Oxaliplatin triggers necrosis as well as apoptosis in gastric cancer SGC-7901 cells
Introduction
Programmed cell death (PCD) is a ubiquitous process to sustain tissue homeostasis and health, and evading PCD is one of the hallmarks of cancer. Conversely, inducing cell death by pharmacological means is the basis of cancer therapy. Unquestionably, apoptosis is the best-characterized form of PCD. However, over the past decade, extensive studies have uncovered a growing number of non-apoptotic cell death pathways, such as lysosomal-mediated cell death, autophagic cell death and necroptosis.
Necrosis, which was thought to be a passive event as a result of overwhelming insults, may be executed through a regulated mechanism termed necroptosis or programmed necrosis. Several stimuli can trigger the necrotic response, such as cytokines, pathogen, alkylating DNA damage, excitotoxins or irradiation [1]. Nowadays the most extensively studied models are tumor necrosis factor-α (TNF-α)-induced receptor interacting protein 1 (RIP1)/receptor interacting protein 3 (RIP3)-dependent pathway and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced poly(ADP-ribose) polymerase-1 (PARP-1) pathway.
Stimulation with TNF-α leads to the activation of RIP1, then RIP3 is recruited to RIP1, the two kinases phosphorylate each other [2]. Necrostatin-1 (Nec-1), a small-molecule inhibitor of necroptosis, is a selective inhibitor of RIP1 kinase [3]. RIP3 in turn phosphorylates mixed lineage kinase domain-like protein (MLKL) [4]. RIP1, RIP3 and MLKL are the core components of the necrosome. Downstream of the necrosome are phosphoglycerate mutase family member 5 (PGAM5), which specifically associates with the necrosome, resulting in PGAM5 phosphorylation and activation. PGAM5 recruits the mitochondrial fission factor dynamin-related protein 1 (Drp1) and activates its GTPase activity by dephosphorylating Drp1. Drp1 activation caused mitochondrial fragmentation and eventually, necroptosis [5].
In response to DNA-alkylating agents such as MNNG, DNA repair enzyme PARP-1 was overactivated, resulting in the massive synthesis of poly(ADP-ribose) (PAR) from NAD+ and, in consequence, to the rapid depletion of intracellular NAD+ and ATP pools [6] and calpains activation [7]. Calpains cleave Bid into tBid, which redistributes from the cytosol to mitochondria, where it facilitates Bax activation. Furthermore, Bax provokes mitochondrial damage and favors the release of apoptosis-inducing factor (AIF) from mitochondria to the cytosol and nucleus [8]. Upon transferring to the nucleus, AIF associates with cyclophilin A (CypA) and H2AX to generate a DNA-degrading complex that promotes chromatinolysis and cell-viability loss [9].
Oxaliplatin is the third generation of platinum anticancer drugs that is widely used in various cancers. As an alkylating agent, oxaliplatin works by binding to DNA leading to oxaliplatin-DNA adducts. The mechanism whereby DNA adducts kill cells is not fully understood. Is oxaliplatin-induced cell death always produced by apoptosis? Whether oxaliplatin could induce necrosis in gastric cancer cells needs to be examined. We report here that although oxaliplatin activates caspase cascade, it kills SGC-7901 cells predominantly by necrosis dependent on RIP1. And PARP-1 overactivation-induced energy depletion, H2AX phosphorylation and AIF translocation are critical in this process.
Section snippets
Cell culture
Human gastric cancer cell SGC-7901 was obtained from Type Culture Collection of Chinese Academy of Sciences. They were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Hyclone).
Reagents
Oxaliplatin was purchased from Jiangsu Hengrui Medicine. PAR antibody and Nec-1 were provided by Enzo Life Sciences. Necrosulfonamide (NSA) and z-VAD-fmk were obtained from Calbiochem. Mdivi-1 was from Sigma–Aldrich. Olaparib was purchased from Selleck. Antibodies against caspase-3, PARP-1, Bcl-2,
Oxaliplatin induces cell death in gastric cancer SGC-7901 cells
To identify the type of cell death induced by oxaliplatin, we first examined the biochemical events in SGC-7901 cells. Dual staining with Annexin V-FITC and PI was used to discriminate apoptosis and necrosis. As depicted in Fig. 1A, the majority of dead cells became positive for both Annexin V and PI, and some only for PI, even at 24 h when only a small proportion of cells died. Furthermore, cell death was associated with reduction in ΔΨm (Fig. 1B), indicating that mitochondrial depolarization
Discussion
During the past decade, cell death researchers have witnessed a gradual conceptual revolution: necrosis, which had been considered as a purely accidental cell death mode, can also be induced by finely regulated signaling pathways. The term necroptosis was firstly introduced by Degterev and his colleagues [10] in 2005 to describe that in the absence of caspases, ligation of TNF-α with its receptor would prime cells to necrotic cell death. At present, it has been established that necrotic cell
Conflict of interest
None.
Acknowledgments
This work was supported by grants from National Natural Science Foundation of China (No. 81071809) and Natural Science Research Project of Anhui Higher Education Institutions, China (No. KJ2014A113).
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