ABSTRACT
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-X(L), 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-X(L), 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.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Membrane Proteins/metabolism , Peptides/metabolism , Proto-Oncogene Proteins/metabolism , bcl-X Protein/antagonists & inhibitors , bcl-X Protein/metabolism , Amino Acid Sequence , Animals , Apoptosis/physiology , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Cells, Cultured , Female , Humans , Leukemia, Myeloid, Acute/metabolism , Lymphocytes/cytology , Lymphocytes/physiology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Sequence Data , Neoplasm Transplantation , Peptides/chemistry , Peptides/genetics , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Transplantation, Heterologous , Tumor Cells, Cultured , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , bcl-X Protein/geneticsABSTRACT
The Bcl-2 family of antiapoptotic proteins is commonly over expressed in many types of human cancer and remains one of the few validated targets. Antiapoptotic family proteins such as Bcl-2 and Bcl-XL function, at least in part, by binding proapoptotic members such as Bax and Bak and thereby prevent release of the apoptotic cascade of events. "BH3-only" members of the family disrupt this interaction by binding, via their BH3 domain, to a hydrophobic pocket on the surface of the antiapoptotic members. Disruption of heterodimerization could be used to modulate cell death reinstating apoptosis in cancer cells. An affinity displacement assay based on Bcl-XL/BH3 interaction has been developed. This assay makes use of soluble His-tagged Bcl-XL and fluorescein tagged BH3. Binding is measured as fluorescence associated with magnetic beads. The assay was miniaturized to 96-well microtiter plates and can be employed in high throughput screening (HTS), in addition it is robust enough to be applied to microbial fermentation extracts.
Subject(s)
Anti-Bacterial Agents/pharmacology , bcl-X Protein/antagonists & inhibitors , Anti-Bacterial Agents/isolation & purification , Apoptosis/drug effects , Bacteria/chemistry , Binding, Competitive , Biological Assay , Dimerization , Drug Design , Fluorescein , Fluorescent Dyes , Fungi/chemistry , Histidine , Humans , In Vitro Techniques , Ligands , Peptide Fragments , Proto-Oncogene Proteins , Recombinant Proteins/antagonists & inhibitors , Reproducibility of Results , Spectrometry, Fluorescence/methods , bcl-X Protein/genetics , bcl-X Protein/metabolismABSTRACT
The role of p53 in modifying sensitivity to cytotoxic drugs has been commonly studied by creating transfection pairs of wt p53 parental cells and altered p53 daughter cells, or vice versa. Authors inevitably tended to extrapolate and generalize their experimental observations, and conflicting reports have been more the rule than the exception. We have performed a meta-analysis of 356 independent studies. Average changes of drug sensitivity after a change of p53 status were observed. E6 transfection predominantly induces sensitization to cytotoxic drugs, whereas p53-/- knockout cells are more drug-resistant than their normal p53+/+ counterpart. Unexpectedly, transfection with a mutated p53 does not change much the drug sensitivity of most wt p53 cancer lines, with the notable exception of A2780, a predominant cell line in the studies analyzed (A2780 cells show increased resistance after transfection with a mutated p53). Rather interestingly, mitotic spindle poisons acted differently from other classes of cytotoxic drugs. A crucial indication of our findings is that the role of p53 alone in determining sensitivity/resistance to cytotoxic drugs is limited: the individual molecular pathology and differentiation of a given cancer line prevail over any average trend, and are causal to a broad spreading of the data. We also identify major "confounding factors", alias independent categorical variables, capable of affecting the average outcome.
