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?NAC inhibition of the FADD-JNK axis plays anti-apoptotic role in multiple cancer cells.

ABSTRACT: Nascent polypeptide-associated complex ? (?NAC) is reportedly overexpressed in several types of cancers and regulates cell apoptosis under hypoxic conditions in HeLa cells. The aim of our study was to investigate the apoptotic function of ?NAC in cancer progression. First, we observed the cellular effects of ?NAC depletion. Mouse ?NAC was used to restore the protein level and verify the effect. An Annexin V assay, a caspase activity reporter assay, an apoptotic molecular marker, and a colony formation assay were used as markers to investigate the mechanisms of cell death caused by ?NAC depletion. The Cancer 10-pathway reporter assay was used to screen downstream pathways. PCR site-directed deletion based on the functional domains of ?NAC was used to construct deletion mutants. Those functional domain deletion mutants were used to recover the apoptotic phenotype caused by ?NAC depletion. Finally, the role of ?NAC in TNF-related apoptosis-inducing ligand (TRAIL) treatment was investigated in vitro. We found that depletion of ?NAC in multiple types of cancer cells induce typical apoptotic cell death. This anti-apoptotic function is mediated by the FADD/c-Jun N-terminal kinase pathway. Intact ?NAC is required for the direct binding of FADD as well as its anti-apoptosis function. Either ?NAC depletion or the deletion of the ubiquitin-associated domain of ?NAC sensitizes L929 cancer cells to mTRAIL treatment. Our study revealed a ?NAC anti-apoptotic function in multiple types of cancer cells and suggested its potential in cancer therapy.

SUBMITTER: Zeng W

PROVIDER: S-EPMC4611707 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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αNAC inhibition of the FADD-JNK axis plays anti-apoptotic role in multiple cancer cells.

Zeng W W Zhang J J Qi M M Peng C C Su J J Chen X X Yuan Z Z

Cell death & disease 20140605

Nascent polypeptide-associated complex α (αNAC) is reportedly overexpressed in several types of cancers and regulates cell apoptosis under hypoxic conditions in HeLa cells. The aim of our study was to investigate the apoptotic function of αNAC in cancer progression. First, we observed the cellular effects of αNAC depletion. Mouse αNAC was used to restore the protein level and verify the effect. An Annexin V assay, a caspase activity reporter assay, an apoptotic molecular marker, and a colony for ...[more]

PMID: 24901053

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Project description:BackgroundSynthetic indolyl- pyridinyl- propenones (IPPs) induce methuosis, a form of non-apoptotic cell death, in glioblastoma and other cancer cell lines. Methuosis is characterized by accumulation of cytoplasmic vacuoles derived from macropinosomes and late endosomes, followed by metabolic failure and rupture of the plasma membrane. However, not all IPPs that cause vacuolization are cytotoxic. The main goals of the present study were to identify key signaling pathways that contribute to methuosis induced by cytotoxic IPPs and to evaluate the anti-tumor potential of a prototype IPP in vivo.MethodsWe utilized metabolic flux analysis, glucose uptake, immunoblotting, and selective pharmacological inhibitors to compare the effects of closely related cytotoxic and non-cytotoxic IPPs in cultured glioblastoma cells. To determine whether the use of methuosis-inducing IPPs might be feasible in a therapeutic context, we quantified the distribution of our lead IPP compound, MOMIPP, in mouse plasma and brain, and tested its ability to inhibit tumor growth in an intracerebral glioblastoma xenograft model.ResultsThe cytotoxic IPP compound, MOMIPP, causes early disruptions of glucose uptake and glycolytic metabolism. Coincident with these metabolic changes, MOMIPP selectively activates the JNK1/2 stress kinase pathway, resulting in phosphorylation of c-Jun, Bcl-2 and Bcl-xL. At the same concentration, the non-cytotoxic analog, MOPIPP, does not activate these pathways. Pharmacologic inhibition of JNK activity promotes survival, even when cells are extensively vacuolated, but suppression of c-Jun transcriptional activity offers no protection. MOMIPP readily penetrates the blood-brain barrier and is moderately effective in suppressing progression of intracerebral glioblastoma xenografts.ConclusionsThe results suggest that interference with glucose uptake and induction of JNK-mediated phosphorylation of pro-survival members of the Bcl-2 family represent key events in the methuosis death process. In addition to providing new insights into the underlying molecular mechanism of methuosis, the results indicate that compounds of the cytotoxic IPP class may have potential for further development as therapeutic agents for brain tumors.

Project description:The low frequency of p53 alterations e.g., mutations/deletions (?10%) in multiple myeloma (MM) makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK) signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP) analysis showed that activated c-Jun binds to the activator protein-1 (AP-1) binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA) against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-? or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with JNK activators in the treatment of MM.

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?NAC inhibition of the FADD-JNK axis plays anti-apoptotic role in multiple cancer cells.

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αNAC inhibition of the FADD-JNK axis plays anti-apoptotic role in multiple cancer cells.

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