Martino Donnini

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)

In vitro blockade of receptor activator of nuclear factor-κB ligand prevents osteoclastogenesis induced by neuroblastoma cells

Proliferation and differentiation of osteoclasts are regulated by a cytokine system that includes RANKL, which binds 2 receptors: RANK, which activates osteoclast differentiation, and osteoprotegerin (OPG), a decoy receptor that limits RANKL action. We investigated the role of the OPG/RANKL/RANK network in the pathogenesis of skeletal metastasis in neuroblastoma. Four different neuroblastoma cell lines (NB100, CHP212, SH‐SY5Y, SJ‐NK‐P) showed a large amount of OPG and RANKL transcripts. Soluble RANKL was detectable in all cell lines, but poor release of OPG was observed. SH‐SY5Y showed the lowest OPG‐to‐RANKL ratio and promoted osteoclastic differentiation of FLG29.1 and peripheral mononuclear cells, inducing expression of the osteoclast markers RANK, c‐src, c‐fos, cathepsin‐K and TRAP. SJ‐N‐KP, which released both OPG and RANKL, did not show the same capability. OPG, neutralizing anti‐RANKL antibody and antisense oligonucleotides were evaluated for their ability to inhibit RANKL activity. The neutralizing antibody hampered osteoclastic differentiation by blocking both the juxtacrine and the paracrine activity of RANKL. Our findings confirm that neuroblastoma cells induce osteoclastogenesis via RANKL and suggest that the RANKL expression associated with lack of the decoy receptor OPG could be a peculiarity of some tumors that makes them able to induce metastatic osteolysis. Moreover, our results suggest that RANKL could be a relevant target in the adjuvant therapy of bone metastatic neuroblastoma as proper neutralization revokes completely osteoclastic differentiation.

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.

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.

Downregulation of bcl-2 expression in lymphoma cells by bcl-2 ARE-targeted modified, synthetic ribozyme

Synthetic ribozymes are catalytic RNA molecules designed to inhibit gene expression by cleaving specific mRNA sequences. We investigated the potential of synthetic ribozymes to inhibit bcl-2 expression in apoptosis defective bcl-2 overexpressing tumors. A chemically stabilized hammerhead ribozyme has been targeted to the A+U-rich regulative element of bcl-2 mRNA that is involved in bcl-2 gene switch-off during apoptosis. The design of the ribozyme was based on the results of probing accessibility of the RNA target in cellular extracts with antisense DNA. The ribozyme was lipotransfected to a bcl-2 overexpressing human lymphoma cell line (Raji). The cellular uptake of this ribozyme resulted in a marked reduction of both bcl-2 mRNA and BCL-2 protein levels and dramatically increased cellular death by apoptosis. Our results suggest a potential therapeutic application of such ribozyme for the treatment of bcl-2 overexpressing tumors.

Concomitant Effect of Topical Ubiquinone Q10 and Vitamin E to Prevent Keratocyte Apoptosis After Excimer Laser Photoablation in Rabbits

PURPOSE To investigate in vivo whether ubiquinone Q10 together with vitamin E protects rabbit corneas from keratocyte apoptosis after excimer laser irradiation. METHODS Photorefractive keratectomy (PRK) was performed in both eyes of three New Zealand white rabbits. During 3 days before surgery, each right eye received four-times-daily instillation of an eye-drop solution containing ubiquinone Q10 0.20% and vitamin E 0.04%; each left eye was treated with a solution that did not contain ubiquinone or vitamin E. The central cornea was analyzed after surgery using the in situ end labelling (ISEL) technique of nicked DNA to detect DNA fragmentation. To determine the number of ISEL positive nuclei, an average of 70 random microscopic fields (five for each de-epithelialized tissue section) of 138,000 mu2 were examined in the right and left cornea samples at 250X by two different observers. RESULTS Light microscopic examination of the sections from corneas treated before PRK showed that cells committed to apoptosis by PRK were about 50% compared to those of untreated controls. CONCLUSION Treatment of rabbit eyes before PRK with ubiquinone Q10 lowered the number of apoptotic events.

Apoptosis and Bax, Bcl-2, Mcl-1 expression in neutrophils of Crohn's disease patients

Background: The etiology of Crohns disease (CD) remains unknown, and the defective function of neutrophils appears to be associated with this pathology. Neutrophils undergo spontaneous apoptosis which, if not tightly regulated, can induce the development of chronic inflammatory disease. The Bcl‐2 protein family is also involved in the regulation of neutrophil apoptosis. Methods: This study investigated the apoptosis and expression of some regulatory factors in CD patient and control polymorphonuclear neutrophils (PMN) in suspension and in adhesion on fibronectin, an extracellular matrix protein. These 2 conditions mimic circulating neutrophils before they are recruited at the intestinal levels, and their adhesion to tissue. Results: Apoptosis in CD patient PMN was delayed in suspension and accelerated in adhesion, which is the opposite of what happens in controls. Higher levels of Bax, Bcl‐2, and Mcl‐1 proteins were registered in freshly isolated CD patient PMN, in contrast to controls, in which Bcl‐2 protein was undetectable. Among the studied pro‐ and antiapoptotic factors, Bax levels seem to be mainly related to the difference in apoptosis between PMN of CD patients and controls. Conclusions: For the first time it has been demonstrated by direct experimental evidence that apoptosis in CD patient PMN is regulated differently from that of control PMN. Abnormal expression of regulating apoptosis proteins is shown in CD patient PMN. These data suggest that the defective functionality of neutrophils can be the early event responsible for the altered mucosal immune response in CD, and that neutrophil apoptosis may offer a new target for specific drugs and therapy tools.

ζ-crystallin is a bcl-2 mRNA binding protein involved in bcl-2 overexpression in T-cell acute lymphocytic leukemia

The human antiapoptotic bcl‐2 gene has been discovered in t(14;18) B‐cell leukemias/lymphomas because of its overexpression caused at a transcriptional control level by the bcl‐2/IgH fusion gene. We were the first to disclose the post‐transcriptional control of bcl‐2 expression mediated by interactions of an adenine + uracil (AU)‐rich element (ARE) in the 3′‐UTR of bcl‐2 mRNA with AU‐binding proteins (AUBPs). Here, we identify and characterize ζ‐crystallin as a new bcl‐2 AUBP, whose silencing or overexpression has impact on bcl‐2 mRNA stability. An increased Bcl‐2 level observed in normal phytohemagglutinin (PHA)‐activated T lymphocytes, acute lymphatic leukemia (ALL) T‐cell lines, and T cells of patients with leukemia in comparison with normal non‐PHA‐activated T lymphocytes was concomitant with an increase in ζ‐crystallin level. The specific association of ζ‐crystallin with the bcl‐2 ARE was significantly enhanced in T cells of patients with ALL, which accounts for the higher stability of bcl‐2 mRNA and suggests a possible contribution of ζ‐crystallin to bcl‐2 overexpression occurring in this leukemia.—Lapucci, A., Lulli, M., Amedei, A, Papucci, L., Witort, E., Di Gesualdo, F., Bertolini, F., Brewer, G., Nicolin, A., Bevilacqua, A., Schiavone, N., Morello, D., Donnini, M., Capaccioli, S. ζ‐Crystallin is a bcl‐2 mRNA binding protein involved in bcl‐2 overexpression in T‐cell acute lymphocytic leukemia. FASEB J. 24, 1852–1865 (2010). www.fasebj.org

B Cell Lymphoma (Bcl)-2 Protein Is the Major Determinant in bcl-2 Adenine-Uridine-rich Element Turnover Overcoming HuR Activity

In the 3′-untranslated region, the destabilizing adenine-uridine (AU)-rich elements (AREs) control the expression of several transcripts through interactions with ARE-binding proteins (AUBPs) and RNA degradation machinery. Although the fundamental role for AUBPs and associated factors in eliciting ARE-dependent degradation of cognate mRNAs has been recently highlighted, the molecular mechanisms underlying the specific regulation of individual mRNA turnover have not yet been fully elucidated. Here we focused on the post-transcriptional regulation of bcl-2 mRNA in human cell lines under different conditions and genetic backgrounds. In the context of an AUBPs silencing approach, HuR knockdown reduced the expression of endogenous bcl-2, whereas unexpectedly, a bcl-2 ARE-reporter transcript increased significantly, suggesting that HuR expression has opposite effects on endogenous and ectopic bcl-2 ARE. Moreover, evidence was provided for the essential, specific and dose-dependent role of the Bcl-2 protein in regulating the decay kinetics of its own mRNA, as ascertained by a luciferase reporter system. Altogether, the data support a model whereby the Bcl-2 protein is the major determinant of its own ARE-dependent transcript half-life in living cells and its effect overcomes the activity of ARE-binding proteins.