Effect of new berberine derivatives on colon cancer cells.

The natural alkaloid berberine has been recently described as a promising anticancer drug. In order to improve its efficacy and bioavailability, several derivatives have been designed and synthesized and found to be even more potent than the lead compound. Among the series of berberine derivatives we have produced, five compounds were identified to be able to heavily affect the proliferation of human HCT116 and SW613-B3 colon carcinoma cell lines. Remarkably, these active compounds exhibit high fluorescence emission property and ability to induce autophagy.

Berberine, an epiphany against cancer.

Alkaloids are used in traditional medicine for the treatment of many diseases. These compounds are synthesized in plants as secondary metabolites and have multiple effects on cellular metabolism. Among plant derivatives with biological properties, the isoquinoline quaternary alkaloid berberine possesses a broad range of therapeutic uses against several diseases. In recent years, berberine has been reported to inhibit cell proliferation and to be cytotoxic towards cancer cells. Based on this evidence, many derivatives have been synthesized to improve berberine efficiency and selectivity; the results so far obtained on human cancer cell lines support the idea that they could be promising agents for cancer treatment. The main properties of berberine and derivatives will be illustrated.

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.

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.

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.

Enzyme-assisted photosensitization activates different apoptotic pathways in Rose Bengal acetate treated HeLa cells.

Photosensitization of tumor cells after incubation with Rose Bengal acetate (RB-Ac) induces multiple organelle photodamage followed by apoptotic cell death. We used immunocytochemical techniques in multicolor fluorescence microscopy to elucidate whether this occurs through the simultaneous activation of different apoptotic pathways, in HeLa cells. We detected in situ the activated forms of caspases 9 and 3, and the translocation from the mitochondria to the nucleus of the apoptosis inducing factor; DNA electrophoretic techniques were also used to assess the occurrence of nuclear DNA cleavage into either high- or low-molecular-weight fragments. Both the caspase-dependent and caspase-independent apoptotic pathways are activated. The genomic DNA is degraded into high molecular weight molecules only, without the formation of oligonucleosome-sized fragments. The ability of RB-Ac to induce the simultaneous release of apoptogenic signals from different photodamaged organelles makes it an especially powerful cytotoxic agent.

Autoantibodies to poly(ADP-ribose) polymerase in centenarians: a reappraisal of Grabar's hypothesis.

BACKGROUND: Centenarians display a characteristic autoantibody profile, this being the absence of organ-specific autoantibodies and an increase in non-organ-specific autoantibodies without any full-blown autoimmune disease. OBJECTIVE: Antibodies directed to the nuclear protein poly(ADP-ribose) polymerase (PARP) were frequently found in the sera of patients affected by autoimmune diseases. This study aims at investigating the presence of circulating autoantibodies directed against PARP-1 in normal subjects, and searching for a possible correlation between level of circulating autoantibodies and age. METHODS: The presence of antibodies to PARP was monitored by ELISA according to a previously developed protocol. Data were analysed by parametric statistics (unpaired t test, chi(2) test). RESULTS: Our study performed on 33 centenarians and 66 subjects of age ranging from 12 to 80 years shows that circulating autoantibodies to the nuclear enzyme PARP, previously described in autoimmune diseases, are present in the sera of normal healthy people and increase with age. CONCLUSIONS: Taking into account the role of PARP-1 in DNA damage and apoptosis, the data are compatible with Grabar's hypothesis, which proposed several decades ago that 'autoreactive antibodies represent a physiological system for disposing the products of metabolism and catabolism', thereby helping to attain longevity.

Arylthioindole inhibitors of tubulin polymerization. 3. Biological evaluation, structure-activity relationships and molecular modeling studies.

The new arylthioindole (ATI) derivatives 10, 14-18, and 21-24, which bear a halogen atom or a small size ether group at position 5 of the indole moiety, were compared with the reference compounds colchicine and combretastatin A-4 for biological activity. Derivatives 10, 11, 16, and 21-24 inhibited MCF-7 cell growth with IC50 values <50 nM. A halogen atom (14-17) at position 5 caused a significant reduction in the free energy of binding of compound to tubulin, with a concomitant reduction in cytotoxicity. In contrast, methyl (21) and methoxy (22) substituents at position 5 caused an increase in cytotoxicity. Compound 16, the most potent antitubulin agent, led to a large increase (56%) in HeLa cells in the G2/M phase at 24 h, and at 48 h, 26% of the cells were hyperploid. Molecular modeling studies showed that, despite the absence of the ester moiety present in the previously examined analogues, most of the compounds bind in the colchicine site in the same orientation as the previously studied ATIs. Binding to beta-tubulin involved formation of a hydrogen bond between the indole and Thr179 and positioning of the trimethoxy phenyl group in a hydrophobic pocket near Cys241.

A new function for miRNAs as regulators of autophagy.

Autophagy is a self-digestive process regulated by an intricate network of factors able either to ensure the prosurvival function of autophagy or to convert it in a death pathway. Recently, the involvement of miRNAs in the regulation of autophagy networks has been reported. This review will summarize the main features of these small noncoding endogenous RNAs, focusing on their relevance in cancer and finally addressing their impact on autophagy.

Oxidative stress response in telomerase-immortalized fibroblasts from a centenarian.

It has been reported that cells with ectopic expression of telomerase are more resistant to apoptotic cell death than their normal counterpart. However, controversial results were obtained when the cellular response to oxidative stress was analyzed. The present research was therefore aimed at defining the effect of the oxidative stress induced by tert-butylhydroperoxide (tBOOH) and 2-deoxy-D-ribose (D-ribose) in human fibroblasts from a centenarian (cen3) and, in parallel, on the same cells after telomerase immortalization (cen3tel cells). By studying different parameters of apoptosis in situ (i.e., chromatin condensation, phosphatidylserine externalization, and DNA fragmentation), we found that both tBOOH and D-ribose induce apoptosis to a greater extent in cen3 than in cen3tel cells, suggesting a protective role of telomerase toward apoptotic death. However, monitoring the cell number during treatment with the drugs, we found a decrease in cell number; since this reduction was lower in cen3 fibroblasts compared to cen3tel cells, it is likely that telomerase does not fully protect cells from drug toxicity.

Solution and Solid-State Analysis of Binding of 13-Substituted Berberine Analogues to Human Telomeric G-quadruplexes.

The interaction between 13-phenylalkyl and 13-diphenylalkyl berberine derivatives (NAX) and human telomeric DNA G4 structures has been investigated by both spectroscopic and crystallographic methods. NAX042 and NAX053 are the best compounds improving the performance of the natural precursor berberine. This finding is in agreement with the X-ray diffraction result for the NAX053-Tel12 adduct, showing the ligand which interacts via π-stacking, sandwiched at the interface of two symmetry-related quadruplex units, with its benzhydryl group contributing to the overall stability of the adduct by means of additional π-stacking interactions with the DNA residues. The berberine derivatives were also investigated for their cytotoxic activity towards a panel of human cancer cell lines. Compounds NAX042 and NAX053 affect the viability of cancer cell lines in a dose-dependent manner.

Poly(ADP-ribosylation) and neurodegenerative disorders.

Impaired mitochondrial structure and function are common features of neurodegenerative disorders, ultimately characterized by the death of neural cells promoted by still unknown signals. Among the possible modulators of neurodegeneration, the activation of poly(ADP-ribosylation), a post-translational modification of proteins, has been considered, being the product of the reaction, poly(ADP-ribose), a signaling molecule for different cell death paradigms. The basic properties of poly(ADP-ribosylation) are here described, focusing on the mitochondrial events; cell death paradigms such as apoptosis, parthanatos, necroptosis and mitophagy are illustrated. Finally, the promising use of poly(ADP-ribosylation) inhibitors to rescue neurodegeneration is addressed.

3D Silicon Microstructures: A New Tool for Evaluating Biological Aggressiveness of Tumor Cells.

In this work, silicon micromachined structures (SMS), consisting of arrays of 3- µ m-thick silicon walls separated by 50- µm-deep, 5- µ m-wide gaps, were applied to investigate the behavior of eight tumor cell lines, with different origins and biological aggressiveness, in a three-dimensional (3D) microenvironment. Several cell culture experiments were performed on 3D-SMS and cells grown on silicon were stained for fluorescence microscopy analyses. Most of the tumor cell lines recognized in the literature as highly aggressive (OVCAR-5, A375, MDA-MB-231, and RPMI-7951) exhibited a great ability to enter and colonize the narrow deep gaps of the SMS, whereas less aggressive cell lines (OVCAR-3, Capan-1, MCF7, and NCI-H2126) demonstrated less penetration capability and tended to remain on top of the SMS. Quantitative image analyses of several fluorescence microscopy fields of silicon samples were performed for automatic cell recognition and count, in order to quantify the fraction of cells inside the gaps, with respect to the total number of cells in the examined field. Our results show that higher fractions of cells in the gaps are obtained with more aggressive cell lines, thus supporting in a quantitative way the observation that the behavior of tumor cells on the 3D-SMS depends on their aggressiveness level.

Involvement of PARPs in cell death.

Poly(ADP-ribosylation), an NAD dependent reaction culminating in the formation of ADP-ribose monomers, and their following polymerization, is activated as an emergency process in crucial situations such as DNA damage and cellular stress; due to this crucial function, the modulation of poly(ADP-ribosylation) during cell death has been investigated. This review will describe the properties of poly(ADP-ribose) as a signalling molecule in different paradigms of cell death, i.e.apoptosis, parthanatos, necroptosis and autophagy.

Poly(ADP-ribose): a signaling molecule in different paradigms of cell death.

Poly(ADP-ribosylation) results from the conversion of NAD(+) into ADP-ribose and the following addition of ADP-ribose units to form polymers, further bound to acceptor proteins; once post-translationally ADP-ribosylated, proteins could change their function in basic processes. Poly(ADP-ribosylation) is activated under critical situations represented by DNA damage and cellular stress, and modulated in different paradigms of cell death. The hallmarks of the main death processes, i.e. apoptosis, parthanatos, necroptosis and autophagy, will be described, focusing on the role of poly(ADP-ribose) as a signaling molecule.

Multiple effects of berberine derivatives on colon cancer cells.

The pharmacological use of the plant alkaloid berberine is based on its antibacterial and anti-inflammatory properties; recently, anticancer activity has been attributed to this compound. To exploit this interesting feature, we synthesized three berberine derivatives, namely, NAX012, NAX014, and NAX018, and we tested their effects on two human colon carcinoma cell lines, that is, HCT116 and SW613-B3, which are characterized by wt and mutated p53, respectively. We observed that cell proliferation is more affected by cell treatment with the derivatives than with the lead compound; moreover, the derivatives proved to induce cell cycle arrest and cell death through apoptosis, thus suggesting that they could be promising anticancer drugs. Finally, we detected typical signs of autophagy in cells treated with berberine derivatives.

Poly(ADP-ribosylation) and neoplastic transformation: effect of PARP inhibitors.

Poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribosylation) play essential roles in several biological processes, among which neoplastic transformation and telomere maintenance. In this paper, we review the poly(ADP-ribosylation) process together with the highly appealing use of PARP inhibitors for the treatment of cancer. In addition, we report our results concerning poly(ADP-ribosylation) in a cellular model system for neoplastic transformation developed in our laboratory. Here we show that PARP-1 and PARP-2 expression increases during neoplastic transformation, together with the basal levels of poly(ADP-ribosylation). Furthermore, we demonstrate a greater effect of the PARP inhibitor 3-aminobenzamide (3AB) on cellular viability in neoplastically transformed cells compared to normal fibroblasts and we show that prolonged 3AB administration to tumorigenic cells causes a decrease in telomere length. Taken together, our data support an active involvement of poly(ADP-ribosylation) in neoplastic transformation and telomere length maintenance and confirm the relevant role of poly(ADP-ribosylation) inhibition for the treatment of cancer.

Morphological Features of Organelles during Apoptosis: An Overview.

An apoptotic program leading to controlled cell dismantling implies perturbations of nuclear dynamics, as well as changes affecting the organelle structure and distribution. In human cancer cells driven to apoptosis by different stimuli, we have recently investigated the morphological properties of several organelles, including mitochondria, lysosomes, endoplasmic reticulum and Golgi apparatus. In this review, we will discuss the body of evidence in the literature suggesting that organelles are generally relocated and/or degraded during apoptosis, irrespectively of the apoptogenic stimulus and cell type.

Regulated forms of cell death are induced by the photodynamic action of the fluorogenic substrate, Hypocrellin B-acetate.

The addition of chemical groups to a photosensitizer makes it to act as a fluorogenic substrate, increasing its ability to enter the cells. In this work, the cytotoxic efficacy of Hypocrellin B modified by addition of two acetate groups (HypB-Ac) was investigated in HeLa cells. Using transmission electron microscopy, cytochemical and immunocytochemical techniques, and flow cytometry we demonstrated that light irradiation of HypB-Ac-loaded cells resulted in either necrosis or apoptosis, depending on the HypB-Ac concentration. Administration of Hyp-Ac at high concentration (1×10(-)(5) M) resulted in massive necrosis, while at low concentration (2.5×10(-)(7) M) apoptosis along with autophagy were induced. Focusing on cells still exhibiting non-apoptotic features, we provide the evidence of early involvement of different organelles in the photodamage, with the frequent presence of autophagic vacuoles already at very short post-irradiation times (30 min, when ultrastructural apoptotic features are rarely found). These findings suggest that the widespread photodamage rather than the target organelle(s) involved is crucial for inducing either a catastrophic or a regulated form of cell death. Fluorogenic substrates such as HypB-Ac have an increased capability to accumulate in cancer cells compared to the native photosensitizing molecules: this would allow to use lower drug doses in vivo, thus decreasing the risk of systemic cytotoxicity in the absence of irradiation improving the efficacy of photodynamic therapy. The ability of HypB-Ac at very low concentration to induce autophagy and apoptosis would additionally be advantageous for therapeutic application, as the preferential induction of regulated forms of cell death entails the rapid phagocytotic removal of dying cells without affecting the tissue and organ structure.

A new cell-selective three-dimensional microincubator based on silicon photonic crystals.

In this work, we show that vertical, high aspect-ratio (HAR) photonic crystals (PhCs), consisting of periodic arrays of 5 µm wide gaps with depth of 50 µm separated by 3 µm thick silicon walls, fabricated by electrochemical micromachining, can be used as three-dimensional microincubators, allowing cell lines to be selectively grown into the gaps. Silicon micromachined dice incorporating regions with different surface profiles, namely flat silicon and deeply etched PhC, were used as microincubators for culturing adherent cell lines with different morphology and adhesion properties. We extensively investigated and compared the proliferative behavior on HAR PhCs of eight human cell models, with different origins, such as the epithelial (SW613-B3; HeLa; SW480; HCT116; HT29) and the mesenchymal (MRC-5V1; CF; HT1080). We also verified the contribution of cell sedimentation into the silicon gaps. Fluorescence microscopy analysis highlights that only cell lines that exhibit, in the tested culture condition, the behavior typical of the mesenchymal phenotype are able to penetrate into the gaps of the PhC, extending their body deeply in the narrow gaps between adjacent silicon walls, and to grow adherent to the vertical surfaces of silicon. Results reported in this work, confirmed in various experiments, strongly support our statement that such three-dimensional microstructures have selection capabilities with regard to the cell lines that can actively populate the narrow gaps. Cells with a mesenchymal phenotype could be exploited in the next future as bioreceptors, in combination with HAR PhC optical transducers, e.g., for label-free optical detection of cellular activities involving changes in cell adhesion and/or morphology (e.g., apoptosis) in a three-dimensional microenvironment.