B2 expression." Journal of Endocrinology 176, no. 1 (January 1, 2003): 69–82. http://dx.doi.org/10.1677/joe.0.1760069.

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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.

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Utriainen, Pauliina, Jianqi Liu, Tiina Kuulasmaa, and Raimo Voutilainen. "Inhibition of DNA methylation increases follistatin expression and secretion in the human adrenocortical cell line NCI-H295R." Journal of Endocrinology 188, no. 2 (February 2006): 305–10. http://dx.doi.org/10.1677/joe.1.06392.

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Activin affects adrenocortical steroidogenesis and increases apoptosis, while follistatin (FS) acts as an activin antagonist by binding to activin, preventing attachment to its receptors. The regulation of FS expression in the adrenal cortex is poorly understood. Adrenocortical tumors often display aberrant methylation. In the present study, we investigated the effect of DNA methylation on FS mRNA expression and peptide secretion in adrenocortical cells. We treated human NCI-H295R adrenocortical cells with the methylation inhibitor 5-Aza-2′deoxycytidine (Azad; 0.1–100 μM for 1, 4 or 7 days) and measured FS mRNA expression by Northern blot and quantitative real time RT-PCR analyses as well as FS secretion by specific ELISA. Methylation-specific PCR showed decreased methylation in the FS promoter region after Azad treatment. A significant (P < 0.05) time- and dose-dependent increase in FS mRNA expression (up to 4.6-fold) and peptide secretion (up to 17.1-fold) was detected after Azad treatment. We conclude that FS gene expression and peptide secretion in NCI-H295R adrenocortical cells are regulated by DNA methylation. Thus, variable methylation in different adrenocortical tumors may influence activin bioactivity and its consequences in steroidogenesis and cell proliferation/apoptosis.

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Subramanian, Chitra, Reid McCallister, Dawn Kuszynski, and Mark S. Cohen. "Re-Evaluation of Combinational Efficacy and Synergy of the Italian Protocol In Vitro: Are We Truly Optimizing Benefit or Permitting Unwanted Toxicity?" Biomedicines 9, no. 9 (September 10, 2021): 1190. http://dx.doi.org/10.3390/biomedicines9091190.

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Introduction: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy, with very poor prognosis as a majority of the patients have advanced disease at the time of diagnosis. Currently, adjuvant therapy for most patients consists of either mitotane (M) alone or in combination with multi-drug chemotherapeutics such as etoposide (E), doxorubicin (D), and cisplatin (P), known as the Italian protocol (IP; EDPM). This multi-drug treatment regimen, however, carries significant toxicity potential for patients. One way to improve toxicity profiles with these drugs in combination is to understand where their synergy occurs and over what dosing range so that lower dose regimens could be applied in combination with equal or improved efficacy. We hypothesize that a better understanding of the synergistic effects as well as the regulation of steroidogenic enzymes during combination therapy may provide more optimized combinational options with good potency and lower toxicity profiles. Methods: Two human ACC cell lines, NCI-H295R (hormonally active) and SW13 (hormonally inactive), were grown in 2D culture in appropriate growth medium. The viability of the cells after treatment with varying concentrations of the drugs (E, D, and P) either alone or in combinations with M was determined using the CellTiter Glow assay after 72 h, and the combination index for each was calculated using Compusyn by the Chou–Talalay method. The expression levels of enzymes associated with steroidogenesis were evaluated by RT-PCR in NCI-H295R. Results: When both cell lines were treated with M (ranging 25–50 μM), +E (ranging 18.75–75 μM), and +D (ranging 0.625–2.5 μM) we observed a synergistic effect (CI < 1) with potency equivalent to the full Italian protocol (IP), whereas combining M + P + D had an antagonistic effect (CI > 1) indicating the negative effect of adding cisplatin in the combination. Comparing the hormonally active and inactive cell lines, M + P + E was antagonistic in NCI-H295R and synergistic in SW13. Treatment of NCI-H295R cells with antagonistic combinations (M + P + D, M + P + E) resulted in a significant decrease in the levels of steroidogenic enzymes STAR, CYP11A1, and CYP21A2 compared to IP (p < 0.05) while M + E + D resulted in increased expression or no significant effect compared to IP across all genes tested. Conclusions: The synergistic effect for M + E + D was significant and equivalent in potency to the full IP in both cell lines and resulted in a steroidogenic gene expression profile similar to or better than that of full IP, warranting further evaluation. Future in vivo evaluation of the combination of M + E + D (with removal of P from the IP regimen) may lower toxicity while maintaining anticancer efficacy in ACC.

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Lichtenauer, U., I. Shapiro, A. Osswald, S. Meurer, A. Kulle, M. Reincke, F. Riepe, and F. Beuschlein. "Characterization of NCI-H295R Cells as an In Vitro Model of Hyperaldosteronism." Hormone and Metabolic Research 45, no. 02 (October 30, 2012): 124–29. http://dx.doi.org/10.1055/s-0032-1323810.

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Saito, Ryuta, Natsuko Terasaki, Makoto Yamazaki, Naoya Masutomi, Naohisa Tsutsui, and Masahiro Okamoto. "Estimation of the Mechanism of Adrenal Action of Endocrine-Disrupting Compounds Using a Computational Model of Adrenal Steroidogenesis in NCI-H295R Cells." Journal of Toxicology 2016 (2016): 1–19. http://dx.doi.org/10.1155/2016/4041827.

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Adrenal toxicity is one of the major concerns in drug development. To quantitatively understand the effect of endocrine-active compounds on adrenal steroidogenesis and to assess the human adrenal toxicity of novel pharmaceutical drugs, we developed a mathematical model of steroidogenesis in human adrenocortical carcinoma NCI-H295R cells. The model includes cellular proliferation, intracellular cholesterol translocation, diffusional transport of steroids, and metabolic pathways of adrenal steroidogenesis, which serially involve steroidogenic proteins and enzymes such as StAR, CYP11A1, CYP17A1, HSD3B2, CYP21A2, CYP11B1, CYP11B2, HSD17B3, and CYP19A1. It was reconstructed in an experimental dynamics of cholesterol and 14 steroids from anin vitrosteroidogenesis assay using NCI-H295R cells. Results of dynamic sensitivity analysis suggested that HSD3B2 plays the most important role in the metabolic balance of adrenal steroidogenesis. Based on differential metabolic profiling of 12 steroid hormones and 11 adrenal toxic compounds, we could estimate which steroidogenic enzymes were affected in this mathematical model. In terms of adrenal steroidogenic inhibitors, the predicted action sites were approximately matched to reported target enzymes. Thus, our computer-aided system based on systems biological approach may be useful to understand the mechanism of action of endocrine-active compounds and to assess the human adrenal toxicity of novel pharmaceutical drugs.

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Haase, Matthias, Matthias Schott, Stefan R. Bornstein, Ludwik K. Malendowicz, Werner A. Scherbaum, and Holger S. Willenberg. "CITED2 is expressed in human adrenocortical cells and regulated by basic fibroblast growth factor." Journal of Endocrinology 192, no. 2 (February 2007): 459–65. http://dx.doi.org/10.1677/joe-06-0083.

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CITED2 gene deletion in mice leads to adrenal agenesis. Therefore, we analyzed CITED2, a CBP/p300 interacting transactivator with transforming activity, in the human adrenal gland. In this study, we examined CITED2 expression in human embryonic and adult adrenal glands as well as adrenocortical carcinomas. As ACTH and basic fibroblast growth factor (bFGF) are connected to the physiology and growth of adrenocortical cells we studied the regulation of CITED2 by these factors in the NCI-H295R adrenocortical carcinoma cell line. We found CITED2 expression in the adult adrenal cortex as well in adrenocortical carcinomas. At an early stage of human adrenal organogenesis CITED2 could be located to the definitive zone of the developing adrenal gland using immunohistochemistry. In NCI-H295R cells, stimulation by bFGF led to a dose-dependent increase in CITED2 promotor activity, mRNA and protein expression while ACTH had no significant effect. The stimulatory effect of bFGF could be reduced by blocking mitogen-activated protein kinase activity using the MAPkinase kinase (MEK1)-inhibitor PD98059. CITED2 is expressed in embryonic and adult human adrenal glands as well as in adrenocortical cancer. It is connected to the signaling cascades of bFGF and its expression is modulated by mitogen-activated protein kinases. This suggests a novel role for CITED2 in human adrenal growth and possibly in adrenal tumorigenesis.

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Knazicka, Zuzana, Norbert Lukac, Zsolt Forgacs, Eva Tvrda, Jana Lukacova, Jana Slivkova, Łukasz Binkowski, and Peter Massanyi. "Effects of mercury on the steroidogenesis of human adrenocarcinoma (NCI-H295R) cell line." Journal of Environmental Science and Health, Part A 48, no. 3 (February 2013): 348–53. http://dx.doi.org/10.1080/10934529.2013.726908.

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Isaka, Tsuyoshi, Keiichi Ikeda, Yuko Takada, Yuri Inada, Katsuyoshi Tojo, and Naoko Tajima. "Azelnidipine inhibits aldosterone synthesis and secretion in human adrenocortical cell line NCI-H295R." European Journal of Pharmacology 605, no. 1-3 (March 2009): 49–52. http://dx.doi.org/10.1016/j.ejphar.2008.12.041.

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Celichowski, Piotr, Karol Jopek, Marta Szyszka, Paulina Milecka, Ludwik K. Malendowicz, Marianna Tyczewska, and Marcin Ruciński. "Nampt (Visfatin) Influence on Proliferative Activity of Normal Rat Adrenocortical Cells and Human Adrenal Corticocarcinoma Nci-H295r Cells." Medical Journal of Cell Biology 6, no. 2 (September 1, 2018): 33–38. http://dx.doi.org/10.2478/acb-2018-0007.

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Abstract Nampt (Nicotinamidephosphoribosyltransferase - also known as visfatin/PBEF) is the enzyme that regulates the NAD+ level, therefore influencing many metabolic pathways within the cells. As circulating cytokine, extracellular Nampt (eNampt) exerts pro-inflammatory, pro-chemotactic, pro-angiogenic and insulin-like effects; however the mechanism of eNampt action is still unclear.Earlier studies have shown that eNampt exerts a stimulating effect on the proliferation of many cancer cell lines. However, the effect of this cytokine on cell proliferation in primary culture is little known. Therefore, the aim of the study was to analyse the influence of eNampt on the proliferation of rat adrenocortical cells in primary culture and to investigate similar influence of eNampt on the line H295R of human adrenal corticocarcinoma cells. Proliferation of the examined cells was assessed using the RTCA (Real Time Cell Analyzer) method. The obtained results indicate that eNampt stimulates the proliferation of H295R cells, but does not change the proliferation of cultured rat adrenocortical cells. In primary culture of rat adrenocortical cells, Fk866 (specific Nampt inhibitor) does not modify the rate of proliferation of tested cells. In H295R cells the addition of Fk866 alone inhibits proliferative activity and stimulates apoptosis. Fk866 also inhibits the stimulating effect of eNampt on H295R cell proliferation.

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Vanderriele, Paul-Emmanuel, Brasilina Caroccia, Teresa Maria Seccia, Maria Piazza, Livia Lenzini, Francesca Torresan, Maurizio Iacobone, Thomas Unger, and Gian Paolo Rossi. "The angiotensin type 2 receptor in the human adrenocortical zona glomerulosa and in aldosterone-producing adenoma: low expression and no functional role." Clinical Science 132, no. 6 (March 20, 2018): 627–40. http://dx.doi.org/10.1042/cs20171593.

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The angiotensin II (Ang II) type 2 receptor (AT2R) and the angiotensin-(1–7) (Ang-(1–7)) receptor (MasR) play a cardiovascular protective role by counter-regulating Ang II type 1 receptor (AT1R)-mediated effects, but whether this involves blunting of adrenocortical hormone secretion is unknown. We investigated the presence of AT1R, AT2R, and MasR in aldosterone-producing adenoma (APA), a condition featuring hyperaldosteronism, and in APA-adjacent tissue. The effect of Compound 21 (C21), an AT2R agonist, on CYP11B1 (cortisol synthase) and CYP11B2 (aldosterone synthase) gene expression in NCI-H295R and HAC15 cell lines, and in APA and APA-adjacent tissue, was also assessed using the AT1R antagonist irbesartan to ascertain the specificity of C21 effect. We found that the AT1R, AT2R, and MasR were expressed in APA and APA-adjacent tissue, albeit heterogeneously. The gene expression of AT1R and AT2R was lower, and that of the MasR higher in APAs than in APA-adjacent tissue. In steroid-producing NCI-H295R and HAC15 cell lines, and in APA and APA-adjacent tissue, C21 was ineffective at nanomolar concentrations, but increased CYP11B1 and CYP11B2 gene expression at micromolar concentrations through AT1R, as this effect was blunted by irbesartan. The scant expression of the AT2R, along with the lack of any effect of C21 at low concentrations on CYP11B2, do not support the contention that the protective arm of renin–angiotensin system (RAS) blunts aldosterone synthase in the normal adrenal cortex and primary aldosteronism.

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Kanczkowski, Waldemar, Piotr Tymoszuk, Monika Ehrhart-Bornstein, Manfred P. Wirth, Kai Zacharowski, and Stefan R. Bornstein. "Abrogation of TLR4 and CD14 Expression and Signaling in Human Adrenocortical Tumors." Molecular Endocrinology 24, no. 10 (October 1, 2010): 2071. http://dx.doi.org/10.1210/mend.24.10.9998.

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Abstract Context: Adrenocortical carcinoma (ACC) is a rare tumor with poor prognosis. The expression of innate immunity receptor Toll-like receptor (TLR)-4 was recently reported in various human tumors, and TLR4 was shown to regulate tumor immune escape processes, proliferation, and resistance to chemotherapeutical agents. Objective: The aim of this study was to investigate TLR4 expression, signaling, and function in the process of tumorigenesis in the human adrenal cortex. Measurements and Main Results: Real-time PCR analysis of human ACC (n = 8), adenoma (n = 8), and ACC cell lines (SW13, NCI-H295R, and HAC15) revealed a significant down-regulation of TLR4, MD2, and CD14 mRNA compared with normal human adrenal cortex and adrenocortical cells in primary culture. Furthermore, immunohistochemistry revealed an abrogation of TLR4 and CD14 expression in ACC but not adenoma tissues. Western blot analysis of MAPK, AKT, activator protein-1, and nuclear factor-κB signaling revealed that the ACC cell lines are unresponsive to lipopolysaccharide action. Restoration of TLR4 signaling by stable transfection of TLR4-CD14 plasmid into NCI-H295R cells sensitized them to lipopolysaccharide incubation as shown by nuclear factor-κB activation and decreased cell viability and induced apoptosis in these cells. Conclusion: Our results demonstrate a significant reduction in the expression of TLR4 and CD14 and an inactivation of TLR4 signaling in ACCs. Furthermore, our data show that reintroduction of TLR4 expression in ACCs may provide a novel therapeutic strategy for adrenal cancer.

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Mikhaylova, Irina V., Tiina Jääskeläinen, Jarmo Jääskeläinen, Jorma J. Palvimo, and Raimo Voutilainen. "Leukemia inhibitory factor as a regulator of steroidogenesis in human NCI-H295R adrenocortical cells." Journal of Endocrinology 199, no. 3 (September 16, 2008): 435–44. http://dx.doi.org/10.1677/joe-08-0377.

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Leukemia inhibitory factor (LIF) is a multiple function cytokine regulating the hypothalamic–pituitary–adrenal axis at the pituitary level. LIF and its receptor are expressed in the adrenal glands, suggesting their potential regulatory role also at the adrenal level. Our aim was to clarify the effects of LIF on adrenal steroidogenesis using cell culture conditions. NCI-H295R human adrenocortical cells were treated with LIF (0.01–100 ng/ml) for 3–48 h with or without 8-bromo-cAMP (8-Br-cAMP; 1 mM). LIF treatment augmented cortisol, dehydroepiandrosterone (DHEA), DHEA sulfate, androstenedione, and aldosterone production (up to 224, 211, 149, 229, and 170% of control respectively, P<0.05 for all). It increased basal steroidogenic acute regulatory protein (STAR) and 17α-hydroxylase/17,20-lyase (CYP17A1) mRNAs (up to 142 and 170% of control respectively, P<0.05) and the respective proteins, but decreased 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) mRNA (down to 72% of control, P<0.05), and protein. LIF also increased 8-Br-cAMP-induced cortisol and DHEA production and STAR mRNA accumulation, while it attenuated 8-Br-cAMP-induced HSD3B2 expression and androstenedione production. It had an additive effect on tumour necrosis factor-induced cortisol production. LIF had no effect on apoptosis, but it increased slightly the number of metabolically active cells (up to 120% of control, P<0.05). These findings indicate that LIF is a potential physiological and/or pathophysiological regulator of steroidogenesis at the adrenal level.

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Bistakova, Jana, Zsolt Forgacs, Zsuzsa Bartos, Maria Racz Szivosne, Tomas Jambor, Zuzana Knazicka, Eva Tvrda, et al. "Effects of 4-nonylphenol on the steroidogenesis of human adrenocarcinoma cell line (NCI-H295R)." Journal of Environmental Science and Health, Part A 52, no. 3 (November 11, 2016): 221–27. http://dx.doi.org/10.1080/10934529.2016.1246936.

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Hasegawa, Eri, Saori Nakagawa, Momoe Sato, Eiichi Tachikawa, and Susumu Yamato. "Effect of Polyphenols on Production of Steroid Hormones from Human Adrenocortical NCI-H295R Cells." Biological and Pharmaceutical Bulletin 36, no. 2 (2013): 228–37. http://dx.doi.org/10.1248/bpb.b12-00627.

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Tkachenko, Irina V., Tiina Jääskeläinen, Jarmo Jääskeläinen, Jorma J. Palvimo, and Raimo Voutilainen. "Interleukins 1α and 1β as regulators of steroidogenesis in human NCI-H295R adrenocortical cells." Steroids 76, no. 10-11 (September 2011): 1103–15. http://dx.doi.org/10.1016/j.steroids.2011.04.018.

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Pagotto, Romina Maria, Elba Nora Pereyra, Casandra Monzón, Carolina Mondillo, and Omar Pedro Pignataro. "Histamine inhibits adrenocortical cell proliferation but does not affect steroidogenesis." Journal of Endocrinology 221, no. 1 (January 14, 2014): 15–28. http://dx.doi.org/10.1530/joe-13-0433.

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Histamine (HA) is a neurotransmitter synthesized in most mammalian tissues exclusively by histidine decarboxylase enzyme. Among the plethora of actions mediated by HA, the modulatory effects on steroidogenesis and proliferation in Leydig cells (LCs) have been described recently. To determine whether the effects on LCs reported could be extrapolated to all steroidogenic systems, in this study, we assessed the effect of this amine on adrenal proliferation and steroidogenesis, using two adrenocortical cell lines as experimental models, murine Y1 cells and human NCI-H295R cells. Even when steroidogenesis was not modified by HA in adrenocortical cells, the biogenic amine inhibited the proliferation of H295R cells. This action was mediated by the activation of HRH1 subtype and an increase in the production of inositol phosphates as second messengers, causing cell-cycle arrest in the G2/M phase. These results indicate a new role for HA in the proliferation of human adrenocortical cells that could contribute to a better understanding of tumor pathology as well as to the development of new therapeutic agents.

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Werth, Stephan, Helge Müller-Fielitz, and Walter Raasch. "Obesity-stimulated aldosterone release is not related to an S1P-dependent mechanism." Journal of Endocrinology 235, no. 3 (December 2017): 251–65. http://dx.doi.org/10.1530/joe-16-0550.

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Aldosterone has been identified as an important factor in obesity-associated hypertension. Here, we investigated whether sphingosine-1-phosphate (S1P), which has previously been linked to obesity, increases aldosterone release. S1P-induced aldosterone release was determined in NCI H295R cells in the presence of S1P receptor (S1PR) antagonists. In vivo release of S1P (100–300 µg/kgbw) was investigated in pithed, lean Sprague Dawley (SD) rats, diet-obese spontaneous hypertensive rats (SHRs), as well as in lean or obese Zucker rats. Aldosterone secretion was increased in NCI H295R cells by S1P, the selective S1PR1 agonist SEW2871 and the selective S1PR2 antagonist JTE013. Treatment with the S1PR1 antagonist W146 or fingolimod and the S1PR1/3 antagonist VPbib2319 decreased baseline and/or S1P-stimulated aldosterone release. Compared to saline-treated SD rats, plasma aldosterone increased by ~50 pg/mL after infusing S1P. Baseline levels of S1P and aldosterone were higher in obese than in lean SHRs. Adrenal S1PR expression did not differ between chow- or CD-fed rats that had the highest S1PR1 and lowest S1PR4 levels. S1P induced a short-lasting increase in plasma aldosterone in obese, but not in lean SHRs. However, 2-ANOVA did not demonstrate any difference between lean and obese rats. S1P-induced aldosterone release was also similar between obese and lean Zucker rats. We conclude that S1P is a local regulator of aldosterone production. S1PR1 agonism induces an increase in aldosterone secretion, while stimulating adrenal S1PR2 receptor suppresses aldosterone production. A significant role of S1P in influencing aldosterone secretion in states of obesity seems unlikely.

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Ferraz-de-Souza, Bruno, Franziska Martin, Delphine Mallet, Rebecca E. Hudson-Davies, Patricia Cogram, Lin Lin, Dianne Gerrelli, et al. "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." Journal of Clinical Endocrinology & Metabolism 94, no. 2 (February 1, 2009): 678–83. http://dx.doi.org/10.1210/jc.2008-1064.

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Abstract Context: 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. Objectives: 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. Design: 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. Results: 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. Conclusions: 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.

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Lichtenauer, Urs D., Igor Shapiro, Klaus Geiger, Marcus Quinkler, Martin Fassnacht, Roland Nitschke, Klaus-Dieter Rückauer, and Felix Beuschlein. "Side Population Does Not Define Stem Cell-Like Cancer Cells in the Adrenocortical Carcinoma Cell Line NCI h295R." Endocrinology 149, no. 3 (December 6, 2007): 1314–22. http://dx.doi.org/10.1210/en.2007-1001.

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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.

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LEHMANN, TOMASZ P., TOMASZ WRZESIŃSKI, and PAWEŁ P. JAGODZIŃSKI. "The effect of mitotane on viability, steroidogenesis and gene expression in NCI-H295R adrenocortical cells." Molecular Medicine Reports 7, no. 3 (December 18, 2012): 893–900. http://dx.doi.org/10.3892/mmr.2012.1244.

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Hirsch, Andrea, Dagmar Hahn, Petra Kempná, Gaby Hofer, Primus E. Mullis, Jean-Marc Nuoffer, and Christa E. Flück. "Role of AMP-Activated Protein Kinase on Steroid Hormone Biosynthesis in Adrenal NCI-H295R Cells." PLoS ONE 7, no. 1 (January 25, 2012): e30956. http://dx.doi.org/10.1371/journal.pone.0030956.

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Bilcikova, Jana, Veronika Fialkova, Hana Duranova, Eva Kovacikova, Zsolt Forgacs, Agnieszka Gren, Peter Massanyi, Norbert Lukac, Shubhadeep Roychoudhury, and Zuzana Knazicka. "Copper affects steroidogenesis and viability of human adrenocortical carcinoma (NCI-H295R) cell line in vitro." Journal of Environmental Science and Health, Part A 55, no. 9 (May 21, 2020): 1070–77. http://dx.doi.org/10.1080/10934529.2020.1769400.

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Qin, Haixia, Patricia Kent, Carlos M. Isales, Peter M. Parker, Mariya V. Wilson, and Wendy B. Bollag. "The role of calcium influx pathways in phospholipase D activation in bovine adrenal glomerulosa cells." Journal of Endocrinology 202, no. 1 (April 16, 2009): 77–86. http://dx.doi.org/10.1677/joe-09-0119.

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The steroid hormone aldosterone maintains sodium homeostasis and is therefore important in the control of blood volume and pressure. Angiotensin II (AngII) and elevated extracellular potassium concentrations ([K+]e), the prime physiologic regulators of aldosterone secretion from adrenal glomerulosa cells, activate phospholipase D (PLD) in these cells. The role of Ca2+ in the activation by these agents is unknown, although nitrendipine, a voltage-dependent Ca2+ channel antagonist, does not inhibit AngII-elicited PLD activation, despite the fact that this compound blocked elevated [K+]e-stimulated PLD activity. PLD activation triggered by AngII was also unaffected by the T-type calcium channel inhibitor nickel. Nevertheless, Ca2+ influx was required for AngII-induced PLD activation in both primary cultures of bovine adrenal glomerulosa cells and a glomerulosa cell model, the NCI H295R adrenocortical carcinoma cell line. The involvement of store-operated Ca2+ (SOC) influx and Ca2+ release-activated Ca2+ (CRAC) influx pathways in PLD activation was investigated using thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor that empties the store to induce SOC influx, and the SOC inhibitor YM-58483 (BTP2), as well as a CRAC inhibitor, tyrphostin A9. In bovine glomerulosa cells, tyrphostin A9 inhibited AngII-induced PLD activation without affecting elevated [K+]e-stimulated enzyme activity. On the other hand, differences were observed between the bovine adrenal glomerulosa and H295R cells in the involvement of Ca2+ influx pathways in PLD activation, with the involvement of the SOC pathway suggested in the H295R cells. In summary, our results indicate that Ca2+ entry only through certain Ca2+ influx pathways is linked to PLD activation.

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Wang, Xiao-Li, Mary Bassett, Yin Zhang, Su Yin, Colin Clyne, Perrin C. White, and William E. Rainey. "Transcriptional Regulation of Human 11β-Hydroxylase (hCYP11B1)." Endocrinology 141, no. 10 (October 1, 2000): 3587–94. http://dx.doi.org/10.1210/endo.141.10.7689.

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Abstract Steroid 11β-hydroxylase is a mitochondrial enzyme that catalyzes the conversion of deoxycortisol to cortisol. The gene encoding human 11β-hydroxylase (hCYP11B1) is expressed in the adrenal cortex under the control of circulating levels of ACTH. The current study was undertaken to define the cis-regulatory elements and transacting factors that regulate hCYP11B1 transcription. The hCYP11B1 5′-flanking DNA was studied using transient transfection of luciferase reporter constructs in NCI-H295R human adrenocortical cells. A cAMP analogue ((Bu)2cAMP) increased expression of a construct containing −1102 bp of hCYP11B1 5′-flanking DNA (pB1–1102). An element at position −71/−64 (TGACGTGA, previously termed Ad1) resembling a consensus cAMP response element (CRE) was required for maximal induction by cAMP. The Ad1 element bound several transcriptional factors in electrophoretic mobility shift assays, including CRE-binding protein, activating transcription factor-1 (ATF-1), and ATF-2, but only the ATF-2 complex migrated similarly to a complex seen using H295R nuclear extract. In addition, Western analysis of H295R and adrenal lysates demonstrated expression of high levels of ATF-2 and ATF-1. CRE-binding protein levels varied among the strains of H295R cells tested. Transcription of CYP11B1 also appeared to be regulated by steroidogenic factor-1 (SF-1). Luciferase reporter gene activity was increased after cotransfection with expression vectors containing SF-1. An element in hCYP11B1 at positions −242/−234 (CCAAGGCTC), previously termed Ad4, was required for maximal induction by SF-1 and was found to bind SF-1 in electrophoretic mobility shift assays. The key role for SF-1 in hCYP11B1 transcription is in contrast to its lack of an effect on expression of the hCYP11B2 (aldosterone synthase) isozyme. The differential effects of SF-1 on transcription of hCYP11B1 and hCYP11B2 may be one of the mechanisms controlling differential expression of these isozymes within the zonae fasciculata and glomerulosa of the human adrenal cortex.

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Zsippai, Adrienn, Diana Rita Szabó, Zsófia Tömböl, Peter M. Szabó, Katalin Éder, Éva Pállinger, Rolf C. Gaillard, et al. "Effects of mitotane on gene expression in the adrenocortical cell line NCI-H295R: a microarray study." Pharmacogenomics 13, no. 12 (September 2012): 1351–61. http://dx.doi.org/10.2217/pgs.12.116.

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Kienitz, Marie-Cécile, Evanthia Mergia, and Lutz Pott. "NCI-H295R cell line as in vitro model of hyperaldosteronism lacks functional KCNJ5 (GIRK4; Kir3.4) channels." Molecular and Cellular Endocrinology 412 (September 2015): 272–80. http://dx.doi.org/10.1016/j.mce.2015.05.013.

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Kempná, Petra, Gaby Hofer, Primus E. Mullis, and Christa E. Flück. "Pioglitazone Inhibits Androgen Production in NCI-H295R Cells by Regulating Gene Expression of CYP17 and HSD3B2." Molecular Pharmacology 71, no. 3 (November 30, 2006): 787–98. http://dx.doi.org/10.1124/mol.106.028902.

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Kempná, Petra, Andrea Hirsch, Gaby Hofer, Primus E. Mullis, and Christa E. Flück. "Impact of Differential P450c17 Phosphorylation by cAMP Stimulation and by Starvation Conditions on Enzyme Activities and Androgen Production in NCI-H295R Cells." Endocrinology 151, no. 8 (June 9, 2010): 3686–96. http://dx.doi.org/10.1210/en.2010-0093.

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CYP17A1 plays a pivotal role in the biosynthesis of androgens in the adrenals and the gonads. Although this enzyme catalyzes two different reactions on one single active site, its specific activities are regulated independently. Although the 17α-hydroxylase activity is rather constant and regulated by gene expression, the 17,20-lyase activity varies significantly with the amount of cofactors or by protein phosphorylation. cAMP increases CYP17A1 expression, P450c17 phosphorylation, and androgen production. However, the exact mechanism(s) and the specific regulators of CYP17A1 remain unknown. Therefore, we studied the regulation of adrenal androgen biosynthesis in human adrenal H295R cells focusing on CYP17A1. We analyzed androgen production and P450c17 activities in H295R cells grown under normal and serum-free conditions and/or after stimulation with 8-bromoadenosine-cAMP. H295R cells grown in starvation medium produced more androgens and had decreased HSD3B2 expression and activity but increased P450c17-17,20-lyase activity and serine phosphorylation. Although starvation increased serine phosphorylation of P450c17 specifically, cAMP stimulation enhanced threonine phosphorylation exclusively. Time-course experiments revealed that a short cAMP stimulation augmented threonine phosphorylation of P450c17 but did not increase 17,20-lyase activity. By contrast, long cAMP stimulation increased androgen production through increased P450c17 activities by enhancing CYP17A1 gene expression. We conclude that serum withdrawal shifts steroidogenesis of H295R cells towards androgen production, providing a suitable model for detailed studies of androgen regulation. In addition, our study shows that starvation and cAMP stimulation regulate P450c17 phosphorylation differentially and that an increase in P450c17 phosphorylation does not necessarily lead to enhanced enzyme activity and androgen production.

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Liu, J., X.-D. Li, A. Vaheri, and R. Voutilainen. "DNA methylation affects cell proliferation, cortisol secretion and steroidogenic gene expression in human adrenocortical NCI-H295R cells." Journal of Molecular Endocrinology 33, no. 3 (December 2004): 651–62. http://dx.doi.org/10.1677/jme.1.01560.

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Aberrant DNA methylation may be involved in human adrenocortical tumorigenesis, which is often accompanied by abnormal hormone production. In this study, we aimed to clarify the effects of DNA methylation on steroidogenesis using the human adrenocortical NCI-H295R cell line as a model. Treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (Azad; 10 μM for 7 days) decreased the proliferation rate to approximately 20% and the cell number to 60% of the control, with a simultaneous increase in the expression of the cyclin-dependent kinase inhibitor p57KIP2 gene. In addition, Azad treatment increased cortisol secretion dose and time dependently, whereas dehydroepiandrosterone sulfate secretion was not affected. Azad treatment decreased basal and (Bu)2cAMP-induced expression of low- and high-density lipoprotein receptor, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme, steroid 17α-hydroxylase/17,20-lyase and steroid 21-hydroxylase mRNA, as well as the StAR protein level. In contrast, Azad treatment increased the basal expression of steroid 11β-hydroxylase and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase genes, although it inhibited the (Bu)2cAMP-induced expression of these two genes. The expression of steroidogenic factor-1 (SF-1) and DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome 1) genes (both harboring putative CpG islands in their promoters) and the methylation degree of the HpaII recognition site(s) in the SF-1 gene promoter region were reduced by Azad treatment. The immunostaining pattern of the methyl-CpG-binding protein MeCP2 was also modified by Azad treatment. These results suggest that DNA methylation may be implicated in the regulation of cell proliferation and steroidogenesis in human adrenocortical cells.

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Cobb, V. J., B. C. Williams, J. I. Mason, and S. W. Walker. "Forskolin treatment directs steroid production towards the androgen pathway in the NCI–H295R adrenocortical tumour cell line." Endocrine Research 22, no. 4 (November 1996): 545–50. http://dx.doi.org/10.1080/07435809609043744.

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Strickland, Janae, Stephen McIlmoil, Brice J. Williams, Dennis C. Seager, James P. Porter, and Allan M. Judd. "Interleukin-6 increases the expression of key proteins associated with steroidogenesis in human NCI-H295R adrenocortical cells." Steroids 119 (March 2017): 1–17. http://dx.doi.org/10.1016/j.steroids.2016.12.014.

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Wei, Xian, Junfeng Zhang, Wei Peng, Hao Xu, Zhewen Wei, Linhao Pang, Jihong Liu, and Tao Wang. "Interleukin‐6 increases adrenal androgen release by regulating the expression of steroidogenic proteins in NCI‐H295R cells." Journal of Cellular Physiology 235, no. 12 (April 28, 2020): 9432–44. http://dx.doi.org/10.1002/jcp.29748.

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Bassett, MH, Y. Zhang, C. Clyne, PC White, and WE Rainey. "Differential regulation of aldosterone synthase and 11beta-hydroxylase transcription by steroidogenic factor-1." Journal of Molecular Endocrinology 28, no. 2 (April 1, 2002): 125–35. http://dx.doi.org/10.1677/jme.0.0280125.

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11beta-Hydroxylase (hCYP11B1) and aldosterone synthase (hCYP11B2) are closely related isozymes with distinct roles in cortisol and aldosterone production respectively. Aldosterone synthase catalyzes the final step in aldosterone biosynthesis and is expressed only in the zona glomerulosa of the normal adrenal. 11beta-Hydroxylase catalyzes the final reaction in the production of cortisol and is expressed at higher levels in the zona fasciculata. The mechanisms causing differential expression of these genes are not well defined. Herein, we demonstrate contrasting roles for the orphan receptor steroidogenic factor-1 (SF-1) in the regulation of human (h) CYP11B1 and hCYP11B2. Human NCI-H295R (H295R) or mouse Y-1 cells were transiently transfected with luciferase reporter constructs containing 5'-flanking regions of hCYP11B1, hCYP11B2, human 17alpha-hydroxylase (hCYP17), human cholesterol side-chain cleavage (hCYP11A1) or mouse (m) cyp11b2 (mcyp11b2). Co-transfection of vectors encoding SF-1 increased expression of hCYP11B1, hCYP11A1 and hCYP17 constructs, but inhibited hCYP11B2 reporter activity. Murine, bovine and human SF-1 were unable to increase transcription of hCYP11B2 in H295R cells. Both hCYP11B2 and mcyp11b2 promoter constructs were inhibited similarly by human SF-1. In mouse Y-1 cells, reporter expression of hCYP11B2 and mcyp11b2 was very low compared with hCYP11B1 constructs, suggesting that this adrenal cell model may not be appropriate for studies of CYP11B2. Electrophoretic mobility shift assay demonstrated that SF-1 interacted with an element from both hCYP11B1 and hCYP11B2. However, mutation of this element, termed Ad4, did not prevent agonist stimulation of hCYP11B2 by angiotensin II or forskolin but blocked activity of hCYP11B1. In some, but not all, reports of genetic linkage analysis, a naturally occurring single nucleotide polymorphism within the Ad4 element of hCYP11B2 (-344C/T) has been associated with cardiovascular disease. Herein, we have demonstrated that this polymorphism influenced binding of SF-1 in electrophoretic mobility shift assays, with the C allele binding SF-1 more strongly than the T allele. However, when hCYP11B2 constructs containing these alleles were transfected into H295R cells, there was no difference in agonist-stimulated expression or the response of either reporter construct to co-expression with human SF-1. Taken together, these data suggest that SF-1 and the Ad4 element are not major regulators of adrenal hCYP11B2 gene expression. Thus far, hCYP11B2 is the first steroid hydroxylase gene which is not positively regulated by SF-1.

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Berruti, Alfredo, Paola Sperone, Anna Ferrero, Antonina Germano, Arianna Ardito, Adriano Massimiliano Priola, Silvia De Francia, et al. "Phase II study of weekly paclitaxel and sorafenib as second/third-line therapy in patients with adrenocortical carcinoma." European Journal of Endocrinology 166, no. 3 (March 2012): 451–58. http://dx.doi.org/10.1530/eje-11-0918.

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BackgroundThere is a strong rationale in the use of antiangiogenic therapy in the management of adrenocortical carcinoma (ACC). Metronomic administration of chemotherapy and antiangiogenic drugs can be synergistic in targeting endothelial cells.ObjectiveWe assessed the activity of sorafenib plus metronomic paclitaxel as second/third-line therapy in advanced ACC patients. We also tested the activity of sorafenib and paclitaxel against NCI-H295R in vitro.DesignMulticenter, prospective phase II trial.SettingReferral centers for ACC.MethodsTwenty-five consecutive metastatic ACC patients who progressed after mitotane plus one or two chemotherapy lines were planned to be enrolled. The patients received a combination of i.v. paclitaxel (60 mg/m2 every week) and oral sorafenib (400 mg twice a day) till progression. The primary aim was to measure the progression-free survival rate after 4 months and the secondary aims were to assess the objective response rate and toxicity.ResultsTumor progression was observed in nine evaluable patients at the first assessment. These results led to the premature interruption of the trial. The treatment was well tolerated. The most relevant toxicities were fatigue, being grade 2 or 3 in four patients, and hypophosphatemia, being grade 3 in three patients. In the in vitro study, sorafenib impaired the viability of H295R cells with dose–response and time–response relationships. The in vitro sorafenib activity was not increased in combination with paclitaxel.ConclusionsDespite the in vitro activity, sorafenib plus weekly paclitaxel is an inactive salvage treatment in patients with advanced ACC and should not be recommended.

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Ikeda, Keiichi, Takatoshi Saito, and Katsuyoshi Tojo. "Efonidipine, a Ca2+-Channel Blocker, Enhances the Production of Dehydroepiandrosterone Sulfate in NCI-H295R Human Adrenocortical Carcinoma Cells." Tohoku Journal of Experimental Medicine 224, no. 4 (2011): 263–71. http://dx.doi.org/10.1620/tjem.224.263.

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Mikhaylova, Irina V., Tiina Kuulasmaa, Jarmo Jääskeläinen, and Raimo Voutilainen. "Tumor Necrosis Factor-α Regulates Steroidogenesis, Apoptosis, and Cell Viability in the Human Adrenocortical Cell Line NCI-H295R." Endocrinology 148, no. 1 (January 1, 2007): 386–92. http://dx.doi.org/10.1210/en.2006-0726.

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TNF-α regulates the hypothalamo-pituitary-adrenal axis at several levels. It has been shown to modify adrenal steroidogenesis in many species, and it is supposed to act as an auto/paracrine factor. However, its significance in human adrenocortical function remains unclear. Therefore, we investigated the effect of TNF-α on adrenal steroidogenesis, expression of the key steroidogenic genes, apoptosis, and cell viability in the human adrenocortical cell line NCI-H295R. TNF-α treatment (1 nm for 48 h) decreased the basal production of cortisol, androstenedione, dehydroepiandrosterone sulfate (DHEAS), and aldosterone (14, 18, 35, and 52%, respectively), and the 8-bromo-cAMP-induced production of cortisol, androstenedione, dehydroepiandrosterone (DHEA), and DHEAS (44, 66, 58, and 48%, respectively). However, when the steroid production data were normalized by the cell number, TNF-α increased the basal production of cortisol, androstenedione, DHEA, DHEAS, and aldosterone (137, 121, 165, 73, and 28%, respectively), and the 8-bromo-cAMP-induced production of cortisol, DHEAS, and aldosterone (122, 121, and 256%, respectively). This was accompanied by a parallel increase in the expression of the genes encoding for the steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase 2, and 17-hydroxylase/17,20-lyase (74, 200, and 50%, respectively; quantitative real-time RT-PCR analysis). TNF-α increased caspase 3/7 activity (an indicator of apoptosis) and decreased cell viability dose and time dependently. The effect of TNF-α on apoptosis was neutralized by a monoclonal TNF-α antibody. These findings indicate that TNF-α is a potent regulator of steroidogenesis and cell viability in adrenocortical cells. TNF-α may have physiological and/or pathophysiological significance as an endocrine and/or paracrine/autocrine regulator of adrenocortical function.

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Kempná, Petra, Nesa Marti, Sameer Udhane, and Christa E. Flück. "Regulation of androgen biosynthesis – A short review and preliminary results from the hyperandrogenic starvation NCI-H295R cell model." Molecular and Cellular Endocrinology 408 (June 2015): 124–32. http://dx.doi.org/10.1016/j.mce.2014.12.015.

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Lundqvist, Johan, Maria Norlin, and Kjell Wikvall. "1α,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells." Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1801, no. 9 (September 2010): 1056–62. http://dx.doi.org/10.1016/j.bbalip.2010.04.009.

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Journal articles on the topic 'NCI-H295R'

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