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

Abstract

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 pK a 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. drug delivery | hydrophilic drugs | cytoplasmic delivery | targeting acidity | targeted chemotherapy

Family of pH (low) insertion peptides for tumor targeting

“…While studying membrane protein folding, we discovered a peptide called pH (low) insertion peptide (pHLIP) that reversibly folds and inserts across membranes in response to pH changes, and this discovery has led to a unique way to target acidic tissue. Our biophysical studies have revealed the molecular mechanism of pHLIP action (1-3), and we have shown that pHLIP can target acidic tissue and selectively translocate polar, cell-impermeable molecules across cell membranes in response to low extracellular pH (1,(4)(5)(6)(7)(8)(9). pHLIP conjugated with fluorescent dyes, PET [ 64 Cu-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and 18 F], single photon emission computed tomography (SPECT; 99 Tc) probes, and gold nanoparticles targets acidic tumors (1,4,5,10,11).…”

pH-controlled delivery of luminescent europium coated nanoparticles into platelets

“…The pHLIP mechanism of action has been determined in liposomes (17) and has been used in cells for the delivery of fluorescent molecules (18). Furthermore, there is significant interest in this peptide as a probe or drug delivery system for cancerous cells and in inflammatory arthritis because of the low extracellular pH associated with these regions (18)(19)(20)(21)(22).…”

Enhancement of radiation effect on cancer cells by gold-pHLIP

“…The pH-sensitive targeting agents we are developing are based on the action of a family of pHLIPs (pH Low-Insertion Peptides), which can "sense" acidity at the surface of cancer cells and deliver diagnostic and therapeutic molecules to tumors of different origins (20)(21)(22)(23)(24)(25). It was shown that pHLIP can promote fusion of liposomes with cancer cells and cellular delivery of various payloads (26,27), including small gold nanoparticles (26).…”