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.

Systemic Delivery of Tumor-Targeted Bax-Derived Membrane-Active Peptides for the Treatment of Melanoma Tumors in a Humanized SCID Mouse Model

“…It was shown that they can induce necrosis after plasma membrane disruption, depending on their L-or D-chirality, 52,53 apoptosis by the upregulation of caspases, 54 the influx of extracellular Ca 2 + , Ca 2 + -mediated DJm disruption and mitochondrial O 2 $ À generation. 55 However, the cationic peptide (KLAKLAK) 2 has been reported to have a potency that is too low for it to be used as an effective anticancer drug. 41 Because BH3-only proteins either directly or indirectly inhibit prosurvival BCL-2 family members, such as BCL-2, BCL-xL, and MCL-1, to increase cellular sensitivity to anticancer agents, efforts were made to develop so-called BH3 mimetics.…”

Antitumor activity of opiorphin, sialorphin and their conjugates with a peptide klaklakklaklak

“…The pathway of tumor cell death after administration of the opio‐klak peptide is similar to that reported by Bouchet et al . . Namely, in a short time (5–15 min), an increase in the count of positive cells stained with annexin solution (apoptosis) was noticed.…”

“…Thus, it seems that the mechanism of the action of the opio‐klak peptide is similar to that suggested by Bouchet et al . . In the first minutes after administration of the peptide to the cells, an increase in the percentage of cells with phosphatidylserine relocated from inside to outside of the cell membrane is observed (Annexin V‐positive staining) followed by increasing of cells with non‐continuous outer membrane (PI Annexin V positive stainings).…”

Recent Advances in Subcellular Targeted Cancer Therapy Based on Functional Materials

“…Therefore, the use of this peptide for anticancer therapy with enhanced apoptotic activity must involve the specific tumor targeting and efficient cellular internalization. Conjugating D (KLAKLAK) 2 with tumor homing moieties, such as NGR motif, RGD peptide, LyP‐1, protein transduction domain (PTD), or antibody, has produced chemical conjugates that are able to accumulate at tumor sites. To further enhance the extracellular and intracellular targeting ability, Zhang and co‐workers designed and prepared a dual‐targeting proapoptotic peptide (DTP) by the conjugation of folic acid (FA) moiety (targeting agent I) and triphenylphosphonium (TPP) cation (targeting agent II) with D (KLAKLAK) 2 to achieve simultaneous tumor cell‐ and mitochondria‐targeted cancer therapy ( Figure a) .…”