HAC15 Cells Research Articles

7888 Mitotane Induces Different Modes Of Cell Death In Treatment Resistant And Sensitive Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. C. Hong: None. C. Hantel: None. W.E. Rainey: None. M.C. Dennedy: None. Adrenocortical carcinoma (ACC) is a rare aggressive cancer with poor survival. Adjuvant mitotane is the only approved drug for treatment of ACC. It improves survival but its use is limited by poor tolerability and drug resistance. For metastatic ACC, combination chemotherapy, using mitotane and etoposide, doxorubicin, and cisplatin improves survival, but efficacy is limited. Better understating the cytotoxic mechanisms of mitotane offers potential to develop improved therapeutic options for ACC. To investigate the underlying mechanisms in vitro, human ACC cells (HAC15 and H295R), in monolayer cell culture were treated with increasing concentrations of mitotane. Cell death was evaluated at 6 and 24 hours using flow cytometry analysis of Annexin V and Sytox Blue. This was complemented by Incucyte ® Live-Cell Analysis System imaging of annexin V and Sytox Blue staining. Expression of the markers of apoptosis, cleaved caspase 3, and necroptosis, phosphorylated MLKL and RIPK1 was measured at 6 and 24 hours. Mitotane significantly reduced the viability of H295R cells at 6 and 24 hours at the therapeutic concentration of 50uM (p>0.0001), thereby demonstrating treatment-sensitivity. In HAC15 cells, a supratherapeutic dose of 200uM was needed to significantly reduce viability (p>0.05) at 24 hours, indicating resistance to mitotane treatment at therapeutic concentrations. There was increasing Annexin V fluorescence with increasing mitotane concentration in H295R and HAC15 cells ar 6 and 24 hours without higher coinciding staining with Sytox Blue. Video analysis demonstrated these findings. Cleaved caspase 3 expression was associated with mitotane-induced cell death in H295R cells at 6 and 24 hours, indicating apoptosis. HAC15 cells demonstrated cleaved caspase 3 expression only at the highest concentration of mitotane exposure (600uM) at 6 hours, without appreciable expression at 24 hours. Phosphorylated-MLKL and RIPK1 expression was seen in controls and all mitotane doses in both cell lines at both timepoints, indicating necroptosis. In this study, H295R cells, representing a treatment-sensitive model of Mitotane undergo both apoptotic and necroptotic cell death at 6 and 24 hours of mitotane exposure. In contrast, HAC15 cells, demonstrated relative treatment resistance to mitotane, and undergo necroptotic cell death only. These results highlight the importance of understanding and targeting non-apoptotic and necroptotic cell death pathways, particularly when evaluating mechanisms of treatment resistance in ACC. Given the difference in the mechanism of cell death in treatment-sensitive and treatment-resistant cells, further exploration of necroptosis is necessary to better understand how ACC cells may manifest treatment resistance. Presentation: 6/1/2024

8468 Development And Characterisation Of Three-Dimensional Cell Culture Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. P. Donlon: None. C. Hantel: None. M.C. dennedy: None. Adrenocortical carcinoma (ACC) is a rare malignancy carrying a poor prognosis and limited treatment options. Surgical resection is the only option for cure; but unsuitable for advanced disease. Mitotane is the approved medical therapy, but associated with treatment resistance and adverse effects. Newer therapies, such as immune checkpoint inhibitors, have limited success. Development of improved therapies is limited by current pre-clinical disease models. No available model reflects the tumour micro-environment. Three-dimensional (3D) cell culture models are increasingly used in cancer research (6), with a paucity of developed models in ACC. In the current study, we developed novel 3D models of ACC using a type-1 collagen matrix. We then characterised these as representative ACC disease models . 3D cell culture models of ACC were optimised by embedding ACC cell lines, MUC-1, HAC15 and H295R within a type-1 Collagen matrix at differing cell numbers. Expression of type-1 collagen in ACC primary tumours was determined using immunohistochemistry (n=4). Cell viability for each model was assessed by evaluating sytox blue staining by Flow Cytometry. Metabolic activity of ACC cells within the matrix was determined using AlamarBlue staining. Prolferative activity was assessed using Ki67. Steroidogenesis was evaluated by measuring cortisol, aldosterone and androstenedione using LC-MS/MS.. Expression of CYP11B1, CYP11B2 CYP17, and StAR was measured using RT-qPCR. Type 1 collagen is strongly expressed in ACC tumour microenvironment. MUC-1, H295R and HAC15 cells were successfully cultured in a type 1 collagen matrix. MUC-1 cells cultured in the collagen matrix remain viable throughout the period of experimentation with optimum viability between 14 and 21 days. H295R cells also remain viable with optimum viability at an earlier stage of 7-14 days. HAC15 cells demonstrate high resilience in 3D cell culture with maximum viability throughout the entire period of experimentation up to 21 days. All three models increase their metabolic and proliferative activity over time. All three steroids are expressed by all three models with an increase in aldosterone secretion observed in 3D HAC15 and H295R models compared to monolayer (p>0.001) at days 7 and 14. All models express key steroidogenic enzymes. 3D ACC cell culture models were developed successfully using collagen type 1 abundant in ACC. Typical characteristics of the ACC tumour environment included (i) the presence of live and necrotic cells (ii) high metabolic activity and proliferation, reflective of cell turnover.. Capacity of cells to spread and proliferate within the 3D cell culture models was limited by available collagen substrate. Steroidogenic capacity was demonstrated in 3D cell culture. This model therefore retained key characteristics of ACC and shows promise as an animal-sparing, pre-clinical model. Presentation: 6/1/2024

OR06-04 Environmental Pollutants Perfluoroalkyl Acids Potently Stimulated Aldosterone Biosynthesis

Abstract Disclosure: B. Caroccia: None. T.M. Seccia: None. G. Pallafacchina: None. M. Piazza: None. I. Caputo: None. R. Rizzuto: None. G. Rossi: None. A large environmental contamination of drinking water by perfluoroalkyl substances (PFAS) markedly increased by 8-fold the plasma levels of pentadecafluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) in a Northern Italy population with that shows a high prevalence of arterial hypertension and cardiovascular disease. As the link between PFAS and arterial hypertension is unknown, we investigated if they could enhance the biosynthesis of aldosterone, the main mineralocorticoid hormone that has pressor effects. We exposed human adrenocortical carcinoma HAC15 cells to 1 µM or 10 µM concentration of PFOA and PFOS, alone or in combination, for 24, 48 or 72 hours and we measured changes in cell viability, aldosterone synthase (CYP11B2) mRNA, cell and mitochondrial reactive oxygen species (ROS), and aldosterone production in the absence or presence of the superoxide scavenger tempol. Experiments were also performed in the presence of 10 or 100 nM Ang II. We found that PFAS increased CYP11B2 mRNA by 3-fold and aldosterone secretion and mitochondrial ROS production by 2-fold over controls (p < 0.01 for all). PFAS also enhanced the effects of the aldosterone secretagogue Ang II on CYP11B2 mRNA and aldosterone secretion: 10 nM or 100 nM Ang II increased CYP11B2 mRNA by 12- and 52-fold, respectively, compared to vehicle (p < 0.001). Moreover, 1 µM PFAS potentiated the effect of 10 nM or 100 nM Ang II on CYP11B2 gene expression by 30- and 80-fold, respectively (p < 0.001). As the ROS scavenger tempol, which decreased ROS, significantly blunted the CYP11B2 gene expression induced by either PFAS alone, or by concomitant exposure to Ang II and PFAS, the effect of PFAS likely occurs via increased ROS generation. These results indicate that at concentrations mimicking those found in human plasma of exposed individuals, PFAS exert a prooxidant effect and act as potent disruptors of human adrenocortical cell function, thus implicating them as causative factors of human arterial hypertension due to aldosteronism. Presentation: Thursday, June 15, 2023

SAT267 BMP4, GDF11 And TGFβ2 Repress Adrenal Cell Cortisol Production By Blocking CYP17A1 And CYP11B1 Expression

Abstract Disclosure: E.C. Fischbach: None. A. Lopez: None. M. Ullenbruch: None. J. Rege: None. W.E. Rainey: None. Introduction: The adrenal cortex is responsible for the biosynthesis of multiple steroid hormones that regulate electrolyte and carbohydrate balance. Cortisol, the principal glucocorticoid in humans, is primarily produced in the middle zone of the cortex known as the zona fasciculata (ZF). The enzymes responsible for cortisol biosynthesis include cytochrome P450c17 (17α-hydroxylase), encoded by CYP17A1 and cytochrome P450c11 (11β-hydroxylase), encoded by CYP11B1. Several paracrine/autocrine regulators, including members of the transforming growth factor beta (TGFβ) superfamily, have been implicated in controlling adrenocortical cell homeostasis and steroid hormone production. Previously, we reported a bone morphogenetic protein 4 (BMP4) expression gradient in the adrenal cortex with higher levels in the zona glomerulosa (ZG) and outer ZF compared to the zona reticularis (ZR). In this study, we sought to compare adrenal expression of members of the TGFβ family including BMP4, growth differentiation factor 11 (GDF11), and transforming growth factor beta 2 (TGFβ2), and determine their effect on adrenal cell cortisol biosynthesis. Methods: Growth factor mRNA levels were studied using microarray analysis of laser captured ZG, ZF and ZR from human adrenals. The adrenal cell line, HAC15 was used to determine the effects of the TGFβ family members alone and in combination. Output measures included cell cortisol production and mRNA levels of steroidogenic enzymes using ELISA and quantitative RT-PCR, respectively. Results: Microarray analysis indicated higher mRNA levels of BMP4, GDF11 and TGFβ2 in the ZG compared with the ZF and ZR regions of the adrenal. Treatment of HAC15 cells with either BMP4 (50 ng/mL), GDF11 (50 ng/mL) or TGFβ2 (10 ng/mL) significantly inhibited cortisol production, as well as CYP11B1 and CYP17A1 mRNA levels. The inhibition was observed under basal and forskolin-stimulated conditions. Furthermore, the inhibitory effects on cortisol production and enzyme expression were additive when factors were added together. Summary: Overall, our findings suggest that the human adrenal has a gradient of multiple members of the TGFβ family. The additive action of these growth factors on adrenal cell differentiation may explain, in part, the inability of the ZG to produce cortisol and its low expression of CYP11B1 and CYP17A1. Presentation: Saturday, June 17, 2023

Exposure to anticancer drugs modulates the expression of ACSL4 and ABCG2 proteins in adrenocortical carcinoma cells

Adrenocortical carcinoma (ACC) is a rare and malignant disease, with more than 50 % of patients developing hormone-secreting tumors. These tumors are genetically heterogeneous and potentially lethal, as metastasis is often underway at the time of diagnosis. While chemoresistance can be multifactorial, Acyl CoA synthetase 4 (ACSL4) is known to contribute to the generation of highly aggressive cellular phenotypes, while increased expression and activity of multidrug transporters such as ATP-binding cassette subfamily G member 2 (ABCG2) are known to play a key role. Therefore, the objective of this work was to determine changes in the expression of ACSL4 and ABCG2 in ACC cell lines after exposure to antitumor drugs. Bioinformatics analysis of public database GSE140818 revealed higher ACSL4 and ABCG2 expression in HAC15 cells resistant to mitotane when compared to wild type cells. In addition, our studies revealed an increase in ACSL4 and ABCG2 expression in lowly aggressive H295R cells undergoing early treatment with non-lethal concentrations of mitotane, doxorubicin and cisplatin. Comparable results were obtained in lowly aggressive breast cancer cells MCF-7. The increase in ACSL4 and ABCG2 expression favored tumor cell viability, proliferation and compound efflux, an effect partially offset by ACSL4 and ABCG2 inhibitors. These results provide relevant data on the undesired molecular effects of antitumor drugs and may fuel future studies on patients’ early response to antitumor treatment.

Endocrine disruptors and arterial hypertension: A developing story

Endocrine disrupting Chemicals (EDCs) are substances that interfere with hormones by several mechanisms including receptor activation or antagonism, changes in gene and protein expression, modification of signal transduction, and/or epigenetic modifications in hormone-producing cells. A survey conducted by the European Union in a Northern Italian region led to the discovery of a large environmental contamination of drinking water by perfluoroalkyl substances (PFAS). As the exposed population showed a high prevalence of arterial hypertension and cardiovascular disease, we decided to investigate if PFAS could enhance the biosynthesis of aldosterone. To this aim, we exposed human adrenocortical carcinoma HAC15 cells to PFAS and found that PFAS markedly increased aldosterone synthase (CYP11B2) gene expression and aldosterone secretion. Moreover, we found that they promoted reactive oxygen species (ROS) production in mitochondria, the organelles where aldosterone biosynthesis takes place. PFAS also enhanced the effects of the aldosterone secretagogue angiotensin II (Ang II) on CYP11B2 gene expression and aldosterone secretion. We also found that not only PFAS but also polychlorinated biphenyl 126 (PCB126), a chemical compound belonging to a different category of EDCs, can increase CYP11B2 gene expression and aldosterone secretion in adrenocortical cells. This novel information needs to be considered in the context of a widespread exposure to the most common EDC, that is excess Na+ intake, whose detrimental effects on human health occur in the setting of aldosterone production exceeding the physiological needs and lead to high blood pressure, congestion, and cardiovascular and renal damage.

Englerin A Inhibits T-Type Voltage-Gated Calcium Channels at Low Micromolar Concentrations.

Englerin A (EA) is a potent agonist of tetrameric transient receptor potential canonical (TRPC) ion channels containing TRPC4 and TRPC5 subunits. TRPC proteins form cation channels that are activated by plasma membrane receptors. They convert extracellular signals such as angiotensin II into cellular responses, whereupon Na+ and Ca2+ influx and depolarization of the plasma membrane occur. Via depolarization, voltage-gated Ca2+ (CaV) channels can be activated, further increasing Ca2+ influx. We investigated the extent to which EA also affects the functions of CaV channels using the high-voltage-activated L-type Ca2+ channel CaV1.2 and the low-voltage-activated T-type Ca2+ channels CaV3.1, CaV3.2, and CaV3.3. After expression of cDNAs in human embryonic kidney (HEK293) cells, EA inhibited currents through all T-type channels at half-maximal inhibitory concentrations (IC50) of 7.5 to 10.3 μM. In zona glomerulosa cells of the adrenal gland, angiotensin II-induced elevation of cytoplasmic Ca2+ concentration leads to aldosterone release. We identified transcripts of low- and high-voltage-activated CaV channels and of TRPC1 and TRPC5 in the human adrenocortical (HAC15) zona glomerulosa cell line. Although no EA-induced TRPC activity was measurable, Ca2+ channel blockers distinguished T- and L-type Ca2+ currents. EA blocked 60% of the CaV current in HAC15 cells and T- and L-type channels analyzed at -30 mV and 10 mV were inhibited with IC50 values of 2.3 and 2.6 μM, respectively. Although the T-type blocker Z944 reduced basal and angiotensin II-induced 24-hour aldosterone release, EA was not effective. In summary, we show here that EA blocks CaV1.2 and T-type CaV channels at low-micromolar concentrations. SIGNIFICANCE STATEMENT: In this study we showed that englerin A (EA), a potent agonist of tetrameric transient receptor potential canonical (TRPC)4- or TRPC5-containing channels and currently under investigation to treat certain types of cancer, also inhibits the L-type voltage-gated Ca2+ (CaV) channel CaV1.2 and the T-type CaV channels CaV3.1, CaV3.2, and CaV3.3 channels at low micromolar concentrations.

Phospholipase D mediates very low-density lipoprotein-induced aldosterone production, in part, via lipin-1.

Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of very low-density lipoprotein (VLDL). VLDL has been shown to induce aldosterone production in multiple adrenal zona glomerulosa models, mediated in part by phospholipase D (PLD). PLD is an enzyme that hydrolyzes phosphatidylcholine to produce phosphatidic acid (PA), a lipid second messenger that can also be dephosphorylated by lipin to yield diacylglycerol (DAG), yet another lipid signal. However, it is unclear which of the two lipid second messengers, PA or DAG, underlies PLD's mediation of aldosterone production. We hypothesized that the key signal produced by PLD (indirectly) is DAG such that PLD mediates VLDL-induced aldosterone production via lipin-mediated metabolism of PA to DAG. To assess the role of lipin in VLDL-induced aldosterone production, lipin-1 was overexpressed (using an adenovirus) or inhibited (using propranolol) in HAC15 cells followed by treatment with or without VLDL. Lipin-1 overexpression enhanced the VLDL-stimulated increase in CYP11B2 expression (by 75%), and lipin-1 inhibition decreased the VLDL-stimulated increase in CYP11B2 expression (by 66%). Similarly, the VLDL-stimulated increase in aldosterone production was enhanced by lipin-1 overexpression (182%) and was decreased by lipin inhibition (80%). Our results are suggestive of DAG being the key lipid signal since manipulating lipin-1 levels/activity affects VLDL-stimulated steroidogenic gene expression and ultimately, aldosterone production. Our study warrants further investigation into VLDL-stimulated steroidogenic signaling pathways which may lead to the identification of novel therapeutic targets, such as lipin-1 and its downstream pathways, to potentially treat obesity-associated hypertension.

PS-B04-9: ELUCIDATING EFFECT OF INHIBITING ALDOSTERONE SYNTHESIS ON HUMAN ADRENOCORTICAL CELLS

Introduction: Modern human diet is high in salt. High salt stimulates decrease of aldosterone by down-regulation of aldosterone synthase (encoded by the gene CYP11B2) in the zona glomerulosa (ZG). Aldosterone regulates blood pressure and volume by regulating salt secretion. Interestingly, adult human adrenal ZG is sparse and confined to discrete islets of cells unlike rodent adrenal where the zone encompasses the whole periphery of the adrenal. We therefore hypothesize that ZG cell function change from synthesis to apoptosis/migration mode when aldosterone is not required due to the high salt diet. Objective: Herein, we intend to characterise human adrenocortical cells silenced for CYP11B2. Methods: We transfected HAC15, a subclone of the H295R human adrenocortical cancer cell line with scrambled siRNA (siScrambled) or siRNA targeting CYP11B2 (siCYP11B2) using the Neon Transfection System. 48-h post-transfection, cell apoptosis rate was assessed using the Pacific Blue Annexin V/SYTOX AADvanced apoptosis kit. The supernatants and cells were harvested for aldosterone and cortisol measurements, and RNA isolation. A representative sample of siCYP11B2 and siScrambled from each independent experiment was selected for RNA-Sequencing (RNAseq). The EZMTT Proliferation Assay was performed on equally seeded HAC15 cells, 72-h post-transfection with either siScrambled or siCYP11B2 using the Lipofectamin 3000 Transfection reagent. Experiments were repeated 3 times independently in triplicates. Results: siCYP11B2 transfection limited the production of aldosterone by 69.8% (p = 0.001) and down-regulated the expression of CYP11B2 by 83.0% (p = 0.001). We confirmed that the silencing was specific to CYP11B2 despite the gene being highly homologous to CYP11B1 as there were no significant change on cortisol production and CYP11B1 mRNA expression. RNAseq analysis found 3 genes, CYP11B2, HNRNPA1, and UHMK1, consistently down-regulated in siCYP11B2 by 32%, 65% and 67% respectively. The most enriched pathways among the differentially expressed genes (DEG) were mitophagy and autophagy. No difference in cell apoptosis was seen in siCYP11B2 cells. Instead, an increase in cell proliferation was detected with the EZMTT assay. Conclusion: Our findings suggest that silencing of CYP11B2 does not affect apoptosis but does affect cell proliferation. Interestingly, the mitophagy and autophagy pathways were enriched in the DEGs of siCYP11B2 cells.

Fatty acids as a direct regulator of aldosterone hypersecretion

Primary hyperaldosteronism is a major cause of secondary hypertension and carries additional cardiovascular risks beyond that of the elevated blood pressure. Primary hyperaldosteronism is more prevalent in obese people, and weight loss reduces aldosterone levels. It needs to be determined whether obesity related factors directly contribute to the pathogenesis of primary hyperaldosteronism. Here we show that the non-esterified fatty acids (NEFA) palmitic acid, and to a lesser extent, linoleic acid significantly stimulated aldosterone production and steroid enzyme induction in adrenocortical HAC15 cells of human origin. Palmitic acid, linoleic acid, and to a much lesser extent, oleic acid induced the expression of aldosterone synthase. Induction of the Steroidogenic Acute Regulatory Protein (StAR) was modest. Increased aldosterone secretion was independent of fatty acid beta-oxidation in the mitochondria but may involve free fatty acid receptor 1 (FFAR1/GPR40) and endoplasmic reticulum (ER) stress. Palmitic acid and linoleic acid induced the expression of C/EBP Homologous Protein (CHOP), a marker of ER stress, correlating with their ability to induce aldosterone synthase gene expression. Palmitic acid, but not linoleic acid decreased mitochondrial potentials and induced uncoupling protein 2 (UCP2). Palmitic acid enhanced, while docosahexaenoic acid (DHA) suppressed aldosterone response to angiotensin II (Ang-II). Our study provides evidence that NEFAs modulate aldosterone production, and further suggests that hyperaldosteronism shares similar pathogenesis with other obesity-related disorders such as metabolic syndrome.

Mammalian Target of Rapamycin Inhibition Decreases Angiotensin II-Induced Steroidogenesis in HAC15 Human Adrenocortical Carcinoma Cells.

Mammalian target of rapamycin (mTOR) inhibitors suppress adrenal cortical carcinoma cell proliferation and cortisol production; the relationship between mTOR and aldosterone production has not been examined. HAC15 cells were incubated with an mTOR activator and several inhibitors including AZD8055 (AZD) in the presence and absence of angiotensin II (AngII). The expression of rapamycin-sensitive adapter protein of mTOR (Raptor) and rapamycin-insensitive companion of mTOR (Rictor), adaptor proteins of mTOR complex 1 and 2, respectively, were studied in the HAC15 cells and deleted by CRISPR/gRNA. The mTOR inhibitors decreased aldosterone induced by AngII. Inhibition of mTOR by AZD significantly suppressed AngII-induced aldosterone and cortisol formation in a dose-dependent manner, whereas the mTOR activator MHY had no effect. AZD did not alter forskolin-induced aldosterone production showing that it is specific to the AngII signaling pathway. AngII-mediated ERK and mTOR activation were suppressed by AZD, along with a concomitant dose-dependent reduction of AngII-induced steroidogenic enzymes including steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase-type 2, CYP17A1, and aldosterone synthase protein. Furthermore, mTOR components ribosomal protein S6 kinase (P70S6K) and protein kinase B phosphorylation levels were decreased by AZD. As mTOR exerts its main effects by forming complexes with adaptor proteins Raptor and Rictor, the roles of these individual complexes were studied. We found an increase in the phosphorylation of Raptor and Rictor by AngII and that their CRISPR/gRNA-mediated knockdown significantly attenuated AngII-induced aldosterone and cortisol production. mTOR signaling has a critical role in transducing the AngII signal initiating aldosterone and cortisol synthesis in HAC15 cells and that inhibition of mTOR could be a therapeutic option for conditions associated with excessive renin-angiotensin system-mediated steroid synthesis.

PSUN17 Magnetic iron oxide nanoparticles for the delivery of thermal therapy for the treatment of primary aldosteronism

Abstract To minimize damage to surrounding tissues, targeted delivery of therapeutics to the tumor is highly desirable, and the development of nanotechnology has shown promising results. Magnetic iron oxide nanoparticles (MIONPs) have been gaining traction over the years for applications such as drug delivery, molecular imaging and delivering hyperthermia for treatment of various cancers (1). MIONPs have great therapeutic potential as they can be produced in various sizes and shapes, with the ability to modify the surface by coating the nanoparticles. MIONPs have the ability to be activated by external magnetic field to generate heat and to cause hyperthermia (2). Translationally, the delivery of thermal therapy offers an option for minimally invasive definitive treatment of primary aldosteronism, an endocrinopathy of aldosterone excess/dysregulation which represents the commonest secondary form of hypertension. In this study, MIONPs have been used at different concentrations to evaluate nanoparticle uptake and rate of uptake by adrenal cortical and endothelial cells, as well as gain understanding of the location of nanoparticles within the cell. Magnetic iron oxide nanoparticles (MIONPs) were provided by Kansas University. Adrenal Cortical cell-lines (MUC1, H295R and HAC15) and Endothelial cell-line (HUVEC) were used in this study. MIONPs were added at concentrations of 0.5, 5, 10, 20 and 50 µg/ml to the cells and incubated overnight. MIONP uptake efficiency, rate of uptake and cytotoxicity was assessed by Flow Cytometry. Confocal Microscopy was used to image the cells following MIONP incubation. Cellular proliferation was assessed by Xcelligence system and alamarBlue. Cellular respiration was assessed by "Seahorse" technology. MIONP location within the cells was assessed by transmission electron microscopy (TEM). Following overnight incubation with MIONPs, Flow Cytometry showed significant uptake by MUC1, HAC15 and HUVEC cells at 10 µg/ml MIONP concentration. Confocal Microscopy and Transmission Electron Microscopy (TEM) images revealed MIONPs in the cytoplasm and in the vesicles for all cell types. Live Confocal imaging showed MIONP phagocytosis specific uptake by the HAC15 cells. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

PSUN14 Elucidating Effect of Modulating Aldosterone Synthesis on Cell Fate in Human Adrenal Cells

Abstract Introduction Aldosterone regulates sodium homeostasis thereby affecting blood volume and blood pressure. Physiologically, aldosterone is synthesized in the zona glomerulosa (ZG) by aldosterone synthase (CYP11B2) when there is a lack of salt. However, in the adult human adrenal ZG is sparse and confined to discrete islets (1). We hypothesize that the high salt in modern diets change ZG cell function from synthesis to apoptosis/migration mode when aldosterone is not required. This hypothesis is based on: [i] CYP11B2−/− mice, where ZG cells migrate and apoptose (2); [ii] the disappearance from most of human ZG of CYP11B2, which became apparent with selective antisera (3); [iii] apoptosis in monkeys following aldosterone synthase inhibition (4). Herein we aim to test our hypothesis by silencing CYP11B2 in human adrenal cells. Methods The HAC15, a subclone of the H295R human adrenocortical cancer cell line, was transfected with scrambled siRNA (siScrambled) or siRNA targeting CYP11B2 (siCYP11B2) using the Neon™ Transfection System (Thermofisher Scientific, USA). An apoptosis assay 48-h post-transfection was performed using the Pacific Blue™ Annexin V/SYTOX™ AADvanced™ apoptosis kit on the BD FACSVerseTM system (BD Biosciences, USA). The supernatants and cells were harvested for aldosterone and cortisol, and RNA isolation. Experiments were repeated 3 times independently in triplicates. RNA-Sequencing (RNAseq) of siCYP11B2 and siScrambled was performed on a representative sample from each independent experiment (BGI Genomics, China). Results Transfected HAC15 cells with siCYP11B2 showed reduction of aldosterone production by 69.8% (p=0.001) and suppression of CYP11B2 mRNA by 8 folds (p=0.001) compared to control, with no significant change on cortisol production and CYP11B1 mRNA expression. This confirmed that the silencing was specific to CYP11B2 despite the gene being highly homologous to CYP11B1. In all 3 independent experiments, 3 genes were significantly downregulated; CYP11B2, HNRNPA1, and UHMK1 expression in siCYP11B2 cells were 32%, 65% and 67% of matched control siScramble cells. Result of KEGG pathway from each independent experiment showed the most enriched pathways among the differentially expressed genes (DEG) were mitophagy and autophagy. However, analysis of flow cytometric apoptosis assay showed silencing of CYP11B2 did not induce cell apoptosis. Conclusion Our finding showed that silencing of CYP11B2 can affect mitophagy and autophagy but does not affect cell apoptosis in the HAC15 human adrenocortical cancer cell line. Further investigation on cell proliferation and in non-cancerous adrenal cells may be needed to identify the effect of modulating aldosterone synthesis on ZG cell fate. Reference (1) Vinson G., Front Neurosci 2016; 10: 238. (2) Lee G, et al., Endocrinology 2005; 146: 2650–2656. (3) Gomez-Sanchez CE, et al., Mol Cell Endocrinol 2014; 383: 111–117. (4) Bogman K, et al., Hypertension. 2017; 69(1): 189–196. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Origin of circulating 18-oxocortisol in the normal human adrenal

18-Oxocortisol is the product of the metabolism of 11-deoxycortisol by the mitochondrial enzyme aldosterone synthase (CYP11B2). The traditional concept is that the CYP11B2 is exclusively expressed in zona glomerulosa cells and the 17α-hydroxylase (CYP17A1) enzyme, required to synthesize 11-deoxycortisol, is in the zona fasciculata of the human adrenal. It has been postulated that the substrate for 18-oxocortisol is either cortisol from the circulation or from zona fasciculata cells adjacent to the zona glomerulosa. P-glycoprotein, which is highly expressed in steroidogenic cells of the adrenal gland, efficiently expels cortisol from the cell. Double immunofluorescence staining for the CYP11B2 and CYP17A1 enzymes in 7 human adrenals demonstrated that a highly variable number of cells in different areas of the zona glomerulosa co-expressed both enzymes. In addition, there were a variable number of cells that exclusively expressed the CYP17A1 embedded within the zona glomerulosa surrounded by CYP11B2-expressing cells.18-Oxocortisol in the media of human adrenocortical HAC15 cells was measured by ELISA after incubation with and without 10 nM of angiotensin II to stimulate CYP11B2 activity, with and without the 3β-hydroxysteroid dehydrogenase (HSD3B) inhibitor trilostane, and with variable amounts of cortisol or 11-deoxycortisol. Cortisol was a poor substrate, while 11-deoxycortisol was a significant substrate for the synthesis of 18-oxocortisol.These data suggest that the biosynthesis of 18-oxocortisol in the human adrenal is likely catalyzed by co-expression of the two crucial enzymes CYP17A1 and CYP11B2 in a small proportion of cells within the zona glomerulosa. It is also possible that 11-deoxycortisol diffusing from cells expressing only CYP17A1 interspersed with cells expressing the CYP11B2 enzyme may be a paracrine substrate in the synthesis of 18-oxocortisol.

A Novel Somatic Mutation of CACNA1H p.V1937M in Unilateral Primary Hyperaldosteronism.

BackgroundSomatic mutations for excess aldosterone production have been frequently identified as important roles in the pathogenesis of unilateral primary hyperaldosteronism (uPA). Although CACNA1H mutation represents a minor etiology in primary aldosteronism, it plays a significant role in causing uPAs in sporadic cases.ObjectiveTo identify novel somatic CACNA1H mutation in patients with uPA and investigate the pathophysiological, immunohistological, and clinical characteristics of the variant.MethodsWe applied a customized and targeted gene panel next-generation sequencing approach to detect mutations from the uPA cohort in Taiwan Primary Aldosteronism Investigation study group. Information from pre-diagnostic to postoperative data was collected, including past history, medications, blood pressure readings, biochemical data, and image studies. The functional role of the variant was confirmed by in vitro studies, demonstrating aldosterone production in variant-transfected human adrenal cell lines.ResultsWe identified a novel somatic CACNA1H mutation c.5809G>A (p.Val1937Met) in a uPA case. The CACNA1H gene encodes the pore-forming alpha-1H subunit of the voltage-dependent T-type calcium channel Cav3.2. This somatic CACNA1H p.V1937M variant showed excellent clinical and biochemical outcomes after ipsilateral adrenalectomy. The functional effect of somatic CACNA1H p.V1937M variant results in increased CYP11B2 expression and aldosterone biosynthesis in HAC15 cells. A distinct heterogeneous foamy pattern of CYP11B2 and CYP17A1 expression was identified in immunohistological staining, supporting the pathological evidence of aldosterone synthesis.ConclusionsThe somatic mutation of CACNA1H p.V1937M might be a pathogenic driver in aldosterone overproduction. This study provides new insight into the molecular mechanism and disease outcomes of uPA.

The Kratom‐Derived Alkaloid Mitragynine Enhances Cortisol Secretion from a Human Adrenocortical Cell Line

Kratom is a tropical evergreen tree indigenous to Southeast Asia whose leaves and decoctions have long been used in traditional folk medicine for the relief of pain and enhancement of vitality. In recent years kratom use has increased in the United States, where an estimated 10‐15 million people use the herb for the self‐management of pain and opioid withdrawal. Kratom leaves contain over 40 active alkaloids, with the most important being mitragynine, 7‐hydroxymitragynine and paynantheine. To further explore the possible anti‐inflammatory actions of these kratom alkaloids, we examined their effects on cortisol secretion in a human adrenocortical cell line. HAC15 cells were exposed to vehicle or mitragynine, 7‐hydroxymitragynine, and paynantheine (10 μM). After 24, 48, 72, and 96 h, cortisol secretion was examined. Basal cortisol secretion was significantly increased by mitragynine at all timepoints, with maximal effect at 96 h (in ng/mL: vehicle, 8.9±1.1; 10 μM mitragynine, 47.3±0.7; n=4). Alternatively, 7‐hydroxymitragynine only significantly increased basal cortisol levels at the 96 h timepoint (in ng/mL: vehicle, 8.9±1.1; 10 μM 7‐hydroxymitragynine, 17.4±1.2; n=4), while paynantheine only significantly increased basal cortisol levels at the 24 h timepoint (in ng/mL: vehicle, 23.4±1.0; 10 μM paynantheine, 32.8±2.2; n=4). Additional studies are currently being done to characterize effects on cellular viability and concentration responses for each of these kratom alkaloids on cortisol secretion. These data indicate that mitragynine can enhance cortisol secretion in vitro and suggest that such a mechanism may contribute to the possible analgesic actions of kratom in vivo.

Hypomethylation associated vitamin D receptor expression in ATP1A1 mutant aldosterone-producing adenoma

DNA methylation alteration is tissue-specific and play a pivotal role in regulating gene transcription during cell proliferation and survival. We aimed to detect genes regulated by DNA methylation, and then investigated whether the gene influenced cell proliferation or survival in adrenal cells. DNA methylation and qPCR analyses were performed in nonfunctioning adrenocortical adenoma (NFA, n = 12) and aldosterone-producing adenoma (APA, n = 35) samples. The VDR gene promoter was markedly hypomethylated in APA with ATP1A1 mutation, and the promoter methylation levels showed a significant inverse association with the transcripts in APA. ATP1A1 mutation led to VDR transcription in HAC15 cells, and VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. We demonstrated that APA with ATP1A1 mutation showed entire hypomethylation in the VDR promoter and abundant VDR mRNA and protein expression. VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. Abundant VDR expression would be essential for ATP1A1 mutation-mediated cell proliferation.

Adrenomedullin: new inhibitory regulator for cortisol synthesis and secretion.

Preterm birth is associated with immaturity of several crucial physiological functions notably those prevailing in the lung and kidney. Recently, a steroid secretion deficiency was identified in very preterm neonates, associated with a partial yet transient deficiency in 11β-hydroxylase activity, sustaining cortisol synthesis. However, the P450c11β enzyme is expressed in preterm adrenal glands, we hypothesized an inhibition of cortisol production by adrenomedullin (ADM), a peptide highly produced in neonates and whose effect on steroidogenesis remains poorly known. We studied the effects of ADM on three models: 104 cord-blood samples of the PREMALDO neonate cohort, genetically targeted mice overexpressing ADM, and two human adrenocortical cell lines (H295R and HAC15 cells). Mid-regional-proADM (MR-proADM) quantification in cord-blood samples showed strong negative correlation with gestational age (P = 0.0004), cortisol production (P < 0.0001), and 11β-hydroxylase activity index (P < 0.0001). Mean MR-proADM was higher in very preterm than in term neonates (1.12 vs 0.60 nmol/L, P < 0.0001). ADM-overexpression mice revealed a lower 11β-hydroxylase activity index (P < 0.05). Otherwise, aldosterone levels measured by LC-MS/MS were higher in ADM-overexpression mice (0.83 vs 0.46 ng/mL, P < 0.05). More importantly, the negative relationship between adrenal ADM expression and aldosterone production found in control was lacking in the ADM-overexpression mice. Finally, LC-MS/MS and gene expression studies on H295R and HAC15 cells revealed an ADM-induced inhibition of both cortisol secretion in cell supernatants and CYP11B1 expression. Collectively, our results converge toward an inhibitory effect of ADM on glucocorticoid synthesis in humans and should be considered to explain the steroid secretion deficiency observed at birth in premature newborns.

Characteristics of a Novel ATP2B3 K416_F418delinsN Mutation in a Classical Aldosterone-Producing Adenoma.

Simple SummaryThe ATP2B3 channel mutation is a rare cause of primary aldosteronism (PA). ATP2B3 gene mutation leads to the dysfunction of calcium channel that pumps calcium ion out of the cell and accumulates intracellular calcium signal to stimulate aldosterone synthesis. In the present study, we found a novel somatic ATP2B3 K416_F418delinsN mutation in a PA patient, and proved its functionality by demonstrating aldosterone hyper-function in the mutant-transfected adrenal cell-line. The ATP2B3 K416_F418delinsN mutation resulted from the deletion from nucleotides 1248 to 1253. The translated amino acid sequence from 416 to 418 as lysine-phenylalanine-phenylalanine was deleted and an asparagine was inserted due to the merging of residual nucleotide sequences.In patients with primary aldosteronism (PA), the prevalence of ATP2B3 mutation is rare. The aim of this study is to report a novel ATP2B3 mutation in a PA patient. Based on our tissue bank of aldosterone-producing adenomas (APA), we identified a novel somatic ATP2B3 K416_F418delinsN mutation. The affected individual was a 53 year-old man with a 4 year history of hypertension. Computed tomography (CT) showed bilateral adrenal masses of 1.6 (left) and 0.5 cm (right) in size. An adrenal venous sampling (AVS) showed a lateralization index (LI) of 2.2 and a contralateral suppression index (CLS) of 0.12; indicating left functional predominance. After a left unilateral adrenalectomy, he achieved partial biochemical and hypertension–remission. This classical adenoma harbored a novel ATP2B3 K416_F418delinsN somatic mutation, which is a deletion from nucleotides 1248 to 1253. The translated amino acid sequence from 416 to 418, reading as lysine-phenylalanine-phenylalanine, was deleted; however, an asparagine was inserted due to merging of residual nucleotide sequences. The CYP11B2 immunohistochemistry staining demonstrated strong immunoreactivity in this classical adenoma. The ATP2B3 K416_F418delinsN mutation is a functional mutation in APA, since HAC15 cells, a human adrenal cell line, transfected with the mutant gene showed increased CYP11B2 expression and aldosterone production.

Genotype-specific cortisol production associated with Cushing's syndrome adenoma with PRKACA mutations

The intracellular molecular mechanisms underlying the genotype of cortisol-producing adenoma (CPA) have not been fully determined. We analyzed gene expressions in CPA and the human adrenocortical cell line (HAC15 cells) with PRKACA mutation. Clustering analysis using a gene set associated with responses to cAMP revealed the possible differences between PRKACA mutant CPAs and GNAS and CTNNB1 mutant CPAs. The levels of STAR, CYP11A1, CYP17A1, CYP21A2, and FDX1 transcripts and cortisol levels per unit area in PRKACA mutant CPAs were significantly higher than those in GNAS mutant CPAs. PRKACA mutations led to an increase in steroidogenic enzyme expression and cortisol production in HAC15 cells. Transcriptome analysis revealed differences between PRKACA mutant CPAs and GNAS and CTNNB1 mutant CPAs. Cortisol production in PRKACA mutant CPAs is increased by the cAMP-PKA signaling pathway-mediated upregulation of steroidogenic enzymes transcription. The intracellular molecular mechanisms underlying these processes would be notably important in PRKACA mutant CPAs.