Ming An

pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation.

We find that pH-(low)-insertion-peptide (pHLIP)-facilitated translocation of phalloidin, a cell-impermeable polar toxin, inhibits the proliferation of cancer cells in a pH-dependent fashion. The monomeric pHLIP inserts its C terminus across a membrane under slightly acidic conditions (pH 6–6.5), forming a transmembrane helix. The delivery construct carries phalloidin linked to its inserting C terminus via a disulfide bond that is cleaved inside cells, releasing the toxin. To facilitate delivery of the polar agent, a lipophilic rhodamine moiety is also attached to the inserting end of pHLIP. After a 3 h incubation at pH 6.1–6.2 with 2–4 μM concentrations of the construct, proliferation in cultures of HeLa, JC, and M4A4 cancer cells is severely disrupted (> 90% inhibition of cell growth). Treated cells also show signs of cytoskeletal immobilization and multinucleation, consistent with the expected binding of phalloidin to F actin, stabilizing the filaments against depolymerization. The antiproliferative effect was not observed without the hydrophobic facilitator (rhodamine). The biologically active delivery construct inserts into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid bilayers with an apparent pKa of ∼6.15, similar to that of the parent pHLIP peptide. Sedimentation velocity experiments show that the delivery construct is predominantly monomeric (> 90%) in solution under the conditions employed to treat cells (pH 6.2, 4 μM). These results provide a lead for antitumor agents that would selectively destroy cells in acidic tumors. Such a targeted approach may reduce both the doses needed for cancer chemotherapy and the side effects in tissues with a normal pH.

pHLIP-Mediated Translocation of Membrane-Impermeable Molecules into Cells

Our goal is to define the properties of cell-impermeable cargo molecules that can be delivered into cells by pH (Low) Insertion Peptide (pHLIP), which can selectively target tumors in vivo based on their acidity. Using biophysical methods and fluorescence microscopy, we show that pHLIP can successfully deliver polar and membrane-impermeable cyclic peptides linked to its C terminus through the membranes of lipid vesicles and cancer cells. Our results also indicate that the translocation of these cargo molecules is pH dependent and mediated by transmembrane helix formation. Since a broad range of cell-impermeable molecules is excluded from discovery efforts because they cannot traverse membranes on their own, we believe that pHLIP has the potential to expand therapeutic options for acidic tissues such as tumors and sites of inflammation.

pHLIP-FIRE, a Cell Insertion-Triggered Fluorescent Probe for Imaging Tumors Demonstrates Targeted Cargo Delivery In Vivo

We have developed an improved tool for imaging acidic tumors by reporting the insertion of a transmembrane helix: the pHLIP-Fluorescence Insertion REporter (pHLIP-FIRE). In acidic tissues, such as tumors, peptides in the pHLIP family insert as α-helices across cell membranes. The cell-inserting end of the pHLIP-FIRE peptide has a fluorophore–fluorophore or fluorophore–quencher pair. A pair member is released by disulfide cleavage after insertion into the reducing environment inside a cell, resulting in dequenching of the probe. Thus, the fluorescence of the pHLIP-FIRE probe is enhanced upon cell-insertion in the targeted tissues but is suppressed elsewhere due to quenching. Targeting studies in mice bearing breast tumors show strong signaling by pHLIP-FIRE, with a contrast index of ∼17, demonstrating (i) direct imaging of pHLIP insertion and (ii) cargo translocation in vivo. Imaging and targeted cargo delivery should each have clinical applications.

Abstract C2: Aspartic acid residues drive the membrane translocation of pHLIP, a therapeutic and imaging agent for solid tumors

The pHLIP peptide is a new therapeutic and imaging agent for cancer treatment. The pHLIP (pH-Low-Insertion Peptide) is soluble in solution but interestingly, it is able to interact with the plasma membrane and insert as a monomeric transmembrane helix (pKa=6.0). The insertion requires an acidic extracellular environment, such as that found in most solid tumors and inflammation sites. The pHLIP, which is nontoxic in mice, is specifically distributed to tumors (with minor labeling of kidney), as imaged by PET and near-infrared fluorescence. The insertion of pHLIP is unidirectional, as the C-terminus is translocated across the membrane, while the N-terminus remains exposed to the extracellular medium. The insertion energy can be employed to translocate into the cytoplasm membrane-impermeable molecules. As a proof of principle, we showed that the polar toxin phalloidin (which binds to F-actin, inhibiting its depolymerization) is translocated in a pH-dependent fashion into HeLa, JC, and M4A4 cancer cells, inhibiting cell growth and causing cytoeskeletal immobilization and multinucleation. Here we study the molecular determinants of pHLIP membrane insertion. The pH-mediated translocation is thought to be triggered by the protonation (loss of negative charge) of carboxyl groups present in pHLIP. Accordingly, the four aspartic acid (Asp) residues of pHLIP were sequentially mutated, and the efficacy of the membrane insertion and exit was studied in a liposome system. We found a correlation between the number and location of Asp with the peptides ability in insert into and exit from the membrane in a pH-dependent manner. We also observed that the number of Asp in the peptide determines the insertion properties (pKa and the cooperativity), suggesting that the tumor labeling properties of pHLIP can be specifically tuned to better match the physiological acidity of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C2.