Gene/Protein Disease Symptom Drug Enzyme Compound
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Cell penetrating peptides (CPPs) have tremendous potential for use in gene and drug delivery applications. The selection of new CPPs with desired capabilities from randomized peptide libraries is challenging, since the CPP phenotype is a complex selection target. Here we report the discovery of an unusual new CPP from a randomized peptide library using a functional selection system based on plasmid display (PD). After four rounds of screening of a 14-mer peptide library over PC12 cells, several peptides were identified and tested for their ability to deliver the green fluorescent protein (GFP). One peptide (SG3) exhibited a cell penetrating phenotype; however, unlike other well-known CPPs such as TAT or Penetratin, the newly identified peptide was not highly cationic. The PD protocol necessitated the addition of a cationic lipid (Lipofectamine2000), and in the presence of this compound, the SG3 peptide significantly outperformed the well-known TAT CPP in the delivery of GFP to PC12 cells and primary astrocytes. When the SG3 peptide was fused to the pro-apoptotic BH3 peptide from the Bak protein, significant cell death was induced in cultured primary astrocytes, indicating relevant, intracellular delivery of a functional cargo. The PD platform is a useful method for identifying functional new CPPs from randomized libraries with unique delivery capabilities.
ACS Chem Biol 2011 May 20
PMID:An unusual cell penetrating peptide identified using a plasmid display-based functional selection platform. 2129 Dec 71

The rapid developments in nanotechnology have brought with them a deep concern over the safety of nanomaterials. Investigating the molecular mechanisms underlying their toxicity in different cell lines will help us better understand and apply nanomaterials appropriately. Poly(ethylene glycol)-phosphoethanolamine (PEG-PE) is an FDA-approved nonionic diblock copolymer and is widely used in drug delivery systems. Here, we find that PEG-PE accumulates in the endoplasmic reticulum (ER) and induces ER stress and that cancer cells and normal cells have different cell fates as a result of this stress. In A549 cancer cells, PEG-PE damages ER functions and triggers apoptosis by activating proapoptotic UPR signaling and high expression of cell death effector CHOP and proapoptotic Bax/Bak. In addition, PEG-PE-induced ER stress also up-regulates lipid synthesis and triggers lipid droplet formation in cancer cells. By contrast, in MRC-5 and 293T cells, high expression of the UPR feedback protein GADD34 which inhibits proapoptotic UPR signaling, and antiapoptotic Bcl-2 and Bcl-xl which down-regulate Bax/Bak, protect these normal cells from PEG-PE-induced apoptosis. When gadd34, bcl-2, or bcl-xl is knocked down, apoptosis occurs in PEG-PE-treated normal cells. In summary, we demonstrate the safety of PEG-PE in normal cells and elaborate the molecular mechanism underlying its nanotoxicity in cancer cells. This study implies PEG-PE-based drug delivery system has the potential to alter the sensitivity of cancer cells to some chemotherapeutic agents by selectively activating unfolded protein response (UPR) in cancer cells, and it also provides a useful foundation for research on ER stress-induced nanotoxicity and other lipid-based nanomaterials.
ACS Nano 2012 Jun 26
PMID:Pegylated phospholipid micelles induce endoplasmic reticulum-dependent apoptosis of cancer cells but not normal cells. 2257 58

The Bcl-2 family of proteins regulates apoptosis at the level of mitochondrial permeabilization. Pro-death members of the family, including Bak and Bax, initiate apoptosis, whereas pro-survival members such as Bcl-x(L) and Mcl-1 antagonize the function of Bak and Bax via heterodimeric interactions. These heterodimeric interactions are primarily mediated by the binding of the helical amphipathic BH3 domain from a pro-death protein to a hydrophobic cleft on the surface of the pro-survival protein. Since high levels of pro-survival Bcl-2 proteins are present in many cancers, peptides corresponding to pro-death BH3 domains hold promise as therapeutics. Here we apply a high-throughput flow cytometry assay to engineer the Bak BH3 domain for improved affinity toward the pro-survival proteins Bcl-x(L) and Mcl-1. Two strategies, engineering the hydrophobic face of the Bak BH3 peptide and increasing its overall helicity, are successful in identifying Bak BH3 variants with improved affinity to Bcl-x(L) and Mcl-1. Hydrophobic face engineering of the Bak BH3 peptide led to variants with up to a 15-fold increase in affinity for Bcl-x(L) and increased specificity toward Bcl-x(L). Engineering of the helicity of Bak BH3 led to modest (3- to 4-fold) improvements in affinity with retention of promiscuous binding to all pro-survival proteins. HeLa cell killing studies demonstrate that the affinity matured Bak BH3 variants retain their expected biological function.
ACS Synth Biol 2012 Mar 16
PMID:A comparison of two strategies for affinity maturation of a BH3 peptide toward pro-survival Bcl-2 proteins. 2365 Oct 73

The Bcl-2 family of proteins is crucial for apoptosis regulation. Members of this family insert through a specific C-terminal anchoring transmembrane domain (TMD) in the mitochondrial outer membrane where they hierarchically interact to determine cell fate. While the mitochondrial membrane has been proposed to actively participate in these protein-protein interactions, the influence of the TMD in the membrane-mediated interaction is poorly understood. Synthetic peptides (TMD-pepts) corresponding to the putative TMD of antiapoptotic (Bcl-2, Bcl-xL, Bcl-w, and Mcl-1) and pro-apoptotic (Bax, Bak) members were synthesized and characterized. TMD-pepts bound more efficiently to mitochondria-like bilayers than to plasma membrane-like bilayers, and higher binding correlated with greater membrane perturbation. The Bcl-2 TMD peptides promoted mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release from isolated mitochondria and different cell lines. TMD-pepts exhibited nonapoptotic pro-death activity when apoptosis stimuli were absent. In addition, the peptides enhanced the apoptotic pathway induced by chemotherapeutic agents in cotreatment. Overall, the membrane perturbation effects of the TMD-pepts observed in the present study open the way for their use as new chemical tools to sensitize tumor cells to chemotherapeutic agents, in accordance with the concept of mitochondria priming.
ACS Chem Biol 2014 Aug 15
PMID:Peptides derived from the transmembrane domain of Bcl-2 proteins as potential mitochondrial priming tools. 2490 60

The photothermal response of plasmonic nanomaterials can be exploited for a number of biomedical applications in diagnostics (biosensing and optoacoustic imaging) and therapy (drug delivery and photothermal therapy). The most common cellular response to photothermal cancer treatment (ablation of solid tumors) using plasmonic nanomaterials is necrosis, a process that releases intracellular constituents into the extracellular milieu producing detrimental inflammatory responses. Here we report the use of laser-induced photothermal therapy employing gold nanoprisms (NPRs) to specifically induce apoptosis in mouse embryonic fibroblast cells transformed with the SV40 virus. Laser-irradiated "hot" NPRs activate the intrinsic/mitochondrial pathway of apoptosis (programmed cell death), which is mediated by the nuclear-encoded proteins Bak and Bax through the activation of the BH3-only protein Bid. We confirm that an apoptosis mechanism is responsible by showing how the NPR-mediated cell death is dependent on the presence of caspase-9 and caspase-3 proteins. The ability to selectively induce apoptotic cell death and to understand the subsequent mechanisms provides the foundations to predict and optimize NP-based photothermal therapy to treat cancer patients suffering from chemo- and radioresistance.
ACS Nano 2015 Jan 27
PMID:Dissecting the molecular mechanism of apoptosis during photothermal therapy using gold nanoprisms. 2786 19

Near-infrared light is an attractive stimulus due to its noninvasive and deep tissue penetration. Particularly, NIR light is utilized for cancer thermotherapy and on-demand release of drugs by the disruption of the delivery carriers. Here we have prepared a novel NIR-responsive DNA-hybrid-gated nanocarrier based on mesoporous silica-coated Cu1.8S nanoparticles. Cu1.8S nanoparticles, possessing high photothermal conversion efficiency under a 980 nm laser, were chosen as photothermal agents. The mesoporous silica structure could be used for drug storage/delivery and modified with aptamer-modified GC-rich DNA-helix as gatekeepers, drug vectors, and targeting ligand. Simultaneously, the as-produced photothermal effect caused denaturation of DNA double strands, which triggered the drug release of the DNA-helix-loaded hydrophilic drug doxorubicin and mesopore-loaded hydrophobic drug curcumin, resulting in a synergistic therapeutic effect. The Cu1.8S@mSiO2 nanocomposites endocytosed by cancer cells through the aptamer-mediated mode are able to generate rational release of doxorubicin/curcumin under NIR irradiation, strongly enhancing the synergistic growth-inhibitory effect of curcumin against doxorubicin in MCF-7 cells, which is associated with a strong mitochondrial-mediated cell apoptosis progression. The underlying mechanism of apoptosis showed a strong synergistic inhibitory effect both on the expression of Bcl-2, Bcl-xL, Mcl-1, and upregulated caspase 3/9 activity and on the expression level of Bak and Bax. Therefore, Cu1.8S@mSiO2 with efficient synergistic therapeutic efficiency is a potential multifunctional cancer therapy nanoplatform.
ACS Appl Mater Interfaces 2015 Sep 23
PMID:DNA-Hybrid-Gated Photothermal Mesoporous Silica Nanoparticles for NIR-Responsive and Aptamer-Targeted Drug Delivery. 2632 85