ABSTRACT
The proapoptotic protein Noxa, a member of the BH3-only Bcl-2 protein family, can effectively induce apoptosis in cancer cells, although the relevant regulatory pathways have been obscure. Previous studies of the cytotoxic effects of α-tocopheryl succinate (α-TOS) on cancer cells identified a mechanism whereby α-TOS caused apoptosis requiring the Noxa-Bak axis. In the present study, ab initio analysis revealed a conserved FoxO-binding site (DBE; DAF-16 binding element) in the NOXA promoter, and specific affinity of FoxO proteins to this DBE was confirmed by fluorescence anisotropy. FoxO1 and FoxO3a proteins accumulated in the nucleus of α-TOS-treated cells, and the drug-induced specific FoxO1 association with the NOXA promoter and its activation were validated by chromatin immunoprecipitation. Using siRNA knockdown, a specific role for the FoxO1 protein in activating NOXA transcription in cancer cells was identified. Furthermore, the proapoptotic kinase Hippo/Mst1 was found to be strongly activated by α-TOS, and inhibiting Hippo/Mst1 by specific siRNA prevented phosphorylation of FoxO1 and its nuclear translocation, thereby reducing levels of NOXA transcription and apoptosis in cancer cells exposed to α-TOS. Thus, we have demonstrated that anticancer drugs, exemplified by α-TOS, induce apoptosis by a mechanism involving the Hippo/Mst1-FoxO1-Noxa pathway. We propose that activation of this pathway provides a new paradigm for developing targeted cancer treatments.
Subject(s)
Forkhead Transcription Factors/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-bcl-2/biosynthesis , alpha-Tocopherol/pharmacology , Apoptosis/physiology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/therapy , Cell Line, Tumor , Forkhead Box Protein O1 , Forkhead Transcription Factors/genetics , Humans , Intracellular Signaling Peptides and Proteins , Jurkat Cells , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/therapy , Lymphoma, T-Cell/genetics , Lymphoma, T-Cell/metabolism , Lymphoma, T-Cell/pathology , Lymphoma, T-Cell/therapy , Promoter Regions, Genetic , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Transcription, GeneticABSTRACT
The role of the death-associated protein Daxx in modulation of apoptosis induced in cardiac myocytes by oxidative stress was studied. Exposure of cultured cardiomyocyte-like cells to oxidative stress or simulated hypoxia increased the level of accumulated ROS and apoptosis. Under conditions of sub-apoptotic stimulation of cardiac myocytes, there was no increase in the level of the Daxx protein, but it translocated from the nucleus to the cytoplasm. Daxx overexpression protected the cells from apoptosis, while they were sensitised to cell death following its down-regulation by siRNA. Moreover, lowering the level of the Daxx protein sensitised cardiac myocytes to spontaneous apoptosis, suggesting that the protein may also have a pro-survival role under physiological conditions. Finally, it was shown that DJ-1, a protein suggested previously to sequester Daxx in the nucleus under conditions of oxidative stress (thereby preventing its cytosolic translocation), was localised solely in the cytoplasm of cardiac myocytes. This indicates that the protein does not modulate the apoptosis regulatory activity of Daxx in cardiac myocytes by its nuclear sequestration. Taken together, Daxx plays a protective role in cultured cardiomyocyte-like cells, at least under the conditions used.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Apoptosis/physiology , Carrier Proteins/physiology , Intracellular Signaling Peptides and Proteins/physiology , Myocytes, Cardiac/cytology , Nuclear Proteins/physiology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis/genetics , Blotting, Western , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Cell Nucleus/metabolism , Co-Repressor Proteins , Cytoplasm/metabolism , Flow Cytometry , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Microscopy, Confocal , Microtubule-Associated Proteins/metabolism , Molecular Chaperones , Myocytes, Cardiac/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oncogene Proteins/metabolism , Peroxiredoxins , Protein Deglycase DJ-1 , RNA, Small Interfering/genetics , Rats , Reactive Oxygen Species/metabolism , TransfectionABSTRACT
Recent evidence suggests that a subset of cells within a tumour have 'stem-like' characteristics. These tumour-initiating cells, distinct from non-malignant stem cells, show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumour cells, resistance to chemotherapy or radiation, and they are often characterised by elevated expression of the stem cell surface marker CD133. Understanding the molecular biology of the CD133(+) cancer cells is now essential for developing more effective cancer treatments. These may include drugs targeting organelles, such as mitochondria or lysosomes, using highly efficient and selective inducers of apoptosis. Alternatively, agents or treatment regimens that enhance sensitivity of these therapy-resistant "tumour stem cells" to the current or emerging anti-tumour drugs would be of interest as well.
