Journal Article

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

An, Ming, Engelman, Donald M., Andreev, Oleg A., Wijesinghe, Dayanjali, Reshetnyak, Yana K.

2010

Overview

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.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: actin / Actins / Actins - metabolism / adverse effects / Amanita - chemistry / antineoplastic agents / Antineoplastics / Biological Sciences / Cancer / Cell growth / Cell Line, Tumor / Cell membranes / cell proliferation / Cell Proliferation - drug effects / Cells / Chemotherapy / cytoskeleton / depolymerization / disulfide bonds / drug therapy / Freight / HeLa cells / Humans / Hydrogen-Ion Concentration / hydrophobicity / lipid bilayers / Medical treatment / Membrane Proteins - metabolism / Membranes / Molecular Structure / Molecules / Mycotoxins - metabolism / Mycotoxins - pharmacology / neoplasm cells / neoplasms / Neoplasms - drug therapy / Peptides / Phalloidine - metabolism / Phalloidine - pharmacology / Phosphatidylcholines - metabolism / Protein Transport - physiology / Sulfide compounds / tissues / Toxins / Translocation / Tumors