Giuseppe Formisano

The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells.

In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was rather due to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild type and MDR tumor cells. In fact, under treatment condition causing about 50% of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro- or anti-apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.

Bone regeneration guided by resorbable collagen membranes in rabbits : a pilot study

Artificial cross-shaped intrabony defects were created in the mandibles of 12 rabbits and the cavities covered with Type I highly cross-linked resorbable collagen membranes for 30 days. Similar cavities were prepared in three control animals and left uncovered for the same time period. Morphological and analytical data were obtained by means of light microscopy, scanning electron microscopy, and x-ray energy-dispersive spectrometry. After the experimental period, the membrane covered cavities were completely filled with regenerated bone. In the control specimens, the artificial cavities were occupied by fibrous connective tissue. (Implant Dent 1993;2:101–105)

The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications.

Evaluation of guided bone regeneration in rabbit femur using collagen membranes.

&NA; The aim of the study was to evaluate the mechanical performance and the structure of neoformed bone around hydroxyapatite‐coated titanium fixtures according to guided bone regeneration techniques. Ten hydroxyapatitecoated titanium fixtures were inserted in the femurs of five rabbits, in which a cortical defect was created and after the insertion of the fixture, covered with a resorbable membrane obtained from bovine Achilles tendon collagen Type I (A implant). In the same femur, a second fixture was inserted in similar cavities without application of the membrane (B implant). After 60 days, the animals were sacrificed, and block sections of the femoral bone con taining the implants were embedded in polymethylmetacrylate and subjected to tensile shear‐stress at break testing. After the detachment of the implants from the bone, their surfaces were examined with a scanning electron microscope. Tensile shear‐stress values for A and B implant specimens were comparable to some extent, but the former had a lower performance. In this regard, scanning electron microscope observations showed that the neoformed cortical bone present cervically around implant A was much thicker than around implant B. (Implant Dent 2000;9:219‐225)

Alteration of organized structure of biofilm formed by Staphylococcus epidermidis on soft contact lenses

The effect of a nonsteroidal antiinflammatory drug commercially available in eye drop form (sodium diclofenac) was assayed for its ability to affect biofilms formed by clinical Staphylococcus epidermidis isolates. Biofilms produced by one strain positive for a slime-associated antigen, suggested to be expressed by more virulent strains, was not affected by sodium diclofenac treatment. On the other hand, biofilm produced by the slime-positive, antigen-negative strain showed dramatic alterations already after short treatments with sodium diclofenac as reported for salicylate and other nonsteroidal drugs. Such results suggest further investigation of the possible use of sodium diclofenac drops in the treatment of ophthalmic infections in soft contact lens wearers.

Perfluorodecalin modifies the pattern of cell arrangement and induces loss of neurites in rat retinal cultures

Perfluorodecalin (PFD), a high specific weight, water-immiscible perfluorocarbon, previously studied as a potential blood substitute, now is used widely in the field of ophthalmic surgery as a tool for maneuvering intraocular tissues and as a short- or medium-term vitreous substitute. In in vivo experiments, several types of lesions in retinal tissue have been described in conjunction with long-term PFD treatment. To better evaluate the biological effects of PFD on retinal cells, we tested it on primary cultures of rat retina seeded on special cyclopore wells that allow the culture to be fed from the bottom side while the top side is in contact with the water-immiscible compound. We found that PFD changed the pattern of cell arrangement and induced loss of neurites. The modification of cell arrangement was less evident at the periphery of the wells where the amount of PFD, and consequently the pressure exerted, was lower. This observation suggests that the changes may be due more to a physical than to a toxic effect of PFD.

Different effects of sequential combinations of N-methylformamide with 5-fluorouracil on human colon carcinoma cells growing in nude mice

SummaryThe effects of the combination ofN-methylformamide (NMF) with 5-fluorouracil (5-FU) on tumor growth and morphological features of human colon carcinoma cells (HT29) implanted in nude mice were assessed. Both agents were administered i.p. at tolerable doses: 5-FU at 19 mg/kg for 5 days and NMF at 200 mg/ kg for 12 days. Four main schedules were tested: 5-FU alone, NMF alone, NMF followed by 5-FU and 5-FU followed by NMF. The last sequence was the most effective, as compared with the other treatment regimens. In particular, the 5-FU → NMF combination induced a tumor inhibition of about 75% at the end of the treatments (17th day) versus an inhibition of 23%–43% in the other schedules. Morphological observations, carried out by light and electron microscopy, indicated a possible relationship between the presence of structural changes and tumor growth inhibition. The results of this study renew interest in the use of NMF in sequential combination confirming sequence as a critical factor for the optimal combination of NMF and 5-FU.

Influence of dacron tissue thickness on the performance of the Pintucci biointegrable keratoprosthesis: an in vitro and in vivo study.

Purpose. In 1979 Pintucci developed a biointegrable keratoprosthesis with polymethylmethacrylate optical cylinder integrated with a Dacron tissue-colonizable supporting element to avoid the complications caused by the interaction between the haptic element and the eye. The purpose of this article is to compare the colonization of three Dacron fabrics (thicknesses of 0.25 mm, 0.6 mm, and 1.4 mm) in vitro and in vivo to optimize the device performance. Methods. In vitro three different Dacron fabrics were cultured for 3 days with 3.5 × 10 5 human fetal lung fibroblasts and observed with a scanning electron microscope. In vivo three different Dacron fabrics were implanted on the sclera near the superior rectus insertion in the right eye of six albino rabbits and were observed after 4 days with light and scanning electron microscopy. Results. In the in vitro experiments, the cells were preserved and their structure was found to be normal. The 0.25-mm thick fabric was coated only on the surface, and the other fabrics were colonized in three dimensions. In the in vivo experiments, the 0.25-mm thick fabric appeared coated only on its surface. The other fabrics were three-dimensionally colonized and the Dacron filaments appeared embedded in neovascularized connective tissue with minimal foreign body reaction. The 1.4-mm thick colonized fabric showed a substantial loss of pliability. Conclusion. Given that the 0.25-mm thick fabric was coated only by connective tissue, that the 0.6-mm and 1.4-mm thick fabrics were perfectly colonized, and that the 1.4-mm thick fabrics showed a substantial loss of pliability, the 0.6-mm thick fabric haptic part of the Pintucci keratoprosthesis is preferred. For 19 years, the 0.6-mm Dacron fabric Pintucci keratoprosthesis was implanted in 159 eyes with good results, overcoming the apparently inseparable difficulties represented by mechanical anchorage and biointegrability of a keratoprosthesis.

Cytoskeletal changes induced in vitro by 2,5-hexanedione: An immunocytochemical study

The effects of 2,5-hexanedione, the main metabolite of the solvents hexane and methyl butyl ketone, have been explored in different in vitro epithelial (CG5 and HEp-2) and melanoma (JR8) cells by means of immunochemistry and electron microscopy. The administration of the toxicant to the cell monolayers at noncytolytic concentrations for 24 and 48 hr exerted several effects on the cell lines studied. Most epithelial and melanoma cells detached from the substrate were in the mitotic phase, whereas cells adhering to the substrate showed time-dependent organelle changes. In fact, after treatment with 2,5-hexanedione, mitochondria appeared swollen, with distorted cristae and rarefied matrix; changes in intracytoplasmic vesicles were also detected. Cytoskeletal components were also investigated. A remarkable rearrangement of microfilaments and intermediate filaments (keratin and vimentin) was detected in a time-dependent manner. In particular, actin ruffles and intermediate filament aggregates were observed. Furthermore, the microtubular apparatus seemed to be less affected. The results here reported seem to indicate cytoskeletal components as probable targets of 2,5-hexanedione cytotoxicity in cultured cells.

In vitro effects of 2,5 hexanedione on a melanoma cell line: a morphological study.

The effect of 2,5 hexanedione (2,5 HD) on a cultured human melanoma cell line (JR8) was explored. The addition of the toxicant at noncytolitic concentrations (0.08-0.16%) to the monolayers for 24 and 48 h, resulted in an irreversible inhibition of cell proliferation. Cessation of melanoma cell proliferation was accompanied by wide changes in morphological features of cells still adhering to the substrate. Incubation with the toxicant seemed to induce a differentiative process characterized mainly by a significant increase in cell protrusions. Melanoma cells, losing their bipolar appearance, often increased cell size and developed long dendritic and axon-like processes sometimes ramified in distal portions. Electron microscopic observations established that a change in the polarized appearance of control cells often occurred with 2,5 HD treatment and that a regular arrangement of organelles and cytoskeletal elements was detectable within these dendritic and axon-like protrusions. Furthermore, immunocytochemical studies confirmed an involvement of microtubules and actin network within cell prolongations. After the differentiative process a necrotizing effect occurred, inducing a progressive loss of viable, dendritic cells after 4 or 5 days. Incubation with cyclic AMP was ineffective in control cells while after 2,5 HD treatment seemed to increase the survival rate of neuronal-like cells. Possible mechanisms for the growth inhibitory and differentiative effects of 2,5 HD were discussed.