ABSTRACT
Here, we report that human lactoferrin (hLF), known for its anticancer properties, induced intracellular activation of the Na+/H+ exchanger (NHE) 7 in human lung cancer PC-9 cells. Compared to non-fused hLF, the fusion of human serum albumin (HSA) with hLF (hLF-HSA) facilitated its internalization into PC-9 cells in a caveolae-mediated manner, thereby exhibiting enhanced anti-proliferative effects. Although hLF alone did not exhibit any discernible effects, hLF-HSA resulted in organelle alkalization as detected using an acidotropic pH indicator. hLF-HSA-induced elevation of organelle pH and inhibition of cancer growth were abolished by NHE7 siRNA. hLF-HSA upregulated NHE7. Thus, upon cellular uptake, hLF-HSA triggers proton leakage through the upregulation of NHE7. This process led to organelle alkalization, probably in the trans-Golgi network (TGN) as suggested by the localization of NHE7 in PC-9 cells, thereby suppressing lung cancer cell growth. Forcing the cellular uptake of hLF alone using a caveolae-mediated endocytosis activator led to an increase in organelle pH. Furthermore, cell entry of hLF also activated proton-loading NHE7, leading to organelle acidification in the pancreatic cancer cell line MIA PaCa-2. Therefore, the intracellularly delivered hLF functions as an activator of NHE7.
Subject(s)
Lactoferrin , Lung Neoplasms , Sodium-Hydrogen Exchangers , Humans , Lactoferrin/metabolism , Lactoferrin/pharmacology , Lung Neoplasms/metabolism , Protons , Sodium-Hydrogen Exchangers/metabolism , trans-Golgi Network/metabolismABSTRACT
The fusion of human serum albumin (HSA) with human lactoferrin (hLF) (designated as hLF-HSA) has improved the pharmacokinetic properties and anti-proliferative activities of hLF against cancer cells. In this study, we evaluated the anti-migratory activities of hLF and hLF-HSA against the human lung adenocarcinoma PC-14 cell line using wound healing and Boyden chamber assays. Despite the unexpected hLF-induced migration, hLF-HSA clearly demonstrated the complete inhibition of PC-14 cell migration. To examine the mechanism underlying the enhanced PC-14 cell migration by hLF alone but suppressed migration by hLF-HSA, we focused on the matrix metalloproteinase (MMP) family of endopeptidases because MMPs are often reported to play important roles in facilitating the migration and metastasis of cancer cells. Furthermore, hLF is a transactivator of MMP1 transcription. As expected, treatment of cells with hLF and hLF-HSA led to the upregulation and downregulation of MMP1, respectively. In contrast, MMP9 expression levels, which are often associated with cancer migration, were unchanged in the presence of either protein. An MMP inhibitor attenuated hLF-induced migration of PC-14 cells. Therefore, specific enhancement and suppression of MMP1 expression by hLF and hLF-HSA have been implicated as causes of a marked increase and decrease in PC-14 cell migration, respectively. In conclusion, the fusion of HSA with hLF (hLF-HSA) promoted its anti-migratory effects against cancer cells. Therefore, hLF-HSA is a promising anti-cancer drug candidate based on its improved anti-migratory activity towards cancer cells.
Subject(s)
Albumins , Lactoferrin , Neoplasms , Recombinant Fusion Proteins , Humans , Albumins/genetics , Albumins/therapeutic use , Cell Movement , Down-Regulation , Lactoferrin/genetics , Lactoferrin/therapeutic use , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 1/metabolism , Neoplasms/therapy , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/therapeutic useABSTRACT
Albumin fusion or conjugation is a well-established technique for tumor delivery and is mainly mediated by albumin-induced caveolae-dependent endocytosis. We report that caveolae-dependent endocytic signaling activated by human serum albumin (HSA) is not sufficiently strong to induce cellular uptake, mainly due to its electrostatic repulsion from the negatively charged cell surface sulfated glycosaminoglycans (GAGs), and fusion of the cell-surface-retained protein with HSA is an effective strategy to activate the HSA-induced endocytic signal, thereby improving its intracellular uptake. In this study, human lactoferrin (hLF), a protein that accumulates on the cell surface along with GAGs, was selected for delivery into human lung adenocarcinoma PC-14 cells. When added exogenously, hLF-fused HSA (hLF-HSA) was successfully endocytosed, whereas the simultaneous addition of HSA and hLF did not result in endocytosis, indicating less efficient activation of endocytic signaling by HSA alone and the importance of its fusion. Importantly, the treatment of cells with chlorate, a known inhibitor of GAG sulfation, dramatically suppressed the endocytosis of hLF-HSA owing to the loss of the hLF-GAG interaction. Therefore, the cell-surface localization of HSA imposed by fusion with the cell-surface-retained protein enhances its binding to the relevant receptor, which improves intracellular delivery as an albumin-fusion platform.