Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma.
BACKGROUND
Our recent work demonstrated that treatment of neuroblastoma with triptolide causes apoptotic cell death in vitro and decreases tumor size in vivo. Triptolide therapy has been associated with reduced expression of Hsp-70, suggesting a mechanism of cell killing involving Hsp-70 inhibition. The principal objective of this study was to investigate the role of Hsp-70 in triptolide-mediated cell death in neuroblastoma.


METHODS
Neuroblastoma cells were transfected with Hsp-70-specific siRNA. Viability, caspase activity, and phosphatidylserine externalization were subsequently measured. An orthotopic, syngeneic murine tumor model was developed, and randomized mice received daily injections of triptolide or vehicle. At 21 d, mice were sacrificed. Immunohistochemisty was used to characterize Hsp-70 levels in residual tumors, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to identify cells undergoing apoptosis.


RESULTS
Targeted silencing of Hsp-70 with siRNA significantly decreased cellular viability, augmented caspase-3 activity, and resulted in increased annexin-V staining. These effects parallel those findings obtained following treatment with triptolide. Residual tumors from triptolide-treated mice showed minimal staining with Hsp-70 immunohistochemistry, while control tumors stained prominently. Tumors from treated mice demonstrated marked staining with the TUNEL assay, while control tumors showed no evidence of apoptosis.


CONCLUSIONS
Use of siRNA to suppress Hsp-70 expression in neuroblastoma resulted in apoptotic cell death, similar to the effects of triptolide. Residual tumors from triptolide-treated mice expressed decreased levels of Hsp-70 and demonstrated significant apoptosis. These findings support the hypothesis that Hsp-70 inhibition plays a significant role in triptolide-mediated neuroblastoma cell death.
Antonoff
Mara B
MB
Department of Surgery, University of Minnesota, Minneapolis, Minnesota 55455, USA. antonoff@umn.edu
Chugh
Rohit
R
Skube
Steven J
SJ
Dudeja
Vikas
V
Borja-Cacho
Daniel
D
Clawson
Kimberly A
KA
Vickers
Selwyn M
SM
Saluja
Ashok K
AK
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D004224:Diterpenes / Q000494:pharmacology / Q000627:therapeutic use*; D004353:Drug Evaluation, Preclinical; D004852:Epoxy Compounds / Q000494:pharmacology / Q000627:therapeutic use; D020868:Gene Silencing; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism; D010616:Phenanthrenes / Q000494:pharmacology / Q000627:therapeutic use*; D034741:RNA, Small Interfering
D018906:Antineoplastic Agents, Alkylating; D004224:Diterpenes; D004852:Epoxy Compounds; D018840:HSP70 Heat-Shock Proteins; D010616:Phenanthrenes; D034741:RNA, Small Interfering; C001899:triptolide