Rationally repurposed nitroxoline inhibits preclinical models of Epstein-Barr virus-associated lymphoproliferation.

Repurposing of currently used drugs for new indications benefits from known experience with those agents. Rational repurposing can be achieved when newly uncovered molecular activities are leveraged against diseases that utilize those mechanisms. Nitroxoline is an antibiotic with metal-chelating activity used to treat urinary tract infections. This small molecule also inhibits the function of bromodomain and extraterminal (BET) proteins that regulate oncogene expression in cancer. Lymphoproliferation driven by the Epstein-Barr virus (EBV) depends on these same proteins. We therefore tested the efficacy of nitroxoline against cell culture and small animal models of EBV-associated lymphoproliferation. Nitroxoline indeed reduces cell and tumor growth. Nitroxoline also acts faster than the prototype BET inhibitor JQ1. We suggest that this rational repurposing may hold translational promise.

Synthesis and evaluation of a large library of nitroxoline derivatives as pancreatic cancer antiproliferative agents.

Pancreatic cancer (PC) is one of the deadliest carcinomas and in most cases, which are diagnosed with locally advanced or metastatic disease, current therapeutic options are highly unsatisfactory. Based on the anti-proliferative effects shown by nitroxoline, an old urinary antibacterial agent, we explored a large library of newly synthesised derivatives to unravel the importance of the OH moiety and pyridine ring of the parent compound. The new derivatives showed a valuable anti-proliferative effect and some displayed a greater effect as compared to nitroxoline against three pancreatic cancer cell lines with different genetic profiles. In particular, in silico pharmacokinetic data, clonogenicity assays and selectivity indexes of the most promising compounds showed several advantages for such derivatives, as compared to nitroxoline. Moreover, some of these novel compounds had stronger effects on cell viability and/or clonogenic capacity in PC cells as compared to erlotinib, a targeted agent approved for PC treatment.

Targeted Nanoparticles for Co-delivery of 5-FU and Nitroxoline, a Cathepsin B Inhibitor, in HepG2 Cells of Hepatocellular Carcinoma.

BACKGROUND: The first choice of treatment in Hepatocellular Carcinoma (HCC) is 5-fluorouracil (5-FU). Nitroxoline (NIT), a potent inhibitor of Cathepsin B, impairs tumor progression by decreased extracellular matrix degradation. The objective of the current project was designed to target nanoparticles for co-delivery of 5-FU and NIT in order to enhance the 5-FU cytotoxic effects and reduce the metastatic properties of HepG2 cells. METHODS: 5-FU and NIT were loaded in chitosan-chondroitin nanoparticles. To target the CD44 receptors of HepG2 cells, Hyaluronic Acid (HA) was conjugated to the chondroitin by adipic acid dihydrazide and the conjugation was confirmed by FTIR and 1HNMR. After physicochemical characterization and optimization of the processing variables, MTT assay was done on HepG2 and NIH3T3 cell lines to determine the cytotoxic properties of HA targeted nanoparticles. Migration of the cells was studied to compare the co-delivery of the drugs with each drug alone. RESULTS: The optimized nanoparticles showed the particle size of 244.7±16.3nm, PDI of 0.30±0.03, drug entrapment efficiency of 46.3±5.0% for 5-FU and 75.1±0.9% for NIT. The drug release efficiency up to 8 hours was about 37.6±0.9% for 5-FU and 62.9±0.7% for NIT. The co-delivery of 5-FU and NIT in targeted nanoparticles showed significantly more cytotoxicity than the mixture of the two free drugs, non-targeted nanoparticles or each drug alone and reduced the IC50 value of 5-FU from 3.31±0.65µg/ml to 0.17±0.03µg/ml and the migration of HepG2 cells was also reduced to five-fold. CONCLUSION: Co-delivery of 5-FU and NIT by HA targeted chitosan-chondroitin nanoparticles may be promising in HCC.

Organoruthenated Nitroxoline Derivatives Impair Tumor Cell Invasion through Inhibition of Cathepsin B Activity.

Lysosomal cysteine peptidase cathepsin B (catB) is an important tumor-promoting factor involved in tumor progression and metastasis representing a relevant target for the development of new antitumor agents. In the present study, we synthesized 11 ruthenium compounds bearing either the clinical agent nitroxoline that was previously identified as potent selective reversible inhibitor of catB activity or its derivatives. We demonstrated that organoruthenation is a viable strategy for obtaining highly effective and specific inhibitors of catB endo- and exopeptidase activity, as shown using enzyme kinetics and microscale thermophoresis. Furthermore, we showed that the novel metallodrugs by catB inhibition significantly impair processes of tumor progression in in vitro cell based functional assays at low noncytotoxic concentrations. Generally, by using metallodrugs we observed an improvement in catB inhibition, a reduction of extracellular matrix degradation and tumor cell invasion in comparison to free ligands, and a correlation with the reactivity of the monodentate halide leaving ligand.

Nitroxoline induces cell apoptosis by inducing MDM2 degradation in small-cell lung cancer.

The proto-oncogene MDM2 is a nuclear-localized E3 ubiquitin ligase, which promotes tumor formation by targeting tumor suppressor proteins, such as p53, for proteasomal degradation. In this study, the anti-infective drug nitroxoline (NXQ) was screened out to effectively inhibit cell survival of small-cell lung cancer (SCLC) cells, and induce SCLC cell apoptosis by suppressing antiapoptotic proteins (such as Bcl-2 and MCL1) and upregulating proapoptotic protein Bim. In the mechanistic study, NXQ was found to downregulate MDM2 expression by inducing its proteasomal degradation, and thus upregulated p53 expression, which was a substrate protein of MDM2. Moreover, overexpression of MDM2 decreased the cytotoxicity of NXQ on SCLC cells. These results demonstrated that NXQ displayed anti-SCLC activity by suppressing MDM2 expression, which suggested that anti-infective NXQ had potential for SCLC treatment by targeting the MDM2/p53 axis.

Rational design of 5-((1H-imidazol-1-yl)methyl)quinolin-8-ol derivatives as novel bromodomain-containing protein 4 inhibitors.

Bromodomain-containing protein 4 (BRD4), an epigenetic reader of acetyl lysine, has emerged as a promising therapeutic target for many diseases including cancer, inflammation and heart failure. Our previous study reported that nitroxoline, an FDA approved antibiotic, showed potential BRD4 inhibitory activity and antiproliferation activity against leukemia cell lines. In this study, we further explored the structure-activity relationship (SAR) around nitroxoline and employed our previously developed machine learning based activity scoring function BRD4LGR for further analysis. To improve the cellular level activity, physico-chemical properties were optimized using computational approaches. Then the candidates were tested for their ADME/T profiles. Finally, based on this rational hit-to-lead optimization strategy, 3 drug-like BRD4 inhibitors were obtained, with different profiles on cell line selectivity for multiple myeloma, leukemia and triple negative breast cancer. Further mechanism study showed these compounds could down-regulate c-Myc to inhibit cancer cell growth. This work illustrates the application of multiple computer-aided drug design techniques in a hit-to-lead optimization scenario, and provides novel potent BRD4 inhibitors with different phenotype propensities for future cancer treatment.

Albumin-Stabilized Metal-Organic Nanoparticles for Effective Delivery of Metal Complex Anticancer Drugs.

Many metal-organic complexes showed potent anticancer efficacy, but their clinical applications were limited by the lack of administration route because of their poor solubility. To make metal-organic nanoparticles (MONPs) comprising metal complex drugs is a new formulation strategy for their administration. Herein, we developed a facile synthesis of an MONP composed of bovine serum albumin (BSA), Cu2+, and an anticancer agent, 5-nitro-8-hydroxyquinoline (NQ) with albumin as a nanoreactor. The resultant BSA/Cu/NQ nanoparticle (BSA/Cu/NQ NP) showed good stability in different physiological buffers and could target tumors through the enhanced permeability and retention effect and receptor-mediated cellular uptake. As the BSA/Cu/NQ NP could be readily and efficiently internalized by cancer cells, it showed much higher cytotoxic cancer cells than the NQ + Cu(II) complex and NQ. Therefore, the treatment with BSA/Cu/NQ NP noticeably enhanced the anticancer efficacy without causing systemic toxicity, indicating that such a facile preparation method has great potential to prepare other metal complex nanoparticles for drug delivery.

Antitrypanosomatid Pharmacomodulation at Position 3 of the 8-Nitroquinolin-2(1H)-one Scaffold Using Palladium-Catalysed Cross-Coupling Reactions.

An antikinetoplastid pharmacomodulation study at position 3 of the recently described hit molecule 3-bromo-8-nitroquinolin-2(1H)-one was conducted. Twenty-four derivatives were synthesised using the Suzuki-Miyaura cross-coupling reaction and evaluated in vitro on both Leishmania infantum axenic amastigotes and Trypanosoma brucei brucei trypomastigotes. Introduction of a para-carboxyphenyl group at position 3 of the scaffold led to the selective antitrypanosomal hit molecule 3-(4-carboxyphenyl)-8-nitroquinolin-2(1H)-one (21) with a lower reduction potential (-0.56 V) than the initial hit (-0.45 V). Compound 21 displays micromolar antitrypanosomal activity (IC50 =1.5 µm) and low cytotoxicity on the human HepG2 cell line (CC50 =120 µm), having a higher selectivity index (SI=80) than the reference drug eflornithine. Contrary to results previously obtained in this series, hit compound 21 is inactive toward L. infantum and is not efficiently bioactivated by T. brucei brucei type I nitroreductase, which suggests the existence of an alternative mechanism of action.

Novel 8-nitroquinolin-2(1H)-ones as NTR-bioactivated antikinetoplastid molecules: Synthesis, electrochemical and SAR study.

To study the antiparasitic 8-nitroquinolin-2(1H)-one pharmacophore, a series of 31 derivatives was synthesized in 1-5 steps and evaluated in vitro against both Leishmania infantum and Trypanosoma brucei brucei. In parallel, the reduction potential of all molecules was measured by cyclic voltammetry. Structure-activity relationships first indicated that antileishmanial activity depends on an intramolecular hydrogen bond (described by X-ray diffraction) between the lactam function and the nitro group, which is responsible for an important shift of the redox potential (+0.3 V in comparison with 8-nitroquinoline). With the assistance of computational chemistry, a set of derivatives presenting a large range of redox potentials (from -1.1 to -0.45 V) was designed and provided a list of suitable molecules to be synthesized and tested. This approach highlighted that, in this series, only substrates with a redox potential above -0.6 V display activity toward L. infantum. Nevertheless, such relation between redox potentials and in vitro antiparasitic activities was not observed in T. b. brucei. Compound 22 is a new hit compound in the series, displaying both antileishmanial and antitrypanosomal activity along with a low cytotoxicity on the human HepG2 cell line. Compound 22 is selectively bioactivated by the type 1 nitroreductases (NTR1) of L. donovani and T. brucei brucei. Moreover, despite being mutagenic in the Ames test, as most of nitroaromatic derivatives, compound 22 was not genotoxic in the comet assay. Preliminary in vitro pharmacokinetic parameters were finally determined and pointed out a good in vitro microsomal stability (half-life > 40 min) and a 92% binding to human albumin.

Cathepsin B inhibitors: Further exploration of the nitroxoline core.

Human cathepsin B is a cysteine protease with many house-keeping functions, such as intracellular proteolysis within lysosomes. Its increased activity and expression have been strongly associated with many pathological processes, including cancers. We present here the design and synthesis of novel derivatives of nitroxoline as inhibitors of cathepsin B. These were prepared either by omitting the pyridine part, or by modifying positions 2, 7, and 8 of nitroxoline. All compounds were evaluated for their ability to inhibit endopeptidase and exopeptidase activities of cathepsin B. For the most promising inhibitors, the ability to reduce extracellular and intracellular collagen IV degradation was determined, followed by their evaluation in cell-based in vitro models of tumor invasion. The presented data show that we have further defined the structural requirements for cathepsin B inhibition by nitroxoline derivatives and provided additional knowledge that could lead to non-peptidic compounds with usefulness against tumor progression.

Discovery of novel BET inhibitors by drug repurposing of nitroxoline and its analogues.

The BET family of bromodomain-containing proteins (BRDs) is believed to be a promising drug target for therapeutic intervention in a number of diseases including cancer, inflammation and cardiovascular diseases. Hence, there is a great demand for novel chemotypes of BET inhibitors. The drug repurposing strategy offers great benefits to find inhibitors with known safety and pharmacokinetic profiles, thus increasing medicinal chemists' interest in recent years. Using the drug repurposing strategy, a BRD4-specific score based virtual screening campaign on an in-house drug library was conducted followed by the ALPHA screen assay test. Nitroxoline, an FDA-approved antibiotic, was identified to effectively disrupt the interaction between the first bromodomain of BRD4 (bromodomain-containing protein 4) and acetylated H4 peptide with IC50 of 0.98 µM. Nitroxoline inhibited all BET family members with good selectivity against non-BET bromodomain-containing proteins, thus it is defined as a selective BET inhibitor. Based on the crystal structure of the nitroxoline-BRD4_BD1 complex, the mechanism of action as well as BET specificity of nitroxoline were determined. Since the anticancer activity of nitroxoline against MLL leukemia, one of the BET related diseases, has not been studied before, we tested whether nitroxoline might serve as a potential repurposing drug candidate for MLL leukemia. Nitroxoline effectively inhibited the proliferation of MLL leukemia cells by inducing cell cycle arrest and apoptosis. The profound efficacy is, at least in part, due to the inhibition of BET and downregulation of target gene transcription. Our discovery of nitroxoline as a BET inhibitor suggests potential application of nitroxoline and its derivatives for clinical translation in BET family related diseases.

Biologically active perspective synthesis of heteroannulated 8-nitroquinolines with green chemistry approach.

A new class of pyrazolo[4,3-c]quinoline (5a-i, 7a-b) and pyrano[3,2-c]quinoline (9a-i) derivatives were designed and synthesized in moderate to good yields by microwave conditions. To enhance the yield of pyrano[3,2-c]quinoline derivatives, multicomponent one-pot synthesis has been developed. The synthesized compounds were identified by spectral and elemental analyses. Compounds 9a and 9i showed good antibacterial activity against Gram-positive and Gram-negative bacterial strains. All of the new compounds exhibited weak to moderate antioxidant activity, compound 9d exerted significant antioxidant power. The cytotoxicity of these compounds were also evaluated against MCF-7 (breast) and A549 (Lung) cancer cell lines. Most of the compounds displayed moderate to good cytotoxic activity against these cell lines. Compound 9i was found to be significantly active in this assay and also induced cell death by apoptosis. Molecular docking studies were carried out using EGFR inhibitor in order to determine the molecular interactions.

A Selective Small Molecule DNA2 Inhibitor for Sensitization of Human Cancer Cells to Chemotherapy.

Cancer cells frequently up-regulate DNA replication and repair proteins such as the multifunctional DNA2 nuclease/helicase, counteracting DNA damage due to replication stress and promoting survival. Therefore, we hypothesized that blocking both DNA replication and repair by inhibiting the bifunctional DNA2 could be a potent strategy to sensitize cancer cells to stresses from radiation or chemotherapeutic agents. We show that homozygous deletion of DNA2 sensitizes cells to ionizing radiation and camptothecin (CPT). Using a virtual high throughput screen, we identify 4-hydroxy-8-nitroquinoline-3-carboxylic acid (C5) as an effective and selective inhibitor of DNA2. Mutagenesis and biochemical analysis define the C5 binding pocket at a DNA-binding motif that is shared by the nuclease and helicase activities, consistent with structural studies that suggest that DNA binding to the helicase domain is necessary for nuclease activity. C5 targets the known functions of DNA2 in vivo: C5 inhibits resection at stalled forks as well as reducing recombination. C5 is an even more potent inhibitor of restart of stalled DNA replication forks and over-resection of nascent DNA in cells defective in replication fork protection, including BRCA2 and BOD1L. C5 sensitizes cells to CPT and synergizes with PARP inhibitors.

Inhibition of endopeptidase and exopeptidase activity of cathepsin B impairs extracellular matrix degradation and tumour invasion.

Cathepsin B is a lysosomal cysteine protease that is implicated in a number of physiological processes, including protein turnover in lysosomes. Changes in its expression are associated with a variety of pathological processes, including cancer. Due to the structural feature, termed the occluding loop, cathepsin B differs from other cysteine proteases in possessing both, endopeptidase and exopeptidase activity. Here we investigated the impact of both cathepsin B activities on intracellular and extracellular collagen IV degradation and tumour cell invasion using new selective synthetic inhibitors, 2-{[(8-hydroxy-5-nitroquinoline-7-yl)methyl]amino}-acetonitrile (1), 8-(4-methylpiperidin-1-yl)-5-nitroquinoline (2) and 7-[(4-methylpiperidin-1yl)methyl]-5-nitroquinolin-8-ol (3). All three compounds (5 µM) reduced extracellular degradation of collagen IV by MCF-10A neoT cells by 45-70% as determined by spectrofluorimetry and they (50 µM) attenuated intracellular collagen IV degradation by 40-60% as measured with flow cytometry. Furthermore, all three compounds (5 µM) impaired MCF-10A neoT cell invasion by 40-80% as assessed by measuring electrical impedance in real time. Compounds 1 and 3 (5 µM), but not compound 2, significantly reduced the growth of MMTV-PyMT multicellular tumour spheroids. Collectively, these data suggest that the efficient strategy to impair harmful cathepsin B activity in tumour progression may include simultaneous and potent inhibition of cathepsin B endopeptidase and exopeptidase activities.

Quinoline-based clioquinol and nitroxoline exhibit anticancer activity inducing FoxM1 inhibition in cholangiocarcinoma cells.

PURPOSE: Fork head box M1 (FoxM1) is an oncogenic transcription factor frequently elevated in numerous cancers, including cholangiocarcinoma (CCA). A growing body of evidence documents its diverse functions contributing to tumorigenesis and cancer progression. As such, discovery of agents that can target FoxM1 would be valuable for the treatment of CCA. The quinoline-based compounds, namely clioquinol (CQ) and nitroxoline (NQ), represent a new class of anticancer drug. However, their efficacy and underlying mechanisms have not been elucidated in CCA. In this study, anticancer activities and inhibitory effects of CQ and NQ on FoxM1 signaling were explored using CCA cells. METHODS: The effects of CQ and NQ on cell viability and proliferation were evaluated using the colorimetric 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-(4-sulfophenyl)-2H-tetrazolium (MTS assay). Colony formation and cell migration affected by CQ and NQ were investigated using a clonogenic and a wound healing assay, respectively. To demonstrate the agents' effects on FoxM1 signaling, expression levels of the target genes were quantitatively determined using real-time polymerase chain reaction. RESULTS: CQ and NQ significantly inhibited cell survival of HuCCT1 and Huh28 in a dose- and a time-dependent fashion. Further investigations using the rapidly proliferating HuCCT1 cells revealed significant suppression of cell proliferation and colony formation induced by low doses of the compounds. Treatment of CQ and NQ repressed expression of cyclin D1 but enhanced expression of p21. Most importantly, upon CQ and NQ treatment, expression of oncogenic FoxM1 was markedly decreased concomitant with downregulation of various FoxM1's downstream targets including cdc25b, CENP-B, and survivin. In addition, the compounds distinctly impaired HuCCT1 migration as well as inhibited expression of matrix metalloproteinase (MMP)-2 and MMP-9. CONCLUSION: Collectively, this study reports for the first time the anticancer effects of CQ and NQ against CCA cells, and highlights new insights into the mechanism of actions of the quinoline-based compounds to disrupt FoxM1 signaling.

Design, microwave-mediated synthesis and biological evaluation of novel 4-aryl(alkyl)amino-3-nitroquinoline and 2,4-diaryl(dialkyl)amino-3-nitroquinolines as anticancer agents.

Design, microwave-assisted synthesis of novel 4-aryl (alkyl)amino-3-nitroquinoline (1a-1l) and 2,4-diaryl (dialkyl)amino-3-nitroquinolines (2a-2k and 3a) via regioselective and complete nucleophilic substitution of 2,4-dichloro-3-nitroquinoline, respectively in water are presented. The newly synthesized compounds were evaluated for the first time for antiproliferative activity against EGFR overexpressing human lung (A-549 and H-460) and colon (HCT-116-wild type and HCT-116-p53 null) cancer cell lines. Some notions about structure-activity relationships (SAR) are presented. Compounds 2e, 2f, 2j and 3a overall exhibited excellent anticancer activity comparable to erlotinib which was used as a positive control. Molecular modeling studies disclosed the recognition pattern of the compounds and also supported the observed SAR.

FSL-61 is a 6-nitroquinolone fluorogenic probe for one-electron reductases in hypoxic cells.

One-electron reductases that reduce nitro compounds in hypoxic human tumour cells are poorly characterized, but are important for targeting hypoxia with nitroaromatic prodrugs. Fluorogenic probes with defined reductase profiles are needed to interrogate the activity of these enzymes in intact cells. In the present paper, we report a 6-nitroquinolone ester (FSL-61) as a fluorogenic probe for POR (NADPH:cytochrome P450 oxidoreductase) activity under hypoxia, and demonstrate its suitability of monitoring POR by flow cytometry. Reduction of FSL-61 by purified recombinant human POR generated the corresponding hydroxylamine, which was non-fluorescent, but was reduced further to the fluorescent amine in cells. Hydrolysis of the ester side chain facilitated cellular entrapment, enabling detection of heterogeneous POR expression in mixed populations of cells. In addition to POR, forced expression of three other diflavin reductases [MTRR (methionine synthase reductase), NDOR1 (NADPH-dependent diflavin oxidoreductase 1) and NOS2A (nitric oxide synthase 2A)] or NADPH:adrenoredoxin oxidoreductase in HCT116 cells significantly increased hypoxic activation of FSL-61. This reductase profile is similar to that for the dinitrobenzamide prodrug PR-104A under hypoxia, and fluorogenic metabolism of FSL-61 correlated significantly with PR-104A activation in a panel of 22 human tumour cell lines. The present study thus demonstrates the utility of FSL-61 for rapid and non-destructive interrogation of the activity of one-electron reductases in hypoxic cells at the single-cell level.

Development of new cathepsin B inhibitors: combining bioisosteric replacements and structure-based design to explore the structure-activity relationships of nitroxoline derivatives.

Human cathepsin B has many house-keeping functions, such as protein turnover in lysosomes. However, dysregulation of its activity is associated with numerous diseases, including cancers. We present here the structure-based design and synthesis of new cathepsin B inhibitors using the cocrystal structure of 5-nitro-8-hydroxyquinoline in the cathepsin B active site. A focused library of over 50 compounds was prepared by modifying positions 5, 7, and 8 of the parent compound nitroxoline. The kinetic parameters and modes of inhibition were characterized, and the selectivities of the most promising inhibitors were determined. The best performing inhibitor 17 was effective in cell-based in vitro models of tumor invasion, where it significantly abrogated invasion of MCF-10A neoT cells. These data show that we have successfully explored the structure-activity relationships of nitroxoline derivatives to provide new inhibitors that could eventually lead to compounds with clinical usefulness against the deleterious effects of cathepsin B in cancer progression.

Discovery of a new antileishmanial hit in 8-nitroquinoline series.

A series of nitrated 2-substituted-quinolines was synthesized and evaluated in vitro toward Leishmania donovani promastigotes. In parallel, the in vitro cytotoxicity of these molecules was assessed on the murine J774 and human HepG2 cell lines. Thus, a very promising antileishmanial hit molecule was identified (compound 21), displaying an IC(50) value of 6.6 µM and CC(50) values ≥ 100 µM, conferring quite good selectivity index to this molecule, in comparison with 3 drug-compounds of reference (amphotericin B, miltefosine and pentamidine). Compound 21 also appears as an efficient in vitro antileishmanial molecule against both Leishmania infantum promastigotes and the intracellular L. donovani amastigotes (respective IC(50) = 7.6 and 6.5 µM). Moreover, hit quinoline 21 does not show neither significant antiplasmodial nor antitoxoplasmic in vitro activity and though, presents a selective antileishmanial activity. Finally, a structure-activity relationships study enabled to define precisely the antileishmanial pharmacophore based on this nitroquinoline scaffold: 2-hydroxy-8-nitroquinoline.

Photochemical and phototoxic properties of ethyl 1,4-dihydro-8-nitro-4-oxoquinoline-3-carboxylate, a new quinoline derivative.

The present study demonstrates photoinduced generation of superoxide radical anion and singlet oxygen upon UVA irradiation of ethyl 1,4-dihydro-8-nitro-4-oxoquinoline-3-carboxylate (DNQC), and its cytotoxic/phototoxic effects on murine leukemia L1210 cells. The formation of reactive oxygen species (ROS) was investigated by EPR spectroscopy using in situ spin trapping technique and 4-hydroxy-2,2,6,6-piperidine (TMP) for singlet oxygen ((1)O(2)) detection. The EPR spectra monitored upon photoexcitation of aerated solutions of DNQC in dimethylsulfoxide evidenced the efficient activation of molecular oxygen via Types I and II mechanisms. The cytotoxic/phototoxic effects of DNQC, analysis of cell cycle, induction of apoptosis/necrosis, DNA damage and molecular mechanism of apoptotic death of L1210 cells in dark and in the presence of UVA irradiation were compared. DNQC induced a different cytotoxic/phototoxic effect, which was concentration- and time-dependent. The four highest tested concentrations of non-photoactivated and photoactivated DNQC induced immediate cytotoxic/phototoxic effect after 24h cultivation of L1210 cells. This effect decreased with the time of treatment. The irradiation increased the sensitivity of leukemia cell line on DNQC, but the cell sensitivity decreased with time of processing. Quinolone derivative DNQC significantly induced direct DNA strand breaks in L1210 cells, which were increased with the irradiation of cells. The DNA damage generated by DNQC alone/with combination of UVA irradiation induced cell arrest in G(0)/G(1) and G(2)/M phases, decrease in the number of L1210 cells in Sphase and apoptotic cell death of certain part of cell population after 24 h of influence. DNQC alone/with combination of UVA irradiation induced apoptosis in L1210 cells through ROS-dependent mitochondrial pathway.