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


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.
Rebbaa
A
A
Children's Memorial Institute for Education and Research, Cancer Biology and Chemotherapy Program, Chicago, Illinois 60614, USA.
Chou
P M
PM
Mirkin
B L
BL
D000818:Animals; D017209:Apoptosis; D017077:Culture Media, Conditioned; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004317:Doxorubicin / Q000627:therapeutic use*; D019008:Drug Resistance, Neoplasm; D006801:Humans; D009447:Neuroblastoma / Q000737:chemistry / Q000188:drug therapy* / Q000378:metabolism*; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D020032:Tyrphostins / Q000494:pharmacology; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D017077:Culture Media, Conditioned; D004268:DNA-Binding Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D020032:Tyrphostins; D051020:bcl-X Protein; D004317:Doxorubicin