Acridin-3,6-dialkyldithiourea hydrochlorides as new photosensitizers for photodynamic therapy of mouse leukemia cells.

Acridin-3,6-dialkyldithiourea hydrochlorides (AcrDTUs) have been evaluated as a new group of photosensitizers (PSs) for photodynamic antitumor therapy (PDT). Mouse leukemia cells L1210 were used for testing of AcrDTUs as the new PSs. The irradiation (UV-A light (365 nm), 1.05 J/cm(2)) increased cytotoxicity of all derivatives against L1210 cells more than ten times. The highest photocytotoxicity was found for propyl-AcrDTU with IC50=0.48±0.03 µM after 48 h incubation. A generation of the superoxide radical anion upon UV-A irradiation of propyl-AcrDTU was confirmed by in situ photochemical EPR experiments. To explain a mechanism of photocytotoxic action of AcrDTUs, an intracellular distribution of propyl-AcrDTU has been studied. It was found that AcrDTU in non-irradiated cells was not present in their nucleus but in the lysosomes and partly in the mitochondria, and sequestration of propyl-AcrDTU was dependent on pH in lysosomes. After irradiation, the cell death was induced by oxidative damage of lysosomal and mitochondrial membranes. Concerning the cell cycle, flow cytometry after PDT with propyl-AcrDTU showed a significant increase of the cells in the subG0 phase. Observed signs of necrosis, apoptosis, and autophagy indicate that PDT/AcrDTU leads to multiple cell death types (caspase independent apoptosis, necrosis, and autophagy).

Intracellular distribution of 3,6-bis(3-alkylguanidino)acridines determines their cytotoxicity.

UNLABELLED: Cytotoxicity of two derivatives of 3,6-bis(3-alkylguanidino)acridines (GNDAs; pentyl- and hexyl-GNDA) was determined against three cell lines: a murine immortalized fibroblast cell line NIH-3T3, a human ovarian carcinoma cell line A2780, and a human neuroblastoma cell line SH-SY5Y. We found out that these GNDAs were cytotoxic against A2780 and NIH-3T3 cells but they showed only a marginal cytotoxicity against neuroblastoma cells SH-SY5Y. To explain differences in cytotoxicity, intracellular distribution of GNDAs was monitored. GNDAs were accumulated in A2780 and NIH-3T3 cells in the nuclei (fluorescence microscopy). In contrast to these cell lines, in SH-SY5Y cells, GNDAs were localized outside of the nuclei, at the plasma membrane and surroundings, extending also to the cytosol. This distribution of GNDAs was confirmed by an ImageStream Flow Cytometer. Acetylcholinesterase (AChE) activity in the SH-SY5Y cells decreased upon incubation with GNDAs. Kinetic studies showed that GNDAs were able to inhibit AChE by the same mode as tacrine (9-amino-1,2,3,4-tetrahydroacridine), a known inhibitor of AChE. A low cytotocity of GNDAs against SH-SY5Y cells could be caused by their affinity to AChE (the enzyme is localized mainly at the plasma membrane). The interaction of GNDAs with AChE may affect their intracellular distribution and consequently the cytotoxicity. KEYWORDS: acetylcholinesterase, acridine, neuroblastoma cell line SH-SY5Y.

Novel 3,6-bis(imidazolidine)acridines as effective photosensitizers for photodynamic therapy.

The photoeffect of new proflavine derivatives with DNA-binding and antitumour activities, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was studied to evaluate them as potential photosensitizers for photodynamic antitumor therapy. EPR measurements showed that superoxide radical anion and singlet oxygen were produced upon irradiation of AcrDIMs with UV-A light (>300nm) in the presence of molecular oxygen. This indicates that AcrDIMs may act as photosensitizers. The most active pentyl-AcrDIM and hexyl-AcrDIM displayed photocytotoxic effect toward the mouse lymphocytic leukemia cell line L1210 and human ovarian cancer cells A2780. Antitumor activity of pentyl-AcrDIM increased as high as about 12 times (72h incubation) after irradiation of A2780 cells (365nm, 1.05J/cm(2)). The photocytotoxicity seems to be associated with oxidative stress. Concerning the cell cycle, flow cytometry showed an arrest in the S-phase already 4h after irradiation. In a comet assay, no genotoxicity of AcrDIMs was found. Typical morphologic changes and formation of DNA-ladders indicated induction of apoptotic cell death, though no activation of caspase-3 was observed. Investigation of intracellular localization of pentyl-AcrDIM confirmed its partial accumulation in mitochondria and lysosomes. After irradiation of the A2780 cells, colocalization of pentyl-AcrDIM with monodansylcadaverine, a lysosomal dye, was proven, suggesting that lysosomes in the irradiated cells may be involved in the cell death.

Subcellular localization of proflavine derivative and induction of oxidative stress--in vitro studies.

Acridines have been studied for several decades because of their numerous biological effects, especially anticancer activity. Recently, cytotoxicity of novel acridine derivatives, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was confirmed for leukemic cell lines [Bioorg. Med. Chem.2011, 19, 1790]. The mechanism of action of the most cytotoxic hexyl-AcrDIM was studied in this paper focusing attention on a subcellular distribution of the drug. Accumulation of hexyl-AcrDIM in mitochondria was confirmed after labeling mitochondria with MitoRED using ImageStream Imaging Flow Cytometer. The derivative significantly decreased intracellular ATP level (reduction of ATP level was decreased by vitamin E), and induced oxidative stress (ROS production detected by DHE assay) as well as cell cycle arrest in the S-phase (flow cytometry analysis) already after short-time incubation and induction of apoptosis. Cytotoxicity of hexyl-AcrDIM is closely connected with induction of oxidative stress in cells.

Interaction of a copper(II)-Schiff base complexes with calf thymus DNA and their antimicrobial activity.

The interaction of a copper complexes containing Schiff bases with calf thymus (CT) DNA was investigated by spectroscopic methods. UV-vis, fluorescence and CD spectroscopies were conducted to assess their binding ability with CT DNA. The binding constants K have been estimated from 0.8 to 9.1×10(4) M(-1). The percentage of hypochromism is found to be over 70% (from spectral titrations). The results showed that the copper(II) complexes could bind to DNA with an intercalative mode. Synergic action of Cu(II) complexes with ascorbic acid against Candida albicans induced the generation of free radicals and increased (more than 60 times) antimicrobial effect of these complexes.

Synthetic isothiocyanate indole-3-ethyl isothiocyanate (homoITC) enhances sensitivity of human ovarian carcinoma cell lines A2780 and A2780/CP to cisplatin.

Isothiocyanates (ITCs), popular chemopreventive agents present in cruciferous vegetables, prove growth-inhibiting and apoptosis-inducing activities in cancer cell lines in vitro. Our study presents a new synthetic ITC derivate indol-3-ethyl isothiocyanate (homoITC) as an effective modulator of cellular proliferation and inducer of apoptosis with potential utility as an anticancer drug or a sensitizer to routinely used chemotherapeutic agent cisplatin (cis-Pt).
We analyzed the growth inhibitory effects of homoITC in the human ovarian carcinoma cell line A2780 and its cisplatin-resistant variant A2780/CP using MTT-test and its apoptosis-inducing properties by flow cytometry and caspase 3 activation. Combination index (CI) values from Calcusyn software were used to characterize the interactions of homoITC and cis-Pt as synergistic (CI1). Significant synergistic effect in growth inhibition of homoITC (5 - 15 microM) and cis-Pt (2.5 - 10 microM) on A2780 parental cell line (CI from 0.42 to 0.85) was also observed on A2780/CP resistant subline (CI from 0.18 to 0.73) for 10-50 microM cis-Pt concentrations and the same concentrations of homoITC. Synergy in growth inhibition correlated with the potential of homoITC to stimulate apoptosis induced by cis-Pt.We conclude that homoITC may be worth of further studies assessing its value in the ovarian cancer treatment and elucidating mechanisms of its action.

Cytotoxicity of copper(II) complexes of N-salicylidene-L-glutamate: modulation by ascorbic acid.

Cytotoxic/cytostatic activity of N-salicylidene-L-glutamato diaqua copper(II) complex (CuC) against mice leukemia cells L1210 has been estimated and their bioactivity was enhanced by addition of ascorbic acid. The Cu-complex with isoquinoline ligand (IQ-CuC) had stronger cytostatic effect (IC50 =15.6 microM) than parental complex (CuC) and its cytotoxicity several times increased in the presence of 0.1 mM ascorbic acid (IC50 =1.0 microM). The cytotoxicity has been caused by oxidative stress, enhanced creation of TBARS has been confirmed, and formation of 2',7'-dichlorofluorescein from 2',7'- dichlorodihydrofluorescein has been observed, also. Some hallmarks of apoptotic/necrotic death of L1210 cells have been observed by fluorescent microscopy after dyeing of cell with propidium iodide and Hoechst 33342. In addition, it was confirmed that both complexes in the presence of ascorbic acid cleavaged of pDNA. Although these copper complexes were initially prepared as substances with antioxidant properties we have showed that combined treatment of L1210 cells with IQCuC and ascorbic acid induced strong oxidative stress and death of cells. Our results confirmed that physiological concentration of ascorbic acid increases the cytostatic/cytotoxic efficiency of N-salicylidene-L-glutamato diaqua copper(II) complexes.

Comparison of inhibition of murine leukaemia cell growth by 9-isothiocyanatoacridine and its cytosine adduct: involvement of thiols.

Cytotoxicity of two fluorescent acridine derivatives - 9-isothiocyanatoacridine (AcITC) and N-(9-acridinylthiocarbamoyl) cytosine (AcTCC) - a novel acridine compound, were investigated. Both substances have cytotoxic activity against the L1210 cellular line, IC50 values were in the micromolar range. Despite the high reactivity of AcITC towards thiols, its effects on leukemia cells were similar to naturally occurring isothiocyanates. AcITC changed the intracellular level of glutathione (GSH), and induced apoptosis. Arrest of cell cycle (G2/M-phase) was also observed. AcITC primarily reacted with -SH groups on cellular surface, and the study of the interaction of the isotiocyanate with human erythrocyte ghosts confirmed that the plasma membrane was the first place where AcITC bound. AcTCC does not react with cellular thiols; images obtained with fluorescent microscopy confirmed interaction of AcTCC with chromatine. Although AcTCC induced cellular arrest in the G2/M phase, apoptosis was not confirmed.

DNA binding properties and evaluation of cytotoxic activity of 9,10-bis-N-substituted (aminomethyl)anthracenes.

The results of DNA binding properties for four selected N-substituted 9,10-bis(aminomethyl)anthracenes are presented. DNA binding affinities were studied using UV-vis and fluorescence spectrophotometric titrations, CD spectroscopy, denaturation transition temperature (Tm) measurements and AM1 quantum chemical calculations. The results obtained indicate that the anthracene products intercalate into the stacked base pairs of DNA with binding constants, K, in the range 1.3-10.9 x 10(5)M(-1) and the binding site size in DNA-base pairs, n, extending over the range 2.4-4.6. Tm values increased in the presence of the anthryl probes, thereby reflecting an increased stability of the calf-thymus (CT) DNA double helix and rendering agreement with the spectrometric titration results. The synthesized compounds were tested against L1210 and HeLa tumor cell lines wherein the HeLa cells appeared to be more sensitive than the L1210 cells. 9,10-Bis{[2-(piperazin-1-yl)ethyl]aminomethyl}anthracene exhibited the highest activity of the tested compounds. Our findings were compared with those of a control drug bisantrene.

Potentiation of doxorubicin-induced apoptosis and differentiation by indomethacin in K-562 leukemia cells.

In this study we have examined the antitumor effect of combined administrations of indomethacin (IND) with doxorubicin (DOX) on growth of K-562 leukemia cells. Although, as single drug treatment, only high concentrations of IND reduced growth (>200 microM) and induced apoptosis (>800 microM) of the K-562 cells, a synergistic effects on DOX-induced cell growth inhibition, apoptosis and differentiation were observed during the co-administration of DOX with 10 microM IND. Cells treated with this combination had elevated GSHt level compare to DOX-treated cells. Modulation of GSHt level of DOX-treated cells with Cd2+ ions or BSO confirmed its important role in processes of DOX-induced differentiation. Results of this study showed that IND has a positive effect on therapeutic efficacy of DOX, and could be a perspective modulator in cancer chemotherapy.

Effects of flavonoids on glutathione and glutathione-related enzymes in cisplatin-treated L1210 leukemia cells.

Connections between the ability of quercetin (Qu)and galangin (Ga) to differentially modulate cis-Pt-induced apoptosis and their effects on glutathione system of murine L1210 leukemia cells were studied. The results showed that total glutathione (GSHt) level is increased significantly (cca 123% of control level), both in cells treated with 10 microM Qu and in cells treated with 4 microM cis-Pt and 10 microM Qu in combination. 10 microM Ga had no effect on GSHt content. Activities of glutathione S-transferase (GST) and glutathione reductase (GR) were not changed significantly when 10 microM flavonoids were used. Significant inhibition of GR activity was observed when flavonoids were used in concentrations higher than 25 microM. The presented data indicate that Qu change the redox state of the cells that is implicated in regulation of apoptosis, due to its ability to increase the GSHt level, and thus may potentiate cis-Pt-induced apoptosis of L1210 cells.

Main targets of tetraaza macrocyclic copper complex on L1210 murine leukemia cells.

Several metal complex agents have already been introduced into clinical tumor therapy and others are subject of antitumor studies. In this study we focused on the tetraaza macrocyclic copper complex (Cu(TAAB)Cl(2)). We studied the influence of the substance on cell growth, cell cycle, membrane integrity, necrosis, apotosis and glutathione level on the leukemic cell line L1210 in 1-day (22 h) and 3-day (72 h) experiments. The metal complex shows a dose-dependent antiproliferative effect, without affecting cell cycle phases. The present results confirm that copper complex can damage plasmatic membranes and trigger apoptosis, and that after treatment of leukemic cells with the copper complex, glutathione levels were increased.

The effect of heparin and pentosan polysulfate on the thermal stability of yeast alcohol dehydrogenase.

Heparin and pentosan polysulfate as organic polyanions inhibit yeast alcohol dehydrogenase (YADH). The aim of this study was to determine the effect of heparin and pentosan polysulfate on the thermostability of alcohol dehydrogenase. Spectral and kinetic analyses showed that these compounds increase the thermal stability of the enzyme and eliminate entirely thermal aggregation. The thermostabilizing effect of unfractionated heparin and pentosan polysulfate was accelerated in the presence of NAD+. The addition of NAD+ (11 microM) to the incubation medium decreased the inhibition of the YADH activity in the presence of pentosan polysulfate (1.32 microM). Moreover, 38% of the residual activity of YADH was found after a 5-min incubation at 70 degrees C. These findings indicate that heparinoids not only modulate the enzyme activity but also can prevent the protein's thermal denaturation.

Interactions between heparinoids and alcohol dehydrogenase.

The interaction between polysulfated polysaecharides (low-molecular-weight heparin LMWH, dextran sulfate DS and pentosan sulfate PS) and yeast alcohol dehydrogenase (YADH) was investigated. The fluorescence and UV spectra of YADH after adding the tested polysaccharides have confirmed the interaction between the enzyme and these compounds. Kinetic studies have shown that LMWH, DS and PS are inhibitors of YADH (mixed type with respect to NAD). The most potent inhibitor is PS (ID50=37.5 ng/ml, Ki=0.6 muM). The inhibition effect depends on the ionic strength (the inhibition decreased by about 50% in the presence of 100 mM Na2SO4) and pH value (the inhibition decreased at pH>7). The results indicate that the inhibition effect of these polyanions is caused by their electrostatic interactions with the NAD-binding region of YADH.

Effect of heparin and dextran sulfate on the activity of glutathione reductase from yeast.

Glutathione reductase (EC 1.6.4.2) is a pivotal enzyme of the glutathione antioxidant system in a cell. The kinetic studies of the interaction of glutathione reductase with unfractionated and low molecular weight heparin and dextran sulfate can contribute to explanation of polyanions effect on the conformation changes of glutathione reductase. The tested polyanions inhibit this enzyme and the inhibition effect depends on the ionic strengths and pH value. The most potent inhibitor is dextran sulfate (ID50 is 4.1 micrograms/ml, pH = 6.8, without NaCl). The ionic strength (> 100 mM) allows the reactivating of GR if the concentration of DS is not higher than 80 micrograms/ml. The inhibition effect of tested polyanions is caused by electrostatic interactions with enzyme; the kinetic analyses indicate that it is a mixed inhibition with respect to oxidized glutathione or NADPH.

Effect of N-tricyanovinylamines on the level of glutathione in hepatocytes.

The effects of N-substituted tricyanovinylamines (N-TCVA; RNHC(CN)=C(CN)2) have been studied on rat hepatocytes and liver mitochondria. Derivatives of N-TCVA act on oxidative phosphorylation as uncouplers, and react with thiols within pH 5.0-8.5. N-Isobutyl-, N-benzyl-, and N-cyclohexyl-TCVA influence the level of GSH and GSSG in isolated hepatocytes. They can act as oxidants, but the level of GSSG increases (about 40%) only if the concentration of N-TCVA is higher than 1 micromol/l. If N-TCVA is added to a final concentration higher than 50 micromol/l a decrease of GSH and GSSG level is observed. Derivatives of N-TCVA also influence the level of GSH and GSSG in mitochondria. At 40-400 micromol/l N-TCVA in the incubation medium the level of GSSG increased and the ratio GSH/GSSG was influenced, but the level of total SH groups did not decrease.

Effects of N-tricyanovinylamines on oxidative phosphorylation and level of SH-groups in rat liver mitochondria.

The effects of N-substituted tricyanovinylamines on oxidative phosphorylation as well as on glutathione and total SH group concentrations in rat liver mitochondria was studied. The N-TCVA derivatives studied (N-cyclohexyl; N-isobutyl; N-benzyl; N-phenyl; N-4-Br-phenyl; N-3-nitrophenyl) had an uncoupling effection on the oxidative phosphorylation. They stimulated the respiration of mitochondria and influenced their membrane potential. In their property as SH agents, the N-TCVA derivatives reduced the level of TSH groups of the mitochondria present in concentrations of 2 mumol/mg protein. The activity of succinate dehydrogenase was decreased by N-TCVA by 13%. N-TCVA derivatives changed the redox state of glutathione in mitochondria. This effect was observed at the concentration 0.3 mumol/mg protein. The results obtained in the present study support the view that the glutathione status is more sensitive than the total level of SH groups to incubation of mitochondria with SH agents such as N-TCVA derivatives.