A new paradigm for aptamer therapeutic AS1411 action: uptake by macropinocytosis and its stimulation by a nucleolin-dependent mechanism.
AS1411 is a first-in-class anticancer agent, currently in phase II clinical trials. It is a quadruplex-forming oligodeoxynucleotide that binds to nucleolin as an aptamer, but its mechanism of action is not completely understood. Mechanistic insights could lead to clinically useful markers for AS1411 response and to novel targeted therapies. Previously, we proposed a model where cell surface nucleolin serves as the receptor for AS1411, leading to selective uptake in cancer cells. Here, we compare uptake of fluorophore-labeled AS1411 (FL-AS1411) in DU145 prostate cancer cells (sensitive to AS1411) and Hs27 nonmalignant skin fibroblasts (resistant to AS1411). Uptake of FL-AS1411 occurred by endocytosis in both cell types and was much more efficient than an inactive, nonquadruplex oligonucleotide. Unexpectedly, uptake of FL-AS1411 was lower in cancer cells compared with Hs27 cells. However, the mechanism of uptake was different, occurring by macropinocytosis in cancer cells, but by a nonmacropinocytic pathway in Hs27 cells. Additionally, treatment of various cancer cells with AS1411 caused hyperstimulation of macropinocytosis, provoking an increase in its own uptake, whereas no stimulation was observed for nonmalignant cells. Nucleolin was not required for initial FL-AS1411 uptake in DU145 cells but was necessary for induced macropinocytosis and FL-AS1411 uptake at later times. Our results are inconsistent with the previous mechanistic model but confirm that nucleolin plays a role in mediating AS1411 effects. The data suggest a new model for AS1411 action as well as a new role for nucleolin in stimulating macropinocytosis, a process with potential applications in drug delivery.
Reyes-Reyes
E Merit
EM
Departments of Medicine and Biochemistry and Molecular Biology, James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40202, USA.
Teng
Yun
Y
Bates
Paula J
PJ
D000970:Antineoplastic Agents / Q000494:pharmacology; D052157:Aptamers, Nucleotide / Q000494:pharmacology; D015153:Blotting, Western; D001940:Breast / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D002478:Cells, Cultured; D016503:Drug Delivery Systems; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010873:Pinocytosis / Q000502:physiology*; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000097796:Nucleolin
D000970:Antineoplastic Agents; D052157:Aptamers, Nucleotide; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D000097796:Nucleolin; C513936:AGRO 100