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

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.