Inhibition of RelA phosphorylation sensitizes apoptosis in constitutive NF-kappaB-expressing and chemoresistant cells

Abstract

The compound 5-(4-methoxyarylimino)-2-N-(3,4-dichlorophenyl)-3-oxo-1,2,4-thiadiazolidine (P 3 -25) is known to possess antibacterial, anti-fungal, anti-tubercular, and local anesthetic activities. We studied the anti-tumorigenic activity of P 3 -25 and the role of nuclear transcription factor kappaB (NF-jB) in this process. In constitutive NF-jB-expressing cells, treatment with P 3 -25 inhibited the expression of NF-jB-dependent reporter gene, adhesion molecules, and cyclooxygenase. It downregulated phosphorylation of p65 by inhibiting upstream kinases, such as protein kinase A and casein kinase II, but did not alter NF-jB DNA-binding activity. Alone, P 3 -25 induced apoptosis in NF-jB-expressing and doxorubicin-resistant breast cancer cells, and in the presence of other chemotherapeutic agents, it potentiated apoptosis. Overall, our results suggest that P 3 -25 exerts antitumorigenic activity by inhibiting phosphorylation of p65, the transcriptionally active subunit of NF-jB by inhibiting its upstream kinases, and potentiates apoptosis mediated by chemotherapeutic agents. These results suggest novel approaches for designing of anticancer drugs for combination chemotherapy.

Ras Puts the Brake on Doxorubicin-mediated Cell Death in p53-expressing Cells

“…Cytotoxicity Assay-The cytotoxicity was measured by MTT assay (15). Briefly, cells (1 ϫ 10 4 cells/well of a 96-well plate) were treated with different agents for the indicated concentrations and times, and thereafter, 25 l of MTT solution (5 mg/ml in PBS) was added.…”

“…As doxorubicin increases reactive oxygen species production, it promotes NF-B activation via activation of IB kinase complex. Aberrantly active NF-B complexes can contribute to tumorigenesis by regulating genes that not only promote the growth but also survival of cancer cells (14,15) and also induce resistance against doxorubicin (16). Doxorubicin has been shown to induce cell death via a nonclassical pathway involving a biphasic induction of NF-B, which in turn expresses interleukin-8 (IL-8), and this IL-8 induces cell death through a sequential process: increase in intracellular Ca 2ϩ release, calcineurin activation, dephosphorylation of NF-AT, nuclear translocation of NF-AT, NF-ATdependent FasL expression, FasL-mediated caspase activation, and induction of cell death (17).…”

Preclinical Pharmacology of AZD5363, an Inhibitor of AKT: Pharmacodynamics, Antitumor Activity, and Correlation of Monotherapy Activity with Genetic Background

“…Therefore, the proapoptotic potential of an AKT inhibitor is more likely to be manifested in combination with chemotherapy, than when dosed as a monotherapy. It is possible that the PKA pharmacology of AZD5363 also contributes to the sensitization to apoptosis that we have observed in combination with chemotherapy; inhibiting PKA can decrease RelA phosphorylation that is able to induce cell death in NF-kB expressing, chemoresistant tumor cell lines (45,46).…”

Novel Derivative of Benzofuran Induces Cell Death Mostly by G2/M Cell Cycle Arrest through p53-dependent Pathway but Partially by Inhibition of NF-κB

“…Transcription Factor Assay by Electrophoretic Mobility Shift Assay (EMSA)-To determine NF-B DNA binding activity, EMSA were conducted essentially as described (22). Briefly, 8 g of nuclear extract proteins were incubated with 32 P endlabeled double-stranded NF-B oligonucleotides for 30 min at 37°C, and the DNA-protein complex was separated from free oligonucleotide on 6.6% native polyacrylamide gels.…”