Repeated treatment with subtoxic doses of TRAIL induces resistance to apoptosis through its death receptors in MDA-MB-231 breast cancer cells (original) (raw)

Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is being evaluated clinically in treating various malignancies. Previous studies have shown that repeated application of high doses of rhTRAIL results in a subpopulation of parental cells that is unresponsive to the death ligand. However, it is not clear whether TRAIL-sensitive cancer cells could acquire resistance to TRAIL treatment. Here, we found that MDA-MB-231 breast cancer cells, which are highly sensitive to TRAIL-induced apoptosis, became resistant to TRAIL killing after a prolonged exposure to subtoxic doses of rhTRAIL. The resulting TRAIL-resistant cells were cross-resistant to antibodies against its death receptors (DR4 and DR5); however, they retained sensitivity to several clinically relevant chemotherapies. Surface expression of DR4 and DR5 was significantly reduced in the selected cells, resulting in failure in death-inducing signaling complex formation and caspase activation. In addition, real-time PCR analysis revealed an upregulation in multiple apoptosis-regulator genes, including c-FLIP, Stat5a, and Stat5b. Inhibition of Janus-activated kinase, an upstream activator of signal transducer and activator of transcription 5 (Stat5), or knockdown of Stat5 itself partially restored cellular sensitivity to TRAIL-induced apoptosis, suggesting that Stat5 signaling is also involved in the development of TRAIL resistance. Furthermore, we showed that acquired TRAIL resistance was effectively eliminated by combination with etoposide, doxorubicin, or paclitaxel. These results suggest that tumor cells could acquire resistance to TRAIL therapy especially when they are repeatedly exposed to low levels of the death ligand, highlighting the necessity of combination with therapies that target the resistance mechanisms. (Mol Cancer Res mixture of three siRNA sequences (see Materials and Methods) targeting different regions of the human c-FLIP transcript. After 24 h, cells were analyzed by immunoblotting for c-FLIP protein expression (top) or treated with rhTRAIL (20 ng/mL) at 37°C for an additional 4 h followed by FACS analysis for apoptosis (bottom). E. The effect of Stat5b knockdown was assessed similarly as in D (top, immunoblots for Stat5 using an anti-pan Stat5 antibody recognizing both Stat5a and Stat5b, Bcl-XL, and cyclin D1; bottom, apoptosis after incubation with TRAIL for 4 h at 20 ng/mL). F. Cells were pretreated with a cell-permeable JAK kinase inhibitor at the indicated concentrations for 1 h, followed by rhTRAIL (20 ng/mL) for an additional 4 h. The results are expressed as JAK inhibitioninduced apoptosis (apoptosis % induced by JAK inhibitor combining with rhTRAIL − apoptosis % by rhTRAIL alone; n = 3, mean ± SD).