KRAS mutation and NF-κB both play crucial role in pancreatic cancer; in addition, defensin, the peptide mediator in innate immunity, can inhibit NF-κB. Assuming a strategy that targets both NF-κB and concomitantly the mutated KRAS indirectly via intensive macropinocytosis, we designed and generated a recombinant protein DF-HSA which consists of two molecules of human beta-defensin 2 (HBD2) and a moiety of human serum albumin (HSA). As shown, the recombinant protein DF-HSA markedly down-regulated NF-κB in both KRAS mutant MIA PaCa-2 cells and wild type BxPC-3 cells. Determined by confocal microscopy, the uptake of DF-HSA in MIA PaCa-2 cells was more intense than that in BxPC-3 cells. The uptake was blocked b... More
KRAS mutation and NF-κB both play crucial role in pancreatic cancer; in addition, defensin, the peptide mediator in innate immunity, can inhibit NF-κB. Assuming a strategy that targets both NF-κB and concomitantly the mutated KRAS indirectly via intensive macropinocytosis, we designed and generated a recombinant protein DF-HSA which consists of two molecules of human beta-defensin 2 (HBD2) and a moiety of human serum albumin (HSA). As shown, the recombinant protein DF-HSA markedly down-regulated NF-κB in both KRAS mutant MIA PaCa-2 cells and wild type BxPC-3 cells. Determined by confocal microscopy, the uptake of DF-HSA in MIA PaCa-2 cells was more intense than that in BxPC-3 cells. The uptake was blocked by the specific inhibitor EIPA, indicating that DF-HSA internalized via macropinocytosis. DF-HSA displayed more potent cytotoxicity to cancer cells than HBD2. DF-HSA induced apoptosis in cancer cells. Notably, DF-HSA inhibited tumor cell spheroid formation, an effect comparable to that of salinomycin. DF-HSA inhibited tumor cell migration and invasion. As detected with scanning electron microscopy, DF-HSA strongly depleted filopodia on cell surface; and salinomycin induced similar changes. By in vivo imaging, DF-HSA displayed intense tumor-site accumulation and lasting retention for over 14 days; however, HBD2 showed much less tumor-site accumulation and a shorter retention time for only 24 h. DF-HSA suppressed the growth of pancreatic cancer MIA PaCa-2 xenograft in athymic mice; and its combination with gemcitabine achieved higher antitumor efficacy. In summary, the recombinant defensin/HSA fusion protein that inhibits NF-κb associated with intensive macropinocytosis is highly effective against pancreatic cancer.
