CLytA-DAAO, Free and Immobilized in Magnetic Nanoparticles, Induces Cell Death in Human Cancer Cells

Abstract

D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids generating hydrogen peroxide, a potential producer of reactive oxygen species. In this study, we used a CLytA-DAAO chimera, both free and bound to magnetic nanoparticles, against colon carcinoma, pancreatic adenocarcinoma, and glioblastoma cell lines. We found that the enzyme induces cell death in most of the cell lines tested and its efficiency increases significantly when it is immobilized in nanoparticles. We also tested this enzyme therapy in non-tumor cells, and we found that there is not cell death induction, or it is significantly lower than in tumor cells. The mechanism triggering cell death is apparently a classical apoptosis pathway in the glioblastoma cell lines, while in colon and pancreatic carcinoma cell lines, CLytA-DAAO-induced cell death is a necrosis. Our results constitute a proof of concept that an enzymatic therapy, based on magnetic nanoparticles-delivering CLytA-DAAO, could constitute a useful therapy against cancer and besides it could be used as an enhancer of other treatments such as epigenetic therapy, radiotherapy, and treatments based on DNA repair.

CLytA-DAAO Chimeric Enzyme Bound to Magnetic Nanoparticles. A New Therapeutical Approach for Cancer Patients?

“…However, immobilization in GNPs and in alginate capsules significantly decreased the cell death induced by the treatment ( Figure 1 A), suggesting that these methods of CLytA-DAAO immobilization are less effective stabilizing the chimeric enzyme than MNPs. We had previously shown that the CLytA-DAAO-bound to MNPs together with D-Ala treatment induces a higher percentage of cells in subG1 phase than the free enzyme in several pancreatic carcinoma, colorectal carcinoma, and glioblastoma cell lines [ 11 ]. Here, a series of cell viability analyses were performed to confirm that, in fact, CLytA-DAAO bound to MNPs induced a significantly higher percentage of cell death compared to the free enzyme in all cancer models tested.…”

“…The differences observed between free and immobilized in MNPs enzyme in viability ( Figure 1 A,B) and cell cycle analysis [ 11 ], were more pronounced than those observed in the MTT assays in pancreatic and colorectal carcinoma cell lines. The viability and cell cycle studies were conducted after 24 h treatment, whereas the MTT assays were carried out after 72 h treatment.…”

“…CLytA-DAAO is an enzymatic chimera formed by the union of the choline-binding domain of N-acetylmuramoyl-L-alanine amidase (CLytA) from Streptococcus pneumoniae with the enzyme D-amino acid oxidase (DAAO) from Rhodotorula gracilis [ 11 ]. This enzyme oxides D-amino acids and, during the reaction, generates hydrogen peroxide as a by-product [ 12 ], which is an important inducer of free radicals.…”

“…Thus, the enzyme is not harmful per se and requires the exogenous addition of D-amino acids to be catalytically active [ 13 , 14 , 15 ]. Previously, we demonstrated the cytotoxic capacity of the enzymatic chimera CLytA-DAAO activated by D-Alanine (D-Ala) in pancreatic carcinoma, colorectal carcinoma, and glioblastoma cell lines [ 11 ]. The therapeutic strategy that we propose is based on targeting the enzyme to the tumor area with an external magnetic field in order to concentrate the nanoparticles in the tumor, and then treating the patients systemically with D-amino acids.…”

“…Although we demonstrated the cytotoxicity induced by CLytA-DAAO in many pancreatic and colorectal carcinoma and glioblastoma cellular models, we found two cell lines resistant to cell death induced by this treatment: Hs766T from pancreatic carcinoma and HT-29 from colorectal carcinoma [ 11 ]. Hs766T resistance to CLytA-DAAO-induced cell death was found to be related to the high expression of antioxidant proteins such as catalase and nuclear factor erythroid 2-related factor 2 (Nrf2) and proteins involved in the inflammatory and stress response such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p38 mitogen-activated protein kinase 11 (p38).…”