Nicola Schiavone

Aponecrosis: Morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis†

A rat fibroblastic cell line (rat‐1/myc‐ER™) was treated with different concentration of Antimycin A, a metabolic poison that affects mitochondrial respiratory chain complex III. The modes of cell death were analyzed by time‐lapse videomicroscopy, in situ end‐labeling (ISEL) technique, and ultrastructural analysis. Intracellular ATP levels were also measured in order to detect whether the energetic stores were determinant for the type of cell death. It was found that while apoptosis was the prevalent cell death in the fibroblasts treated with low doses, 100 or 200 μM Antimycin A, a new type of cell demise that shared dynamic, molecular, and morphological features with both apoptosis and necrosis represents the most common cell death when the cells were exposed to high doses, 300 or 400 μM, of the hypoxic stimulus. This new type of cell death has been chimerically termed aponecrosis. The inhibition of caspase 3, an enzyme critical for the apoptotic DNA degradation, caused a clear shift from aponecrosis to necrosis in the cell culture, suggesting that this new type of cell death could account for an incomplete execution of the apoptotic program and the following degeneration in necrosis. After being treated with higher doses, i.e., 1000 μM Antimycin A, almost all of the cells died by true necrosis. The analysis of the cellular energetic stores showed that the levels of ATP were a primary determinant in directing toward active cell death (apoptosis), aponecrosis, or necrosis. We conclude that chemically induced hypoxia produces different types of cell death depending on the intensity of the insult and on the ATP availability of the cell, and that the classic apoptosis and necrosis may represent only two extremes of a continuum of intermediate forms of cell demise. J. Cell. Physiol. 182:41–49, 2000.

A conserved AU-rich element in the 3′ untranslated region of bcl-2 mRNA is endowed with a destabilizing function that is involved in bcl-2 down-regulation during apoptosis

The control of mRNA stability is becoming recognized as a crucial point of gene expression regulation. A common element responsible for mRNA decay modulation is the adenine‐ and uracil‐rich element that is found in the 3’ untranslated region of numerous mRNAs subjected to fast expression changes in response to various stimuli. Previously we identified a post‐transcriptional regulation level for the antiapoptotic bcl‐2 gene, which could be involved in t(14;18) lymphoma‐associated bcl‐2 overexpression. Here we demonstrate that bcl‐2 mRNA is endowed with an adenine‐ and uracil‐rich element (ARE) characterized by high evolutionary conservation not only among all chordates examined, but even between chordates and the nematode Caeno‐rhabditis elegans (ced‐9 gene). As for other well‐established destabilizing AREs, the insertion of the bcl‐2 ARE downstream from stable β‐globin mRNA causes an enhanced decay of the β‐globin transcript, which proves its functional role. This possibility is corroborated by the fact that the pathway leading to the modulating activity of bcl‐2 ARE is influenced by PKC, since the addition of DAG and TPA markedly attenuated the bcl‐2 ARE destabilizing potential. Conversely, it is noteworthy that when C2‐ceramide is added to the culture medium as the apoptotic agent, the β‐globin transcript harboring the bcl‐2 ARE undergoes a dramatic increase in decay. This observation clearly indicates that the destabilizing function of bcl‐2 ARE is enhanced by apoptotic stimuli and suggests that this element could be involved in a post‐transcriptional mechanism of bcl‐2 down‐regulation during apoptosis. The half‐life of the mRNA of bcl‐2 in Jurkat cells is prolonged by PKC stimulation and shortened by C2‐ceramide addition, strongly supporting the view that bcl‐2 mRNA stability plays a physiological role in modulating bcl‐2 expression, particularly in its down‐regulation during apoptosis. Thus, this element becomes a new candidate for mediating those bcl‐2 gene expression changes— from apoptosis‐associated down‐regulation to tumor‐associated overexpression—observed thus far that profoundly influence single cell fate and tissue ho‐meostasis. Schiavone, N., Rosini, P., Quattrone, A., Donnini, M., Lapucci, A., Citti, L., Bevilacqua, A., Nicolin, A., Capaccioli, S. A conserved AU‐rich element in the 3* untranslated region of bcl‐2 mRNA is endowed with a destabilizing function that is involved in bcl‐2 down‐regulation during apoptosis. FASEB J. 14, 174–184(2000)

Inducible Nitric Oxide Synthase Expression in Human Colorectal Cancer : Correlation with Tumor Angiogenesis

To investigate the potential involvement of the nitric oxide (NO) pathway in colorectal carcinogenesis, we correlated the expression and the activity of inducible nitric oxide synthase (iNOS) with the degree of tumor angiogenesis in human colorectal cancer. Tumor samples and adjacent normal mucosa were obtained from 46 surgical specimens. Immunohistochemical expression of iNOS, vascular endothelial growth factor (VEGF), and CD31 was analyzed on paraffin-embedded tissue sections. iNOS activity and cyclic GMP levels were assessed by specific biochemical assays. iNOS protein expression was determined by Western blot analysis. iNOS and VEGF mRNA levels were evaluated using Northern blot analysis. Both iNOS and VEGF expressions correlated significantly with intratumor microvessel density (r(s) = 0.31, P = 0.02 and r(s) = 0.67, P < 0.0001, respectively). A significant correlation was also found between iNOS and VEGF expression (P = 0.001). iNOS activity and cyclic GMP production were significantly higher in the cancer specimens than in the normal mucosa (P < 0.0001 and P < 0.0001, respectively), as well as in metastatic tumors than in nonmetastatic ones (P = 0.002 and P = 0.04, respectively). Western and Northern blot analyses confirmed the up-regulation of the iNOS protein and gene in the tumor specimens as compared with normal mucosa. NO seems to play a role in colorectal cancer growth by promoting tumor angiogenesis.

Inhibition of 5-lipoxygenase by MK886 augments the antitumor activity of celecoxib in human colon cancer cells

Cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LOX) are key enzymes involved in arachidonic acid metabolism. Their products, prostaglandins and leukotrienes, are involved in colorectal tumor development. We aimed at evaluating whether combined blocking of the COX-2 and 5-LOX pathways might have additive antitumor effects in colorectal cancer. The expression/activity of COX-2 and 5-LOX were assessed in 24 human colorectal cancer specimens. The effects of the COX-2 inhibitor celecoxib and the 5-LOX inhibitor MK886 on prostaglandin E2 and cysteinyl leukotriene production, tumor cell proliferation, cell apoptosis, and Bcl-2/Bax expression were evaluated in the Caco-2 and HT29 colon cancer cells. We also investigated the effect of the enzymatic inhibition on mitochondrial membrane depolarization, one of the most important mechanisms involved in ceramide-induced apoptosis. Up-regulation of the COX-2 and 5-LOX pathways was found in the tumor tissue in comparison with normal colon mucosa. Inhibition of either COX-2 or 5-LOX alone resulted in activation of the other pathway in colon cancer cells. Combined treatment with 10 μmol/L celecoxib and MK886 could prevent this activation and had additive effects on inhibiting tumor cell proliferation, inducing cell apoptosis, decreasing Bcl-2 expression, increasing Bax expression, and determining mitochondrial depolarization in comparison with treatment with either inhibitor alone. The administration of the ceramide synthase inhibitor fumonisin B1 could prevent some of these antineoplastic effects. In conclusion, our study showed that inhibition of 5-LOX by MK886 could augment the antitumor activity of celecoxib in human colorectal cancer. [Mol Cancer Ther 2006;5(11):2716–26]

LMW-PTP is a positive regulator of tumor onset and growth

Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are an enzyme family that plays a key role in cell proliferation control by dephosphorylating/inactivating both tyrosine kinase receptors (such as PDGF, insulin, and ephrin receptors) and docking proteins (such, as β-catenin) endowed with both adhesion and transcriptional activity. Besides being a frequent event in human tumors, overexpression of LMW-PTP has been recently demonstrated to be sufficient to induce neoplastic transformation. We recently demonstrated that overexpression of LMW-PTP strongly potentiates the stability of cell–cell contacts at the adherens junction level, which powerfully suggests that LMW-PTP may also contribute to cancer invasivity. Focusing on mechanisms by which LMW-PTP is involved in cancer onset and progression, the emerging picture is that LMW-PTP strongly increases fibronectin-mediated cell adhesion and mobility but, paradoxically, decreases cell proliferation. Nevertheless, LMW-PTP-transfected NIH3T3 fibroblasts engrafted in nude mice induce the onset of larger fibrosarcomas, which are endowed with higher proliferation activity as compared to mock-transfected controls. Quite opposite effects have been obtained with engrafted fibroblasts transfected with a dominant-negative form of LMW-PTP. Notably, in sarcoma extracts, LMW-PTP overexpression greatly influences the ephrin A2 (EphA2) but not PDGF receptor or β-catenin tyrosine phosphorylation. The high association of dephosphorylated EphA2 overexpression with most human cancers and our observation that cell growth stimulation by LMW-PTP overexpression is restricted to the in vivo model, strongly suggest that LMW-PTP oncogenic potential is mediated by its EphA2 tyrosine dephosphorylating activity.

The Role of Cyclooxygenase-2 in Mediating the Effects of Histamine on Cell Proliferation and Vascular Endothelial Growth Factor Production in Colorectal Cancer

Purpose: Activity of histidine decarboxylase, the key enzyme in the synthesis of histamine, has been shown to be increased in several types of human tumors. We attempted to establish whether the possible involvement of histidine decarboxylase and histamine in colorectal carcinogenesis might be mediated by the activation of the cyclooxygenase-2 (COX-2) pathway. Experimental Design: Expression/activity of histidine decarboxylase, histamine content, and prostaglandin E2 (PGE2) production were analyzed in 33 colorectal cancer samples and in the HT29, Caco-2, and HCT116 colon cancer cell lines. The effects of histamine, celecoxib, and H1, H2, and H4 receptor antagonists on COX-2 expression/activity, cell proliferation, and vascular endothelial growth factor (VEGF) production were assessed in the three colon cancer lines that showed different constitutive COX-2 expression. Results: We showed the up-regulation of histidine decarboxylase protein expression and activity in the tumor specimens when compared with normal colonic mucosa. Histidine decarboxylase activity and histamine content were also significantly higher in metastatic tumors than in nonmetastatic ones. These variables significantly correlated with tumor PGE2 production. The administration of histamine increased COX-2 expression/activity, cell proliferation, and VEGF production in the COX-2-positive HT29 and Caco-2 cells. Treatment with either H2/H4 receptor antagonists or celecoxib prevented these effects. Histamine had no effect on both the COX-2 pathway and VEGF production in the COX-2-negative HCT116 cells. Conclusions: Our data showed that histamine exerts both a proproliferative and a proangiogenic effect via H2/H4 receptor activation. These effects are likely to be mediated by increasing COX-2-related PGE2 production in COX-2-expressing colon cancer cells.

Down-regulation of nitric oxide synthase-2 and cyclooxygenase-2 pathways by p53 in squamous cell carcinoma

The goal of this study was to analyze the correlation between inducible nitric oxide synthase (iNOS) and COX-2 activities and p53 gene status in head and neck squamous cell carcinomas (HNSCCs) in vivo and in vitro. In a series of 43 HNSCCs we observed an up-regulation of both iNOS and COX-2 pathways in tumor tissues and both activities were correlated each other (rs = 0.612 and P = 0.0002). We also found that p53-mutated HNSCCs (25 cases, 58.1%) showed higher levels of iNOS activity and cGMP in comparison with wild-type p53 tumors (18 cases, 41.9%) (P = 0.0005 and P = 0.01), as well as higher iNOS immunohistochemical expression (P = 0.03). Analogously, higher PgE2 levels were documented in p53-mutated HNSCCs when compared with wild-type p53 tumors (P = 0.015) and COX-2 protein expression was higher in p53-mutated HNSCCs (P = 0.007). A431 cancer cells expressing a p53 temperature-sensitive mutant showed an approximately 1.9- and 2.6-fold decrease in spontaneous NO(2-)/NO(3-) and PgE2 synthesis at permissive temperature, respectively, when compared with the same cells at nonpermissive temperature (P <or= 0.001). Basal levels of iNOS and COX-2 proteins and mRNAs were markedly suppressed by restoration of p53 activity. Our results indicate that p53 gene mutation(s) may be responsible for iNOS and COX-2 up-regulation frequently observed in HNSCCs and suggest that restoration of wild-type p53 expression may interfere with tumor growth by inhibiting iNOS and COX-2 pathways.

Prevention of corneal keratocyte apoptosis after argon fluoride excimer laser irradiation with the free radical scavenger ubiquinone Q10.

Purpose To assess in vitro the potential of the free radical scavenger ubiquinone Q10 in preventing keratocyte apoptosis after argon fluoride (ArF) excimer laser irradiation. Methods Cultured rabbit keratocytes were irradiated at very low single-pulse laser fluences. The cumulative effects generated by three total fluence doses between 12 and 45 mJ/cm2, representative of single-pulse subablative doses during photorefractive keratectomy (PRK) in humans, were evaluated. We employed the following parameters to compare pretreated (10 μM ubiquinone Q10) and untreated samples: 1) number and morphology of living cells by Trypan blue test and ultramicroscopy, respectively; 2) level of free-radical formation assessed by malonaldehyde quantitation; 3) cellular energy level evaluated by ATP assay. Results Excimer laser irradiation kills cultured keratocytes by inducing apoptosis. The effect increases with the cumulative fluence dose. In the samples pretreated with ubiquinone Q10 there were significantly fewer cumulative apoptotic events than in the untreated ones. Quantitative analysis of malonaldehyde cellular levels suggested this protective action of ubiquinone Q10 was connected with its ability to scavenge laser-generated free radicals. ATP assay also confirmed that it raised cellular energy levels. Conclusions The treatment of corneal keratocytes with relatively low concentrations of ubiquinone Q10 can prevent apoptosis after ArF excimer laser irradiation. If these findings are confirmed on human keratocytes this treatment could be usefully exploited in the PRK surgical procedure. That might lead to a reduction in the occurrence of haze and curvature regression triggered by programmed cell death.

Lipid-polyethylene glycol based nano-ocular formulation of ketoconazole.

Ophthalmic mycoses including corneal keratitis or endophthalmitis affects 6-million persons/year and can cause blindness. Its management requires antifungals to penetrate the ocular tissue. Oral use of Ketoconazole (KTZ), the first broad-spectrum antifungal to be marketed, is now restricted to life-threatening infections due to severe adverse effects and drug-interactions. Local use of KTZ loaded nanocarrier system can address its toxicity, poor solubility, photodegradation, permeation and bioavailability issues. Solid lipid nanoparticles (SLNs) comprising Compritol(®) 888 ATO and PEG 600 matrix, were presently prepared using hot high-pressure homogenization. Employing extensive characterization: TEM, NMR, DSC, XRD and FTIR, it is proposed that SLNs comprise of a polyethylene glycol (PEG) core into which KTZ is dissolved. PEG endows the lipid matrix with amorphousness and imperfections; rigidity; and, stability to aggregation, on storage and autoclaving. PEG is a simple, cost-effective and safe polymer with superior solubilizing and surfactant-supporting properties. Without its inclusion KTZ could not be loaded into SLNs. It ensured high incorporation efficiency (70%) of KTZ; small size (126 nm); and, better permeation into the eye. Pharmacokinetic studies indicated 2.5 and 1.6 fold higher bioavailability (AUC) in aqueous and vitreous humor, respectively. Biocompatibility and in vitro (both in corneal and retinal cell lines) and in vivo (in rabbits) ocular safety is the other highlight of developed formulation.

Autologous lipofilling: coenzyme Q10 can rescue adipocytes from stress-induced apoptotic death.

Background: Autologous fat transplantation (or lipofilling) is an excellent technique for correction of cosmetic defects. The success of the procedure relies strongly on the techniques of harvesting and transferring viable adipocytes. The purpose of this study was to evaluate effects of two harvesting methods and coenzyme Q10 on the viability and apoptotic death of adipocytes collected for autologous lipofilling. Methods: Human adipose tissue from six patients was collected by Luer-Lok syringe according to Coleman’s technique or by means of an aspirator with a 680-mmHg vacuum. Half of each sample collected using Coleman’s technique was treated with 10 &mgr;M Coenzyme Q10, and the other half served as untreated control. Viability and apoptosis were assessed by immunoenzymatic, biochemical, and morphological methods. Results: The harvesting of adipose tissue by aspirator reduced the viability and increased apoptotic death significantly more than harvesting tissue using Coleman’s technique. Biochemical and morphological analyses confirmed that treatment of adipose tissue with coenzyme Q10 reduced and even inhibited apoptotic death of harvested adipocytes. Conclusion: Coenzyme Q10 can rescue adipocytes from stress-induced apoptotic death.