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.

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.

Leydig cell tumorigenesis - implication of G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and xenoestrogen exposure. In vivo and in vitro appraisal.

The etiology and molecular characteristics of Leydig cell tumor (LCT) are scarcely known. From the research data stems that estrogen can be implicated in LCT induction and development, however it is not investigated in detail. Considering the above, herein we analyzed the relation between G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and insulin-like family peptides (insulin-like 3 peptide; INSL3 and relaxin; RLN) expressions as well as estrogen level with impact of xenoestrogen (bisphenol A; BPA, tetrabromobisphenol A; TBBPA, and tetrachlorobisphenol A; TCBPA). While in our previous studies altered GPER-PPAR partnership was found in human LCT being a possible cause and/or additionally effecting on LCT development, here mouse testes with experimentally induced LCT and mouse tumor Leydig cell (MA-10) treated with BPA chemicals were examined. We revealed either diverse changes in expression or co-expression of GPER and PPAR in mouse LCT as well as in MA-10 cells after BPA analogues when compared to human LCT. Relationships between expression of INSL3, RLN, including co-expression, and estrogen level in human LCT, mouse LCT and MA-10 cells xenoestrogen-treated were found. Moreover, involvement of PI3K-Akt-mTOR pathway or only mTOR in the interactions of examined receptors and hormones was showed. Taken together, species, cell of origin, experimental system used and type of used chemical differences may result in diverse molecular characteristics of LCT. Estrogen/xenoestrogen may play a role in tumor Leydig cell proliferation and biochemical nature but this issue requires further studies. Experimentally-induced LCT in mouse testis and MA-10 cells after BPA exposure seem to be additional models for understanding some aspects of human LCT biology.

Ligands of peroxisome proliferator-activated receptor-gamma increase the generation of vascular endothelial growth factor in vascular smooth muscle cells and in macrophages.

Peroxisome proliferator-activated receptors-gamma (PPARgamma) are ligand-inducible transcription factors of the nuclear hormone receptor superfamily. We examined the effect of PPARgamma activation on the generation of vascular endothelial growth factor (VEGF), one of the major angiogenic agents. Rat vascular smooth muscle cells (VSMC) and murine macrophages RAW264.7 were incubated for 24 h with PPARgamma activators: prostaglandin J2 and ciglitazone. PPARgamma were expressed in VSMC and RAW cells and their activity was upregulated in the presence of PGJ2 and ciglitazone. Incubation of the cells with PPARgamma activators significantly augmented the release of VEGF protein into the media, both in resting and in IL-1beta- or LPS-stimulated cultures. The higher protein generation was connected with the increased expression of mRNA and transcriptional activation of VEGF promoter. We conclude that the activation of PPARgamma upregulates the generation of VEGF and may be involved in the regulation of angiogenesis.