Cezary Dobosz

Ethanolic extract of Brazilian green propolis sensitizes prostate cancer cells to TRAIL-induced apoptosis

Prostate cancer represents an ideal disease for chemopreventive intervention. Propolis possesses immuno-modulatory, anti-tumour and chemopreventive properties. The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is an important endogenous anti-cancer agent that induces apoptosis selectively in tumour cells. However, some cancer cells are resistant to TRAIL-mediated apoptosis. Naturally occurring phenolic and polyphenolic compounds sensitize TRAIL-resistant cancer cells and augment the apoptotic activity of TRAIL. The ethanolic extract of Brazilian green propolis (EEP) is rich in phenolic components. Our in vitro results indicate the potential targets in the TRAIL-induced apoptotic pathway for the cancer chemopreventive activity of Brazilian propolis. We examined the cytotoxic and apoptotic effects of Brazilian EEP and its bioactive components in combination with TRAIL on LNCaP prostate cancer cells. The chemical composition of Brazilian green propolis was determined by high performance liquid chromatography-diode array detection. The cytotoxicity was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl-tetrazolium and lactate dehydrogenase assays. Apoptosis was detected using annexin V-FITC by flow cytometry and fluorescence microscopy. The mitochondrial membrane potential (∆Ψm) was evaluated using DePsipher staining by fluorescence microscopy. Flow cytometry was used to analyse death receptor (TRAIL-R1 and TRAIL-R2) expression in LNCaP cells. The inhibition of nuclear factor-κB (NF-κB) (p65) activation in cancer cells was confirmed by the ELISA-based TransAM NF-κB kit. The LNCaP cells were shown to be resistant to TRAIL-induced apoptosis. Our study demonstrates that EEP sensitizes TRAIL-resistant prostate cancer cells. The main phenolic components detected in Brazilian green propolis are artepillin C, quercetin, kaempferol and p-coumaric acid. Brazilian propolis and its bioactive components markedly augmented TRAIL-mediated apoptosis and cytotoxicity in prostate cancer cells. Brazilian EEP enhanced the expression of TRAIL-R2 and the activity of NF-κB in LNCaP cells. The co-treatment of prostate cancer cells with 100 ng/ml TRAIL and 50 µg/ml EEP increased the percentage of apoptotic cells to 65.8 ± 1.2% and caused a significant disruption of ∆Ψm in LNCaP cells. We show that Brazilian EEP helped cells overcome TRAIL resistance by engaging both intrinsic and extrinsic apoptotic pathways and regulating NF-κB activity. The data demonstrate the important role of Brazilian green propolis and its bioactive compounds in prostate cancer chemoprevention through the enhancement of TRAIL-mediated apoptosis.

Medicine students and exposure to environmental tobacco smoke

ObjectiveAlthough medicine students express positive attitudes toward providing lifestyle counseling, they require more instruction in many areas of health behavior in order to be helpful to their patients. The presented study included the students’ questionnaires analysis regarding their lifestyle and exposure to tobacco smoke. The aim of this study was to examine students’ exposure to chosen xenobiotics by determination of selected biomarkers in urine samples, which underlay the basis for exposure assessment towards tobacco smoke.Materials and MethodsThe investigated group consisted of first- and second-year medicine students from the Silesian Medical University (N = 133). Data obtained from a questionnaire survey was compared with the results of chosen biomarkers determined in urine samples. The analyses of the main nicotine metabolites were carried out firstly with use of ELISA, followed by the TLC technique with densitometry.ResultsAccording to questionnaires, every third student examined was exposed to passive smoking. The mean concentration of the main nicotine metabolites determined by ELISA in urine samples of smoking students was 1293.52±396.70 μg/g creatinine. The results of the TLC analysis in the group of smoking students were as follows: for cotinine − 523.10±68.10 μg/g creatinine and for trans-3’-hydroxycotinine − 653.81±62.30 μg/g creatinine.ConclusionsMedicine students, regardless of their area of study, are a highly-exposed part of the population to tobacco smoke, not only actively but also passively. Tobacco smoke exposure can be assessed by ELISA as a screening method as well as by more specific TLC technique with densitometry.

Determination of 7-ketocholesterol and 7-hydroxycholesterol in meat samples by TLC with densitometric detection

Concentrations of two oxycholesterols: 7-ketocholesterol and 7- hydroxycholesterol in meat samples (raw and cooked) were determined by TLC with densitometric detection. To obtain the oxycholesterols fraction, a multistage clean-up procedure based on liquid extraction and solid-phase extraction (SPE) was performed on silica gel and Florisil columns. The oxycholesterol fractions isolated from meat samples were separated by TLC on silica gel in an horizontal chamber. Quantitation was performed densitometrically directly after the TLC separation for 7-ketocholesterol and after Liebermann—Burchard reaction for 7-hydroxycholesterol. The presence of oxycholesterols determined by TLC was confirmed by GC—MS analysis of oxysterols derivatized to trimethylsilyl ethers. The investigations showed that the 7-ketocholesterol and 7-hydroxycholesterol content of the meat eaten most often in Poland ranged from approximately 40 ng g−1 to 3.5 µg g−1 of the sample.

Densitometric thin-layer chromatography of polycyclic aromatic sulfur compounds

Thin-layer chromatography has been used to separate a mixture of sulfur polynuclear aromatic hydrocarbons (S-PAH), heterocyclic compounds with mutagenic and carcinogenic properties. S-PAH were oxidized to give the sulfone derivatives and then S-PAH standards, their oxidized forms, and PAH were applied to silica gel and RP-18 plates and developed in a horizontal chamber with different mobile phases. After chromatography the plates were observed in UV light at λ = 254 nm and scanned densitometrically at the same wavelength. RF values were determined for the compounds.

Application of a modified high-performance thin-layer chromatographic-densitometric technique to evaluate tobacco smoke exposure of multiple sclerosis patients

Multiple sclerosis (MS) is a serious, one of the most common disorders of the nervous system and is still uncharted course and etiology. Modern medicine characterized MS as a disease of multifactorial, complex pathogenesis. The studies were designed to test some new dependencies and determine exposure to selected environmental factors, including passive and active smoking using separation techniques in biological materials sampled nonin vasively. The aim of this research was to determine by high-performance thin-layer chromatography (HPTLC) with densito metry major nicotine metabolites in urine samples taken from people with multiple sclerosis in earlier described conditions, but using new version of sandwich chamber, allowing to achieve better separation results. Urine samples (28) were collected noninvasively from patients with multiple sclerosis living in the region of Upper Silesia, Poland. The control samples (21) came from healthy people — family and friends of people with MS. Including active and passive smoking, 42.86% of the respondents were exposed to ingredients of tobacco smoke. The concentration of cotinine in the urine samples analyzed by HPTLC—densitometry ranged from 16.1 ng mL−1 to 453.15 ng mL−1, while the concentration of trans-3′-hydroxycotinine was relatively higher, 25.5–1011 ng mL−1. There were no significant changes in the excretion of the main nicotine metabolites concentrations in both tested group, but the average concentration of trans-3′-hydroxycotinine determined in urine was higher in the urine of MS smokers and various profiles of urinary main nicotine metabolites were observed.