Search

Adrenocortical carcinoma (ACC) is a rare aggressive malignancy with a poor outcome largely due to limited treatment options. Here, we propose a novel therapeutic approach through modulating intracellular free cholesterol via the liver X receptor alpha (LXRα) in combination with current first line pharmacotherapy, mitotane. H295R and MUC-1 ACC cell lines were pretreated with LXRα inhibitors in combination with mitotane. In H295R, mitotane (20, 40, 50µM) induced dose-dependent cell death, however, in MUC-1 this only occurred at a supratherapeutic concentration (200µM). LXRα inhibition potentiated mitotane-induced cytotoxicity in both cell lines. This was confirmed through use of the CompuSyn model which showed moderate pharmacological synergism and was indicative of apoptotic cell death via an increase in annexinV and cleaved-caspase 3 expression. Inhibition of LXRα was confirmed through downregulation of cholesterol efflux pumps ABCA1 and ABCG1, however, combination treatment with mitotane attenuated this effect. Intracellular free cholesterol levels were associated with increased cytotoxicity in H295R (r2=0.5210) and MUC-1 (r2=0.9299) cells. While both cell lines exhibited similar levels of free cholesterol at baseline, H295R were cholesterol ester rich whereas MUC-1 were cholesterol ester poor. We highlight the importance of LXRα mediated cholesterol metabolism in the management of ACC, drawing attention to its role in the therapeutics of mitotane sensitive tumours. We also demonstrate significant differences in cholesterol storage between mitotane sensitive and resistant disease.

Introduction: Adrenocortical Carcinoma (ACC) is a rare aggressive cancer which carries a poor prognosis. Adjuvant mitotane improves survival but is limited by poor response rates and resistance following tumour recurrence. Mitotane’s efficacy has been attributed to intracellular accumulation of toxic free cholesterol (FC) predominantly through inhibition of cholesterol storage through SOAT1. Yet SOAT1 specific inhibitors demonstrate inferior efficacy to mitotane in inducing ACC cell death. We hypothesize that mitotane’s efficacy to induce toxic FC accumulation in ACC cells is also mediated through enhanced breakdown of stored cholesterol within intracellular lipid droplets (LDs). Methodology: ATCC-H295R (mitotane sensitive) and MUC-1 (mitotane resistant) ACC cells were evaluated for neutral lipid content using BODIPY493/503 under baseline and cholesterol loaded conditions using Amnis ImageStream, additionally cells were treated with mitotane (H295R - 20, 40, 50µM; MUC1 - 50, 100, 200µM) for 6hr. Analysis of LDs using CE-BODIPY and FA-BODIPY identified cholesterol ester (CE) and triacylglycerol (TAG)-containing LDs, respectively. Lipid droplet-associated proteins (LDAPs) Perilipin (PLIN) 1–4 and hormone sensitive lipase (HSL) were evaluated using western blotting and PCR. Lipid uptake receptors; SRB1, LDLR, LRP1 and CD36 were measured by flow cytometry. Results: Mitotane treatment, within therapeutic range, decreased staining for LDs significantly in H295R. This was also reflected by decreased expression of LDAPs, PLIN1 and PLIN3. The decrease in H295R LDs was associated with increased activation of HSL (pHSL and LIPE). However, this effect was only evident in MUC-1 at supratherapeutic mitotane (200µM). H295R and MUC-1 demonstrated similar overall LD numbers at baseline and under cholesterol supplementation. Expression of PLIN3 was high in both cell lines, while PLIN1, PLIN2 and PLIN4 demonstrated distinct LD profiles in each. Investigation of LD content showed that H295R preferentially store CEs while MUC-1 store only TAG, irrespective of cholesterol-loading. Mitotane treatment significantly reduces both CE and TAG LDs in H295R and MUC-1. Expression of lipid uptake receptors also demonstrated significant variability between cell lines including SRB1 and LRP1. Conclusion: We highlight that lipolysis through LD breakdown and activation of HSL represents a putative additional mechanism for mitotane induced FC cytotoxicity in ACC. We also demonstrate significant differences in cholesterol handling and LDAPs between mitotane sensitive and mitotane resistant models, in particular, the absence of CE LDs in MUC-1. We therefore propose a mechanism of resistance to mitotane through absent CE storage. Further understanding of cholesterol and lipid handling in ACC offers novel therapeutic exploitation, especially in the setting of mitotane resistant disease.

Human monocytes are classified as classical, intermediate and non-classical according to their surface expression of CD14 and CD16. They also represent precursors to atherogenic foam cells and macrophages in metabolically adverse crown-like structures. Monocyte distribution, phenotype and function were investigated in obese individuals with and without type 2 diabetes in relation to clinical parameters of metabolic and cardiovascular risk. Peripheral monocytes obtained from obese and non-obese subjects were enumerated and their phenotype investigated using flow cytometry and q-RT-PCR. Multivariate analyses were performed against validated clinical markers of cardiovascular and metabolic risk. When compared to their non-obese counterparts, obese individuals demonstrated decreased total and classical monocyte numbers with proportionately increased intermediate monocytes and unchanged non-classical monocytes. Surface staining of intermediate monocytes for HLA-DR identified two distinct subpopulations with mid (DR mid) and high (DR hi) level expression respectively. Expansion of the intermediate subset of monocytes in obese individuals occurred specifically within the DRmid subpopulation and this correlated with key indices of lipid metabolism and glycemia. DRmid intermediate monocytes displayed higher surface expression of pro-inflammatory and lipid receptors and higher uptake of acetylated-LDL. A distinct subpopulation of intermediate monocytes sharing phenotypic features with atherogenic foam cells and macrophages in crown-like structures is expanded in the blood of obese individuals with and without type 2 diabetes. Direct interactions between modified lipoproteins and circulating monocyte subpopulations may represent an early pathogenic driver of obesity-associated atherosclerosis and adipose tissue inflammation.

Introduction Adrenocortical carcinoma (ACC) is a rare aggressive cancer with low overall survival. Adjuvant mitotane improves survival but is limited by poor response rates and resistance. Mitotane’s efficacy is attributed to the accumulation of toxic free cholesterol, predominantly through cholesterol storage inhibition. However, targeting this pathway has proven unsuccessful. We hypothesize that mitotane-induced free-cholesterol accumulation is also mediated through enhanced breakdown of lipid droplets. Methodology ATCC-H295R (mitotane-sensitive) and MUC-1 (mitotane-resistant) ACC cells were evaluated for lipid content using specific BODIPY dyes. Protein expression was evaluated by immunoblotting and flow cytometry. Cell viability was measured by quantifying propidium iodide-positive cells following mitotane treatment and pharmacological inhibitors of lipolysis. Results H295R and MUC-1 cells demonstrated similar neutral lipid droplet numbers at baseline. However, evaluation of lipid machinery demonstrated distinct profiles in each model. Analysis of intracellular lipid droplet content showed H295R cells preferentially store cholesteryl esters, whereas MUC-1 cells store triacylglycerol. Decreased lipid droplets were associated with increased lipolysis in H295R and in MUC-1 at toxic mitotane concentrations. Pharmacological inhibition of lipolysis attenuated mitotane-induced toxicity in both models. Conclusion We highlight that lipid droplet breakdown and activation of lipolysis represent a putative additional mechanism for mitotane-induced cytotoxicity in ACC. Further understanding of cholesterol and lipids in ACC offers potential novel therapeutic exploitation, especially in mitotane-resistant disease.

Primary aldosteronism (PA) accounts for 5–12% of all hypertension and confers a higher risk for cardiovascular and cerebrovascular complications compared to age and blood pressure (BP) matched essential hypertension. PA is caused by autonomous aldosterone secretion from unilateral (40%) or bilateral (60%) sources. The current definitive management for unilateral PA is surgical laparoscopic adrenalectomy. Bilateral disease is not currently definitively managed but rather treated pharmacologically using mineralocorticoid receptor antagonists.. Currently, the option of surgery is limited to 30% of individuals with unilateral disease due to the collateral resection of normal cortex. Adrenalectomy is very rarely considered for those with bilateral disease. Therefore, there exists a need for improved definitive options for management of bilateral and unilateral PA which selectively removes or disrupts the diseased tissue causing aldosterone excess, while also preserving normal adrenocortical tissue. Proposed herein, is a novel image-guided adrenal thermal therapy approach to definitively manage PA. The hyperthermia dosimetry that induces cell death in both H295R and HAC15 adrenocortical cancer cell lines was assessed to inform ablation therapy applications. In both cell lines cell death via necrosis occurred following 500C exposure for 15 and 30 minutes. In those cells exposed to sublethal exposures of hyperthermia a heat shock response was induced, indicated by increased protein expression of HSP90 and HSP70. The effects of sublethal hyperthermia at 420C and 450C on steroidogenesis was next investigated in H295R and HAC15 cell lines. This study assessed which steroidogenic pathways and enzymes are most sensitive to hyperthermia. Hyperthermia of 450C exposure for 30 minutes was found to decrease CYP17A1 and HSD3B2 protein expression in both cell lines. While under the same conditions StaR was decreased in H295Rs and increased in HAC15 cells. CYP11B2 remained stable under all treatments. Sublethal hyperthermia was found to significantly inhibit cortisol output but aldosterone output remained intact. Finally, an in-vivo swine model was used to investigate the feasibility and safety of subtotal adrenal ablation. From this it was found that subtotal ablation of the adrenal could be achieved whereby the selectively ablated zone was disrupted while the nontargeted tissue remained unaffected. The only safety concern raised was due to transient elevated blood pressure during the procedure caused by medullary degranulation and the release of catecholamines. Ultimately, these data identify a novel strategy to provide definitive adrenocortical sparing treatment to patients with primary aldosteronism, and additionally provides a molecular understanding as to how this is achieved. 2025-10-19

Adrenocortical carcinoma (ACC) is a rare aggressive malignancy with a poor outcome largely due to limited treatment options. Here, we propose a novel therapeutic approach through modulating intracellular free cholesterol via the liver X receptor alpha (LXRα) in combination with current first-line pharmacotherapy, mitotane. H295R and MUC-1 ACC cell lines were pretreated with LXRα inhibitors in combination with mitotane. In H295R, mitotane (20, 40 and 50 µM) induced dose-dependent cell death; however, in MUC-1, this only occurred at a supratherapeutic concentration (200 µM). LXRα inhibition potentiated mitotane-induced cytotoxicity in both cell lines. This was confirmed through use of the CompuSyn model which showed moderate pharmacological synergism and was indicative of apoptotic cell death via an increase in annexinV and cleaved-caspase 3 expression. Inhibition of LXRα was confirmed through downregulation of cholesterol efflux pumps ABCA1 and ABCG1; however, combination treatment with mitotane attenuated this effect. Intracellular free-cholesterol levels were associated with increased cytotoxicity in H295R (r2 = 0.5210) and MUC-1 (r2 = 0.9299) cells. While both cell lines exhibited similar levels of free cholesterol at baseline, H295R were cholesterol ester rich, whereas MUC-1 were cholesterol ester poor. We highlight the importance of LXRα mediated cholesterol metabolism in the management of ACC, drawing attention to its role in the therapeutics of mitotane sensitive tumours. We also demonstrate significant differences in cholesterol storage between mitotane sensitive and resistant disease.

Introduction: Aldosterone-producing adenoma (APA) is the most common cause of endocrine-related hypertension but surgery is not always feasible. Current medical interventions are associated with significant side effects and poor patient compliance. New APA animal models that replicate basic characteristics of APA and give physical and biochemical feedback are needed to test new non-surgical treatment methods, such as image-guided thermal ablation., Methods: A model of APA was developed in nude mice using HAC15 cells, a human adrenal carcinoma cell line. Tumor growth, aldosterone production, and sensitivity to angiotensin II were characterized in the model. The utility of the model was validated via treatment with microwave ablation and characterization of the resulting physical and biochemical changes in the tumor., Results: The APA model showed rapid and relatively homogeneous growth. The tumors produced aldosterone and steroid precursors in response to angiotensin II challenge, and plasma aldosterone levels were significantly higher in tumor bearing mice two hours after challenge verses non-tumor bearing mice. The model was useful for testing microwave ablation therapy, reducing aldosterone production by 80% in treated mice., Conclusion: The HAC15 model is a useful tumor model to study and develop localized treatment methods for APA.