Abstract
Poly(ADP-ribose)polymerase-1 (PARP-1) is thought to be required for apoptosis-inducing factor (AIF) release from mitochondria in caspase-independent apoptosis. The mechanism by which AIF is released through PARP-1 remains unclear. Here, we provide evidence that PARP-1-independent AIF release and cell death are induced by a trienoic fatty acid, alpha-eleostearic acid (alpha-ESA). Alpha-ESA induced the caspase-independent and AIF-initiated apoptotic death of neuronal cell lines, independently of PARP-1 activation. The cell death was inhibited by the MEK inhibitor U0126 and by knockdown of MEK using small interfering RNA. However, inhibitors for JNK, p38 inhibitors, calpain, phospholipase A(2), and phosphatidylinositol 3-kinase, did not block cell death. AIF was translocated to the nucleus after the induction of apoptosis by alpha-ESA in differentiated PC12 cells without activating caspase-3 and PARP-1. The alpha-ESA-mediated cell death was not inhibited by PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinoline and by knockdown of PARP-1 using small interfering RNA. Unlike N-methyl-N'-nitro-N-nitrosoguanidine treatment, histone-phosphorylated histone 2AX was not phosphorylated by alpha-ESA, which suggests no DNA damage. Overexpression of Bcl-2 did not inhibit the cell death. alpha-ESA caused a small quantity of superoxide production in the mitochondria, resulting in the reduction of mitochondrial membrane potential, both of which were blocked by a trace amount of alpha-tocopherol localized in the mitochondria. Our results demonstrate that alpha-ESA induces PARP-1-independent AIF release and cell death without activating Bax, cytochrome c, and caspase-3. MEK is also a key molecule, although the link between ERK, AIF release, and cell death remains unknown. Finding molecules that regulate AIF release may be an important therapeutic target for the treatment of neuronal injury.
Highlights
Apoptosis is a mode of programmed cell death that is used by multicellular organisms to remove surplus and unwanted cells in the immune and nervous systems [1,2,3,4,5]
Our results demonstrate that ␣-ESA induces Poly(ADP-ribose) polymerase-1 (PARP-1)-independent apoptosis-inducing factor (AIF) release and cell death without activating Bax, cytochrome c, and caspase-3
Apoptosis is activated through two main pathways as follows: the extrinsic pathway, which originates from the activation of cell-surface death receptors, such as Fas and tumor necrosis factor-receptor 1, and results in the activation of caspase-8; and the intrinsic pathway, which originates from the mitochondrial release of cytochrome c and results in the activation of caspase-9 through the Cyt-c2/apoptotic protease-activating factor-1/procaspase-9 heptamer [5, 8, 9]
Summary
Cyt-c, cytochrome c; ␣-ESA, ␣-eleostearic acid; MEK, mitogen-activated protein kinase kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; ␥-H2AX, phosphorylated histone 2AX; AIF, apoptosis-inducing factor; Bak, Bcl-2-antagonist/killer; Bax, Bcl-2-associated X protein; CPT, camptothecin; DPQ, 3,4-dihydro-5-[4(1-piperidinyl)butoxyl]-1(2H)-isoquinoline; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase kinase; MNNG, N-methyl-NЈ-nitro-N-nitrosoguanidine; NGF, nerve growth factor; PARP-1, poly(ADP-ribose) polymerase-1; Z-, N-benzyloxycarbonyl; fmk, fluoromethyl ketone; ROS, reactive oxygen species; ␣-ESA, ␣-eleostearic acid; Ab, antibody; siRNA, small interfering RNA; TUNEL, terminal dUTP nick endlabeling; ␣-Toc, ␣-tocopherol; NMDA, N-methyl-D-aspartic acid; STS, staurosporine; JNK, c-Jun N-terminal kinase; PAR, polymer of ADP-ribose; CM-H2DCF-DA, 5(and 6)-chloromethyl-2Ј,7Ј-dichlorodihydrofluorescein diacetate; NBD, 7-nitro-2,1,3-benzoxadiazol-4-yl. ␣-ESA has been reported to suppress tumor growth through caspase-3 and peroxisome proliferator-activated receptor-␥ activation accompanied by DNA fragmentation [22,23,24]. We have found that ␣-ESA induces caspase-independent apoptosis that is not associated with nucleosomal DNA fragmentation in neuronal cells. ␣-ESA-mediated apoptotic cell death is accompanied by AIF translocation to the nucleus and prolonged ERK phosphorylation that lasts for more than 16 h, but not by PARP-1 activation, in rat adrenal pheochromocytoma PC12 cells. We show that ␣-ESA causes PARP-1-independent AIF release and the cell death through the superoxide production in a small quantity in the mitochondria and the prolonged ERK1/2 phosphorylation without inducing other apoptotic molecules such as Bax, Bcl-2, Cyt-c, caspase-3, and PARP-1
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