Guido Cimoli

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.

Sister-chromatid exchanges, chromosomal aberrations and cytotoxicity produced by topoisomerase II-targeted drugs in sensitive (A2780) and resistant (A2780-DX3) human ovarian cancer cells: Correlations with the formation of DNA double-strand breaks

Doxorubicin, ellipticine and etoposide are antineoplastic drugs with topoisomerase II inhibitory activity. The relationship between drug-induced sister-chromatid exchanges (SCEs) or chromosomal aberrations (CAs) and cytotoxicity, or drug-induced DNA double-strand breaks (DSBs) and cytotoxicity, or drug-induced SCEs and DSBs was investigated in human ovarian cancer cells sensitive (A2780) and resistant (A2780-DX3) to topoisomerase II inhibitors. 30-min drug treatments produced SCEs, CAs and DSBs in sensitive cells, doxorubicin being more potent than etoposide at equimolar concentrations. The same treatments of resistant (A2780-DX3) cells did not produce chromosomal damage (SCEs, CAs, DSBs) and no cytotoxicity was observed. A plot of cytotoxicity versus SCEs indicated a good correlation between these two parameters for topoisomerase II inhibitors and not for mytomicin C. The plot of DSBs versus SCEs also showed a very good correlation.

Effect of recombinant human tumor necrosis factor on A2774 human ovarian cancer cell line: Potentiation of mitoxantrone cytotoxicity

Recombinant human tumor necrosis factor (rHuTNF) is a macrophage-secretory protein with antitumor activity. In vivo and in vitro cytotoxicity studies have been carried out to test the effectiveness of rHuTNF alone or in combination with chemotherapeutic agents. We have evaluated the direct cytotoxic effect of rHuTNF on a human epithelial ovarian cancer cell line in vitro (A2774), alone, or in combination with mitoxantrone (Mit), a topoisomerase II (Topo II) targeted drug. Our results not only suggest that rHuTNF is directly cytotoxic, but also that it is able to induce a very strong potentiation of Mit cytotoxicity.

Interactions between taxol and camptothecin.

Taxol is an antitumor drug which, as its mechanism of action, promotes microtubule assembly in vitro. Camptothecin (CPT) is an anticancer agent with the peculiar mechanism of poisoning eukaryotic DNA topoisomerase I. Both drugs are in clinical trials and their chemotherapeutic efficacy seems promising in refractory human ovarian cancer. We studied the molecular and cellular pharmacology of the two drugs when administered simultaneously toward human ovarian cancer cell line A2780. Taxol inhibits CPT-induced single-strand breaks as well as CPT-induced cytotoxicity. Taken together, our experiments indicate that the two drugs might interact with the same class of nuclear enzyme, i.e. DNA topoisomerase I.

Interferon-α or β potentiate platinum analogous in human glioblastoma cell lines

Abstract The effect of interferon-α or β on platinum analogues [cisplatin (CDDP) and carboplatin] cytotoxicity was studied in four glioblastoma cell lines (U373MG, T98G, A172 and U118Mg). All cell lines were strongly resistant to the cytotoxic effect of CDDP or carboplatin. Although both interferons were not cytotoxic in all cell lines, they were able to significantly increase the cell platinum-sensitivity. Specifically interferon-α increased the magnitude of CDDP-induced DNA interstrand crosslinks Our findings suggest that interferons are able to induce a very strong potentiation of platinum analogues cytotoxicity in drug-resistant human glioma cell lines

“Atypical” multidrug resistance in human ovarian cancer cell line A2780 selected for resistance to doxorubicin (A2780 DX3)

Human ovarian cancer cells A2780, selected for resistance to doxorubicin (A2780-DX3), are crossresistant to various other topoisomerase-II-targeted drugs but not to vinblastine. The parental cell line was very sensitive to doxorubicin-, mitoxantrone- or etoposide (VP16)-induced DNA single-strand breaks, under deproteinizing conditions. In contrast, little or no DNA strand breakage was seen in resistant A2780-DX3 cells, even at very high concentrations, indicating a good correlation, with cytotoxicity. No significant alterations in cellular drug uptake were observed in DX3 cells. Further studies showed that the nuclei isolated from resistant cells were also resistant to mitoxantroneor VP16-induced single-strand breaks, indicating that nuclear modifications in resistant cells are responsible for this resistance. Catalytic activity in crude nuclear extracts from wild-type and DX3 cells was almost equal. However, an assay that specifically measures generation of 5′-protein-linked breaks in32P-labeled 3 DNA revealed that, DNA cleavage activity in nuclear extract from the DX3 cell line is profoundly resistant to a stimulation by VP16. These data indicate that stimulation of topoisomerase-II-mediated DNA cleavage is responsible for topoisomerase-II-targeted drugcytotoxicity rather than loss of normal topoisomerase catalytic function. These data support the hypothesis that A2780-DX3 cells display an “atypical” multidrug resistance.