Diet-derived phytochemicals: from cancer chemoprevention to cardio-oncological prevention.

Cardiovascular diseases and cancer are the leading causes of death in most countries. These diseases share many common risk factors as well as pathogenetic determinants, and their incidence is related to age in an exponential manner. Furthermore, it has become apparent that several treatments used in therapy or even in prevention of cancer can impair the structural and functional integrity of the cardiovascular system, giving rise to an interdisciplinary field: cardio-oncology. However, tumors and cardiovascular diseases also share common protective factors: they can be prevented either by avoiding exposure to recognized risk factors, and/or by favoring the intake of protective compounds and by modulating the host defense machinery. These latter approaches are generally known as chemoprevention. A great variety of dietary and pharmacological agents have been shown to be potentially capable of preventing cancer in preclinical models, most of which are of plant origin. Phytochemicals, in particular diet-derived compounds, have therefore been proposed and applied in clinical trials as cancer chemopreventive agents. There is now increasing evidence that some phytochemicals can be also protective for the heart, having the potential to reduce cancer, cardiovascular disease and even anticancer drug-induced cardiotoxicity. We introduce the concept that these compounds induce pre-conditioning, a low level cellular stress that induces strong protective mechanisms conferring resistance to toxins such as cancer chemotherapeutics. Cancer cells and cardiomyocytes have fundamental differences in their metabolism and sensitivity to preconditioning, autophagy and apoptosis, so that dosage of the prevention compounds is important. Here we discuss the mechanisms responsible for the cardiotoxicity of anticancer drugs, the possibility to prevent them and provide examples of diet-derived phytochemicals and other biological substances that could be exploited for protecting the cardiovascular system according to a joint cardio-oncological preventative approach.

Use of IgG Avidity test in case definitions of toxoplasmosis in pregnancy.

A survey network for congenital toxoplasmosis (TOXO-NET) was set up in December 1996 in Piedmont (Italy). Participants were asked to classify the infections in pregnant mothers and newborns by the criteria of the European Network on Congenital Toxoplasmosis published by Lebech in 1996. Because the IgG Avidity test is largely employed as a 2nd level test in toxoplasmosis diagnosis and it could be helpful to date infection, the co-ordinators of TOXO-NET suggested including it in the "case definition" of "probable" infection and "unlikely" infection. 117 cases of toxoplasmosis in pregnancy divided into the risk categories under Lebech's criteria were re-examined using the "new" case definitions. 77 out of 117 (65.8%) Toxoplasma gondii infections during pregnancy could be defined with only one serum sample using the IgG Avidity test. The IgG Avidity test proved a useful method to classify the Toxoplasma gondii infections in pregnancy, especially when we had only one serum sample.

Anti-angiogenesis and angioprevention: mechanisms, problems and perspectives.

The recognition that angiogenesis is a key early event in tumor progression and metastasis has led to the development of new strategies for cancer therapy. The generation of a new blood vessel network under physiological conditions is regulated by the concerted action of activators and inhibitors. Perturbation of this balance, as it occurs in solid tumor growth and metastasis, appears to be a critical point in tumorigenesis. This has led to the "angiogenic switch" hypothesis: the point at which a tumor acquires the potential to induce angiogenesis is a critical step towards malignancy. Based on experimental evidence, prevention of blood vessel development appears to be the mechanism of action of many successful chemopreventive drugs of natural or synthetic origin: a novel concept that we termed "angioprevention". The hypothesis that anti-angiogenesis is at the basis of tumor prevention also suggests that many anti-angiogenic drugs could be used for chemoprevention in higher risk populations or in early intervention. There is a growing body of experimental evidence that anti-angiogenic strategies will contribute to the future therapy of cancer, several compounds with anti-angiogenic properties are now under clinical investigation including anti-inflammatory compounds, as inflammation may play a key role in angiogenesis. We must persevere in the development of novel, powerful and safer angiogenesis inhibitors and in the use of anti-angiogenic drugs in combination with other natural or synthetic anti-cancer agents in a biological therapy strategy.

Growth factor supplemented matrigel improves ectopic skeletal muscle formation--a cell therapy approach.

Following damage to skeletal muscle, satellite cells become activated, migrate towards the injured area, proliferate, and fuse with each other to form myotubes which finally mature into myofibers. We tested a new approach to muscle regeneration by incorporating myoblasts, with or without the exogenous growth factors bFGF or HGF, into three-dimensional gels of reconstituted basement membrane (matrigel). In vitro, bFGF and HGF induced C2C12 myoblast proliferation and migration and were synergistic when used together. In vivo, C2C12 or primary i28 myoblasts were injected subcutaneously together with matrigel and growth factors in the flanks of nude mice. The inclusion of either bFGF or HGF increased the vascularization of the gels. Gels supplemented with bFGF showed myogenesis accompanied by massive mesenchymal cell recruitment and poor organization of the fascicles. Samples containing HGF showed delayed differentiation with respect to controls or bFGF, with increased myoblast proliferation and a significantly higher numbers of cells in myotubes at later time points. HGF samples showed limited mesenchymal cell infiltration and relatively good organization of fascicles. The use of both bFGF and HGF together showed increased numbers of nuclei in myotubes, but with bFGF-mediated fibroblast recruitment dominating. These studies suggest that an appropriate combination of basement membrane components and growth factors could represent a possible approach to enhance survival dispersion, proliferation, and differentiation of myogenic cells during muscle regeneration and/or myoblast transplantation. This model will help develop cell therapy of muscle diseases and open the future to gene therapy approaches.

Ocular effects of fenretinide, a vitamin A analog, in a chemoprevention trial of bladder cancer.

Fenretinide is a vitamin A derivative under investigation in cancer prevention trials. Because all available pharmacologic and toxicologic data were obtained from breast cancer patients, we measured plasma drug, metabolite, and vitamin A levels and studied their relationship with visual and ocular symptoms in a cohort formed mostly by male subjects belonging to a bladder cancer prevention trial. After 1 year, the mean plasma retinol levels (+/- standard deviation [SD]) were 168.2 +/- 75.8 ng/ml in 31 subjects treated with fenretinide and 594.5 +/- 168.4 ng/ml in 36 control subjects (P < .001). Plasma retinol levels were correlated inversely to drug and metabolite concentrations, which in turn were correlated inversely to the interval from last drug intake. The decline of plasma vitamin A levels accounted for a 41.7% cumulative incidence of diminished dark adaptability in the retinoid arm as compared to 6.8% in the control arm (odds ratio = 13.8; 95% confidence interval, 2.9-66.1). Although compliance as assessed by capsule count was high, three subjects originally assigned to the treatment group who proved to be noncompliers (8.8%, or 3 of 34) had no detectable plasma drug or metabolite levels. Our data confirm the specific pharmacologic and visual effects of fenretinide also in a male population and strengthen the importance of multiple blood measurements to monitor treatment compliance in prevention trials.

Studies on the cellular uptake of retinol binding protein and retinol.

The uptake and release of (125)I-RBP and of holoRBP labeled with [(3)H]retinol ((3)H-ROH) were studied in two cell lines which synthesize and secrete RBP, the HepG2 hepatocarcinoma cell line and the Caki-1 kidney adenocarcinoma cell line, and in HeLa cells that do not express the endogenous RBP gene. In all three cell lines a part of endocytosed (125)I-RBP is recycled to the extracellular medium and part is degraded. Nonspecific endocytosis of (125)I-RBP was estimated to be approximately 10% of total endocytosed (125)I-RBP. In HepG2 cells the (3)H-ROH from the [(3)H]retinol-RBP complex ((3)H-ROH-RBP) is recycled bound to RBP into serum-free chase medium. This (3)H-ROH recycling is blocked in HepG2 cells by cyclohexymide and by brefeldin A, an inhibitor of protein export from the main secretory route, and is absent in HeLa cells, which do not synthesize RBP. These data suggest that at least part of retinol taken up from exogenous holoRBP is delivered to newly synthesized RBP. (3)H-ROH recycled by HeLa cells is bound to serum albumin, as is a portion of that recycled by HepG2 cells. Transfer of (3)H-ROH from RBP to serum albumin does not occur in the absence of cells. We conclude that RBP is endocytosed through a specific pathway and that the RBP-associated retinol is transferred to newly synthesized RBP or to serum albumin.

Retinol binding protein and transthyretin are secreted as a complex formed in the endoplasmic reticulum in HepG2 human hepatocarcinoma cells.

Retinol binding protein (RBP), the retinol-specific carrier, circulates in blood as a 1:1 complex with the homotetrameric protein transthyretin (TTR). Both RBP and TTR are synthesized and secreted by the hepatocyte. In this work we have demonstrated, using HepG2 cells as a model system, that the association between the two proteins occurs inside the cell before secretion. The intracellular complex was detected only when metabolically labeled cells were lysed under mild detergent conditions (1.5% octylglucoside), followed by immunoprecipitation and SDS-PAGE. Alternatively, the immunoprecipitates from unlabeled cells lysed with the same buffer were analyzed by Western blotting. This finding was confirmed using the cross-linking agent dithiobis(succinimidyl) propionate before cell lysis. Moreover, we found that in cells treated with brefeldin A to block the exit of proteins from the endoplasmic reticulum (ER), the complex was present in the microsomal fraction. Thus, we can conclude that the RBP-TTR complex is formed inside the cell, more precisely within the ER. As RBP and TTR both lack an ER retention signal, we considered the possible involvement of chaperones in RBP and TTR retention in the ER and in complex formation. We found that calnexin, an ER integral membrane protein which functions as a chaperone, coprecipitates with RBP and TTR when cell lysis and immunoprecipitation are performed under mild conditions (1% Triton X-100). This result strongly suggests that calnexin may be involved in RBP and TTR retention in the ER, in TTR tetramer assembly, and possibly in complex formation.

A retinoic acid resistant HL-60 cell clone sensitive to N-(4-hydroxyphenyl) retinamide-mediated clonal growth inhibition.

Among the Retinoic Acid (RA) derivatives, retinamides, and in particular N-(4-hydroxyphenyl) retinamide (4-HPR), are currently being investigated in selected cases of cancer chemoprevention. The cellular target range, however, seems to be limited, as cells of hemopoietic origin are virtually incapable of terminal differentiation upon addition of the compound. We have reconsidered the effect of 4-HPR on HL-60 cells by taking advantage of a mutant clone, generated in our laboratory, unresponsive to RA but highly responsive to dimethylsulfoxide (DMSO). We show here that this clone, upon addition of 4-HPR, although unable of undergoing full differentiation, shows considerable reduction of clonal growth. Moreover, the combination of 4-HPR and RA resulted in a much greater effect than the administration of 4-HPR alone. We suggest that 4-HPR and RA, at least in terms of mediating growth inhibition, may follow different metabolic pathways.

An improved RT-PCR protocol for the quantitation of human retinoic acid receptor RNA.

A quantitative reverse transcriptase polymerase chain reaction (RT-PCR) system has been developed to calculate the level of expression of human retinoic acid receptors (hRAR) alpha, beta, and gamma. Starting from a single cDNA preparation, the system allows the measurement of the number of molecules of each mRNA receptor. This is made possible by a synthetic internal standard mRNA which is added in known concentrations at the beginning of the reaction. The system is tested in a rhabdomyosarcoma cell line (A-673) where we have measured the upregulation of beta and gamma receptor mRNAs following treatment with retinoic acid.

Immunization of rabbits with proteins reacted with malonic dialdehyde (MDA): kinetics and specificity of the immune response.

MDA-modified casein, lysozyme or polylysine (MC, ML and MP respectively), was intradermically injected to rabbits in the presence of complete Freund's adjuvant (cFA). Two other animal sets received either cFA alone, or MDA alone. MDA, cFA and MP did not induce any antibody response. Both ML and MC produced an increase of antibody reactivity towards ML, but reactivity towards native lysozyme (L) was increased only by ML and not by MC. According to these results, it was concluded that the epitopes recognized by antibodies reacting with ML and not with L are AIP bridges and possibly the two surrounding aminoacyl (especially lysyl) residues.

Regulation of plasma retinol binding protein secretion in human HepG2 cells.

Retinol binding protein (RBP) is the plasma transport protein of retinol. Mobilization of RBP from the liver stores is stimulated by retinol. During vitamin A deficiency, RBP secretion is specifically inhibited while its rate of biosynthesis is unaffected. As a consequence, RBP, as apoprotein, accumulates inside the endoplasmic reticulum (ER) of the hepatocyte, and a new elevated steady-state concentration is reached. We have studied the role of degradation on the regulation of RBP metabolism in retinol deficient HepG2 cells and determined the intracellular site where RBP degradation takes place. Pulse-chase experiments show that RBP half-life is ca.9 h in retinol-depleted cells. RBP degradation is slow and is insensitive to the treatment with NH4Cl, which inactivates lysosomal proteases and to the drug brefeldin A, which prevents protein export from the ER. The data obtained suggest that RBP degradation occurs, at least in part, in a pre-Golgi compartment. 2-Mercaptoethanol, at millimolar concentration, induces RBP secretion, suggesting a possible role for sulfhydryl-mediated apo-RBP retention by resident ER proteins.

Immunological relevance of malonic dialdehyde (MDA): IV. Further evidences about the epitope recognized by antibodies obtained from rabbits immunized with MDA-modified lysozyme.

Reactions of MDA with primary amino groups produce inter- or intra-molecular 1-amino-3-imino-propene (AIP) bridges, leading to structural modifications of biological molecules. In this work, applying electrophoresis followed by transfer onto nitrocellulose membranes, we observed that serum of a rabbit immunized with MDA-modified lysozyme (ML) reacts not only with ML and native lysozyme (L), but also with MDA-modified ribonuclease, cytochrome c or polylysine (MR, MC and MP respectively), while it does not react with native ribonuclease, cytochrome c or polylysine (R, C and P respectively). These results confirm previous ones indicating that sera of rabbits immunized with ML contain antibodies reacting specifically with epitopes containing AIP bridges.

Histone acetylation in conjugating Tetrahymena thermophila.

We have monitored histone acetylation during conjugation of the ciliated protozoan Tetrahymena thermophila using antibodies against the tetraacetylated form of H4 histone (Pfeffer, U., N. Ferrari, and G. Vidali. 1986. J. Biol. Chem. 261:2496-2498). During meiosis, the three prezygotic divisions, fertilization, and the first postzygotic division, micronuclei, do not contain highly acetylated forms of H4 histone. However, after the second postzygotic division, when anteriorly located micronuclei begin to develop into new macronuclei, they are strongly stained by the anti-tetraacetylated H4 histone antibody. In the old macronucleus, histones are actively deacetylated when it has ceased to transcribe but before it is eliminated. Histone acetylation processes analyzed here appear to be correlated to the commitment to transcription rather than to the transcription process itself. This is in good correlation with evidence we have obtained in chick erythrocyte nuclei during reactivation upon fusion with mammalian cells (Pfeffer, U., N. Ferrari, F. Tosetti, and G. Vidali. 1988. Exp. Cell Res. 178:25-30). Furthermore, it becomes clear from our data that histone acetylation occurs in close correlation to the position of nuclei within the cytoplasm of T. thermophila. Mechanisms that control differential histone acetylation and deacetylation are discussed.

Histone hyperacetylation is induced in chick erythrocyte nuclei during reactivation in heterokaryons.

Transcriptionally inactive avian red blood cell nuclei were reactivated by Sendai virus-induced fusion of chicken erythrocytes with HeLa cells. We have used antibodies which specifically recognize the tetraacetylated form of H4 histone to show that histone hyperacetylation is an event required for chromatin reorganization leading to a transcriptionally competent chromatin structure.

Serial determination of platinum, protein content and free sulfhydryl levels in plasma of patients treated with cisplatin or carboplatin.

We report here the results of serial determinations of free-sulfhydryl, protein content and platinum levels in plasma of cancer patients following either cisplatin (cis-DDP, 3 patients, 60 mg/m2) or carboplatin (JM-8, 3 patients, 300 mg/m2) i.v. administration. After treatment with cis-DDP, significant platinum binding to plasma components with MW greater than 25 kD was observed; the ratio free-sulfhydryls/protein content decreased during the first two hours, returning to normal values at 24 hours after injection. In contrast, no evidence of platinum binding to plasma components with MW greater than 25 kD was noted after JM-8 i.v. administration, and the ratio free sulfhydryls/protein content did not change significantly after treatment. In vitro experiments show that at a molar ratio cis-DDP:JM-8 1:10, the two compounds bind to low MW thiols with the same kinetics. These data seems to suggest different interactions at the plasma level of these drugs, which may be correlated with their different toxicity.

Denitrosation of N-nitrosodimethylamine and N-nitrosomethylurea by liver microsomes from trout (Salmo gairdneri Rich.).

N-Nitrosodimethylamine and N-nitrosomethylurea are partially metabolized to nitrite and formaldehyde by trout liver microsomes. Results show that the denitrosation pathway of both N-nitroso derivatives is NADPH-dependent, requires molecular oxygen, and is inhibited by common inhibitors of microsomal enzyme activities.

The whole blood oxygen affinity in normal human newborns: II) P50, n value and related parameters.

The cord blood from 82 normal newborns (48 males and 34 females) has been extensively investigated, to check p50, cooperativity and related parameters. The most important results are the following: Neonatal p50 is slightly above 20 torr, in good agreement with the literature data, but this value refers to the p50 "CO-free" (T50); otherwise, the original p50 is slightly less than 22 torr, a value which is not indicative enough, because neonatal blood contains a consistent excess of Hb-CO. Either this CO-excess and the low p50 value seem to be mainly depending on the low binding capacity of Hb-F with DPG, even if further studies are still necessary at regard. The empirical factor correcting adult p50 to T50 is -0.27, a value which must be increased to -0.36 for neonatal blood. Multiple gas analyses of the samples indicate that oxygen transport is a very complicate equilibrium involving pH, CO2, bicarbonate balance and other factors. A complete statistical analysis of the data has been carried out and the main result indicates that oxygen assumption favours CO2 releasing and vice versa, a phenomenon already well known and globally called "Bohr effect".