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

María Fuentes-Baile,María P Ventero,Pilar García-Morales,Miguel Saceda,Jesús M Sanz,Elizabeth Pérez-Valenciano,Víctor M Barberá,Cristina Alenda,Camino De Juan Romero

doi:10.3390/ijms21228522

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.

Highlights

Cell death is a necessary phenomenon for the normal development of vertebrate and invertebrate organisms
To examine whether the entry of calcium into the mitochondria could be responsible for an membrane potential (MMP) decrease, we analyzed the role of calcium in CLytA-DAO-induced cell death
CLytA-D-amino acid oxidase (DAAO)-induced cell death was blocked upon BAPTA/AM treatment whereas cells pretreated with EGTA did not show a decrease in cell death, confirming the importance of intracellular calcium release in this process

Summary

Introduction

Cell death is a necessary phenomenon for the normal development of vertebrate and invertebrate organisms. Through this process, organisms discard damaged cells in pathological conditions such as ischemic diseases, viral infections and radiation damage, among others. A few years ago, it was only possible to differentiate between two forms of death: apoptosis and necrosis [1]. Researchers have extensively studied the different mechanisms by which cells die in response to different stimuli [2,3,4] allowing the development of a wide range of well-characterized cell death mechanisms. The main forms of cell death have been divided into subclasses, as variations in the mechanism have been described, providing a better understanding of how this phenomenon works. All forms of cell death have different morphological and molecular characteristics, they are closely related and, in many cases, interconnected

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