Discovery of a new class of potent pyrrolo[3,4-c]quinoline-1,3-diones based inhibitors of human dihydroorotate dehydrogenase: Synthesis, pharmacological and toxicological evaluation.

Twenty N-substituted pyrrolo[3,4-c]quinoline-1,3-diones 3a-t were synthesized by a cyclization reaction of Pfitzinger's quinoline ester precursor with the selected aromatic, heteroaromatic and aliphatic amines. The structures of all derivatives were confirmed by IR, 1H NMR, 13C NMR and HRMS spectra, while their purity was determined using HPLC techniques. Almost all compounds were identified as a new class ofpotent inhibitors against hDHODH among which 3a and 3t were the most active ones with the same IC50 values of 0.11 µM, about seven times better than reference drug leflunomide. These two derivatives also exhibited very low cytotoxic effects toward healthy HaCaT cells and the optimal lipophilic properties with logP value of 1.12 and 2.07 respectively, obtained experimentally at physiological pH. We further evaluated the comparative differences in toxicological impact of the three most active compounds 3a, 3n and 3t and reference drug leflunomide. The rats were divided into five groups and were treated intraperitoneally, control group (group I) with a single dose of leflunomide (20 mg/kg) group II and the other three groups, III, IV and V were treated with 3a, 3n and 3t (20 mg/kg bw) separately. The investigation was performed in liver, kidney and blood by examining serum biochemical parameters and parameters of oxidative stress.

Thymus zygis, Valuable Antimicrobial (In Vitro and In Situ) and Antibiofilm Agent with Potential Antiproliferative Effects.

With the growing issues of food spoilage, microbial resistance, and high mortality caused by cancer, the aim of this study was to evaluate T. zygis essential oil (TZEO) as a potential solution for these challenges. Here, we first performed GC/MS analysis which showed that the tested TZEO belongs to the linalool chemotype since the abundance of linalool was found to be 38.0%. Antioxidant activity assays showed the superiority of TZEO in neutralizing the ABTS radical cation compared to the DPPH radical. The TZEO was able to neutralize 50% of ABTS•+ at the concentration of 53.03 ± 1.34 µg/mL. Antimicrobial assessment performed by employing disc diffusion and minimal inhibitory concentration assays revealed TZEO as a potent antimicrobial agent with the highest inhibition activity towards tested gram-negative strains. The most sensitive on the treatment with TZEO was Enterobacter aerogenes showing an MIC 50 value of 0.147 ± 0.006 mg/mL and a MIC 90 value of 0.158 ± 0.024 mg/mL. Additionally, an in situ analysis showed great effects of TZEO in inhibiting gram-negative E. coli, P. putida, and E. aerogenes growing on bananas and cucumbers. Treatment with the TZEO vapor phase in the concentration of 500 µg/mL was able to reduce the growth of these bacteria on the food models to the extent > 90%, except for E. coli growth on the cucumber, which was reduced to the extent of 83.87 ± 4.76%. Furthermore, a test on the antibiofilm activity of the tested essential oil revealed its biofilm prevention effects against Salmonella enterica which forms biofilms on plastic and stainless-steel surfaces. Performed tests on the TZEO effects towards cell viability showed no effects on the normal MRC-5 cell line. However, the results of MTT assay of TZEO effects on three cancer cell lines (MDA-MB-231, HCT-116, and K562) suggest that TZEO exerted the strongest effects on the inhibition of the viability of MDA-MB-231 cells, especially after long-term treatment in the highest concentration applied with reducing the viability of the cells to 57%. Additionally, results of NBT and Griess assays suggest that TZEO could be a convenient candidate for future testing for developing novel antitumor therapies.

Biological Activity of Cupressus sempervirens Essential Oil.

The aim of this study was to evaluate the antioxidant, antibiofilm, antimicrobial (in situ and in vitro), insecticidal, and antiproliferative activity of Cupressus sempervirens essential oil (CSEO) obtained from the plant leaf. The identification of the constituents contained in CSEO was also intended by using GC and GC/MS analysis. The chemical composition revealed that this sample was dominated by monoterpene hydrocarbons α-pinene, and δ-3-carene. Free radical scavenging ability, performed by using DPPH and ABTS assays, was evaluated as strong. Higher antibacterial efficacy was demonstrated for the agar diffusion method compared to the disk diffusion method. The antifungal activity of CSEO was moderate. When the minimum inhibitory concentrations of filamentous microscopic fungi were determined, we observed the efficacy depending on the concentration used, except for B. cinerea where the efficacy of lower concentration was more pronounced. The vapor phase effect was more pronounced at lower concentrations in most cases. Antibiofilm effect against Salmonella enterica was demonstrated. The relatively strong insecticidal activity was demonstrated with an LC50 value of 21.07% and an LC90 value of 78.21%, making CSEO potentially adequate in the control of agricultural insect pests. Results of cell viability testing showed no effects on the normal MRC-5 cell line, and antiproliferative effects towards MDA-MB-231, HCT-116, JEG-3, and K562 cells, whereas K562 cells were the most sensitive. Based on our results, CSEO could be a suitable alternative against different types of microorganisms as well as suitable for the control of biofilms. Due to its insecticidal properties, it could be used in the control of agricultural insect pests.

Green Synthesis of Silver Nanoparticles Using Salvia verticillata and Filipendula ulmaria Extracts: Optimization of Synthesis, Biological Activities, and Catalytic Properties.

The study's objective was to obtain silver nanoparticles (SVAgNP and FUAgNP) using aqueous extracts of Salvia verticillata and Filipendula ulmaria. The optimal conditions for nanoparticle synthesis were determined and obtained; nanoparticles were then characterized using UV-Vis, Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), Dynamic Light Scattering (DLS), Scanning Electron Microscopy with Energy Dispersive Spectroscopy (SEM/EDS). SVAgNP and FUAgNP possessed a crystalline structure with 48.42% and 60.41% silver weight, respectively. The highest percentage of nanoparticles in the solution had a diameter between 40 and 70 nm. In DPPH˙ and ABTS˙+ methods, FUAgNP (IC50 15.82 and 59.85 µg/mL, respectively) demonstrated a higher antioxidant capacity than SVAgNP (IC50 73.47 and 79.49 µg/mL, respectively). Obtained nanoparticles also showed pronounced antibacterial activity (MIC ˂ 39.1 µg/mL for most of the tested bacteria), as well as high biocompatibility with the human fibroblast cell line MRC-5 and significant cytotoxicity on some cancer cell lines, especially on the human colon cancer HCT-116 cells (IC50 31.50 and 66.51 µg/mL for SVAgNP and FUAgNP, respectively). The nanoparticles demonstrated high catalytic effectiveness in degrading Congo red dye with NaBH4. The results showed a rapid and low-cost methodology for the synthesis of AgNPs using S. verticillata and F. ulmaria with promising biological potential.

GC, GC/MS Analysis, and Biological Effects of Essential Oils from Thymus mastchina and Elettaria cardamomum.

Spanish marjoram (Thymus mastichina) and cardamom (Elettaria cardamomum) are traditional aromatic plants with which several pharmacological properties have been associated. In this study, the volatile composition, antioxidative and antimigratory effects on human breast cancer (MDA-MB-468 cell line), antimicrobial activity, and antibiofilm effect were evaluated. Results obtained via treatment of human breast cancer cells generally indicated an inhibitory effect of both essential oils (EOs) on cell viability (after long-term treatment) and antioxidative potential, as well as the reduction of nitric oxide levels. Antimigratory effects were revealed, suggesting that these EOs could possess significant antimetastatic properties and stop tumor progression and growth. The antimicrobial activities of both EOs were determined using the disc diffusion method and minimal inhibition concentration, while antibiofilm activity was evaluated by means of mass spectrometry. The best antimicrobial effects of T. mastichina EO were found against the yeast Candida glabrata and the G+ bacterium Listeria monocytogenes using the disc diffusion and minimal inhibitory concentration methods. E. cardamomum EO was found to be most effective against Pseudomas fluorescens biofilm using both methods. Similarly, better effects of this oil were observed on G- compared to G+ bacterial strains. Our study confirms that T. mastichina and E. cardamomum EOs act to change the protein structure of older P. fluorescens biofilms. The results underline the potential use of these EOs in manufactured products, such as foodstuffs, cosmetics, and pharmaceuticals.

Chemical Composition and Biological Activity of Tanacetum balsamita Essential Oils Obtained from Different Plant Organs.

The aim of this study is to evaluate the chemical composition of Tanacetum balsamita L. essential oils (EOs) obtained from different plant organs, flowers (FEO), leaves (LEO), and stems (SEO), as well as to assess their biological properties. The results obtained by using GC and GC/MS analysis indicate that this plant belongs to the carvone chemotype. Moreover, we examined the oil's antimicrobial and antitumor potential. Antimicrobial effects were determined using minimum inhibitory concentrations assay and the vapor phase method. Obtained results indicate better antimicrobial activity of investigated EO samples compared to the commercially available antibiotics. On the treatment with FEO, Y. enterocolitica and H. influenzae showed high sensitivity, while treatment with LEO and SEO showed the highest effects against S. aureus. The vapor phase method, as an in situ antibacterial analysis, was performed using LEO. Obtained results showed that this EO has significant activity toward S. pneumoniae in the apple and carrot models, L. monocytogenes in the pear model, and Y. enterocolitica in the white radish model. The potential antitumor mechanisms of FEO, LEO, and SEO were determined by the means of cell viability, redox potential, and migratory capacity in the MDA-MB-231 and MDA-MB-468 cell lines. The results show that these EOs exert antiviability potential in a time- and dose-dependent manner. Moreover, treatments with these EOs decreased the levels of superoxide anion radical and increased the levels of nitric oxide in both tested cell lines. The results regarding total and reduced glutathione revealed, overall, an increase in the levels of total glutathione and a decrease in the levels of reduced glutathione, indicating strong antioxidative potential in tested cancer cells in response to the prooxidative effects of the tested EOs. The tested EOs also exerted a drop in migratory capacity, which indicates that they can be potentially used as chemotherapeutic agents.

Chemical Composition, Antitumor Potential, and Impact on Redox Homeostasis of the Essential Oils of Orlaya grandiflora from Two Climate Localities.

It is well known that abiotic components can affect biosynthetic pathways in the production of certain volatile compounds. The aim of this study was to compare the chemical composition of essential oils obtained from Orlaya grandiflora (L.) Hoffm. collected from two localities in Serbia (continental climate, OG1) and Montenegro (Mediterranean climate, OG2) and to assess their antitumor potential on the human colon cancer HCT-116 and breast cancer MDA-MB-231 cell lines. EOs obtained by hydrodistillation were analyzed using GC-MS and GC-FID methods. The results indicate considerable differences in the chemical compositions of the two samples. Although in both samples the main class of volatiles observed was sesquiterpenes (47.5% for OG1 and 70.1% for OG2), the OG1 sample was characterized by a high amount of monoterpene hydrocarbons (29.3%), and sesquiterpene germacrene D (29.5%) as the most abundant compound. On the other hand, the OG2 sample contained a high quantity of oxygenated sesquiterpenes (20.6%), and ß-elemene (22.7%) was the major constituent. The possible antitumor mechanisms of these EOs in the HCT-116 and MDA-MB-231 cell lines were examined by means of cell viability, apoptosis, redox potential, and migratory capacity. The antiviability potential appeared to be dose dependent, since the results showed that both EOs decreased the viability of the tested cells. Stronger antitumor effects were shown in MDA-MB-231 cells after short-term treatment, especially at the highest applied concentration, where the percentage of viability was reduced by over 40%. All tested concentrations of EOs exhibited proapoptotic activity and elevated activity of caspase-3 in a dose- and time-dependent manner. The results also showed decreased concentrations of superoxide anion radical in the treated cells, which indicates their significant antioxidative role. Long-term treatments showed mild recovery effects on cell viability in both cell lines, probably caused by the balancing of redox homeostasis. Elevated levels of nitrites indicate high levels of nitric oxide (NO) production and suggest its higher bioavailability due to the antioxidative environment. The tested EOs also induced a drop in migratory capacity, especially after short-time treatments. Taken together, these results suggest considerable antitumor activity of both EOs, which could have potential therapeutic applications.

Antiproliferative, antimigratory, and prooxidative potential of novel platinum(IV) complexes and resveratrol on breast cancer (MDA-MB-231) and choriocarcinoma (JEG-3) cell lines.

Platinum(IV) complexes offer the potential to overcome cisplatin resistance of cancer cells, with possible improved selectivity. Resveratrol, a natural polyphenol with anticancer and antioxidant capacity, could limit the possible side effects of chemotherapeutics on healthy cells. This study investigates the effects of platinum(IV) complexes containing some esters of the ethylenediamine-N,N'-di-S,S-(2,2'-dibenzyl)acetate acid (H2 -S,S-eddba), and resveratrol on proliferation, migration, and redox balance of breast cancer (MDA-MB-231), choriocarcinoma (JEG-3), and human lung fibroblast (MRC-5) cell line. According to IC50 values, all complexes exhibited a significantly stronger antiproliferative effect on tested cell lines compared to cisplatin. Due to reduced adverse effects on MRC-5 cells, the complex containing ethyl-substituent (10 µM) was selected for further examination with resveratrol (25 µM) cotreatment. Resveratrol enhanced the survival of MRC-5 cells while diminished the viability of both used cancer cell lines when applied combined with selected complex. Furthermore, cotreatment of these two compounds decreased the migratory potential of tested cancer cell lines. The examined platinum(IV) complex was able to induce oxidative stress in all tested cell lines. Resveratrol proved to be efficient in protecting MRC-5 cells from complex-induced oxidative damage, while it significantly amplified antiproliferative, antimigratory, and prooxidative effects of platinum(IV) complex on both examined cancer cell lines. These findings may be valuable in elucidating the mechanism of action of platinum(IV) drugs, which should be further investigated.

First report of Head blight of wheat caused by Fusarium vorosii in Serbia.

The cosmopolitan species Fusarium graminearum Schwabe directly reduces yield, as well as grain quality of cereals, due to its ability to synthesize mycotoxins. Previously it was considered to be one species occurring on all continents. However, phylogenetic analysis employing the GCPSR method (Genealogical Concordance Phylogenetic Species Recognition) revealed the existence of 15 phylogenetic species within what is now recognised as the Fusarium graminearum Species Complex (FGSC) (Sarver et al. 2011). During 1996-2008, a MRIZP collection of FGSC isolates was established and isolates originating from wheat (5), maize (3) and barely (2) were selected for further study. Morphological features including the appearance of colonies and macroconidia (average size 38.5-53.1 × 4.6-5.4 µm, No 50) of all 10 isolates on PDA were consistent with descriptions of F. graminearum (O'Donnell et al. 2004, Leslie and Summerell 2006). Total DNA was isolated from mycelium removed from 7-day old colonies of single-spore isolates grown on PDA using the DNeasy Plant Mini Kit (Qiagen, Hilden). Further identification was based on amplification and sequencing of elongation factor TEF-1α, histone H3 and ß-tubulin in both directions, with primers ef1/ef2, H3-1a/H3-1b and T1/T22, respectively (Jacobs et al. 2010). The sequences were deposited in NCBI under accession numbers MF974399 - MF974408 (TEF-1α), MG063783 - MG063792 (ß-tubulin) and MF999139 - MF999148 (histone H3). Sequence analysis was performed using BLAST while genetic similarity was calculated using MEGA 6.0 software. Isolate 1339 originating from wheat (collected at the locality of Kikinda in 2006), shared 100% nucleotide identity with TEF-1α (DQ459745), histone H3 (DQ459728) and ß-tubulin (DQ459643) of F. vorosii isolate NRRL37605 (Starkey et al. 2007). The remaining nine isolates were identified as F. graminearum as they shared 99% to 100% nucleotide similarity with F. graminearum NRRL 28439 (O'Donnell et al. 2004). Pathogenicity was tested using artificial inoculations of spikes during wheat flowering (Mesterhazy et al. 1999). Thirty classes were inoculated with each isolate, in three replicates. Inoculum was prepared from 7-day colonies on PDA, and 30 ml of a conidia suspension (1x105 conidia/ml) was used. Control plants were inoculated with sterile water. Three weeks after inoculation, typical Fusarium head blight symptoms were visible on inoculated plants, from which all 10 isolates were successfully reisolated. Control spikes remained symptomless. Disease severity was estimated on the 1-7 scale (Blandino et al. 2012). Average pathogenicity of the F. vorosii isolate 1339 was 1.9, and 2.4 -5.1 of F. graminearum isolates. Toxin production was determined using gas chromatography-tandem mass spectrometry. Kernels inoculated with the 10 isolates were ground and tested for the presence of deoxynivalenol (DON) and its acetyl derivatives 3ADON, 15ADON and NIV. F. vorosii isolate 1339 possessed the 15ADON chemotype, as well as eight F. graminearum isolates, while only one F. graminearum isolate was 3ADON chemotype. To date, F. vorosii has only been detected in Hungary on wheat (Toth et al. 2005) and Korea on barley, corn and rice (Lee et al. 2016). This is the first report of F. vorosii in Serbia, which is of great importance, because it indicates the spread of this toxigenic species. Further studies should be focused on determining the distribution, aggressiveness and toxicological profile of F. vorosii.

Antiproliferative and antimigratory effects of 3-(4-substituted benzyl)-5- isopropyl-5-phenylhydantoin derivatives in human breast cancer cells.

In this study, a series of synthesized 3-(4-substituted benzyl)-5-isopropyl-5-phenylhydantoin derivatives as a potential antiproliferative and antimigratory agents were investigated. The possible antitumor mechanisms of investigated hydantoin derivatives were examined on human breast cancer cell line MDA-MB-231. The cells were treated with different concentrations of compounds (from 0.01 µM to 100 µM) during 24 h and 72 h. The proliferation index, nitric oxide production, apoptosis rate, and migration capacity were measured. The cell invasion potential was examined by measuring the level of MMP-9 and COX-2 gene expression. All tested compounds expressed antiproliferative activity and induced dose- and time-dependent increase in the level of nitrites. The investigated molecules significantly decreased cell survival rate, migration capacity and the expression levels of genes included in the process of tumor invasion. Obtained data suggest that the tested hydantoin derivatives express considerable antitumor activity by reducing cell division rate, elevating apoptosis level, and inhibiting the motility and invasiveness of breast cancer cells. The results obtained in this study indicate that investigated compounds express potential as a novel chemotherapeutic agents against breast cancer growth and progression.

Redox status, DNA and HSA binding study of naturally occurring naphthoquinone derivatives.

In the present work we modified the procedure for isolation of naphthoquinones α-methylbutyrylshikon ( 1 ), acetylshikonin ( 2 ) and ß-hydroxyisovalerylshikonin ( 3 ) from Onosma visianii Clem. We also investigated possible mechanisms of 1 , 2 and 3 as antitumor agents. Accordingly, we estimated concentrations of superoxide anion radical (O2 .-), nitrite (NO2 -) and glutathione in HCT-116 and MDA-MB-231 cell lines. Compounds 1 and 3 expressed significant prooxidative activity, while all tested compounds exhibited significant increase in nitrite levels. Also, all examined compounds significantly increased the concentration of oxidized glutathione (GSSG), suggesting significant prooxidative disbalance. The levels of reduced glutathione (GSH) were also elevated as a part of antioxidative cell response. The data indicate that induced oxidative imbalance could be one of the triggers for previously recorded decreased viability of HCT-116 and MDA-MB-231 cells exposed to tested naphthoquinone derivatives. Moreover, we examined interactions mode of compounds 1 , 2 and 3 with CT-DNA as one of the crucial targets of many molecules that express cytotoxic activity. The results obtained by UV-visible, fluorescence and molecular docking study revealed that 1 , 2 and 3 bound to CT-DNA through minor groove binding. Furthermore, the interactions between HSA and 1 , 2 and 3 were examined employing the same methods as for the CT-DNA interaction study. Based on the obtained results, it can be concluded that naphthoquinones 1 , 2 and 3 could be effectively transported by human serum albumin. As a conclusion, this study provides further insight of antitumor activity of selected naphthoquinones.

Anti-Tumor Mechanisms of Novel 3-(4-Substituted Benzyl)-5-Isopropil-5- Phenylhydantoin Derivatives in Human Colon Cancer Cell Line.

BACKGROUND: Hydantoin and its newly synthesized derivatives have recently become a focus of interest due to their numerous biological activities and newly emerging beneficial effects in different pathological conditions, including cancer. OBJECTIVE: The aim of this study was to evaluate the possible anti-tumor mechanisms of a series of newly synthesized 3-(4-substituted benzyl)-5-isopropyl-5-phenylhydantoin derivatives in different aspects of cell physiology of human colon cancer cell line, HCT-116. METHODS: The increasing concentrations of derivatives (0.01µM up to 100µM) were applied to cells during 24h, 48h, and 72h after which the evaluation of proliferation, apoptosis, oxidative/anti-oxidative status, nitrite production, and migration/invasion potential of treated cells was performed. RESULTS: All tested compounds expressed the dose- and time-dependent anti-proliferative and pro-apoptotic activities against HCT-116 cells. The investigated derivatives induced a decrease in levels of oxidative stress parameters and an increase in levels of nitrite production by treated cells suggesting their significant antioxidative effects. The cell migration index and expression level of tumor invasion-promoting metalloproteinase- 9 (MMP-9) gene were significantly decreased after treatment with the tested hydantoin derivatives implicating their inhibitory role in colon cancer cell motility and invasion processes. The mRNA level of cyclooxygenase-2 (COX-2) gene as a pro-inflammatory gene related to colorectal carcinogenesis was reduced compared to values in the non-treated control cells indicating the significant anti-inflammatory/anti-tumor effects of these compounds. CONCLUSION: The obtained results show the significant anti-tumor potential of tested derivatives, especially 3- benzyl-5-isopropyl-5-phenylhydantoin and 3-(4-chlorobenzyl)-5-isopropyl-5-phenylhydantoin, suggesting their potential usage in the development of more effective chemotherapies.

Proapoptotic and Antimigratory Effects of Pseudevernia furfuracea and Platismatia glauca on Colon Cancer Cell Lines.

The aim of this study is to investigate cytotoxic, proapoptotic, antimigratory and pro-antioxidant effects of methanol, acetone and ethyl acetate extracts of lichens Pseudevernia furfuracea and Platismatia glauca on colorectal cancer (HCT-116 and SW-480) cell lines. We compared the cytotoxic effects on colorectal cancer cells with the effects obtained from normal human fibroblast (MRC-5) cell line. Tetrazolium (MTT) test evaluated the cytotoxic effects, Transwell assay evaluated cell migration, acridine orange/ethidium bromide (AO/EB) fluorescent method followed the apoptosis, while prooxidant/antioxidant effects were determined spectrophotometrically through concentration of redox parameters. The tested extracts showed considerable cytotoxic effect on cancer cells with no observable cytotoxic effect on normal cells. Ethyl acetate and acetone extract of P. furfuracea induced the highest cytotoxicity (IC50=(21.2±1.3) µg/mL on HCT-116, and IC50=(51.3±0.8) µg/mL on SW-480 cells, respectively, after 72 h), with noteworthy apoptotic and prooxidant effects, and antimigratory potential of methanol extract. P. glauca extracts induced cytotoxic effects on HCT-116 cells after 72 h (IC50<40 µg/mL), while only methanol and acetone extracts had cytotoxic effects on SW-480 cells after 24 h, with proapoptotic/necrotic activity, as a consequence of induced oxidative stress. In conclusion, lichen extracts changed to a great extent cell viability and migratory potential of colorectal cancer cell lines. HCT-116 cells were more sensitive to treatments, P. furfuracea had better proapoptotic and antimigratory effects, and both investigated lichen species might be a source of substances with anticancer activity.

Trichothecene Genotypes of Fusarium graminearum Populations Isolated from Winter Wheat Crops in Serbia.

Fusarium graminearum as the main causal agent of Fusarium head blight (FHB) and its ability to produce trichothecenes was investigated by molecular techniques. A total of 37 strains isolated from the wheat, harvested in Serbia in 2005, 2008 and 2015, and previously designated by morphological observation as F. graminearum, were used for trichothecene genotypes characterization. The strains were identified using the species-specific primer set FG16R/FG16F while genotypic characterization was done using specific TRI13 and TRI3 sequences of the trichothecene gene clusters. The PCR assays identified all strains as species of F. graminearum sensu stricto with the DON/15-ADON genotype. The quantification of the mycotoxin (DON) was performed using the biochemical assay. The high levels of DON (>20,000 µg kg-1) were recorded in all of the strains from 2005, four strains from 2008 and two strains from 2015. Weather data of the investigated seasons, showed that the optimal temperature, frequent rains and high relative humidity (RH) was very favourable for the development of F. graminearum, affecting the DON biosynthesis.

Cytotoxic, proapoptotic and antioxidative potential of flavonoids isolated from propolis against colon (HCT-116) and breast (MDA-MB-231) cancer cell lines.

Isolated and structurally confirmed, eleven flavonoids from propolis were examined for their cytotoxicity toward human colon cancer and human breast cancer cells. Their effect on induction of apoptosis and their antioxidative activities were also evaluated. Six flavonoids induced cytotoxic effects in both cell lines. Luteolin had a marked effect on both cell lines, especially on HCT-116 cells (IC50 72h, 66.86µM). Also, luteolin was observed to have the highest apoptotic potential after 72h treatment of examined cell lines (27.13% and 37.09%, respectively). Myricetin exhibited selective inhibition of cell growth (IC50 114.75µM) and induced apoptosis in MDA-MB-231 cells only. Luteolin and galangin exhibited prooxidative properties 24h after the treatment in HCT-116 cells, while myricetin induced prooxidative effects in MDA-MB-231 cells. On the other hand, selected flavonoids exhibited antioxidative properties 72h after the treatment, decreasing superoxide anion radical and nitrite levels in both cell lines. Cytotoxic and proapoptotic effects on colon and breast cancer cell lines and the influence on their redox status make tested flavonoids good candidates for developing new anticancer drugs.

Naphthoquinone rich Onosma visianii Clem (Boraginaceae) root extracts induce apoptosis and cell cycle arrest in HCT-116 and MDA-MB-231 cancer cell lines.

In the present study, five root extracts of Onosma visianii Clem were investigated for their in vitro cytotoxic activity. On the basis of HPLC-PDA analysis, these extracts have proved to be a rich source of naphthoquinones as natural colourants for food and cosmetic industry. All investigated root extracts contain acetylshikonin, isobutyrylshikonin and α-methylbutyrylshikonin as major compounds. As the most abundant source of active compounds for antitumour therapy, acetone, chloroform and ethyl acetate extracts showed strong cytotoxic activity towards HCT-116 and MDA-MB-231 cancer cell lines. Also, these extracts induced apoptosis and cell cycle arrest in HCT-116 and MDA-MB-231 cancer cell lines.

Energy production and redox status of rat red blood cells after reticulocytosis induced by various treatments.

Stimulated erythropoiesis and reticulocytosis can be induced by daily bleeding, or by phenylhydrazine (PHZ) treatment. We compared the in vivo effects of PHZ and bleeding treatment on haematological, energy and redox status parameters in red blood cells (RBC) of rats. The results showed that all followed haematological parameters were significantly lower in bleeding, compared to PHZ-treated rats. PHZ induced even 2.58-fold higher reticulocytosis as compared to bleeding treatment. Although PHZ induced higher reticulocytosis, respiration intensity and energy production was lower than in bleeding-induced reticulocytes. These alterations were the consequence of increased superoxide anion and peroxynitrite concentrations in PHZ-treated rats. Bleeding treatment resulted in increased activity of an antioxidative enzyme, superoxide dismutase. In conclusion, differences in these two experimental models for reticulocytosis may be used as tools for appropriate pharmacological testing of redox-active substances considering energy and redox processes, as well as apoptosis pathways.

Effects of acute in vivo cisplatin and selenium treatment on hematological and oxidative stress parameters in red blood cells of rats.

Although cisplatin (cisPt) is one of the most often used cytotoxic drugs in the treatment of cancer, its clinical application is associated with nephrotoxicity and a cumulative anemia. In this study, we evaluated posible protective effects of selenium (Se) on hematological and oxidative stress parameters in rats, acutely treated with cisPt. Four groups of Wistar albino rats included control rats, cisPt-treated (7.5 mg/kg of body weight of cisPt, i.p.), Se-treated (6 mg/kg of body weight of Na(2)SeO(4), i.p.), and Se and cisPt co-treated rats. The rats were killed 72 h after treatment; hematological and oxidative stress parameters were followed in red blood cells. The results showed depletion in platelet number induced by high acute doses of cisPt and strong utilization of reduced glutathione, resulting in elevation of GSSG/2 GSH ratio. Se treatment was followed by stimulated erythropoiesis, increased lipid peroxidation, and GSH depletion. Se and cisPt co-treatment were followed by stimulated erythropoiesis and significant recovery of reduced glutathione status when compared with cisPt-treated rats. In conclusion, acute doses of Se and cisPt primarily act as pro-oxidants. CisPt influenced antioxidative properties of exogenous Se and their synergistic effects may partially participate in protection against cisPt-induced toxicity.