Proapoptotic effects of halogenated bis-phenylthiourea derivatives in cancer cells.

New halogenated thiourea derivatives were synthesized via the reaction of substituted phenylisothiocyanates with aromatic amines. Their cytotoxic activity was examined in in vitro studies against solid tumors (SW480, SW620, PC3), a hematological malignance (K-562), and normal keratinocytes (HaCaT). Most of the compounds were more effective against SW480 (1a, 3a, 3b, 5j), K-562 (2b, 3a, 4a), or PC3 (5d) cells than cisplatin, with favorable selectivity. Their anticancer mechanisms were studied by Annexin V-fluorescein-5-isothiocyanate apoptosis, caspase-3/caspase-7 assessment, cell cycle analysis, interleukin-6 (IL-6) release inhibition, and reactive oxygen species (ROS) generation assay. Thioureas 1a, 2b, 3a, and 4a were the most potent activators of early apoptosis in K-562 cells, and substances 1a, 3b, 5j triggered late-apoptosis or necrosis in SW480 cells. This proapoptotic effect was proved by the significant increase of caspase-3/caspase-7 activation. Cell cycle analysis revealed that derivatives 1a, 3a, 5j increased the number of SW480 and K-562 cells in the sub-G1 and/or G0/G1 phases, and one evoked cycle arrest at the G2 phase. The most potent thioureas inhibited IL-6 cytokine secretion from PC3 cells and both colon cancer cell lines. Apoptosis-inducing compounds also increased ROS production in all tumor cell cultures, which may enhance their anticancer properties.

Anticancer activity and metabolic alteration in colon and prostate cancer cells by novel moxifloxacin conjugates with fatty acids.

The positive and pro-economic trend in the management of cancer treatment is the search for the antineoplastic potential of known, widely used and safe drugs with a different clinical purpose. A good candidate seems to be moxifloxacin with broad-spectrum antibacterial activity, which as the member of the fourth generation fluoroquinolone is known to affect not only bacterial but also eukaryotic DNA topoisomerases, however at high concentration. Due to the fact that the modification of parent drug with lipid component can improve anticancer potential by increasing of bioavailability, selectivity, and cytotoxic efficiency, we evaluated the mechanisms of cytotoxic activity of novel moxifloxacin conjugates with fatty acids and verified metabolic profile in SW480, SW620 and PC3 cell lines. Our study revealed that cytotoxic potential of moxifloxacin conjugates was stronger than free moxifloxacin, moreover, they remained non-toxic to normal HaCaT cells. PC3 were more sensitive to MXF conjugates than colon cancer cells. The most promising cytotoxic activity exhibited conjugate 4m and 16m with oleic and stearic acid reducing viability of PC3 and SW620 cells. Tested conjugates activated caspases 3/7 and induced late-apoptosis, mainly in PC3 and SW620 cells. However, the most pronounced inhibition of NF-κB activation and IL-6 secretion was observed in SW480. Metabolomic analysis indicated influence of the moxifloxacin conjugates on intensity of lipid derivatives with the most successful metabolite profile in PC3. Our findings suggested the cytotoxic potential of moxifloxacin conjugates, especially with oleic and stearic acid can be beneficial in oncological therapy, including their possible anti-inflammatory and known antibacterial effect.

Comparative Study of Chemical Stability of a PK20 Opioid-Neurotensin Hybrid Peptide and Its Analogue [Ile9]PK20-The Effect of Isomerism of a Single Amino Acid.

Chemical stability is one of the main problems during the discovery and development of potent drugs. When ignored, it may lead to unreliable biological and pharmacokinetics data, especially regarding the degradation of products' possible toxicity. Recently, two biologically active drug candidates were presented that combine both opioid and neurotensin pharmacophores in one entity, thus generating a hybrid compound. Importantly, these chimeras are structurally similar except for an amino acid change at position 9 of the peptide chain. In fact, isoleucine (C6H13NO2) was replaced with its isomer tert-leucine. These may further lead to various differences in hybrids' behavior under specific conditions (temperature, UV, oxidative, acid/base environment). Therefore, the purpose of the study is to assess and compare the chemical stability of two hybrid peptides that differ in nature by way of one amino acid (tert-leucine vs. isoleucine). The obtained results indicate that, opposite to biological activity, the substitution of tert-leucine into isoleucine did not substantially influence the compound's chemical stability. In fact, neither hydrolysis under alkaline and acidic conditions nor oxidative degradation resulted in spectacular differences between the two compounds-although the number of potential degradation products increased, particularly under acidic pH. However, such a modification significantly reduced the compound's half-life from 204.4 h (for PK20 exposed to 1M HCl) to 117.7 h for [Ile9]PK20.

Magnesium and Morphine in the Treatment of Chronic Neuropathic Pain-A Biomedical Mechanism of Action.

The effectiveness of opioids in the treatment of neuropathic pain is limited. It was demonstrated that magnesium ions (Mg2+), physiological antagonists of N-methyl-D-aspartate receptor (NMDAR), increase opioid analgesia in chronic pain. Our study aimed to determine the molecular mechanism of this action. Early data indicate the cross-regulation of µ opioid receptor (MOR) and NMDAR in pain control. Morphine acting on MOR stimulates protein kinase C (PKC), while induction of NMDAR recruits protein kinase A (PKA), leading to a disruption of the MOR-NMDAR complex and promoting functional changes in receptors. The mechanical Randall-Selitto test was used to assess the effect of chronic Mg2+ and morphine cotreatment on streptozotocin-induced hyperalgesia in Wistar rats. The level of phosphorylated NMDAR NR1 subunit (pNR1) and phosphorylated MOR (pMOR) in the periaqueductal gray matter was determined with the Western blot method. The activity of PKA and PKC was examined by standard enzyme immunoassays. The experiments showed a reduction in hyperalgesia after coadministration of morphine (5 mg/kg intraperitoneally) and Mg2+ (40 mg/kg intraperitoneally). Mg2+ administered alone significantly decreased the level of pNR1, pMOR, and activity of both tested kinases. The results suggest that blocking NMDAR signaling by Mg2+ restores the MOR-NMDAR complex and thus enables morphine analgesia in neuropathic rats.

Comprehensive Insight into Chemical Stability of Important Antidiabetic Drug Vildagliptin Using Chromatography (LC-UV and UHPLC-DAD-MS) and Spectroscopy (Mid-IR and NIR with PCA).

During forced degradation, the intrinsic stability of active pharmaceutical ingredients (APIs) could be determined and possible impurities that would occur during the shelf life of the drug substance or the drug product could be estimated. Vildagliptin belongs to relatively new oral antidiabetic drugs named gliptins, inhibiting dipeptidyl peptidase 4 (DPP-4) and prolonging the activities of the endogenous incretin hormones. At the same time, some gliptins were shown as prone to degradation under specific pH and temperature conditions, as well as in the presence of some reactive excipients. Thus, forced degradation of vildagliptin was performed at high temperature in extreme pH and oxidative conditions. Then, selective LC-UV was used for quantitative determination of non-degraded vildagliptin in the presence of its degradation products and for degradation kinetics. Finally, identification of degradation products of vildagliptin was performed using an UHPLC-DAD-MS with positive ESI. Stability of vildagliptin was also examined in the presence of pharmaceutical excipients, using mid-IR and NIR with principal component analysis (PCA). At 70 °C almost complete disintegration of vildagliptin occurred in acidic, basic, and oxidative media. What is more, high degradation of vildagliptin following the pseudo first-order kinetics was observed at room temperature with calculated k values 4.76 × 10-4 s-1, 3.11 × 10-4 s-1, and 1.73 × 10-4 s-1 for oxidative, basic and acidic conditions, respectively. Next, new degradation products of vildagliptin were detected using UHPLC-DAD-MS and their molecular structures were proposed. Three degradants were formed under basic and acidic conditions, and were identified as [(3-hydroxytricyclo- [3.3.1.13,7]decan-1-yl)amino]acetic acid, 1-{[(3-hydroxytricyclo[3.3.1.13,7]decan-1-yl)amino]acetyl}-pyrrolidine-2-carboxylic acid and its O-methyl ester. The fourth degradant was formed in basic, acidic, and oxidative conditions, and was identified as 1-{[(3-hydroxytricyclo[3.3.1.13,7]-decan-1-yl)amino]acetyl}pyrrolidine-2-carboxamide. When stability of vildagliptin was examined in the presence of four excipients under high temperature and humidity, a visible impact of lactose, mannitol, magnesium stearate, and polyvinylpirrolidone was observed, affecting-NH- and CO groups of the drug. The obtained results (kinetic parameters, interactions with excipients) may serve pharmaceutical industry to prevent chemical changes in final pharmaceutical products containing vildagliptin. Other results (e.g., identification of new degradation products) may serve as a starting point for qualifying new degradants of vildagliptin as it is related to substances in pharmacopoeias.

The anti-inflammatory potential of cefazolin as common gamma chain cytokine inhibitor.

A continuing quest for specific inhibitors of proinflammatory cytokines brings promise for effective therapies designed for inflammatory and autoimmune disorders. Cefazolin, a safe, first-generation cephalosporin antibiotic, has been recently shown to specifically interact with interleukin 15 (IL-15) receptor subunit α (IL-15Rα) and to inhibit IL-15-dependent TNF-α and IL-17 synthesis. The aim of this study was to elucidate cefazolin activity against IL-2, IL-4, IL-15 and IL-21, i.e. four cytokines sharing the common cytokine receptor γ chain (γc). In silico, molecular docking unveiled two potential cefazolin binding sites within the IL-2/IL-15Rß subunit and two within the γc subunit. In vitro, cefazolin decreased proliferation of PBMC (peripheral blood mononuclear cells) following IL-2, IL-4 and IL-15 stimulation, reduced production of IFN-γ, IL-17 and TNF-α in IL-2- and IL-15-treated PBMC and in IL-15 stimulated natural killer (NK) cells, attenuated IL-4-dependent expression of CD11c in monocyte-derived dendritic cells and suppressed phosphorylation of JAK3 in response to IL-2 and IL-15 in PBMC, to IL-4 in TF-1 (erythroleukemic cell line) and to IL-21 in NK-92 (NK cell line). The results of the study suggest that cefazolin may exert inhibitory activity against all of the γc receptor-dependent cytokines, i.e. IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.

Changes in global gene expression indicate disordered autophagy, apoptosis and inflammatory processes and downregulation of cytoskeletal signalling and neuronal development in patients with Niemann-Pick C disease.

Changes in gene expression profiles were investigated in 23 patients with Niemann-Pick C1 disease (NPC). cDNA expression microarrays with subsequent validation by qRT-PCR were used. Comparison of NPC to control samples revealed upregulation of genes involved in inflammation (MMP3, THBS4), cytokine signalling (MMP3), extracellular matrix degradation (MMP3, CTSK), autophagy and apoptosis (CTSK, GPNMB, PTGIS), immune response (AKR1C3, RCAN2, PTGIS) and processes of neuronal development (RCAN2). Downregulated genes were associated with cytoskeletal signalling (ACTG2, CNN1); inflammation and oxidative stress (CNN1); inhibition of cell proliferation, migration and differentiation; ERK-MAPK pathway (COL4A1, COL4A2, CPA4); cell adhesion (IGFBP7); autophagy and apoptosis (CDH2, IGFBP7, COL4A2); neuronal function and development (CSRP1); and extracellular matrix stability (PLOD2). When comparing NPC and Gaucher patients together versus controls, upregulation of SERPINB2 and IL13RA2 and downregulation of CSRP1 and CNN1 were characteristic. Notably, in NPC patients, the expression of PTGIS is upregulated while the expression of PLOD2 is downregulated when compared to Gaucher patients or controls and potentially could serve to differentiate these patients. Interestingly, in NPC patients with (i) jaundice, splenomegaly and cognitive impairment/psychomotor delay-the expression of ACTG2 was especially downregulated; (ii) ataxia-the expression of ACTG2 and IGFBP5 was especially downregulated; and (iii) VSGP, dysarthria, dysphagia and epilepsy-the expression of AKR1C3 was especially upregulated while the expression of ACTG2 was downregulated. These results indicate disordered apoptosis, autophagy and cytoskeleton remodelling as well as upregulation of immune response and inflammation to play an important role in the pathogenesis of NPC in humans.

The bioactivity of flavonoid glucuronides and free aglycones in the context of their absorption, II phase metabolism and deconjugation at the inflammation site.

One of the most spread group of phenolics are flavonoids. Many studies focusing on the digestion and bioavailability of flavonoids have been carried out. Several possible directions of flavonoid metabolism are suspected and described in the literature. The aim of the present study was to evaluate the bioactivity of 8 flavonoid 3-O- and 7-O- glucuronides and 7 free aglycones on inflammatory response of PMNs and HUVECs in the context of their fate in humans after oral intake. The present study for the first time compared the activity of several most popular in plant flavonol and flavone aglycones and their beta-glucuronides. The results showed that in all in vitro experiments only aglycones have anti-inflammatory activity in PMNs and HUVECs models in the concentration range 1-50 µM. The most significant influence on the inflammatory response was observed in the case of HUVECs. Compounds were able to down-regulate levels of adhesion molecules (ICAM, VCAM and E-selectin). The possible deconjugation phenomenon at the inflammation site was evaluated using enzymes produces by stimulated PMNs. This is the first report suggesting the role of ß-glucuronidase in the inflammatory process taking place on the inflammation site. Additionally, the anti-inflammatory effect was significantly better for flavones.

Eupatoriopicrin Inhibits Pro-inflammatory Functions of Neutrophils via Suppression of IL-8 and TNF-alpha Production and p38 and ERK 1/2 MAP Kinases.

During chronic inflammation, neutrophils acting locally as effector cells not only activate antibacterial defense but also promote the inflammatory response. Interleukin 8 (IL-8), the main cytokine produced by activated neutrophils, positively correlates with the severity of respiratory tract diseases. By screening European plants traditionally used for treating respiratory tract diseases, we found that extracts of aerial parts of Eupatorium cannabinum inhibit IL-8 release from neutrophils. Using bioassay-guided fractionation, we identified five sesquiterpene lactones, eupatoriopicrin (1), 5'-deoxyeupatoriopicrin (2), hiyodorilactone A (3), 3-hydroxy-5'- O-acetyleupatoriopicrin = hiyodorilactone D (4), and hiyodorilactone B (5), that efficiently (IC50 < 1 µM) inhibited IL-8 and TNF-α release in lipopolysaccharide (LPS)-stimulated human neutrophils. Moreover, all these sesquiterpene lactones suppressed the adhesion of human neutrophils to an endothelial monolayer by downregulating the expression of the ß2 integrin CD11b/CD18 on the neutrophil surface. Furthermore, eupatoriopicrin efficiently suppressed LPS-induced phosphorylation of p38 MAPK and ERK and attenuated neutrophil infiltration in the thioglycolate-induced peritonitis model in mice. Altogether, these results demonstrate the potential of the sesquiterpene lactone eupatoriopicrin as a lead substance for targeting inflammation.

Lignans From Forsythia x Intermedia Leaves and Flowers Attenuate the Pro-inflammatory Function of Leukocytes and Their Interaction With Endothelial Cells.

Aim of the study: Taking into account that overactivated leukocytes are an important factor in the development of many chronic diseases, we investigated the activity of phytochemically characterized (HPLC-DAD-MSn) extracts from forsythia leaves and flowers on the pro- and anti-inflammatory functions of leukocytes (effects on IL-1ß, IL-8, TNF-α, and TGFß release) and their adherence to endothelial cells. Using bio-guided fractionation, we isolated the active compounds and determined their biological activity, and we included the positive control quercetin. Methods: The effect on IL-1ß, TNF-α, IL-8, and TGF-α production by leukocytes was measured by enzyme-linked immunosorbent assay (ELISA). The surface expression of adhesion molecules was analyzed with flow cytometry, and the neutrophil attachment to the endothelial cells was assessed fluorimetrically. The effects on p38MAPK, ERK1/2 and JNK phosphorylation were determined using western blots. Results: Leaf extracts had the effect of decreasing TNF-α production in neutrophils and monocyte/macrophage cells. The bio-guided fractionation led to the isolation of the following lignan aglycones: (+)-pinoresinol, (+)-epipinoresinol, (-)-matairesinol, (+)-phillygenin, and (-)-arctigenin. Only phillygenin was able to stimulate the anti-inflammatory function of macrophages by inducing TGF-ß release and IL-10 receptor surface expression. Arctigenin, phillygenin, and a metabolite produced by the gut microbiota, enterolactone, decreased TNF-α and IL-1ß production and neutrophil adhesion to endothelial cells, probably by attenuating the p38 and ERK kinase pathways. Conclusion:Forsythia x intermedia is a valuable source of active lignans, which may be potential candidates for treating inflammatory diseases that are associated with the excessive production of cytokines such as TNF-α and IL-1ß.

Pharmacophore guided discovery of small-molecule interleukin 15 inhibitors.

Upregulation of interleukin 15 (IL-15) contributes directly i.a. to the development of inflammatory and autoimmune diseases. Selective blockade of IL-15 aimed to treat rheumatoid arthritis, psoriasis and other IL-15-related disorders has been recognized as an efficient therapeutic method. The aim of the study was to identify small molecules which would interact with IL-15 or its receptor IL-15Rα and inhibit the cytokine's activity. Based on the crystal structure of IL-15Rα·IL-15, we created pharmacophore models to screen the ZINC database of chemical compounds for potential IL-15 and IL-15Rα inhibitors. Twenty compounds with the highest predicted binding affinities were subjected to in vitro analysis using human peripheral blood mononuclear cells to validate in silico data. Twelve molecules efficiently reduced IL-15-dependent TNF-α and IL-17 synthesis. Among these, cefazolin - a safe first-generation cephalosporin antibiotic - holds the highest promise for IL-15-directed therapeutic applications.

Novel stilbenoids, including cannabispiradienone glycosides, from Tragopogon tommasinii (Asteraceae, Cichorieae) and their potential anti-inflammatory activity.

A phytochemical investigation of Tragopogon tommasinii Sch.Bip. (Asteraceae, Cichorieae) yielded a total of 21 natural products, two simple phenolic acids (4-hydroxybenzoic acid and p-coumaric acid), four caffeic acid derivatives (chlorogenic acid, 3-O-caffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid), six flavonoids (luteolin, luteolin 7-O-glucoside, vitexin, orientin, quercetin 3-O-glucoside, and isorhamnetin 3-O-glucoside), three simple bibenzyls [2-carboxyl-5-hydroxy-3-methoxy-4'-ß-glucopyranosyl-oxybibenzyl, 3-caffeoyl-(9→5)-ß-apiosyl-(1→6)-ß-glucopyranosyloxy-5,4'-dihydroxy-3'-methoxybibenzyl, 3-caffeoyl-(9→5)-ß-apiosyl-(1→6)-ß-glucopyranosyloxy-4'-dihydroxy-5,3'-dimethoxybibenzyl], three phtalides [3-(4-ß-glucopyranosyloxybenzyl)-7-hydroxy-5-methoxyphtalide, 7-ß-glucopyranosyloxy-(S)-3-(4-hydroxybenzyl)-5-methoxyphtalide, and 7-(1→6)-α-rhamnosyl-ß-glucopyranosyloxy-(S)-3-(4-hydroxybenzyl)-5-methoxyphtalide], two cannabispiradienone derivatives [3-O-ß-glucopyranosyldemethoxycannabispiradienone and 3-caffeoyl-(9→5)-ß-apiosyl-(1→6)-ß-glucopyranosyloxydemethoxycannabispiradienone], and tetra-N-coumaroyl spermine. The three bibenzyls, the latter two benzylphthalides, and both cannabispiradienone derivatives represent new natural compounds and all compounds, except the caffeic acid derivatives and the flavonoids were new for T. tommasinii. The structures were established by HR mass spectrometry, extensive 1D and 2D NMR spectroscopy, and CD spectroscopy. Moreover, the potential anti-inflammatory activities of the new compounds were assayed using human neutrophils and their production of IL-1b, IL-8, TNF-α and MMP-9 as well as the expression of TLR-4, respectively.

Antioxidant and anti-inflammatory flavonol glucuronides from Polygonum aviculare L.

A series of 11 flavonol glucuronides were isolated from the herb of Polygonum aviculare L. (Ph.Eur) of which 8 were reported for the first time from the Polygonum species. Three acetylated kaempferol and isorhamnetin glucuronides were isolated from a natural source for the first time. All compounds, including the new ones, were characterized using 1D and 2D NMR spectra as well as high resolution mass spectrometry. The influence of all isolated compounds on the production of reactive oxygen species, as well as on elastase release by human neutrophils, was evaluated in in vitro studies. The results showed that all investigated compounds at physiologically achievable concentrations within the range of 0.5-10 µM significantly inhibited the production of reactive oxygen species as well as elastase release in human neutrophils model and should be considered as responsible for anti-inflammatory activity of the P. aviculare herb. The chemotaxonomic value of isolated compounds was also discussed.

Effects of penta-O-galloyl-ß-D-glucose on human neutrophil function: significant down-regulation of L-selectin expression.

Penta-O-galloyl-ß-D-glucose (PGG) occurrs in high concentrations in medicinal herbs such as Rhus chinensis, Paeonia suffruticosa, Acer truncatum and Terminalia chebula, which demonstrate anti-inflammatory activity. We investigated the effect of PGG on stimulated and non-stimulated neutrophils in processes which included reactive oxygen species generation (ROS), metalloproteinase-9 and interleukin-8 secretion (IL-8), ß2 integrin (CD11b) and L-selectin (CD62L) expression and apoptosis. In concentrations of 5 µM-20 µM, PGG demonstrated statistically significant inhibition of ROS generation, IL-8 secretion and ß2 integrin expression in stimulated neutrophils. The inhibition of L-selectin expression by PGG resulted in prevention in neutrophils' endothelial attachment. The result obtained may explain the anti-inflammatory activity of this compound and underline the contribution of PGG in the activity of PGG rich plant extracts.

Contribution of endothelial cells to human bone-derived cells expansion in coculture.

Creating a functional vascularized bone tissue remains one of the main goals of bone tissue engineering. Recently, a growing interest in the crosstalk between endothelial cells (EC) and osteoblasts (OB), the two main players in a new bone formation, has been observed. However, only a few reports have addressed a mutual influence of OB and EC on cell proliferation. Our study focuses on this issue by investigating cocultures of human bone-derived cells (HBDC) and human umbilical vein endothelial cells (HUVEC). Three various proportions of cells have been used that is, HBDC:HUVEC 1:1, 1:4, and 4:1 and the cocultures were investigated on day 1, 4, and 7, while HUVEC and HBDC monocultures served as reference. We have detected enhanced alkaline phosphatase (ALP) activity in a direct HBDC-HUVEC coculture. This effect was not observed when cells were separated by an insert, which is consistent with other reports on various OB-EC lineages. The appearance of gap-junctions in coculture was confirmed by a positive staining for connexin 43. The number of cells of both phenotypes has been determined by flow cytometry: CD-31-positive cells have been considered EC, while CD-31-negative have been counted as OB. We have observed an over 14-fold increase in OB number after a week in the 1:4 HBDC:HUVEC coculture as compared with less than fourfold in monoculture. The increase in HBDC number in 1:1 coculture has been less pronounced and has reached the value of about sevenfold. These results correspond well with the cell proliferation rate, which has been measured by 5-bromo-2'-deoxyuridine incorporation. Moreover, at day 7 EC have been still present in the coculture, which is inconsistent with some other reports. Real-time polymerase chain reaction analysis has revealed the upregulation of ALP and collagen type I genes, but not osteocalcin gene, in all the cocultures grown without pro-osteogenic additives. Our study indicates that HUVEC significantly promote HBDC expansion and upregulate collagen I gene expression in these cells. We believe that these findings have application potency in bone tissue engineering.

Identification of suitable reference genes for real-time PCR analysis of statin-treated human umbilical vein endothelial cells.

Proper data normalization in quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) is of critical importance for reliable mRNA expression analysis. Due to a diversity in putative reference genes expression stability in different in vitro models, a validation of an internal control gene should be made for each particular tissue or cell type and every specific experimental design. A few approaches have been proposed for reference gene selection, including pair-wise comparison approach and model-based approach. In this article we have assessed the expression stability of eight putative reference genes: ACTB, B2M, GADD45A, GAPDH, HPRT1, PES1, PSMC4, YWHAZ, in human umbilical vein endothelial cells (HUVEC) treated with different statins and with TNF-α. The analysis was performed with three reference gene validation programs: geNorm, NormFinder and BestKeeper. We have shown that hypoxanthine phosphoribosyltransferase 1 gene (HPRT1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) are the most stably expressed genes among the analyzed ones. Furthermore, our results show that ß-actin gene (ACTB) is downregulated by statins and thus should not be used as a normalizing gene in a discussed experimental setup. A ranking of candidate reference genes stability values is provided and might serve as a valuable guide for future gene expression studies in endothelial cells. This is the first report on reference gene selection for RT-qPCR applications in statin-treated HUVEC model.