title: Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane.
abstract: 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.
authors.affiliation: Department of Neurosciences, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 323K, P.O. Box 250606, Charleston, SC 29425, USA.
authors.forename: S
authors.initials: S
authors.lastname: Karmakar
authors.forename: M S
authors.initials: MS
authors.lastname: Weinberg
authors.forename: N L
authors.initials: NL
authors.lastname: Banik
authors.forename: S J
authors.initials: SJ
authors.lastname: Patel
authors.forename: S K
authors.initials: SK
authors.lastname: Ray
mesh_terms: 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
chemical_list: 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