Chromatin-modifying agents in anti-cancer therapy

Epigenetic alterations are involved in every step of carcinogenesis. The development of chromatin-modifying agents (CMAs) has provided the ability to fight cancer by reversing these alterations. Currently, four CMAs have been approved for cancer treatment; two DNA demethylating agents and two deacetylase inhibitors. A number of promising CMAs are undergoing clinical trials in several cancer types. Moreover, already approved CMAs are still under clinical investigation to improve their efficacy and to extend their use to a broader panel of cancers. Combinatorial treatments with CMAs are already considered a promising strategy to improve clinical benefits and to limit side effects. The real mechanisms by which these CMAs allow the improvement and remission of patients are still obscure. A deeper analysis of the molecular features expressed by responding patients should be performed to reveal this information. In this review, we focus on clinical trials with CMAs, discussing the success and the pitfalls of this new class of anti-cancer drugs.     

Isolation and partial characterization of three isoamylases of Rhyzopertha dominica F. (Coleoptera: Bostrichidae)

Three isoamylases of Rhyzopertha dominica (termed RdA70, RdA79, and RdA90 according to their relative mobility in gel electrophoresis) were isolated by ammonium sulfate fractionation and hydrophobic interaction chromatography. RdA70 and RdA79 showed an optimal pH of 7.0, whereas for RdA90 the optimal pH was 6.5. The three isoamylases remained stable at 50 °C for 1 h, but at 60 °C, all lost 50% of their activity in 20 min and were completely inactivated in 1 h. RdA70 and RdA79 were inhibited by albumin extracts from wheat samples varying widely in amylase inhibitory activity; however, RdA90 was highly resistant to inhibition. β-Mercaptoethanol up to 30 mM increased the activity of the three isoamylases by 2.5-fold. The action pattern of the three isoamylases was typical of endoamylases; however, differences were observed on the hydrolytic efficiency rates measured as V_max/K_m ratio on starch, amylopectin, and amylose. The hydrolyzing action of RdA90 on starch and amylopectin (V_max/K_m = 90.4 ± 2.3 and 78.9 ± 6.6, respectively) was less efficient than that on amylose (V_max/K_m = 214 ± 23.2). RdA79 efficiently hydrolyzed both amylopectin and amylose (V_max/K_m = 260.6 ± 12.9 and 326.5 ± 9.4, respectively). RdA70 hydrolyzed starch and amylose at similar rates (V_max/K_m = 202.9 ± 5.5 and 215.9 ± 6.2, respectively), but amylopectin was a poor substrate (V_max/K_m = 124.2 ± 7.4). The overall results suggest that RdA70 and RdA79 appear to belong to a group of saccharifying isoamylases that breaks down long fragments of oligosaccharide chains produced by the hydrolytic action of RdA90. The simultaneous action of the three isoamylases on starch, aside from the high resistance of RdA90 to wheat amylase inhibitors, might allow R. dominica to feed and reproduce successfully on the wheat kernel.     

Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes

The redox properties of a periplasmic triheme cytochrome, PpcB from Geobacter sulfurreducens, were studied by NMR and visible spectroscopy. The structure of PpcB was determined by X-ray diffraction. PpcB is homologous to PpcA (77% sequence identity), which mediates cytoplasmic electron transfer to extracellular acceptors and is crucial in the bioenergetic metabolism of Geobacter spp. The heme core structure of PpcB in solution, probed by 2D-NMR, was compared to that of PpcA. The results showed that the heme core structures of PpcB and PpcA in solution are similar, in contrast to their crystal structures where the heme cores of the two proteins differ from each other. NMR redox titrations were carried out for both proteins and the order of oxidation of the heme groups was determined. The microscopic properties of PpcB and PpcA redox centers showed important differences: (i) the order in which hemes become oxidized is III-I-IV for PpcB, as opposed to I-IV-III for PpcA; (ii) the redox-Bohr effect is also different in the two proteins. The different redox features observed between PpcB and PpcA suggest that each protein uniquely modulates the properties of their co-factors to assure effectiveness in their respective metabolic pathways. The origins of the observed differences are discussed.     

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 μM, GE prevents oxidative stress and MPT induced by supraphysiological Ca²⁺ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe-S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca²⁺. GE also inhibits Ca²⁺ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca²⁺ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe-S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.     

Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+) measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, omega-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of approximately 50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.     

Protective<Emphasis Type="Italic">in vivo</Emphasis> effect of curcumin on copper genotoxicity evaluated by comet and micronucleus assays

Curcumin is a phytochemical with antiinflammatory, antioxidant and anticarcinogenic activities. Apparently, curcumin is not genotoxic in vivo, but in vitro copper and curcumin interactions induce genetic damage. The aim of this study was to test if in vivo copper excess induces DNA damage measured by comet and micronucleus assays in the presence of curcumin. We tested 0.2% curcumin in Balb-C mice at normal (13 ppm) and high (65, 130 and 390 ppm) copper ion concentrations. The comet and micronucleus assays were performed 48 hr after chemical application. Comet tail length in animals treated with 0.2% curcumin was not significantly different from the control. Animals exposed to copper cations (up to 390 ppm) exhibited higher oxidative DNA damage. Curcumin reduced the DNA damage induced by 390 ppm copper. We observed statistically significant increase in damage in individuals exposed to 390 ppm copper versus the control or curcumin groups, which was lowered by the presence of curcumin. Qualitative data on comets evidenced that cells from individuals exposed to 390 ppm copper had longer tails (categories 3 and 4) than in 390 ppm copper + curcumin. A statistically significant increase in frequency of micronucleated erythrocytes (MNE/10000TE) was observed only in 390 ppm copper versus the control and curcumin alone. Also cytotoxicity measured as the frequency of polychromatic erythrocytes (PE/1000TE) was attributable to 390 ppm copper. The lowest cytotoxic effect observed was attributed to curcumin. In vivo exposure to 0.2% curcumin for 48 hr did not cause genomic damage, while 390 ppm copper was genotoxic, but DNA damage induced by 390 ppm copper was diminished by curcumin. Curcumin seems to exert a genoprotective effect against DNA damage induced by high concentrations of copper cations. The comet and micronucleus assays prove to be suitable tools to detect DNA damage by copper in the presence of curcumin.     

Redox-linked conformational changes of a multiheme cytochrome from Geobacter sulfurreducens

Multiheme c-type cytochromes from members of the Desulfovibrionacea and Geobactereacea families play crucial roles in the bioenergetics of these microorganisms. Thermodynamic studies using NMR and visible spectroscopic techniques on tetraheme cytochromes c(3) isolated from Desulfovibrio spp. and more recently on a triheme cytochrome from Geobacter sulfurreducens showed that the properties of each redox centre are modulated by the neighbouring redox centres enabling these proteins to perform energy transduction and thus contributing to cellular energy conservation. Electron/proton transfer coupling relies on redox-linked conformational changes that were addressed for some multiheme cytochromes from the comparison of protein structure of fully reduced and fully oxidised forms. In this work, we identify for the first time in a multiheme cytochrome the simultaneous presence of two different conformations in solution. This was achieved by probing the different oxidation stages of a triheme cytochrome isolated from G. sulfurreducens using 2D-NMR techniques. The results presented here will be the foundations to evaluate the modulation of the redox centres properties by conformational changes that occur during the reoxidation of a multiheme protein.     

Synergistic inhibition of pancreatic adenocarcinoma cell growth by trichostatin A and gemcitabine

We investigated the ability of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to interact with gemcitabine (GEM) in inducing pancreatic cancer cell death. The combined treatment with TSA and GEM synergistically inhibited growth of four pancreatic adenocarcinoma cell lines and induced apoptosis. This effect was associated with the induction of reactive oxygen species (ROS) by GEM, increased expression of the pro-apoptotic BIM gene by both TSA and GEM and downregulation of the 5'-nucleotidase UMPH type II gene by TSA. The expression of other genes critical for GEM resistance (nucleoside transporters, deoxycytidine kinase, cytidine deaminase, and ribonucleotide reductase genes) was not affected by TSA. The functional role of ROS in cell growth inhibition by GEM was supported by (i) a significantly reduced GEM-associated growth inhibition by the free radical scavenger N-acetyl-L-cysteine, and (ii) a positive correlation between the basal level of ROS and sensitivity to GEM in 10 pancreatic cancer cell lines. The functional role of both Bim and 5'-nucleotidase UMPH type II in cell growth inhibition by TSA and GEM was assessed by RNA interference assays. In vivo studies on xenografts of pancreatic adenocarcinoma cells in nude mice showed that the association of TSA and GEM reduced to 50% the tumour mass and did not cause any apparent form of toxicity, while treatments with TSA or GEM alone were ineffective. In conclusion, the present study demonstrates a potent anti-tumour activity of TSA/GEM combination against pancreatic cancer cells in vitro and in vivo, strongly supporting the use of GEM in combination with an HDAC inhibitor for pancreatic cancer therapy.