Antiproliferative effect of pHLIP-amanitin.
Toxins could be effective anticancer drugs, if their selective delivery into cancer cells could be achieved. We have shown that the energy of membrane-associated folding of water-soluble membrane peptides of the pHLIP (pH low insertion peptide) family could be used to move cell-impermeable cargo across the lipid bilayer into the cytoplasm of cancer cells. Here we present the results of a study of pHLIP-mediated cellular delivery of a polar cell-impermeable toxin, α-amanitin, an inhibitor of RNA polymerase II. We show that pHLIP can deliver α-amanitin into cells in a pH-dependent fashion and induce cell death within 48 h. Translocation capability could be tuned by conjugating amanitin to the C-terminus of pHLIP via linkers of different hydrophobicities that could be cleaved in the cytoplasm. pHLIP-SPDP-amanitin, which exhibits 4-5 times higher antiproliferative ability at pH 6 than at pH 7.4, was selected as the best construct. The major mechanism of amanitin delivery is direct translocation (flip) across a membrane by pHLIP and cleavage of the S-S bond in the cytoplasm. The antiproliferative effect was monitored on four different human cancer cell lines. pHLIP-mediated cytoplasmic delivery of amanitin could create great opportunities to use the toxin as a potent pH-selective anticancer agent, which predominantly targets highly proliferative cancer cells at low extracellular pH values.
Physics Department, University of Rhode Island, 2 Lippitt Road, Kingston, RI 02881, USA.
Anna
A
Moshnikova
Valentina
V
Moshnikova
Oleg A
OA
Andreev
Yana K
YK
Reshetnyak
D053959:Alpha-Amanitin / Q000737:chemistry / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry; D049109:Cell Proliferation / Q000187:drug effects; D004337:Drug Carriers / Q000737:chemistry / Q000494:pharmacology*; D016503:Drug Delivery Systems / Q000379:methods; D004354:Drug Screening Assays, Antitumor; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000494:pharmacology; D008969:Molecular Sequence Data; D021381:Protein Transport
D053959:Alpha-Amanitin; D000970:Antineoplastic Agents; D004337:Drug Carriers; D008565:Membrane Proteins; C533201:pHLIP protein