ABSTRACT
Nuclear localization signals are short amino acid sequences that target proteins for nuclear import. In this manuscript, we have generated a chimeric tri-functional peptide composed of a cell penetrating peptide (CPP), a nuclear localization sequence and an interfering peptide blocking the interaction between TEAD and YAP, two transcription factors involved in the Hippo signalling pathway, whose deregulation is related to several types of cancer. We have validated the cell penetration and nuclear localization by flow cytometry and fluorescence microscopy and shown that the new generated peptide displays an apoptotic effect in tumor cell lines thanks to the specific nuclear delivery of the cargo, which targets a protein/protein interaction in the nucleus. In addition, the peptide has an anti-tumoral effect in vivo in xenograft models of breast cancer. The chimeric peptide designed in the current study shows encouraging prospects for developing nuclear anti- neoplastic drugs.
Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Breast Neoplasms/drug therapy , DNA-Binding Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Peptides/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Nucleus/metabolism , Drug Delivery Systems , Female , Hippo Signaling Pathway , Humans , Male , Mice , Mice, Inbred C3H , Nuclear Localization Signals/metabolism , Nuclear Proteins/metabolism , Protein Transport/drug effects , Signal Transduction/drug effects , TEA Domain Transcription Factors , Xenograft Model Antitumor Assays , YAP-Signaling ProteinsABSTRACT
Apoptotic signaling pathways are altered in numerous pathologies such as cancer. In this scenario, caspase-9/PP2Acα interaction constitutes a key target with pharmacological interest to re-establish apoptosis in tumor cells. Very recently, a short peptide (C9h) known to disrupt caspase-9/PP2Acα interaction with subsequent apoptosis induction was described. Here, we prepared two sets of mesoporous silica nanoparticles loaded with safraninâ O (S2) or with C9h peptide (S4) and functionalized with ϵ-polylysine as capping unit. Aqueous suspensions of both nanoparticles showed negligible cargo release whereas in the presence of pronase, a marked delivery of safraninâ O or C9h was observed. Confocal microscopy studies carried out with HeLa cells indicated that both materials were internalized and were able to release their entrapped cargos. Besides, a marked decrease in HeLa cell viability (ca. 50 %) was observed when treated with C9h-loaded S4 nanoparticles. Moreover, S4 provides peptide protection from degradation additionally allowing for a dose reduction to observe an apoptotic effect when compared with C9h alone or in combination with a cell-penetrating peptide (i.e., Mut3DPT-C9h). Flow cytometry studies, by means of Annexin V-FITC staining, showed the activation of apoptotic pathways in HeLa as a consequence of S4 internalization, release of C9h peptide and disruption of caspase-9/PP2Acα interaction.
Subject(s)
Nanoparticles/chemistry , Peptides/chemistry , Polylysine/chemistry , Silicon Dioxide/chemistry , Amino Acid Sequence , Apoptosis/drug effects , Caspase 9/chemistry , Caspase 9/metabolism , Circular Dichroism , Drug Carriers/chemical synthesis , Drug Carriers/chemistry , HeLa Cells , Humans , Microscopy, Confocal , Peptides/toxicity , Phenazines/chemistry , Phenazines/toxicity , Porosity , Protein Phosphatase 2/chemistry , Protein Phosphatase 2/metabolismABSTRACT
BACKGROUND: Disruption of alternative splicing in apoptotic factors has been associated to chronic lymphocytic leukemia among other cancers and hematological malignancies. The proapoptotic proteins Caspase-9 and PP2Acα are functionally related in a direct interaction, which constitutes a promising target for cancer therapy. Both proteins present aberrant mRNA splicing variants that are antiapoptotic (Caspase-9b) and catalytically inactive (PP2Acα2), respectively. RESULTS: In this work we have analyzed the relative abundance of the aberrant spliced forms Caspase-9b and PP2Acα2 in several cell lines and chronic lymphocytic leukemia patients and correlated it with several parameters of the disease. Despite 40 % of the patients presented Caspase-9b dysregulation, there was no direct association between alterations in Caspase-9b relative abundance and the parameters analyzed in medical records. More importantly, PP2Acα2 dysregulation was observed in 88 % of CLL patients and was related with advanced stages of the malignancy. CONCLUSIONS: Caspase-9b dysregulation seemed to be associated with the disease, although the differences between healthy donors and CLL patients were not statistically significant. However, PP2Acα2 dysregulation was significantly different between healthy donors and CLL patients and correlated with Binet B and C stages; therefore, we propose the use of PP2Acα2 dysregulation as a potential biomarker for advanced stages of chronic lymphocytic leukemia.