Cell Death Mechanisms Induced by CLytA-DAAO Chimeric Enzyme in Human Tumor Cell Lines

Abstract

The combination of the choline binding domain of the amidase N-acetylmuramoyl-L-alanine (CLytA)-D-amino acid oxidase (DAAO) (CLytA-DAAO) and D-Alanine induces cell death in several pancreatic and colorectal carcinoma and glioblastoma cell lines. In glioblastoma cell lines, CLytA-DAAO-induced cell death was inhibited by a pan-caspase inhibitor, suggesting a classical apoptotic cell death. Meanwhile, the cell death induced in pancreatic and colon carcinoma cell lines is some type of programmed necrosis. In this article, we studied the mechanisms that trigger CLytA-DAAO-induced cell death in pancreatic and colorectal carcinoma and glioblastoma cell lines and we acquire a further insight into the necrotic cell death induced in pancreatic and colorectal carcinoma cell lines. We have analyzed the intracellular calcium mobilization, mitochondrial membrane potential, PARP-1 participation and AIF translocation. Although the mitochondrial membrane depolarization plays a crucial role, our results suggest that CLytA-DAAO-induced cell death is context dependent. We have previously detected pancreatic and colorectal carcinoma cell lines (Hs766T and HT-29, respectively) that were resistant to CLytA-DAAO-induced cell death. In this study, we have examined the putative mechanism underlying the resistance in these cell lines, evaluating both detoxification mechanisms and the inflammatory and survival responses. Overall, our results provide a better understanding on the cell death mechanism induced by CLytA-DAAO, a promising therapy against cancer.

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

“…This result agrees with previous data showing that CLytA-DAAO induces cell death by a different mechanism depending on the tumor model. A traditional apoptosis was described in glioblastoma cell lines, whereas pancreatic and colorectal carcinoma cell lines displayed a necrotic-like cell death [ 11 , 16 ]. The results showed slight changes between the immobilized and free CLytA-DAAO, suggesting that CLytA-DAAO bound to MNPs acts faster than free CLytA-DAAO.…”

“…The resistance mechanisms against CLytA-DAAO-induced cell death in Hs766T cell line from pancreatic carcinoma and in HT-29 cell line from colorectal carcinoma were determined in our previously published article. Resistance mechanisms were related to the expression of some genes involved in the response to oxidative stress and cell survival [ 16 ].…”

“…In previous studies, it was determined that cell death is dependent on the intracellular ROS increase caused by the reaction catalyzed by CLytA-DAAO [ 11 ]. These ROS caused DNA damage and plasma membrane rupture [ 11 ], as well as a decrease in the mitochondrial membrane potential during the first hours of treatment with CLytA-DAAO [ 16 ]. Besides, here we have demonstrated that this effect is not equivalent to the direct administration of H 2 O 2 on tumor cell lines.…”

“…In cell model studies, two cell lines resistant to cell death induced by CLytA-DAAO were mainly detected: Hs766T from exocrine pancreatic carcinoma and HT-29 from colorectal carcinoma. Subsequently, the resistance mechanisms present in both cell lines were studied [ 16 ]. The resistance of Hs766T is related to a high antioxidant response dependent on the high expression of CAT and NFE2L2 genes, but also on a high stress response mediated by p38 and NF-κB [ 16 ].…”

“…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). Regarding HT-29 resistance to CLytA-DAAO-induced cell death, it is mediated by glutathione peroxidase 2 (GPX2), an antioxidant protein, but also by NF-κB and p38 [ 16 ].…”