Three-dimensional cell cultures as a new tool in drug discovery

Background and purpose: Producing of reliable information about pharmacological activity of new chemical entities is essential in early stages of drug discovery and development. There is a continuous need for improvement of existing in vitro technologies, in order to get more accurate and more predictive biological data (and for compounds selection) in pre-clinical screening methods and models. Materials and methods: Two-dimensional (2D) cell cultures, in comparison with original tissues, does not fully reproduce in vivo cell growth and differentiation. Therefore, significant efforts have been made toward the development of more realistic three-dimensional (3D) in vitro cell culture models that would better mimic tissue physiology. Results: Two-dimensional (2D) cell cultures, in comparison with original tissues, does not fully reproduce in vivo cell growth and differentiation. Therefore, significant efforts have been made toward the development of more realistic three-dimensional (3D) in vitro cell culture models that would better mimic tissue physiology. Basic concepts and advantages of 3D cell cultures, as well as different approaches in technologies that enable the cell growth in 3D will be presented here. Possible applications of 3D cell culture in drug discovery will be discussed, and example of formation of spherical growth of three different human breast cancer cells (MDA-MB-231, SK-BR-3 and T-47D cells) in 3D format will be shown. Conclusions: Although biological significance of obtained data from 2D and 3D cell cultures is still poorly understood, discrepancy of compunds activity illustrated importance of implementation 3D cell culture assays in early part of drug discovery process

Comparison of Antitumor Activity of Some Benzothiophene and Thienothiophene Carboxanilides and Quinolones in 2D and 3D Cell Culture System

One of the main reasons for the high drug attrition rate in oncology is the poor clinical predictive power of 2D cancer cell lines cultures in vitro which are the standard assay used as screening assay to select new chemical entities (NKE) with potent anticancer activity. Therefore, there is increasing interest in developing 3D in vitro cell culture systems, as a primary screening assays which would represent a biologically more relevant assay system, due to similarity to physiological conditions in which tumors growth in living organism. Very important is to develop reproducible 3D cell line culture in vitro assays, feasible for the primary screening of NKEs platforms. Within this manuscript we have tested small platform of selected benzo[b]thieno, thieno[2,3-b]thiophene, and thieno[3,2-b]thiophene 2-carboxanilides, as well as their cyclic derivatives [2,3-c]quinolones, which already showed anti-proliferative activity on other cancer cell lines in 2D system. Platform was tested in 2D and 3D cancer cell culture assays on three human breast cancer cell lines (SK-BR-3, MDA-MB-231, T-47D). Those cell lines were selected on the basis of ability to growth and form cell spheres and also on different sensitivity to chemotherapeutic agents. We used doxorubicin as control compound due similar mode of action, (specific intercalation with the DNA double helix), as tested compound probably have. Obtained results in some cases showed significant disagreement, indicating that in early screening selection of active compounds only on 2D activity basis we could pick up false positive compounds (active only on 2D cell culture lines and not on 3D cell lines for which it is clamed that are more similar to real physiological conditions for tumor growth). One possibility for obtained discrepancy of results obtained on 2D and 3D cell cultures could be physico-chemical properties of compounds. Therefore, we analyzed some physico-chemical properties of active compounds as chrom logD values and calculated structural parameters: number of hydrogen bond donors and acceptors, calculated logP and logD values, molecular weight, ionization constants (pKa), number of aromatic rings, number of rotatable bonds and polar surface area. Association of physico-chemical descriptors with observed anti-proliferative activity has been investigated. Basicity, molecular weight and number of H-bond donors are found to be main factors contributing to the anti-proliferative effect of investigated compounds for both 2D and 3D cell cultures. This work is licensed under a Creative Commons Attribution 4.0 International License

Synthesis and Anti-inflammatory Activity of Novel Furochromenes

A series of variously substituted furochromenes, hemiacetals 2, acetals 3, and rearranged compounds 4, were synthesized from variously substituted 4-hydroxycoumarins and evaluated in several in vitro assays, inhibition of mast cell degranulation induced by the activation of Fcε receptor type I or calci-um ionophore and leukotriene B4 (LTB4) inhibition. The most active derivatives, 3p and 4p (8-iso-propyl substitution in coumarin ring) and 3r (5-methyl-8-chloro substitution), showed significant inhibition of mast cell degranulation (Fctriggered) and LTB4, and exhibited significant local anti-inflammatory activity in PMA induced ear edema in CD1 mice, with potency equal (compounds 3p and 4p) or better (compound 3r) in comparison with zileuton, a reference drug used. It might be a promising direction for developing novel drugs as potential agents for the treatment of allergies and other inflammatory diseases.(doi: 10.5562/cca2240

Biological Activity of Newly Synthesized Benzimidazole and Benzothizole 2,5‐Disubstituted Furane Derivatives

Newly designed and synthesized cyano, amidino and acrylonitrile 2, 5‐disubstituted furane derivatives with either benzimidazole/benzothiazole nuclei have been evaluated for antitumor and antimicrobial activity. For potential antitumor activity, the compounds were tested in 2D and 3D cell culture methods on three human lung cancer cell lines, A549, HCC827 and NCI‐H358, with MTS cytotoxicity and BrdU proliferation assays in vitro. Compounds 5, 6, 8, 9 and 15 have been proven to be compounds with potential antitumor activity with high potential to stop the proliferation of cells. In general, benzothiazole derivatives were more active in comparison to benzimidazole derivatives. Antimicrobial activity was evaluated with Broth microdilution testing (according to CLSI (Clinical Laboratory Standards Institute) guidelines) on Gram‐negative Escherichia coli and Gram‐positive Staphylococcus aureus. Additionally, Saccharomyces cerevisiae was included in testing as a eukaryotic model organism. Compounds 5, 6, 8, 9 and 15 showed the most promising antibacterial activity. In general, the compounds showed antitumor activity, higher in 2D assays in comparison with 3D assays, on all three cell lines in both assays. In natural conditions, compounds with such an activity profile (less toxic but still effective against tumor growth) could be promising new antitumor drugs. Some of the tested compounds showed antimicrobial activity. In contrast to ctDNA, the presence of nitro group or chlorine in selected furane‐benzothiazole structures did not influence the binding mode with AT‐DNA. All compounds dominantly bound inside the minor groove of AT‐DNA either in form of monomers or dimer and higher‐order aggregates

Comparison of systemic inflammatory and hematology parameters in normal C57BI/6 and genetically diabetic db/db mice during local wound repair

Uvod: Upala je početni odgovor domaćina na ozljedu. Ona nije ograničena samo na mjesto rane, nego izaziva sustavne promjene uključujući raznovrsne fiziološke i biokemijske promjene koje se skupno nazivaju odgovorom akutne faze. Ove se promjene nastavljaju tijekom rješavanja upale i procesa cijeljenja rane. U ovom ispitivanju smo usporedili serumski amiloid A protein (SAA), hematološke parametre (ukupna bijela krvna slika, postotak neutrofila i lim-focita) te koncentracije interferona-gama (IFN-γ) u serumu tijekom cijeljenja neokludirane, ekscizijske kožne rane u punoj debljini kod genetski dijabetičnih db/db miševa i nedijabetičnih C57Bl/6 miševa iz istoga legla. Materijal i metode: Područje rane izazvane „punch" biopsijom (promjera 8 mm) kod svakog je miša analizirano planimetrijski uz računalnu potporu. Trećeg, 6., 9. i 13. dana od ranjavanja SAA i IFN-g mjereni su u plazmi testovima ELISA, a hematološki parametri u punoj krvi na automatskom hematološkom analizatoru Sysmex SF 3000. Rezultati: Šestog i devetog dana jasno je zabilježeno kašnjenje u zatvaranju rane kod db/db miševa u usporedbi sa zdravim miševima. Ukupna bijela krvna slika bila je značajno viša u db/db miševa 9. i 13. dana. Kroz čitavo razdoblje obnove rane, diferencijalni broj neutrofila bio je viši, a broj limfocita niži kod db/db miševa u usporedbi s C57Bl/6 miševima. Vršne koncentracije SAA zabilježene su 3. dana kod C57Bl/6 miševa i db/db miševa (368,7 mg/L odnosno 173,5 mg/L), s težnjom prema nižim vrijednostima kod db/db miševa. Razine IFN-γ bile su značajno više (P < 0,05) 9. i 13. dana kod db/db miševa (75,3 pg/mLodnosno 89,9 pg/mL) u usporedbi s razinama kod C57Bl/6 miševa (66,6 pg/mL odnosno 57,2 pg/mL). Zaljučak: Lokalni proces tkivne regeneracije kod miševa nakon lokalne kožne ozljede uzrokuje sustavne promjene u perifernoj krvi. Niti određivanje koncentracije SAA niti IFN-γ nije se moglo rabiti za motrenje dinamike cijeljenja rane u ovim vremenskim točkama.Introduction: Inflammation is the initial host response to injury. It is not only localized to the wound site but also causes systemic changes, including a variety of physiological and biochemical changes collectively called the acute phase response. These changes continue during the resolution of inflammation and the wound healing process. In this study we compared serum amyloid A protein (SAA), hematological parameters (total white blood cell count, neutrophil and lymphocyte percentage) and interferon-gamma (IFN-γ) concentrations in serum during healing of non-occluded, excisional, full-thickness dermal wounds in genetically diabetic db/db mice and non-diabetic C57Bl/6 littermates. Materials and Methods: Area of a punch biopsy (8 mm in diameter) wound in each mouse was analyzed by computer-assisted planimetry. On days 3,6, 9 and 13 after wounding, SAA and IFN-γ were measured in plasma by ELISA assays and hematological parameters in whole blood by SysmexSF 3000 automatic hematology analyzer. Results: A delay in the closure of wounds in db/db in comparison to normal mice was clearly seen on days 6 and 9. Total white blood cell count was significantly higher on days 9 and 13 in db/db mice. Differential neutrophil counts were higher and lymphocyte counts lower in db/db mice in comparison to C57BL/6 mice throughout the wound repair period. Peak SAA concentrations were seen on day 3 in C57Bl/6 and db/db mice (368.7 mg/L and 173.5 mg/L, respectively), but tended to be lower in db/db mice. IFN-γ levels were significantly higher (P < 0.05) on days 9 and 13 in db/db (75.3 pg/mL and 89.9 pg/mL, respectively) in comparison to those in C57Bl/6 mice (66.6 pg/mL and 57.2 pg/mL, respectively). Conclusion. The local tissue regeneration process in mice after local skin injury causes systemic changes in peripheral blood. Determination of neither SAA nor IFN-γ concentrations could be used to monitor wound healing dynamics at these time points

Utjecaj tvrdokorne infekcije bakterijom helicobacter pylori na izraženost bcl-2 u upalnim stanicama želučane sluznice

Chronic Helicobacter (H.) pylori infection is an etiological factor related to gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The expression of bcl-2 protein significantly decreases as the grade of MALT lymphoma advances. The aim of this study was to evaluate bcl-2 expression in inflammatory cells in lamina propria in gastric biopsy samples collected from two groups of patients with chronic gastritis divided on the basis of the success or failure of H. pylori eradication. Sixty-five patients with chronic gastritis were divided into two groups of 45 and 20 patients according to their therapeutic response. The gastric mucosa samples were analyzed histologically in both groups of patients before and after standard therapy (for eradicated, after one therapeutic cycle; and for non-eradicated, after three therapeutic cycles) for H. pylori density, urease activity and bcl-2 expression. In the eradicated group of patients, H. pylori eradication was accompanied by significantly lower grades of bacterial colonization and lower urease activity in the corpus and antrum. Bcl-2 expression in inflammatory cells showed no statistically significant changes in either patient group at either location. There was no between-group difference in bcl-2 expression either. In conclusion, persistent long-lasting H. pylori infection is associated with higher grades of bacterial colonization and higher urease activity but not with bcl-2 expression in inflammatory cells.Kronična infekcija bakterijom Helicobacter (H.) pylori je etiološki čimbenik želučanog adenokarcinoma i limfoma limfoidnog tkiva povezanog sa sluznicom (MALT limfoma). Izraženost proteina bcl-2 značajno se smanjuje s napredovanjem stupnja MALT limfoma. Cilj ove studije bio je procijeniti izraženost bcl-2 u upalnim stanicama lamine proprije u uzorcima dobivenim želučanom biopsijom u dvjema skupinama bolesnika s kroničnim gastritisom podijeljenim prema uspješnoj ili neuspješnoj eradikaciji H. pylori. Ukupno je 65 bolesnika s kroničnim gastritisom podijeljeno u dvije skupine od po 45 i 20 bolesnika prema terapijskom odgovoru. U objema skupinama su uzorci želučane sluznice analizirani histološki prije i nakon standardne terapije (kod onih s uspješnom eradikacijom nakon jednog terapijskog ciklusa, a u onih s neuspješnom eradikacijom nakon tri terapijska ciklusa) na gustoću H. pylori, aktivnost ureaze i izraženost bcl-2. Eradikacija H. pylori u skupini bolesnika s uspješnom eradikacijom bila je praćena značajno nižim stupnjem bakterijske kolonizacije i nižom aktivnošću ureaze u korpusu i antrumu. Izraženost bcl-2 nije se statistički značajno promijenila ni na jednoj lokaciji ni u jednoj skupini bolesnika. Isto tako, nije bilo nikakve razlike među dvjema skupinama bolesnika u izraženosti bcl-2. Zaključuje se kako je dugotrajna ustrajna infekcija bakterijom H. pylori povezana s višim stupnjem bakterijske kolonizacije i višom aktivnošću ureaze, ali nije povezana s izraženošću bcl-2 u upalnim stanicama