The CNGRC-GG- D (KLAKLAK) 2 peptide induces a caspase-independent, Ca 2+ -dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13

// Sandrine Bouchet 1, 2 , Ruoping Tang 3, 4 , Fanny Fava 3, 4 , Ollivier Legrand 3, 4 , Brigitte Bauvois 1 1 Centre de Recherche des Cordeliers, INSERM UMRS1138, Sorbonne Universites UPMC Paris 06, Universite Paris Descartes Sorbonne Paris Cite, Paris, France 2 Assistance Publique des Hopitaux de Paris, Paris, France 3 Centre de Recherche de Saint-Antoine, INSERM UMRS 938, Service d’Hematologie, Hopital St Antoine, Paris, France 4 Sorbonne Universites UPMC Paris 06, Paris, France Correspondence to: Brigitte Bauvois, e-mail: brigitte.bauvois@crc.jussieu.fr Keywords: calcium, leukemia, metalloproteinase, necrosis, superoxide radical Received: July 30, 2015      Accepted: November 16, 2015      Published: December 09, 2015 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 (O 2 − ), (ii) N-acetyl-L-cysteine (NAC) and extra/intracellular Ca 2+ chelators (BAPTA) prevent both O 2 − production and cell death, (iii) the Ca 2+ -channel blocker nifedipine prevents cell death (indicating that Ca 2+ influx is the initial death trigger), and (iv) BAPTA, but not NAC, prevents ΔΨm loss (suggesting O 2 − 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 O 2 − . 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 Ca 2+ -mitochondria-O 2 .-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.