Lactoferrin selectively triggers apoptosis in highly metastatic breast cancer cells through inhibition of plasmalemmal V-H+-ATPase

Abstract

Breast cancer is the most common type of cancer affecting women. Despite the good prognosis when detected early, significant challenges remain in the treatment of metastatic breast cancer. The recruitment of the vacuolar H+-ATPase (V-H+-ATPase) to the plasma membrane, where it mediates the acidification of the tumor microenvironment (TME), is a recognized feature involved in the acquisition of a metastatic phenotype in breast cancer. Therefore, inhibitors of this pump have emerged as promising anticancer drugs. Lactoferrin (Lf) is a natural pro-apoptotic iron-binding glycoprotein with strong anticancer activity whose mechanism of action is not fully understood. Here, we show that bovine Lf (bLf) preferentially induces apoptosis in the highly metastatic breast cancer cell lines Hs 578T and MDA-MB-231, which display a prominent localisation of V-H+-ATPase at the plasma membrane, but not in the lowly metastatic T-47D or in the non-tumorigenic MCF-10-2A cell lines. We also demonstrate that bLf decreases the extracellular acidification rate and causes intracellular acidification in metastatic breast cancer cells and, much like the well-known proton pump inhibitors concanamycin A and bafilomycin A1, inhibits V-H+-ATPase in sub-cellular fractions. These data further support that bLf targets V-H+-ATPase and explain the selectivity of bLf for cancer cells, especially for highly metastatic breast cancer cells. Altogether, our results pave the way for more rational in vivo studies aiming to explore this natural non-toxic compound for metastatic breast cancer therapy.

Lactoferrin’s Anti-Cancer Properties: Safety, Selectivity, and Wide Range of Action

“…Except for the indirect immunomodulatory pathway, the other mechanisms require the direct recognition of and selection between cancerous and normal cells by Lf, possibly involving a primary interaction with peculiar cancer cell surface receptors or a secondary one via the regulation of differential intracellular networks. To date, only few evidences have been reported for the primary recognition between Lf and cancer cell surface receptors [120][121][122]. In this respect, most of cancerous cells have a high content of proteoglycans, glycosaminoglycans (GAGs, mainly heparan and chondroitin sulfates), as well as of sialic acids, all these molecules being well-known interactors for Lfs [120,123,124].…”

“…Recently, the vacuolar H + -ATPase (V-H + -ATPase) was described as a potential target for bLf in highly metastatic breast cancer cell lines [121,122]. BLf was found to induce apoptosis, along with a significant decrease in V-H + -ATPase-mediated extracellular acidification rate, in two highly metastatic breast cancer cell lines, Hs 578T and MDA-MB-231, displaying a prominent localization of V-H + -ATPase at the plasma membrane, but not in the lowly metastatic T-47D or in the non-tumorigenic MCF-10-2A cell lines [121]. By a similar approach, bLf was described to selectively act as pro-apoptotic agent on highly metastatic prostate PC-3 and osteosarcoma MG-63 cell lines, both displaying aberrant plasma membrane localization of V-H + -ATPase [122].…”

The Nonapeptide RWQWRWQWR: A Promising Molecule for Breast Cancer Therapy

“…The cytotoxic effect of the palindrome against breast cancer cell lines MCF-7 and MDA-MB-231 is fast and induces severe cellular damage and morphological changes, like shrinkage and acquiring rounded forms ( Figure 2). These results suggest that LfcinB (21)(22)(23)(24)(25) Pal is a selective peptide, due to the non-significant viability reduction and unchanged morphology against non-cancerous cells observed at the evaluated concentrations. Also we evaluated if the peptide will maintain its activity up to 24 h after the stimuli (Supporting information Figure S2), it was observed that the cytotoxic effect was similar with IC 50 of 135 and 61 μM for MDA-MB-231 and MCF-7 cell lines, respectively.…”

“…The results of the scaling and the methodology for the synthesis of 200 mg and 1 g of palindromic peptide LfcinB (21)(22)(23)(24)(25) Pal : H-1 RWQWRWQWR 9 -NH 2 through the SPPS Fmoc-tBu strategy was carried out under controlled conditions and documentation based on the GMP management system. It was established the minimum time required for the coupling of each residue and the minimum volume of washing solvents, piperidine 5%, and solvent for the mixture of amino acid/DCC/ HOBt required to obtain the expected product.…”

“…[15] Previous studies have shown that the 20 RRWQWR 25 sequence is the minimum motif with anticancer activity, and palindromic peptides derived from this motif have exhibited a significant increase in anticancer activity against oral and breast cancer cell lines while maintaining their selectivity against trophoblast, fibroblasts, and HEK-293 cells. [16,17] We have previously reported that the palindromic peptide LfcinB (21)(22)(23)(24)(25) Pal exerts a cytotoxic effect on CAL-27 cells in a rapid, concentration-dependent manner with IC 50 = 23 μM and has no effect on the viability of Het-1 A cells. [16] In addition, the palindromic peptide was evaluated against the MDA-MB-468 breast cancer line, where a similar cytotoxic effect was observed with IC 50 = 72 μM and with morphological changes such as apparent decrease in cell size, shrinkage, and rounding.…”