Multiple effects of the Na(+)/H (+) antiporter inhibitor HMA on cancer cells.

Amiloride derivatives are a class of new promising chemotherapeutic agents. A representative member of this family is the sodium-hydrogen antiporter inhibitor HMA (5-(N,N-hexamethylene amiloride), which has been demonstrated to induce cellular intracytosolic acidification and cell death through the apoptotic pathway(s). This work aims at characterizing drug response of human cancer cell lines to HMA. After a first screening revealing that HMA interferes with cancer cell survival, we focused our attention on SW613-B3 colon carcinoma cells, which are intrinsically resistant to a panel of drugs. Searching for the activation of canonical apoptosis, we found that this process was abortive, given that the final steps of this process, i.e. PARP-1 cleavage and DNA ladder, were not detectable. Thus, we addressed caspase-independent paradigms of cell death and we observed that HMA promotes the induction of the LEI/L-DNase II pathway as well as of parthanatos. Finally, we explored the possible impact of autophagy of cell response to HMA, providing the evidence that autophagy is activated in our experimental system. On the whole, our results defined the biochemical reactions triggered by HMA, and elucidated its multiple effects, thus adding further complexity to the intricate network leading to drug resistance.

From "Click" to "Fenton" chemistry for 5-bromo-2'-deoxyuridine determination.

Ascorbic acid (AA) and copper have been increasingly employed in flow cytometry (FCM) and high content analysis (HCA) since the introduction of "click chemistry" as a non-destructive alternative to classical 5-bromo-2'-deoxyuridine (BrdU) immunodetection for DNA synthesis and proliferation assays. Mixtures of ascorbate and catalytic copper, under certain experimental conditions, act as oxidizing agent, catalyzing the formation of reactive hydroxyl radicals through hydrogen peroxides decomposition via Fenton reaction. We developed a procedure for BrdU incorporation detection based on the use of AA and cupric ions as DNA damaging agents. Optimal DNA damaging conditions were identified and found to provide results comparable with "click" 5-ethynyl-deoxyuridine (EdU) cycloaddition approach and classical BrdU immunodetection. Scavenger agents were found to prevent hydroxyl-induced DNA damages, providing the proof-of-concept for the use of this procedure for DNA denaturation prior to BrdU detection. We demonstrated hydroxyl radicals' reaction to be readily applicable to HCA and FCM assays, for both classical BrdU immunostaining and EdU cycloaddition procedure. This technique was successfully employed for BrdU pulse-chase experiments and in multiparametric immunofluorescence assays for the simultaneous detection of labile phosphoproteins in intact cells. The use of AA/Cu prior to immunodetection for BrdU incorporation assays is a viable alternative to chemical/physical DNA denaturing agents (acids or heat), since it allows preservation of labile epitopes such as phosphoproteins, and over enzymatic agents (digestion with DNases) for its lower cost.

Mitochondrial fusion: a mechanism of cisplatin-induced resistance in neuroblastoma cells?

Cisplatin induces apoptosis through different pathways. The intrinsic apoptotic pathway is mediated by mitochondria, which, as a result of cisplatin treatment, undergo morphological alterations. The aim of this study was to investigate cisplatin-induced mitochondrial functional and morphological long-term effects in neuroblastoma B50 rat cells. To this purpose, we followed evaluated different several apoptotic markers by means of flow cytometry, confocal and electron microscopy and western blotting techniques. We applied different treatment protocols based on the incubation of the neuroblastoma B50 rat cells with 40 µM cisplatin: (i) for 48 h and harvesting of the cells at the end of the treatment; (ii) further recovery in drug-free medium for 7 days post-treatment; (iii) conditions as in (ii) followed by re-seeding in normal medium and growth for a further 4 days. We observed apoptosis induction after the first treatment and after the recovery from cell death after long-term culture in drug-free medium. Interestingly, the latter phenomenon was characterized by mitochondrial elongation and mitochondrial protein rearrangement. In recovered and re-seeded cells, mitochondrial equilibrium moved toward fusion, possibly protecting cells from apoptosis.

Characterization of stress response in human retinal epithelial cells.

The pathogenesis of age-related macular degeneration (AMD) involves demise of the retinal pigment epithelium and death of photoreceptors. In this article, we investigated the response of human adult retinal pigmented epithelial (ARPE-19) cells to 5-(N,N-hexamethylene)amiloride (HMA), an inhibitor of Na(+) /H(+) exchangers. We observed that ARPE-19 cells treated with HMA are unable to activate 'classical' apoptosis but they succeed to activate autophagy. In the first 2 hrs of HMA exposure, autophagy is efficient in protecting cells from death. Thereafter, autophagy is impaired, as indicated by p62 accumulation, and this protective mechanism becomes the executioner of cell death. This switch in autophagy property as a function of time for a single stimulus is here shown for the first time. The activation of autophagy was observed, at a lesser extent, with etoposide, suggesting that this event might be a general response of ARPE cells to stress and the most important pathway involved in cell resistance to adverse conditions and toxic stimuli.

Impaired osteoblastogenesis in a murine model of dominant osteogenesis imperfecta: a new target for osteogenesis imperfecta pharmacological therapy.

The molecular basis underlying the clinical phenotype in bone diseases is customarily associated with abnormal extracellular matrix structure and/or properties. More recently, cellular malfunction has been identified as a concomitant causative factor and increased attention has focused on stem cells differentiation. Classic osteogenesis imperfecta (OI) is a prototype for heritable bone dysplasias: it has dominant genetic transmission and is caused by mutations in the genes coding for collagen I, the most abundant protein in bone. Using the Brtl mouse, a well-characterized knockin model for moderately severe dominant OI, we demonstrated an impairment in the differentiation of bone marrow progenitor cells toward osteoblasts. In mutant mesenchymal stem cells (MSCs), the expression of early (Runx2 and Sp7) and late (Col1a1 and Ibsp) osteoblastic markers was significantly reduced with respect to wild type (WT). Conversely, mutant MSCs generated more colony-forming unit-adipocytes compared to WT, with more adipocytes per colony, and increased number and size of triglyceride drops per cell. Autophagy upregulation was also demonstrated in mutant adult MSCs differentiating toward osteogenic lineage as consequence of endoplasmic reticulum stress due to mutant collagen retention. Treatment of the Brtl mice with the proteasome inhibitor Bortezomib ameliorated both osteoblast differentiation in vitro and bone properties in vivo as demonstrated by colony-forming unit-osteoblasts assay and peripheral quantitative computed tomography analysis on long bones, respectively. This is the first report of impaired MSC differentiation to osteoblasts in OI, and it identifies a new potential target for the pharmacological treatment of the disorder.

Autophagy and cancer.

Autophagy is a housekeeping survival mechanism with a protective function against stress conditions. However, when stress severity or duration increases, it may promote cell death. Paradoxically, autophagy favors cancer development, since cancer cells could enhance their proliferation potential (thus becoming able to resist anticancer therapy) thanks to the energetic supply provided by organelle degradation typically driven by autophagy following a stepwise pathway. The main actors of the autophagic machinery as well as the features shared with apoptosis will be described. Special attention will be paid to the effects of autophagy manipulation.

Killing of tumor cells: a drama in two acts.

Cancer still represents a major health problem worldwide, which urges the development of more effective strategies. Resistance to chemotherapy, a major obstacle for cancer eradication, is mainly related to an intrinsic failure to activate the apoptotic pathways. However, a protective effect of autophagy toward cancer cells has been recently observed, thus adding further complexity to the development of an effective approach counteracting cancer cell growth and improving the response to therapy.

Conversation between apoptosis and autophagy: "Is it your turn or mine?".

Drug resistance of cancer cells is often correlated with apoptosis evasion; however, an active involvement of autophagy in this scenario has been recently proposed, based on the evidence that autophagy could exert a protective role toward the activation of apoptosis in cancer cells. In this review, we briefly review the basic features of apoptosis, and we describe in details the molecular patterns of autophagy, with a special emphasis on its still controversial physiological function(s). The crucial factors governing the cross talk between autophagy and apoptosis will be illustrated.

Expression of antioxidant defense and poly(ADP-ribose) polymerase-1 in rat developing Sertoli cells.

Sertoli cells play an essential role in the development of a functional testis. ROS (reactive oxygen species) are normally produced by the developing testicular cells and may be dangerous to spermatogenesis. The aim of this study was to investigate the developmental expression of genes involved in antioxidant defense as well as in the DNA damage response in rat Sertoli cells. As revealed by quantitative RT-PCR analysis, the expression pattern of the antioxidant enzymes GST (glutathione-S-transferase), CAT (catalase) and SOD (superoxide dismutase) showed a progressive decrease from birth to puberty. The expression level of the oncosuppressor p53 revealed a net reduction as well. We next focused on PARP-1 [poly(ADP-ribose) polymerase-1], a 'guardian of the genome' that combats stress conditions. At both the mRNA and protein level, PARP-1 expression was low at the early stage of development and increased later on. Maximal PARP-1 expression was preceded by a rise in the transcript level for MTs (metallothioneins), which provide zinc to zinc-dependent enzymes and proteins, including PARP-1. Our results showed an increased expression of PARP-1 during Sertoli cell development, together with a decrease in the expression of antioxidant enzymes. In conclusion, a role of PARP-1 in protecting the testicular differentiation is suggested.

PARP inhibitors: new tools to protect from inflammation.

Poly(ADP-ribosylation) consists in the conversion of ß-NAD(+) into ADP-ribose, which is then bound to acceptor proteins and further used to form polymers of variable length and structure. The correct turnover of poly(ADP-ribose) is ensured by the concerted action of poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) enzymes, which are responsible for polymer synthesis and degradation, respectively. Despite the positive role of poly(ADP-ribosylation) in sensing and repairing DNA damage, generated also by ROS, PARP over-activation could allow NAD depletion and consequent necrosis, thus leading to an inflammatory condition in many diseases. In this respect, inhibition of PARP enzymes could exert a protective role towards a number of pathological conditions; i.e. the combined treatment of tumors with PARP inhibitors/anticancer agents proved to have a beneficial effect in cancer therapy. Thus, pharmacological inactivation of poly(ADP-ribosylation) could represent a novel therapeutic strategy to limit cellular injury and to attenuate the inflammatory processes that characterize many disorders.

Arylthioindoles: Promising compounds against cancer cell proliferation.

Drugs that are able to modulate the microtubule dynamics either by inhibiting tubulin polymerization or by blocking microtubule disassembly are of great interest in anti-cancer therapy; a number of them are currently applied in clinical development. Tubulin polymerization inhibitors, including arylthioindoles, are characterized by the presence of an indole nucleus and have been obtained from natural sources or prepared by semi-synthesis. We characterized the effect of 5-bromo-3-[(3,4,5-trimetoxyphenyl)thio]-1H-indole (RS 2518) on the metabolism of human cell lines derived from solid tumors. We found that this new compound impairs cell adhesion, arrests the cells in the G(2)/M cell cycle phase and inhibits cell proliferation, thus leading to apoptosis. The described effects of RS 2518 on cancer cells have led to its selection as a lead compound for further studies. Some analogues have been developed and tested on a panel of cancer cell lines.

New arylthioindoles and related bioisosteres at the sulfur bridging group. 4. Synthesis, tubulin polymerization, cell growth inhibition, and molecular modeling studies.

New arylthioindoles along with the corresponding ketone and methylene compounds were potent tubulin assembly inhibitors. As growth inhibitors of MCF-7 cells, sulfur derivatives were superior or sometimes equivalent to the ketones, while methylene derivatives were substantially less effective. Esters 24, 27-29, 36, 39, and 41 showed approximately 50% of inhibition on human HeLa and HCT116/chr3 cells at 0.5 microM, and these compounds inhibited the growth of HEK, M14, and U937 cells with IC(50)'s in the 78-220 nM range. While murine macrophage J744.1 cell growth was significantly less affected (20% at higher concentrations), four other nontransformed cell lines remained sensitive to these esters. The effect of drug treatment on cell morphology was examined by time-lapse microscopy. In a protocol set up to evaluate toxicity on the Saccharomyces cerevisiae BY4741 wild type strain, compounds 24 and 54 strongly reduced cell growth, and 29, 36, and 39 also showed significant inhibition.

Study of the effects of a new pyrazolecarboxamide: changes in mitochondria and induction of apoptosis.

Drug resistance of cancer cells is often correlated with the evasion of apoptosis, thus a major goal in cancer research is to search for compounds able to counteract cancer by promoting apoptosis. A variety of compounds with anticancer activity are characterised by the presence of the pyrazole as core nucleus. We synthesised a panel of pyrrolyl-pyrazole-carboxamides and we focused on the new compound RS 2780 (N-2-phenylethyl 1-(4-chlorophenyl)-3-methyl-5-pyrrolylpyrazole-4-carboxamide). The biological effects of RS 2780 on cell proliferation and viability were first evaluated on human HeLa cancer cells. As revealed by cell growth and viability experiments, a 24-h treatment of HeLa cells with increasing concentrations of RS 2780 (ranging from 0.1 to 100 microM) proved to inhibit cell proliferation and to affect cell viability. Notably, the new compound was effective also on colon carcinoma SW613-B3 cells, which are extremely resistant to most drugs, while it does not alter the proliferation of normal fibroblasts. We observed that RS 2780 interferes with the structural and functional properties of mitochondria, leading to the activation of the mitochondria-dependent apoptotic pathway. Apoptosis occurrence was supported by a number of morphological and biochemical hallmarks, including chromatin condensation, internucleosomal DNA fragmentation, PARP-1 cleavage and caspase activation. In conclusion, our results demonstrate for the first time the antiproliferative properties of the new compound RS 2780 on HeLa and SW613-B3 cancer cells and show that its effects on mitochondria lead to apoptosis.