Davide Malacarne

Drug metabolism polymorphisms as modulators of cancer susceptibility

Recently, several molecular genetic bases of polymorphic enzyme activities involved in drug activation and detoxification have been elucidated. Many molecular epidemiology studies based on these premises have sought to gather information on the association of genetically determined metabolic variants with different risks of environmentally induced cancer. While rare alterations of tumor suppressor genes dramatically raise cancer risk for the single affected subjects, far more common and less dramatic differences in genes encoding for drug metabolism enzymes can be responsible for a relatively small, but rather frequent increase of cancer risk at the population level. This increase could be especially important in specific cases of occupational, pharmacological or environmental exposure. Examination of the current literature reveals that the most extensively investigated metabolic polymorphisms are those of P450 1A1 and P450 2D6 cytochromes, glutathione S-transferases (GSTs; M1 and, to a lesser extent, M3, P1 and T1) and N-acetyltransferases (NATs; NAT1 and NAT2). Making reference to these enzymes, we have assayed the current knowledge on the relations among polymorphisms of human xenobiotic-metabolizing enzymes and cancer susceptibilities. We have found intriguing models of susceptibility toward different types of cancer. We have reviewed and commented these models on light of the complex balance among different enzyme activities that, in each individual, determines the degree of each cancer susceptibility. Moreover, we have found techniques of molecular genetic analysis, more suitable than previous ones on phenotypic expression, now allowing better means to detect individuals at risk of cancer. According to the models presently available, a systematic screening of individuals at risk seems to make sense only in situations of well defined carcinogenic exposures and when performed by the polymorphism analysis of coordinated enzyme activities concurring to the metabolism of the carcinogen(s) in question. Genetic polymorphism analysis can allow for the detection of patients more prone to some types of specific cancers, or to the adverse effects of specific pharmaceutical agents. Considering the increasingly confirmed double-edged sword nature of metabolism polymorphism (both wild-type and variant alleles can predispose to cancer, albeit in different situations of exposure), individual susceptibility to cancer should be monitored as a function of the nature, and mechanism of action, of the carcinogen(s) to which the individual under study is known to be exposed, and with reference to the main target organ of the considered type of exposure.

A retro-inverso peptide homologous to helix 1 of c-Myc is a potent and specific inhibitor of proliferation in different cellular systems

In 1998 we reported that an L‐peptide derived from H1 of c‐Myc (Int‐H1‐S6A,F8A), linked to an internalization sequence from the third α‐helix of Antennapedia, was endowed with an antiproliferative and proapoptotic activity toward a human mammary cancer cell line: The activity apparently depends upon the presence of the Myc motif. In the present work we have added new dimensions to our original findings. It is known that short retro‐inverso (RI‐) peptides can assume a 3D conformation very close to their corresponding L‐forms and can be recognized by the same monoclonal antibody. We synthesized a RI‐peptide form of our original L‐peptide: It was much more resistant to serum peptidases than the original molecule (a half life of days rather than hours); in addition, the RI‐form of the original Antennapedia internalization sequence was perfectly capable of carrying a D‐peptide into human cells. We have studied three different potentially active peptides. L‐peptides: Int‐H1wt, Int‐H1‐S6A,F8A. D‐peptides: RI‐Int‐H1‐S6A,F8A. We have also studied three presumed control peptides: Int and RI‐Int (no H1 motif), H1‐S6A,F8A (no internalization sequence). Both ‘active’ and ‘control’ peptides have essentially confirmed our expectations, however, in cells treated with the higher concentration (10 μM) of the control peptide RI‐Int, non‐Myc related side effects could be detected. In order to investigate whether the antiproliferative activities displayed by some of our molecules were indeed related to an interference with the role of c‐Myc (and molecules of the family), we chose an iso‐amphipathic modified peptide of the H1 motif, with a proximity coefficient >50% and where the major change was at position 7 (F→A). From a family of 73 H1 motifs belonging to (H1‐Loop‐H2) human sequences, the smallest evolutionary distance from our reference peptide was observed for the H1 of N‐Myc, L‐Myc, c‐Myc, H1‐S6A,F8A of c‐Myc, and Max, in that order. Our reference peptide was therefore appropriate as a check of whether we were indeed observing activities related to Myc functions. Both Int‐H1isoamph and the corresponding RI‐Int‐H1isoamph peptide were synthesized and studied. In terms of biological targets, we added to the human mammary cancer line of our previous work (MCF‐7 cells) a colon cancer line (HCT‐116 cells) and also a system of normal cells: human peripheral blood lymphocytes (PBLs) stimulated with phytohemoagglutinin (PHA). Peptides carrying an iso‐amphipathic‐modified H1 sequence were always very clearly (3‐10 times) less active than the corresponding peptides carrying a conserved “H1 of Myc” motif. This finding was noted in five independent situations (all the cellular models considered at the present time): MCF‐7 cells treated with L‐peptides; MCF‐7 cells treated with RI‐peptides; HCT‐116 cells treated with L‐peptides; PBLs treated with L‐peptides; PBLs treated with RI‐peptides. Modulation of transcription levels of ornithine decarboxylase (ODC), p53, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), in PBLs treated with our different molecules, was well compatible with an interference by our active peptides at the level of Myc transcriptional activity. We had already reported a similar observation in MCF‐7 cells. On a molar basis, RI‐peptides were about 5–10 times more potent and 30–35 times more stable in complete culture medium, than their corresponding L‐forms. RI‐Int can probably internalize longer peptido‐mimetic molecules (for instance molecules mimetic of (H1‐Loop‐H2), or even more. These possibilities open the way to rodent studies and to more potent/selective Myc inhibitors—two steps closer to a potential drug.

A novel Bim-BH3-derived Bcl-XL inhibitor: biochemical characterization, in vitro, in vivo and ex-vivo anti-leukemic activity.

BH3-only members of the Bcl-2 family exert a fundamental role in apoptosis induction. This work focuses on the development of a novel peptidic molecule based on the BH3 domain of Bim. The antiapoptotic molecule Bcl-XL, involved in cancer development/progression and tumour resistance to cytotoxic drugs, is a target for Bim. According to a rational study of the structural interactions between wt Bim-BH3 and Bcl-XL, we replaced specific residues of Bim-BH3 with natural and non-natural aminoacids and added an internalizing sequence, thus increasing dramatically the inhibitory activity of our modified Bim-BH3 peptide, called 072RB. Confocal microscopy and flow cytometry demonstrated cellular uptake and internalization of 072RB, followed by co-localization with mitochondria. Multiparameter flow cytometry demonstrated that the 072RB dose-dependent growth inhibition of leukaemia cell lines was due to apoptotic cell death. No effect was observed when cells were treated with the internalizing vector alone or a mutated control peptide (single aminoacid substitution L94A). Ex-vivo derived leukemic cells from acute myeloid leukaemia (AML) patients underwent cell death when cultured in vitro in the presence of 072RB. Conversely, no significant cytotoxic effect was observed when 072RB was administered to cultures of peripheral blood mononuclear cells, either resting or PHA-stimulated, and bone marrow cells of normal donors. Xenografts of human AML cells in NOD/SCID mice displayed a significant delay of leukemic cell growth upon treatment with 072RB administered intravenously (15 mg/Kg three times, 48 hours after tumour cell injection). Altogether, these observations support the therapeutic potentials of this novel BH3 mimetic.

A computerized connectivity approach for analyzing the structural basis of mutagenicity in Salmonella and its relationship with rodent carcinogenicity.

We have applied a new software program, based on graph theory and developed by our group, to predict mutagenicity in Salmonella. The software analyzes, as information in input, the structural formula and the biological activities of a relatively large database of chemicals to generate any possible molecular fragment with size ranging from two to ten nonhydrogen atoms, and detects (as predictors of biological activity) those fragments statistically associated with the biological property investigated. Our previous work used the program to predict carcinogenicity in small rodents. In the current work we applied a modified version of the program, which bases its predictions solely on the most important fragment present in a given molecule, considering as practically negligible the effects of additional less important fragments. For Salmonella mutagenicity we used a database of 551 compounds, and the program achieved a level of predictivity (73.9%) comparable to that obtained by other authors using the Computer Automated Structure Evaluation (CASE) program. We evaluated the relative contributions of biophores and biophobes to overall predictivity: biophores tended to be more important than biophobes, and chemicals containing both biophores and biophobes were more difficult to predict. Many of the molecular fragments identified by the program as being strongly associated with mutagenic activity were similar to the structural alerts identified by the human experts Ashby and Tennant. Our results tend to confirm that structural alerts useful to predict Salmonella mutagenicity are generally not very strong predictors of rodent carcinogenicity. Although the predictivity level achieved for oncogenic activity improved when the program was directly trained with carcinogenicity data, carcinogenicity as a biological endpoint was still more difficult to predict than Salmonella mutagenicity.

Methods for predicting carcinogenic hazards: new opportunities coming from recent developments in molecular oncology and SAR studies

Without epidemiological evidence, and prior to either short-term tests of genotoxicity or long-term tests of carcinogenicity in rodents, an initial level of information about the carcinogenic hazard of a chemical that perhaps has been designed on paper, but never synthesized, can be provided by structure-activity relationship (SAR) studies. Herein, we have reviewed the interesting strategies developed by human experts and/or computerized approaches for the identification of structural alerts that can denote the possible presence of a carcinogenic hazard in a novel molecule. At a higher level of information, immediately below epidemiological evidence, we have discussed carcinogenicity experiments performed in new types of genetically engineered small rodents. If a dominant oncogene is already mutated, or if an allele of a recessive oncogene is inactivated, we have a model animal with (n-1) stages in the process of carcinogenesis. Both genotoxic and receptor-mediated carcinogens can induce cancers in 20-40% of the time required for classical murine strains. We have described the first interesting results obtained using these new artificial animal models for carcinogenicity studies. We have also briefly discussed other types of engineered mice (lac operon transgenic mice) that are especially suitable for detecting mutagenic effects in a broad spectrum of organs and tissues and that can help to establish mechanistic correlations between mutations and cancer frequencies in specific target organs. Finally, we have reviewed two complementary methods that, while obviously also feasible in rodents, are especially suitable for biomonitoring studies. We have illustrated some of the advantages and drawbacks related to the detection of DNA adducts in target and surrogate tissues using the 32P-DNA postlabeling technique, and we have discussed the possibility of biomonitoring mutations in different human target organs using a molecular technique that combines the activity of restriction enzymes with polymerase chain reaction (RFLP/PCR). Prediction of carcinogenic hazard and biomonitoring are very wide-ranging areas of investigation. We have therefore selected five different subfields for which we felt that interesting innovations have been introduced in the last few years. We have made no attempt to systematically cover the entire area: such an endeavor would have produced a book instead of a review article.

Molecular fragments associated with non-genotoxic carcinogens, as detected using a software program based on graph theory : their usefulness to predict carcinogenicity

We assembled 390 chemicals with a structure non-alerting to DNA-reactivity (145 carcinogens and 245 non-carcinogens) for which rodent carcinogenicity data were available. These non-alerting chemicals were defined by the absence in their molecules of DNA-reactive (directly or after metabolic activation) alerting structures, as described by Ashby and coworkers (Mutat. Res., 204 (1988) 17-115; Mutat. Res., 223 (1989) 73-103; Mutat. Res., 257 (1991) 209-227; Mutat. Res., 286 (1993) 3-74). Using our software program based on graph theory we analyzed the compounds in order to estimate the programs ability to predict nonalerting carcinogens. Our software fragmented the structural formula of the chemicals into all possible fragments of contiguous atoms with size between 2 and 8 (non-hydrogen) atoms and learned about statistically significant fragments from a training set of chemicals. These fragments were used to predict carcinogenicity or lack thereof in a verification set of compounds. For 390 runs of the software program we used (n - 1) of the chemicals as a training set, to predict the excluded chemical at each run (as a test set). Using two different probability thresholds to select significant fragments (P = 0.05 and P = 0.125 1-tailed according to binomial distribution), we performed two analyses: in the better one (P = 0.05) 19% of the molecules tested lacked significant fragments, for the remaining 81% the observed level of accuracy of the prediction was 66.0% against an expected level of accuracy of 51.7%. The difference was highly significant (P < 0.0001). We also examined the more significant activating fragments (biophores) and discussed at length both their biological plausibility and the working hypothesis that additional alerting structures for carcinogenicity (not only those related to genotoxicity) can be detected using this type of SAR approach. This new class of alerting structures could identify subfamilies of congeneric analogs active through mechanisms of receptor mediated carcinogenesis.

DNA aneuploidy relationship with patient age and tobacco smoke in OPMDs/OSCCs

The aim of this study was to investigate the relationship between tobacco smoke habit, patient age, DNA aneuploidy and genomic DNA copy number aberrations (CNAs) in oral potentially malignant disorder (OPMD) and oral squamous cell carcinoma (OSCC) patients. DNA aneuploidy was detected by high-resolution DNA flow cytometry (hr DNA-FCM) on DAPI stained nuclei obtained from multiple tissue samples from OPMDs/OSCCs in 220 consecutive patients. Nuclear genomic aberrations were determined in a subset of 65 patients by genome-wide array comparative genomic hybridization (aCGH) using DNA extracted from either diploid or aneuploid nuclei suspension sorted by FCM. DNA aneuploidy and mean nuclear genomic aberrations were associated with patients’ age. In particular, DNA aneuploidy strongly associated with age in non-smoker OPMDs/OSCCs patients. OSCCs from smokers showed a lower prevalence of DNA aneuploidy compared to OSCCs from non-smokers. A higher occurrence of DNA aneuploidy (particularly in smokers’ OPMDs) was observed in patients characterized by involvement of a single oral subsite. Our study suggests that: 1) DNA aneuploidy in non-smokers is mainly related to aging; 2) OPMDs/OSCCs involving multiple oral subsites in smokers are less likely to develop DNA aneuploidy compared to non-smokers; 3) OSCC development is characterized by both CIN and CIN-independent mechanisms and that the latter are more relevant in smokers. This study provides evidence that DNA diploid OPMDs may be considered at lower risk of cancerization than DNA aneuploid ones in non-smokers but not in smokers.

Innovative leads for antineoplastic drugs suggested by a more intimate familiarity with structural motifs of oncoproteins

Innovative leads for antineoplastic drugs suggested by a more intimate familiarity with structural motifs of oncoproteins

A decision support system for the prediction of carcinogenic activity of organic chemical compounds

A decision support system which analyses the topological properties of molecular fragments to predict carcinogenic activity of organic chemical compounds is described in this work. Such a system, given a set of organic compound of known carcinogenic activity, detects the submolecular structures that could interact with biological sites involved in the biological process analyzed. These fragments are then used to predict the activity of compounds of unknown biological behavior.<<ETX>>

Dose-Response Relationships for Benzene: Human and Experimental Carcinogenicity Data

In two previous papers we have discussed problems related to dose-response relationships for benzene in animals and humans.1,2