Down-regulation of vinculin upon MK886-induced apoptosis in LN18 glioblastoma cells.
Glioblastomas are a type of malignant brain tumor and are among the most difficult cancers to treat. One strategy to treat aggressive cancers is the use of drugs that target multiple signaling pathways. MK886 is a drug known to inhibit both 5- lipoxygenase-activating-protein (FLAP) and peroxisome proliferator activated receptor-alpha (PPAR-alpha). The objectives of this study were to investigate the ability of MK886 to induce apoptotic cell death in LN18 glioblastoma cells and to characterize the cell death mechanisms. MK886 induced massive apoptotic LN18 cell death that was manifested by the release of nucleosomes, annexinV binding to phosphatidylserine in the absence of nuclear staining, and changes in the fluorescent intensity of Mito Tracker Deep Red 633 indicating changes in mitochondrial oxidative function and mass. The alteration of the mitochondrial function implied that MK886 induced apoptosis in LN18 cells via a mitochondrial pathway. The broad caspases inhibitor ZVAD-FMK inhibited MK886-induced nucleosome release, but not annexinV binding or MK886-altered mitochondrial function. Real time RT-PCR demonstrated that LN18 cells expressed significant levels of FLAP and PPAR- alpha mRNAs. A low level of arachidonate 5-lipoxygenase (ALOX-5) mRNA was detected, but little, if any, arachidonate 12- lipoxygenase (ALOX-12) mRNA was present. In addition, MK886-induced apoptosis in LN18 cells was accompanied by a decrease in the protein and mRNA levels of vinculin, but not other focal adhesion proteins. In summary, the data presented here indicate that disruption of the actin-vinculin-cell-cytoskeleton matrix of the LN18 glioblastoma is a component of the MK886 induced apoptosis. In addition, MK886 treated LN18 cells could provide one model in which to investigate drugs that target lipoxygenase and PPAR-alpha pathways in the chemotherapeutic treatment of glioblastomas.
Magro
A M
AM
Department of Biology, Fairmont University, Fairmont, WV 26554, USA. amagro@fairmontstate.edu
Magro
A D
AD
Cunningham
C
C
Miller
M R
MR
D058927:5-Lipoxygenase-Activating Proteins; D000199:Actins / Q000096:biosynthesis / Q000187:drug effects; D017209:Apoptosis / Q000187:drug effects*; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology*; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects; D047493:PPAR alpha / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016596:Vinculin / Q000096:biosynthesis / Q000187:drug effects*; D002722:Chlorobenzenes
D058927:5-Lipoxygenase-Activating Proteins; D000199:Actins; D002352:Carrier Proteins; D007211:Indoles; D016859:Lipoxygenase Inhibitors; D008565:Membrane Proteins; D047493:PPAR alpha; D012333:RNA, Messenger; D016596:Vinculin; C548592:ALOX5AP protein, human; C060893:MK-886; D002722:Chlorobenzenes