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Cholestane-3β,5α,6β-triol (CT) and its analogues are abundant in natural sources and are reported to demonstrate cytotoxicity toward different kinds of tumor cells without a deep probe into their mechanism of action. CT is also one of the major metabolic oxysterols of cholesterol in mammals and is found to accumulate in various diseases. An extensive exploration of the biological roles of CT over the past few decades has established its identity as an apoptosis inducer. In this study, the effects of CT on A549 cell death were investigated through cell viability assays. RNA-sequencing analysis and western blot of CT-treated A549 cells revealed the role of CT in inducing endoplasmic reticulum (ER) stress response and enhancing autophagy flux, suggesting a putative mechanism of CT-induced cell-death activation involving reactive oxygen species (ROS)-mediated ER stress and autophagy. It is reported for the first time that the upregulation of autophagy induced by CT can serve as a cellular cytotoxicity response in accelerating CT-induced cell death in A549 cells. This research provides evidence for the effect of CT as an oxysterol in cell response to oxidative damage and allows for a deep understanding of cholesterol in its response in an oxidative stress environment that commonly occurs in the progression of various diseases. [ABSTRACT FROM AUTHOR]

Cholestane-3β,5α,6β-triol (CT) and its analogues are abundant in natural sources and are reported to demonstrate cytotoxicity toward different kinds of tumor cells without a deep probe into their mechanism of action. CT is also one of the major metabolic oxysterols of cholesterol in mammals and is found to accumulate in various diseases. An extensive exploration of the biological roles of CT over the past few decades has established its identity as an apoptosis inducer. In this study, the effects of CT on A549 cell death were investigated through cell viability assays. RNA-sequencing analysis and western blot of CT-treated A549 cells revealed the role of CT in inducing endoplasmic reticulum (ER) stress response and enhancing autophagy flux, suggesting a putative mechanism of CT-induced cell-death activation involving reactive oxygen species (ROS)-mediated ER stress and autophagy. It is reported for the first time that the upregulation of autophagy induced by CT can serve as a cellular cytotoxicity response in accelerating CT-induced cell death in A549 cells. This research provides evidence for the effect of CT as an oxysterol in cell response to oxidative damage and allows for a deep understanding of cholesterol in its response in an oxidative stress environment that commonly occurs in the progression of various diseases.

Oncosterone (6-oxo-cholestane-3β,5α-diol; OCDO) is an oncometabolite and a tumor promoter on estrogen receptor alpha-positive breast cancer (ER(+) BC) and triple-negative breast cancers (TN BC). OCDO is an oxysterol formed in three steps from cholesterol: 1) oxygen addition at the double bond to give α- or β- isomers of 5,6-epoxycholestanols (5,6-EC), 2) hydrolyses of the epoxide ring of 5,6-ECs to give cholestane-3β,5α,6β-triol (CT), and 3) oxidation of the C6 hydroxyl of CT to give OCDO. On the other hand, cholesterol can be hydroxylated by CYP27A1 at the ultimate methyl carbon of its side chain to give 27-hydroxycholesterol ((25R)-Cholest-5-ene-3beta,26-diol, 27HC), which is a tumor promoter for ER(+) BC. It is currently unknown whether OCDO and its precursors can be hydroxylated at position C27 by CYP27A1, as is the impact of such modification on the proliferation of ER(+) and TN BC cells. We investigated, herein, whether 27H-5,6-ECs ((25R)-5,6-epoxycholestan-3β,26-diol), 27H-CT ((25R)-cholestane-3β,5α,6β,26-tetrol) and 27H-OCDO ((25R)-cholestane-6-oxo-3β,5α,26-triol) exist as metabolites and can be produced by cells expressing CYP27A1. We report, for the first time, that these compounds exist as metabolites in humans. We give pharmacological and genetic evidence that CYP27A1 is responsible for their production. Importantly, we found that 27-hydroxy-OCDO (27H-OCDO) inhibits BC cell proliferation and blocks OCDO and 27-HC-induced proliferation in BC cells, showing that this metabolic conversion commutes the proliferative properties of OCDO into antiproliferative ones. These data suggest an unprecedented role of CYP27A1 in the control of breast carcinogenesis by inhibiting the tumor promoter activities of oncosterone and 27-HC.

Sickle cell disease (SCD) causes anemia, oxidative stress, chronic inflammation, and lipid abnormalities. Oxysterols are oxidized derivatives of cholesterol and affect cholesterol metabolism and eryptosis. Our aim was to determine whether the plasma concentrations of 7-ketocholesterol (7-KC) and cholestane-3β,5α,6β-triol (C-triol) were associated with hemolysis and lipid profile in patients with SCD. A total of 32 steady-state pediatric patients with SCD (22 HbSS and 10 HbSß+) and 25 healthy controls were included in the study. Hemolysis parameters, ferritin, serum iron, lipids, 7-KC and C-triol concentrations of all subjects were measured. Oxysterols were quantified with N,N-dimethylglycine derivatization via LC-MS/MS. 7-KC and C-triol concentrations were found to be increased in SCD patients, while there was no difference between the HbSS and HbSß+ subgroups. 7-KC concentrations s were correlated negatively with hemoglobin and positively with lactate dehydrogenase concentrations, while C-triol concentrations were negatively correlated with HDL cholesterol. Furthermore, while 7-KC and C-triol concentrations were highly correlated among controls, there was no correlation in patients. The findings of our study suggest that 7-KC and C-triol may have a role in SCD pathophysiology. The lack of correlation in patients' 7-KC and C-triol concentrations suggest alterations in oxysterol production in patients with SCD. [ABSTRACT FROM AUTHOR]

Oncosterone (6-oxo-cholestane-3β,5α-diol; OCDO) is an oncometabolite and a tumor promoter on estrogen receptor alpha-positive breast cancer (ER(+) BC) and triple-negative breast cancers (TN BC). OCDO is an oxysterol formed in three steps from cholesterol: 1) oxygen addition at the double bond to give α- or β- isomers of 5,6-epoxycholestanols (5,6-EC), 2) hydrolyses of the epoxide ring of 5,6-ECs to give cholestane-3β,5α,6β-triol (CT), and 3) oxidation of the C6 hydroxyl of CT to give OCDO. On the other hand, cholesterol can be hydroxylated by CYP27A1 at the ultimate methyl carbon of its side chain to give 27-hydroxycholesterol ((25R)-Cholest-5-ene-3beta,26-diol, 27HC), which is a tumor promoter for ER(+) BC. It is currently unknown whether OCDO and its precursors can be hydroxylated at position C27 by CYP27A1, as is the impact of such modification on the proliferation of ER(+) and TN BC cells. We investigated, herein, whether 27H-5,6-ECs ((25R)-5,6-epoxycholestan-3β,26-diol), 27H-CT ((25R)-cholestane-3β,5α,6β,26-tetrol) and 27H-OCDO ((25R)-cholestane-6-oxo-3β,5α,26-triol) exist as metabolites and can be produced by cells expressing CYP27A1. We report, for the first time, that these compounds exist as metabolites in humans. We give pharmacological and genetic evidence that CYP27A1 is responsible for their production. Importantly, we found that 27-hydroxy-OCDO (27H-OCDO) inhibits BC cell proliferation and blocks OCDO and 27-HC-induced proliferation in BC cells, showing that this metabolic conversion commutes the proliferative properties of OCDO into antiproliferative ones. These data suggest an unprecedented role of CYP27A1 in the control of breast carcinogenesis by inhibiting the tumor promoter activities of oncosterone and 27-HC., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Niemann-Pick type C (NPC) is a neurological disease caused by an intracellular cholesterol accumulation. Cholesterol oxidation product cholestane-3β,5α,6β-triol (C-triol) serves as diagnostic biomarker for NPC, but its measurement in the routine laboratory remains difficult. We developed an isotope dilution gas chromatography–mass spectrometry (GC–MS) method permitting screening for NPC in plasma. 1440 plasma samples obtained from clinically suspicious patients were subjected to alkaline saponification. C-triol was extracted with carbon tetrachloride, transformed into the trimethylsilylethers, separated on a fused silica capillary column with a nonpolar silicone stationary phase, and analyzed by GC–MS. NPC diagnosis was confirmed by DNA sequencing. The method was linear over a concentration range of 0.03–200 ng/mL with a mean recovery rate of 98.6%. The intra- and inter-day variation coefficients assessed at two concentrations were below 15%. Limits of quantification (LOQ) and detection (LOD) were 0.03 ng/mL and 0.01 ng/mL, respectively. Receiver operating characteristic (ROC) analysis estimated that the area under curve was 0.997 implying a significant discriminatory power to identify subjects with NPC. Nevertheless, 13 NPC patients and 29 control subjects confirmed by sequencing showed false negative or positive results, respectively. Two patients with cerebrotendinous xanthomatosis showed a 5–10-fold increase in C-triol levels. We developed a quick and sensitive GC–MS method for determination of C-triol, which may serve as a simple and inexpensive diagnostic tool aiding NPC diagnosis in a routine hospital laboratory. As C-triol elevation is not limited to NPC, the NPC diagnosis has to be confirmed by DNA sequencing. [ABSTRACT FROM AUTHOR]

Background Niemann–Pick type C (NPC) is a treatable genetic disorder, mainly characterized by neurological dysfunction and liver damage. Its diagnosis is based on an invasive test which requires a skin biopsy to demonstrate the cholesterol accumulation in culture fibroblasts of affected patients. In the last years, new biomarkers have been investigated aiming to facilitate the diagnosis and screening of NPC; two of these possible candidates are the oxysterol cholestane-3β,5α,6β-triol (triol), a product of non-enzymatic oxidation of cholesterol, and the enzyme chitotriosidase (CT), a fully active chitinase (EC-3.2.1.14) synthesized by activated macrophages. Methods In this work, we investigated the potential use of the combined analysis of triol levels and CT activity for diagnosis, screening and monitoring of NPC in Brazilian patients, correlating with the results of Filipin staining and genetic analysis. We studied 122 untreated individuals with clinical suspicion of NPC who were separated in two groups according their concentrations of triol (higher or lower than the cutoff value of 100 ng/mL). We also analyzed blood samples from 5 patients with previous diagnosis of NPC who were under treatment with miglustat. Results The results of this work demonstrated that patients with higher concentrations of triol (group A) also presented a high activity of CT and most of them had also a positive Filipin test. Two patients of this group presented an inconclusive Filipin test, being one eventually diagnosed as NPC by molecular investigation and the other eventually diagnosed as Niemann–Pick type A or B (NPA/B) by the low acid sphingomyelinase activity presented. Three patients with high triol concentrations who had a negative result in the Filipin test presented low activities of acid sphingomyelinase, being diagnosed as NPA/B. On the other hand, triol concentrations were normal in NPC patients treated with miglustat, although CT activity in these individuals remained abnormal. In the patients with triol lower than 100 ng/mL (group B), most presented a normal activity of CT. No patient of this group had a positive Filipin test and the few patients with inconclusive Filipin test did not present pathogenic mutations in the NPC1 or NPC2 genes. Conclusions In conclusion, our data demonstrated that the combined analysis of triol and CT is quite sensitive and specific for the identification of NPC patients. Although the number of analysis in NPC patients treated with miglustat was small, the data indicate that the measurement of triol could also be potentially useful for treatment monitoring. [ABSTRACT FROM AUTHOR]

Abstract: Reactive oxygen species (ROS) have been implicated in oxysterol-induced apoptosis. However, the mechanism of ROS production induced by oxysterols within cells is not clear. Considering that mitochondria is the main source of intracellular ROS, and play a key role in oxysterol-induced apoptosis, we investigated the effect of oxysterol cholestane-3β,5α,6β-triol (Triol) on ROS production and mitochondrial function in isolated mice liver mitochondria. Triol at higher concentrations (10–50μM) enhanced the production of O2 − and H2O2 in isolated mitochondria, which might be due to its stimulation to the activities of complexes I and II of mitochondrial electron transfer chain, and its inhibition to glutathione peroxidase activity. The same concentrations of Triol induced obviously oxidative damage of mitochondrial membrane lipids and proteins, as demonstrated by the increased MDA level and the decreased protein thiols content. Furthermore, Triol caused mitochondrial dysfunction, including the opening of mitochondrial permeability transition pore, the decrease of mitochondrial membrane potential (ΔΨ m), and the release of cytochrome c. Antioxidant butylated hydroxytoluene significantly inhibited oxidative damage, the decrease of ΔΨ m, and the release of cytochrome c, implying that ROS might mediate mitochondrial dysfunction induced by Triol. We concluded that Triol-induced mitochondrial ROS production and subsequently oxidative damage, leading to the mitochondrial dysfunction, thus suggesting a putative mechanism of apoptosis activation by oxysterols in vascular cells. [Copyright &y& Elsevier]

To evaluate the cytoprotection mechanism of selenium against cholestane-3β,5α,6β-triol (3-triol)-induced vascular smooth muscle cells (VSMCs) damage, cell viability was analyzed by 3-(4,5-dimethylthiazol-2 -yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell count, the percentage release of lactate dehydrogenase (LDH) from the cell was assessed, and apoptosis was detected by DNA laddering and flow cytometric analysis. Meanwhile, the activity of glutathione peroxidase (GPx) of VSMCs was measured. The results showed that 3-triol could inhibit proliferation of VSMCs time-dependently and dose-dependently, increase the percentage release of LDH and induce VSMCs apoptosis. While the cytotoxicity and cells apoptosis induced by 3-triol was attenuated by pretreatment of cells with low concentration of sodium selenite, and the longer the pretreated time was, the stronger the inhibition was. Preincubation of cells with sodium selenite (50 nM) for 12 or 24 h before 1, 5, 10, 25, or 50 μM 3-triol exposure, the cell viabilities increased 28.5% (P<0.05), 18.3%, 197.6% (P<0.01), 66.7%, 50.0% or 35.1% (P<0.05), 62.3% (P<0.05), 329.6% (P<0.01), 221.3% (P<0.05), 74.0% compared with the control cells, respectively. When the cells were preincubated with sodium selenite (50 nM) for 12 or 24 h before exposure to 3-triol (10 μM), the percent of apoptotic cells reduced from 30.47±15.34% to 26.88±17.32% or 7.41±5.46% (P<0.05). With preincubation of sodium selenite (50 nM) for 24 h, the GPx activity of VSMCs increased 18.5% compared with control (P<0.05). In conclusion, the results suggested that incubated VSMCs could absorb and transfer selenite as selenoprotrein, such as GPx, if the time is long enough and VSMCs selenoproteins can protect markedly against apoptosis and damage induced by 3-triol in VSMCs. [Copyright &y& Elsevier]

Suspension cultures of isolated rat hepatocytes were used to investigate whether 7-ketocholesterol and cholestane-3β,5α,6β-triol exert oxidative stress in cells as manifested by increased lipid peroxidation and the induction of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase. The oxysterols were found to increase the levels of both superoxide dismutase and catalase and to have variable effects on glutathione peroxidase activity. Increased lipid peroxidation was not observed, indicating that the endogenous antioxidant defense system was capable of protecting against any oxidative stress that might otherwise by exerted by 7-ketocholesterol or cholestane-3β,5α,6β-triol. Covi-ox, a natural tocopherol blend reduced the effects of both oxysterols on the antioxidant enzymes. A concurrent reduction in the production of thiobarbituric acid-reactive substances in Covi-ox-treated cells is indirect evidence that reactive oxygen species were produced by oxysterols in hepatocyte suspension cultures. [ABSTRACT FROM AUTHOR]

• Oxygenated cholesterol metabolites (oxysterols) can contribute to cancer progression. • Circulating levels of 7 oxysterols were assessed in luminal breast carcinoma patients. • High cholestane-3β,5α,6β-triol levels were associated with poor disease-free survival. • These observations provide new insight into the role of oxysterols in breast cancer. Oxygenated metabolites of cholesterol (oxysterols) have been previously demonstrated to contribute to progression of various cancers and to modulate resistance to breast cancer endocrine therapy in vitro. We measured prognostic roles of circulating levels of seven major oxysterols in the progression of luminal subtype breast carcinoma. Liquid chromatography coupled with tandem mass spectrometry was used for determination of levels of non-esterified 25-hydroxycholesterol, 27-hydroxycholesterol, 7 α -hydroxycholesterol, 7-ketocholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide, and cholestane-3β,5α,6β-triol in plasma samples collected from patients (n = 58) before surgical removal of tumors. Oxysterol levels were then associated with clinical data of patients. All oxysterols except cholesterol-5α,6α-epoxide were detected in patient plasma samples. Circulating levels of 7 α -hydroxycholesterol and 27-hydroxycholesterol were significantly lower in patients with small tumors (pT1) and cholesterol-5β,6β-epoxide and cholestane-3β,5α,6β-triol were lower in patients with stage IA disease compared to larger tumors or more advanced stages. Patients with higher than median cholestane-3β,5α,6β-triol levels had significantly worse disease-free survival than patients with lower levels (p = 0.037 for all patients and p = 0.015 for subgroup treated only with tamoxifen). In conclusion, this study shows, for the first time, that circulating levels of oxysterols, especially cholestane-3β,5α,6β-triol, may have prognostic roles in patients with luminal subtype breast cancer. [ABSTRACT FROM AUTHOR]

The formation of 5α,6α- and 5β,6β-epoxides of cholesterol and β-sitosterol in rat liver subcellular fractions has been studied. The results show that the epoxidation seems to occur only in connection with the nonspecific tissue oxidation of the sterols. The β-epoxides were formed in three- to fourfold excess over the α-epoxides. Both cholesterol epoxides were efficiently converted by a microsomal hydrolase into the 3β,5α,6β-triol. The conversion was less extensive with β-sitosterol epoxides, especially the β-epoxide. The possible biological significance in the formation of the sterol epoxides and the triols was evaluated by their ability to inhibit the microsomal cholesterol 7α-hydroxylase. Only the cholesterol epoxides and especially the β-epoxide were active in this respect.

Background: A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque., Method: In vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80 μl plasma. For preparation of 10mg atherosclerotic plaques an additional liquid-liquid extraction was included. Free 7-keto-, 7-α/ß-hydroxy-, 5,6-α-epoxy-, 5,6-β-epoxycholesterol, cholestane-3ß,5α,6ß-triol and cholesterol were separated within 7 min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization., Results: The detection limit was 0.1 ng/ml and the linearity ranged from 0.5 to 0.75 to 2000 ng/ml for the oxysterols and from 50 to 1000 μg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47 ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n=5)., Conclusion: This rapid LC-MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions., (© 2013. Published by Elsevier B.V. All rights reserved.)