Protein kinase C‐ERK1/2 signal pathway switches glucose depletion‐induced necrosis to apoptosis by regulating superoxide dismutases and suppressing reactive oxygen species production in A549 lung cancer cells

Abstract

Cells typically die by either apoptosis or necrosis. However, the consequences of apoptosis and necrosis are quite different for a whole organism. In the case of apoptosis, the cell content remains packed in the apoptotic bodies that are removed by macrophages, and thereby inflammation does not occur; during necrosis, the cell membrane is ruptured, and the cytosolic constituents are released into the extracellular space provoking inflammation. Recently, inflammation and necrosis have been suggested to promote tumor growth. We investigated the molecular mechanism underlying cell death in response to glucose depletion (GD), a common characteristic of the tumor microenvironment. GD induced necrosis through production of reactive oxygen species (ROS) in A549 lung carcinoma cells. Inhibition of ROS production by N-acetyl-L-cysteine and catalase prevented necrosis and switched the cell death mode to apoptosis that depends on mitochondrial death pathway involving caspase-9 and caspase-3 activation, indicating a critical role of ROS in determination of GD-induced cell death mode. We demonstrate that protein kinase C-dependent extracellular regulated kinase 1/2 (ERK1/2) activation also switched GD-induced necrosis to apoptosis through inhibition of ROS production possibly by inducing manganese superoxide dismutase (SOD) expression and by preventing GD-induced degradation of copper zinc SOD. Thus, these results suggest that GD-induced cell death mode is determined by the protein kinase C/ERK1/2 signal pathway that regulates MnSOD and CuZnSOD and that these antioxidants may exert their known tumor suppressive activities by inducing necrosis-to-apoptosis switch.

Role of reactive oxygen species-dependent protein aggregation in metabolic stress-induced necrosis

“…Previously, we have shown that metabolic stress induces necrosis and HMGB1 release through mitochondrial ROS production in several cancer cells (10). Here we show that a specific set of cellular proteins including pro-apoptotic molecules p53, caspase-3, and caspase-9 and a pro-autophagic molecule beclin 1 are aggregated in a ROS-dependent manner during GD-induced necrotic cell death.…”

“…A549, MDA-MB231, and MCF-7 cells were obtained from American Type Culture Collection and maintained in RPMI-1640 or DMEM containing 10% FBS and treated with GD or chemicals as described previously (10).…”

“…HO/PI staining was performed as described previously (10). For immunofluorescence and confocal microscopy, cells were fixed with 100% methanol for 10 min at -20˚C and then incubated with blocking buffer (PBS containing 0.1% Triton X-100 and anti-goat antibody) for 1 h at RT.…”

“…Unlike in apoptosis, where the cellular contents remain packed in the apoptotic bodies that are removed by phagocytes or neighboring cells, thereby resulting in cellular deletion without inflammation (5), during necrosis the cell membrane is ruptured, and many cellular proteins including the nuclear protein high mobility group box 1 (HMGB1) and the S100 family of molecules are relased into the extra-cellular space, thus provoking the inflammatory response and facilitating tumour progression (6)(7)(8). Previously, we also showed that CuZnSOD, but not the antioxidants MnSOD and catalase, is released into the extracellular space in an active form upon glucose depletion (GD), one of metabolic stresses, thereby accelerating ROS damage and facilitating necrotic cell death (9,10). Unlike tumour suppressive apoptosis (11)(12)(13) or autophagic cell death (14)(15), necrosis has been implicated in tumour growth and aggressiveness (16)(17)(18)(19)(20).…”

Implication of PI3K-dependent HSP27 and p53 expression in mild heat shock-triggered switch of metabolic stress-induced necrosis to apoptosis in A549 cells

“…5G and H). We have previously shown that in A549 cells, GD was accompanied by release of cytosolic CuZnSOD into extracellular medium, resulting in strong oxidative stress (39). In this regard, aggregation of p53 and HSP27 appears to be closely associated with incurrence of excessive oxidative stress.…”