Targeting the tumor suppressor p53 to the mitochondria triggers a rapid apoptotic response as efficiently as transcription-dependent p53.1, 2 p53 forms a complex with the anti-apoptotic Bcl-XL, which leads to Bak and Bax oligomerization resulting in apoptosis via mitochondrial outer membrane permeabilization.3, 4 Although p53 performs its main role in the mitochondrial outer membrane it also interacts with different proteins in the mitochondrial inner membrane and matrix.5, 6 To further investigate mitochondrial activity of p53, EGFP-p53 was fused to different mitochondrial targeting signals (MTSs) directing it to the mitochondrial outer membrane (“XL-MTS” from Bcl-XL; “TOM-MTS” from TOM20), the inner membrane (“CCO-MTS” from cytochrome c oxidase) or matrix (“OTC-MTS” from ornithine transcarbamylase). Fluorescence microscopy and a p53 reporter dual luciferase assay demonstrated that fusing MTSs to p53 increased mitochondrial localization and nuclear exclusion depending on which MTS was used. To examine if the MTSs initiate mitochondrial damage, we fused each individual MTS to EGFP (a non-toxic protein) as negative controls. We performed caspase-9, TUNEL, Annexin-V, and 7-AAD apoptosis assays on T47D breast cancer cells transfected with mitochondrial constructs. Except for EGFP-XL, apoptotic potential was observed in all MTS-EGFP-p53 and MTS-EGFP constructs. In addition, EGFP-p53-XL showed the greatest significant increase in programmed cell death compared to its non-toxic MTS control (EGFP-XL). The apoptotic mechanism for each construct was further investigated using pifithrin-α (an inhibitor of p53 transcriptional activity), pifithrin-μ (a small molecule that reduces binding of p53 to Bcl-2 and Bcl-XL), and over-expressing the anti-apoptotic Bcl-XL. Unlike the MTSs from TOM, CCO, and OTC, which showed different apoptotic mechanisms, we conclude that p53 fused to the MTS from Bcl-XL performs its apoptotic potential exclusively through p53/Bcl-XL specific pathway.

The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).1,2 The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.3, 4 We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.5 A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homo-dimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homo-dimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications.

The current status of peptides that target the mitochondria in the context of cancer is the focus of this review. Chemotherapy and radiotherapy used to kill tumor cells are principally mediated by the process of apoptosis that is governed by the mitochondria. The failure of anticancer therapy often resides at the level of the mitochondria. Therefore, the mitochondrion is a key pharmacological target in cancer due to many of the differences that arise between malignant and healthy cells at the level of this ubiquitous organelle. Additionally, targeting the characteristics of malignant mitochondria often rely on disruption of protein-protein interactions that are not generally amenable to small molecules. We discuss anticancer peptides that intersect with pathological changes in the mitochondrion.The mitochondrion holds importance among cellular organelles in consideration of selective anticancer therapy because it is the nexus for propagating malignant transformation and controls cell death. The mitochondrion is a universal target in all cancer cells, and new information about functional and structural differences between healthy and malignant cells continues to emerge [1]. Peptides that specifically target malignant mitochondria offer advantages of low toxicity, high specificity and generally increase the range of interactions that are difficult to target with small molecules (e.g., protein-protein, protein-lipid and protein-DNA) [2]. However, the benefits of peptides are offset by difficulties in delivery, such as degradation by proteases and rapid clearance. As such, the delivery of peptide/ protein therapeutics is almost exclusively by the parenteral route, but the status quo is actively being challenged by broad efforts (e.g., liposomes, microparticles, nanoparticles, 'smart' polymers, hydrogels and chemical modifications to the peptide/protein) to achieve more convenient routes of administration (i.e., oral, nasal and transdermal) and improved pharmacokinetics [3][4][5]. However, peptides, in many cases, are far from passive in the delivery process and through the maze that is drug delivery (from route of administration to perhaps a target residing within a specific organelle), peptides are employed as the vehicle, homing motif and trans-/intra-cellular passport [6].There is an emerging view in cancer therapy that the way in which a cancer cell dies (i.e., immunogenic cell death) is important for a durable response [7]. However, traditional chemo therapy and immune response are in conflict because chemotherapy (e.g., DNA-© 2012 Future Science Ltd * Author for correspondence: Tel.: +1 801 581 7120 Fax: +1 801 585 3614 carol.lim@pharm.utah.edu. Financial & competing interests disclosureThe authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript....

Altering the subcellular localization of signal transducing proteins is a novel approach for therapeutic intervention. Mislocalization of tumor suppressors, oncogenes, or factors involved in apoptosis result in aberrant functioning of these proteins, leading to disease. In the case of chronic myelogenous leukemia (CML), cytoplasmic Bcr-Abl causes oncogenesis/proliferation. On the other hand, nuclear entrapment of endogenous Bcr-Abl (in K562 human leukemia cells) causes apoptosis. The goal of this study was to determine whether plasmid expressed Bcr-Abl could cause apoptosis of K562 cells when specifically directed to the nucleus via strong nuclear localization signals (NLSs). A single NLS from SV40 large T-antigen or four NLSs were subcloned to Bcr-Abl (1NLS-Bcr-Abl or 4NLS-Bcr-Abl). When transfected into K562 cells, only 4NLS-Bcr-Abl translocated to the nucleus. BcrAbl alone was found to localize in the cell cytoplasm, colocalizing with actin due to its actin binding domain. 1NLS-Bcr-Abl also localized with actin. Apoptosis induced by 4NLS-Bcr-Abl was evaluated 24 hours post-transfection by morphologic determination, DNA staining, and caspase-3 assay. This is the first demonstration that altering the location of plasmid expressed Bcr-Abl can kill leukemia cells. Multiple NLSs are required to overcome Bcr-Abl binding to actin, thus driving it into the nucleus and causing apoptosis.

Invasive fungal infections are a significant cause of morbidity and mortality in children. Successful management of these systemic infections requires identification of the causative pathogen, appropriate antifungal selection, and optimisation of its pharmacokinetic and pharmacodynamic properties to maximise its antifungal activity and minimise toxicity and the emergence of resistance. This review highlights salient scientific advancements in paediatric antifungal pharmacotherapies and focuses on pharmacokinetic and pharmacodynamic studies that underpin current clinical decision making. Four classes of drugs are widely used in the treatment of invasive fungal infections in children, including the polyenes, triazoles, pyrimidine analogues and echinocandins. Several lipidic formulations of the polyene amphotericin B have substantially reduced the toxicity associated with the traditional amphotericin B formulation. Monotherapy with the pyrimidine analogue flucytosine rapidly promotes the emergence of resistance and cannot be recommended. However, when used in combination with other antifungal agents, therapeutic drug monitoring of flucytosine has been shown to reduce high peak flucytosine concentrations, which are strongly associated with toxicity. The triazoles feature large inter-individual pharmacokinetic variability, although this pattern is less pronounced with fluconazole. In clinical trials, posaconazole was associated with fewer adverse effects than other members of the triazole family, though both posaconazole and itraconazole display erratic absorption that is influenced by gastric pH and the gastric emptying rate. Limited data suggest that the clinical response to therapy may be improved with higher plasma posaconazole and itraconazole concentrations. For voriconazole, pharmacokinetic studies among children have revealed that children require twice the recommended adult dose to achieve comparable blood concentrations. Voriconazole clearance is also affected by the cytochrome P450 (CYP) 2C19 genotype and hepatic impairment. Therapeutic drug monitoring is recommended as voriconazole pharmacokinetics are highly variable and small dose increases can result in marked changes in plasma concentrations. For the echinocandins, the primary source of pharmacokinetic variability stems from an age-dependent decrease in clearance with increasing age. Consequently, young children require larger doses per kilogram of body weight than older children and adults. Routine therapeutic drug monitoring for the echinocandins is not recommended. The effectiveness of many systemic antifungal agents has been correlated with pharmacodynamic targets in in vitro and in murine models of invasive candidiasis and aspergillosis. Further study is needed to translate these findings into optimal dosing regimens for children and to understand how these agents interact when multiple antifungal agents are used in combination.