Bovine lactoferricin selectively induces apoptosis in human leukemia and carcinoma cell lines

Abstract

Bovine lactoferricin (LfcinB) is a cationic, amphipathic peptide that is cytotoxic for human and rodent cancer cells. However, the mechanism by which LfcinB causes the death of cancer cells is not well understood. Here, we show that in vitro treatment with LfcinB rapidly induced apoptosis in several different human leukemia and carcinoma cell lines as determined by DNA fragmentation assays and phosphatidylserine headgroup inversion detected by Annexin V binding to the surface of cancer cells. Importantly, LfcinB treatment did not adversely affect the viability of untransformed human lymphocytes, fibroblasts, or endothelial cells. Studies with different LfcinB-derived peptide fragments revealed that the cytotoxic activity of LfcinB resided within the amino acid sequence FKCRRWQWRM. Treatment of Jurkat T leukemia cells with LfcinB resulted in the production of reactive oxygen species followed by caspase-2-induced dissipation of mitochondrial transmembrane potential and subsequent activation of caspase-9 and caspase-3. Selective inhibitors of caspase-2 (Z-VDVAD-FMK), caspase-9 (Z-LEHD-FMK), and caspase-3 (Z-DEVD-FMK) protected both leukemia and carcinoma cells from LfcinB-induced apoptosis. Conversely, a caspase-8 inhibitor (Z-IETD-FMK) had no effect, which argued against a role for caspase-8 and was consistent with the finding that death receptors were not involved in LfcinB-induced apoptosis. Furthermore, Jurkat T leukemia cells that overexpressed Bcl-2 were less sensitive to LfcinB-induced apoptosis, which was characterized by mitochondrial swelling and the release of cytochrome c from mitochondria into the cytosolic compartment. We conclude that LfcinB kills cancer cells by triggering the mitochondrial pathway of apoptosis at least in part through the generation of reactive oxygen species. [Mol Cancer Ther 2005;4(4):612 -24]

Bovine Lactoferricin Inhibits Basic Fibroblast Growth Factor- and Vascular Endothelial Growth Factor165-Induced Angiogenesis by Competing for Heparin-Like Binding Sites on Endothelial Cells

“…44 LfcinB did not exhibit any cytotoxic activity against HUVECs, excluding the possibility that LfcinB simply caused HUVECs to undergo apoptosis, as occurs when various cancer cell lines are exposed to LfcinB. 24,27 Because both bovine lactoferrin and LfcinB are known to bind heparin, 29,30 our findings led us to hypothesize that LfcinB competed with bFGF and VEGF 165 for heparin-like binding sites on heparan sulfate proteoglycans on the surface of HUVECs. Heparan sulfate proteoglycans are required for bFGF and VEGF 165 binding and signaling through their respective cell-surface receptors.…”

“…Interestingly, glypican-1 is overexpressed by human breast and pancreatic cancer cells, 51,52 which might promote the binding of LfcinB to these tumor cell types. We therefore speculate that the selective cytotoxic activity that LfcinB exhibits against several different human breast carcinoma cell lines 27 may be, at least in part, attributable to interactions between LfcinB and cell-surface glypican-1.…”

“…Because LfcinB induces apoptosis in a variety of human cancer cell lines as early as 1 hour after exposure to the peptide, 24,27 we addressed the possibility that the antiangiogenic activity of LfcinB was the result of a cytotoxic effect by LfcinB on endothelial cells. Figure 4a shows that 51 Cr-labeled HUVECs that were exposed to medium, bFGF, VEGF 165 , or LfcinB for 6 hours released similar amounts of 51 Cr into culture supernatant.…”

“…26 Recently, we have shown that LfcinB selectively induces apoptosis in a range of human leukemia and carcinoma cell lines via the reactive oxygen species-dependent loss of mitochondrial transmembrane potential and the sequential activation of caspase-2, -9, and -3. 27 Interestingly, LfcinB treatment of tumor-bearing mice leads to a reduction in the number of tumor-induced blood vessels, 25 suggesting a possible antiangiogenic role for LfcinB. However, whether this effect is a consequence of LfcinB-induced apoptosis of endothelial cells or LfcinB-mediated inhibition of tumor blood vessel development remains to be determined.…”

Future Challenges of Whey Proteins

“…Breast cancer in human is inhibited by several commercial BSA preparations during in vitro cell culture (Solak and Akin, 2012). Mader et al (2005) also demonstrated that the nature of both synthetic and pepsin-generated lactoferricin B is against human leukemia and carcinoma cell lines (Colon, breast and ovary). In an acidic environment, α-lactalbumin forms the HAMLET (human α-lactalbumin made lethal to tumor cells) complex with oleic acid which inhibits the proliferation of various tumors by an apoptosis like mechanism (Svensson et al, 2003).…”