Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.

Fungal biotransformation of carvone and camphor by Aspergillus flavus and investigation of cytotoxic activities of naturally obtained essential oils.

In this study, the biotransformation of carvone and camphor by Aspergillus flavus and the products were investigated. The biotransformation reaction of carvone by A. flavus resulted in the production of neodihydrocarveol, dihydrocarvone, 2-cyclohexene-1-one,2-methyl-5-(1-methylethenyl), limonene-1,2-diol, trans-p-mentha-1(7),8-dien-2-ol, p-menth-8(10)-ene-2,9-diol, and the biotransformation reaction of camphor resulted in the production of 2 -campholenic acid, 2-cyclohexene-1-one,2-hydroxy-4,4,6,6-tetramethyl, α-campholene aldehyde. The naturally produced essential oils by biotransformation of carvone and camphor were observed to be cytotoxic to breast cancer cells while no significant inhibition was seen in the healthy cell line. Additionally, biotransformation products had the highest inhibition (74%) against aflatoxin B1. The bioactivities of biotransformation products are promising, and they can be further investigated for their therapeutic potential as active agents.

Simultaneous measurement of perillyl alcohol and its metabolite perillic acid in plasma and lung after inhalational administration in Wistar rats.

The inhalational administration of drugs is a practical and non-invasive approach with the potential to reduce side effects and with a quick onset of therapeutic activity. Perillyl alcohol (POH) is a monoterpene with antitumor activity that currently is undergoing clinical evaluation as an inhalational anticancer agent. A detection method was developed that will be applicable to pharmacokinetic studies of not only POH, but also its longer-lived main metabolite, perillic acid (PA), in lung tissue and plasma after inhalational delivery. The anticancer activity of POH was investigated in vitro with the use of various lung cancer cell lines. Toxicity was established by a standard MTT assay, and apoptosis markers were analyzed by Western blot. For the detection of POH and PA in lungs and plasma, albino Wistar rats were used that were exposed to POH inhalation. Tissues were subjected to chromatographic separation on an Agilent Zorbax Eclipse XDB C18 column, followed by detection of absorption in the ultraviolet (UV) range. In vitro, POH exerted cytotoxic activity against six different lung tumor cell lines, and apoptotic cell death was indicated by induction of active caspase 3 and cleavage of poly (ADP-ribose) polymerase 1 (PARP1). These results demonstrate that inhalational delivery of POH results in effective biodistribution and metabolism of POH in the systemic circulation. In addition, our study introduces a simple, rapid HPLC-UV method with high accuracy for simultaneous detection of POH and its metabolite PA in plasma, and for sensitive detection of PA in lung tissue, which should prove useful for applications in clinical studies.

UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus).

Saffron, a spice derived from the dried red stigmas of Crocus sativus, is one of the oldest natural food additives. The flowers have long red stigmas, which store significant quantities of the glycosylated apocarotenoids crocins and picrocrocin. The apocarotenoid biosynthetic pathway in saffron starts with the oxidative cleavage of zeaxanthin, from which crocins and picrocrocin are derived. In the processed stigmas, picrocrocin is converted to safranal, giving saffron its typical aroma. By a targeted search for differentially expressed uridine diphosphate glycosyltransferases (UGTs) in Crocus transcriptomes, a novel apocarotenoid glucosyltransferase (UGT709G1) from saffron was identified. Biochemical analyses revealed that UGT709G1 showed a high catalytic efficiency toward 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene (HTCC), making it suited for the biosynthesis of picrocrocin, the precursor of safranal. The role of UGT709G1 in picrocrocin/safranal biosynthesis was supported by the absence or presence of gene expression in a screening for HTCC and picrocrocin production in different Crocus species and by a combined transient expression assay with CsCCD2L in Nicotiana benthamiana leaves. The identification of UGT709G1 completes one of the most highly valued specialized metabolic biosynthetic pathways in plants and provides novel perspectives on the industrial production of picrocrocin to be used as a flavor additive or as a pharmacological constituent.

Binding of safranal to whey proteins in aqueous solution: Combination of headspace solid-phase microextraction/gas chromatography with multi spectroscopic techniques and docking studies.

The objective of this work was to study molecular binding of safranal to whey proteins by taking advantage of headspace solid-phase microextraction combined with gas chromatography (HS-SPME/GC), fluorescence and circular dichroism (CD) spectroscopies, and docking studies. The results of HS-SPME/GC indicated that bovine serum albumin (BSA) had the highest affinity toward safranal, with binding constant of 3.196 × 103 M-1. Also, binding strength was reduced in the order of α-lactalbumin (α-Lact), whey protein isolate (WPI), and ß-lactoglobulin (ß-Lg). Although there was a good agreement between results of HS-SPME/GC and fluorescence spectroscopy regarding the safranal binding site on whey proteins, the order of their binding affinity toward safranal was not consistent for both techniques. According to docking studies, conformational alterations in secondary and tertiary structures of whey proteins induced by safranal association resulted from hydrophobic interactions and hydrogen bonds.

Alpha-terpineol affects synthesis and antitumor activity of triterpenoids from Antrodia cinnamomea mycelia in solid-state culture.

To enhance production of Antrodia cinnamomea triterpenoids (ACTs) from mycelia in solid-state culture, α-terpineol was added to the medium as an elicitor at an optimal concentration of 0.05 mL L-1. Multi-stage solvent extraction and HPLC analysis were performed, and the compositions of ACTs-E (from culture with elicitor) and ACTs-NE (from culture without elicitor) were found to be quite different. In assays of in vitro antitumor activity, ACTs-E, in comparison with ACTs-NE, produced stronger viability reduction in several tumor cell lines and stronger apoptosis induction in HeLa in a dose-dependent manner. Several related proteins involved in the mitochondrial pathway of apoptosis (p53, Bax, caspase-3) did not show expression upregulation by ACTs-E, suggesting that apoptosis induction occurred through a p53-independent process. Further analysis revealed that ACTs-E strongly inhibited synthesis of topoisomerase I (TOP1) and tyrosyl-DNA phosphodiesterase I (TDP1), which are involved in DNA repair, at both transcriptional and protein levels. Our findings suggest that ACTs-E have potential for applications in the pharmaceutical, clinical, and functional food industries, as a novel antitumor agent and a dual TOP1/TDP1 inhibitor.

Biochemical significance of limonene and its metabolites: future prospects for designing and developing highly potent anticancer drugs.

Monocyclic monoterpenes have been recognized as useful pharmacological ingredients due to their ability to treat numerous diseases. Limonene and perillyl alcohol as well as their metabolites (especially perillic acid and its methyl ester) possess bioactivities such as antitumor, antiviral, anti-inflammatory, and antibacterial agents. These therapeutic properties have been well documented. Based on the aforementioned biological properties of limonene and its metabolites, their structural modification and development into effective drugs could be rewarding. However, utilization of these monocyclic monoterpenes as scaffolds for the design and developments of more effective chemoprotective agents has not received the needed attention by medicinal scientists. Recently, some derivatives of limonene metabolites have been synthesized. Nonetheless, there have been no thorough studies on their pharmacokinetic and pharmacodynamic properties as well as their inhibition against isoprenylation enzymes. In this review, recent research progress in the biochemical significance of limonene and its metabolites was summarized with emphasis on their antitumor effects. Future prospects of these bioactive monoterpenes for drug design and development are also highlighted.

Spectroscopic and computational evaluation on the binding of safranal with human serum albumin: Role of inner filter effect in fluorescence spectral correction.

For determining the pharmacological properties of medicinal compounds, their binding with serum albumins is very crucial. Herein, we have selected safranal, a major constituent of saffron which is known to retain a number of medicinal properties including antioxidant, anti-inflammatory, tumoricidal, anti-genotoxic, and anti-aging activities; and studied its mechanism of binding with human serum albumin at physiological pH using various spectroscopic methods along with computational approach using molecular docking. A change in the difference UV-visible spectrum of HSA in presence of safranal was found which is due to the complex formation. Owing to the strong absorption of safranal at the fluorescence excitation wavelength of HSA (295 nm) and in the whole range of emission, the fluorescence spectra of HSA in presence of safranal were corrected for the inner filter effect. After the correction the spectra were free from the safranal absorption effect and it was found that addition of safranal causes the quenching of HSA fluorescence and a blue shift of the emission maximum which are attributed to the binding of safranal to the protein and dominance of hydrophobic forces in the interaction, respectively. It was evident from the comparison of observed and corrected fluorescence spectra that before correction there was a large red shift while after correction appearance of blue shift was occurred. The involvement of hydrophobic interaction was also found from the extrinsic fluorescence measurements using ANS dye as well as from the analyzed thermodynamic parameters. Safranal was found to partially induce the secondary structure of HSA as construed from the CD measurements. The size of the HSA was also decreased as evident from the DLS and RLS measurements. Both site marker studies and molecular docking simulations suggested that the primary binding site of the safranal in the HSA is Sudlow's site 1 located in the subdomain IIA. Hydrophobic interaction provides the major contribution to the binding forces along with a little amount of hydrogen bonding.

Analysis of changes in volatile constituents and expression of genes involved in terpenoid metabolism in oleocellosis peel.

Oleocellosis is a serious physiological disorder in citrus fruit that mainly results in appearance and quality deterioration. It has been well established that the occurrence of oleocellosis is highly correlated with the release of peel oil from citrus fruit, while there is little information on the dynamic changes in the content of the volatile constituents and the expression of genes involved in terpenoid metabolism during oleocellosis development. In the present research, large changes in the volatile profiles and gene expression in terpenoid metabolism were observed in oleocellosis peels compared to healthy ones. Among volatiles, the decreased contents of α-pinene, d-limonene, ß-myrcene, linalool, ß-caryophyllene, α-terpineol, nonanal, neryl acetate and (-)-carvone played a major role in these changes. For gene expressions in terpenoid metabolism, the up-regulated genes aldehyde dehydrogenase (NAD+) (ALDH) and the down-regulated genes ß-caryophyllene synthase 1 (BCS1), α-terpineol synthase 2 (TES2) and myrcene synthase (MS) were the main differences in oleocellosis peels.

Biogenesis and iron-dependency of ribosomal RNA hydroxylation.

Post-transcriptional modifications of ribosomal RNAs (rRNAs) are involved in ribosome biogenesis and fine-tuning of translation. 5-Hydroxycytidine (ho5C), a modification of unknown biogenesis and function, is present at position 2501 of Escherichia coli 23S rRNA. We conducted a genome-wide screen in E. coli to identify genes required for ho5C2501 formation, and found a previously-uncharacterized gene, ydcP (renamed rlhA), iron-sulfur cluster (isc) genes, and a series of genes responsible for prephenate biosynthesis, indicating that iron-sulfur clusters and prephenate are required for ho5C2501 formation. RlhA interacted with precursors of the 50S ribosomal subunit, suggesting that this protein is directly involved in formation of ho5C2501. RlhA belongs to a family of enzymes with an uncharacterized peptidase U32 motif and conserved Cys residues in the C-terminal region. These elements were essential for ho5C2501 formation. We also found that the frequency of ho5C2501 is modulated by environmental iron concentration. Together, our results reveal a novel biosynthetic pathway for RNA hydroxylation and its response to iron.

Production of flavor compounds from olive mill waste by Rhizopus oryzae and Candida tropicalis

Abstract The purpose of this study was to investigate the production of flavor compounds from olive mill waste by microbial fermentation of Rhizopus oryzae and Candida tropicalis. Olive mill waste fermentations were performed in shake and bioreactor cultures. Production of flavor compounds from olive mill waste was followed by Gas Chromatography–Mass spectrometry, Gas chromatography- olfactometry and Spectrum Sensory Analysis ®. As a result, 1.73-log and 3.23-log cfu/mL increases were observed in the microbial populations of R. oryzae and C. tropicalis during shake cultures, respectively. C. tropicalis can produce a higher concentration of d-limonene from olive mill waste than R. oryzae in shake cultures. The concentration of d-limonene was determined as 185.56 and 249.54 µg/kg in the fermented olive mill waste by R. oryzae and C. tropicalis in shake cultures respectively. In contrast, R. oryzae can produce a higher concentration of d-limonene (87.73 µg/kg) d-limonene than C. tropicalis (11.95 µg/kg) in bioreactor cultures. Based on sensory analysis, unripe olive, wet towel, sweet aromatic, fermented aromas were determined at high intensity in olive mill waste fermented with R. oryzae meanwhile olive mill waste fermented with C. tropicalis had only a high intensity of unripe olive and oily aroma.

O2-(6-Oxocyclohex-1-en-1-yl)methyl diazen-1-ium-1,2-diolates: a new class of nitric oxide donors activatable by GSH/GSTπ with both anti-proliferative and anti-metastatic activities against melanoma.

The new nitric oxide (NO) donor O2-(6-oxocyclohex-1-en-1-yl)methyl diazen-1-ium-1,2-diolate 3c could simultaneously liberate NO as well as an active 3-glutathionyl-2-exomethylene-cyclohexanone 2 in the presence of GSH/GSTπ; exhibit potent antiproliferative activity; repress migration, invasion, and lateral migration of metastatic B16-BL6 cells; and significantly decrease hetero-adhesion of B16-BL6 cells to human umbilical vein endothelial cells.

Glass bottle sampling solid phase microextraction gas chromatography mass spectrometry for breath analysis of drug metabolites.

Breath analysis is a non-invasive approach which may be applied to disease diagnosis and pharmacokinetic study. In the case of offline analysis, the exhaled gas needs to be collected and the sampling bag is often used as the storage vessel. However, the sampling bag usually releases some extra compounds, which may interfere with the result of the breath test. In this study, a novel breath sampling glass bottle was developed with a syringe needle sampling port for solid phase microextraction (SPME). Such a glass bottle scarcely liberates compounds and can be used to collect exhaled gas for ensuing analysis by gas chromatography-mass spectrometry (GC-MS). The glass bottle sampling SPME-GC-MS analysis was carried out to investigate the breath metabolites of myrtol, a multicompound drug normally used in the treatment of bronchitis and sinusitis. Four compounds, α-pinene, 2,3-dehydro-1,8-cineole, d-limonene and 1,8-cineole were found in the exhaled breath of all eight volunteers who had taken the myrtol. While for other ten subjects who had not used the myrtol, these compounds were undetectable. In the SPME-GC-MS analysis of the headspace of myrtol, three compounds were detected including α-pinene, d-limonene and 1,8-cineole. Comparing the results of breath and headspace analysis, it indicates that 2,3-dehydro-1,8-cineole in the breath is the metabolite of 1,8-cineole. It is the first time that this metabolite was identified in human breath. The study demonstrates that the glass bottle sampling SPME-GC-MS method is applicable to exhaled gas analysis including breath metabolites investigation of drugs like myrtol.

Safranal and its analogs inhibit Escherichia coli ATP synthase and cell growth.

Safranal, a dominant component of saffron, is known to have antitumor, cytotoxic, and antibacterial properties. In this study, we examined safranal and its structural analogs-thymol, carvacrol, damascenone, cuminol, 2,6,6-trimethyl-2-cyclohexene-1,4-dione (TMCHD), 4-isopropylbenzyl bromide (IPBB), and 4-tert-butylphenol (TBP) induced inhibition of Escherichia coli membrane bound F1Fo ATP synthase. Safranal and its analogs inhibited wild-type enzyme to variable degrees. While safranal caused 100% inhibition of wild-type F1Fo ATP synthase, only about 50% inhibition occurred for αR283D mutant ATP synthase. Moreover, safranal, thymol, carvacrol, damascenone, cuminol, TMCHD, IPBB, and TBP all fully abrogated the growth of wild-type E. coli cells and had partial or no effect on the growth of null and mutant E. coli strains. Therefore, the antimicrobial properties of safranal, thymol, carvacrol, damascenone, cuminol, TMCHD, IPBB, and TBP can be linked to their binding and inhibition of ATP synthase. Total loss of growth in wild-type and partial or no growth loss in null or mutant E. coli strains demonstrates that ATP synthase is a molecular target for safranal and its structural analogs. Partial inhibition of the αArg-283 mutant enzyme establishes that αArg-283 residue is required in the polyphenol binding pocket of ATP synthase for the binding of safranal. Furthermore, partial growth loss for the null and mutant strains in the presence of inhibitors also suggests the role of other targets and residues in the process of inhibition.

Saffron: An Old Medicinal Plant and a Potential Novel Functional Food.

The spice saffron is made from the dried stigmas of the plant Crocus sativus L. The main use of saffron is in cooking, due to its ability to impart colour, flavour and aroma to foods and beverages. However, from time immemorial it has also been considered a medicinal plant because it possesses therapeutic properties, as illustrated in paintings found on the island of Santorini, dated 1627 BC. It is included in Catalogues of Medicinal Plants and in the European Pharmacopoeias, being part of a great number of compounded formulas from the 16th to the 20th centuries. The medicinal and pharmaceutical uses of this plant largely disappeared with the advent of synthetic chemistry-produced drugs. However, in recent years there has been growing interest in demonstrating saffron's already known bioactivity, which is attributed to the main components-crocetin and its glycosidic esters, called crocins, and safranal-and to the synergy between the compounds present in the spice. The objective of this work was to provide an updated and critical review of the research on the therapeutic properties of saffron, including activity on the nervous and cardiovascular systems, in the liver, its antidepressant, anxiolytic and antineoplastic properties, as well as its potential use as a functional food or nutraceutical.

Enhanced limonene production in cyanobacteria reveals photosynthesis limitations.

Terpenes are the major secondary metabolites produced by plants, and have diverse industrial applications as pharmaceuticals, fragrance, solvents, and biofuels. Cyanobacteria are equipped with efficient carbon fixation mechanism, and are ideal cell factories to produce various fuel and chemical products. Past efforts to produce terpenes in photosynthetic organisms have gained only limited success. Here we engineered the cyanobacterium Synechococcus elongatus PCC 7942 to efficiently produce limonene through modeling guided study. Computational modeling of limonene flux in response to photosynthetic output has revealed the downstream terpene synthase as a key metabolic flux-controlling node in the MEP (2-C-methyl-d-erythritol 4-phosphate) pathway-derived terpene biosynthesis. By enhancing the downstream limonene carbon sink, we achieved over 100-fold increase in limonene productivity, in contrast to the marginal increase achieved through stepwise metabolic engineering. The establishment of a strong limonene flux revealed potential synergy between photosynthate output and terpene biosynthesis, leading to enhanced carbon flux into the MEP pathway. Moreover, we show that enhanced limonene flux would lead to NADPH accumulation, and slow down photosynthesis electron flow. Fine-tuning ATP/NADPH toward terpene biosynthesis could be a key parameter to adapt photosynthesis to support biofuel/bioproduct production in cyanobacteria.

Limonene in exhaled breath is elevated in hepatic encephalopathy.

Breath samples were taken from 31 patients with liver disease and 30 controls in a clinical setting and proton transfer reaction quadrupole mass spectrometry (PTR-Quad-MS) used to measure the concentration of volatile organic compounds (VOCs). All patients had cirrhosis of various etiologies, with some also suffering from hepatocellular cancer (HCC) and/or hepatic encephalopathy (HE). Breath limonene was higher in patients with No-HCC than with HCC, median (lower/upper quartile) 14.2 (7.2/60.1) versus 3.6 (2.0/13.7) and 1.5 (1.1/2.3) nmol mol-1 in controls. This may reflect disease severity, as those with No-HCC had significantly higher UKELD (United Kingdom model for End stage Liver Disease) scores. Patients with HE were categorized as having HE symptoms presently, having a history but no current symptoms and having neither history nor current symptoms. Breath limonene in these groups was median (lower/upper quartile) 46.0 (14.0/103), 4.2 (2.6/6.4) and 7.2 (2.0/19.1) nmol mol-1, respectively. The higher concentration of limonene in those with current symptoms of HE than with a history but no current symptoms cannot be explained by disease severity as their UKELD scores were not significantly different. Longitudinal data from two patients admitted to hospital with HE show a large intra-subject variation in breath limonene, median (range) 18 (10-44) and 42 (32-58) nmol mol-1.

Genetic and correlation analysis of oleoresin chemical components in slash pine.

This is the first comprehensive study of the genetic analysis of the majority of oleoresin components of slash pine (Pinus elliottii). Pine oleoresin, the resin secreted from the pine tree, is a raw material widely used in industrial products. The objective of this study was to explore the genetic variation and correlation between the major oleoresin components of 50 open pollinated families of slash pine. The individual narrow-sense heritability of the 23 oleoresin components and genetic correlations between them were estimated using the residual maximum likelihood in the flexible mixed modeling program, ASReml-R. A high heritability of 0.424 was observed for ß-pinene. Moderate levels of heritability were estimated for ß-phellandrene, methyl abietate, estragole, 15-hydroxy-dehydroabietic acid, and isopimaric acid methyl ester at 0.303, 0.294, 0.27, 0.258, and 0.2, respectively. The heritabilities for pimaric acid methyl ester, abieta-8, 13-diene-18-oic acid methyl ester, sandaracopimaric acid, methyl ester, and camphene were relatively low and ranged from 0.11 to 0.17. Many negative genetic correlations were observed as unfavorable while the corresponding phenotypic correlations presented no significant relationships or positive phenotypic correlations. However, the heritabilities and genetic correlations showed that single or multiple component selections and improvement, directly or indirectly, were effective. We postulate that genetic parameters estimated in this study will work as a reference in breeding programs of oleoresin components, especially in slash pine.

The α-Terpineol to 1,8-Cineole Cyclization Reaction of Tobacco Terpene Synthases.

Flowers of Nicotiana species emit a characteristic blend including the cineole cassette monoterpenes. This set of terpenes is synthesized by multiproduct enzymes, with either 1,8-cineole or α-terpineol contributing most to the volatile spectrum, thus referring to cineole or terpineol synthase, respectively. To understand the molecular and structural requirements of the enzymes that favor the biochemical formation of α-terpineol and 1,8-cineole, site-directed mutagenesis, in silico modeling, and semiempiric calculations were performed. Our results indicate the formation of α-terpineol by a nucleophilic attack of water. During this attack, the α-terpinyl cation is stabilized by π-stacking with a tryptophan side chain (tryptophan-253). The hypothesized catalytic mechanism of α-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (histidine-502 and glutamate-249), acting as a base, and a threonine (threonine-278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (S)-(-)-α-terpineol, which is the favored enantiomer product of the recombinant enzymes. Furthermore, by site-directed mutagenesis, we were able to identify amino acids at positions 147, 148, and 266 that determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase. Since amino acid 266 is more than 10 Å away from the active site, an indirect effect of this amino acid exchange on the catalysis is discussed.

Study of Bioreductive Anticancer Agent RH-1-Induced Signals Leading the Wild-Type p53-Bearing Lung Cancer A549 Cells to Apoptosis.

Aziridinylquinone RH-1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-cyclohexa-2,5-diene-1,4-dione) is a potential anticancer agent. RH-1 action is associated with NAD(P)H: quinone oxidoreductase (NQO1) which reduces this diaziridinylbenzoquinone into DNA-alkylating hydroquinone and is overexpressed in many tumors. Another suggested mechanism of RH-1 toxicity is the formation of reactive oxygen species (ROS) arising from its redox cycling. In order to improve anticancer action of this and similar antitumor quinones, we investigated the involvement of different signaling molecules in cytotoxicity induced by RH-1 by using wild-type tumor suppressor p53 bearing nonsmall cell lung carcinoma A549 cells as a model. Gradual and prolonged increase of mitogen-activated protein kinases (MAPK) ERK, P38, and JNK phosphorylation was observed during 24-h RH-1 treatment. In parallel, activation of DNA damage-sensing ATM kinase, upregulation, and phosphorylation of TP53 (human p53) took place. Inhibition studies revealed that RH-1-induced A549 apoptosis involved the NQO1-ATM-p53 signaling pathway and ROS generation. TP53 participated in ROS- and DNA damage-induced cell death differently. Moreover, MAP kinase JNK was another TP53 activator and death inducer in A549 cells. At the same time, rapid and prolonged activation of AKT kinase during RH-1 treatment was found, and it proved to be antiapoptotic kinase in our model system. Therefore, we identified that different and opposite cell death regulating signaling pathways, which may counteract one another, are induced in cancer cells during chemotherapeutic RH-1 treatment.