Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane.
Glioblastoma is the most malignant and prevalent brain tumor that still remains incurable. Recent studies reported anti-cancer effect of the broccoli-derived compound sulforaphane. We explored the mechanisms of sulforaphane-mediated apoptosis in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed apoptosis in glioblastoma cells treated with sulforaphane. Increase in intracellular free Ca2+ was detected by fura-2 assay, suggesting activation of Ca2+-dependent pathways for apoptosis. Western blotting was used to detect changes in expression of Bax and Bcl-2 proteins resulting in increased Bax:Bcl-2 ratio that indicated a commitment of glioblastoma cells to apoptosis. Upregulation of calpain, a Ca2+-dependent cysteine protease, activated caspase-12 that in turn caused activation of caspase-9. With the increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol for sequential activation of caspase-9 and caspase-3. Increased calpain and caspase-3 activities generated 145 kD spectrin breakdown product and 120 kD spectrin breakdown product, respectively. Activation of caspase-3 also cleaved the inhibitor-of-caspase-activated-DNase. Accumulation of apoptosis-inducing-factor in cytosol suggested caspase-independent pathway of apoptosis as well. Two of the inhibitor-of-apoptosis proteins were downregulated because of an increase in 'second mitochondrial activator of caspases/Direct inhibitor-of-apoptosis protein binding protein with low pI.' Decrease in nuclear factor kappa B and increase in inhibitor of nuclear factor kappa B alpha expression favored the process of apoptosis. Collectively, our results indicated activation of multiple molecular mechanisms for apoptosis in glioblastoma cells following treatment with sulforaphane.
Karmakar
S
S
Department of Neurosciences, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 323K, P.O. Box 250606, Charleston, SC 29425, USA.
Weinberg
M S
MS
Banik
N L
NL
Patel
S J
SJ
Ray
S K
SK
D000704:Analysis of Variance; D016588:Anticarcinogenic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D016257:Fura-2; D018303:Ganglioglioma / Q000188:drug therapy*; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods; D017879:Isothiocyanates; D008954:Models, Biological; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013454:Sulfoxides; D013861:Thiocyanates / Q000627:therapeutic use*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D016588:Anticarcinogenic Agents; D017879:Isothiocyanates; D019253:Proto-Oncogene Proteins c-bcl-2; D013454:Sulfoxides; D013861:Thiocyanates; D051028:bcl-2-Associated X Protein; D020169:Caspases; C016766:sulforaphane; D002118:Calcium; D016257:Fura-2