Mario Pavani

Buthionine Sulfoximine Increases the Toxicity of Nifurtimox and Benznidazole to Trypanosoma cruzi

ABSTRACT l-Buthionine (S,R)-sulfoximine (BSO) increased the toxicity of nifurtimox and benznidazole toward the epimastigote, trypomastigote, and amastigote forms of Trypanosoma cruzi. BSO at 500 μM decreased total glutathione-derived thiols by 70 to 80% in 48 h. In epimastigotes, 500 μM BSO decreased the concentration of nifurtimox needed to inhibit constant growth of the parasites by 50%, from 14.0 to 9.0 μM, and decreased that of benznidazole from 43.6 to 24.1 μM. The survival of epimastigotes or trypomastigotes treated with nifurtimox or benznidazole, as measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction, was significantly decreased by 500 μM BSO. In Vero cells infected with amastigotes, 25 μM BSO was able to potentiate the effect of nifurtimox and benznidazole as measured by the percentage of infected Vero cells multiplied by the average number of intracellular amastigotes (endocytic index). At 0.5 μM nifurtimox, the proportion of Vero cells infected decreased from 27 to 20% and the endocytic index decreased from 2,500 to 980 when 25 μM BSO was added. Similar results were obtained with benznidazole- and BSO-benznidazole-treated cells. This study indicates that potentiation of nifurtimox or benznidazole by BSO could decrease the clinical dose of both drugs and diminish the side effects or the length of therapy.

Effects and mode of action of 1,4-naphthoquinones isolated from Calceolaria sessilis on tumoral cells and Trypanosoma parasites

The naphthoquinones 2-hydroxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone (CS-1), (-)-2,3,3-trimethyl-2-3-dihydronaphtho[2,3-b]furan-4,9-quinone (CS-3), and 2-acetoxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone (CS-5) isolated from Calceolaria sessilis were tested against Trypanosoma cruzi epimastigotes, the TA3 tumor cell line and the methotrexate-resistant subline TA3-MTX-R. Naphthoquinone CS-3 was the most active; the 50% culture growth inhibition (I50) on T. cruzi (Tulahuén and LQ strain and DM28c clone) was at concentrations ranging from 2.1 to 5.2 mumolar. Also CS-3 inhibited TA3 and TA3-MTX-R culture growth with an I50 of 2.1 and 3.8 mumolar, respectively. Naphthoquinone CS-3 inhibited the respiration of the tumor cells by interfering with the electron transport at some point between NADH and ubiquinone. The respiration of T. cruzi was not inhibited by naphthoquinone CS-3. Naphthoquinone CS-3 produced a temporary increase of oxygen consumption in T. cruzi and tumor cells, suggesting the generation and participation of free radicals.

Inhibition of tumoral cell respiration and growth by nordihydroguaiaretic acid

The effects of nordihydroguaiaretic acid (NDGA), best known as an inhibitor of lipoxygenase activities, on the culture growth, oxygen consumption, ATP level, viability, and redox state of some electron carriers of intact TA3 and 786A ascites tumor cells have been studied. NDGA inhibited the respiration rate of these two tumor cell lines by preventing electron flow through the respiratory chain. Consequently, ATP levels, cell viability and culture growth rates were decreased. NDGA did not noticeably inhibit electron flow through both cytochrome oxidase and ubiquinone-cytochrome b-c1 complex. Also, the presence of NDGA changed to redox state of NAD(P)+ to a more reduced level, and the redox states of ubiquinone, cytochrome b and cytochromes c + c1 changed to a more oxidized level. These observations suggest that the electron transport in the tumor mitochondria was inhibited by NDGA at the NADH-dehydrogenase-ubiquinone level (energy-conserving site 1). As a consequence, mitochondrial ATP synthesis would be interrupted. This event could be related to the cytotoxic effect of NDGA.

Synthesis, Characterization and Antitumor Activity ofcis-bis(acylthioureato) platinum(II) Complexes, cis-[PtL2] [HL1=N,N-Diphenyl-N'-Benzoylthiourea or HL2=N,N-diphenyl-N'-(p-nitrobenzoyl)thiourea]

A low-molecular weight chromium-containing fraction of the material resulting from dichromate reduction by bovine liver homogenate was investigated by NMR and ES-MS. The ES-MS spectrum showed a readily detectable peak at m/z 786.1. The same molecular weight reasonably agreed with the relatively low diffusion coefficient measured by NMR-DOSY experiments on the main species observed in the 1H NMR spectrum. At least two downfield shifted and broad paramagnetic signals were apparent in the 1H NMR spectrum. Temperature dependence of chemical shift was exploited in order to estimate the diamagnetic shift of the signals in the diamagnetic region of the spectrum. 2D TOCSY, NOESY, COSY and 1H-13C HMQC spectra revealed the presence of aromatic protons (which were assigned as His residues), Gly and some other short chain amino-acids. Combinations of the molecular masses of such components together with acetate (which is present in the solution) and chromium atoms allowed a tentative proposal of a model for the compound.

An ortho-carbonyl substituted hydroquinone derivative is an anticancer agent that acts by inhibiting mitochondrial bioenergetics and by inducing G2/M-phase arrest in mammary adenocarcinoma TA3

Tumor cells present a known metabolic reprogramming, which makes them more susceptible for a selective cellular death by modifying its mitochondrial bioenergetics. Anticancer action of the antioxidant 9,10-dihydroxy-4,4-dimethyl-5,8-dihydroanthracen-1(4H)-one (HQ) on mouse mammary adenocarcinoma TA3, and its multiresistant variant TA3-MTXR, were evaluated. HQ decreased the viability of both tumor cells, affecting slightly mammary epithelial cells. This hydroquinone blocked the electron flow through the NADH dehydrogenase (Complex I), leading to ADP-stimulated oxygen consumption inhibition, transmembrane potential dissipation and cellular ATP level decrease, without increasing ROS production. Duroquinol, an electron donor at CoQ level, reversed the decrease of cell viability induced by HQ. Additionally, HQ selectively induced G₂/M-phase arrest. Taken together, our results suggest that the bioenergetic dysfunction provoked by HQ is implicated in its anticancer action.

Inhibitory effect of vanillin-like compounds on respiration and growth of adenocarcinoma TA3 and its multiresistant variant TA3-MTX-R

The effects of some imine and amine derivatives of vanillin on the respiration rate of mouse mammary adenocarcinoma TA3 line, its multiresistant variant TA3-MTX-R line and mouse hepatocytes, together with their respective mitochondrial fractions, are described. These derivatives inhibit respiration in both tumour cell lines more effectively than vanillin in the absence or presence of the uncoupler CCCP. Since both types of derivatives block the electron flow, mainly through the NADH-CoQ span, they behave as oxidative phosphorylation inhibitors. Thus, they prevent ATP synthesis and alter cellular processes requiring energy, which would lead to cellular death. Amine derivatives of vanillin present a similar effect on both tumour cell lines, being amine C the most efficient inhibitor. Moreover, mouse hepatocytes are about 4-fold less sensitive to amine C than tumour cells. These amine derivatives are better inhibitors than the corresponding imines; probably because they should interact better with the respiratory chain reaction site.

Tumor cell death induced by the inhibition of mitochondrial electron transport: The effect of 3-hydroxybakuchiol

Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependence of the first step of glycolysis on mitochondrial ATP. The oxidative phosphorylation (OXPHOS) system is responsible for the synthesis of approximately 90% of the ATP in normal cells and up to 50% in most glycolytic cancers; therefore, inhibition of the electron transport chain (ETC) emerges as an attractive therapeutic target. We studied the effect of a lipophilic isoprenylated catechol, 3-hydroxybakuchiol (3-OHbk), a putative ETC inhibitor isolated from Psoralea glandulosa. 3-OHbk exerted cytotoxic and anti-proliferative effects on the TA3/Ha mouse mammary adenocarcinoma cell line and induced a decrease in the mitochondrial transmembrane potential, the activation of caspase-3, the opening of the mitochondrial permeability transport pore (MPTP) and nuclear DNA fragmentation. Additionally, 3-OHbk inhibited oxygen consumption, an effect that was completely reversed by succinate (an electron donor for Complex II) and duroquinol (electron donor for Complex III), suggesting that 3-OHbk disrupted the electron flow at the level of Complex I. The inhibition of OXPHOS did not increase the level of reactive oxygen species (ROS) but caused a large decrease in the intracellular ATP level. ETC inhibitors have been shown to induce cell death through necrosis and apoptosis by increasing ROS generation. Nevertheless, we demonstrated that 3-OHbk inhibited the ETC and induced apoptosis through an interaction with Complex I. By delivering electrons directly to Complex III with duroquinol, cell death was almost completely abrogated. These results suggest that 3-OHbk has antitumor activity resulting from interactions with the ETC, a system that is already deficient in cancer cells.

Destabilization of mitochondrial functions as a target against breast cancer progression: Role of TPP+-linked-polyhydroxybenzoates

Mitochondrion is an accepted molecular target in cancer treatment since it exhibits a higher transmembrane potential in cancer cells, making it susceptible to be targeted by lipophilic-delocalized cations of triphenylphosphonium (TPP(+)). Thus, we evaluated five TPP(+)-linked decyl polyhydroxybenzoates as potential cytotoxic agents in several human breast cancer cell lines that differ in estrogen receptor and HER2/neu expression, and in metabolic profile. Results showed that all cell lines tested were sensitive to the cytotoxic action of these compounds. The mechanism underlying the cytotoxicity would be triggered by their weak uncoupling effect on the oxidative phosphorylation system, while having a wider and safer therapeutic range than other uncouplers and a significant lowering in transmembrane potential. Noteworthy, while the TPP(+)-derivatives alone led to almost negligible losses of ATP, when these were added in the presence of an AMP-activated protein kinase inhibitor, the levels of ATP fell greatly. Overall, data presented suggest that decyl polyhydroxybenzoates-TPP(+) and its derivatives warrant future investigation as potential anti-tumor agents.

Derivatives of alkyl gallate triphenylphosphonium exhibit antitumor activity in a syngeneic murine model of mammary adenocarcinoma

&NA; We previously demonstrated that alkyl gallates coupled to triphenylphosphine have a selective and efficient antiproliferative effect by inducing mitochondrial uncoupling in vitro due to the increased mitochondrial transmembrane potential of tumor cells. Therefore, in this work, the in vivo antitumor activities of alkyl gallate triphenylphosphonium derivatives (TPP+ C8, TPP+ C10 and TPP+ C12) were evaluated in a syngeneic murine model of breast cancer. We found that TPP+ C10 increased the cytosolic ADP/ATP ratio and significantly increased the AMP levels in a concentration‐dependent manner in TA3/Ha murine mammary adenocarcinoma cells. Interestingly, TPP+ C10 induced a decrease in the levels of cellular proliferation markers and promoted caspase‐3 activation in tumor‐bearing mice. Additionally, TPP+ C10 inhibited tumor growth in the syngeneic mouse model. Importantly, 30 days of intraperitoneal (i.p.) administration of the combination of TPP+ C10 (10 mg/kg/48 h) and the antibiotic doxycycline (10 mg/kg/24 h) completely eliminated the subcutaneous tumor burden in mice (n = 6), without any relapses at 60 days post‐treatment. This enhancement of the individual activities of TPP+ C10 and doxycycline is due to the uncoupling of oxidative phosphorylation by TPP+ C10 and the inhibition of mitochondrial biogenesis by doxycycline, as demonstrated by loss of mitochondrial mass and overexpression of PGC1‐&agr; as an adaptive response. Moreover, i.p. administration of TPP+ C10 (10 mg/kg/24 h) to healthy mice did not produce toxicity or damage in organs important for drug metabolism and excretion, as indicated by hematological, biochemical and histological assessments. These findings suggest that the combination of TPP+ C10 with doxycycline is a valuable candidate therapy for breast cancer management. Graphical abstract Figure. No caption available. HighlightsTPP+ C10 produces a metabolic stress by altering the levels of ATP, ADP and AMP.TPP+ C10 enhanced the inhibition of mitochondrial biogenesis induced by doxycycline.TPP+ C10 prevent tumor growth in a syngeneic murine model without systemic toxicity.Combination of TPP+ C10 and doxycycline completely eradicates the tumor burden.Effects explain by mitochondrial uncoupling and inhibition of mitochondrial biogenesis.

FR58P1a; a new uncoupler of OXPHOS that inhibits migration in triple-negative breast cancer cells via Sirt1/AMPK/β1-integrin pathway

Highly malignant triple-negative breast cancer (TNBC) cells rely mostly on glycolysis to maintain cellular homeostasis; however, mitochondria are still required for migration and metastasis. Taking advantage of the metabolic flexibility of TNBC MDA-MB-231 cells to generate subpopulations with glycolytic or oxidative phenotypes, we screened phenolic compounds containing an ortho-carbonyl group with mitochondrial activity and identified a bromoalkyl-ester of hydroquinone named FR58P1a, as a mitochondrial metabolism-affecting compound that uncouples OXPHOS through a protonophoric mechanism. In contrast to well-known protonophore uncoupler FCCP, FR58P1a does not depolarize the plasma membrane and its effect on the mitochondrial membrane potential and bioenergetics is moderate suggesting a mild uncoupling of OXPHOS. FR58P1a activates AMPK in a Sirt1-dependent fashion. Although the activation of Sirt1/AMPK axis by FR58P1a has a cyto-protective role, selectively inhibits fibronectin-dependent adhesion and migration in TNBC cells but not in non-tumoral MCF10A cells by decreasing β1-integrin at the cell surface. Prolonged exposure to FR58P1a triggers a metabolic reprograming in TNBC cells characterized by down-regulation of OXPHOS-related genes that promote cell survival but comprise their ability to migrate. Taken together, our results show that TNBC cell migration is susceptible to mitochondrial alterations induced by small molecules as FR58P1a, which may have therapeutic implications.