Coumarins-lipophilic cations conjugates: Efficient mitocans targeting carbonic anhydrases.

Being aware of the need to develop more efficient therapies against cancer, herein we disclose an innovative approach for the design of selective antiproliferative agents. We have accomplished the conjugation of a coumarin fragment with lipophilic cations (triphenylphosphonium salts, guanidinium) for providing mitochondriotropic agents that simultaneously target also carbonic anhydrases IX and XII, involved in the development and progression of cancer. The new compounds prepared herein turned out to be strong inhibitors of carbonic anhydrases IX and XII of human origin (low-to-mid nM range), also endowed with high selectivity, exhibiting negligible activity towards cytosolic CA isoforms. Key interactions with the enzyme were analysed using docking and molecular dynamics simulations. Regarding their in vitro antiproliferative activities, an increase of the tether length connecting both pharmacophores led to a clear improvement in potency, reaching the submicromolar range for the lead compounds, and an outstanding selectivity towards tumour cell lines (S.I. up to >357). Cytotoxic effects were also analysed on MDR cell lines under hypoxic and normoxic conditions. Chemoresistance exhibited by phosphonium salts, and not by guanidines, against MDR cells was based on the fact that the former were found to be substrates of P-glycoprotein (P-gp), the pump responsible for extruding foreign chemicals; this situation was reversed by administrating tariquidar, a third generation P-gp inhibitor. Moreover, phosphonium salts provoked a profound depolarization of mitochondria membranes from tumour cells, thus probably compromising their oxidative metabolism. To gain insight into the mode of action of title compounds, continuous live cell microscopy was employed; interestingly, this technique revealed two different antiproliferative mechanisms for both families of mitocans. Whereas phosphonium salts had a cytostatic effect, blocking cell division, guanidines led to cell death via apoptosis.

Factors Associated with Length of Hospitalization in Patients with Diabetes and Mild COVID-19: Experiences from a Tertiary University Center in Serbia.

Background and Objectives: During the COVID-19 pandemic, there was an increased number of hospitalized COVID-19-positive patients suffering from type 2 diabetes mellitus (T2DM). The objective of this research study was to explore factors associated with the length of hospitalization of patients with T2DM and the mild form of COVID-19. Material and Methods: This retrospective cohort study involved all patients who tested positive for COVID-19 and those who were treated in the dedicated COVID-19 department of the University Clinical Center (UCC) in Nis between 10 September 2021 and 31 December 2021. Upon admission, patients underwent blood tests for biochemical analysis, including blood count, kidney and liver function parameters (C-reactive protein (CRP), creatinine kinase, and D-dimer), and glycemia and HbA1c assessments. Additionally, all patients underwent lung radiography. Univariate and multivariate regression analyses were employed to assess the impact of specific factors on the length of hospitalization among patients with T2DM. Results: Out of a total of 549 treated COVID-19-positive patients, 124 (21.0%) had T2DM, while 470 (79.0%) did not have diabetes. Among patients with T2DM, men were significantly younger than women (60.6 ± 16.8 vs. 64.2 ± 15.3, p < 0.01). The average hospitalization length of patients with diabetes was 20.2 ± 9.6 (5 to 54 days), and it was significantly longer than for patients without diabetes, at 15.0 ± 3.4, which ranged from 3 days to 39 (t-test ≈ 5.86, p < 0.05). According to the results of the univariate regression analysis, each year of age is associated with an increase in the length of hospital stay of 0.06 days (95% CI: 0.024 to 0.128, p = 0.004). Patients who received oxygen therapy were treated for 2.8 days longer than those who did not receive oxygen treatment (95% CI: 0.687 to 4988, p = 0.010), and each one-unit increase in CRP level was associated with a 0.02-day reduction in the length of hospitalization (95% CI: 0.004 to 0.029, p = 0.008). Based on the results of the multivariate regression analysis, each year of age is associated with an increase in the length of hospitalization by 0.07 days (95% CI: 0.022 to 0.110, p = 0.003). Patients who received oxygen therapy were treated for 3.2 days longer than those who did not receive oxygen therapy (95% CI: 0.653 to 5726, p = 0.014), and each unit increase in CRP level was associated with a 0.02-day reduction in the length of hospitalization (95% CI: 0.005 to 0.028, p = 0.004). Conclusions: Based on the presented results, COVID-19-positive patients with diabetes had, on average, longer hospitalizations than COVID-19 patients without diabetes. The hospital treatment of patients with T2DM and a milder form of COVID-19 was associated with older age, the use of oxygen therapy, and elevated CRP values. Patients who received oxygen therapy were treated approximately 3 days longer than those who did not receive this therapy.

Significance of Duodenal Prolactin Receptor Modulation by Calcium and Vitamin D in Sulpiride-Induced Hyperprolactinemia.

Background and Objectives: Hyperprolactinemia, as a potential side-effect of some antipsychotic medications, is associated with decreased bone density and an increased risk of fractures. This study investigates whether calcium and vitamin D supplementation affects prolactin receptor (Prlr) gene expression in the duodenum, vertebrae, and kidneys of female rats with sulpiride-induced hyperprolactinemia. Materials and Methods: Twenty-one-week-old female Wistar rats were assigned to three groups: Group S consisted of ten rats who received sulpiride injections (10 mg/kg) twice daily for 6 weeks; Group D (10 rats) received daily supplementation of 50 mg calcium and 500 IU vitamin D along with sulpiride for the last 3 weeks; and Group C consisting of seven age-matched nulliparous rats serving as a control group. Real-time PCR was used to assess Prlr gene expression in the duodenum, vertebrae, and kidneys. Results: In Group S, Prlr gene expression was notably decreased in the duodenum (p < 0.01) but elevated in the vertebrae and kidneys compared to Group C. Conversely, Group D exhibited significantly increased Prlr expression in the duodenum (p < 0.01) alongside elevated expression in the vertebrae and kidneys. Conclusions: In sulpiride-induced hyperprolactinemia, decreased Prlr gene expression in the duodenum may lead to reduced intestinal calcium absorption. Consequently, prolactin may draw calcium from the skeletal system to maintain calcium balance, facilitated by increased Prlr gene expression in the vertebrae. However, vitamin D supplementation in sulpiride-induced hyperprolactinemia notably enhances Prlr gene expression in the duodenum, potentially ameliorating intestinal calcium absorption and mitigating adverse effects on bone health.

Novel hybrids of sclareol and 1,2,4-triazolo[1,5-a]pyrimidine show collateral sensitivity in multidrug-resistant glioblastoma cells.

The synthesis of 24 hybrid molecules, consisting of naturally occurring sclareol (SCL) and synthetic 1,2,4-triazolo[1,5-a]pyrimidines (TPs), is described. New compounds were designed with the aim of improving the cytotoxic properties, activity, and selectivity of the parent compounds. Six analogs (12a-f) contained 4-benzylpiperazine linkage, while 4-benzyldiamine linkage was present in eighteen derivatives (12g-r and 13a-f). Hybrids 13a-f consist of two TP units. After purification, all hybrids (12a-r and 13a-f), as well as their precursors (9a-e and 11a-c), were tested on human glioblastoma U87 cells. More than half of the tested synthesized molecules, 16 out of 31, caused a significant reduction of U87 cell viability (more than 75% reduction) at 30 µM. The concentration-dependent cytotoxicity of these 16 compounds was also examined on U87 cells, corresponding multidrug-resistant (MDR) U87-TxR cells with increased P-glycoprotein (P-gp) expression and activity, and normal lung fibroblasts MRC-5. Importantly, 12l and 12r were active in the nanomolar range, while seven compounds (11b, 11c, 12i, 12l, 12n, 12q, and 12r) were more selective towards glioblastoma cells than SCL. All compounds except 12r evaded MDR, showing even better cytotoxicity in U87-TxR cells. In particular, 11c, 12a, 12g, 12j, 12k, 12m, 12n, and SCL showed collateral sensitivity. Hybrid compounds 12l, 12q, and 12r decreased P-gp activity to the same extent as a well-known P-gp inhibitor - tariquidar (TQ). Hybrid compound 12l and its precursor 11c affected different cellular processes including the cell cycle, cell death, and mitochondrial membrane potential, and changed the levels of reactive oxygen and nitrogen species (ROS/RNS) in glioblastoma cells. Collateral sensitivity towards MDR glioblastoma cells was caused by the modulation of oxidative stress accompanied by inhibition of mitochondria.

Biotinylated selenocyanates: Potent and selective cytostatic agents.

Most of the currently available cytotoxic agents for tackling cancer are devoid of selectivity, thus causing severe side-effects. This situation stimulated us to develop new antiproliferative agents with enhanced affinity towards tumour cells. We focused our attention on novel chalcogen-containing compounds (thiosemicarbazones, disulfides, selenoureas, thio- and selenocyanates), and particularly on selenium derivatives, as it has been documented that this kind of compounds might act as prodrugs releasing selenium-based reactive species on tumour cells. Particularly interesting in terms of potency and selectivity was a pharmacophore comprised by a selenocyanato-alkyl fragment connected to a p-phenylenediamine residue, where the nature of the second amino moiety (free, Boc-protected, enamine-protected) provided a wide variety of antiproliferative activities, ranging from the low micromolar to the nanomolar values. The optimized structure was in turn conjugated through a peptide linkage with biotin (vitamin B7), a cellular growth promoter, whose receptor is overexpressed in numerous cancer cells; the purpose was to develop a selective vector towards malignant cells. Such biotinylated derivative behaved as a very strong antiproliferative agent, achieving GI50 values in the low nM range for most of the tested cancer cells; moreover, it was featured with an outstanding selectivity, with GI50 > 100 µM against human fibroblasts. Mechanistic studies on the mode of inhibition of the biotinylated selenocyanate revealed (Annexin-V assay) a remarkable increase in the number of apoptotic cells compared to the control experiment; moreover, depolarization of the mitochondrial membrane was detected by flow cytometry analysis, and with fluorescent microscopy, what supports the apoptotic cell death. Prior to the apoptotic events, cytostatic effects were observed against SW1573 cells using label-free cell-living imaging; therefore, tumour cell division was prevented. Multidrug resistant cell lines exhibited a reduced sensitivity towards the biotinylated selenocyanate, probably due to its P-gp-mediated efflux. Remarkably, antiproliferative levels could be restored by co-administration with tariquidar, a P-gp inhibitor; this approach can, therefore, overcome multidrug resistance mediated by the P-gp efflux system.

Okara-Enriched Gluten-Free Bread: Nutritional, Antioxidant and Sensory Properties.

The aim of this study was to produce an eco-innovative gluten-free bread with a pleasant taste and a unique formulation that includes the highest quality grains and pseudocereals (buckwheat; rice; and millet); and okara; a by-product of soy milk production. The mixture of pseudocereal and cereal flour contained buckwheat flour 45%, rice flour 33%, and millet flour 22%. Three gluten-free breads; each containing different contents of gluten-free flour (90%, 80%, and 70%, respectively); okara (10%, 20%, and 30%, respectively); and a control sample (without okara); were prepared and subjected to sensory evaluation. The okara-enriched gluten-free bread with the highest sensory score was selected for further analysis of physico-chemical (total proteins; total carbohydrates; insoluble fiber; soluble fiber; sugars; total lipids; saturated fatty acids; and salt) and functional properties (total phenolic content and antioxidant properties). The highest sensory scores were obtained for 30% okara-enriched gluten-free bread including taste; shape; odor; chewiness; and cross-section properties; classifying this bread in the category of very good quality and excellent quality (mean score 4.30 by trained evaluators and 4.59 by consumers). This bread was characterized by a high content of dietary fiber (14%), the absence of sugar; low content of saturated fatty acids (0.8%), rich source of proteins (8.8%) and certain minerals (e.g.,; iron; zinc); and low energy value (136.37 kcal/100g DW). Total phenolic content was 133.75 mgGAE/100g FW; whereas ferric reducing power; ABTS radical cation; and DPPH radical scavenging activity were 119.25 mgAA/100g FW; 86.80 mgTrolox/100g FW; and 49.92 mgTrolox/100g FW; respectively. Okara addition in gluten-free bread production enables the formulation of high-nutritive; good antioxidative; low-energy bread; and better soy milk waste management.

Stochastic Fluctuations Drive Non-genetic Evolution of Proliferation in Clonal Cancer Cell Populations.

Evolutionary dynamics allows us to understand many changes happening in a broad variety of biological systems, ranging from individuals to complete ecosystems. It is also behind a number of remarkable organizational changes that happen during the natural history of cancers. These reflect tumour heterogeneity, which is present at all cellular levels, including the genome, proteome and phenome, shaping its development and interrelation with its environment. An intriguing observation in different cohorts of oncological patients is that tumours exhibit an increased proliferation as the disease progresses, while the timescales involved are apparently too short for the fixation of sufficient driver mutations to promote explosive growth. Here, we discuss how phenotypic plasticity, emerging from a single genotype, may play a key role and provide a ground for a continuous acceleration of the proliferation rate of clonal populations with time. We address this question by combining the analysis of real-time growth of non-small-cell lung carcinoma cells (N-H460) together with stochastic and deterministic mathematical models that capture proliferation trait heterogeneity in clonal populations to elucidate the contribution of phenotypic transitions on tumour growth dynamics.

In vitro biomimetic models for glioblastoma-a promising tool for drug response studies.

The poor response of glioblastoma to current treatment protocols is a consequence of its intrinsic drug resistance. Resistance to chemotherapy is primarily associated with considerable cellular heterogeneity, and plasticity of glioblastoma cells, alterations in gene expression, presence of specific tumor microenvironment conditions and blood-brain barrier. In an attempt to successfully overcome chemoresistance and better understand the biological behavior of glioblastoma, numerous tri-dimensional (3D) biomimetic models were developed in the past decade. These novel advanced models are able to better recapitulate the spatial organization of glioblastoma in a real time, therefore providing more realistic and reliable evidence to the response of glioblastoma to therapy. Moreover, these models enable the fine-tuning of different tumor microenvironment conditions and facilitate studies on the effects of the tumor microenvironment on glioblastoma chemoresistance. This review outlines current knowledge on the essence of glioblastoma chemoresistance and describes the progress achieved by 3D biomimetic models. Moreover, comprehensive literature assessment regarding the influence of 3D culturing and microenvironment mimicking on glioblastoma gene expression and biological behavior is also provided. The contribution of the blood-brain barrier as well as the blood-tumor barrier to glioblastoma chemoresistance is also reviewed from the perspective of 3D biomimetic models. Finally, the role of mathematical models in predicting 3D glioblastoma behavior and drug response is elaborated. In the future, technological innovations along with mathematical simulations should create reliable 3D biomimetic systems for glioblastoma research that should facilitate the identification and possibly application in preclinical drug testing and precision medicine.

Advanced technological tools to study multidrug resistance in cancer.

The complexity of cancer biology and its clinical manifestation are driven by genetic, epigenetic, transcriptomic, proteomic and metabolomic alterations, supported by genomic instability as well as by environmental conditions and lifestyle factors. Although novel therapeutic modalities are being introduced, efficacious cancer therapy is not achieved due to the frequent emergence of distinct mechanisms of multidrug resistance (MDR). Advanced technologies with the potential to identify and characterize cancer MDR could aid in selecting the most efficacious therapeutic regimens and prevent inappropriate treatments of cancer patients. Herein, we aim to present technological tools that will enhance our ability to surmount drug resistance in cancer in the upcoming decade. Some of these tools are already in practice such as next-generation sequencing. Identification of genes and different types of RNAs contributing to the MDR phenotype, as well as their molecular targets, are of paramount importance for the development of new therapeutic strategies aimed to enhance drug response in resistant tumors. Other techniques known for many decades are in the process of adaptation and improvement to study cancer cells' characteristics and biological behavior including atomic force microscopy (AFM) and live-cell imaging. AFM can monitor in real-time single molecules or molecular complexes as well as structural alterations occurring in cancer cells induced upon treatment with various antitumor agents. Cell tracking methodologies and software tools recently progressed towards quantitative analysis of the spatio-temporal dynamics of heterogeneous cancer cell populations and enabled direct monitoring of cells and their descendants in 3D cultures. Besides, novel 3D systems with the advanced mimicking of the in vivo tumor microenvironment are applicable to study different cancer biology phenotypes, particularly drug-resistant and aggressive ones. They are also suitable for investigating new anticancer treatment modalities. The ultimate goal of using phenotype-driven 3D cultures for the investigation of patient biopsies as the most appropriate in vivo mimicking model, can be achieved in the near future.

Nano-Motion Analysis for Rapid and Label Free Assessing of Cancer Cell Sensitivity to Chemotherapeutics.

Background and Objectives: Optimization of chemotherapy is crucial for cancer patients. Timely and costly efficient treatments are emerging due to the increasing incidence of cancer worldwide. Here, we present a methodology of nano-motion analysis that could be developed to serve as a screening tool able to determine the best chemotherapy option for a particular patient within hours. Materials and Methods: Three different human cancer cell lines and their multidrug resistant (MDR) counterparts were analyzed with an atomic force microscope (AFM) using tipless cantilevers to adhere the cells and monitor their nano-motions. Results: The cells exposed to doxorubicin (DOX) differentially responded due to their sensitivity to this chemotherapeutic. The death of sensitive cells corresponding to the drop in signal variance occurred in less than 2 h after DOX application, while MDR cells continued to move, even showing an increase in signal variance. Conclusions: Nano-motion sensing can be developed as a screening tool that will allow simple, inexpensive and quick testing of different chemotherapeutics for each cancer patient. Further investigations on patient-derived tumor cells should confirm the method's applicability.

A perspective view on the nanomotion detection of living organisms and its features.

The insurgence of newly arising, rapidly developing health threats, such as drug-resistant bacteria and cancers, is one of the most urgent public-health issues of modern times. This menace calls for the development of sensitive and reliable diagnostic tools to monitor the response of single cells to chemical or pharmaceutical stimuli. Recently, it has been demonstrated that all living organisms oscillate at a nanometric scale and that these oscillations stop as soon as the organisms die. These nanometric scale oscillations can be detected by depositing living cells onto a micro-fabricated cantilever and by monitoring its displacements with an atomic force microscope-based electronics. Such devices, named nanomotion sensors, have been employed to determine the resistance profiles of life-threatening bacteria within minutes, to evaluate, among others, the effect of chemicals on yeast, neurons, and cancer cells. The data obtained so far demonstrate the advantages of nanomotion sensing devices in rapidly characterizing microorganism susceptibility to pharmaceutical agents. Here, we review the key aspects of this technique, presenting its major applications. and detailing its working protocols.

Analysis of association of ADORA2A and ADORA3 polymorphisms genotypes/haplotypes with efficacy and toxicity of methotrexate in patients with Rheumatoid arthritis.

Adenosine receptors ADORA2A and ADORA3 are part of the adenosine-mediated antiinflammatory pathway and are overexpressed in patients with Rheumatoid arthritis (RA). Methotrexate (MTX) antiinflammatory effects are partially mediated via increased release of adenosine into extracellular space. Polymorphisms in ADORA2A and ADORA3 genes may have an impact on the efficacy and toxicity of MTX in RA patients. The study included 127 RA patients. Treatment efficacy was estimated using the changes in Disease activity score (DAS28) after 6 months of MTX monotherapy, according to EULAR response criteria. Patients with good and moderate response were classified as "responders", and with a poor response as "nonresponders". Adverse effects were collected during the follow-up period. Genotyping for polymorphisms within ADORA2A gene (rs2298383, rs2236624, rs5751876, rs17004921) and ADORA3 gene (rs2298191, rs1544223, rs3393) was performed using the KASPar assays. Among patients 112 (88.19%) were responders (18.8% good, 81.2% moderate). We observed no association between analyzed genotypes or alleles and MTX response by EULAR criteria but carriers of ADORA2A rs17004921 T allele (CT + TT) had a higher DAS28 decrease after 6 months of treatment than patients with CC genotype (p = 0.013). Adverse effects were reported in 31 patients (24.41%). Bone erosions were present in 82 (64.6%) patients. Haplotype block was observed among all 3 analyzed polymorphisms within ADORA3 gene and TAA haplotype was associated with bone erosions (29% vs 15.6%, p = 0.023) and hepatotoxicity (51.3% vs 21.6%, p = 0.013). According to our study, ADORA3 TAA haplotype may be associated with bone erosions and hepatotoxicity in RA patients treated with MTX.

CKT0353, a novel microtubule targeting agent, overcomes paclitaxel induced resistance in cancer cells.

Microtubule targeting agents (MTAs) are extensively used in cancer treatment and many have achieved substantial clinical success. In recent years, targeting microtubules to inhibit cell division has become a widespread pharmaceutical approach for treatment of various cancer types. Nevertheless, the development of multidrug resistance (MDR) in cancer remains a major obstacle for successful application of these agents. Herein, we provided the evidence that CKT0353, α-branched α,ß-unsaturated ketone, possesses the capacity to successfully evade the MDR phenotype as an MTA. CKT0353 induced G2/M phase arrest, delayed cell division via spindle assembly checkpoint activation, disrupted the mitotic spindle formation and depolymerized microtubules in human breast, cervix, and colorectal carcinoma cells. Molecular docking analysis revealed that CKT0353 binds at the nocodazole binding domain of ß-tubulin. Furthermore, CKT0353 triggered apoptosis via caspase-dependent mechanism. In addition, P-glycoprotein overexpressing colorectal carcinoma cells showed higher sensitivity to this agent when compared to their sensitive counterpart, demonstrating the ability of CKT0353 to overcome this classic MDR mechanism involved in resistance to various MTAs. Taken together, these findings suggest that CKT0353 is an excellent candidate for further optimization as a therapeutic agent against tumors with MDR phenotype.

Population pharmacokinetic analysis of bisoprolol in type 2 diabetic patients with hypertension.

PURPOSE: Given that it has been reported that type 2 diabetes mellitus may affect the pharmacokinetics of a large number of drugs and that there are still no published population pharmacokinetic (PopPK) analyses in routinely treated patients with hypertension and type 2 diabetes mellitus as comorbid condition, the aim of this study was to determine PK variability of bisoprolol in 70 Serbian patients using the PopPK approach. METHODS: PopPK analysis was conducted using a nonlinear mixed effects model (NONMEM), version 7.3.0 (Icon Development Solutions). In our patients, a total daily dose of bisoprolol ranged from 1.25 to 10 mg. The drug was administrated orally as a single daily dose or in two divided doses per day. RESULTS: A wide range of the drug concentrations were noted (1-103 ng/mL) in the population consisted of the adult patients with type 2 diabetes mellitus. From a total of 21 separately assessed covariates, our results indicated that only creatinine clearance could have a potential impact on the variability of the clearance of bisoprolol. CONCLUSION: Routine assessment of renal function should be carried out before the initiation of treatment with bisoprolol in order to individualize the dose and to prevent possible accumulation and adverse drug reactions.

What sustains the multidrug resistance phenotype beyond ABC efflux transporters? Looking beyond the tip of the iceberg.

Identification of multidrug (MDR) efflux transporters that belong to the ATP-Binding Cassette (ABC) superfamily, represented an important breakthrough for understanding cancer multidrug resistance (MDR) and its possible overcoming. However, recent data indicate that drug resistant cells have a complex intracellular physiology that involves constant changes in energetic and oxidative-reductive metabolic pathways, as well as in the molecular circuitries connecting mitochondria, endoplasmic reticulum (ER) and lysosomes. The aim of this review is to discuss the key molecular mechanisms of cellular reprogramming that induce and maintain MDR, beyond the presence of MDR efflux transporters. We specifically highlight how cancer cells characterized by high metabolic plasticity - i.e. cells able to shift the energy metabolism between glycolysis and oxidative phosphorylation, to survive both the normoxic and hypoxic conditions, to modify the cytosolic and mitochondrial oxidative-reductive metabolism, are more prone to adapt to exogenous stressors such as anti-cancer drugs and acquire a MDR phenotype. Similarly, we discuss how changes in mitochondria dynamics and mitophagy rates, changes in proteome stability ensuring non-oncogenic proteostatic mechanisms, changes in ubiquitin/proteasome- and autophagy/lysosome-related pathways, promote the cellular survival under stress conditions, along with the acquisition or maintenance of MDR. After dissecting the complex intracellular crosstalk that takes place during the development of MDR, we suggest that mapping the specific adaptation pathways underlying cell survival in response to stress and targeting these pathways with potent pharmacologic agents may be a new approach to enhance therapeutic efficacy against MDR tumors.

Analysis of the Association Between Polymorphisms within PAI-1 and ACE genes and Ischemic Stroke Outcome After rt-PA Therapy.

PURPOSE: Treatment of Ischemic stroke (IS) in acute phase is based on the use of thrombolytic rt-PA therapy. We aimed to determine whether different alleles and genotypes of I/D ACE gene and 4G/5G PAI-1 gene polymorphisms may influence outcome of rt-PA therapy in patients with IS and the occurrence of haemorrhagic transformation (HT). METHODS: Our study included 94 consecutive patients with IS treated with rt-PA. Modified Rankin Scale (mRS) at 3rd month after IS was used to determine the stroke outcome, with scores 0-1 defining the favourable outcome, and scores 2-6 defining poor outcome. Genotypisation of the ACE-1 I/D polymorphism was performed by polymerase chain reaction and of the PAI-1 4G/5G polymorphism by polymerase chain reaction - restriction fragment length analysis. RESULTS: Regarding PAI-I 4G/5G polymorphism, 44 patients (46.8%) were heterozygotes, and the number of 4G/4G and 5G/5G homozygotes was the same - 25 each (26.6%). Number of heterozygotes for the ACE I/D polymorphism was 54 (57.4%), 9 patients (9.6%) had II, and 31 (33%) DD genotypes. A favourable outcome was recorded in 26 (28.0%) and the poor outcome in 67 (72.0%) patients. Favourable and poor outcome groups did not differ significantly in PAI-1 4G/5G and ACE I/D polymorphisms genotype or allele frequencies. There was a statistically significant difference in the occurrence of HT between patients with ACE II and patients with ACE ID or DD genotypes (p=0.035). CONCLUSION: Results of our study suggest that stroke patients with ACE II genotype, treated with rt-PA, may be at risk of HT.

Design, synthesis, and biological evaluation of novel derivatives of dithiodiglycolic acid prepared via oxidative coupling of thiols.

Human thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing enzyme which plays a crucial role in regulating numerous redox signalling pathways within the cell. While its functioning is important in all cells, levels of TrxR1 expression are higher in cancer cells, possibly as an adaptation to much higher levels of reactive oxygen species and the need for more extensive DNA synthesis. This makes TrxR1 an attractive target for cancer therapy development. Inspired by the structure of disulphide compounds which have advanced through various stages of clinical development, we designed a series of dithiodiglycolic acid derivatives. These were prepared from respective thiol synthons using an iodine- or benzotriazolyl chloride-promoted oxidative disulphide bond formation. Inhibition of TrxR present in cell lysates from human neuroblastoma cells (SH-SY5Y) and rat liver cells indicated several compounds with a potential for TrxR inhibition. Some of these compounds were also tested for growth inhibition against two human cancer cell lines and normal human keratinocytes.

Novel Heat Shock Protein 90 Inhibitors Suppress P-Glycoprotein Activity and Overcome Multidrug Resistance in Cancer Cells.

Heat Shock Protein 90 (Hsp90) chaperone interacts with a broad range of client proteins involved in cancerogenesis and cancer progression. However, Hsp90 inhibitors were unsuccessful as anticancer agents due to their high toxicity, lack of selectivity against cancer cells and extrusion by membrane transporters responsible for multidrug resistance (MDR) such as P-glycoprotein (P-gp). Recognizing the potential of new compounds to inhibit P-gp function and/or expression is essential in the search for effective anticancer drugs. Eleven Hsp90 inhibitors containing an isoxazolonaphtoquinone core were synthesized and evaluated in two MDR models comprised of sensitive and corresponding resistant cancer cells with P-gp overexpression (human non-small cell lung carcinoma and colorectal adenocarcinoma). We investigated the effect of Hsp90 inhibitors on cell growth inhibition, P-gp activity and P-gp expression. Structure-activity relationship analysis was performed in respect to cell growth and P-gp inhibition. Compounds 5, 7, and 9 directly interacted with P-gp and inhibited its ATPase activity. Their potential P-gp binding site was identified by molecular docking studies. In addition, these compounds downregulated P-gp expression in MDR colorectal carcinoma cells, showed good relative selectivity towards cancer cells, while compound 5 reversed resistance to doxorubicin and paclitaxel in concentration-dependent manner. Therefore, compounds 5, 7 and 9 could be promising candidates for treating cancers with P-gp overexpression.

Chokeberry juice supplementation in type 2 diabetic patients - impact on health status.

Recent literature evidence indicates the potential use of chokeberry preparations in the prevention and treatment of some chronic noncommunicable diseases. The aim of the present study was to evaluate the effects of the three months oral chokeberry juice supplementation in type 2 diabetic patients, as well as its influence on hematological parameters and certain parameters of the renal dysfunction. The study was designed as an open-label trial, which included 35 patients who have received the herbal supplement, polyphenol-rich chokeberry juice (150 ml/day, three times a day for 50 ml), in addition to their standard therapy. Chokeberry juice as a rich source of polyphenol compounds could be an effective preventive and therapeutic agent in diabetes mellitus type 2. Hematological and biochemical parameters were measured at baseline, after 3 months with the chokeberry juice supplementation and after the next 3 months without the chokeberry juice supplementation (follow-up period). Significant difference was noticed in the levels of LDL-cholesterol, glycated hemoglobin and serum creatinine (p < 0.05), as well as in the levels of some hematological parameters, such as white blood cell and lymphocyte count (p < 0.01), hematocrit, blood hemoglobin, mean corpuscular volume, hemoglobin and hemoglobin concentration and red blood cell count (p < 0.05). The daily consumption of the chokeberry juice could improve the health status in patients with type 2 diabetes mellitus, in combination with their standard therapy.

Comparative toxicity evaluation of targeted anticancer therapeutics in embryonic zebrafish and sea urchin models.

Cancer drug resistance and poor selectivity towards cancer cells demand the constant search for new therapeutics. PI3K-Akt-mTOR and RAS-MAPK-ERK signaling pathways are key mechanisms involved in cell survival, proliferation, differentiation, and metabolism and their deregulation in cancer can promote development of therapy resistance. We investigated the effects of targeted inhibitors (wortmannin, GSK690693, AZD2014 and tipifarnib) towards these two pathways on early zebrafish and sea urchin development to assess their toxicity in normal, fast proliferating cells. PI3K inhibitor wortmannin and RAS inhibitor tipifarnib displayed highest toxicity while GSK690693, a pan-Akt kinase inhibitor, exhibited a less significant impact on embryo survival and development. Moreover, inhibition of the upstream part of the PI3K-Akt-mTOR pathway (wortmannin/GSK690693 co-treatment) produced a synergistic effect and impacted zebrafish embryo survival and development at much lower concentrations. Dual mTORC1/mTORC2 inhibitor AZD2014 showed no considerable effects on embryonic cells of zebrafish in concentrations substantially toxic in cancer cells. AZD2014 also caused the least prominent effects on sea urchin embryo development compared to other inhibitors. Significant toxicity of AZD2014 in human cancer cells, its capacity to sensitize resistant cancers, lower antiproliferative activity against human normal cell lines and fast proliferating embryonic cells could make this agent a promising candidate for anticancer therapy.