Cancer Cell Proliferation is Inhibited by Phlip Mediated Delivery of Membrane Impermeable Toxin Phalloidin

We wish to use the pH-(Low)-Insertion-Peptide (pHLIP) to transport therapeutic agents to acidic tumors, with the ultimate goal of improving the treatment of cancer. pHLIP inserts into a lipid bilayer under slightly acidic conditions (pH 6-6.5), forming a transmembrane helix. We demonstrate here that pHLIP-mediated translocation of a cell-impermeable, polar toxin phalloidin can inhibit the proliferation of cancer cells. The delivery constructs, pHLIP-K(rho)C(aph) and pHLIP-C(aph), both carry the phalloidin toxin at the inserting C-terminus, via a disulfide linkage that could be cleaved in cells. The constructs differ in that a lipophilic rhodamine moiety is also attached to the inserting end, near the phalloidin cargo, in pHLIP-K(rho)C(aph). After a brief incubation with 2-4 μM of pHLIP-K(rho)C(aph) at pH 6.1-6.2 (for 1-3 h), proliferation of HeLa, JC, and M4A4 cancer cells were severely disrupted (> 90% inhibitions). Cells treated with pHLIP-K(rho)C(aph) also showed signs of cytoskeletal immobilization, consistent with the knowledge that phalloidin binds to F-actin and stabilizes the filament against depolymerization. However, the antiproliferative effect was not observed with pHLIP-C(aph). The insertion behavior of both constructs were studied in POPC liposomes using Trp fluorescence: pHLIP-K(rho)C(aph) and pHLIP-C(aph) insert with the same apparent pK of 6.1-6.2, similar to that of pHLIP (without any cargo). However, kinetic experiments suggest that pHLIP-C(aph) inserts much slower than pHLIP-K(rho)C(aph), possibly accounting for its lack of antiproliferative effects in cell assays. In short, our results obtained with pHLIP-K(rho)C(aph) lay the foundation for the development of a new class of anti-tumor agents that would selectively enter and destroy cancer cells while not affecting normal cells. Such pHLIP-mediated delivery of otherwise cell-impermeable agents may enhance the efficacy of treatment, as well as significantly reducing the side effect.