Factors Secreted by Human Neuroblastoma Mediate 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-apoptolic gene Bcl-x_L. This investigation analyzes the relationship between the development of resistance to doxorubicinmediated 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-x_L expression, increased nuclear translocation of phospho-Stat3, and binding to DNA occurred only in resistant SKN-SH/Dox6 cells. The expression of Bcl-x_L was inhibited by AG490, an inhibitor of the JAK/Stat3 signaling pathway, suggesting that the regulation of Bcl-x_L 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.