Betulinic acid as new activator of NF-κB:: molecular mechanisms and implications for cancer therapy

Recent evidence demonstrates that the anticancer activity of betulinic acid (BetA) can be markedly increased by combination protocols, for example with chemotherapy, ionizing radiation or TRAIL. Since nuclear factor kappaB (NF-kappa B), a key regulator of stress-induced transcriptional activation, has been implicated in mediating apoptosis resistance, we investigated the role of NF-kappa B in BetA-induced apoptosis. Here, we provide for the first time evidence that BetA activates NF-kappa B in a variety of tumor cell lines. NF-kappa B DNA-binding complexes induced by BetA consisted of p50 and p65 subunits. Nuclear translocation of p65 was also confirmed by immunofluoresence microscopy. BetA-induced NF-kappa B activation involved increased IKK activity and phosphorylation of I kappa B-alpha at serine 32/36 followed by degradation of I kappa B-alpha. Reporter assays revealed that NF-kappa B activated by BetA is transcriptionally active. Interestingly, inhibition of BetA-induced NF-kappa B activation by different chemical inhibitors ( proteasome inhibitor, antioxidant, IKK inhibitor) attenuated BetA-induced apoptosis. Importantly, specific NF-kappa B inhibition by transient or stable expression of I kappa B-alpha super-repressor inhibited BetA-induced apoptosis in SHEP neuroblastoma cells, while transient expression of I kappa B-alpha super-repressor had no influence on BetA-induced apoptosis in two other cell lines. Thus, our. ndings that activation of NF-kappa B by BetA promotes BetA-induced apoptosis in a cell type-specific fashion indicate that NF-kappa B inhibitors in combination with BetA would have no therapeutic benefit or could even be contraproductive in certain tumors, which has important implications for the design of BetA-based combination protocols.