Factors secreted by human neuroblastoma mediated doxorubicin resistance by activating STAT3 and inhibiting apoptosis

Mol Med. 2001 Jun;7(6):393-400.


Background: The transcription factor Stat3 has been reported to play a key role in protecting cells against apoptosis by up-regulating expression of the anti-apoptotic gene BCl-xL. This investigation analyzes the relationship between the development of resistance to doxorubicin-mediated apoptosis in neuroblastoma cells (SKN-SH) and activation of the Stat3 signaling pathway.

Materials and methods: A drug-resistant cell line (SKN-SH/Dox6) was generated by continuous exposure to incremental concentrations of doxorubicin. Specific antibodies were utilized for Western blots and confocal microscopy to determine the nuclear localization of activated Stat3.

Results: Doxorubicin-mediated DNA fragmentation was inhibited and caspase-3 activity decreased in SKN-SH/Dox6 cells. Up-regulation of Stat3 phosphorylation and Bcl-xL expression, increased nuclear translocation of phospho-Stat3, and binding to DNA occurred only in resistant SKN-SH/Dox6 cells. The expression of Bcl-xL was inhibited by AG490, an inhibitor of the JAK/Stat3 signaling pathway, suggesting that the regulation of Bcl-xL and Stat3 involved a common mechanism. Activation of Stat3 in SKN-SH/Dox6 cells was contingent upon stimulation evoked by ligands secreted by the drug-resistant cells. Evidence to support this hypothesis was provided by experiments in which doxorubicin-sensitive SKN-SH cells were preincubated with conditioned media obtained from doxorubicin-resistant SKN-SH/Dox6 cells. This treatment increased Stat3 activation. It also rendered SKN-SH cells resistant to doxorubicin as demonstrated by a sharp decrease in doxorubicin-induced DNA degradation and cytotoxic potency.

Conclusions: These findings suggest that the resistance of SKN-SH/Dox6 cells to doxorubicin may be mediated by anti-apoptotic factor(s) that are synthesized and secreted by tumor cells in response to cytotoxic agents.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis*
  • Culture Media, Conditioned
  • DNA-Binding Proteins / metabolism*
  • Doxorubicin / therapeutic use*
  • Drug Resistance, Neoplasm*
  • Humans
  • Neuroblastoma / chemistry
  • Neuroblastoma / drug therapy*
  • Neuroblastoma / metabolism*
  • Phosphorylation
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • STAT3 Transcription Factor
  • Signal Transduction
  • Trans-Activators / metabolism*
  • Tumor Cells, Cultured
  • Tyrphostins / pharmacology
  • bcl-X Protein


  • BCL2L1 protein, human
  • Culture Media, Conditioned
  • DNA-Binding Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • STAT3 Transcription Factor
  • STAT3 protein, human
  • Trans-Activators
  • Tyrphostins
  • bcl-X Protein
  • Doxorubicin