PU-H71 effectively induces degradation of IκB kinase β in the presence of TNF-α

This study is to determine if PU-H71, a heat shock protein inhibitor, induces killing of malignant breast cells together with treatment of tumor necrosis factor-alpha (TNF-alpha). The related molecular mechanisms were also studied. A primary mammary epithelial cell line HMEC2595 cells and the highly metastatic breast cell line MDA-MB-231, the HER2-positive BT-474 cells, and the ER-positive MCF7 cells were treated with PU-H71 in the presence or absence of TNF-alpha. The effects of PU-H71 and TNF-alpha treatments on cells viabilities and on intracellular signaling pathway proteins were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, apoptosis assays, immunoblot assays, and luciferase assays. It was found that TNF-alpha enhances the toxic effects of PU-H71 on tumor cells but not normal cells. PU-H71 treatments lead to degradation of IKK beta. Moreover, PU-H71 down-regulates the NF-kappa B transcriptional activity induced by TNF-alpha treatment. The experimental results indicated PU-H71 effectively induces cell killing of malignant breast cells in the presence of TNF-alpha, possibly through a mechanism related to degradation of IKKb. It is suggested that combination of PU-H71 and TNF-alpha treatments might be an effective therapeutic strategy of breast malignancies.