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Polyoxo-noble-metalates (PONMs), a class of molecular noble metal-oxo nanoclusters that combine features of both polyoxometalates and noble metals, are a promising platform for the development of next-generation antitumor metallodrugs. This study aimed to evaluate the antitumor potential against human neuroblastoma cells (SH-SY5Y), as well as toxicity towards healthy human peripheral blood cells (HPBCs), of five polyoxopalladates(II): (Na8[Pd13As8O34(OH)6]·42H2O (Pd13), Na4[SrPd12O6(OH)3(PhAsO3)6(OAc)3]·2NaOAc·32H2O (SrPd12), Na6[Pd13(AsPh)8O32]·23H2O (Pd13L), Na12[SnO8Pd12(PO4)8]·43H2O (SnPd12), and Na12[PbO8Pd12(PO4)8]·38H2O (PbPd12)), as the largest subset of PONMs. A pure inorganic, Pd13, was found as the most potent and selective antineuroblastoma agent with IC50 values (µM) of 7.2 ± 2.2 and 4.4 ± 1.2 for 24 and 48 h treatment, respectively, even lower than cisplatin (28.4 ± 7.4 and 11.6 ± 0.8). The obtained IC50 values (µM) for 24/48 h treatment with SrPd12 and Pd13L were 75.8 ± 6.7/76.7 ± 22.9 and 63.8 ± 3.6/21.4 ± 10.8, respectively, whereas SnPd12 and PbPd12 did not remarkably affect the SH-SY5Y viability (IC50 > > 100 µM). Pd13 caused depolarisation of inner mitochondrial membrane prior to superoxide ion hyperproduction, followed by caspase activation, DNA fragmentation and cell cycle arrest, all hallmarks of apoptotic cell death, and accompanied by an increase in acidic vesicles content, suggestive of autophagy induction. Importantly, Pd13 demonstrated the antitumor effect at concentrations not cytogenotoxic for normal HPBCs. On the contrary, SrPd12 and Pd13L at concentrations ≥ 1/3 IC50 (24 h) decreased HPBC viability and increased % tail DNA up to 42% and 3.05 times, respectively, related to control. SnPd12 and PbPd12, previously confirmed promising antileukemic agents, did not exhibit cytogenotoxicity to HPBCs, and thus could be regarded as tumor cell specific and selective drug candidates.

Polyoxopalladates (POPs) are a subclass of polyoxometalates (POMs) comprising discrete, anionic palladium(II)-oxo complexes. Although numerous studies have been conducted on the biological activity of POMs, their toxicity (e.g. nonselectivity) is frequently a limitation for real-world biomedical applications. Thus, the aim of this study was to evaluate the in vitro safety of the three POPs Pd13 As8 , SrPd12 As6 , and Pd13 (PhAs)8 , which exhibited strong antitumour activity against human neuroblastoma cell line SH-SY5Y, by performing a cyto/genotoxicity study on human healthy blood. Blood samples obtained from a healthy female donor were treated with three different concentrations (12.5, 25, and 50 µmol/L) of the POPs, and incubated at 37 oC for 4 and 24 h. A cytotoxicity (cell viability) assay was performed on isolated human peripheral blood lymphocytes stained with acridine orange and ethidium bromide. A genotoxicity test was carried out on whole blood by alkaline comet assay (microgel electrophoresis), and the percentage of tail DNA was used to assess the level of DNA damage. Pd13 As8 did not affect neither cell viability nor DNA damage, related to the control, at either of the investigated concentrations (after both 4 and 24 h). On the contrary, higher concentrations (25 and 50 µmol/L) of both SrPd12 As6 and Pd13 (PhAs)8 induced a statistically significant decrease in cell viability after 24 h (up to 42 %), and a relative increase of tail DNA (up to 3×) was observed at 50 µmol/L, after 24 h. Therefore, Pd13 As8 could be regarded as non-toxic to human healthy cells, whereas SrPd12 As6 and Pd13 (PhAs)8 require additional toxicity analysis.

Polyoxopalladates (POPs) are discrete, anionic palladium(II)- oxo nanoclusters combining properties of polyoxometalates and palladium(II), and thus are highly promising for the development of novel antitumor metallodrugs. The aim of this study was to investigate in vitro the influence of three POP salts with approved anti-neuroblastoma action, Na8[Pd13As8O34(OH)6]·42H2O (Pd13), Na4[SrPd12O6(OH)3(PhAsO3)6(OAc)3]·2NaOAc·32H2O (SrPd12), and Na6[Pd13O8(PhAsO3)8]·23H2O (Pd13L), on E-NTPDase activity using rat synaptic plasma membranes (SPMs) isolated from Wistar brain as a model system. Dose-dependent inhibition of ENTPDases was obtained within concentration range 2 × 10-6 - 1 × 10-3 mol/L for all investigated POPs. Inhibition parameters, IC50 value and Hill's coefficient, nH, were determined by sigmoidal fitting the experimental results. The calculated IC50 values were (1.08 ± 0.25) × 10-4 , (1.19 ± 0.13) × 10-4 , and (2.06 ± 0.88) × 10-4 mol/L for Pd13, SrPd12, and Pd13L, respectively, indicating their similar inhibitory strengths. The nH values were determined to be < 1, indicating negatively cooperative binding for all POPs studied. The observed inhibitory effect of these anti-neuroblastoma POPs on ENTPDase activity suggest that the inhibition of E-NTPDases, the enzymes representing the major part of purinergic signaling, could be considered as a putative mechanism of antitumor action and a new strategy in the development of novel antitumor therapeutics.

Polyoxopalladates(II) (POPs) are the largest subset of polyoxo-noble-metalates (PONMs), representing a class of discrete, anionic noble metal-oxo nanoclusters. In this study, the in vitro effects of two isostructural, fully inorganic POP salts containing tetravalent metal ions (SnIV and PbIV) incorporated inside the cubic Pd12-oxo host-shell, Na12[SnIVO8Pd12(PO4)8]·43H2O (SnPd12) and Na12[PbIVO8Pd12(PO4)8]·38H2O (PbPd12), which were found to exhibit considerable antileukemic effects, on E-NTPDase activity were investigated using rat synaptic plasma membranes (SPMs) as a model system. Concentration-dependent inhibition of E-NTPDases was observed within the concentration range 5 × 10-6 - 2 × 10-4 mol/L for both POPs. Inhibition parameters, half-maximum inhibitory concentrations (IC50 values) and Hill's coefficients, nH, were determined by sigmoidal fitting the experimental results and Hill's analysis. The calculated IC50 values were (6.59 ± 1.09) × 10-5 and (9.88 ± 3.83) × 10-5 mol/L for SnPd12 and PbPd12, respectively. The calculated nH values were < 1, indicating negatively cooperative enzyme-inhibitor binding for both POPs. Accordingly, the confirmed antileukemic activities of SnPd12 and PbPd12 could be associated with the observed inhibition of E-NTPDases as a potential target of the antileukemic action of these promising drug candidates.

Polyoxometalates (POMs) are discrete, negatively charged metal-oxo clusters of early transition metal ions in high oxidation states. Their biological significance has greatly increased in recent years because of their approved anticancer, antibiotic, and antidiabetic properties. However, toxicity studies have reported adverse effects after in vivo POM studies, which limits their potential application in biomedicine. The aim of this study is to evaluate the in vitro cyto- and genotoxic properties of two polyoxopalladates(II) containing tetravalent metal ion guests, SnPd12 and PbPd12, that were found to possess potent antitumor activities against the human acute promyelocytic cell line HL-60. For this purpose, blood samples obtained from a healthy female donor were treated with three different concentrations (12.5, 25, and 50 µmol/L) of the tested POPs, and incubated at 37 o C for 4 and 24 h, respectively. Cytotoxicity studies were performed on isolated human peripheral blood lymphocytes which were stained with acridine orange and ethidium bromide, and then viewed under a fluorescence microscope. The genotoxicity was tested in whole blood by alkaline comet assay (microgel electrophoresis). The percentage of tail DNA was used to determine the level of DNA damage. The obtained cytotoxicity results indicated that neither SnPd12 nor PbPd12 induced statistically significant alterations of cell viability related to the control, at all of the investigated concentrations. Moreover, the results of the comet assay showed that none of the tested POPs resulted in a statistically significant relative increase of tail DNA. Accordingly, both SnPd12 and PbPd12 could be considered as safe promising antileukaemic drugs from a cyto/genotoxicity point of view.

Polyoxo-noble-metalates (PONMs), a class of molecular noble metal-oxo nanoclusters that combine features of both polyoxometalates and noble metals, are a promising platform for the development of next-generation antitumor metallodrugs. This study aimed to evaluate the antitumor potential against human neuroblastoma cells (SH-SY5Y), as well as toxicity towards healthy human peripheral blood cells (HPBCs), of five polyoxopalladates(II): (Na8[Pd13As8O34(OH)6]·42H2O (Pd13), Na4[SrPd12O6(OH)3(PhAsO3)6(OAc)3]·2NaOAc·32H2O (SrPd12), Na6[Pd13(AsPh)8O32]·23H2O (Pd13L), Na12[SnO8Pd12(PO4)8]·43H2O (SnPd12), and Na12[PbO8Pd12(PO4)8]·38H2O (PbPd12)), as the largest subset of PONMs. A pure inorganic, Pd13, was found as the most potent and selective antineuroblastoma agent with IC50 values (µM) of 7.2 ± 2.2 and 4.4 ± 1.2 for 24 and 48 h treatment, respectively, even lower than cisplatin (28.4 ± 7.4 and 11.6 ± 0.8). The obtained IC50 values (µM) for 24/48 h treatment with SrPd12 and Pd13L were 75.8 ± 6.7/76.7 ± 22.9 and 63.8 ± 3.6/21.4 ± 10.8, respectively, whereas SnPd12 and PbPd12 did not remarkably affect the SH-SY5Y viability (IC50 > > 100 µM). Pd13 caused depolarisation of inner mitochondrial membrane prior to superoxide ion hyperproduction, followed by caspase activation, DNA fragmentation and cell cycle arrest, all hallmarks of apoptotic cell death, and accompanied by an increase in acidic vesicles content, suggestive of autophagy induction. Importantly, Pd13 demonstrated the antitumor effect at concentrations not cytogenotoxic for normal HPBCs. On the contrary, SrPd12 and Pd13L at concentrations ≥ 1/3 IC50 (24 h) decreased HPBC viability and increased % tail DNA up to 42% and 3.05 times, respectively, related to control. SnPd12 and PbPd12, previously confirmed promising antileukemic agents, did not exhibit cytogenotoxicity to HPBCs, and thus could be regarded as tumor cell specific and selective drug candidates.

Background: The discovery of cisplatin and the subsequent research revealed the importance of dinitrogen-containing moiety for the anticancer action of metal complexes. Moreover, certain diamine ligands alone display cytotoxicity that contributes to the overall activity of corresponding complexes. Objective: To summarize the current knowledge on the anticancer efficacy, selectivity, and the mechanisms of action of metal complexes with various types of diamine ligands. Methods: The contribution of aliphatic acyclic, aliphatic cyclic, and aromatic diamine ligands to the anticancer activity and selectivity/toxicity of metal complexes with different metal ions were analyzed by comparison with organic ligand alone and/or conventional platinum-based chemotherapeutics. Results: The aliphatic acyclic diamine ligands are present mostly in complexes with platinum. Aliphatic cyclic diamines are part of Pt(II), Ru(II) and Au(III) complexes, while aromatic diamine ligands are found in Pt(II), Ru(II), Pd(II) and Ir(III) complexes. The type and oxidation state of metal ions greatly influences the cytotoxicity of metal complexes with aliphatic acyclic diamine ligands. Lipophilicity of organic ligands, dependent on alkyl-side chain length and structure, determines their cellular uptake, with edda and eddp/eddip ligands being most useful in this regard. Aliphatic cyclic diamine ligands improved the activity/toxicity ratio of oxaliplatin-type complexes. The complexes with aromatic diamine ligands remain unexplored regarding their anticancer mechanism. The investigated complexes mainly caused apoptotic or necrotic cell death. Conclusion: Metal complexes with diamine ligands are promising candidates for efficient and more selective alternatives to conventional platinum-based chemotherapeutics. Further research is required to reveal the chemico-physical properties and molecular mechanisms underlying their biological activity.

The first two examples of polyoxopalladates(II) (POPs) containing tetravalent metal ion guests, [MO8Pd12(PO4)8]12- (M = SnIV, PbIV), have been prepared and structurally characterized in the solid state, solution, and gas phase. The interactions of the metal ion guests and the palladium-oxo shell were studied by theoretical calculations. The POPs were shown to possess anticancer activity by causing oxidative stress inducing caspase activation and consecutive apoptosis of leukemic cells.

The first two examples of polyoxopalladates(II) (POPs) containing tetravalent metal ion guests, [MO8Pd12(PO4)8]12- (M = SnIV, PbIV), have been prepared and structurally characterized in the solid state, solution, and gas phase. The interactions of the metal ion guests and the palladium-oxo shell were studied by theoretical calculations. The POPs were shown to possess anticancer activity by causing oxidative stress inducing caspase activation and consecutive apoptosis of leukemic cells.

Prevremeno rođena deca su posebno ugrožena zbog skromnijeg antioksidativnog potencijala u kombinaciji sa većom izloženošću oksidativnom stresu. Naime, oksidativni stres izazivaju visoke koncentracije kiseonika, fototerapija i intravenska primena lipida koji su deo nužnog terapijskog pristupa ovoj populaciji dece, kao i učestale infekcije. Humano mleko (HM) predstavlja složeni biološki fluid koji ima antioksidaciona svojstva. Antioksidativni enzimi prisutni u HM su superoksid dismutaza (SOD), katalaza (CAT), glutation peroksidaza (GSH-Px) i glutation reduktaza (GR). Neenzimski antioksidansi u HM su vitamin C, E, beta-karoten, glutation, laktoferin, koenzim Q, beta-endorfini i drugi. Ishrana humanim mlekom kod prevremeno rođene dece ima važnu ulogu u prevenciji oksidativnog stresa. Cilj: Određivanje ukupnog antioksidativnog kapaciteta (UAK) mleka majki prevremeno rođene dece tokom laktacije (kolostrum, prelazno i zrelo mleko). Određivanje UAK zrelog svežeg HM i poređenje sa zrelim HM koje je termički obrađeno (skladištenjem u frižideru, zamrzavanjem ili pasterizacijom ), sa HM u koga je dodat obogaćivač, kao i adaptiranom mlečnom formulom za ishranu prevremeno rođene dece. Upoređen je i antioksidativnni status zrelog obogaćenog mleka i adaptirane mlečne formule za ishranu prevremeno rođene dece. Materijal i metode istraživanja: Ova kohortna studija je obuhvatila 30 majki prevremeno rođene dece (porođaj pre 37. gestacijske nedelje), kod kojih je uspostavljena laktacija, prema redosledu prijema u Institut za neonatologiju u Beogradu, u periodu od 01.12.2014. do 31.05.2016. Uzorci HM su od svake majke uzimani tri puta. Naime UAK je određivan u svežem HM uzorkovanom u prva 3 dana nakon porođaja (kolostrum), zatim od 5-14. dana nakon porođaja i u zrelom HM uzorkovanom nakon 21. dana od porođaja. Kriterijum za isključivanje iz studije je bio prekidanje laktacije, potreba za primenom terapije koja je kontraindikovana za vreme laktacije, kao i pušenje duvana..., Preterm infants are especially exposed to oxidative stress because of poorer antioxidative defense mechanism. Oxidative stress is induced by frequent infections, as well as by high oxygen concentrations, phototherapy and intravenous lipid infusions, all of which are part of necessary therapy in this population of children. Human milk (HM) represents a complex biological fluid with antioxidative properties. Antioxidative enzymes of HM are superoxide-dismutase (SOD), catalase (CAT), glutathione-peroxidase (GSH-Px), glutathione-reductase (GR). Non enzyme antioxidants in HM are vitamin C, vitamin E, beta-carothen, glutathione, lactoferrin, coenzyme Q, beta-endorphins, and others. Human milk nutrition in preterm infants has an important role in prevention of oxidative stress. Goal: Determination of total antioxidant capacity (TAC) of milk of mothers of preterm infants during lactation period (colostrums, early milk, mature milk). TAC of mature fresh milk is determined and compared with milk exposed to thermal processing (refrigeration, freezing or pasteurization), with fortified HM, and preterm milk formula. Antioxidative status of mature fortified milk and preterm milk formula was also compared. Material and methods: This cohort study included 30 mothers of preterm infants (delivery before 37 weeks of gestation), with lactation according to admission in Institute for neonatology, during the period from December 1st 2014. to May 31st 2016. Sample of HM were collected from each mother three times. The TAC was determined in fresh HM collected in the first 3 days after delivery (colostrum), then between days 5 and 14 after the delivery, and in mature HM after 21 days from the delivery. Criteria for exclusion were cessation of lactation, need for therapy contraindicated during breast feeding and smoking. Determination of TAC in HM was performed by different non-enzyme: spectrophotometric (ability of catching DPPH radicals, determination of total phenol content with Folin-Cio

Melanom je visoko agresivni maligni tumor kože. Incidenca melanoma se značajno povećala u poslednjih 30 godina, stope morataliteta su visoke, a i pored napretka u hemio-/imuno-/ i radioterapiji odgovor pacijenata sa uznapredovalim stadijumom bolesti je svega 15-25%. Veliki napori se ulažu u otkrivanje, sintezu, karakterizaciju i ispitivanje mehanizma delovanja novih jedinjenja - potencijalnih antitumorskih agenasa. Među njima se izdvajaju derivati etilendiamin dipropanske kiseline, posebno etil estar cikloheksil analog etilendiamin dipropanske kiseline (EE), čiji antimelanomski efekat do sada nije detaljnije objašnjen. Cilj ovog rada je bio da se ispita citotoksični potencijal EE na ćelijskom i animalnom modelu melanoma kao i molekularni mehanizam dejstva koji EE pokreće. U eksperimentima su korišćene ćelijske linije mišjeg melanoma (B16), mišjih makrofaga (RAW264.7), humanog melanoma (518A2), humanih plućnih fibroblasta (MRC-5) i humanih keratinocita (HaCaT). Vijabilitet ćelija je ispitan testom aktivnosti kisele fosfataze, sulforodamin B testom, MTT testom i bojenjem tripan plavim, dok je integritet ćelijske membrane utvrđen testom oslobađanja laktat dehidrogenaze. Molekularni mehanizam dejstva EE na melanom miša je ispitan utvrđivanjem produkcije slobodnih kiseoničnih radikala, depolarizacije mitohondrija, aktivacije kaspaza, eksternalizacije fosfatidil serina, fragmentacije DNK i prisustva kiselih vezikula u ćelijama, korišćenjem odgovarajućih fluorohroma i analizom na protočnom citofluorimetru. Nivoi proteina značajnih za apoptozu i autofagiju su mereni imunoblot metodom. Morfološke karakteristike ćelija su ispitane fluorescentnom i transmisionom elektronskom mikroskopijom. U in vivo eksperimentima miševima soja C57Bl/6 je indukovan primarni i metastatski melanom injektiranjem B16 ćelija supkutano, odnosno intravenski. Sistemska toksičnost EE je ispitana praćenjem funkcije kostne srži, jetre i bubrega. Ispitan je efekat EE na progresiju supkutanog melanoma u po, Melanoma is a highly aggressive skin malignant tumor. The incidence of melanoma is increasing for the last 30 years, mortality is high, and despite all the improvements in chemo-/immuno- and radiotherapy the response of patients with advanced disease is as low as 15-25 %. Great efforts are put into the discovering, synthesis, characterization and investigation of molecular mechanisms of action of these compounds - potential antitumor agents. Promising new potential cytotoxic agents are derivatives of ethylene diamine dipropanoic acid, specifically ethyl ester cyclohexyl analog of ethylene diamine dipropanoic acid (EE). Its antimelanoma effect was not previously investigated and thus the aim of this research was to assess cytotoxic potential and molecular mechanism of EE's action using cell and animal melanoma models. Mouse melanoma (B16), mouse macrophages (RAW264.7), human melanoma (518A2), human pulmonary fibroblast (MRC-5) and human keratinocyte (HaCaT) cell lines were used in the experiments. Cell viability was assessed using acid phosphatase, sulphorhodamin B, MTT and trypan blue assays, whereas lactate dehydrogenase release assay was used for measuring cell membrane integrity. Molecular mechanism of action was investigated by measuring the production of reactive oxygen species, mitochondrial membrane depolarization, caspase activation, phosphatidyl serine externalization, DNA fragmentation, and number of acidic vesicles, using appropriate fluorochromes followed by flow cytometry analysis. Levels of apoptosis- and autophagy- related proteins were measured using immunoblot. Cell morphology was investigated using fluorescent and transmission electron microscopy. C57Bl/6 mice were used for in vivo experiments. Primary and metastatic melanoma were established by injecting B16 cells subcutaneously and into the tail vein of the mice. The bone marrow, liver, and kidney function were assessed for systemic toxicity of EE. The effect of EE on growth of subcutaneous melan

Melanoma, an aggressive skin tumor with high metastatic potential, is associated with high mortality and increasing morbidity. Multiple available chemotherapeutic and immunotherapeutic modalities failed to improve survival in advanced disease, and the search for new agents is ongoing. The aim of this study was to investigate antimelanoma effects of O, O-diethyl-(S, S)-ethylenediamineN, N'di-2-(3-cyclohexyl) propanoate dihydrochloride (EE), a previously synthesized and characterized organic compound. Mouse melanoma B16 cell viability was assessed using acid phosphatase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, sulforhodamine B, and lactate dehydrogenase assays. Apoptosis and autophagy were investigated using flow cytometry, fluorescence and electron microscopy, and western blotting. In vivo antitumor potential was assessed in subcutaneous mouse melanoma model after 14 days of treatment with EE. Tumor mass and volume were measured, and RT-PCR was used for investigating the expression of autophagy-related, proapoptotic, and antiapoptotic molecules in tumor tissue. Investigated organic compound exerts significant cytotoxic effect against B16 cells. EE induced apoptosis, as confirmed by phosphatidyl serine externalisation, caspase activation, and ultrastructural features typical for apoptosis seen on fluorescence and electron microscopes. The apoptotic mechanism included prompt disruption of mitochondrial membrane potential and oxidative stress. No autophagy was observed. Antimelanoma action and apoptosis induction were confirmed in vivo, as EE decreased mass and volume of tumors, and increased expression of several proapoptotic genes. EE possesses significant antimelanoma action and causes caspasedependent apoptosis mediated by mitochondrial damage and reactive oxygen species production. Decrease in tumor growth and increase in expression of proapoptotic genes in tumor tissue suggest that EE warrants further investigation as a candidate agent in tre

Melanoma, an aggressive skin tumor with high metastatic potential, is associated with high mortality and increasing morbidity. Multiple available chemotherapeutic and immunotherapeutic modalities failed to improve survival in advanced disease, and the search for new agents is ongoing. The aim of this study was to investigate antimelanoma effects of O,O-diethyl-(S,S)-ethylenediamine-N,N'di-2-(3-cyclohexyl) propanoate dihydrochloride (EE), a previously synthesized and characterized organic compound. Mouse melanoma B16 cell viability was assessed using acid phosphatase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, sulforhodamine B, and lactate dehydrogenase assays. Apoptosis and autophagy were investigated using flow cytometry, fluorescence and electron microscopy, and western blotting. In vivo antitumor potential was assessed in subcutaneous mouse melanoma model after 14 days of treatment with EE. Tumor mass and volume were measured, and RT-PCR was used for investigating the expression of autophagy-related, proapoptotic, and antiapoptotic molecules in tumor tissue. Investigated organic compound exerts significant cytotoxic effect against B16 cells. EE induced apoptosis, as confirmed by phosphatidyl serine externalisation, caspase activation, and ultrastructural features typical for apoptosis seen on fluorescence and electron microscopes. The apoptotic mechanism included prompt disruption of mitochondrial membrane potential and oxidative stress. No autophagy was observed. Antimelanoma action and apoptosis induction were confirmed in vivo, as EE decreased mass and volume of tumors, and increased expression of several proapoptotic genes. EE possesses significant antimelanoma action and causes caspase-dependent apoptosis mediated by mitochondrial damage and reactive oxygen species production. Decrease in tumor growth and increase in expression of proapoptotic genes in tumor tissue suggest that EE warrants further investigation as a candidate agent in trea

Uvod: Uprkos uspehu u lečenju različitih vrsta tumora, klinička efikasnost cisplatine je veoma ograničena razvijanjem rezistencije tumora na lek. Kompleksi rutenijuma kao antitumorski agensi najviše obećavaju, pokazujući citotoksičnu aktivnost kod tumora rezistentnih na cisplatinu. Smatra se da ove osobine jedinjenja rutenijuma potiču od njihove sposobnosti da imitiraju način vezivanja gvožđa za biomolekule kao što su humani serum albumin i transferin. Budući da tumorske ćelije na svojim membranama povećano eksprimiraju transferinske receptore zbog povećane potrebe za gvožđem, dopremanje kompleksa rutenijuma do kancerskih ćelija je efikasnije u odnosu na druge komplekse metala. Cilj: Studija se bavi ispitivanjem potencijalne citotoksičnosti dva novosintetisana kompleksa rutenijuma: Ru-1, [Ru(Cl-Ph-tpy)(o-pda)Cl]Cl (Cl-Ph-tpy = 4-(4'-hlorofenil)- 2,2',6',2''-terpiridin, o-pda = o-fenilendiamin) i Ru-2, [Ru(Cl-Ph-tpy)(phen)Cl]Cl(Cl-Ph-tpy = 4- (4'-hlorofenil)-2,2',6',2''-terpiridin,phen = 1,10-fenantrolin) na sledećim tumorskim ćelijama: A549 – adenokarcinom pluća, MCF7 – karcinom dojke, HeLa – karcinom cerviksa, Hs 294T – maligni melanom i na ćelijskoj liniji zdravih fibroblasta MRC-5 (kontrola). Materijal i metod: Citotoksičnost kompleksa rutenijuma Ru-1 i Ru-2 na tumorskim i kontrolnim, zdravim ćelijama je određivana MTT metodom. Tip ćelijske smrti kao i relativni odnos nekrotične i apoptotične smrti tumorskih ćelija izazvane testiranim kompleksima ispitivan je metodom protočne citometrije ćelija bojenih Annexin-om V i 7- aminoaktinomicinom D. Faze ćelijskog ciklusa su određivane metodom bojenja propidijum-jodidom. Aktivacija i lokalizacija ključnih proteina uključenih u proces apoptoze je određena protočnom citometrijom bojenjem specifičnim antitelima. Rezultati: Citotoksičnost kompleksa rutenijuma Ru-1 i Ru-2 je ispitana na četiri tumorske ćelijske linije (A549, MCF7, HeLa i Hs294T) i na zdravim fibroblastima (MRC- 5). Naši rezultati jasno pokazuju značajno smanj, Introduction: Ruthenium complexes might be very promising candidates as antitumor agents, showing activity in tumors which had developed resistance to cisplatin or in which cisplatin is inactive. This is believed to be due to the ability of ruthenium to mimic iron in binding to biomolecules, such as human serum albumin and transferrin. As cancer cells overexpress transferrin receptors, to satisfy their increased demand for iron, ruthenium-based drugs may be delivered more efficiently to cancer cells compared to other metal-based drugs. Objective: The aim of our study was to investigate antitumor effect and the mechanism of action of two newly sinthesized ruthenium complexes: Ru-1, [Ru(Cl-Ph-tpy)(o-pda)Cl]Cl (Cl-Ph-tpy = 4- (4'-hlorofenil)-2,2',6',2''-terpiridin, o-pda = o-fenilendiamin) and Ru-2, [Ru(Cl-Phtpy)( phen)Cl]Cl(Cl-Ph-tpy = 4-(4'-hlorofenil)-2,2',6',2''-terpiridin,phen = 1,10-fenantrolin) against human cancer cell lines: A549 - lung cancer, MCF-7 - breast cancer, HeLa - cervical cancer, Hs 294T- melanoma and one non-cancerous cell line MRC-5 - healthy fibroblasts (control). Material and Methods: The cytotoxicity of two newly synthesized Ru(II) polypyridyl complexes Ru-1 and Ru-2 on experimental and control group of cells was determined by the MTT assay. Apoptosis of both control and experimental group of tumor cells was estimated by annexin Vfluorescein isothiocyanate (FITC)/7-amino-actinomycin D (7-AAD) staining. The cell cycle distribution was determined using propidium iodide staining. The localization and activation of key apoptotic proteins (bax, bcl-2, cytochrome c and caspase-3) was determined by flowcytometry. Results: The cytotoxicity of two ruthenium(II) complexes 1 and 2 was evaluated in four human cancer cell lines (A549, MCF7, HeLa and Hs294T) and in one non-cancer cell line (MRC-5). Our results clearly showed significant decrease of cancer cells’ viability compared to the viability of control, non-cancerous cells, with specific individual cel

This study presents the synthesis, characterization, and antitumor action of five new Pt(II) halogenido, chlorido, and iodido complexes with edda type of ligands. (S,S)-Ethylenediamine-N,N-di-2-(3-cyclohexyl)propanoic acid dihydrochloride and its methyl, ethyl, and n-propyl esters were prepared according to the previously reported procedure. All investigated complexes were characterized by IR, ESI-MS (H-1, C-13, and HMBC) NMR spectroscopy, and elemental analysis. Their cytotoxic action was investigated in four human tumor cell lines: promyelocytic (HL-60) and lymphocytic (REH) leukemia, glioma (U251), and lung carcinoma (H460). Cell viability was assessed by acid phosphatase and LDH assay, while oxidative stress and cell death parameters were analyzed by flow cytometry. The results showed that novel Pt(II) complexes exhibited antitumor action superior to precursor ligands, with iodido complexes being more efficient than corresponding chlorido complexes. Human promyelocytic cell line (HL-60) was the most sensitive to antitumor action of all investigated substances and was used for investigation of the underlying mode of antileukemic action. The investigated Pt(II) complexes showed more potent antileukemic action than corresponding Pt(IV) complex, through induction of oxidative stress and apoptosis, evidenced by caspase (8, 9, and 3) activation and phosphatidylserine externalization.

Hronična limfocitna leukemija (HLL) nastaje zbog poremećaja ravnoteže između apoptoze i proliferacije B limfocita. Preko 70% supstanci koje se koriste u lečenju tumora su ili supstance prirodnog porekla ili supstance dobijene modifikacijom prirodnih supstanci. Osnovni cilj ovog istraživanja bio je da se ispita efekat dve različite vrste propolisa, hrizina i metanolskih ekstrakata biljaka Ligustrum vulgare i Teucrium pollium i gljiva Phellinus linteus i Cordyceps sinensis na vijabilnost ćelija leukemijskih ćelijskih linija MOLT-4 i JVM-13, na vijabilnost limfocita dobijenih iz krvi pacijenata obolelih od HLL-a, kao i na vijabilnost mononuklearnih leukocita izolovanih iz krvi zdravih osoba (kontrola). Uticaj navedenih bioaktivnih supstanci na vijabilnost ćelija MOLT-4 ćelija i JVM-13 ćelija, HLL limfocita, kao i na vijabilnost zdravih mononuklearnih leukocita je ispitan MTT testom i LDH testom. Tip ćelijske smrti izazvan dejstvom ispitivanih supstanci je određen Annexin V/7AAD testom. Ćelijska lokalizacija i količina proteina uključenih u proces apoptoze Bax, Bcl-2, citohroma s i kaspaze-3 je određena metodama protočne citometrije i fluorescentnom mikroskopijom. Istraživanje je pokazalo da hrizin, metanolski ekstrakti lista i ploda biljke Ligustrum vulgare i biljke Teucrium pollium selektivno smanjuju vijabilnost leukemijskih ćelija a ne utiču na zdrave mononuklearne leukocite. Propolisi i metanolski ekstrakt gljive Cordyceps sinensis smanjuju vijabilnost leukemijskih ćelija, ali utiču na vijabilnost i zdravih mononuklearnih leukocita, a metanolski ekstrakt gljive Phellinus linteus smanjuje vijabilnost ćelija leukemijskih ćelijskih linija MOLT-4 i JVM-13, ali povećava vijabilnost limfocita hronične limfocitne leukemije i zdravih mononuklearnih leukocita. Hrizin, metanolski ekstrakti lista i ploda biljke Ligustrum vulgare i metanolski ekstrakt biljke Teucrium pollium indukuju apoptozu limfocita hronične limfocitne leukemije. Hrizin i metanolski ekstrakt biljke Teucrium, Chronic lymphocytic leukemia (CLL) develops due to an imbalance between apoptosis and proliferation of B lymphocytes. More than 70% of compounds used in the treatment of different types of cancers are derived from natural compounds or produced by modification of natural compounds. Therefore, in our investigation we examined the cytotoxicity of two different types of propolis, chrysin, methanol extracts of plants Ligustrum vulgare and Teucrium pollium and fungi Phellinus linteus и Cordyceps sinensis against two leukemia cell lines, MOLT-4 and JVM-13, peripheral blood lymphocytes isolated from CLL patients and peripheral blood mononuclear cells (PBMCs) from healthy individuals (control) in vitro. The effects of these bioactive compounds on viability of MOLT-4 and JVM-13 cell lines, CLL cells derived from CLL patients and PBMCs from healthy subjects were determined by MTT assay and LDH assay. The type of cell death induced by examined compounds was verified by Annexin V/7AAD assay. Intracellular localization and endogenic expression of apoptotic proteins including Bax, Bcl-2, cytochrome c and caspase-3 were determined by flow cytometry and fluorescent microscopy. Our results demonstrated that exposure of MOLT-4 and JVM-13 cell lines and CLL cells to chrysin, leaf and fruit methanol extracts of Ligustrum vulgare and methanol extract of Teucrium pollium, selectively decreased viability of these cells populations, but not in the PBMCs derived from healthy subjects. Both types of propolis and methanol extract of Cordyceps sinensis decreased viability of leukemic cells, but also influenced on viability of PBMCs from healthy subjects. Methanol extract of Phellinus linteus decreased viability of MOLT-4 and JVM-13 cell lines, but increased viability of CLL cells and PBMCs from healthy subjects. Chrysin, leaf and fruit methanol extracts of Ligustrum vulgare and methanol extract of Teucrium pollium induced apoptosis of CLL cells. Our findings demonstrated that chrysin and methan

Copper(II) complexes with the condensation derivative of methyl hydrazinoacetate and 2-acetylpyridine were synthesized. The X-ray crystal structures for both complexes revealed that they are polymerized isomers. A common feature of both complexes is the bidentate coordination of the ligand by one hydrazone and one pyridine nitrogen atom. In the monomeric complex, the copper(II) center is tetracoordinate, whereas dimerization through chlorido bridges results in a pentacoordinate arrangement about the metal ions in the dimer. The electronic and magnetic properties of both complexes are discussed on the basis of their X-ray structures, electron paramagnetic resonance (EPR) spectroscopy studies, and superconducting quantum interference device (SQUID) magnetization measurements combined with DFT calculations. Magnetostructural comparisons with structurally similar copper(II) complexes are also provided, and a possible correlation has been established. The antitumor activities of the Cu-II complexes were investigated against six different cancer cell lines, and the results suggest that the antiglioma action of the dimeric species is based on oxidative-stress-mediated phosphatidylserine externalization and caspase activation, which indicate apoptosis.

U ovom radu su opisani sinteza, karakterizacija i biološka aktivnost nove (S,S)-1,3-propandiamin-N,N’-di-2-(3-cikloheksil)propanske kiseline i njenih estara (metil,etil, n-propil, n-butil, izobutil, n-pentil i izopentil), koji su korišćeni za sintezukvadratno-planarnih Pt(II) jodido kompleksa, opšte formule [PtI2{(S,S)-R2pddchxp}].Takođe je opisana sinteza, karakterizacija i biološka aktivnost novihorganorutenijum(II) kompleksa sa estrima (S,S)-etandiamin-N,N’-di-2-(3-cikloheksil)propanske kiseline (metil, etil, n-propil, n-butil, izobutil i izopentil), opšteformule [(η6-p-cimen)RuCl{(S,S)-R2eddchxp}]PF6.Polazna kiselina je dobijena u reakciji (S)-2-amino-3-cikloheksilpropanskekiseline i 1,3-dibrompropana u vodenom rastvoru, dok je njena esterifikacija izvršenapomoću tionil-hlorida i odgovarajućeg alkohola. Jodido kompleksi platine(II) sudobijeni reakcijom kalijum-tetrahloridoplatinata(II) i odgovarajućih estara, uz dodatakkalijum-jodida, dok su odgovarajući rutenijum(II) kompleksi sintetisani u reakcijiizmeđu [(η6-p-cimen)RuCl2]2 i estara (S,S)-etandiamin-N,N’-di-2-(3-cikloheksil)propanske kiseline.Karakterizacija liganada je urađena klasičnim metodama: elementalnomanalizom, IC, (1H i 13C) NMR spektroskopijom i masenom spektrometrijom, dok sukompleksi pored navedenih metoda, okarakterisani i elektronskom apsorpcionomspektroskopijom, rendgenskom strukturnom analizom za kompleks [(η6-pcimen)RuCl{(S,S)-iBu2eddchxp}]PF6 i 195Pt NMR spektroskopijom za kompleks[PtI2{(S,S)-iBu2pddchxp}]. Na osnovu rezultata dobijenih karakterizacijom, utvrđeno jeda kompleksi platine(II) imaju kvadratno-planarnu strukturu, sa N,N’ bidentatnovezanim estarskim ligandima. Na osnovu rendgenske-strukturne analize kompleksa[(η6-p-cimen)RuCl{(S,S)-iBu2eddchxp}]PF6, potvrđena je pseudo-oktaedarskageometrija sintetisanih Ru(II) kompleksa, sa koordinacijom estarskog liganda preko..., In this work, the synthesis, characterization and biological activity of novel(S,S)-1,3-propanediamine-N,N’-di-2-(3-cyclohexyl)propanoic acid, its esters (methyl,ethyl, n-propyl, n-butyl, i-butyl, n-pentyl and i-pentyl) and corresponding square-planariodido Pt(II) complexes, of general formula [PtI2{(S,S)-R2pddchxp}], are presented. Inaddition, the synthesis, characterization and biological activity of novelorganorutenium(II) complexes with esters of (S,S)-ethanediamine-N,N’-di-2-(3-cyclohexyl)propanoic acid (methyl, ethyl, n-propyl, n-butyl, i-butyl and i-pentyl),general formula, of [(η6-p-cimen)RuCl{(S,S)-R2eddchxp}]PF6, were reported.Starting acid was obtained by reaction of (S)-2-amino-3-cyclohexyl-propanoicacid and 1,3-dibromopropane, in water solution, while esterification was performedwith thionyl chloride and corresponding alcohol. Iodido complexes of platinum(II) wereobtained by reaction of potassium tetrachloridoplatinate(II) and synthesized esters, withadding of potassium iodide. Ruthenium(II) complexes were synthesized by reactionbetween [(η6-p-cymene)RuCl2]2 and different esters of (S,S)-ethanediamine-N,N’-di-2-(3-cyclohexyl)propanoic acid. Ligands were characterized by elemental analysis, IR,(1H and 13C) NMR spectroscopy and mass spectrometry, while the complexes, inaddition to these methods, were characterized and UV/Vis, single crystal X-raystructure diffraction analysis for [(η6-p-cymene)RuCl{(S,S)-iBu2eddchxp}]PF6 and 195PtNMR spectroscopy for [PtI2{(S,S)-iBu2pddchxp}]. Based on these results, it is foundthat complexes platinum(II) have square-planar geometry with coordination of ligandsvia nitrogen donor atoms of ester ligands. Single crystal X-ray structure diffractionanalysis of [(η6-p-cymene)RuCl{(S,S)-iBu2eddchxp}]PF6 showed the usual piano-stoolgeometry of complexes, with N,N’ coordination of ester ligands. The stability of thecomplexes of platinum and ruthenium in different solvents was investigated, as well asthe ability of these complexes

New square-planar Pd(II) and Pt(II) complexes with 8-quinolinecarboxaldehyde selenosemicarbazone have been synthesized and characterized by use of elemental analysis, molar conductivity measurements, and IR and NMR spectroscopy. The cytotoxic activity of the ligand, new Pt(II) and Pd(II) compounds, and previously synthesized Pd(II), Pt(II), Cd(II), and Ni(II) complexes with the analogous ligand, 2-quinolinecarboxaldehyde selenosemicarbazone, was tested against two human cancer cell lines: lung carcinoma (H460) and glioma (U251). The potential of these compounds to induce perturbations of the H460 cell cycle was also evaluated. These substances had an excellent radical-scavenging effect against ABTS radical cations. The best antimicrobial activity, among two yeasts and eight bacterial strains tested, was against Bacillus cereus.

This study involves the synthesis and characterization of novel cyclohexyl 1,3-propanediamine-N, N'-diacetate molecules as well as investigation of their cytotoxic action. New acid 1a was synthesized by reaction between (S)-2-amino-3-cyclohexylpropanoic acid and 1,3-dibromopropane, while the esters (1b-1e) derived from this acid were obtained by reaction of the corresponding absolute alcohol, thionyl chloride and synthesized acid. All compounds were characterized by IR, ESI-MS, (H-1, C-13 and HSQC) NMR spectroscopy and elemental analysis. The cytotoxic activity of all compounds was tested on several tumour cell lines: human (U251) and rat (C6) glioma, human promyelocytic leukaemia (HL-60), human neuroblastoma (SHSY-5Y) and mouse fibrosarcoma (L929) as well as primary rat astrocytes. The present study reveals potent antitumour activity of novel purely organic compounds (1a-1e), which was most pronounced in human glioma (U251) cells. The esterification is required for the novel compounds' cytotoxic action since the n-butyl ester 1e was the most efficient compound. Importantly, n-butyl ester 1e was more toxic to glioma cells in comparison to rat astrocytes, with 24-h IC50 values lower than those for cisplatin. n-Butyl ester 1e induced production of reactive oxygen species (ROS) and caused an oxidative- stress-derived accumulation of glioma cells in the G(0)/G(1) phase of the cell cycle, as well as caspase activation and DNA fragmentation, suggesting that apoptosis induction plays an important role in the novel compounds' antiglioma action. (C) 2014 Elsevier Inc. All rights reserved.

Cilj ovog istraživanja je bio da ispita molekularne mehanizme u osnovi citotoksičnog dejstva estara (S,S)-etilendiamin-N,N’-di-2-(3- cikloheksil)propanske kiseline na leukemijskim ćelijama. Vijabilitet ćelija je procenjivan testom aktivnosti kisele fosfataze i laktat dehidrogenaze, parametri apoptotske smrti ćelija, kao i diferencijacije su analizirani protočnom citometrijom i elektronskom mikroskopijom. Intranukleusna lokalizacija faktora pokretača apoptoze (AIF) je utvrđena tehnikom imunoblota. Pokazano je da metil-, etil- i propil-estar deluju citotoksično na HL-60, REH, MOLT-4, KG-1, JVM-2 i K-562 leukemijske ćelijske linije, dok ishodno jedinjenje (kiselina) i butil-estar nisu pokazali toksičnost u in vitro uslovima. Citotoksičnost etil-estra je bila najveća (IC50: 10,7 – 45,4 μM), i uporediva sa cisplatinom, tipičnim proapoptotskim antitumorskim lekom. U osnovi antileukemijskog delovanja etil-estra na HL-60 ćelijsku liniju, najosetljiviju od ispitivanih, nalazila se hiperprodukcija superoksidnog anjona i depolarizacija mitohondrijalne membrane. Oštećenje mitohondrija je za posledicu imalo eksternalizaciju fosfatidil-serina, fragmentaciju DNK i sledstvenu apoptozu HL-60 ćelija. Fragmentacija DNK je bila posredovana translokacijom AIF-a iz mitohondrija u jedro, i prethodila je aktivaciji kaspaza, što je ukazalo na apoptozu nezavisnu od kaspaza. Ćelije tretirane subtoksičnim dozama etil-estra su pokazale morfološke znake granulocitne diferencijacije (segmentacija jedra i primarne granule u citoplazmi) kao i porast u ekspresiji markera diferencijacije CD11b i CD15. Sposobnost da uzrokuje apoptozu i diferencijaciju leukemijskih ćelija čini cikloheksil analoge etilendiamindipropanske kiseline po tipu estara potencijalnim kandidatima za dalja predklinička istraživanja sa ciljem ispitivanja mogućnosti ulaska u buduće kliničke studije..., The aim of the present study was to investigate antileukemic action of recently synthesized (S,S)-ethylenediamine-N,N’-di-2-(3-cyclohexyl)propanoic acid esters. Cell viability was assessed by the acid phosphatase and LDH release assay, apoptosis- and differentiation-related parameters were analyzed by flow cytometry/electron microscopy, while intracellular localization of apoptosis-inducing factor (AIF) was determined by immunoblotting. It was demonstrated that methyl, ethyl and propyl ester were toxic to HL-60, REH, MOLT-4, KG-1, JVM-2 and K-562 leukemic cell lines, while the non-esterified parental compound and butyl ester were devoid of in vitro antileukemic action. The cytotoxic activity of the ethyl ester was the highest (IC50: 10.7 - 45.4 μM) and comparable to that of the prototypical anticancer drug cisplatin. Its cytotoxic effect in HL-60 cells was associated with an increase in superoxide production and mitochondrial membrane depolarization, leading to apoptotic cell death characterized by phosphatidylserine externalization and DNA fragmentation. DNA fragmentation preceded caspase activation and followed AIF translocation from mitochondria to nucleus, indicating that the observed apoptotic death was caspase-independent. Surviving cells displayed morphological signs of granulocytic differentiation (nuclear indentations and presence of cytoplasmic primary granules), as well as an increased expression of differentiation markers CD11b and CD15. The ability to induce both apoptosis and differentiation of leukemic cells makes the investigated cyclohexyl analogues plausible candidates for further preclinical examination and probably clinical trials...

We investigated the cytotoxicity of recently synthesized (S,S)-ethyleridiamine-N,N'-di-2-(3-cyclohexyl)propanoic acid esters toward human leukemic cell lines and healthy blood mononuclear cells. Cell viability was assessed by acid phosphatase assay, apoptosis, and differentiation were analyzed by flow cytometry and electron microscopy, while intracellular localization of apoptosis-inducing factor (AIF) was determined by immunoblotting. It was demonstrated that methyl, ethyl, and n-propyl esters were toxic to HL-60, REH, MOLT-4, KG-1, JVM-2, and K-562 leukemic cell lines, while the nonesterified parental compound and n-butyl ester were devoid of cytotoxic action. The ethyl ester exhibited the highest cytotoxic activity (IC50 10.7 mu M-45.4 mu M), which was comparable to that of the prototypical anticancer drug cisplatin. The observed cytotoxic effect in HL-60 cells was associated with an increase in superoxide production and mitochondrial membrane depolarization, leading to apoptotic cell death characterized by phosphatidylserine externalization and DNA fragmentation in the absence of autophagic response. DNA fragmentation preceded caspase activation and followed AIF translocation from mitochondria to nucleus, which was indicative of caspase-independent apoptotic cell death. HL-60 cells treated with subtoxic concentration of the compound displayed morphological signs of granulocytic differentiation (nuclear indentations and presence of cytoplasmic primary granules), as well as an increased expression of differentiation markers CD11b and CD15. The cyclohexyl analogues of ethylenediamine dipropanoic acid were also toxic to peripheral blood mononuclear cells of both healthy controls and leukemic patients, the latter being more sensitive. Our data demonstrate that the toxicity of the investigated cyclohexyl compounds against leukemic cell lines is mediated by caspase-independent apoptosis associated with oxidative stress, mitochondrial dysfunction, and AIF translocation.

Herein we describe the synthesis, characterization, and anticancer activity of novel p-cymeneruthenium(II) complexes containing methyl, ethyl, n-propyl, and n-butyl esters of (S, S)-ethylenediamine-N,N'-di-2-(3-cyclohexyl)propanoic acid. The results of IR, UV/Vis, ESIMS, (1)H, and (13)C NMR characterization reveal that ligand coordination occurs through nitrogen donor atoms of the ester ligands, with the organoruthenium moiety being kept in complex. These ruthenium(II) complexes are cytotoxic toward various cancer cell lines including leukemic HL-60, K562, and REH cells (IC(50) : 1.0-20.2 mu m), with the n-butyl ester complex being the most effective. It causes apoptotic cell death associated with mitochondrial depolarization, caspase activation, phosphatidylserine exposure, and DNA fragmentation. Importantly, the n-butyl ester complex is more effective against leukemic patients' blood mononuclear cells relative to those from healthy control subjects, thus indicating a fairly selective antileukemic action of Ru(II)-based compounds.

New polymeric copper(II) complexes with two tridentate ONS thiosemicarbazone ligands containing substituted pyrazolone moiety were synthesized and characterized by means of spectroscopic, electrochemical and crystallographic techniques. While both ligands exist as different tautomers in the solid state and DMSO-d(6) solution, Cu(II) ion coordinates the ligands from the same tautomeric form with square-pyramidal geometry around each Cu atom. In the crystal structures, the copper(II) complex cation forms polymeric chains {[Cu(L)Cl(+)]}(n) with a bridging chlorine atom. One of the complexes was found to have a significantly higher cytotoxic potential in comparison with cisplatin in inhibition of several cell lines (HL60, REH, C6, L929 and B16). The results obtained on the basis of flow cytometry indicated that apoptosis could be possible mechanism of cell death. (C) 2011 Elsevier Inc. All rights reserved.