α-Lipoic acid prevents p53 degradation in colon cancer cells by blocking NF-κB induction of RPS6KA4.

α-Lipoic acid (α-LA) is a biogenic antioxidant that has been used successfully in the treatment of diabetic polyneuropathy and its application to many oxidative stress-associated chronic diseases has increased. In this study, we investigated the effect of α-LA on colorectal cancer cell growth and its underlying mechanism. α-LA treatment resulted in a marked reduction in the growth of HCT116 colon cancer cells in a dose-dependent manner through the G1 arrest of the cell cycle and apoptosis induction. α-LA treatment significantly increased tumor cell response to various apoptotic stresses, such as etoposide, 5-fluorouracil, UVC, γ-irradiation, hypoxia, and tumor necrosis factor α (TNFα). Interestingly, α-LA increased p53 protein stability and its apoptosis-enhancing effect was more evident in wild-type p53-carrying cells compared with p53-deficient cells, suggesting that the proapoptotic role of α-LA is associated with its p53-stabilizing function. On the basis of our microarray data showing α-LA downregulation of the ribosomal protein p90S6K (RPS6KA4), which has been reported to inhibit p53 function, we tested whether α-LA regulation of RPS6KA4 is associated with its proapoptotic function. α-LA treatment led to a marked reduction in the RPS6KA4 mRNA level in multiple colorectal cancer cells and restoration of RPS6KA4 expression markedly attenuated α-LA induction of apoptosis in a p53-dependent manner. In addition, we observed that RPS6KA4 expression is activated by TNFα whereas both basal and TNFα induction of RPS6KA4 are inhibited by the nuclear factor-κB (NF-κB) inhibitor BAY11-7082 or transfection of a dominant-negative mutant of NF-κB, indicating that NF-κB plays a crucial role in RPS6KA4 gene expression. Finally, we found that α-LA exerts an inhibitory effect on the nuclear translocation of NF-κB triggered by TNFα. Collectively, our study shows that α-LA suppresses colorectal tumor cell growth at least partially by preventing RPS6KA4-mediated p53 inhibition through blockade of NF-κB signaling.

Characterization of a novel anti-human TNF-alpha murine monoclonal antibody with high binding affinity and neutralizing activity.

In order to develop an anti-human TNF-alpha mAb, mice were immunized with recombinant human TNF-alpha. A murine mAb, TSK114, which showed the highest binding activity for human TNF-alpha was selected and characterized. TSK114 specifically bound to human TNF-alpha without cross-reactivity with the homologous murine TNF-alpha and human TNF-beta. TSK114 was found to be of IgG1 isotype with kappa light chain. The nucleotide sequences of the variable regions of TSK114 heavy and light chains were determined and analyzed for the usage of gene families for the variable (V), diversity (D), and joining (J) segments. Kinetic analysis of TSK114 binding to human TNF-alpha by surface plasmon resonance technique revealed a binding affinity (K(D)) of approximately 5.3 pM, which is about 1,000- and 100-fold higher than those of clinically relevant infliximab (Remicade) and adalimumab (Humira) mAbs, respectively. TSK114 neutralized human TNF-alpha-mediated cytotoxicity in proportion to the concentration, exhibiting about 4-fold greater efficiency than those of infliximab and adalimumab in WEHI 164 cells used as an in vitro model system. These results suggest that TSK114 has the potential to be developed into a therapeutic TNF-alpha-neutralizing antibody with picomolar affinity.

A hot pepper cDNA encoding ascorbate peroxidase is induced during the incompatible interaction with virus and bacteria.

Capsicum annuum L. is infected by a number of viruses, including the tobacco mosaic virus (TMV). To study the defense-related genes that are induced by TMV in hot peppers, the pepper plant, which is susceptible to P1.2 but resistant to the P0 pathotype of TMV, was inoculated with TMV-P0. Differential screening isolated the genes that were specifically up- or down-regulated during the hypersensitive response (HR). The CaAPX1 cDNA clone that putatively encodes a polypeptide of cytosolic ascorbate peroxidase was selected as an up-regulated gene. It was isolated for further study. The full-length cDNA for CaAPX1, which is 972 bp long, contained the open-reading frame of 250-amino acid residues. A genomic Southern blot analysis showed that there were only limited copies of the CaAPX1 gene in the hot pepper genome. In hot pepper cv. Bugang, which is resistant to TMV-P0 and susceptible to TMV-P1.2, the CaAPX1 gene transcript was accumulated by TMV-P0, but not by TMV-P1.2 inoculation. CaAPX1 transcripts began to accumulate 24 h post-inoculation of TMV-P0, and increased gradually until 96 h. To investigate whether each transcript is induced by other stimuli, the plants were treated with various chemicals and wounding. A striking induction of the CaAPX1 transcript was observed at 2 h. It subsided 12 h after salicylic acid (SA), ethephon, and methyl jasmonate (MeJA) treatments. The response of the gene upon other pathogen infection was also examined by a bacterial pathogen (Xanthomonas campestris pv. vesicatoria race 3) inoculation. The CaAPX1 gene was induced in a hot pepper (C. annuum cv. ECW 20R) that was resistant to this bacterial pathogen, but not in a susceptible hot pepper (C. annuum cv. ECW). These results suggest the possible role(s) for the CaAPX1 gene in plant defense against viral and bacterial pathogen.

CD81 is a candidate tumor suppressor gene in human gastric cancer.

BACKGROUND: CD81 is a transmembrane protein that serves as a putative receptor for hepatitis C virus. In addition, CD81 has been suggested to be involved in a broad range of other cellular functions. Its putative implication in tumorigenesis has so far, however, remained largely unexplored. To assess the candidacy of CD81 as a tumor suppressor in gastric cancer development, we investigated its expression and function in a series of primary gastric tumors and gastric tumor-derived cell lines. METHODS: The expression and concomitant methylation status of the CD81 gene and its effect on tumor development and cellular signaling were evaluated. RESULTS: CD81 mRNA levels were found to be low in 16 of 40 (40 %) primary tumors and 9 of 14 (64.2 %) cell lines, and these low expression levels were found to correlate with the stage and grade of the tumors. Genomic alterations of CD81 were not encountered, whereas its expression could be re-activated in low expressing cells upon 5-aza-dC treatment. Bisulfite DNA sequencing analysis of 10 CpG sites within the 5' proximal region of the CD81 gene promoter revealed that the observed transcriptional silencing was tightly associated with aberrant hypermethylation. Subsequent restoration of CD81 expression induced a G1 cell cycle arrest and apoptosis, whereas siRNA-mediated CD81 down-regulation promoted cell proliferation and attenuated cellular responses to various apoptotic stress stimuli. Also the colony-forming ability of the tumor cells could be inhibited and enhanced through CD81 up- and down-regulation, respectively. CD81 was found to inhibit p38 (but not ERK, JNK and AKT) phosphorylation and its growth suppressive effect could be abolished through p38 up- and down-regulation. CONCLUSION: From our data we conclude that epigenetic inactivation of CD81 is a common feature of gastric tumors and that this inactivation may render growth and survival advantages to the tumor cells, at least partially through p38 signaling.

Comparative analyses of complex formation and binding sites between human tumor necrosis factor-alpha and its three antagonists elucidate their different neutralizing mechanisms.

Tumor necrosis factor-alpha (TNFalpha)-blocking therapy, using biologic TNFalpha antagonists, has been approved for the treatment of several diseases including rheumatoid arthritis, psoriasis and Crohn's disease. There have been few detailed studies of binding characterizations for the complex formation by TNFalpha and clinically relevant antagonists, particularly Infliximab (Remicade) and Etanercept (Enbrel). Here we characterized the binding stoichiometry and size of soluble TNFalpha-antagonist complexes and identified energetically important binding sites on TNFalpha for the three antagonists, Etanercept, Infliximab, and the recently developed humanized TNFalpha neutralizing monoclonal antibody, YHB1411-2. Size-exclusion chromatography and dynamic light scattering analyses revealed that the three antagonists formed distinct thermodynamically stable TNFalpha-antagonist complexes that exhibited differences in their size and composition. Energetically important binding residues on TNFalpha were identified for each antagonist by a sequence of experiments that consisted of competition binding assays, fragmentations, loop mutations, and single-point mutations using yeast surface-displayed TNFalpha, which was further confirmed for solubly purified TNFalpha mutants by surface plasmon resonance technique. Analyses of the binding geometry based on binding site location, spatial constraints, and valency satisfaction allowed us to interpret the thermodynamically stable complexes as follows: one molecule of Etanercept and one molecule of trimeric TNFalpha (Etanercept1-TNFalpha1), Infliximab6-TNFalpha3, and YHB1411-2(4)-TNFalpha2. The distinct features of the soluble antagonist-TNFalpha complex formation among the antagonists may give further insights into their different neutralizing mechanisms and pharmacokinetic profiles.

Ornithine decarboxylase gene (CaODC1) is specifically induced during TMV-mediated but salicylate-independent resistant response in hot pepper.

A gene encoding putative ornithine decarboxylase (ODC) has been isolated by differential screening of a cDNA library from the resistant hot pepper (Capsicum annuum L.) inoculated with avirulent tobacco mosaic virus (TMV) pathotype P0. In hot pepper plants, transcripts of the CaODC1 (C. annuum ODC1) gene started to accumulate at 24 h post-inoculation of TMV-P0 and the signal was spread systemically. The transcript level of CaODC1 was increased rapidly in a hot pepper resistant to Xanthomonas campestris pv. vesicatoria (Xcv) but not in a susceptible hot pepper after inoculation. These results suggest possible role(s) for CaODC1 in plant defense against a broad range of pathogens including viruses and bacteria.