Immunosuppressive viruses cause substantial economic losses to the poultry industry. Chicken anaemia virus (CAV) causes severe disease in young chickens, whereas subclinical infection in older birds ...causes immunosuppression. In this study, we addressed the ability of CAV to interfere with production of antimicrobial molecule nitric oxide (NO) by macrophages. NO production in chicken macrophage cell line HD11 was induced using both Toll-like receptor 4 agonist, bacterial lipopolysaccharide, and an immune modulator, interferon-γ. In addition, we treated macrophages with CAV propagated in chicken lymphoblastoid cells. The levels of NO were measured by the Griess reaction. Addition of CAV decreased both the interferon-γ and the lipopolysaccharide associated induction of NO. Observed effect was not caused by CAV-related cytotoxicity, as no decrease in number of viable cells was observed. Although CAV could not completely abrogate NO production, attenuation of NO induction was clearly present. We have previously shown that CAV interferes with the expression of interferons in chickens during subclinical infection. Since the signalling pathways of expression of interferons and type 2 nitric oxide synthase, enzyme involved in NO formation, overlap, we conclude that measured decrease in NO levels is a consequence of CAV interference with interferon and NO synthase signalling. Regardless of the fact whether the attenuation of NO serves as a viral primary defence, or is only a secondary effect, it could impair the immune response to other pathogens and contribute to the global immunosuppression in chicken houses.Key words: chicken; immunosuppression; chicken anaemia virus (CAV); macrophage; nitric oxide (NO) VIRUS PIŠČANČJE ANEMIJE VPLIVA NA PROIZVODNJO DUŠIKOVIH OKSIDOV V MAKROFAGIH PIŠČANEV HD11 Povzetek: Imunosupresivni virusi povzročajo velike gospodarske izgube v perutninski industriji. Virus piščančje anemije (CAV) pri mladih piščancih povzroča hudo bolezen, medtem ko subklinična okužba pri starejših pticah povzroča oslabljen imunski odziv. V tej raziskavi je bil spremljan vpliv CAV na proizvodnjo dušikovih oksidov (NO) v makrofagih. Proizvodnja NO v piščančjih makrofagih v celični liniji HD11 je bila sprožena z uporabo agonista Toll-u podobnega receptorja 4, bakterijskega lipopolisaharida in imunskega modulatorja interferona-γ, makrofagi pa so bili okuženi s CAV, razmnoženim v piščančjih limfoblastoidnih celicah. Ravni NO so izmerili po Griessovi reakciji. Prisotnost CAV je zmanjšala proizvodnjo NO, spodbujeno tako z interferonom-γ, kot z lipopolisaharidom. Opaženega učinka ni povzročila citotoksičnost, povezana s CAV, saj ni bilo opaziti zmanjšanja števila živih celic. Čeprav CAV ni popolnoma zavrla nastajanja NO, je bilo očitno prisotno zmanjšanje nastajanja NO. Pred tem so pokazali, da CAV moti izražanje interferonov pri piščancih med subklinično okužbo. Ker se poti znotrajceličnega prenosa urejanja izražanja interferonov in sintaze dušikovih oksidov tipa 2, encima, ki sodeluje pri tvorbi NO, prekrivajo, predvidevamo, da je izmerjeno znižanje ravni NO posledica motenj CAV pri znotrajceličnem prenosu sporočila interferona do sintaze dušikovih oksidov. Ne glede na to, ali zaviranje nastajanja NO služi kot primarna virusna obramba ali je le sekundarni učinek, lahko poslabša imunski odziv na druge patogene in prispeva k splošnemu zmanjšanju imunskega odziva v kurnikih ali na kokošjih farmah.Ključne besede: piščanci; zmanjšanje imunskega odziva; virus piščančje anemije (CAV); makrofagi; dušikov oksid (NO)
The synthesis of 5-amino substituted benzimidazo1,2-aquinolines prepared by microwave assisted amination from halogeno substituted precursor was described. The majority of compounds were active at ...micromolar concentrations against colon, lung and breast carcinoma cell lines in vitro. The N,N-dimethylaminopropyl 9 and piperazinyl substituted derivative 19 showed the most pronounced activity towards all of the three tested tumor cell lines, which could be correlated to the presence of another N heteroatom and its potential interactions with biological targets. The DNA binding studies, consisting of UV/Visible absorbency, melting temperature studies, and fluorescence and circular dichroism titrations, revealed that compounds 9, 19 and 20 bind to DNA as strong intercalators. The cellular distribution analysis, based on compounds’ intrinsic fluorescence, showed that compound 20 does not enter the cell, while compounds 9 and 19 do, which is in agreement with their cytotoxic effects. Compound 9 efficiently targets the nucleus whereas 19, which also showed DNA intercalating properties in vitro, was mostly localised in the cytoplasm suggesting that the antitumor mechanism of action is DNA-independent.
Evaluation of the mechanism of action of 5-amino substituted benzimidazo1,2-aquinolines revealed binding of strong DNA intercalators. N,N-dimethylaminopropyl substituted intercalator entered the cell localize in the nucleus. Display omitted
•5-Aminobenzimidazo1,2-aquinolines with prominent antiproliferative activity.•Compounds 9, 19 and 20 bind to DNA as strong intercalators.•Compounds 9 and 19 enter the cell according to cellular distribution.•Compound 9 efficiently targets the nucleus whereas 19 is mostly cytoplasmic.
In this paper design and synthesis of a scaffold comprising primaquine (PQ) motif and cinnamic acid derivatives (CADs) bound directly (compounds
-
) or via a spacer (compounds
-
) are reported. In ...the first series of compounds, PQ and various CADs were connected by amide bonds and in the second series by acylsemicarbazide functional groups built from the PQ amino group, CONHNH spacer and the carbonyl group originating from the CADs. PQ-CAD amides
-
were prepared by a simple one-step condensation reaction of PQ with a series of CAD chlorides (method A) or benzotriazolides
(method B). The synthesis of acylsemicarbazides
-
included activation of PQ with benzotriazole, preparation of PQ-semicarbazide
and its condensation with CAD chlorides
. All synthesized PQ-CAD conjugates were evaluated for their anticancer, antiviral and antioxidative activities. Almost all compounds from series
were selective towards the MCF-7 cell line and active at micromolar concentrations. The
-fluoro derivative
showed high activity against HeLa, MCF-7 and in particular against the SW 620 cell line, while acylsemicarbazide
with a benzodioxole ring and
,
and especially
with methoxy-, chloro- or trifluoromethyl-substituents in the
position showed high selectivity and high inhibitory activity against MCF-7 cell line at micromolar (
,
,
) and nanomolar (
) levels. Acylsemicarbazide derivatives with trifluoromethyl group(s)
,
and 7
showed specific activity against human coronavirus (229E) at concentrations which did not alter the normal cell morphology. The same compounds exerted the most potent reducing activity in the DPPH test, together with
and
, while methoxy (compounds
-
), benzodioxole (
),
-Cl (
) and
-CF₃ (
) acylsemicarbazides and amide
presented the highest LP inhibition (83%-89%). The dimethoxy derivative
was the most potent LOX inhibitor (IC
= 10 μΜ). The performed biological tests gave evidence of acylsemicarbazide functional group as superior binding group in PQ-CAD conjugates.
3,5-Disubstituted hydantoin (1,3-imidazolidinedione) derivatives 5a-h were prepared by base induced cyclization of the corresponding N-(1-benzotriazolecarbonyl)-L- and D-amino acid amides 4a-h. ...Compounds 5a-h were evaluated for their cytostatic and antiviral activities. Among all the compounds evaluated, 3-benzhydryl-5-isopropyl hydantoin (5a) showed a weak but selective inhibitory effect against vaccinia virus (EC(50) = 16 microg/mL; selectivity index: 25). 3-Cyclohexyl-5-phenyl hydantoin (5g) showed inhibitory activity against cervical carcinoma (HeLa, IC(50) = 5.4 microM) and breast carcinoma (MCF-7, IC(50) = 2 microM), but also cytotoxic effects towards human normal fibroblasts (WI 38). On the contrary, the 3-benzhydryl-5-phenyl substituted hydantoin derivative 5h showed moderate inhibitory activity towards HeLa, MCF-7, pancreatic carcinoma (MiaPaCa-2), lung carcinoma (H 460) and colon carcinoma (SW 620) (IC(50) = 20-23 microM), but no effect on WI 38.
Background and purpose: PEI is a cationic polymer able to neutralise negative DNA charges and to condense large genes which makes it suitable for gene delivery in human cells. Despite its low cost, ...simplicity of use and moderate toxicity, there is still room for broader usage and experimental adjustments, especially in cell lines that are difficult to transfect. In the presented research, we used PEI for the delivery of plasmid DNA into TNBC cell line SUM159 and breast CSC model HMLE-Twist.Material and methods: Cultured cells were transfected with GFP-expressing plasmid using both PEI and Lipofectamin. Transfection efficiency was determined by flow cytometry measurements of the intensity of the green fluorescence, while viability was determined by measuring intensity of the red fluorescence after propidium iodide staining. Results: In SUM159 and HMLE-Twist cells we obtain transfection efficiency between 30-40% using PEI, while cytotoxicity was generally low to moderate. PEI caused 10% of cell death in SUM159 and 20 % in HMLE-Twist. Transfection efficiency of PEI was comparable and even higher than the efficiency of the Lipofectamine in both SUM159 and HMLE-Twist. In mammary epithelia (control HMLE) we obtained only 20 % transfection efficiency using both carriers.Conclusions: We demonstrated for the first time that PEI represents a suitable nanocarrier for gene delivery into breast CSC model. We successfully transfected both breast CSC model HMLE-Twist and TNBC line SUM159. Since PEI is inexpensive and easy to use, we recommend it for further exploitations of these cell lines in TNBC research.
Novel heterocyclic 2-substituted-5-amidino-benzimidazoles were prepared. Compounds having imidazolinylamidine-substituent showed reasonable antiproliferative activity, while all show noticeable ...selectivity toward breast cancer cells. Strong antiviral activity was obtained by pyridyl-substituted compounds toward coxsackieviruses and echoviruses. Distinct effect toward adenoviruses was also shown.
We have prepared a set of heterocyclic benzimidazole derivatives bearing amidino substituents at C-5 of benzimidazole ring, by introducing various heterocyclic nuclei (pyridine,
N-methyl-pyrrole or imidazole) at C-2, and evaluated their antitumor and antiviral activities. The most pronounced antiproliferative activity was shown with compounds
6 and
9, having imidazolinylamidino-substituent. Interestingly, all compounds show noticeable selectivity toward breast cancer cell line MCF-7. The most distinct and selective antiviral activity toward coxsackieviruses and echoviruses was observed with compounds having pyridine ring at C-2. Especially interesting was fairly strong activity of
4 and
8 toward adenoviruses, which could be considered as leads against adenoviral replication.
Multidrug resistance (MDR) is a widespread phenomenon exhibited by many cancers and represents a fundamental obstacle for successful cancer treatments. Tumour cells commonly achieve MDR phenotype ...through overexpression and/or increased activity of ABC transporters. P-glycoprotein transporter (P-gp, ABCB1) is a major cause of MDR and therefore represents a valuable target for MDR reversal. Several naturally occurring potassium ionophores (e.g. salinomycin) were shown to inhibit P-gp effectively. We have previously shown antitumour activity of a number of 18-crown-6 ether compounds that transport potassium ions across membranes. Here we present data on P-gp inhibitory activity of 16 adamantane-substituted monoaza- and diaza-18-crown-6 ether compounds, and their effect on MDR reversal in model cell lines. We show that crown ether activity depends on their lipophilicity as well as on the linker to adamantane moiety. The most active crown ethers were shown to be more effective in sensitising MDR cells to paclitaxel and adriamycin than verapamil, a well-known P-gp inhibitor. Altogether our data demonstrate a novel use of crown ethers for inhibition of P-gp and reversal of MDR phenotype.
Primaquine (PQ) is a commonly used drug that can prevent the transmission of Plasmodium falciparum malaria, however toxicity limits its use. We prepared five groups of PQ derivatives: amides 1a-k, ...ureas 2a-k, semicarbazides 3a,b, acylsemicarbazides 4a-k and bis-ureas 5a-v, and evaluated them for antimalarial activity in vitro against the erythrocytic stage of P. falciparum NF54. Particular substituents, such as trityl (in 2j and 5r) and methoxybenzhydryl (in 3b and 5v) were associated with a favorable cytotoxicity-to-activity ratio. To systematically link structural features of PQ derivatives to antiplasmodial activity, we performed a quantitative structure-activity relationship (QSAR) study using the Support Vector Machines machine learning method. This yielded a highly accurate statistical model (R2 = 0.776 in cross-validation), which was used to prioritize novel candidate compounds. Seven novel PQ-ureidoamides 10a-g were synthesized and evaluated for activity, highlighting the benzhydryl ureidoamides 10e and 10f derived from p-chlorophenylglycine. Further experiments on human cell lines revealed that 10e and 10f are an order of magnitude less toxic than PQ in vitro while having antimalarial activity indistinguishable from PQ. The toxicity profile of novel compounds 10 toward human cells was particularly favorable when the glucose-6-phosphate dehydrogenase (G6PD) was inhibited, while toxicity of PQ was exacerbated by G6PD inhibition. Our work therefore highlights promising lead compounds for the development of effective antimalarial drugs that may also be safer for G6PD-deficient patients. In addition, we provide computational inferences of antimalarial activity and cytotoxicity for thousands of PQ-like molecular structures.
Display omitted
•Trityl and methoxybenzhydryl primaquine (PQ) derivatives have activity against Plasmodium.•QSAR model based on Support Vector Machines predicts activity with R2 = 0.776•Novel PQ-ureidoamides are active, but an order of magnitude less cytotoxic in vitro than PQ.•Low toxicity is retained in G6PD-inhibited conditions, unlike previous PQ compounds.
New bifunctional quinone methide (QM) precursors, bisphenols 2a-2e, and monofunctional QM precursor 7 were synthesized. Upon treatment with fluoride, desilylation triggers formation of reactive ...intermediates, QMs, which was demonstrated by trapping QM with azide or methanol. The ability of QMs to alkylate and cross-link DNA was assayed by investigation of the effects of QMs to DNA denaturing, but without conclusive evidence. Furthermore, treatment of a plasmid DNA with compounds 2a-2e and KF, followed by the analysis by alkaline denaturing gel electrophoresis, did not provide evidence for the DNA cross-linking. MTT test performed on two human cancer cell lines (MCF7 breast adenocarcinoma and SUM159 pleomorphic breast carcinoma), with and without fluoride, indicated that 2a-2e or the corresponding QMs did not exhibit cytotoxic activity, in line with the lack of ability to cross-link DNA. The lack of reactivity with DNA and biological activity were explained by sequential formation of QMs where bifunctional cytotoxic reagent is probably never produced. Instead, the sequential generation of monofunctional QM followed by a faster hydrolysis leads to the destruction of biologically active reagent. The findings described here are particularly important for the rational design of new generation of QM precursor molecules that will attain desirable DNA reactivity and cytotoxicity. Keywords: antiproliferative activity, DNA alkylation, Grignard reaction, nucleic acid, quinone methide, structure-based drug design.
Excessive expression of subunit 1 of GIRK1 in ER
breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating ...properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10A
with the control (MCF10A
). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.
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