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Purpose: To investigate the anticancer effects and underlying mechanisms of parthenolide on HepG2 human hepatocellular carcinoma cells., Materials and Methods: Cell viability was assessed by MTT assay and cell apoptosis through DAPI, TUNEL staining and Western blotting. Monodansylcadaverin(MDC) and AO staining were used to detect cell autophagy. Cell proliferation was assessed by Ki67 immunofluorescence staining., Results: Parthenolide induced growth inhibition in HepG2 cells. DAPI and TUNEL staining showed that parthenolide could increase the number of apoptotic nuclei, while reducing the expression of the anti-apoptotic protein Bcl-2 and elevating the expression of related proteins, like p53, Bax, cleaved caspase9 and cleaved caspase3. Parthenolide could induce autophagy in HepG2 cells and inhibited the expression of proliferation-related gene, Ki-67., Conclusions: Parthenolide can exert anti-cancer effects by inducing cell apoptosis, activating autophagy and inhibiting cell proliferation.

The high biological activity of dehydroabietylamine derivatives has been reported previously. In this study, we aimed to screen 73 dehydroabietylamine derivatives as potential candidate inhibitors in liver cancer cells. Initially, the compounds structural activity relationship analysis was explored and N-benzoyl-12-nitrodehydroabietylamine-7-one (compound 81) was shown to have significant growth inhibitory activity in the human liver carcinoma cell line, HepG2. Further research into the anti-proliferative effect on HepG2 cells mediated by compound 81 was undertaken. The results suggest that compound 81 effectively induced apoptosis in HepG2 cells characterized by nuclear staining of DAPI, TUNEL assay and the activation of caspase-3. A decreased level of anti-apoptotic protein Bcl-2 and increased apoptotic Bax were also observed. Furthermore, Ki-67 protein staining and the BrdU incorporation assay showed that compound 81 significantly inhibited the proliferation of HepG2 cells. Cell cycle components analysis found that expression of cyclin D1 and cyclin B1 was reduced in HepG2 cells with compound 81 treatment, whereas the content of p21(Waf1/Cip1) was increased. Taken together, our data indicate that compound 81 induces apoptosis and inhibits proliferation in HepG2 cells, and may be a promising candidate in the development of a novel class of antitumor agents., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

TSP50 (testes-specific protease 50) is a testis-specific expression protein, which is expressed abnormally at high levels in breast cancer tissues. This makes it an attractive molecular marker and a potential target for diagnosis and therapy; however, the biological function of TSP50 is still unclear. In the present study, we show that overexpression of TSP50 in CHO (Chinese-hamster ovary) cells markedly increased cell proliferation and colony formation. Mechanistic studies have revealed that TSP50 can enhance the level of TNFα (tumour necrosis factor α)- and PMA-induced NF-κB (nuclear factor κB)-responsive reporter activity, IκB (inhibitor of NF-κB) α degradation and p65 nuclear translocation. In addition, the knockdown of endogenous TSP50 in MDA-MB-231 cells greatly inhibited NF-κB activity. Co-immunoprecipitation studies demonstrated an interaction of TSP50 with the NF-κB-IκBα complex, but not with the IKK (IκB kinase) α/β-IKKγ complex, which suggested that TSP50, as a novel type of protease, promoted the degradation of IκBα proteins by binding to the NF-κB-IκBα complex. Our results also revealed that TSP50 can enhance the expression of NF-κB target genes involved in cell proliferation. Furthermore, overexpression of a dominant-negative IκB mutant that is resistant to proteasome-mediated degradation significantly reversed TSP50-induced cell proliferation, colony formation and tumour formation in nude mice. Taken together, the results of the present study suggest that TSP50 promotes cell proliferation, at least partially, through activation of the NF-κB signalling pathway.

Earlier studies identified testes-specific protease 50 (TSP50), which encodes a threonine protease, and showed that it was abnormally reactivated in many breast cancer biopsies. Further, it was shown to be negatively regulated by the p53 gene. However, little is known about the biological function of TSP50. In this study, we applied RNA interference to knockdown TSP50 gene expression in P19 murine embryonal carcinoma stem cells and tested whether this modulated the cell phenotype. The results showed that downregulation of TSP50 expression not only reduced cell proliferation, colony formation, and migration but also induced cell apoptosis. Further investigation revealed that knockdown of TSP50 resulted in greater sensitivity to doxorubicin-induced apoptosis and that activation of caspase-3 was involved in this process.

Background: Interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) are multifunctional cytokines that regulate immune responses, cell proliferation, and tumour development and progression, which frequently have functionally opposing roles. The cellular responses to both cytokines are activated via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. During the past 10 years, the crosstalk mechanism between the IFN-gamma and IL-6 pathways has been studied widely and several biological hypotheses have been proposed, but the kinetics and detailed crosstalk mechanism remain unclear. Results: Using established mathematical models and new experimental observations of the crosstalk between the IFN-gamma and IL-6 pathways, we constructed a new crosstalk model that considers three possible crosstalk levels: (1) the competition between STAT1 and STAT3 for common receptor docking sites; (2) the mutual negative regulation between SOCS1 and SOCS3; and (3) the negative regulatory effects of the formation of STAT1/3 heterodimers. A number of simulations were tested to explore the consequences of cross-regulation between the two pathways. The simulation results agreed well with the experimental data, thereby demonstrating the effectiveness and correctness of the model. Conclusion: In this study, we developed a crosstalk model of the IFN-gamma and IL-6 pathways to theoretically investigate their cross-regulation mechanism. The simulation experiments showed the importance of the three crosstalk levels between the two pathways. In particular, the unbalanced competition between STAT1 and STAT3 for IFNR and gp130 led to preferential activation of IFN-gamma and IL-6, while at the same time the formation of STAT1/3 heterodimers enhanced preferential signal transduction by sequestering a fraction of the activated STATs. The model provided a good explanation of the experimental observations and provided insights that may inform further research to facilitate a better understanding of the cross-regulation mechanism between the two pathways. [ABSTRACT FROM AUTHOR]

Background: Testes-specific protease 50 (TSP50), a newly discovered threonine enzyme, has similar amino acid sequences and enzymatic structures to those of many serine proteases. It may be an oncogene. TSP50 is up-regulated in breast cancer epithelial cells, and ectopic expression of TSP50 in TSP50-deficient Chinese hamster ovary (CHO) cells has been found to promote cell proliferation. However, the mechanisms by which TSP50 exerts its growth-promoting effects are not yet fully understood. Methodology/Principal Findings: To delineate whether the threonine protease activity of TSP50 is essential to its function in cell proliferation, we constructed and characterized a mutant TSP50, called TSP50 T310A, which was identified as a protease-dead mutant of TSP50. By a series of proliferation analyses, colony formation assays and apoptosis analyses, we showed that T310A mutation significantly depresses TSP50-induced cell proliferation in vitro. Next, the CHO stable cell line expressing either wild-type or T310A mutant TSP50 was injected subcutaneously into nude mice. We found that the T310A mutation could abolish the tumorigenicity of TSP50 in vivo. A mechanism investigation revealed that the T310A mutation prevented interaction between TSP50 and the NF-κBIkBa complex, which is necessary for TSP50 to perform its function in cell proliferation. Conclusion: Our data highlight the importance of threonine 310, the most critical protease catalytic site in TSP50, to TSP50- induced cell proliferation and tumor formation. [ABSTRACT FROM AUTHOR]

Testes-specific protease 50 (TSP50) is a novelly identified pro-oncogene and it shares a similar enzymatic structure with many serine proteases. Our previous results suggested that TSP50 could promote tumorigenesis through degradation of IκBα protein and activating NF-κB signaling, and the threonine mutation in its catalytic triad could depress TSP50-mediated cell proliferation. However, whether the two other residues in the catalytic triad of TSP50 play a role in maintaining protease activity and tumorigenesis, and the mechanisms involved in this process remain unclear. Here, we constructed and characterized three catalytic triad mutants of TSP50 and found that all the mutants could significantly depress TSP50-induced cell proliferation and colony formation in vitro and tumor formation in vivo, and the aspartic acid at position 206 in the catalytic triad played a more crucial role than threonine and histidine in this process. Mechanistic studies revealed that the mutants in the catalytic triad abolished the enzyme activity of TSP50, but did not change the cellular localization. Furthermore, our data indicated that all the three mutants suppressed activation of NF-κB signal by preventing the interaction between TSP50 and the NF-κB:IκBα complex. Most importantly, we demonstrated that TSP50 could interact with IκBα protein and cleave it directly as a new protease in vitro. [ABSTRACT FROM AUTHOR]