Characterization of sonic hedgehog as a novel NF-kappaB target gene that promotes NF-kappaB-mediated apoptosis resistance and tumor growth in vivo.

To explore mechanisms controlling sonic hedgehog (Shh) expression in human cancers, we investigated regulation of Shh by the transcription factor NF-kappaB. We identify putative NF-kappaB binding sites in the human Shh promoter region that specifically bind NF-kappaB complexes. Further, NF-kappaB activation by tumor necrosis factor alpha (TNF-alpha) or p65 overexpression stimulates Shh promoter activity and p65 binds to Shh promoter in vivo. NF-kappaB-mediated transcriptional activation of Shh is mapped to a minimal NF-kappaB consensus site at position +139 of Shh promoter. NF-kappaB activation results in increased Shh mRNA and protein expression in vitro and, notably, also in vivo in a genetic mouse model of inducible NF-kappaB activity. Specific NF-kappaB inhibition by inhibitory NF-kappaBalpha (Ikappa-Balpha) superrepressor or p65 knockdown inhibits NF-kappaB-induced Shh promoter activation and Shh expression. NF-kappaB-mediated Shh expression promotes proliferation and confers resistance to TRAIL-induced apoptosis. Silencing of Shh prevents NF-kappaB-stimulated proliferation, while the addition of Shh rescues the proliferation defect imposed by NF-kappaB inhibition. Notably, NF-kappaB-stimulated tumor growth is significantly impaired by Shh knockdown in an in vivo model of pancreatic cancer. By demonstrating that NF-kappaB regulates Shh expression, which contributes to NF-kappaB-mediated proliferation and apoptosis resistance in vitro and in vivo, our findings have important implications to target aberrant Shh expression in human cancers.

Sensitization of neuroblastoma cells for TRAIL-induced apoptosis by NF-kappaB inhibition.

The transcription factor nuclear factor-kappaB (NF-kappaB) plays a central role in stress-induced transcriptional activation and has been implicated in chemoresistance of cancers. In the present study, we investigated the role of NF-kappaB in inducible chemoresistance of neuroblastoma. Doxorubicin, VP16 and the cytotoxic ligand TRAIL trigger NF-kappaB activation, whereas cisplatin and taxol have no impact on NF-kappaB activity. Specific inhibition of NF-kappaB activation by overexpression of dominant-negative mutant IkappaBalpha-super-repressor does not alter cell death upon doxorubicin or VP16 treatment, although it prevents doxorubicin- or VP16-mediated NF-kappaB activation. By comparison, inhibition of TRAIL-stimulated NF-kappaB activation by IkappaBalpha-superrepressor or the small molecule NF-kappaB inhibitor BMS-345541 significantly enhances TRAIL-induced apoptosis, pointing to an antiapoptotic function of NF-kappaB in TRAIL-mediated apoptosis. Analysis of signaling pathways reveals that NF-kappaB inhibition prevents TRAIL-triggered up-regulation of Mcl-1, promoting TRAIL-induced cytochrome c release and activation of caspases. Accordingly, knockdown of Mcl-1 by RNA interference significantly enhances TRAIL-induced apoptosis and also increases sensitivity of neuroblastoma cells to CD95- or chemotherapy-induced apoptosis. In conclusion, NF-kappaB regulates apoptosis in a stimulus-specific manner in neuroblastoma cells and confers protection against TRAIL-induced apoptosis. By demonstrating that NF-kappaB inhibition sensitizes neuroblastoma cells for TRAIL-induced apoptosis, our findings have important implications. Thus, NF-kappaB inhibitors may open new perspectives to potentiate the efficacy of TRAIL-based protocols in the treatment of neuroblastoma.

The repair enzyme peptide methionine-S-sulfoxide reductase is expressed in human epidermis and upregulated by UVA radiation.

Recently, we reported that photoaging correlates well with the amount of oxidized protein accumulated in the upper dermis, while protein oxidation levels in the viable epidermis are very low. We hypothesized that this might be due to epidermal expression of the repair enzymes methionine sulfoxide reductases (MSRs). The expression of human methionine sulfoxide reductase A (MSRA) was investigated in HaCaT cells, primary human keratinocytes, and in human skin. High MSRA mRNA and protein levels as well as MSR activity were found in cultured human keratinocytes. MSRA was expressed in human epidermis, as shown by immunohistochemistry in healthy human skin. Repetitive in vivo exposure of human skin to solar-simulated light on 10 consecutive days (n=10 subjects) significantly increased epidermal MSRA expression. To further assess the functional relevance of the enzyme, its expression in response to UVB, UVA, and H(2)O(2) was investigated in HaCaT cells. While UVB lowered protein expression of MSRA, an upregulation was observed in response to low doses of UVA and H(2)O(2). In summary, MSRA represents the only enzyme so far identified in human skin that is capable of repairing oxidative protein damage. In addition to melanogenesis and DNA repair systems, a wavelength-specific activation of epidermal MSRA may be involved in epidermal photoprotection.

Sensitization for gamma-irradiation-induced apoptosis by second mitochondria-derived activator of caspase.

Resistance to current treatment regimens, such as radiation therapy, remains a major concern in oncology and may be caused by defects in apoptosis programs. Because inhibitor of apoptosis proteins (IAPs), which are expressed at high levels in many tumors, block apoptosis at the core of the apoptotic machinery by inhibiting caspases, therapeutic modulation of IAPs could target a key control point in resistance. Here, we report for the first time that full-length or mature second mitochondria-derived activator of caspase (Smac), an inhibitor of IAPs, significantly enhanced gamma-irradiation-induced apoptosis and reduced clonogenic survival in neuroblastoma, glioblastoma, or pancreatic carcinoma cells. Notably, Smac had no effect on DNA damage/DNA repair, activation of nuclear factor-kappaB, up-regulation of p53 and p21 proteins, or cell cycle arrest following gamma-irradiation, indicating that Smac did not alter the initial damage and/or cellular stress response. Smac enhanced activation of caspase-2, caspase-3, caspase-8, and caspase-9, loss of mitochondrial membrane potential, and cytochrome c release on gamma-irradiation. Inhibition of caspases also blocked gamma-irradiation-induced mitochondrial perturbations, indicating that Smac facilitated caspase activation, which in turn triggered a mitochondrial amplification loop. Interestingly, mitochondrial perturbations were completely blocked by the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or the relatively selective caspase-2 inhibitor N-benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone, whereas caspase-8 or caspase-3 inhibitors only inhibited the increased drop of mitochondrial membrane potential provided by Smac, suggesting that caspase-2 was acting upstream of mitochondria after gamma-irradiation. In conclusion, our findings provide evidence that targeting IAPs (e.g., by Smac agonists) is a promising strategy to enhance radiosensitivity in human cancers.

Betulinic acid as new activator of NF-kappaB: 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-kappaB), a key regulator of stress-induced transcriptional activation, has been implicated in mediating apoptosis resistance, we investigated the role of NF-kappaB in BetA-induced apoptosis. Here, we provide for the first time evidence that BetA activates NF-kappaB in a variety of tumor cell lines. NF-kappaB DNA-binding complexes induced by BetA consisted of p50 and p65 subunits. Nuclear translocation of p65 was also confirmed by immunofluorescence microscopy. BetA-induced NF-kappaB activation involved increased IKK activity and phosphorylation of IkappaB-alpha at serine 32/36 followed by degradation of IkappaB-alpha. Reporter assays revealed that NF-kappaB activated by BetA is transcriptionally active. Interestingly, inhibition of BetA-induced NF-kappaB activation by different chemical inhibitors (proteasome inhibitor, antioxidant, IKK inhibitor) attenuated BetA-induced apoptosis. Importantly, specific NF-kappaB inhibition by transient or stable expression of IkappaB-alpha super-repressor inhibited BetA-induced apoptosis in SH-EP neuroblastoma cells, while transient expression of IkappaB-alpha super-repressor had no influence on BetA-induced apoptosis in two other cell lines. Thus, our findings that activation of NF-kappaB by BetA promotes BetA-induced apoptosis in a cell type-specific fashion indicate that NF-kappaB 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.

Activity, tissue distribution and site-directed mutagenesis of a human peptide methionine sulfoxide reductase of type B: hCBS1.

Human CBS1 is a methionine sulfoxide reductase of type B (MSRB) as it specifically reduced Met-R-SO in peptides with dithiothreitol or the thioredoxin system as reductants. Mutation C169S in the active site completely abolished enzymatic activity, while mutation W110A only reduced activity and C105S had no effect. Like human MSRA, hCBS1 showed in vivo reducing activity coexpressed with the Drosophila ShC/B potassium channel in oocytes, by accelerating the overall inactivation time course. hCBS1-encoding mRNA is most abundant in muscle tissues, especially in the heart and thereby shows an expression pattern different to the human MSRA.