Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma

Mara B. Antonoff, Rohit Chugh, Steven J. Skube, Vikas Dudeja, Daniel Borja-Cacho, Kimberly A. Clawson, Selwyn M. Vickers, Ashok Saluja

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Background: Our recent work demonstrated that treatment of neurobastoma 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. Materials and 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.

Original languageEnglish (US)
Pages (from-to)72-78
Number of pages7
JournalJournal of Surgical Research
Volume163
Issue number1
DOIs
StatePublished - Sep 1 2010
Externally publishedYes

Fingerprint

Neuroblastoma
Cell Death
Residual Neoplasm
Small Interfering RNA
Neoplasms
Apoptosis
Staining and Labeling
triptolide
DNA Nucleotidylexotransferase
Annexin A5
Phosphatidylserines
Caspases
Transferases
Caspase 3
Therapeutics
Immunohistochemistry
Injections

Keywords

  • apoptosis
  • Hsp-70
  • neuroblastoma, heat shock protein
  • triptolide

ASJC Scopus subject areas

  • Surgery

Cite this

Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma. / Antonoff, Mara B.; Chugh, Rohit; Skube, Steven J.; Dudeja, Vikas; Borja-Cacho, Daniel; Clawson, Kimberly A.; Vickers, Selwyn M.; Saluja, Ashok.

In: Journal of Surgical Research, Vol. 163, No. 1, 01.09.2010, p. 72-78.

Research output: Contribution to journalArticle

Antonoff, MB, Chugh, R, Skube, SJ, Dudeja, V, Borja-Cacho, D, Clawson, KA, Vickers, SM & Saluja, A 2010, 'Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma', Journal of Surgical Research, vol. 163, no. 1, pp. 72-78. https://doi.org/10.1016/j.jss.2010.04.047
Antonoff, Mara B. ; Chugh, Rohit ; Skube, Steven J. ; Dudeja, Vikas ; Borja-Cacho, Daniel ; Clawson, Kimberly A. ; Vickers, Selwyn M. ; Saluja, Ashok. / Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma. In: Journal of Surgical Research. 2010 ; Vol. 163, No. 1. pp. 72-78.
@article{8efc3a8bf5034ede9ee77bcf0b4aa985,
title = "Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma",
abstract = "Background: Our recent work demonstrated that treatment of neurobastoma 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. Materials and 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.",
keywords = "apoptosis, Hsp-70, neuroblastoma, heat shock protein, triptolide",
author = "Antonoff, {Mara B.} and Rohit Chugh and Skube, {Steven J.} and Vikas Dudeja and Daniel Borja-Cacho and Clawson, {Kimberly A.} and Vickers, {Selwyn M.} and Ashok Saluja",
year = "2010",
month = "9",
day = "1",
doi = "10.1016/j.jss.2010.04.047",
language = "English (US)",
volume = "163",
pages = "72--78",
journal = "Journal of Surgical Research",
issn = "0022-4804",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma

AU - Antonoff, Mara B.

AU - Chugh, Rohit

AU - Skube, Steven J.

AU - Dudeja, Vikas

AU - Borja-Cacho, Daniel

AU - Clawson, Kimberly A.

AU - Vickers, Selwyn M.

AU - Saluja, Ashok

PY - 2010/9/1

Y1 - 2010/9/1

N2 - Background: Our recent work demonstrated that treatment of neurobastoma 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. Materials and 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.

AB - Background: Our recent work demonstrated that treatment of neurobastoma 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. Materials and 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.

KW - apoptosis

KW - Hsp-70

KW - neuroblastoma, heat shock protein

KW - triptolide

UR - http://www.scopus.com/inward/record.url?scp=77955919366&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77955919366&partnerID=8YFLogxK

U2 - 10.1016/j.jss.2010.04.047

DO - 10.1016/j.jss.2010.04.047

M3 - Article

C2 - 20638672

AN - SCOPUS:77955919366

VL - 163

SP - 72

EP - 78

JO - Journal of Surgical Research

JF - Journal of Surgical Research

SN - 0022-4804

IS - 1

ER -