Maja Antunović

Cytotoxic Activity of Novel Palladium-Based Compounds on Leukemia Cell Lines

Effective treatment methods for human leukemia are under development, but so far none of them have been found to be completely satisfactory. It was recently reported that palladium complexes have significant anticancer activity as well as lower toxicity compared with some clinically used chemotherapeutics. The anticancer activities of two novel palladium(II) complexes, [Pd(sac)(terpy)](sac)·4H2O and [PdCl(terpy)](sac)·2H2O, were tested against three human leukemia cell lines, Jurkat, MOLT-4, and THP-1, in comparison with cisplatin and adriamycin. The cytotoxic effect of the drugs was determined using the MTT assay. Cell death was assessed using fluorescein isothiocyanate-annexin/propidium iodide staining for flow cytometry. Furthermore, p53 phosphorylation, poly(ADP-ribose) polymerase cleavage, and Bax and Bcl-2 mRNA levels were examined to elucidate the mechanism of cell death induction. Both complexes exhibited a significant dose-dependent antigrowth effect in vitro. The complexes predominately induced apoptosis, but necrosis was also observed. In-vitro results have shown that palladium(II) complexes may be regarded as potential anticancer agents for treating human leukemia. Therefore, further analysis to determine the putative mechanism of action and in-vivo studies on animal models are warranted.

Human Mesenchymal Stem Cells Differentiation Regulated by Hydroxyapatite Content within Chitosan-Based Scaffolds under Perfusion Conditions

The extensive need for hard tissue substituent greatly motivates development of suitable allogeneic grafts for therapeutic recreation. Different calcium phosphate phases have been accepted as scaffold’s components with positive influence on osteoinduction and differentiation of human mesenchymal stem cells, in terms of their higher fraction within the graft. Nevertheless, the creation of unlimited nutrients diffusion through newly formed grafts is of great importance. The media flow accomplished by perfusion forces can provide physicochemical, and also, biomechanical stimuli for three-dimensional bone-construct growth. In the present study, the influence of a different scaffold’s composition on the human mesenchymal stem cells (hMSCs) differentiation performed in a U-CUP bioreactor under perfusion conditioning was investigated. The histological and immunohistochemical analysis of cultured bony tissues, and the evaluation of osteogenic genes’ expression indicate that the lower fraction of in situ formed hydroxyapatite in the range of 10–30% within chitosan scaffold could be preferable for bone-construct development.

Biomimetic design of bone substitutes based on cuttlefish bone-derived hydroxyapatite and biodegradable polymers: Biomimetic design of bone

Being a major component of bone tissue, hydroxyapatite is the most investigated calcium phosphate in the design and development of bone implants. The high brittleness and poor load-bearing properties have led researchers to manipulate hydroxyapatite performance by applying polymer or metal materials. The present study focuses on biomimetic approach of the hydroxyapatite synthesis from the cuttlefish bone in order to preserve highly porous structure. The low stiffness of hydroxyapatite scaffold was altered by thin polycaprolactone/poly(lactic acid) coating, resulting in remarkably 18-fold increase of Youngs modulus. The mechanical test revealed that poly(lactic acid) increases the stiffness of composite scaffolds which depends on the polycaprolactone/poly(lactic acid) volume ratio. The composite scaffolds are bioactive supporting the deposition of new calcium phosphates when incubated in simulated physiological medium for 21 days. Moreover, the culture of human embryonic kidney cells indicated non-cytotoxicity of the composite scaffolds with emphasis on the cell proliferation during three days of culture.

SAHAquines, Novel Hybrids Based on SAHA and Primaquine Motifs, as Potential Cytostatic and Antiplasmodial Agents

Abstract We report the synthesis of SAHAquines and related primaquine (PQ) derivatives. SAHAquines are novel hybrid compounds that combine moieties of suberoylanilide hydroxamic acid (SAHA), an anticancer agent with weak antiplasmodial activity, and PQ, an antimalarial drug with low antiproliferative activity. The preparation of SAHAquines is simple, cheap, and high yielding. It includes the following steps: coupling reaction between primaquine and a dicarboxylic acid monoester, hydrolysis, a new coupling reaction with O‐protected hydroxylamine, and deprotection. SAHAquines 5 a–d showed significant reduction in cell viability. Among the three human cancer cell lines (U2OS, HepG2, and MCF‐7), the most responsive were the MCF‐7 cells. The antibodies against acetylated histone H3K9/H3K14 in MCF‐7 cells revealed a significant enhancement following treatment with N‐hydroxy‐N′‐{4‐[(6‐methoxyquinolin‐8‐yl)amino]pentyl}pentanediamide (5 b). Ethyl (2E)‐3‐({4‐[(6‐methoxyquinolin‐8‐yl)amino]pentyl}carbamoyl)prop‐2‐enoate (2 b) and SAHAquines were the most active compounds against both the hepatic and erythrocytic stages of Plasmodium parasites, some of them at sub‐micromolar concentrations. The results of our research suggest that SAHAquines are promising leads for new anticancer and antimalarial agents.

PO-084 Ascorbic acid selectively targets glucose metabolism of osteosarcoma stem cells

Introduction Osteosarcoma (OS) is the most common primary bone sarcoma that mainly occurs in children and adolescents. The existence of drug resistant cancer stem cells (CSCs) with progenitor properties is responsible for OS relapse and metastasis. Thus, development of specific therapies targeting OS-CSCs is necessary to increase the long-term survival rate. Although ascorbic acid (AA) has controversial history as anticancer agent, recently it has been re-evaluated revealing more cytotoxic effect to cancer than normal cells. The aim of the study was to analyse AA as potential therapeutic for selective targeting of OS-CSCs. Material and methods To establish primary tumour cultures, tumour samples were mechanically dissected and enzymatically digested. Sarcosphere assay was used to isolate OS-CSCs. The cytotoxic effect of AA was determined by MTT assay as well as relationship between cell concentration and AA. OS-CSCs were treated with different concentrations of AA (2.5–55 µg/ml) during 72 hour. Concentrations of AA used for further experiments were 30 µg/ml and 40 µg/ml, respectively. Effect of AA on sarcosphere-forming ability was measured under low-attachment condition during 28 days. Cell death type was determined by Annexin V/PI staining using flow cytometry. Levels of GAPDH were determined by western blot while ROS were measured by DCFH-DA assay. Seahorse XF analyzer was used to measure glycolysis and oxidative phosphorylation. Results and discussions While AA did not have any effect on hMSCs, U2OS and Hek 293, respectively, AA efficiently induced dose-dependent viability reduction of OS-CSCs. Further, it can be concluded that IC50 values of AA depend on the number of seeded OS-CSCs. AA successfully reduced sarcosphere formation on 6th day. High cytotoxicity of AA was further confirmed by Annexin V/PI staining. Prevalent death mode induced by AA was apoptotic since more than 70% of Annexin V-positive cells were detected. In addition, AA inhibited the activity of the key glycolytic enzyme GAPDH and induced ROS levels. Following the treatment with AA, extracellular acidification rate as a measure of glycolysis, was reduced significantly. Moreover, AA increased metabolic potential of OS-CSCs implying cells’ ability to meet an energy demand via respiration and glycolysis. Conclusion Based on the obtained results, it can be concluded that AA selectively targets OS-CSCs. The death mechanism is based on the blockage of glycolytic cycle and increased intracellular levels of ROS.

Effect of UVC radiation on mouse fibroblasts deficient for FAS-associated protein with death domain

Abstract Purpose: Ultraviolet (UV) radiation-induced apoptosis enabled us to study the mechanism of DNA damage and to investigate how cells avoid consequences of damaged DNA. Cells with extensive DNA damage activate extrinsic and intrinsic pathways of apoptosis. The extrinsic pathway is coupled to a FAS-associated protein with death domain (FADD), an adaptor protein molecule necessary for mediating apoptotic signals through the cell. Materials and methods: Viability and apoptosis of wild-type and FADD-deficient mouse embryonic fibroblasts were investigated 1, 3, 24 and 48 h after exposure to three doses (50, 75 and 300 J/m2) of UVC radiation. Morphological changes were observed using DNA binding dyes (Hoechst and propidium iodide) while biochemical changes were monitored using immunodetection of the poly (ADP-ribose) polymerase (PARP) protein cleavage and caspase-3 activity assay. Results: Results showed that the difference in cell death response between wild-type and FADD-deficient cells depended on dose and incubation time after exposure to UVC radiation. FADD-deficient cells are more sensitive to UVC radiation. Even though FADD-deficient cells lack an adapter protein of apoptotic extrinsic pathway, higher doses of UVC triggered their apoptotic response, while wild-type cells die mainly due to necrosis. A different pattern of caspase 3 activity and PARP cleavage was observed 24 h after radiation between two cell lines confirming higher apoptotic response in FADD-deficient cells. Conclusions: Wild-type cells can execute apoptosis via both, the mitochondrial and the receptor-mediated pathway whereas FADD-deficient cells can only activate the intrinsic pathway. There is a difference in UVC radiation response between two cell lines indicating the role of FADD in the selection of cell death modality.

Bioreactor-Based Bone Tissue Engineering

The aim of this chapter is to describe the main issues of bone tissue engineering. Bone transplants are widely used in orthopedic, plastic and reconstructive surgery. Current technologies like autologous and allogenic transplantation have several disadvantages making them relatively unsatisfactory, like donor site morbidity, chronic pain, and immunogenicity and risk hazard from infectious disease. Therefore, regenerative orthopedics seeks to establish a successful protocol for the healing of severe bone damage using engineered bone grafts. The optimization of protocols for bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreactor, should be able to solve the current limitations in managing bone injuries. We discuss mesenchymal stem cells as the most suitable cell type for bone tissue engineering. They can be isolated from a variety of mesenchymal tissues and can differentiate into osteoblasts when given appropriate mechanical support and osteoinductive signal. Mechanical support can be provided by different cell scaffolds based on natural or synthetic biomaterials, as well as combined composite materials. Three-dimensional support is enabled by bioreactor systems providing several advantages as mechanical loading, homogeneous distribution of cells and adequate nutrients/waste exchange. We also discuss the variety of osteoinductive signals that can be applied in bone tissue engineering. The near future of bone healing and regeneration is closely related to advances in tissue engineering. The optimization of protocols of bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreac‐ tor, should be able to solve the current limitations in managing bone injuries.

561 Renin-angiotensin system expression in myeloproliferative diseases

Myeloproliferative diseases present the group of clonal malignant diseases of hematopoietic stem cell. Somatic mutation of JAK2 gene (JAK2V617F+) is present in most of the patients (>90%) with polycythemia vera (PV), and 50% of patients with essential thrombocythemia (ET). This mutation causes the constitutive activation of tyrosine kinase and the consequence is cytokine independent proliferation of cells. Signaling pathway JAK2/STAT5/BclxL is essential for erythropoiesis, controlling cell proliferation and survival. In JAK2V617F+ PV and ET, growth of erythroid progenitors is erythropoietin independent. There is a lot of evidence of local renin-angiotensin system (RAS) presence in bone marrow affecting cell proliferation and differentiation. There is an increase of mRNA expression of angiotensinogen (AGT), rennin (REN) and angiotensin II receptor 1 (AT2R1) in bone marrow of JAK2V617F+ PV and ET patients. Our research is focused on understanding the correlation of these two pathways in order to find the control points that can be used as a drug targets for myeloproliferative disorders. Bone marrow mononuclear cells from PV and ET patients were seeded on MethoCult (StemCell) in medium with and without erythropoietin (EPO). At day 13 erythroid colonies were observed for morphology differences, colony density and isolation of DNA, RNA and proteins (Trizol, Invitrogene). DNA is used to determine JAK2 status by allele-specific PCR, RNA for real-time PCR detection of RAS components and proteins for Western detection of AT2R1 protein. We analyzed 5 different bone marrow samples by now – 3 PV (JAK2V617F+), 1 PV (JAK2V617F−) and 1 ET (JAK2V617F+). JAK2V617F+ PV and ET erythroid colonies grown without EPO were smaller (50–100 cells), paler and in lower density then erythroid colonies grown with EPO (>200 cells). No erythroid colonies were noticed in JAK2V617F− PV sample without EPO. We collected high quality DNA, RNA and protein from cca 104 cells. We modified protein extraction method to gain better solubilization of proteins by resuspending them in 2% DEA. Preliminary results suggest increase of AT2R1 expression in JAK2V617F+ patients when compared with JAK2V617F− patients. Western and RT-PCR data are in preparation. Confirimation of higher expression of RAS components in patients that have constitutive activation of JAK2 will enable us to use drugs like ACE inhibitors and AT2R1 antagonists in assessing erythroid proliferation-differentiation process.