Mauro Castelli

Resveratrol exhibits a strong cytotoxic activity in cultured cells and has an antiviral action against polyomavirus: potential clinical use

BackgroundResveratrol is a non flavonoid polyphenol compound present in many plants and fruits and, at especially high concentrations, in the grape berries of Vitis vinifera. This compound has a strong bioactivity and its cytoprotective action has been demonstrated, however at high concentrations the drug exhibits also an effective anti-proliferative action. We recently showed its ability to abolish the effects of oxidative stress in cultured cells. In this work we assayed the bioactivity of resveratrol as antiproliferative and antiviral drug in cultured fibroblasts. Studies by other Authors showed that this natural compound inhibits the proliferation of different viruses such as herpes simplex, varicella-zoster and influenza A. The results presented here show an evident toxic activity of the drug at high concentrations, on the other hand at sub-cytotoxic concentrations, resveratrol can effectively inhibit the synthesis of polyomavirus DNA. A possible interpretation is that, due to the damage caused by resveratrol to the plasma membrane, the transfer of the virus from the endoplasmic reticulum to the nucleus, may be hindered thus inhibiting the production of viral DNA.MethodsThe mouse fibroblast line 3T6 and the human tumor line HL60 were used throughout the work. Cell viability and vital cell count were assessed respectively, by the MTT assay and Trypan Blue staining. Cytotoxic properties and evaluation of viral DNA production by agarose gel electrophoresis were performed according to standard protocols.ResultsOur results show a clear dose dependent both cytotoxic and antiviral effect of resveratrol respectively at high and low concentrations. The cytotoxic action is exerted towards a stabilized cell-line (3T6) as well as a tumor-line (HL60). Furthermore the antiviral action is evident after the phase of virion entry, therefore data suggest that the drug acts during the synthesis of the viral progeny DNA.ConclusionResveratrol is cytotoxic and inhibits, in a dose dependent fashion, the synthesis of polyomavirus DNA in the infected cell. Furthermore, this inhibition is observed at non cytotoxic concentrations of the drug. Our data imply that cyto-toxicity may be attributed to the membrane damage caused by the drug and that the transfer of polyomavirus from the endoplasmic reticulum to the cytoplasm may be hindered. In conclusion, the cytotoxic and antiviral properties of resveratrol make it a potential candidate for the clinical control of proliferative as well as viral pathologies.

Identification of genes down-regulated during lung cancer progression: A cDNA array study

BackgroundLung cancer remains a major health challenge in the world. Survival for patients with stage I disease ranges between 40–70%. This suggests that a significant proportion of patients with stage I NSCLC may actually be under-staged.MethodsIn order to identify genes relevant for lung cancer development, we carried out cDNA array experiments employing 64 consecutive patients (58 men and 6 women) with a median age of 58 years and stage 1 or stage 2 non-small-cell lung cancer (NSCLC).ResultsBasic cDNA array data identified 14 genes as differentially regulated in the two groups. Quantitative RT-PCR analysis confirmed an effective different transcriptional regulation of 8 out of 14 genes analyzed. The products of these genes belong to different functional protein types, such as extra-cellular matrix proteins and proteases (Decorin and MMP11), genes involved in DNA repair (XRCC1), regulator of angiogenesis (VEGF), cell cycle regulators (Cyclin D1) and tumor-suppressor genes (Semaphorin 3B, WNT-5A and retinoblastoma-related Rb2/p130). Some previously described differences in expression patterns were confirmed by our array data. In addition, we identified and validated for the first time the reduced expression level of some genes during lung cancer progression.ConclusionComparative hybridization by means of cDNA arrays assisted in identifying a series of novel progression-associated changes in gene expression, confirming, at the same time, a number of previously described results.

Preclinical evaluation of KIT/PDGFRA and mTOR inhibitors in gastrointestinal stromal tumors using small animal FDG PET

BackgroundPrimary and secondary drug resistance to imatinib and sunitinib in patients with gastrointestinal stromal tumors (GISTs) has led to a pressing need for new therapeutic strategies such as drug combinations. Most GISTs are caused by mutations in the KIT receptor, leading to upregulated KIT tyrosine kinase activity. Imatinib and nilotinib directly inhibit the kinase activity of KIT, while RAD001 (everolimus) inhibits mTOR. We report a preclinical study on drug combinations in a xenograft model of GIST in which effects on tumor dimensions and metabolic activity were assessed by small animal PET imaging.MethodsRag2-/-; γcommon -/- male mice were injected s.c. into the right leg with GIST 882. The animals were randomized into 6 groups of 6 animals each for different treatment regimens: No therapy (control), imatinib (150 mg/kg b.i.d.) by oral gavage for 6 days, then once/day for another 7 days, everolimus (10 mg/kg/d.) by oral gavage, everolimus (10 mg/kg/d.) + imatinib (150 mg/kg b.i.d.) by oral gavage for 6 days, then once/day for another 7 days, nilotinib (75 mg/kg/d.) by oral gavage, nilotinib (75 mg/kg/d.) + imatinib (150 mg/kg b.i.d) by oral gavage for 6 days, then once/day for another 7 days. Tumor growth control was evaluated by measuring tumor volume (cm3). Small animal PET (GE Explore tomography) was used to evaluate tumor metabolism and performed in one animal per group at base-line then after 4 and 13 days of treatment.ResultsAfter a median latency time of 31 days, tumors grew in all animals (volume 0,06-0,15 cm3) and the treatments began at day 38 after cell injection. Tumor volume control (cm3) after 13 days of treatment was > 0.5 for imatinib alone and nilotinib alone, and < 0.5 for the 2 combinations of drugs and for everolimus alone. The baseline FDG uptake was positive in all animals. FDG/SUV/TBR was strongly reduced over time by everolimus both as a single agent and in combination with imatinib respectively: 3.1 vs. 2.3 vs. 1.9 and 2.5 vs 2.3 vs 0.ConclusionsAs single agents, all drugs showed an anti-tumor effect in GIST xenografts but everolimus was superior. The everolimus plus imatinib combination appeared to be the most active regimen both in terms of inhibiting tumor growth and tumor metabolism. The integration of everolimus in GIST treatment merits further investigation.

Immunohistochemical evaluation of inflammatory and proliferative markers in adjacent normal thyroid tissue in patients undergoing total thyroidectomy: results of a preliminary study

BackgroundTotal thyroidectomy is the treatment of choice in the majority of thyroid malignancies, preventing the risk of reoperative surgery due to recurrences. In order to assess the usefulness of such an approach, expression levels of inflammatory and proliferative markers were evaluated immunohistochemically in non-lesional adjacent thyroid tissues from a group of patients who underwent total thyroidectomy for different thyroid diseases.MethodsNineteen consecutive patients treated by total thyroidectomy for different thyroid diseases entered the study. IL-6Rb gp130 component of the IL-6 cytokine family members receptor complexes, STAT3 cytokine signalling transduction and transcription activation factor, p53 as tumour suppressor and CK19 cytokeratin as proliferation marker were analyzed in non-lesional thyroid tissues.ResultsGp 130 expression was detected in all tissue samples with a scattered distribution while STAT3 and p53 positivity was observed in 17 out of 19 patients with a prevailing cytoplasmic localization. Cytokeratin 19 positivity was found in patients with papillary carcinoma, in one case of follicular adenoma, 3 multinodular goiters and one Basedow disease.ConclusionBased on the results of this preliminary study, it may be concluded that the presence of a persisting cytokine-mediated activation associated with cytoplasmic localization of p53 is frequently observed in different thyroid diseases. Such a process seems to occur in the thyroid gland as a whole. Moreover, STAT3 activation as well as mutant p53 are risk factors for the development of neoplastic diseases. Total thyroidectomy may be supported as an adequate therapeutic approach for all the patients in whom overexpression of cytokine-dependent markers is detected.

The synthetic inhibitor of Fibroblast Growth Factor Receptor PD166866 controls negatively the growth of tumor cells in culture

BackgroundMany experimental data evidence that over-expression of various growth factors cause disorders in cell proliferation. The role of the Fibroblast Growth Factors (FGF) in growth control is indisputable: in particular, FGF1 and its tyrosine kinase receptor (FGFR1) act through a very complex network of mechanisms and pathways. In this work we have evaluated the antiproliferative activity effect of PD166866, a synthetic molecule inhibiting the tyrosin kinase action of FGFR1.MethodsCells were routinely grown in Dulbecco Modified Eagles medium supplemented with newborn serum and a penicillin-streptomycin mixture.Cell viability was evaluated by Mosmann assay and by trypan blue staining. DNA damage was assessed by in situ fluorescent staining with Terminal Deoxynucleotidyl Transferase dUTP nick end labeling (TUNEL assay).Assessment of oxidative stress at membrane level was measured by quantitative analysis of the intra-cellular formation of malonyl-dialdheyde (MDA) deriving from the decomposition of poly-unsaturated fatty acids.The expression of Poly-ADP-Ribose-Polymerase (PARP), consequent to DNA fragmentation, was evidenced by immuno-histochemistry utilizing an antibody directed against an N-terminal fragment of the enzyme.ResultsThe bioactivity of the drug was investigated on Hela cells. Cytoxicity was assessed by the Mosmann assay and by vital staining with trypan blue. The target of the molecule is most likely the cell membrane as shown by the significant increase of the intracellular concentration of malonyl-dihaldheyde. The increase of this compound, as a consequence of the treatment with PD166866, is suggestive of membrane lipoperoxidation. The TUNEL assay gave a qualitative, though clear, indication of DNA damage. Furthermore we demonstrate intracellular accumulation of poly-ADP-ribose polymerase I. This enzyme is a sensor of nicks on the DNA strands and this supports the idea that treatment with the drug induces cell death.ConclusionsData presented in this work show that PD166866 has clear antiproliferative effects. The negative control of cell proliferation may be exerted through the activation of the apoptotic pathway. The results of experiments addressing this specific point, such as: evaluation of DNA damage, lipoperoxidation of the cell membrane and increase of expression of PARP, an enzyme directly involved in DNA repair. Results suggest that cells exposed to PD16866 undergo apoptosis. However, concomitant modes of cell death cannot be ruled out. The possible use of this drug for therapeutic purposes is discussed.