Julio Benites

Biological evaluation of donor-acceptor aminonaphthoquinones as antitumor agents.

Several members of the phenylamino-1,4-naphthoquinone series were prepared in order to investigate structure-activity relationships (SAR) and to explore the antitumor effects associated with this scaffold. The cytotoxic effects of the aminoquinones (EC50) against a panel of cancer cell lines (MCF7, DU145 and T24 cells) and healthy fibroblasts (BALB/3T3) were assessed in vitro using the MTT reduction assay 48 h after drug exposure. SAR analysis of the aminonaphthoquinone series showed that insertion of a chlorine atom in the acceptor quinone nucleus and/or insertion of a methyl group at the nitrogen atom of the donor phenylamino group induced significant changes in cytotoxic activity. Quinones 7 and 9, which exhibited the highest selective indexes (5.73 and 6.29, respectively), were further characterized using the following assays: Colony formation, caspase-3 activity, and ATP content. The results showed that aminoquinone 7 strongly influenced ATP levels and impaired the proliferative capacity of T24 cells without activating caspase-3.

Redox-active quinones and ascorbate: an innovative cancer therapy that exploits the vulnerability of cancer cells to oxidative stress.

Cancer cells are particularly vulnerable to treatments impairing redox homeostasis. Reactive oxygen species (ROS) can indeed play an important role in the initiation and progression of cancer, and advanced stage tumors frequently exhibit high basal levels of ROS that stimulate cell proliferation and promote genetic instability. In addition, an inverse correlation between histological grade and antioxidant enzyme activities is frequently observed in human tumors, further supporting the existence of a redox dysregulation in cancer cells. This biochemical property can be exploited by using redox-modulating compounds, which represent an interesting approach to induce cancer cell death. Thus, we have developed a new strategy based on the use of pharmacologic concentrations of ascorbate and redox-active quinones. Ascorbate-driven quinone redox cycling leads to ROS formation and provoke an oxidative stress that preferentially kill cancer cells and spare healthy tissues. Cancer cell death occurs through necrosis and the underlying mechanism implies an energetic impairment (ATP depletion) that is likely due to glycolysis inhibition. Additional mechanisms that participate to cell death include calcium equilibrium impairment and oxidative cleavage of protein chaperone Hsp90. Given the low systemic toxicity of ascorbate and the impairment of crucial survival pathways when associated with redox-active quinones, these combinations could represent an original approach that could be combined to standard cancer therapy.

Studies on quinones. Part 35: Access to antiprotozoal active euryfurylquinones and hydroquinones

(+)-Euryfuran adds regiospecifically to activated monosubstituted 1,4-benzoquinones under mild conditions to give the corresponding Michael adducts which, depending on the quinone substituent, undergo in situ redox reactions to the respective euryfurylbenzoquinones. One of these Michael adducts undergoes a facile stereoselective cyclisation under oxidant conditions to afford a naphthofuro[4,3-c]benzopyran derivative. The in vitro activities of the obtained euryfurylquinones and hydroquinones against Leishmania amazonensis are described.

Antioxidant Capacities and Analysis of Phenolic Compounds in Three Endemic Nolana Species by HPLC-PDA-ESI-MS

The antioxidant features, polyphenolic composition and chromatographic fingerprints of the aerial parts from three Chilean endemic plants from the Paposo Valley located on the cost of the Atacama Desert were investigated for the first time using high pressure liquid chromatography coupled with photodiode array detector and electrospray ionization mass analysis (HPLC-PDA-ESI-MS) and spectroscopic methods. The phenolic fingerprints obtained for the plants were compared and correlated with the antioxidant capacities measured by the bleaching of the DPPH radical, the ferric reducing antioxidant power (FRAP) and quantification of the total content of phenolics and flavonoids measured by spectroscopic methods. Thirty phenolics were identified for the first time for these species, mostly phenolic acids, flavanones, flavonols and some of their glycoside derivatives, together with three saturated fatty acids (stearic, palmitic and arachidic acids). Nolana ramosissima showed the highest antioxidant activity (26.35 ± 1.02 μg/mL, 116.07 ± 3.42 μM Trolox equivalents/g dry weight and 81.23% ± 3.77% of inhibition in the DPPH, FRAP and scavenging activity (SA) assays, respectively), followed by N. aplocaryoides (85.19 ± 1.64 μg/mL, 65.87 ± 2.33 μM TE/g DW and 53.27% ± 3.07%) and N. leptophylla (124.71 ± 3.01, 44.23 ± 5.18 μM TE/g DW and 38.63% ± 1.85%).

COMPOSITION AND BIOLOGICAL ACTIVITY OF THE ESSENTIAL OIL OF PERUVIAN LANTANA CAMARA

The composition of the essential oil from Lantana camara L. (Verbenaceae) obtained by hydrodistillation of the aerial parts was examined by GC, GC/ MS, and 13C-NMR. The GC analysis showed that carvone is the most abundant monoterpene 75.9%, together with limonene 16.9%, accounting for 92.8% of the oil. The major components were also tested by 13C-NMR analysis of the essential oil. The L. camara oil was assayed against several microorganisms, showing moderate antibacterial activity against the human pathogen Staphylococcus aureus (MIC 200 µg/ml). High antioxidant activity evaluated by the Trolox equivalent antioxidant capacity assay (TEAC) was found (29.0 mmol Trolox/kg) and relative low anti-inflammatory activity due to its weak ability for inhibiting lipoxygenase (IC 50 = 81.5 µg/ml).

Inhibition of cell proliferation and migration by oxidative stress from ascorbate-driven juglone redox cycling in human bladder-derived T24 cells

The effects of juglone on T24 cells were assessed in the presence and absence of ascorbate. The EC(50) value for juglone at 24 h decreased from 28.5 μM to 6.3 μM in the presence of ascorbate. In juglone-treated cells, ascorbate increased ROS formation (4-fold) and depleted GSH (65%). N-acetylcysteine or catalase restricted the juglone/ascorbate-mediated effects, highlighting the role of oxidative stress in juglone cytotoxicity. Juglone alone or associated with ascorbate did not cause caspase-3 activation or PARP cleavage, suggesting necrosis-like cell death. DNA damage and the mild ER stress caused by juglone were both enhanced by ascorbate. In cells treated with juglone (1-5 μM), a concentration-dependent decrease in cell proliferation was observed. Ascorbate did not impair cell proliferation but its association with juglone led to a clonogenic death state. The motility of ascorbate-treated cells was not affected. Juglone slightly restricted motility, but cells lost their ability to migrate most noticeably when treated with juglone plus ascorbate. We postulate that juglone kills cells by a necrosis-like mechanism inhibiting cell proliferation and the motility of T24 cells. These effects are enhanced in the presence of ascorbate.

Part 1: effect of vitamin C on the biological activity of two euryfurylbenzoquinones on TLT, a murine hepatoma cell line

2-euryfuryl- and 2-euryfuryl-3-nitro-1,4-benzoquinone Q2 and Q3, prepared via oxidative coupling reactions of sesquiterpene euryfuran 1 to 2-nitro-1,4-benzoquinone and 1,4-benzoquinone, were tested for their cytotoxicity towards TLT cells (a murine hepatoma cell line) in the absence and in the presence of vitamin C. Their cytotoxic profile was completely different. In cells incubated with Q2 (from 1 to 50 microg/ml), cell survival was not modified, both GSH and ATP were depleted to about 50% of control values (at 50 microg/ml); and caspase-3 was activated in a dose-dependent manner. These effects were observed whatever cells were incubated or not in the presence of vitamin C. In the case of Q3, the cytotoxicity was rather unrelated to its concentration but the association of vitamin C plus the highest Q3 concentration (50 microg/ml) results in a strong cell death (more than 60%). At such a concentration, a complete lack of caspase-3 activity was observed, probably due to cell lysis. At lower concentrations of Q3 (1 and 10 microg/ml), caspase-3 activity was lower than that observed in the absence of vitamin C or even under control conditions. Both GSH and ATP were kept fairly constant as compared to control values but in the presence of vitamin C and Q3, at 50 microg/ml, a decrease in their amounts was observed.

Antiproliferative effects of phenylaminonaphthoquinones are increased by ascorbate and associated with the appearance of a senescent phenotype in human bladder cancer cells.

Quinone-containing molecules have been developed against cancer mainly for their redox cycling ability leading to reactive oxygen species (ROS) formation. We have previously shown that donor-acceptor phenylaminonaphthoquinones are biologically active against a panel of cancer cells. In this report, we explored the mechanisms involved in cancer cell growth inhibition caused by two phenylaminonaphthoquinones, namely Q7 and Q9, with or without ascorbate (ASC). The results show that Q7 and Q9 are both redox cyclers able to form ROS, which strongly inhibit the proliferation of T24 cells. Q9 was a better redox cycler than Q7 because of marked stabilization of the semiquinone radical species arising from its reduction by ascorbate. Indeed, ASC dramatically enhances the inhibitory effect of Q9 on cell proliferation. Q9 plus ASC impairs the cell cycle, causing a decrease in the number of cells in the G2/M phase without involving other cell cycle regulating key proteins. Moreover, Q9 plus ASC influences the MAPK signaling pathways, provoking the appearance of a senescent cancer cell phenotype and ultimately leading to necrotic-like cell death. Because cellular senescence limits the replicative capacity of cells, our results suggest that induction of senescence may be exploited as a basis for new approaches to cancer therapy.

Biological Evaluation of 3-Acyl-2-Arylamino-1,4-Naphthoquinones as Inhibitors of Hsp90 Chaperoning Function

Hsp90 is a chaperone that plays a key function in cancer cells by stabilizing proteins responsible of cell growth and survival. Disruption of the Hsp90 chaperone machinery leads to the proteasomal degradation of its client proteins. Hsp90 appears then as an attractive target for the development of new anticancer molecules. We have shown that ascorbate- driven menadione-redox cycling inhibits Hsp90 activity by provoking an N-terminal cleavage of the protein, inducing the degradation of several of its client proteins. Since the mechanism involves an oxidative stress, we explored the effect of a series of diverse donor-acceptor 3-acyl-2-phenylamino 1,4-naphthoquinones on Hsp90 integrity, in the presence of ascorbate. Results show that quinone-derivatives that bear two electroactive groups (namely quinone and nitro) exhibit the highest inhibitory activity (Hsp90 cleavage and cell death). The biological activity of the series mainly relies on their redox capacity and their lipophilicity, which both modulate the ability of these compounds to induce a cytotoxic effect in K562 cells. As observed with other redox cycling quinones, the protein cleavage is blocked in the presence of N-terminal Hsp90 inhibitors suggesting that the availability or occupancy of nucleotide binding site in the N-terminal pocket of Hsp90 plays a critical role. In addition the survival of cancer cells and their metabolic and redox homeostasis were strongly impaired by the presence of ascorbate. Since these effects were similar to that obtained by ascorbate/menadione and they were blocked by the antioxidant N-acetylcyteine (NAC), it appears that oxidative stress is a major component of this cytotoxicity.

COMPOSITION AND ANTIMICROBIAL SCREENING OF THE ESSENTIAL OIL FROM THE LEAVES AND STEMS OF Senecio atacamensis Phil. FROM CHILE

An essential oil from Senecio atacamensis Phil. (Asteraceae) was obtained by hydrodistillation of its aerial parts (leaves and stems) and its composition was determined by GC and GC/MS analysis. The identification by GC of the essential oil components, in both leaves and stems respectively, showed α-terpinene (36.05% and 20.57%); α-phellandrene (27.79% and 25.37%), and p-cymene (11.85% and 22.55%) as the most abundant monoterpenes. Furthermore, the oil was tested for its antimicrobial activity using paper disc diffusion and the dilution broth method, exhibiting moderate inhibition of human pathogenic bacteria.