Garlic compounds generate reactive oxygen species leading to activation of stress kinases and cysteine proteases for apoptosis in human glioblastoma T98G and U87MG cells.
BACKGROUND
Garlic-derived organosulfur compounds such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS) provide significant protection against carcinogenesis.


METHODS
Dose-dependent cytotoxic effects of the garlic compounds (DAS, DADS, and DATS) were tested in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed induction of apoptosis. Measurements showed that production of reactive oxygen species (ROS) and an increase in intracellular free [Ca(2+)] promoted apoptosis. Western blot analysis indicated that increased expression and activities of the stress kinases and cysteine proteases caused apoptosis. Use of JC-1 showed changes in mitochondrial membrane potential (Delta psi(m)) for mediation of apoptosis. Use of the specific inhibitors monitored the activation of different kinases and proteases in apoptosis.


RESULTS
Treatment of glioblastoma cells with garlic compounds triggered production of ROS that induced apoptosis with the phosphorylation of p38 MAPK and activation of the redox-sensitive JNK1 pathway. Pretreatment of cells with ascorbic acid attenuated ROS production, p38 MAPK phosphorylation, and JNK1 activation. Pretreatment with JNK1 inhibitor I also significantly reduced cell death. Increases in intracellular free [Ca(2+)], expression of calreticulin, and activation of caspase-4 indicated involvement of endoplasmic reticulum (ER) stress in apoptosis. Other events in apoptosis included overexpression of Bax, down-regulation of Bcl-2 and some BIRC proteins, mitochondrial release of cytochrome c and Smac into the cytosol, and activation of calpain, caspase-9, and caspase-3.


CONCLUSIONS
Garlic compounds induced apoptosis in glioblastoma cells due toproduction of ROS, increase in ER stress, decrease in Delta psi(m), and activation of stress kinases and cysteine proteases.
Das
Arabinda
A
Department of Neurosciences, Medical University of South Carolina, Charleston, SC 29425, USA.
Banik
Naren L
NL
Ray
Swapan K
SK
D000498:Allyl Compounds / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004220:Disulfides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005737:Garlic / Q000737:chemistry*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006801:Humans; D048055:Mitogen-Activated Protein Kinase 8 / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D013440:Sulfides / Q000494:pharmacology
D000498:Allyl Compounds; D051017:Apoptosis Regulatory Proteins; D004220:Disulfides; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013440:Sulfides; C042577:diallyl trisulfide; C028009:diallyl disulfide; C038491:allyl sulfide; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D003546:Cysteine Endopeptidases; D002118:Calcium