Sergio Capaccioli

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.

Post-Transcriptional Regulation of Gene Expression by Degradation of Messenger RNAs

Recent evidence suggests that gene expression may be regulated, at least in part, at post‐transcriptional level by factors inducing the extremely rapid degradation of messenger RNAs. These factors include reactions between adenyl‐uridyl‐rich elements (AREs) of the relevant mRNA and either specific proteins that bind to these elements or exosomes. This review deals with examples of the proteins (AU‐rich binding proteins, AUBPs) and exosomes, which have been shown to form complexes with AREs and bring about rapid degradation of the relevant mRNA, and with certain other factors, which protect the RNA from such degradation. The biochemical and physiological factors underlying the stability of messenger RNAs carrying the ARE motifs will be reviewed in the light of their emerging significance for cell physiology, human pathology, and molecular medicine. We also consider the possible application of the results of recent insights into the mechanisms to pharmacological interventions to prevent or cure disorders, especially developmental disorders, which the suppression of gene expression may bring about. Molecular targeting of specific steps in protein degradation by synthetic compounds has already been utilized for the development of pharmacological therapies.

Natural compounds for cancer treatment and prevention.

We describe here the main natural compounds used in cancer therapy and prevention, the historical aspects of their application and pharmacognosy. Two major applications of these compounds are described: as cancer therapeutics and as chemopreventive compounds. Both natural compounds, extracted from plants or animals or produced by microbes (antibiotics), and synthetic compounds, derived from natural prototype structures, are being used. We also focus on the molecular aspects of interactions with their recognized cellular targets, from DNA to microtubules. Some critical aspects of current cancer chemotherapy are also discussed, focusing on genetics and genomics, and the recent revolutionary theory of cancer: aneuploidy as the primum movens of cancer.

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)

Bcl-2 phosphorylation and apoptosis activated by damaged microtubules require mTOR and are regulated by Akt

The serine/threonine kinase mTOR, the major sensor of cell growth along the PI3K/Akt pathway, can be activated by agents acting on microtubules. Damaged microtubules induce phosphorylation of the Bcl-2 protein and lower the threshold of programmed cell death, both of which are inhibited by rapamycin. In HEK293 cells expressing Akt mutants, the level of Bcl-2 phosphorylation and the threshold of apoptosis induced by taxol or by nocodazole are significantly modified. In cells expressing dominant-negative Akt (DN-Akt), Bcl-2 phosphorylation and p70S6KThr421/Ser424 phosphorylation induced by taxol or nocodazole were significantly enhanced as compared to cells expressing constitutively active Akt (CA-Akt) and inhibited by rapamycin. Moreover, DN-Akt cells were more sensitive to antitubule agents than CA-Akt cells. In nocodazole-treated HEK293 cells sorted according to cell cycle, the p70S6KThr421/Ser424 phosphorylation was associated to the G2/M fraction. More relevant, nocodazole inhibited, in a dose–response manner, mTOR phosphorylation at Ser2448. This activity, potentiated in DN-Akt cells, was not detectable in CA-Akt cells. Our results suggest that death signals originating from damaged microtubules in G2/M can compete with G1 survival pathways at the level of mTOR. These findings have implications for cancer therapy and drug resistance.

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.

Vitamin E protects human skeletal muscle from damage during surgical ischemia-reperfusion.

PURPOSE The biochemical and morphological alterations induced in lower limb skeletal muscle by ischemia-reperfusion (I-R) during aortic surgery and the effect of vitamin E pretreatment were investigated. METHODS Two groups of patients undergoing aortic aneurysm resection, one untreated and one treated with vitamin E, were examined. Quadricep muscle biopsies were taken after induction of anesthesia, at the end of ischemia, and after reperfusion. The malondialdehyde (MDA) content and morphology of biopsies were examined to assess peroxidative processes. RESULTS Ischemia did not induce an increase in MDA content but did increase neutrophil infiltration in muscle fibers of untreated patients. Reperfusion led to a significant increase in MDA content and to intermyofibrillar edema and mitochondrial swelling. The MDA content was not increased during ischemia and neutrophil infiltration was minimal in vitamin E treated patients. At reperfusion, the MDA content, the ultrastructural injuries and neutrophil infiltration were significantly reduced by the treatment. CONCLUSIONS Vitamin E is effective in reducing the oxidative muscle damage occurring after a period of I-R.