A method of isolating and analysing drugs from cancer cells for preclinical research.

The presented paper describes a new isolation method of recovery and analysis of selected drugs developed for preclinical research. The method uses the RP-HPLC technique (in a single chromatographic separation) and serves the recovery and analysis of selected drugs from neoplastic cells. It enables the determination of cytostatics statins, fibrates, and pioglitazone. Chromatographic separations of the tested compounds were carried out on a Gemini-NX 5 µ C18 (4.6 × 150 mm i.d.) column, in a gradient system with a mobile phase consisting of ACN (0.1% TFA) and water (0.1% TFA) at ambient temperature. The separations were carried out at a flow of 1 ml/min and UV detection of 220 nm. The inter-day and intra-day precision and accuracy of the method were determined. Extending the extraction time at reduced temperature resulted in a significant increase in the recovery of the pharmaceuticals in comparison with traditional extraction methods. The presence of the tested pharmaceuticals at defined retention times was confirmed by mass spectrometry. A recovery procedure for the tested compounds from biological material (medium, cell pellets) was developed at a level ranging between 93 and 99%. The utility of the new HPLC method has been confirmed in drug absorption studies as screening tests for the analysis of the new therapeutic compositions on melanoma cell lines.

Property of a new Prussian blue-bounded iron complex able to peroxidate non-saturated fatty acids with a tendency to create conditions that may encourage ferroptosis.

Ferroptosis is a cell death process caused by redox imbalance in the cell environment. However, the cell death pathway proves beneficial in anticancer therapy, so compounds inducing ferroptosis are sought. The paper presents a newly synthesized iron complex named FeT, composed of ferricyanide and tartrate, which seems to meet these expectations. It is relatively stable, easily soluble in water and capable of peroxidating unsaturated fatty acids. T24 bladder cells were used as model cells. Preliminary studies demonstrated a strong inhibitory effect of this compound on cell proliferation. The cytotoxicity of FeT was assessed. Independently, it initiates caspase activity, indicating the complex cellular impact of this compound. This effect is compellingly the result of FeT penetration into the cell's interior with possible direct damage to mitochondria, thus explaining the involvement of apoptosis in cell death. At the same time, after penetrating into the cell, it causes an increase in reactive oxygen species (ROS), lipid peroxidation and a decrease in reduced glutathione, which is interpreted as to cause ferroptosis. In turn, reducing mitochondrial potential may indicate both ferroptosis and an internal pathway to apoptosis.

Simultaneous detection of melatonin and six metabolites of L-tryptophan pathway in rat gastric mucosa.

Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine synthesized in vertebrates mainly in the pineal gland, and is known to be involved mainly in thermoregulation and control of the circadian rhythm. That indoleamine can affect the auto-, para- and endocrine pathways, regulating body functions and affecting the metabolism of animals and humans. In addition to the pineal gland, melatonin can be synthesized in many extra-pineal tissues, mainly in the gastrointestinal tract. Previous studies have shown that melatonin plays an important role in the defense system of the gastrointestinal mucosa, demonstrating a protective effect on the gastrointestinal tract and the acceleration of healing of chronic ulcers through the scavenging of reactive oxygen metabolites (ROS) and the activation of protective nitric oxide (NO) and vasodilator neuropeptides released from the sensory afferent neurons. The process of converting the melatonin precursor L-tryptophan into melatonin is already known, but not all aspects of this process for the synthesis of other metabolites of this pathway have been fully elucidated and this issue remains poorly understood. In this study, the conversion of L-tryptophan to melatonin and other metabolites was determined in gastric mucosa collected from rats with or without intragastric (i.g.) melatonin or L-tryptophan administration, both administered at a single dose of 50 mg/kg. For the determination of five metabolites of L-tryptophan: kynurenine, 5-hydroxytryptamine, 5-hydroxytryptophan, anthranilic acid, indole-3-acetic acid together with melatonin, we have modified the previously developed high-performance liquid chromatography (HPLC) method using a native fluorescence detection system and UV-VIS. The obtained results show that: 1) L-tryptophan is converted into melatonin in the gastric mucosa during the day, e.g. after eating a meal containing L-tryptophan, as it was imitated and confirmed by our study, in which this amino acid was administered directly to the stomach, 2) the gastric mucosa is capable of producing melatonin in much greater amounts than those recorded in the blood serum of rats given a single dose of L-tryptophan, and 3) apart from melatonin, the only serum levels of these five metabolites of the L-tryptophan metabolic pathway are detectable, while their level in the gastric mucosa is low and barely detectable under physiological conditions. Our present observations support the notion that the gastric mucosa is one of the main sources of melatonin production from L-tryptophan outside the pineal gland.

Small-molecule inhibitor - tyrphostin AG1296 regulates proliferation, survival and migration of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most commonly occurring malignant soft tissue tumor in children. Despite improving its treatment methods, the current outcome in the advanced stages of this tumor is not satisfactory. RMS cells are characterized by abnormal cellular signaling due to the changes in the activity of the tyrosine kinases. Thus, substances blocking the mitogenic signal transmitted by receptors with tyrosine kinase activity raise hopes for inhibition of the uncontrolled cell growth. In this study, we examined the anticancer activity of tyrphostin AG1296, a tyrosine kinase inhibitor that binds to the intracellular domain of the PDGF (platelet-derived growth factor) receptor in human RMS alveolar and embryonal cell lines. We have discovered that tyrphostin AG1296 completely inhibited cell proliferation and effectively inhibited cell viability. Tyrphostin AG1296 induced apoptosis of the RMS cells and significantly inhibited their migration. Additionally, investigated inhibitor slightly inhibited expression of AKT and phosphorylation of ERK in alveolar RMS cells. Importantly, the inhibitor exerted also potent effects on the nanomechanical properties and cytoskeleton organization of RMS cells. To conclude, tyrphostin AG1296 is a promising compound in the treatment of alveolar RMS. Undoubtedly, a better knowledge of receptor pathomechanism of tyrosine kinases may contribute to developing new, more effective ways of RMS treatment.

Conversion L-tryptophan to melatonin in the gastrointestinal tract: the new high performance liquid chromatography method enabling simultaneous determination of six metabolites of L-tryptophan by native fluorescence and UV-VIS detection.

Melatonin is a major biosynthetic product of pineal gland exerting a potent antioxidant and the reactive oxygen metabolites scavenging activities but the mechanism of formation of this indole at extrapineal sources has not been fully elucidated. It is known that the gastrointestinal (GI)-tract plays an important role as a source of melatonin synthesis but the conversion of L-tryptophan into melatonin in the GI-tract of experimental animals and humans should be further examined. In this study, the conversion of L-tryptophan to melatonin was determined in the serum collected from rats administered intragastrically with this amino acid acting as melatonin precursor. For this purpose, a simple, sensitive and reliable method was developed for simultaneous determination of six L-tryptophan metabolites in rat serum, namely, 5-hydroxytryptamnie (5-HT), 5-hydroksytryptophan (5-HTR), kynurenin (KYN), antranilic acid (AA), indole-3-acetic acid (IAA) and melatonin that were analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with UV and native fluorimetric detection with multiple wavelengths. We used nucleosil Supelco C18 5 µm 4.6 mm x 250 nm column with the standard mobile phase consisting of solvent A (water/0.1% trifluoroacetic acid (TFA) and solvent B (methanol/0.1% TFA) in gradient elution. Fifty five rats received vehicle (saline) of L-tryptophan (50 mg/kg) or melatonin (50 mg/kg) by means of intragastric gavage and they were anesthetized and sacrificed at 0, 10, 20, 30, 60, 120 or 240 min upon L-tryptophan or melatonin administration for the venous blood withdrawal. The serum collected samples were kept on ice for the HPLC determination. The average recovery of 5-HT, 5-HRT, KYN, AA, TRP, IAA, and melatonin were 99±3%, 97±1.5%, 94±2.5%, 99±2.46, 98±1.5 and 98±2%, respectively. We conclude that 1) L-tryptophan is converted to melatonin in the GI-tract during the day when the pineal gland synthesis is inhibited, and 2) the reverse phase high performance liquid chromatography (RP-HPLC) is a new sensitive and reliable method that could be successfully applied to the study of kinetics and metabolism of L-tryptophan in GI-tract.

Proangiogenic activity of beta-carotene is coupled with the activation of endothelial cell chemotaxis.

Endothelial cells play an important role in angiogenesis (formation of new vessels from preexisting ones), which is essential for organogenesis, tissue remodeling but also inflammatory response, carcinogenesis in all periods of our life. Beta-carotene (BC) in non-toxic concentrations (up to 3 microM) had no detectable effect on HUVECs (human umbilical vein endothelial cells) proliferation or apoptosis, despite significant changes of the expression patterns of pro- and anti-apoptotic genes. However beta-carotene did not change the tubulogenic activity of HUVEC in the in vitro angiogenesis model, it potently accelerated the bFGF-induced development of microcapillaries, as well as the migration of endothelial cells, in matrigel plug injected subcutaneously to mice. Potent activation of endothelial cell migration in the in vitro model of chemotaxis was also observed. According to the microarray data, genes involved in cell/cell and cell/matrix adhesion, matrix reorganization, activation of chemotaxis, the G-protein regulated intracellular signaling as well as genes involved in the rapid remodeling of protein cytoskeleton were the most affected by BC in HUVEC. We conclude that beta-carotene in the physiological concentration range stimulates early steps of angiogenesis by the activation of cellular migration as well as matrix reorganization and decrease of cell adhesion.