Applications of Cell-Penetrating Peptides as Signal Transduction Modulators for the Selective Induct

The discovery of cell-penetrating peptides (CPP) has provided the scientific community with relatively small and increasingly cost-effective molecular agents that readily cross the normally impermeable cell membrane. Thus, as either inert delivery vectors or biologically active agents, CPP can be used to selectively modulate intracellular signal transduction events. Indeed, the survival of many cancer cells is associated with alterations in the function of key intracellular signalling proteins. Accordingly, CPP constructs have been developed to access intracellular target loci in both normal and transformed cells. Thus, CPP are a novel, generic class of signal transduction modulator which can be utilized to specifically induce apoptosis in tumour cells as a potential therapeutic option. However, and particularly at higher concentrations, CPP can induce non-specific membrane perturbations, thus leading to cell death by necrotic mechanisms. This chapter, therefore, focuses on methodologies for the assessment of apoptotic events, including in situ TUNEL analysis, activation of caspase-3, and the MTT assay, whilst also discussing dual Annexin V and propidium iodide staining, an assay used for the quantification of cell populations undergoing apoptosis and/or necrosis.