Adrenocortical Carcinoma Cell Line H295R Research Articles

Quercetin-Rich Extracts from Onions (Allium cepa) Play Potent Cytotoxicity on Adrenocortical Carcinoma Cell Lines, and Quercetin Induces Important Anticancer Properties.

Adrenocortical carcinoma (ACC) is a rare subtype of cancer, with a poor prognosis in children and adults. Mitotane is the only approved adrenolytic drug for the treatment of ACC, which has controversies regarding its efficacy and side effects on patients. Onion (Allium cepa), a worldwide consumed food, is associated with many health benefits. Along with its glycosides, the flavonoid quercetin is abundant in onions. After evaluating the cytotoxicity of A. cepa extracts on adrenocortical carcinoma cell line (H295R), the rich quercetin fractions had better results. Then, we aimed to compare the quercetin vs. mitotane effectiveness, using adrenocortical carcinoma cell lines H295R and SW-13. Quercetin showed a higher cytotoxicity response on both cancerous cell lines after 10 µM concentration, while mitotane only after 30 µM. Cell cycle dynamics were altered upon quercetin treatments, with G2 phase increase with 30 µM of quercetin on H295R cell line and G1 arrest on SW-13 cell line with 15 µM. Early and late apoptosis, alongside intracellular calcium, were increased on SW-13 treated with 30 µM of quercetin, and ROS rates were reduced by quercetin on H295R. Therefore, quercetin-rich onions have the potential to be a natural source of anticancer agents for adrenocortical carcinoma.

Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched.

Despite the development of new therapeutic strategies, cancer remains one of the leading causes of mortality worldwide. One of the current major challenges is the resistance of cancers to chemotherapy treatments inducing metastases and relapse of the tumor. The Hedgehog receptor Patched (Ptch1) is overexpressed in many types of cancers. We showed that Ptch1 contributes to the efflux of doxorubicin and plays an important role in the resistance to chemotherapy in adrenocortical carcinoma (ACC), a rare cancer which presents strong resistance to the standard of care chemotherapy treatment. In the present study, we isolated and characterized a subpopulation of the ACC cell line H295R in which Ptch1 is overexpressed and more present at the cell surface. This cell subpopulation is more resistant to doxorubicin, grows as spheroids, and has a greater capability of clonogenicity, migration, and invasion than the parental cells. Xenograft experiments performed in mice and in ovo showed that this cell subpopulation is more tumorigenic and metastatic than the parental cells. These results suggest that this cell subpopulation has cancer stem-like or persistent cell properties which were strengthened by RNA-seq. If present in tumors from ACC patients, these cells could be responsible for therapy resistance, relapse, and metastases.

ARMC5 modifies cell redox state to regulate steroidogenesis and lipid metabolism in the adrenal cortex

ARMC5 is a putative tumor suppressor gene that is frequently mutated in primary bilateral macronodular adrenal hyperplasia (PBMAH), a rare cause of Cushing's syndrome. The function of ARMC5 is poorly known, aside the fact that it regulates cell apoptosis and adrenal steroidogenesis in by mechanisms still unknown. Tumor suppressor genes play an important role in oxidative stress. In this study we used as model the adrenocortical carcinoma cell line H295R. In order to investigate ARMC5 response to stress, cells were treated with Menadione and reactive oxygen species (ROS) production was measured by Cellrox assays with flow cytometry. Oxidative response genes and steroidogenic enzymes expression were investigated by qPCR, whereas p38 phosphorylation was investigated by western blotting. ARMC5 protein is differentially regulated in response to menadione-induced stress in H295R adrenocortical cells. Moreover, ARMC5 depletion increases total intracellular ROS production (P < 0.05) and causes an imbalanced transcription of pro and anti-oxidant genes leading to increased phosphorylation of p38 (P < 0.05). ROS production and p38 pathway activation alter steroidogenesis. These effects are partially reversed by the anti-oxidant compound N-acetylcysteine (NAC) or the p38 inhibitor (SB203580). Altogether, this study describes a new function of ARMC5 as regulator of the redox homeostasis in adrenocortical cells.

MiR-486-3p was downregulated at microRNA profiling of adrenals of multiple endocrine neoplasia type 1 mice, and inhibited human adrenocortical carcinoma cell lines

Adrenocortical carcinoma is a rare aggressive disease commonly recurring regardless of radical surgery. Although data on genomic alterations in malignant tumors are accumulating, knowledge of molecular events of importance for initiation of adrenocortical transformation is scarce. In an attempt to recognize early molecular alterations, we used adrenals from young multiple endocrine neoplasia type 1 conventional knock-out mice (Men1+/−) closely mimicking the human MEN1 trait (i.e. transformation of pituitary, parathyroid, endocrine pancreatic, and adrenocortical cells). MicroRNA array and hierarchical clustering showed a distinct pattern. Twenty miRNAs were significantly upregulated and eleven were downregulated in Men1+/− compared to wild type littermates. The latter included the known suppressor miRNA miR-486-3p, which was chosen for transfection in human adrenocortical carcinoma cell lines H295R and SW13. Cell growth decreased in miR-486-3p overexpressing clones and levels of the predicted target gene fatty acid synthase (FASN) and its downstream product, palmitic acid, were lowered. In conclusion, heterozygous inactivation of Men1 in adrenals results in distinct miRNA profile regulating expression of genes with impact on tumorigenesis, e.g. transcription, nucleic acid and lipid metabolism. Low levels of miR-486-3p in the early stages of transformation may contribute to proliferation by increasing FASN and thus fatty acid production. FASN as a potentially druggable target for treatment of the devastating disease adrenocortical carcinoma warrants further studies.

Effects of Sorafenib, a Tyrosin Kinase Inhibitor, on Adrenocortical Cancer.

The lack of an effective medical treatment for adrenocortical carcinoma (ACC) has prompted the search for better treatment protocols for ACC neoplasms. Sorafenib, a tyrosine kinase inhibitor has exhibited effectiveness in the treatment of different human tumors. Therefore, the aim of this study was to understand the mechanism through which sorafenib acts on ACC, especially since treatment with sorafenib alone is sometimes unable to induce a long-lasting antiproliferative effect in this tumor type. The effects of sorafenib were tested on the ACC cell line H295R by evaluating cell viability, apoptosis and VEGF receptor signaling which was assessed by analyzing VE-cadherin and β-catenin complex formation. We also tested sorafenib on an in vitro 3D cell culture model using the same cell line. Apoptosis was observed after sorafenib treatment, and coimmunoprecipitation data suggested that the drug prevents formation VEGFR-VE-cadherin and β-catenin proteins complex. These results were confirmed both by ultrastructural analysis and by a 3D model where we observed a disaggregation of spheres into single cells, which is a crucial event that represents the first step of metastasis. Our findings suggest that although sorafenib induces apoptotic cell death a small portion of cells survive the treatment and have characteristics of a malignancy. Based on our data we recommend against the use of sorafenib in patients with ACC.

Abstract 6681: Inhibition of LSD1 using the reversible inhibitor SP2577 promotes Interferon dependent anti-tumor response in adrenocortical carcinoma

Abstract Purpose: Adrenocortical carcinoma (ACC) is a rare and aggressive disease with poor prognosis. The first line of treatment is surgery followed by chemotherapy (Mitotane), though these therapies still result in a dismal 15-44% survival rate after 5 years. Currently, there is no effective second line of treatment. Efforts have been made to treat ACC with immune therapy agents such as Avelumab, a PD-L1 antibody, but a recent study showed that there was only an overall response rate of 6% in a phase 1 clinical trial, further exhibiting a need for the development of novel therapeutics. Further research suggests that the current first line ACC treatments may benefit from the addition of immune therapies that induce an Interferon Beta (IFN-β) response in the tumor microenvironment. Previous studies have shown that treatment with IFN-β induces cell death and sensitizes human ACC lines to Mitotane, implying that combination of therapies that induce an IFN-β response may improve the anti-tumor response to Mitotane. Recently, inhibition of Lysine-Specific histone Demethylase 1 (LSD-1) was shown to stimulate an IFN-dependent anti-tumor response driven by the expression of Endogenous Retroviral Elements (ERVs) in a variety of cancer types. In addition, PD-L1 expression has also been shown to increase after LSD-1 inhibition. Yang, et al. reported that SP-2509, a preclinical analog of SP-2577 (Seclidemstat, currently in clinical phase I trials, Salarius Pharmaceuticals, Houston, TX) was able to induce the expression and release of interferons and cytokines in triple negative breast cancer models. In this study we investigate the ability of SP-2577 to promote the expression of IFN-β in human ACC cell lines, resulting in an anti-tumor immune response. Experimental Design: qRT-PCR was performed on ACC cell lines H295R and SW-13 at 48 and 72 hours post treatment to verify the ability of SP-2577 to promote IFN-β expression. ACC cells were co-cultured with peripheral blood mononuclear cells (PBMCs) in a 3D platform to evaluate lymphocyte infiltration following SP-2577 treatment. Interferons, chemokines, and cytokines were quantified in the culture supernatant after treatment with SP-2577 using the Meso Scale Discovery platform. Results: qRT-PCR showed that SP-2577 promotes ERV-mediated immune response in ACC cell lines via the expression and release of interferons, chemokines, and cytokines. Additionally, SP-2577 stimulates lymphocyte infiltration in an ACC derived 3D-spheroid platform further resulting in a cytotoxic effect. Conclusion: Our data suggests that the LSD-1 inhibitor SP-2577 can be used as a single agent in ACC patients to promote immune response and can potentially sensitize the tumor to first line of therapy, Mitotane. This study was supported by funding from the TGen Foundation. Citation Format: Sunil Sharma, Raffaella Soldi, Alexis Weston, Samuel Sampson, Sherin Daniel Ampanattu, Scott Celinski, Haiyong Han, Mohan Kaadige, Michael Berens, Jeffrey Trent. Inhibition of LSD1 using the reversible inhibitor SP2577 promotes Interferon dependent anti-tumor response in adrenocortical carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6681.

Intermediate frequency magnetic field at 250.8 kHz does not induce DNA damage or “Adaptive Response” in vitro

The aim of this study was to examine whether intermediate frequency (IF)magnetic field (MF) induces genotoxic effects or play a role in the induction ofadaptive response after combined exposure to IF MF and ionizing radiation inleukocytes and in adrenocortical carcinoma cell line H295R. Cells were exposedto 250.8 kHz at the magnetic field strength of 80 A/m (equivalent to 100 µTmagnetic flux density) for 20 hours, or exposed to IF MF for 20 hours and 24hours later challenged with ionizing radiation (1.5 Gy X-ray). Evaluation of theDNA damage was performed with alkaline comet assay. Our results showed thatthere was no significant genotoxic effect of IF MF exposure compared to thecontrols in both cell types. Furthermore, results did not indicate a statisticallysignificant change in DNA strand breaks in IF MF pre-exposed cells when theywere subsequently exposed to 1.5 Gy. Consequently no adaptive response wasdetected.

Targeting the multidrug transporter Patched potentiates chemotherapy efficiency on adrenocortical carcinoma in vitro and in vivo

One of the crucial challenges in the clinical management of cancer is the resistance to chemotherapeutics. We recently demonstrated that the Hedgehog receptor Patched, which is overexpressed in many recurrent and metastatic cancers, is a multidrug transporter for chemotherapeutic agents such as doxorubicin. The present work provides evidences that Patched is expressed in adrenocortical carcinoma (ACC) patients, and is a major player of the doxorubicin efflux and the doxorubicin resistance in the human ACC cell line H295R. We discovered that methiothepin inhibits the doxorubicin efflux activity of Patched. This drug-like molecule enhances the cytotoxic, pro-apoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells which endogenously overexpress Patched, and thereby mitigates the resistance of these cancer cells to doxorubicin. Moreover, we report that in mice the combination of methiothepin with doxorubicin prevents the development of xenografted ACC tumors more efficiently than doxorubicin alone by enhancing the accumulation of doxorubicin specifically in tumors without obvious undesirable side effects. Our results suggest that the use of an inhibitor of Patched drug efflux such as methiothepin in combination with doxorubicin could be a promising therapeutic option for adrenocortical carcinoma, and most likely also for other Patched-expressing cancers.

Synthetic high-density lipoprotein nanodisks for targeted withalongolide delivery to adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy and has a 5-year survival rate of <35%. ACC cells require cholesterol for steroid hormone production, and this requirement is met via expression on the cell surface of a high level of SRB1, responsible for the uptake of high-density lipoproteins (HDLs), which carry and transport cholesterol in vivo. Here, we describe how this natural lipid carrier function of SRB1 can be utilized to improve the tumor-targeted delivery of a novel natural product derivative – withalongolide A 4,19,27-triacetate (WGA-TA) – which has shown potent antitumor efficacy, but poor aqueous solubility. Our strategy was to use synthetic HDL (sHDL) nanodisks, which are effective in tumor-targeted delivery due to their smallness, long circulation half-life, documented safety, and ability to bind to SRB1. In this study, we prepared sHDL nanodisks using an optimized phospholipid composition combined with ApoA1 mimetic peptide (22A), which has previously been tested in clinical trials, to load WGA-TA. Following optimization, WGA-TA nanodisks showed drug encapsulation efficiency of 78%, a narrow particle size distribution (9.81±0.41 nm), discoid shape, and sustained drug release in phosphate buffered saline. WGA-TA-sHDL nanodisks exhibited higher cytotoxicity in the ACC cell line H295R half maximal inhibitory concentration ([IC50] 0.26±0.045 μM) than free WGA-TA (IC50 0.492±0.115 μM, P<0.05). Fluorescent dye-loaded sHDL nanodisks efficiently accumulated in H295R adrenal carcinoma xenografts 24 hours following dosing. Moreover, daily intraperitoneal administration of 7 mg/kg WGA-TA-loaded sHDL nanodisks significantly inhibited tumor growth during 21-day administration to H295R xenograft-bearing mice compared to placebo (P<0.01). Collectively, these results suggest that WGA-TA-loaded nanodisks may represent a novel and beneficial therapeutic strategy for the treatment of ACC.

Targeting polo-like kinase 1, a regulator of p53, in the treatment of adrenocortical carcinoma.

BackgroundAdrenocortical carcinoma (ACC) is an aggressive cancer with a 5 year survival rate of 20–30 %. Various factors have been implicated in the pathogenesis of ACC including dysregulation of the G2/M transition and aberrant activity of p53 and MDM2. Polo-like kinase 1 (PLK-1) negatively modulates p53 functioning, promotes MDM2 activity through its phosphorylation, and is involved in the G2/M transition. Gene expression profiling of 44 ACC samples showed that increased expression of PLK-1 in 29 % of ACC. Consequently, we examined PLK-1’s role in the modulation of the p53 signaling pathway in adrenocortical cancer.MethodsWe used siRNA knock down PLK-1 and pharmacological inhibition of PLK-1 and MDM2 ACC cell lines SW-13 and H295R. We examined viability, protein expression, p53 transactivation, and induction of apoptosis.ResultsKnocking down expression of PLK-1 with siRNA or inhibition of PLK-1 by a small molecule inhibitor, BI-2536, resulted in a loss of viability of up to 70 % in the ACC cell lines H295R and SW-13. In xenograft models, BI-2536 demonstrated marked inhibition of growth of SW-13 with less inhibition of H295R. BI-2536 treatment resulted in a decrease in mutant p53 protein in SW-13 cells but had no effect on wild-type p53 protein levels in H295R cells. Additionally, inhibition of PLK-1 restored wild-type p53’s transactivation and apoptotic functions in H295R cells, while these functions of mutant p53 were restored only to a smaller extent. Furthermore, inhibition of MDM2 with nutlin-3 reduced the viability of both the ACC cells and also reactivated wild-type p53′s apoptotic function. Inhibition of PLK-1 sensitized the ACC cell lines to MDM2 inhibition and this dual inhibition resulted in an additive apoptotic response in H295R cells with wild-type p53.ConclusionsThese preclinical studies suggest that targeting p53 through PLK-1 is an attractive chemotherapy strategy warranting further investigation in adrenocortical cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-015-0080-3) contains supplementary material, which is available to authorized users.

Adhesion to type V collagen enhances staurosporine-induced apoptosis of adrenocortical cancer cells.

Adrenocortical carcinoma (ACC) is a rare and aggressive tumor characterized by poor prognosis and resistance to conventional chemotherapy. Many chemotherapy agents act determining apoptosis, therefore, studying the responsiveness of ACC to apoptosis inducing molecules, can help to identify possible conditions to promote cancer cell death. Tumor progression is strictly related to the interaction between cancer cells and stroma; yet, extracellular matrix remodeling regulates tumor cell proliferation and apoptosis. At this purpose, we have studied staurosporine-induced apoptosis of ACC cell line H295R adherent to different extracellular matrix molecules. H295R cells grown on plastic showed a low responsiveness to staurosporine, with an apoptotic rate of 24 %, as compared to breast cancer MCF7 cells, with an apoptotic rate of 60 %. The adhesion of H295R cells to type V collagen induced a significant increase of apoptosis up to 52 %; this effect was inhibited by anti-integrin alpha2 antibody. At the same time, the adhesion of H295R cells on polylysine, matrigel, lamimin, fibronectin, and type I-III collagens didn't modify staurosporine-induced apoptosis. Staurosporine-treated H295R cells showed an increase of PARP cleavage and of annexin-V expression, when adherent to type V collagen. Yet, staurosporine induced Akt and Erk activation on H295R cells: the adhesion on type V collagen didn't modify Akt activation, while determined a dramatic inhibition of Erk activation. The described data demonstrate that the adhesion to type V collagen specifically increases the responsiveness of ACC cells to staurosporine-induced apoptosis and that this is probably obtained through the inhibition of Erk activation.

Exposure to the Three Structurally Different PCB Congeners (PCB 118, 153, and 126) Results in Decreased Protein Expression and Altered Steroidogenesis in the Human Adrenocortical Carcinoma Cell Line H295R

Polychlorinated biphenyls (PCB), synthetic, persistent organic pollutants (POP), are detected ubiquitously, in water, soil, air, and sediments, as well as in animals and humans. PCB are associated with range of adverse health effects, such as interference with the immune system and nervous system, reproductive abnormalities, fetotoxicity, carcinogenicity, and endocrine disruption. Our objective was to determine the effects of three structurally different PCB congeners, PCB118, PCB 126, and PCB 153, each at two concentrations, on the steroidogenic capacity and proteome of human adrenocortical carcinoma cell line cultures (H295R) . After 48 h of exposure, cell viability was monitored and estradiol, testosterone, cortisol and progesterone secretion measured to quantify steroidogenic capacity of the cells. Two-dimensional (2D) gel-based proteomics was used to screen for proteome alterations in H295R cells in response to the PCB. Exposure to PCB 118 increased estradiol and cortisol secretion, while exposure to PCB 153 elevated estradiol secretion. PCB 126 was the most potent congener, increasing estradiol, cortisol, and progesterone secretion in exposed H295R cells. Seventy-three of the 711 spots analyzed showed a significant difference in normalized spot volumes between controls (vehicle only) and at least one exposure group. Fourteen of these protein spots were identified by liquid chromatography with mass spectroscopy (LC-MS/MS). Exposure to three PCB congeners with different chemical structure perturbed steroidogenesis and protein expression in the H295R in vitro model. This study represents an initial analysis of the effects on proteins and hormones in the H295R cell model, and additional studies are required in order to obtain a more complete understanding of the pathways disturbed by PCB congeners in H295R cells. Overall, alterations in protein regulation and steroid hormone synthesis suggest that exposure to PCB disturbs several cellular processes, including protein synthesis, stress response, and apoptosis.

Effects of single pesticides and binary pesticide mixtures on estrone production in H295R cells

The aim of the present study was to determine whether the human adrenocortical carcinoma cell line H295R can be used as an in vitro test system to investigate the effects of binary pesticide combinations on estrone production as biological endpoint. In the first step ten pesticides selected according to a tiered approach were tested individually. The anilinopyrimidines cyprodinil and pyrimethanil as well as the dicarboximides iprodione and procymidone increased estrone concentration, while the triazoles myclobutanil and tebuconazole as well as the strobilurins azoxystrobin and kresoxim-methyl decreased estrone concentration in the supernatant of H295R cells. The N-methylcarbamate methomyl did not show any effects, and the phthalimide captan reduced estrone concentration unspecifically due to its detrimental impact on cellular viability. When cyprodinil and pyrimethanil, which belong to the same chemical group and increase estrone production, were combined, in most of the cases the overall effect was solely determined by the most potent compound in the mixture (i.e., cyprodinil). When cyprodinil and procymidone, which belong to different chemical groups but increase estrone production, were combined, in most cases an additive effect was observed. When cyprodinil, which increased estrone production, was combined with either myclobutanil or azoxystrobin, which decreased estrone production, the overall effect of the mixture was in most cases either entirely determined by myclobutanil or at least partially modulated by azoxystrobin. In conclusion, H295R cells appear to be an adequate in vitro test system to study the effect of combining two pesticides affecting estrone production.

Mechanistic Computational Model of Steroidogenesis in H295R Cells: Role of Oxysterols and Cell Proliferation to Improve Predictability of Biochemical Response to Endocrine Active Chemical—Metyrapone

The human adrenocortical carcinoma cell line H295R is being used as an in vitro steroidogenesis screening assay to assess the impact of endocrine active chemicals (EACs) capable of altering steroid biosynthesis. To enhance the interpretation and quantitative application of measurement data in risk assessments, we are developing a mechanistic computational model of adrenal steroidogenesis in H295R cells to predict the synthesis of steroids from cholesterol (CHOL) and their biochemical response to EACs. We previously developed a deterministic model that describes the biosynthetic pathways for the conversion of CHOL to steroids and the kinetics for enzyme inhibition by the EAC, metyrapone (MET). In this study, we extended our dynamic model by (1) including a cell proliferation model supported by additional experiments and (2) adding a pathway for the biosynthesis of oxysterols (OXY), which are endogenous products of CHOL not linked to steroidogenesis. The cell proliferation model predictions closely matched the time-course measurements of the number of viable H295R cells. The extended steroidogenesis model estimates closely correspond to the measured time-course concentrations of CHOL and 14 adrenal steroids both in the cells and in the medium and the calculated time-course concentrations of OXY from control and MET-exposed cells. Our study demonstrates the improvement of the extended, more biologically realistic model to predict CHOL and steroid concentrations in H295R cells and medium and their dynamic biochemical response to the EAC, MET. This mechanistic modeling capability could help define mechanisms of action for poorly characterized chemicals for predictive risk assessments.

Computational Model of Steroidogenesis in Human H295R Cells toPredict Biochemical Response to Endocrine-Active Chemicals: Model Developmentfor Metyrapone

Background: An in vitro steroidogenesis assay using the human adrenocortical carcinoma cell line H295R is being evaluated as a possible screening assay to detect and assess the impact of endocrine-active chemicals (EACs) capable of altering steroid biosynthesis. Data interpretation and their quantitative use in human and ecological risk assessments can be enhanced with mechanistic computational models to help define mechanisms of action and improve understanding of intracellular concentrationresponse behavior.Objectives: The goal of this study was to develop a mechanistic computational model of the metabolic network of adrenal steroidogenesis to estimate the synthesis and secretion of adrenal steroids in human H295R cells and their biochemical response to steroidogenesis-disrupting EAC.Methods: We developed a deterministic model that describes the biosynthetic pathways for the conversion of cholesterol to adrenal steroids and the kinetics for enzyme inhibition by metryrapone (MET), a model EAC. Using a nonlinear parameter estimation method, the model was fitted to the measurements from an in vitro steroidogenesis assay using H295R cells.Results: Model-predicted steroid concentrations in cells and culture medium corresponded well to the time-course measurements from control and MET-exposed cells. A sensitivity analysis indicated the parameter uncertainties and identified transport and metabolic processes that most influenced the concentrations of primary adrenal steroids, aldosterone and cortisol.Conclusions: Our study demonstrates the feasibility of using a computational model of steroidogenesis to estimate steroid concentrations in vitro. This capability could be useful to help define mechanisms of action for poorly characterized chemicals and mixtures in support of predictive hazard and risk assessments with EACs.

Resorcylic Acid Lactone L-783,277 Inhibits the Growth of the Human Adrenal Cancer Cell Line H295R in Vitro

The resorcylic acid lactone L-783,277, isolated from a Phoma sp. (ATCC 74403), is a potent and specific inhibitor of MEK (Map kinase kinase) that exerts very interesting pharmacological activities including anti-neoplastic properties. However, the role of this compound in the regulation of endocrine-related cancer cell growth and tumor progression remains unknown. In the present study we have evaluated the effect of L-783,277 on the viability, proliferation and cell cycle of the human adrenocortical carcinoma cell line H295R. L-783,277 inhibited viability (IC50 of 22 microM) and cell proliferation (IC50 of 21 microM) of H295R. At concentrations of 10(-6)-10(-8)M this effect was associated with the accumulation of H295R cells in S-phase, whereas at concentrations of 10(-9)-10(-10)M a prolonged G1-phase and reduced transition into S-phase were observed. Our findings demonstrate for the first time the anti-proliferative action of L-783,277 on the human adrenocortical H295R cell line.

Comparative effects of prolactin versus ACTH, estradiol, progesterone, testosterone, and dihydrotestosterone on cortisol release and proliferation of the adrenocortical carcinoma cell line H295R

In this study, using the H295R cell line as a model system, we investigated the role of prolactin (PRL) and steroid hormones in the growth regulation and cortisol release of adrenocortical cells. H295R cells were treated with increasing doses (10(-13)-10(-6) M) of PRL, adrenocorticotropic hormone (ACTH), 17beta-estradiol (E(2)), progesterone (P(4)), testosterone (T), and dihydrotestosterone (DHT). As expected, ACTH raised cortisol secretion and increased the proliferation rate of cultured cells. Incubation with T, DHT, E(2), and P(4) for 24 h did not significantly increase cortisol release. Conversely, PRL concentrations of 10(-8)-10(-6) M caused a significant increase in the release of cortisol. Long-term (5 days) stimulation of H295R cells with E(2), P(4), and PRL was a trigger to increased cell proliferation, while T and DHT did not alter H295R cell proliferation. Taken together, these results indicate that steroid hormones exert differential effects on adrenocortical function. Additionally, the present study demonstrates that PRL had biphasic actions in regulating adrenocortical function. PRL may form a novel regulatory system for steroid hormone secretion and cell proliferation in the adrenal cortex.

Alteration of steroidogenesis in H295R cells by organic sediment contaminants and relationships to other endocrine disrupting effects

A novel bioassay with the human adrenocortical carcinoma cell line H295R can be used to screen for endocrine disrupting chemicals that affect the expression of genes important in steroidogenesis. This assay was employed to study the effects of organic contaminants associated with the freshwater pond sediments collected in the Ostrava-Karvina region, Czech Republic. The modulation of ten major genes involved in the synthesis of steroid hormones (CYP11A, CYP11B2, CYP17, CYP19, 17βHSD1, 17βHSD4, CYP21, 3βHSD2, HMGR, StAR) after exposure of H295R cells to sediment extracts was investigated using quantitative real-time polymerase chain reaction (PCR). Crude sediment extracts, containing high concentrations of polycyclic aromatic hydrocarbons (PAHs) and moderate amounts of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) significantly stimulated expression of the CYP11B2 gene (up to 10-fold induction), and suppressed expression of 3βHSD2 and CYP21 genes. A similar pattern was observed with the extracts after treatment with concentrated sulfuric acid to remove labile chemicals (including PAHs) leaving only persistent PCBs, OCPs and potentially PCDD/Fs. Comparison of the results with other mechanistically based bioassays (arylhydrocarbon receptor, AhR, mediated responses in H4IIE-luc cells, and estrogen receptor mediated effects in MVLN cells) revealed significant endocrine disrupting potencies of organic contaminants present in the sediments (most likely antiestrogenicity). Pronounced effects were observed particularly in sediment extracts from the Pilnok Pond which harbors an unusual intersexual population of the narrow-cawed crayfish Pontastacus leptodactylus (Decapoda, Crustacea). This pilot study provided the first experimental evidence of the wider application of the H295R bioassay for screening complex environmental samples, and the results support the hypothesis of chemical-induced endocrine disruption in intersexual crayfish.

Forskolin up-regulates aromatase (CYP19) activity and gene transcripts in the human adrenocortical carcinoma cell line H295R.

A number of conditions related to sex-reversal in boys and men and precocious puberty in girls are caused by estrogen-secreting adrenal tumors. In these tumors, cytochrome P450 aromatase (aromatase) that is encoded in the CYP19 gene is expressed at high levels. To investigate the molecular mechanism of aromatase expression in these adrenal tumors, we characterized the activity, gene transcript and genomic promoter region of aromatase in the human adrenocortical carcinoma cell line H295R. Aromatase activity and the transcript of the CYP19 gene were highly up-regulated by forskolin, but not by dexamethasone. The results from exon I-specific reverse transcriptase (RT)-PCR and the transfection of reporter constructs suggested that promoter I.3 and promoter II were activated in H295R. Deletion and mutation analysis suggested that cAMP response element-like sequence (CLS) and steroidogenic factor-1 (SF-1) motif, were critical for the activation of promoter II. The results of this work should provide the basis for the molecular analysis of aromatase expression in adrenocortical cells.

Comparative Effect of Pituitary Adenylate Cyclase-Activating Polypeptide on Aldosterone Secretion in Normal Bovine and Human Tumorous Adrenal Cells*

The purpose of this study was to investigate the mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in stimulating aldosterone production in two different models: bovine adrenal zona glomerulosa (ZG) cells in primary culture and the human adrenocortical carcinoma cell line H295R. PACAP binds to two major groups of receptors: type I, which prefers PACAP38 and PACAP27 over vasoactive intestinal peptide (VIP); and type II, which has approximately equal affinity for PACAP38, PACAP27, and VIP. The type I subclass comprises multiple splice variants that can be distinguished by their specificity to PACAP38 and PACAP27 in their activation of adenylate cyclase and phospholipase C. Type II PACAP/ VIP receptors couple only to AC. In bovine ZG cells, PACAP38 and PACAP27 stimulated aldosterone production in a dose-dependent manner, whereas VIP was ineffective. In H295R cells, PACAP38, PACAP27, and VIP dose-dependently stimulated aldosterone production with roughly the same ED50. In bovine ZG cells, PACAP38 and PACAP27 stimulated cAMP production with similar efficacy, whereas VIP had no effect. In H295R cells, all three peptides stimulated cAMP accumulation. PACAP38 and PACAP27 also activated PLC in bovine ZG cells as they induced an increase in Ins(1,4,5)Ps production. In H295R cells, neither of these peptides was able to stimulate IP turnover. These results indicate that PACAP stimulation of aldosterone production is mediated by the PVR1s or the PVR1hop splice variants of the type I PACAP-specific receptor subtype in bovine ZG cells, whereas only type II PACAP/VIP receptors seemed to occur in the human H295R cell line. In addition, PACAP-stimulated aldosterone production was inhibited by atrial natriuretic peptide in bovine and human adrenocortical cells, however not by the same mechanism. This further supports species-specific and/or cell type-specific signaling pathways for PACAP in the regulation of aldosterone production.