Abstract
The anthracyclines, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we showed that these drugs could activate the transcription factor, nuclear factor kappaB, in a DNA damage-dependent manner. We now show that these drugs can potentiate the activation of signal transducer and activator of transcription 1 (STAT1) in MDA-MB 435 breast cancer cells treated with IFN-gamma. We observed that key markers of STAT1 activation, including tyrosine 701 and serine 727 phosphorylation, were enhanced in the presence of doxorubicin. This potentiation resulted in enhanced nuclear localization of activated STAT1 and led to an increase in the nuclear binding of activated STAT complexes. The observed potentiation was specific for STAT1 and IFN-gamma, as no effects were observed with either STAT3 or STAT5. Furthermore, the type I IFNs (alpha and beta) had little or no effect. The observed effects on STAT1 phosphorylation have previously been linked with maximal transcriptional activation and apoptosis. Cell viability was assessed by crystal violet staining followed by analysis with CalcuSyn to determine combination index values, a measure of synergy. We confirmed that significant synergy existed between IFN-gamma and doxorubicin (combination index = 0.34) at doses lower than IC(50) values for this drug (0.67 micromol/L). In support of this, we observed that apoptotic cell death was also enhanced by measuring poly(ADP-ribose) polymerase and caspase-3 cleavage. Finally, suppression of STAT1 expression by small-interfering RNA resulted in a loss of synergistic apoptotic cell death compared with cells, where no suppression of STAT1 expression was attained with scrambled small-interfering RNA control. We conclude that doxorubicin potentiates STAT1 activation in response to IFN-gamma, and that this combination results in enhanced apoptosis in breast cancer cells.
Highlights
Members of the signal transducer and activator of transcription (STAT) family of transcription factors regulate the expression of a variety of genes involved in proliferation, differentiation, survival, and apoptosis [1, 2]
Doxorubicin Potentiates the Activation of signal transducer and activator of transcription 1 (STAT1) in MDA-MB 435 Breast Cancer Cells Treated with IFN-␥
We observed that IFN-␥ (500 units) treatment of MDA-MB 435 breast cancer cells resulted in the expected increase in the tyrosine 701 phosphorylated form of STAT1 (Fig. 1A), required for STAT dimerization and nuclear translocation [1, 24]
Summary
Members of the signal transducer and activator of transcription (STAT) family of transcription factors regulate the expression of a variety of genes involved in proliferation, differentiation, survival, and apoptosis [1, 2]. Our overall aim was to investigate the link between STAT1 potentiation and enhanced apoptotic cell death, observed concurrently in breast cancer cells treated with topoisomerase II-targeted drugs and the type II cytokine, IFN-␥.
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