Organization of Carotenoid Aggregates in Membranes Studied Selectively using Resonance Raman Optical Activity.

In living organisms, carotenoids are incorporated in biomembranes, remarkably modulating their mechanical characteristics, fluidity, and permeability. Significant resonance enhancement of Raman optical activity (ROA) signals of carotenoid chiral aggregates makes resonance ROA (RROA), a highly selective tool to study exclusively carotenoid assemblies in model membranes. Hence, RROA is combined with electronic circular dichroism (ECD), dynamic light scattering (DLS), molecular dynamics, and quantum-chemical calculations to shed new light on the carotenoid aggregation in dipalmitoylphosphatidylcholine (DPPC) liposomes. Using representative members of the carotenoid family: apolar α-carotene and more polar fucoxanthin and zeaxanthin, the authors demonstrate that the stability of carotenoid aggregates is directly linked with their orientation in membranes and the monomer structures inside the assemblies. In particular, polyene chain distortion of α-carotene molecules is an important feature of J-aggregates that show increased orientational freedom and stability inside liposomes compared to H-assemblies of more polar xanthophylls. In light of these results, RROA emerges as a new tool to study active compounds and drugs embedded in membranes.

Does the pancreas of gekkotans differentiate similarly? Developmental structural and 3D studies of the mourning gecko (Lepidodactylus lugubris) and the leopard gecko (Eublepharis macularius).

This study investigated the pancreas differentiation of two species of gekkotan families-the mourning gecko Lepidodactylus lugubris (Gekkonidae) and the leopard gecko Eublepharis macularius (Eublepharidae)-based on two-dimensional (2D) histological samples and three-dimensional (3D) reconstructions of the position of the pancreatic buds and the surrounding organs. The results showed that at the moment of egg laying, the pancreas of L. lugubris is composed of three distinct primordia: one dorsal and two ventral. The dorsal primordium differentiates earlier than either ventral primordium. The right ventral primordium is more prominent and distinctive, starting to form earlier than the left one. Moreover, at this time, the pancreas of the leopard gecko is composed of the dorsal and right ventral primordium and the duct of the left ventral primordium. It means that the leopard gecko's left primordium is a transitional structure. These results indicate that the early development of the gekkotan pancreas is species specific. The pancreatic buds of the leopard and mourning gecko initially enter the duodenum by separate outlets, similar to the pancreas of other vertebrates. The pancreatic buds (3 of the mourning gecko and 2 of the leopard gecko) fuse quickly and form an embryonic pancreas. After that, the structure of this organ changes. After fusion, the pancreas of both gekkotans comprises four parts: the head of the pancreas (central region) and three lobes: upper, splenic, and lower. This organ develops gradually and is very well distinguished at hatching time. In both gekkotan species, cystic, hepatic, and pancreatic ducts enter the duodenum within the papilla. During gekkotan pancreas differentiation, the connection between the common bile duct and the dorsal pancreatic duct is associated with intestinal rotation, similar to other vertebrates.

Quantitative Analysis of Isopimpinellin from Ammi majus L. Fruits and Evaluation of Its Biological Effect on Selected Human Tumor Cells.

Ammi majus L. (Apiaceae) is a medicinal plant with a well-documented history in phytotherapy. The aim of the present work was to isolate isopimpinellin (5,8-methoxypsoralen; IsoP) from the fruit of this plant and evaluate its biological activity against selected tumor cell lines. The methanol extract obtained with the use of an accelerated solvent extraction (ASE) method was the most suitable for the quantitative analysis of coumarins in the A. majus fruit matrix. The coumarin content was estimated by RP-HPLC/DAD, and the amount of IsoP was found to be 404.14 mg/100 g dry wt., constituting 24.56% of the total coumarin fraction (1.65 g/100 g). This, along with the presence of xanthotoxin (368.04 mg/100 g, 22.36%) and bergapten (253.05 mg/100 g, 15.38%), confirmed A. majus fruits as an excellent source of these compounds. IsoP was isolated (99.8% purity) by combined liquid chromatography/centrifugal partition chromatography (LC/CPC) and tested for the first time on its antiproliferative activity against human colorectal adenocarcinoma (HT29, SW620), osteosarcoma (Saos-2, HOS), and multiple myeloma (RPMI8226, U266) cell lines. MTT assay results (96 h incubation) demonstrated a dose- and cell line-dependent decrease in cell proliferation/viability, with the strongest effect of IsoP against the Saos-2 cell line (IC50; 42.59 µM), medium effect against U266, HT-29, and RPMI8226 (IC50 = 84.14, 95.53, and 105.0 µM, respectively), and very weak activity against invasive HOS (IC50; 321.6 µM) and SW620 (IC50; 711.30 µM) cells, as well as normal human skin fibroblasts (HSFs), with IC50; 410.7 µM. The mechanistic study on the Saos-2 cell line showed that IsoP was able to reduce DNA synthesis and trigger apoptosis via caspase-3 activation. In general, IsoP was found to have more potency towards cancerous cells (except for HOS and SW620) than against healthy cells. The Selective Index (SI) was determined, underlining the higher selectivity of IsoP towards cancer cells compared to healthy cells (SI = 9.62 against Saos-2). All these results suggest that IsoP might be a promising molecule in the chemo-prevention and treatment of primary osteosarcoma.

Thalidomide derivatives as nanomolar human neutrophil elastase inhibitors: Rational design, synthesis, antiproliferative activity and mechanism of action.

Here, we rationally designed a human neutrophil elastase (HNE) inhibitors 4a-4f derived from thalidomide. The HNE inhibition assay showed that synthesized compounds 4a, 4b, 4e and 4f demonstrated strong HNE inhibiton properties with IC50 values of 21.78-42.30 nM. Compounds 4a, 4c, 4d and 4f showed a competitive mode of action. The most potent compound 4f shows almost the same HNE inhibition as sivelestat. The molecular docking analysis revealed that the strongest interactions occur between the azetidine-2,4-dione group and the following three aminoacids: Ser195, Arg217 and His57. A high correlation between the binding energies and the experimentally determined IC50 values was also demonstrated. The study of antiproliferative activity against human T47D (breast carcinoma), RPMI 8226 (multiple myeloma), and A549 (non-small-cell lung carcinoma) revealed that designed compounds were more active compared to thalidomide, pomalidomide and lenalidomide used as the standard drugs. Additionally, the most active compound 4f derived from lenalidomide induces cell cycle arrest at the G2/M phase and apoptosis in T47D cells.

Pseudomonas aeruginosa exotoxin A induces apoptosis in Galleria mellonella hemocytes.

The cellular immune response of the greater wax moth Galleria mellonella to Pseudomonas aeruginosa exotoxin A was investigated for the first time. The insects were challenged with a sublethal dose of exoA, and then hemocyte parameters were assessed. The analysis showed a statistically significant decrease in the total hemocyte count (THC), which was associated with significant decreases in the number of granulocytes and plasmatocytes. In turn, no statistically significant changes were observed in the number of spherulocytes and oenocytoides. Fluorescent staining indicated that cells collected from the exoA-challenged larvae exhibited features characteristic for apoptotic and autophagic cell death, e.g. cytoplasm vacuolization and chromatin condensation. The flow cytometry analysis revealed a significant increase in the number of phosphatidylserine- and active caspase 3-positive hemocytes challenged with exoA, which proved apoptosis induction. Our results will help in understanding the role of exotoxin A during P. aeruginosa infections not only in insects but also in mammals, including humans.

Evaluation of the Biological Effect of Non-UV-Activated Bergapten on Selected Human Tumor Cells and the Insight into the Molecular Mechanism of Its Action.

There is some evidence that non-photoactivated psoralens may be active against breast and colon tumor cells. Therefore, we evaluated the antiproliferative, proapoptotic, and anti-migrative effect of 5-methoxypsoralen (5-MOP) isolated from Peucedanum tauricum MB fruits in human colorectal adenocarcinoma (HT-29 and SW620), osteosarcoma (Saos-2 and HOS), and multiple myeloma (RPMI8226 and U266). Dose- and cell-line-dependent effects of 5-MOP on viability and proliferation were observed, with the strongest inhibitory effect against Saos-2 and a moderate effect against the HOS, HT-29, and SW620 cells. Multiple myeloma showed low sensitivity. The high viability of human normal cell cultures (HSF and hFOB) in a wide range of 5-MOP concentrations tested (6.25-100 µM) was confirmed. Moreover, the migration of treated Saos-2, SW620, and HT-29 cell lines was impaired, as indicated via a wound healing assay. Flow cytometry analysis conducted on Saos-2 cells revealed the ability of 5-MOP to block the cell cycle in the G2 phase and trigger apoptosis, which was accompanied by a loss of mitochondrial membrane potential, caspases (-9 and -3) activation, the altered expression of the Bax and Bcl-2 proteins, and decreased AKT phosphorylation. This is the first report evaluating the antiproliferative and antimigratory impact of non-UV-activated bergapten on the abovementioned (except for HT-29) tumor cells, which provides new data on the potential role of 5-MOP in inhibiting the growth of various types of therapeutic-resistant cancers.

Time-Dependent Hydrogen Bond Network Formation in Glycerol-Based Deep Eutectic Solvents.

The front cover artwork is provided by Patryk Palenque Marcinkowski. The image shows a glycerol-choline network accommodating water molecules over time. The glycerol molecules are represented as pandas adapting to the change. Read the full text of the Research Article at 10.1002/cphc.202100806.

Simulations of electric field gradient fluctuations and dynamics around sodium ions in ionic liquids.

The T1 relaxation time measured in nuclear magnetic resonance experiments contains information about electric field gradient (EFG) fluctuations around a nucleus, but computer simulations are typically required to interpret the underlying dynamics. This study uses classical molecular dynamics (MD) simulations and quantum chemical calculations, to investigate EFG fluctuations around a Na+ ion dissolved in the ionic liquid 1-ethyl 3-methylimidazolium tetrafluoroborate, [Im21][BF4], to provide a framework for future interpretation of NMR experiments. Our calculations demonstrate that the Sternheimer approximation holds for Na+ in [Im21][BF4], and the anti-shielding coefficient is comparable to its value in water. EFG correlation functions, CEFG(t), calculated using quantum mechanical methods or from force field charges are roughly equivalent after 200 fs, supporting the use of classical MD for estimating T1 times of monatomic ions in this ionic liquid. The EFG dynamics are strongly bi-modal, with 75%-90% of the de-correlation attributable to inertial solvent motion and the remainder to a highly distributed diffusional processes. Integral relaxation times, ⟨τEFG⟩, were found to deviate from hydrodynamic predictions and were non-linearly coupled to solvent viscosity. Further investigation showed that Na+ is solvated by four tetrahedrally arranged [BF4]- anions and directly coordinated by ∼6 fluorine atoms. Exchange of [BF4]- anions is rare on the 25-50 ns timescale and suggests that motion of solvent-shell [BF4]- is the primary mechanism for the EFG fluctuations. Different couplings of [BF4]- translational and rotational diffusion to viscosity are shown to be the source of the non-hydrodynamic scaling of ⟨τEFG⟩.

Discovery of New 3,3-Diethylazetidine-2,4-dione Based Thiazoles as Nanomolar Human Neutrophil Elastase Inhibitors with Broad-Spectrum Antiproliferative Activity.

A series of 3,3-diethylazetidine-2,4-dione based thiazoles 3a-3j were designed and synthesized as new human neutrophil elastase (HNE) inhibitors in nanomolar range. The representative compounds 3c, 3e, and 3h exhibit high HNE inhibitory activity with IC50 values of 35.02-44.59 nM, with mixed mechanism of action. Additionally, the most active compounds 3c and 3e demonstrate high stability under physiological conditions. The molecular docking study showed good correlation of the binding energies with the IC50 values, suggesting that the inhibition properties are largely dependent on the stage of ligand alignment in the binding cavity. The inhibition properties are correlated with the energy level of substrates of the reaction of ligand with Ser195. Moreover, most compounds showed high and broad-spectrum antiproliferative activity against human leukemia (MV4-11), human lung carcinoma (A549), human breast adenocarcinoma (MDA-MB-231), and urinary bladder carcinoma (UMUC-3), with IC50 values of 4.59-9.86 µM. Additionally, compounds 3c and 3e can induce cell cycle arrest at the G2/M phase and apoptosis via caspase-3 activation, leading to inhibition of A549 cell proliferation. These findings suggest that these new types of drugs could be used to treat cancer and other diseases in which immunoreactive HNE is produced.

New Borane-Protected Derivatives of α-Aminophosphonous Acid as Anti-Osteosarcoma Agents: ADME Analysis and Molecular Modeling, In Vitro Studies on Anti-Cancer Activities, and NEP Inhibition as a Possible Mechanism of Anti-Proliferative Activity.

Many organophosphorus compounds (OPs), especially various α-aminophosphonates, exhibit anti-cancer activities. They act, among others, as inhibitors of the proteases implicated in cancerogenesis. Thesetypes of inhibitors weredescribed, e.g., for neutral endopeptidase (NEP) expressed in different cancer cells, including osteosarcoma (OS). The aim of the present study isto evaluate new borane-protected derivatives of phosphonous acid (compounds 1-7) in terms of their drug-likeness properties, anti-osteosarcoma activities in vitro (against HOS and Saos-2 cells), and use as potential NEP inhibitors. The results revealed that all tested compounds exhibited the physicochemical and ADME properties typical for small-molecule drugs. However, compound 4 did not show capability of blood-brain barrier penetration (Lipinski and Veber rules;SwissAdme tool). Moreover, the α-aminophosphonite-boranes (compounds 4-7) exhibited stronger anti-proliferative activity against OS cells than the other phosphonous acid-borane derivatives (compounds 1-3),especially regarding HOS cells (MTT assay). The most promising compounds 4 and 6 induced apoptosis through the activation of caspase 3 and/or cell cycle arrest at the G2 phase (flow cytometry). Compound 4 inhibited the migration and invasiveness of highly aggressive HOS cells (wound/transwell and BME-coated transwell assays, respectively). Additionally, compound 4 and, to a lesser extent, compound 6 inhibited NEP activity (fluorometric assay). This activity of compound 4 was involved in its anti-proliferative potential (BrdU assay). The present study shows that compound 4 can be considered a potential anti-osteosarcoma agent and a scaffold for the development of new NEP inhibitors.

Alpha Ketoglutarate Downregulates the Neutral Endopeptidase and Enhances the Growth Inhibitory Activity of Thiorphan in Highly Aggressive Osteosarcoma Cells.

Since natural substances are widely explored as epigenetic modulators of gene expression and epigenetic abnormalities are important causes of cancerogenesis, factors with pro-tumor activities subjected to epigenetic control, e.g., neutral endopeptidase (NEP, neprilysin), are promising anticancer targets for potential therapies acting via epigenetic regulation of gene expression. Alpha-ketoglutarate (AKG) is a naturally occurring co-substrate for enzymes involved in histone and DNA demethylation with suggested anti-cancer activity. Hence, we investigated a potential effect of AKG on the NEP expression in cells derived from various cancers (cervical, colon, osteosarcoma) and normal epithelial cells and osteoblasts. Moreover, the overall methylation status of histone H3 was explored to establish the molecular target of AKG activity. Additionally, it was investigated whether AKG in combination with thiorphan (NEP specific inhibitor) exhibited enhanced anticancer activity. The results revealed that AKG downregulated the expression of NEP at the protein level only in highly aggressive osteosarcoma HOS cells (flow cytometry and fluorometric assays), and this protease was found to be involved in AKG-induced growth inhibition in osteosarcoma cells (siRNA NEP silencing, BrdU assay, flow cytometry). Unexpectedly, AKG-induced hypermethylation of H3K27 in HOS cells, which was partially dependent on EZH2 activity. However, this effect was not implicated in the AKG-induced NEP downregulation (flow cytometry). Finally, the combined treatment with AKG and thiorphan was shown to significantly enhance the growth inhibitory potential of each one towards HOS cells (BrdU assay). These preliminary studies have shown for the first time that the downregulation of NEP expression is a promising target in therapies of NEP-implicating HOS cells. Moreover, this therapeutic goal can be achieved via AKG-induced downregulation of NEP and synergistic activity of AKG with thiorphan, i.e., a NEP specific inhibitor. Furthermore, this study has reported for the first time that exogenous AKG can influence the activity of histone methyltransferase, EZH2. However, this issue needs further investigation to elucidate the mechanisms of this phenomenon.

Architecture of the Pancreatic Islets and Endocrine Cell Arrangement in the Embryonic Pancreas of the Grass Snake (Natrix natrix L.). Immunocytochemical Studies and 3D Reconstructions.

During the early developmental stages of grass snakes, within the differentiating pancreas, cords of endocrine cells are formed. They differentiate into agglomerates of large islets flanked throughout subsequent developmental stages by small groups of endocrine cells forming islets. The islets are located within the cephalic part of the dorsal pancreas. At the end of the embryonic period, the pancreatic islet agglomerates branch off, and as a result of their remodeling, surround the splenic "bulb". The stage of pancreatic endocrine ring formation is the first step in formation of intrasplenic islets characteristics for the adult specimens of the grass snake. The arrangement of endocrine cells within islets changes during pancreas differentiation. Initially, the core of islets formed from B and D cells is surrounded by a cluster of A cells. Subsequently, A, B, and D endocrine cells are mixed throughout the islets. Before grass snake hatching, A and B endocrine cells are intermingled within the islets, but D cells are arranged centrally. Moreover, the pancreatic polypeptide (PP) cells are not found within the embryonic pancreas of the grass snake. Variation in the proportions of different cell types, depending on the part of the pancreas, may affect the islet function-a higher proportion of glucagon cells is beneficial for insulin secretion.

Assessment of Paroxetine Molecular Interactions with Selected Monoamine and γ-Aminobutyric Acid Transporters.

Thus far, many hypotheses have been proposed explaining the cause of depression. Among the most popular of these are: monoamine, neurogenesis, neurobiology, inflammation and stress hypotheses. Many studies have proven that neurogenesis in the brains of adult mammals occurs throughout life. The generation of new neurons persists throughout adulthood in the mammalian brain due to the proliferation and differentiation of adult neural stem cells. For this reason, the search for drugs acting in this mechanism seems to be a priority for modern pharmacotherapy. Paroxetine is one of the most commonly used antidepressants. However, the exact mechanism of its action is not fully understood. The fact that the therapeutic effect after the administration of paroxetine occurs after a few weeks, even if the levels of monoamine are rapidly increased (within a few minutes), allows us to assume a neurogenic mechanism of action. Due to the confirmed dependence of depression on serotonin, norepinephrine, dopamine and γ-aminobutyric acid levels, studies have been undertaken into paroxetine interactions with these primary neurotransmitters using in silico and in vitro methods. We confirmed that paroxetine interacts most strongly with monoamine transporters and shows some interaction with γ-aminobutyric acid transporters. However, studies of the potency inhibitors and binding affinity values indicate that the neurogenic mechanism of paroxetine's action may be determined mainly by its interactions with serotonin transporters.

Carbon Monoxide and Nitric Oxide as Examples of the Youngest Class of Transmitters.

The year 2021 is the 100th anniversary of the confirmation of the neurotransmission phenomenon by Otto Loewi. Over the course of the hundred years, about 100 neurotransmitters belonging to many chemical groups have been discovered. In order to celebrate the 100th anniversary of the confirmation of neurotransmitters, we present an overview of the first two endogenous gaseous transmitters i.e., nitric oxide, and carbon monoxide, which are often termed as gasotransmitters.

Paroxetine-Overview of the Molecular Mechanisms of Action.

In the 21st century and especially during a pandemic, the diagnosis and treatment of depression is an essential part of the daily practice of many family doctors. It mainly affects patients in the age category 15-44 years, regardless of gender. Anxiety disorders are often diagnosed in children and adolescents. Social phobias can account for up to 13% of these diagnoses. Social anxiety manifests itself in fear of negative social assessment and humiliation, which disrupts the quality of social functioning. Treatment of the above-mentioned disorders is based on psychotherapy and pharmacotherapy. Serious side effects or mortality from antidepressant drug overdose are currently rare. Recent studies indicate that paroxetine (ATC code: N06AB), belonging to the selective serotonin reuptake inhibitors, has promising therapeutic effects and is used off-label in children and adolescents. The purpose of this review is to describe the interaction of paroxetine with several molecular targets in various points of view including the basic chemical and pharmaceutical properties. The central point of the review is focused on the pharmacodynamic analysis based on the molecular mechanism of binding paroxetine to various therapeutic targets.

Antiepileptic Drug Tiagabine Does Not Directly Target Key Cardiac Ion Channels Kv11.1, Nav1.5 and Cav1.2.

Tiagabine is an antiepileptic drug used for the treatment of partial seizures in humans. Recently, this drug has been found useful in several non-epileptic conditions, including anxiety, chronic pain and sleep disorders. Since tachycardia-an impairment of cardiac rhythm due to cardiac ion channel dysfunction-is one of the most commonly reported non-neurological adverse effects of this drug, in the present paper we have undertaken pharmacological and numerical studies to assess a potential cardiovascular risk associated with the use of tiagabine. A chemical interaction of tiagabine with a model of human voltage-gated ion channels (VGICs) is described using the molecular docking method. The obtained in silico results imply that the adverse effects reported so far in the clinical cardiological of tiagabine could not be directly attributed to its interactions with VGICs. This is also confirmed by the results from the isolated organ studies (i.e., calcium entry blocking properties test) and in vivo (electrocardiogram study) assays of the present research. It was found that tachycardia and other tiagabine-induced cardiac complications are not due to a direct effect of this drug on ventricular depolarization and repolarization.

Experimental and Theoretical Analysis of Metal Complex Diffusion through Cell Monolayer.

The study of drugs diffusion through different biological membranes constitutes an essential step in the development of new pharmaceuticals. In this study, the method based on the monolayer cell culture of CHO-K1 cells has been developed in order to emulate the epithelial cells barrier in permeability studies by laser interferometry. Laser interferometry was employed for the experimental analysis of nickel(II) and cobalt(II) complexes with 1-allylimidazole or their chlorides' diffusion through eukaryotic cell monolayers. The amount (mol) of nickel(II) and cobalt(II) chlorides transported through the monolayer was greater than that of metals complexed with 1-allylimidazole by 4.34-fold and 1.45-fold, respectively, after 60 min. Thus, laser interferometry can be used for the quantitative analysis of the transport of compounds through eukaryotic cell monolayers, and the resulting parameters can be used to formulate a mathematical description of this process.

Gastropathy in patients with Wilson disease.

Background: Gastrointestinal symptoms are common in patients with Wilson disease (WD) and may be related to the disease itself or to adverse drug reactions (ADRs).Aim: To investigate gastroscopy findings in patients with WD and to analyze the risk of gastropathy in the context of different manifestations and treatments of WD as well as Helicobacter pylori infection status.Methods: This cross-sectional study included patients diagnosed or monitored for WD between 2007 and 2017. All enrolled patients were examined with gastroscopy and checked for infection with a urease test. Based on predominant manifestations, WD was classified as pre-symptomatic, hepatic (only liver symptoms) or neurological. Patients were divided into three treatment groups: untreated, treated with d-penicillamine (DPA) or zinc sulfate therapy.Results: Of 115 patients, 58 were male and the median age was 30 years. Gastropathy was observed in 65.2% of all patients. Factors that increased the risk of gastropathy were zinc sulfate (odds ratio [OR] = 3.01; 95% confidence interval [CI]: 1.12-8.09, p = .03), H. pylori infection (OR = 2.96; 95%CI: 1.34-6.56, p = .01) and neurological manifestations (OR = 2.55; 95%CI: 1.16-5.60, p = .02). In total, 9.6% of patients had gastric or duodenal ulcers and 29.6% had esophageal varices but no difference was seen by treatment status. In multivariate analysis, zinc sulfate remained associated with higher risk of gastropathy compared with no treatment (OR = 4.57; 95%CI: 1.21-17.19; p = .03) and DPA (OR = 6.28; 95%CI: 1.43-27.56; p = .01).Conclusions: Our results show that gastropathy in WD may be influenced by the treatment used.KeypointsIn a retrospective study of 115 patients with Wilson's disease, gastric injury was frequent.Patients receiving zinc sulfate had increased gastropathy risk compared with those receiving no treatment or d-penicillamine.

KV11.1, NaV1.5, and CaV1.2 Transporter Proteins as Antitarget for Drug Cardiotoxicity.

Safety assessment of pharmaceuticals is a rapidly developing area of pharmacy and medicine. The new advanced guidelines for testing the toxicity of compounds require specialized tools that provide information on the tested drug in a quick and reliable way. Ion channels represent the third-largest target. As mentioned in the literature, ion channels are an indispensable part of the heart's work. In this paper the most important information concerning the guidelines for cardiotoxicity testing and the way the tests are conducted has been collected. Attention has been focused on the role of selected ion channels in this process.

Alpha Ketoglutarate Exerts In Vitro Anti-Osteosarcoma Effects through Inhibition of Cell Proliferation, Induction of Apoptosis via the JNK and Caspase 9-Dependent Mechanism, and Suppression of TGF-ß and VEGF Production and Metastatic Potential of Cells.

Osteosarcoma (OS) is the most common type of primary bone tumor. Currently, there are limited treatment options for metastatic OS. Alpha-ketoglutarate (AKG), i.e., a multifunctional intermediate of the Krebs cycle, is one of the central metabolic regulators of tumor fate and plays an important role in cancerogenesis and tumor progression. There is growing evidence suggesting that AKG may represent a novel adjuvant therapeutic opportunity in anti-cancer therapy. The present study was intended to check whether supplementation of Saos-2 and HOS osteosarcoma cell lines (harboring a TP53 mutation) with exogenous AKG exerted an anti-cancer effect. The results revealed that AKG inhibited the proliferation of both OS cell lines in a concentration-dependent manner. As evidenced by flow cytometry, AKG blocked cell cycle progression at the G1 stage in both cell lines, which was accompanied by a decreased level of cyclin D1 in HOS and increased expression of p21Waf1/Cip1 protein in Saos-2 cells (evaluated with the ELISA method). Moreover, AKG induced apoptotic cell death and caspase-3 activation in both OS cell lines (determined by cytometric analysis). Both the immunoblotting and cytometric analysis revealed that the AKG-induced apoptosis proceeded predominantly through activation of an intrinsic caspase 9-dependent apoptotic pathway and an increased Bax/Bcl-2 ratio. The apoptotic process in the AKG-treated cells was mediated via c-Jun N-terminal protein kinase (JNK) activation, as the specific inhibitor of this kinase partially rescued the cells from apoptotic death. In addition, the AKG treatment led to reduced activation of extracellular signal-regulated kinase (ERK1/2) and significant inhibition of cell migration and invasion in vitro concomitantly with decreased production of pro-metastatic transforming growth factor ß (TGF-ß) and pro-angiogenic vascular endothelial growth factor (VEGF) in both OS cell lines suggesting the anti-metastatic potential of this compound. In conclusion, we showed the anti-osteosarcoma potential of AKG and provided a rationale for a further study of the possible application of AKG in OS therapy.