In vitro investigating of anticancer activity of new 7-MEOTA-tacrine heterodimers.

A combination of biochemical, biophysical and biological techniques was used to study calf thymus DNA interaction with newly synthesized 7-MEOTA-tacrine thiourea 12-17 and urea heterodimers 18-22, and to measure interference with type I and II topoisomerases. Their biological profile was also inspected in vitro on the HL-60 cell line using different flow cytometric techniques (cell cycle distribution, detection of mitochondrial membrane potential dissipation, and analysis of metabolic activity/viability). The compounds exhibited a profound inhibitory effect on topoisomerase activity (e.g. compound 22 inhibited type I topoisomerase at 1 µM concentration). The treatment of HL-60 cells with the studied compounds showed inhibition of cell growth especially with hybrids containing thiourea (14-17) and urea moieties (21 and 22). Moreover, treatment of human dermal fibroblasts with the studied compounds did not indicate significant cytotoxicity. The observed results suggest beneficial selectivity of the heterodimers as potential drugs to target cancer cells.

Influence of Kartogenin on Chondrogenic Differentiation of Human Bone Marrow-Derived MSCs in 2D Culture and in Co-Cultivation with OA Osteochondral Explant.

Articular cartilage has limited capacity for natural regeneration and repair. In the present study, we evaluated kartogenin (KGN), a bioactive small heterocyclic molecule, for its effect on in vitro proliferation and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBMSCs) in monolayer culture and in co-culture models in vitro. OA osteochondral cylinders and hBMSCs were collected during total knee replacement. The effect of KGN on hBMSCs during 21 days of culture was monitored by real-time proliferation assay, immunofluorescence staining, histological assay, scanning electron microscopy (SEM) (imaging and multiplex enzyme-linked immunosorbent assay) ELISA assay. The rate of proliferation of hBMSCs was significantly increased by treatment with 10 µM KGN during nine days of culture. Histological and SEM analyses showed the ability of hBMSCs in the presence of KGN to colonize the surface of OA cartilage and to produce glycosaminoglycans and proteoglycans after 21 days of co-culture. KGN treated hBMSCs secreted higher concentrations of TIMPs and the secretion of pro-inflammatory molecules (MMP 13, TNF-α) were significantly suppressed in comparison with control without hBMSCs. Our preliminary results support the concept that 10 µM KGN enhances proliferation and chondrogenic differentiation of hBMSCs and suggest that KGN is a potential promoter for cell-based therapeutic application for cartilage regeneration.

Tacrine derivatives as dual topoisomerase I and II catalytic inhibitors.

This study examines the binding properties of a series of newly synthetized tacrine derivatives 1-4 and their anticancer effects. Spectroscopic techniques (UV-Vis, fluorescence spectroscopy, thermal denaturation, and linear spectropolarimetry) and viscometry were used to study DNA binding properties and to determine the types of DNA interaction with the studied derivatives. The binding constants for the complexes with DNA were obtained using UV-Vis spectroscopic titrations (K = 1.6 × 10(4)-4.0 × 10(5) M(-1)) and electrophoretic methods were used to determine the effect of the derivatives on topoisomerase I and II activity. Monotacrine derivative 1 showed evidence of topoisomerase Irelaxation activity at a concentration of 30 × 10(-6) M, while bistacrine derivatives 2-4 produced a complete inhibition of topoisomerase Iat a concentration of 5 × 10(-6) M. The biological activities of the derivatives were studied using MTT-assay and flow cytometric methods (detection of mitochondrial membrane potential and measurement of cell viability) following incubation of 24 and 48 h with human leukemic cancer cell line HL60. The ability of the derivatives to impair cell proliferation was also tested through the analysis of cell cycle distribution.

Novel trisubstituted acridines as human telomeric quadruplex binding ligands.

A novel series of trisubstituted acridines were synthesized with the aim of mimicking the effects of BRACO19. These compounds were synthesized by modifying the molecular structure of BRACO19 at positions 3 and 6 with heteroacyclic moieties. All of the derivatives presented in the study exhibited stabilizing effects on the human telomeric DNA quadruplex. UV-vis spectroscopy, circular dichroism, linear dichroism and viscosimetry were used in order to study the nature of the DNA binding in more detail. The results show that all of the novel derivatives were able to fold the single-stranded DNA sequences into antiparallel G-quadruplex structures, with derivative 15 exhibiting the highest stabilizing capability. Cell cycle analysis revealed that a primary trend of the "braco"-like derivatives was to arrest the cells in the S- and G2M-phases of the cell cycle within the first 72h, with derivative 13 and BRACO19 proving particularly effective in suppressing cell proliferation. All studies derivatives were less toxic to human fibroblast cell line in comparison with HT 29 cancer cell line.

Cytotoxic 3,6-bis((imidazolidinone)imino)acridines: synthesis, DNA binding and molecular modeling.

New acridine derivatives bearing two symmetrical imidazolidinone rings, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides 6a-6e (alkyl=ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl), have been prepared and their interactions with calf thymus DNA and selected cell lines were studied. The DNA-binding of 6a-6e to ctDNA was examined by UV-vis, fluorescence, and CD spectroscopy. The binding constants determined by UV-vis spectroscopy were found in the range 1.9×10(5)-7.1×10(5) M(-1). An electrophoretic separation proved that ligands 6a-6e inhibited topoisomerase I in 40 µM concentration although only those with longer alkyl chains were able to penetrate the membranes and efficiently suppress the cell proliferation. The highest activity in cytotoxic tests was found for 3,6-bis((1-n-hexyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochloride (6e) with IC(50)=2.12 µM (HL 60) and 5.28 µM (L1210) after 72 h incubation. Molecular dynamics simulations and calculations of solvent-accessible surface areas (SASAs) were used to explore the intercalation mechanism. MD simulations favor stacking between adjacent C:G base pairs from the minor groove side. MD and SASAs calculations indicate that the decrease of K with alkyl extension is due to negative entropic change upon binding.

3D printed Polylactid Acid based porous scaffold for bone tissue engineering: an in vitro study.

PURPOSE: The objective of this study was to fabricate PLA-based porous scaffold by 3D printing technology and to evaluate their cytotoxicity and biocompatibility under in vitro conditions in respect to bone tissue engineering. MATERIAL AND METHODS: Pure PLA in filamentous form was processed via 3D printing technology of fused filament fabrication into porous scaffolds. The structure and porosity of scaffolds were measured by metrotomography. PLA scaffolds were pre-treated by human serum, foetal bovine serum and complete cell culture medium to enhance bio-attractivity of the scaffold's surface for the adherence of the cells. Cells were enzymatically isolated from the periosteum of the proximal tibia and then expanded in monolayer. Periosteum-derived osteoprogenitors (PDOs) were seeded on the pre-treated PLA scaffolds and subsequent cell proliferation was measured by commercially available cell proliferation assay. Adherence of PDOs on the PLA scaffold was confirmed by scanning electron microscopy (SEM). RESULTS: Prepared scaffolds had well-defined structure and were characterized by uniform distribution of pores. They were non-toxic and biocompatible with PDOs, however, PLA scaffold with the periosteum-derived progenitor cells was significantly better in the group of scaffolds pre-treated with normal human serum. CONCLUSIONS: The obtained PLA porous scaffolds favored attachment of periosteum derived progenitors and proliferation, furthermore, cells penetrated into the scaffold through the interstitial pores which was meaningful for cytocompatibility evaluation.

The neuroprotective effect of rat adipose tissue-derived mesenchymal stem cell-conditioned medium on cortical neurons using an in vitro model of SCI inflammation.

Objectives In this study, a new approach was used with an in vitro model in which neural cells were exposed to conditioned media from the injured spinal cord (SCI-CM) mimicking a local inflammatory microenvironment . Subsequently, the neuroprotective effect of rat adipose tissue-derived msesenchymal stem cell-conditioned media (ATMSC-CM) was investigated through a cell-free based therapy, which was used to treat cortical neurons and astrocytes under inflammation. Methods Primary cell cultures isolated from postnatal day (P6) Wistar rat brain cortex were exposed to SCI-CM derived from the central lesion, rostral and caudal segments of injured spinal cord. After 48 h incubation, the SCI-CM was replaced and primary cultures were cultivated either in DMEM media alone or in ATMSC-CM for 72 h. The impact of ATMSC-CM on the viability of neurons and astrocytes was assessed using a CyQUANT® Direct Cell Proliferation Assay Kit as well as immunocytochemistry analysis. Results Immunocytochemical analysis revealed significant decrease in the number of MAP2 positive neurons exposed to SCI-CM compared to Control. Protection by ATMSC-CM was associated with increased survival of neurons compared to primary culture cultivated in DMEM media alone. The ATMSC-CM effect on astrocytes was more variable and without any significant impact. Conclusion The results demonstrate that SCI-CM mimicking inflammation can reduce cortical neuron survival, and subsequent exposure to ATMSC-CM can stabilize the neuronal population most likely via released neuroprotective and trophic factors. In addition, astrogliosis was not affected by ATMSC-CM.

A Preliminary Study Comparing Microfracture and Local Adherent Transplantation of Autologous Adipose-Derived Stem Cells Followed by Intraarticular Injection of Platelet-Rich Plasma for the Treatment of Chondral Defects in Rabbits.

Objective This study aimed to compare microfracture and application of adipose-derived stem cells (ADSCs) by local adherent technique enhanced by platelet-rich plasma (PRP) to provide a new approach for the repair of cartilage defect. Design Full-thickness cylindrical defects were created in the medial femoral condyle in 9 New Zealand White rabbits (5 months old, 4.65 ± 0.20 kg). Two groups of rabbits ( n = 3) were either treated with ADSCs (Group 1) or the microfracture technique (Group 2) following intraarticular injection of PRP 3 times in weekly intervals. Rabbits in control group ( n = 3) remained untreated. The outcome was assessed macroscopically, histologically, and immunohistochemically. Results At the end of week 12, Group 1 showed better defect filling compared with Group 2. Specimens treated with the combination of ADSCs and PRP exhibited significant differences from the other groups in all criteria of International Cartilage Repair Society macroscopic scoring system. Conclusions Intraarticular injection of autologous PRP in combination with transplantation of autologous ADSCs by local adherent technique enhances the quality of cartilage defect repair with better results in comparison with microfracture surgery in a rabbit model.

Inhibition of DNA topoisomerases I and II and growth inhibition of HL-60 cells by novel acridine-based compounds.

HL-60 cancer cells were treated with a series of novel acridine derivatives (derivatives 1-4) in order to test the compounds' ability to inhibit both cancer cell growth and topoisomerase I and II activity. Binding studies of derivatives 1-4 with calf thymus DNA were also performed using a number of techniques (UV-Vis and fluorescence spectroscopy, thermal denaturation, linear dichroism and viscometry) to determine the nature of the interaction between the compounds and ctDNA. The binding constants for the complexes of the studied acridine derivatives with DNA were calculated from UV-Vis spectroscopic titrations (K=3.1×10(4)-2.0×10(3)M(-1)). Some of the compounds showed a strong inhibitory effect against Topo II at the relatively low concentration of 5µM. Topo I/II inhibition mode assays were also performed and verified that the novel compounds are topoisomerase suppressors rather than poisons. The biological activities of derivatives were studied using MTT assay and flow cytometric methods (detection of mitochondrial membrane potential, measurement of cell viability) after 24 and 48h incubation. The ability of derivatives to impair cell proliferation was tested by an analysis of cell cycle distribution.

Isolation, cultivation and characterisation of pigeon osteoblasts seeded on xenogeneic demineralised cancellous bone scaffold for bone grafting.

Avian osteoblasts have been isolated particularly from chicken embryo, but data about other functional tissue sources of adult avian osteoblast precursors are missing. The method of preparation of pigeon osteoblasts is described in this study. We demonstrate that pigeon cancellous bone derived osteoblasts have particular proliferative capacity in vitro in comparison to mammalian species and developed endogenous ALP. Calcium deposits formation in vitro was confirmed by alizarin red staining. Only a few studies have attempted to investigate bone grafting and treatment of bone loss in birds. Lack of autologous bone grafts in birds has prompted investigation into the use of avian xenografts for bone augmentation. Here we present a method of xenografting of ostrich demineralised cancellous bone scaffold seeded with allogeneic adult pigeon osteoblasts. Ostrich demineralised cancellous bone scaffold supported proliferation of pigeon osteoblasts during two weeks of co - cultivation in vitro. Scanning electron microscopy demonstrated homogeneous adult pigeon osteoblasts attachment and distribution on the surface of xenogeneic ostrich demineralised cancellous bone. Our preliminary in vitro results indicate that demineralised cancellous bone from ostrich tibia could provide an effective biological support for growth and proliferation of allogeneic osteoblasts derived from cancellous bone of pigeons.

Interaction of cholinesterase modulators with DNA and their cytotoxic activity.

This research was focused on a study of the binding properties of a series of cholinesterase reactivators compounds K075 (1), K027 (2) and inhibitors compounds K524, K009 and 7-MEOTA (3-5) with calf thymus DNA. The nature of the interactions between compounds 1-5 and DNA were studied using spectroscopic techniques (UV-vis, fluorescence spectroscopy and circular dichroism). The binding constants for complexes of cholinesterase modulators with DNA were determined from UV-vis spectroscopic titrations (K=0.5 × 10(4)-8.9 × 10(5)M(-1)). The ability of the prepared analogues to relax topoisomerase I was studied with electrophoretic techniques and it was proved that ligands 4 and 5 inhibited this enzyme at a concentration of 30 µM. The biological activity of the novel compounds was assessed through an examination of changes in cell cycle distribution, mitochondrial membrane potential and cellular viability. Inhibitors 3-5 exhibited a cytotoxic effect on HL-60 (human acute promyelocytic leukaemia) cell culture, demonstrated a tendency to affect mitochondrial physiology and viability, and also forced cells to accumulate in the G1/G0-phase of the cell cycle. The cholinesterase reactivators 1 and 2 were found relatively save from the point of view of DNA binding, whereas cholinesterase inhibitors 3-5 resulted as strong DNA binding agents that limit their plausible use.

Inhibition of GSK-3ß reverses the pro-apoptotic effect of proadifen (SKF-525A) in HT-29 colon adenocarcinoma cells.

Proadifen (SKF-525A) is a well-known inhibitor of cytochrome P450 monooxygenases. Besides the prevention of drug metabolism it affects the proliferation of cancer cells, although the mechanisms of possible anti-cancer activity of proadifen have not been fully understood yet. The aim of this study therefore was to evaluate the potential anti-proliferative effect of proadifen on HT-29 colon cancer cells. Our results show that proadifen inhibited the growth of HT-29 cells by the accumulation of cells in the G1 phase of the cell cycle, reduction of metabolic activity and colony formation and by the induction of apoptosis. Analyses of Western blots and flow cytometry revealed time- and dose-dependent phosphatidylserine externalization, caspase-3 activation and PARP cleavage. Intense upregulation of NAG-1 and ATF3 and downregulation of Mcl-1 and Egr-1 were also observed. Further investigation showed that NAG-1 gene silencing by siRNA had no effect on the pro-apoptotic action of proadifen. In contrast, we found that AR-A014418, the specific inhibitor of glycogen synthase kinase-3 ß (GSK-3ß), significantly decreased proadifen-induced apoptosis. Inactivation of GSK-3ß (phosphorylation at serine 9) resulted in changes in phosphatidylserine externalization and caspase-3 activation. These data suggest that GSK-3ß is an important factor in the induction of apoptosis in HT-29 colon cancer cells treated with proadifen.

3,6-bis(3-alkylguanidino)acridines as DNA-intercalating antitumor agents.

A series of 3,6-bis(3-alkylguanidino) acridines was prepared and the interaction of these novel compounds with calf thymus DNA was investigated with UV-vis, fluorescence and circular dichroism spectroscopy, in addition to DNA melting techniques. The binding constants K were estimated to range from 1.25 to 5.26 × 10(5) M(-1), and the percentage of hypochromism was found to be 17-42% (from spectral titration). UV-vis, fluorescence and circular dichroism measurements indicated that the compounds act as effective DNA-intercalating agents. Electrophoretic separation proved that ligands 6a-e relaxed topoisomerase I at a concentration of 60 µM, although only those with longer alkyl chains were able to penetrate cell membranes and suppress cell proliferation effectively. The biological activity of novel compounds was assessed using different techniques (cell cycle distribution, phosphatidylserine externalization, caspase-3 activation, changes in mitochondrial membrane potential) and demonstrated mostly transient cytostatic action of the ethyl 6c and pentyl 6d derivatives. The hexyl derivative 6e proved to be the most cytotoxic. Different patterns of cell penetration were also observed for individual derivatives. Principles of molecular dynamics were applied to explore DNA-ligand interactions at the molecular level.