title: The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13.
abstract: The CD13 antigen's binding site for the Asn-Gly-Arg (NGR) motif enables NGR-containing chemotherapeutic drugs to be delivered to CD13-positive tumours. Human CD13-positive acute myeloid leukemia (AML) cells proliferate abnormally and escape death. Here, we show that the CNGRC-GG-D(KLAKLAK)2 peptide induces death in AML cell lines (U937, THP-1, NB4, HL-60) and primary blood cells from AML patients. Cell death was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization and membrane disruption. Our results demonstrate in U937 cells that (i) the NGR-peptide triggers the loss of mitochondrial potential(ΔΨm) and generates superoxide anion (O2-), (ii) N-acetyl-L-cysteine (NAC) and extra/intracellular Ca2+ chelators (BAPTA) prevent both O2- production and cell death, (iii) the Ca2+-channel blocker nifedipine prevents cell death (indicating that Ca2+ influx is the initial death trigger), and (iv) BAPTA, but not NAC, prevents ΔΨm loss (suggesting O2- is a mitochondrial downstream effector). AML cell lines and primary blasts responding to the lethal action of NGR-peptide express promatrix metalloproteinase-12 (proMMP-12) and its substrate progranulin (an 88 kDa cell survival factor). A cell-free assay highlighted proMMP-12 activation by O2-. Accordingly, NGR-peptide's downregulation of 88 kDa progranulin protein was prevented by BAPTA and NAC. Conversely, AML blast resistance to NGR-peptide is associated with the expression of a distinct, 105 kDa progranulin isoform. These results indicate that CNGRC-GG-D(KLAKLAK)2 induces death in AML cells through the Ca2+-mitochondria-O2.-pathway, and support the link between proMMP-12 activation and progranulin cleavage during cell death. Our findings may have implications for the understanding of tumour biology and treatment.
authors.affiliation: Centre de Recherche des Cordeliers, INSERM UMRS1138, Sorbonne Universités UPMC Paris 06, Université Paris Descartes Sorbonne Paris Cité, Paris, France.|Assistance Publique des Hôpitaux de Paris, Paris, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Bouchet
authors.affiliation: Centre de Recherche de Saint-Antoine, INSERM UMRS 938, Service d'Hématologie, Hôpital St Antoine, Paris, France.|Sorbonne Universités UPMC Paris 06, Paris, France.
authors.forename: Ruoping
authors.initials: R
authors.lastname: Tang
authors.affiliation: Centre de Recherche de Saint-Antoine, INSERM UMRS 938, Service d'Hématologie, Hôpital St Antoine, Paris, France.|Sorbonne Universités UPMC Paris 06, Paris, France.
authors.forename: Fanny
authors.initials: F
authors.lastname: Fava
authors.affiliation: Centre de Recherche de Saint-Antoine, INSERM UMRS 938, Service d'Hématologie, Hôpital St Antoine, Paris, France.|Sorbonne Universités UPMC Paris 06, Paris, France.
authors.forename: Ollivier
authors.initials: O
authors.lastname: Legrand
authors.affiliation: Centre de Recherche des Cordeliers, INSERM UMRS1138, Sorbonne Universités UPMC Paris 06, Université Paris Descartes Sorbonne Paris Cité, Paris, France.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Bauvois
mesh_terms: D000208:Acute Disease; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects*; D018826:CD13 Antigens / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D007951:Leukemia, Myeloid / Q000378:metabolism / Q000473:pathology; D053508:Matrix Metalloproteinase 12 / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D010455:Peptides / Q000494:pharmacology*; D000077153:Progranulins; D013481:Superoxides / Q000378:metabolism; D020298:U937 Cells
chemical_list: C492314:GRN protein, human; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D000077153:Progranulins; D013481:Superoxides; D018826:CD13 Antigens; D020169:Caspases; D053508:Matrix Metalloproteinase 12; D002118:Calcium
keywords: calcium; leukemia; metalloproteinase; necrosis; superoxide radical