Lactoferrin treatment activates acetylcholinesterase, decreasing acetylcholine levels in non-small cell lung cancer (NSCLC) cell culture supernatants, inhibiting cell survival.

Lactoferrin (Lf) is a multifunctional glycoprotein of the transferrin family which has shown to efficiently block cell migration and/or invasion in a wide range of cancer cell models. The objective of this study was to further understand how Lf targets cancer cells by examining the effect of acetylcholine (ACh) levels on Lf signaling using A549 (p53 wild-type) and H1299 (p53-null) nonsmall cell lung cancer (NSCLC) cell lines. Treatment with Lf reduced cell viability more effectively in A549 cells than in H1299 cells. The half maximal inhibitory concentration (IC₅₀) of Lf for A549 and H1299 was 8.97 ± 1.4 and 35.03 ± 4.2 mg·mL^-1, respectively. To uncover the potential molecular mechanism involved in the decreased cell viability observed in A549 cell following Lf treatment, the activity of tumor suppressor (p53), acetylcholinesterase (AChE), and ACh levels were measured. Treatment of A549 cells with Lf led to ~ 1.50-fold activation of p53, ~ 1.60-fold activation of AChE, and ~ 1.80-fold decrease in ACh levels. Vascular endothelial growth factor (VEGF) levels also decreased in cell culture supernatants upon treatment with Lf in both cell lines, and in A549 cells, the decrease occurred in a manner dependent on p53 and AChE. Given previous reports on the role of Lf in apoptosis induction, we examined AKT activity following Lf treatment and showed that AKT activity decreased ~ 1.95-fold in A549 cells and ~ 1.50-fold in H1299 cells. Furthermore, Lf-induced activation of caspase-3 was diminished by A549 cell cotreatment with siRNA targeted against p53 and/or AChE and increased by inhibiting the function of VEGF and/or AKT in both cell lines. In conclusion, this study identifies a mechanism wherein ACh concentrations in the cell culture supernatant attenuate the impact of Lf on NSCLC cell viability. These findings provide preliminary insight into the complex actions of Lf and suggest that the Lf-AChE-ACh pathway may warrant further study as a potential target in NSCLC.