Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane

Neuroscience. 2006 Sep 1;141(3):1265-80. doi: 10.1016/j.neuroscience.2006.04.075. Epub 2006 Jun 12.

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Analysis of Variance
  • Anticarcinogenic Agents / therapeutic use*
  • Apoptosis / drug effects*
  • Blotting, Western / methods
  • Calcium / metabolism
  • Caspases / metabolism
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Cell Survival / physiology
  • Dose-Response Relationship, Drug
  • Fura-2
  • Ganglioglioma / drug therapy*
  • Humans
  • In Situ Nick-End Labeling / methods
  • Isothiocyanates
  • Models, Biological
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Signal Transduction / drug effects*
  • Signal Transduction / physiology
  • Thiocyanates / therapeutic use*
  • bcl-2-Associated X Protein / metabolism

Substances

  • Anticarcinogenic Agents
  • Isothiocyanates
  • Proto-Oncogene Proteins c-bcl-2
  • Thiocyanates
  • bcl-2-Associated X Protein
  • Caspases
  • sulforaphane
  • Calcium
  • Fura-2