New Thiosemicarbazide Derivatives with Multidirectional Biological Action.

Over the years, several new medicinal substances have been introduced for the treatment of diseases caused by bacteria and parasites. Unfortunately, due to the production of numerous defense mechanisms by microorganisms and parasites, they still pose a serious threat to humanity around the world. Therefore, laboratories all over the world are still working on finding new, effective methods of pharmacotherapy. This research work aimed to synthesize new compounds derived from 3-trifluoromethylbenzoic acid hydrazide and to determine their biological activity. The first stage of the research was to obtain seven new compounds, including six linear compounds and one derivative of 1,2,4-triazole. The PASS software was used to estimate the potential probabilities of biological activity of the newly obtained derivatives. Next, studies were carried out to determine the nematocidal potential of the compounds with the use of nematodes of the genus Rhabditis sp. and antibacterial activity using the ACCT standard strains. To determine the lack of cytotoxicity, tests were performed on two cell lines. Additionally, an antioxidant activity test was performed due to the importance of scavenging free radicals in infections with pathogenic microorganisms. The conducted research proved the anthelmintic and antibacterial potential of the newly obtained compounds. The most effective were two compounds with a 3-chlorophenyl substituent, both linear and cyclic derivatives. They demonstrated higher efficacy than the drugs used in treatment.

Exosome secretion and cellular response of DU145 and PC3 after exposure to alpha radiation.

Exosomes are spherical membrane nanovesicles secreted from cells, and they play an important role in tumor immune response, metastasis, angiogenesis, and survival. Studies investigating exosomes isolated from cells exposed to photon radiation commonly used in conventional radiotherapy demonstrate the influence of this type of radiation on exosome characteristics and secretion. There is currently no research investigating the effects of densely ionizing particles such as protons and alpha radiation on exosomes. Thus we have evaluated the cellular response of human prostate cancer cells exposed to 0, 2, and 6 Gy of alpha radiation emitted from the Am-241 source. Irradiated PC3 and DU145 cell lines, characterized by differences in radiosensitivity, were studied using apoptosis, LDH, and IL-6 assays. Additionally, the corresponding concentration and size of isolated exosomes were measured using NTA. We found that exposure to ionizing radiation resulted in gross changes in viability and cell damage. There were increased amounts of apoptotic or necrotic cells as a function of radiation dose. We demonstrated that irradiated PC3 cells secrete higher quantities of exosomes compared to DU145 cells. Additionally, we also found no statistical difference in exosome size for control and irradiated cells.

Alterations in Blood Plasma Metabolome of Patients with Lesniowski-Crohn's Disease Shortly after Surgical Treatment-Pilot Study.

Lesniowski-Crohn's disease (CD) is a type of chronic inflammatory bowel disease (IBD) of uncertain etiology. Initially, pharmacological management is undertaken; however, surgical intervention is necessary to improve life quality and relieve symptoms in most cases. Here changes are reported in blood metabolome that occurred three days after the ileo-colic region resection in the case of seven patients. Alterations are observed in levels of metabolites associated with multiple mitochondrial pathways, based on the Metabolite Set Enrichment Analysis, reflecting a high energy demand in the post-operative period. As most of these metabolites are also essential nutrients supplied from foods, we believe that our results might contribute to the discussion on perioperative nutrition's role in enhanced recovery.

Effect of Hydroxyl Groups Esterification with Fatty Acids on the Cytotoxicity and Antioxidant Activity of Flavones.

Flavonoids and polyunsaturated fatty acids due to low cytotoxicity in vitro studies are suggested as potential substances in the prevention of diseases associated with oxidative stress. We examined novel 6-hydroxy-flavanone and 7-hydroxy-flavone conjugates with selected fatty acids (FA) of different length and saturation and examined their cytotoxic and antioxidant potential. Our findings indicate that the conjugation with FA affects the biological activity of both the original flavonoids. The conjugation of 6-hydroxy-flavanone increased its cytotoxicity towards prostate cancer PC3 cells. The most noticeable effect was found for oleate conjugate. A similar trend was observed for 7-hydroxy-flavone conjugates with the most evident effect for oleate and stearate. The cytotoxic potential of all tested conjugates was not specific towards PC3 because the viability of human keratinocytes HaCaT cells decreased after exposure to all conjugates. Additionally, we showed that esterification of the two flavonoids decreased their antioxidant activity compared to that of the original compounds. Of all the tested compounds, only 6-sorbic flavanone showed a slight increase in antioxidant potential compared to that of the original compound. Our data show that conjugated flavonoids are better absorbed and enhance cytotoxic effects, but the presence of FA lowered the antioxidant potential.

The Effect of Fatty Acids on Ciprofloxacin Cytotoxic Activity in Prostate Cancer Cell Lines-Does Lipid Component Enhance Anticancer Ciprofloxacin Potential?

PURPOSE: To assess cytotoxic effect of ciprofloxacin conjugates with fatty acids on prostate cancer cells (LNCaP and DU-145) with different hormone sensitivity, based on previous promising results from the PC3 cells. METHODS: Cytotoxicity were estimated using MTT and LDH tests, whereas its mechanisms were estimated by apoptosis and IL-6 assays. The intensity of proteins involved in lipid metabolism was determined using ML-CS assay. RESULTS: The hormone insensitive DU-145 cells were more vulnerable than the hormone sensitive LNCaP cells. The IC50 values for oleic (4), elaidic (5) and docosahexaenoic acid (8) conjugates were 20.2 µM, 17.8 µM and 16.5 µM, respectively, in DU-145 cells, whereas in LNCaP cells IC50 exceeded 20 µM. The strong conjugate cytotoxicity was confirmed in the LDH test, the highest (70.8%) for compound (5) and 64.2% for compound (8) in DU-145 cells. This effect was weaker for LNCaP cells (around 60%). The cytotoxic effect of unconjugated ciprofloxacin and fatty acids was weaker. The early apoptosis was predominant in LNCaP while in DU-145 cells both early and late apoptosis was induced. The tested conjugates decreased IL-6 release in both cancer cell lines by almost 50%. Proteomic analysis indicated influence of the ciprofloxacin conjugates on lipid metabolic proteins in prostatic cancer. CONCLUSION: Our findings suggested the cytotoxic potential of ciprofloxacin conjugates with reduction in proteins involved in prostate cancer progress.

Exosomes in Angiogenesis and Anti-angiogenic Therapy in Cancers.

Angiogenesis is the process through which new blood vessels are formed from pre-existing ones. Exosomes are involved in angiogenesis in cancer progression by transporting numerous pro-angiogenic biomolecules like vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and microRNAs. Exosomes promote angiogenesis by suppressing expression of factor-inhibiting hypoxia-inducible factor 1 (HIF-1). Uptake of tumor-derived exosomes (TEX) by normal endothelial cells activates angiogenic signaling pathways in endothelial cells and stimulates new vessel formation. TEX-driven cross-talk of mesenchymal stem cells (MSCs) with immune cells blocks their anti-tumor activity. Effective inhibition of tumor angiogenesis may arrest tumor progression. Bevacizumab, a VEGF-specific antibody, was the first antiangiogenic agent to enter the clinic. The most important clinical problem associated with cancer therapy using VEGF- or VEFGR-targeting agents is drug resistance. Combined strategies based on angiogenesis inhibitors and immunotherapy effectively enhances therapies in various cancers, but effective treatment requires further research.

Matrix Metalloproteinases as Biomarkers of Atherosclerotic Plaque Instability.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases responsible for tissue remodeling and degradation of extracellular matrix (ECM) proteins. MMPs may modulate various cellular and signaling pathways in atherosclerosis responsible for progression and rupture of atherosclerotic plaques. The effect of MMPs polymorphisms and the expression of MMPs in both the atherosclerotic plaque and plasma was shown. They are independent predictors of atherosclerotic plaque instability in stable coronary heart disease (CHD) patients. Increased levels of MMPs in patients with advanced cardiovascular disease (CAD) and acute coronary syndrome (ACS) was associated with future risk of cardiovascular events. These data confirm that MMPs may be biomarkers in plaque instability as they target in potential drug therapies for atherosclerosis. They provide important prognostic information, independent of traditional risk factors, and may turn out to be useful in improving risk stratification.

Tumor-Derived Exosomes in Immunosuppression and Immunotherapy.

Tumor-derived exosomes (TEX) are involved in cancer development, metastasis, and disease progression. They can modulate angiogenesis to elevate the malignant degree of tumor cells. TEX carry immunosuppressive factors affecting the antitumor activities of immune cells. Tumor cells as well as immune cells secrete immunologically active exosomes which affect intercellular communication, antigen presentation, activation of immune cells, and immune surveillance. Cell proliferation and immune response suppression create a favorable microenvironment for tumor. TEX can inhibit immune cell proliferation, induce apoptosis of activated CD8+ Teffs, suppress NK cell activity, interfere with monocyte differentiation, and promote Treg as well as MDSC expansion. Exosomes of microenvironment cells may also contribute to the development of drug resistance in cancer therapy. An important role of TEX in modulating the sensitivity of tumor cells to immunotherapy is a promising area of research to make the cancer therapy more successful.

Molecular mechanisms of ethanol biotransformation: enzymes of oxidative and nonoxidative metabolic pathways in human.

Ethanol, as a small-molecule organic compound exhibiting both hydrophilic and lipophilic properties, quickly pass through the biological barriers. Over 95% of absorbed ethanol undergoes biotransformation, the remaining amount is excreted unchanged, mainly with urine and exhaled air.The main route of ethyl alcohol metabolism is its oxidation to acetaldehyde, which is converted into acetic acid with the participation of cytosolic NAD+ - dependent alcohol (ADH) and aldehyde (ALDH) dehydrogenases. Oxidative biotransformation pathways of ethanol also include reactions catalyzed by the microsomal ethanol oxidizing system (MEOS), peroxisomal catalase and aldehyde (AOX) and xanthine (XOR) oxidases. The resulting acetic acid can be activated to acetyl-CoA by the acetyl-CoA synthetase (ACS).It is also possible, to a much smaller extent, non-oxidative routes of ethanol biotransformation including its esterification with fatty acids by ethyl fatty acid synthase (FAEES), re-esterification of phospholipids, especially phosphatidylcholines, with phospholipase D (PLD), coupling with sulfuric acid by alcohol sulfotransferase (SULT) and with glucuronic acid using UDP-glucuronyl transferase (UGT, syn. UDPGT).The intestinal microbiome plays a significant role in the ethanol biotransformation and in the initiation and progression of liver diseases stimulated by ethanol and its metabolite - acetaldehyde, or by lipopolysaccharide and ROS.

Exosomes in Prostate Cancer Diagnosis, Prognosis and Therapy.

Prostate cancer (PCa) is the second most common cause of cancer-related mortality among men in the developed world. Conventional anti-PCa therapies are not effective for patients with advanced and/or metastatic disease. In most cases, cancer therapies fail due to an incomplete depletion of tumor cells, resulting in tumor relapse. Exosomes are involved in tumor progression, promoting the angiogenesis and migration of tumor cells during metastasis. These structures contribute to the dissemination of pathogenic agents through interaction with recipient cells. Exosomes may deliver molecules that are able to induce the transdifferentiation process, known as "epithelial to mesenchymal transition". The composition of exosomes and the associated possibilities of interacting with cells make exosomes multifaceted regulators of cancer development. Extracellular vesicles have biophysical properties, such as stability, biocompatibility, permeability, low toxicity and low immunogenicity, which are key for the successful development of an innovative drug delivery system. They have an enhanced circulation stability and bio-barrier permeation ability, and they can therefore be used as effective chemotherapeutic carriers to improve the regulation of target tissues and organs. Exosomes have the capacity to deliver different types of cargo and to target specific cells. Chemotherapeutics, natural products and RNA have been encapsulated for the treatment of prostate cancers.

Synthesis and Biological Evaluation of Novel Indole-Derived Thioureas.

A series of 2-(1H-indol-3-yl)ethylthiourea derivatives were prepared by condensation of 2-(1H-indol-3-yl)ethanamine with appropriate aryl/alkylisothiocyanates in anhydrous media. The structures of the newly synthesized compounds were confirmed by spectroscopic analysis and the molecular structures of 8 and 28 were confirmed by X-ray crystallography. All obtained compounds were tested for antimicrobial activity against Gram-positive cocci, Gram-negative rods and for antifungal activity. Microbiological evaluation was carried out over 20 standard strains and 30 hospital strains. Compound 6 showed significant inhibition against Gram-positive cocci and had inhibitory effect on the S. aureus topoisomerase IV decatenation activity and S. aureus DNA gyrase supercoiling activity. Compounds were tested for cytotoxicity and antiviral activity against a large panel of DNA and RNA viruses, including HIV-1 and other several important human pathogens. Interestingly, derivative 8 showed potent activity against HIV-1 wild type and variants bearing clinically relevant mutations. Newly synthesized tryptamine derivatives showed also a wide spectrum activity, proving to be active against positive- and negative-sense RNA viruses.

Disubstituted 4-Chloro-3-nitrophenylthiourea Derivatives: Antimicrobial and Cytotoxic Studies.

4-Chloro-3-nitrophenylthioureas 1⁻30 were synthesized and tested for their antimicrobial and cytotoxic activities. Compounds exhibited high to moderate antistaphylococcal activity against both standard and clinical strains (MIC values 2⁻64 µg/mL). Among them derivatives with electron-donating alkyl substituents at the phenyl ring were the most promising. Moreover, compounds 1⁻6 and 8⁻19 were cytotoxic against MT-4 cells and various other cell lines derived from human hematological tumors (CC50 ≤ 10 µM). The influence of derivatives 11, 13 and 25 on viability, mortality and the growth rate of immortalized human keratinocytes (HaCaT) was observed.

Synthesis, Structural Studies and Biological Evaluation of Connections of Thiosemicarbazide, 1,2,4-Triazole and 1,3,4-Thiadiazole with Palmitic Acid.

Thirty new derivatives of palmitic acid were efficiently synthesized. All obtained compounds can be divided into three groups of derivatives: Thiosemicarbazides (compounds 1-10), 1,2,4-triazoles (compounds 1a-10a) and 1,3,4-thiadiazoles (compounds 1b-10b) moieties. ¹H-NMR, 13C-NMR and MS methods were used to confirm the structure of derivatives. All obtained compounds were tested in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative rods and Candida albicans. Compounds 4, 5, 6, 8 showed significant inhibition against C. albicans. The range of MIC values was 50-1.56 µg/mL. The halogen atom, especially at the 3rd position of the phenyl group was significantly important for antifungal activity. The biological activity against Candida albicans and selected molecular descriptors were used as a basis for QSAR models, that have been determined by means of multiple linear regression. The models have been validated by means of the Leave-One-Out Cross Validation. The obtained QSAR models were characterized by high determination coefficients and good prediction power.

Cytochrome P450 polymorphism--molecular, metabolic, and pharmacogenetic aspects. IV. Application of long polymerase chain reaction for identification of CYP2D6 gene duplication.

In the human organism 58 cytochrome P450 (CYP) isoenzymes belonging to 18 families have been described. Isoenzyme CYP2D6 is an important human xenobiotic-metabolizing enzyme. CYP2D6 biotransforms a significant number of drugs, widely used in clinical practice, such as antidepressants, neuroleptics, antiarrhythmics, analgesics, antiemetics and anticancer agents. The occurrence of polymorphic variants of the enzyme results in different metabolic capacity ranging from poor to ultrarapid. Ultrarapid metabolizer phenotype explains lack of response and decreased levels of drugs which are metabolized by CYP2D6. Therefore, the identification of ultrarapid metabolizers as potential non-compliance cases requiring dose adjustment, has serious clinical importance. In this study we evaluate a long-PCR procedure for detecting CYP2D6 gene duplication.

Cytochrome P450 polymorphism--molecular, metabolic and pharmacogenetic aspects. I. Mechanisms of activity of cytochrome P450 monooxygenases.

Cytochrome P450, initially perceived as a type of cell pigment, was soon identified as a hemoprotein with an enzymatic activity characteristic for monooxygenases with an affinity for differentiated endo- or exogenous substrates, including drugs. So far in the human organism 58 CYP isoenzymes belonging to 18 families have been described. Most from the CYP monooxygenases superfamily turned out to be integral elements of hepatocytic reticular monooxygenase complexes which also contain NADPH-dependent cytochrome P450 reductase (CPR). Later investigations indicated the possibility of the participation in electron transport for reticular CYP isoenzymes, alternative NADH-dependent reticular system composed of cytochrome b5 reductase (CBR) and cytochrome b5. The demonstration of the activity of some CYP superfamily isoenzymes not only in hepatocytes but also in many other cells of the human organism, numerous plant and animal tissues and even in cells of fungi, protists and prokaryotes has contributed to the significantly increased understanding of the role of CYP in biological systems. In addition, some CYP isoenzymes were found to be characteristic for the inner mitochondrial membrane monooxygenase complexes which contain NADPH-dependent adrenodoxin reductase (AR) and adrenodoxin (Ad), which is identical with ferredoxin-1 (Fd-1) and hepatoredoxin (Hd).

Cytochrome P450 polymorphism--molecular, metabolic, and pharmacogenetic aspects. II. Participation of CYP isoenzymes in the metabolism of endogenous substances and drugs.

In the human organism 58 cytochrome P450 (CYP) isoenzymes belonging to 18 families have been described. These hemoproteins, with enzymatic activity characteristic for monooxygenases, show a broad affinity for chemically differentiated endo- or exogenous compounds, including drugs. CYP isoenzymes participate in metabolic pathways important for proper physiological functioning of the human organism, i.e.: cholesterol, bile acid and oxysterol biosynthesis; metabolism of fatty acids, prostaglandins, prostacyclins, leukotrienes, steroid hormones, ketone bodies, vitamines A and D. CYP isoenzymes participate in the metabolism of over 80% of drugs and other xenobiotic substances which can be present in the human organism. Differences in molecular structure and kinetics of conformational changes of particular isoenzymes of CYP superfamily monooxygenases on the one hand determine their affinity direction for chemically differentiated groups of compounds susceptible to oxidation, on the other hand determine the mechanism and position of the oxidative change of their molecules. Drugs and their metabolites and other endogenous and xenobiotic compounds may be acting not only as substrates, but also as competitive and non- competitive inhibitors, suicide inhibitors and inducers of CYP isoenzymes as well as repressors of CYP genes. These relationships are the metabolic basis of numerous multidirectional interactions between drugs, drug metabolites, food components, stimulants, environmental toxins and metabolites of these xenobiotics.

Cytochrome P450 polymorphism--molecular, metabolic, and pharmacogenetic aspects. III. Influence of CYP genetic polymorphism on population differentiation of drug metabolism phenotype.

In the human genome 684 alleles of CYP genes, and additionally 30 complete CYP pseudogenes, have been identified. So far 388 isoforms of 58 human CYP isoenzymes have been described at the phenotypic level. The molecular forms of many CYP isoenzymes responsible for drug biotransformation show a differentiated degree of specific catalytic activity - from increased, through normal and decreased to various extent, to trace or even absent. Depending on the homo- or heterozygous genotype, a broad palette of phenotypic forms may be present, differentiated in respect to biotransformation dynamics of specific drugs. The progress of molecular biology with particular consideration of genotyping and DNA microarray technologies has created a basis for the dynamic progress of pharmacogenetics, allowing fast and sensitive determination of the individual pharmacogenetic profile, encompassing a large set of CYP alleles extended by allelic variants of genes encoding other enzymes participating in drug metabolism. The possibility to evaluate the pharmacogenetic profile of patients together with the increasing knowledge about the mechanisms of inhibition, repression and also induction of enzymes participating in biotransformation of xenobiotics and endogenous compounds create increasing possibilities of elaborating optimal individualized pharmacotherapeutic strategies.