In vitro and in vivo cancer cell apoptosis triggered by competitive binding of Cinchona alkaloids to the RING domain of TRAF6.

TRAF6 is highly expressed in many tumors and plays an important role in the immune system. The aim of this study is to confirm anti-tumor activities of all naturally occurring Cinchona alkaloids that have been screened using computational docking program, and to validate the accuracy and specificity of the RING domain of TRAF6 as a potential anti-tumor target, and to explore their effect on the immune system. Results reported herein would demonstrate that Cinchona alkaloids could induce apoptosis in HeLa cells, inhibit the ubiquitination and phosphorylation of both AKT and TAK1, and up-regulate the ratio of Bax/Bcl-2. In addition, these compounds could induce apoptosis in vivo, and increase the secretion of TNF-α, IFN-γ, and IgG, while not significantly impacting the ratio of CD4+T/CD8+T. These investigations suggest that the RING domain of TRAF6 could serve as a de novo biological target for therapeutic treatment in cancers.

N6-Isopentenyladenosine promoted HeLa cell apoptosis through inhibitions of AKT and transforming growth factor ß-activated kinase 1 activation.

N6-Isopentenyladenosine, a member of the family of plant hormones, possesses anti-cancer activities on a number of cancer cell lines. However, its mode of action in cervical cancer cell remains poorly understood. Our computational docking studies showed that N6-Isopentenyladenosine could bind with the really interesting new gene domain of tumor necrosis factor receptor-associated factor 6, which is an ubiquitination E3 ligase. Tumor necrosis factor receptor-associated factor 6-mediated ubiquitination is known to activate both protein kinase B (also known as AKT) and transforming growth factor ß-activated kinase 1, and the really interesting new gene domain comprises the core of the ubiquitin ligase catalytic domain. First, we evaluated the effects of iPA on cervical cancer cell line HeLa using MTT and flow cytometry. Second, we examined the effects of iPA on activation of tumor necrosis factor receptor-associated factor 6-mediated downstream targets using western blot or immunoprecipitation. iPA could reduce HeLa cell proliferation through apoptosis, and such anti-cancer activity is associated with inhibitions of both AKT and transforming growth factor ß-activated kinase 1 signaling pathways. In addition, suppression of the anti-apoptotic protein Bcl-2 and elevation of the pro-apoptotic protein Bax were also observed. Anti-proliferation properties of iPA are likely due to its binding at the really interesting new gene domain of tumor necrosis factor receptor-associated factor 6 and loss of AKT and transforming growth factor ß-activated kinase 1 activities as a result of functional modulations of tumor necrosis factor receptor-associated factor 6. These results support the emerging notion that tumor necrosis factor receptor-associated factor 6 could serve as a viable target for developing new cancer therapeutics.

Emodin extends lifespan of Caenorhabditis elegans through insulin/IGF-1 signaling pathway depending on DAF-16 and SIR-2.1.

The naturally occurring anthraquinone emodin has been serving primarily as an anti-bacterial and anti-inflammatory agent. However, little is known about its potential on anti-aging. This investigation examined the effect of emodin on lifespan and focused on its physiological molecular mechanisms in vivo. Using Caenorhabditis elegans (C. elegans) as an animal model, we found emodin could extend lifespan of worms and improve their antioxidant capacity. Our mechanistic studies revealed that emodin might function via insulin/IGF-1 signaling (IIS) pathway involving, specifically the core transcription factor DAF-16. Quantitative RT-PCR results illustrated that emodin up-regulated transcription of DAF-16 target genes which express antioxidants to promote antioxidant capacity and lifespan of worms. In addition, attenuated effect in sir-2.1 mutants suggests that emodin likely functioned in a SIR-2.1-dependent manner. Our study uncovers a novel role of emodin in prolonging lifespan and supports the understanding of emodin being a beneficial dietary supplement.

Transformation of graphene oxide affects photodegradation of imidacloprid in the aquatic environment: Mechanism and implication.

Graphene oxide (GO) is a representative novel carbonaceous nanomaterial, and neonicotinoid insecticides (NEOs) are currently the insecticides with the highest market share in the world. Their widespread application deservedly leads to their release to the environment. Thus, the complex interactions of these two types of organic compounds have attracted extensive attention. In this study, the effects of GO and its derivatives, reduced GO (RGO) and oxidized GO (OGO), on the photolysis of imidacloprid (IMD) (a typical NEO) under ultraviolet (UV) irradiation were systematically investigated. The results showed that the presence of the graphene-based nanomaterials (GNs) largely depressed the photodegradation of IMD, and the inhibition degree followed the order of RGO > GO > OGO. This was because the sp2 π-conjugated structure in the GNs caused light-shielding effect and attenuated the direct photolysis of IMD, even though the GNs-generated reactive oxygen species (ROS) promoted the indirect photodegradation of IMD to a certain extent. Additionally, the rich O-functionalized GO and OGO altered the photolysis pathway of IMD and induced more toxic intermediate products. These results highlight the implication of carbonaceous nanomaterials on the behavior, fate and potential risk of NEOs in aqueous systems.

Benzoyl-xanthone derivative induces apoptosis in MCF-7 cells by binding TRAF6.

TNF receptor-associated factor 6 (TRAF6) has been reported to be associated with the development of cancer. Nevertheless, the exact role of TRAF6 in cancer remains unclear. The purpose of the present study was to explore the mechanism of 2-benzoyl-3-hydroxy-4-methyl-9H-xanthen-9-one leading to the inhibition of the activation of AKT and TGF-ß-activated kinase 1 (TAK1), and to the apoptosis of MCF-7 cells. Using a computational docking program and examination of AKT and TAK1 level changes, a new small molecule was identified, 2-benzoyl-3-hydroxy-4-methyl-9H-xanthen-9-one, which competitively bound to TRAF6. Next, the effect of this new compound on MCF-7 cells' biological behavior was studied in vitro. MTT assays were used to investigate cell viability; flow cytometry and invasion assays were performed to detect early apoptosis and invasion in MCF-7 cells, respectively. Immunoprecipitation, western blotting and caspase-3/9 activity assays were carried out to explore changes in protein expression. Briefly, the present data indicated that 2-benzoyl-3-hydroxy-4-methyl-9H-xanthen-9-one could suppress proliferation, induce early apoptosis and inhibit invasion in MCF-7 cells by suppressing the expression of Bcl-2 and promoting the expression of Bax, caspase-9, and caspase-3. These findings indicated that 2-benzoyl-3-hydroxy-4-methyl-9H-xanthen-9-one could induce apoptosis by inhibiting the activation of AKT and TAK1, and affecting the Bcl-2/Bax-caspase-9-caspase-3 pathway by competitively binding with TRAF6.

Calycosin promotes lifespan in Caenorhabditis elegans through insulin signaling pathway via daf-16, age-1 and daf-2.

The naturally occurring calycosin is a known antioxidant that prevents redox imbalance in organisms. However, calycosin's effect on lifespan and its physiological molecular mechanisms are not yet well understood. In this study, we demonstrated that calycosin could prolong the lifespan of Caenorhabditis elegans, and that such extension was associated with its antioxidant capability as well as its ability to enhance stress resistance and reduce ROS (reactive oxygen species) accumulation. To explore mechanisms of this longevity effect, we assessed the impact of calycosin on lifespans of insulin-signaling impaired worms: daf-2, age-1, and daf-16 mutants. We found that calycosin did not alter the lifespan of all three mutants, thereby suggesting that calycosin requires insulin signaling to promote lifespan extension. On the other hand, we observed that calycosin could enhance the nuclear translocation of the core transcription factor DAF-16/FoXO instead of the conserved stress-responsive transcription factor SKN-1/Nrf-2. This observation is consistent with the understanding that the nuclear localized DAF-16 up-regulates its downstream targets sod-3, ctl-1, and hsp-16.2. Lastly, it is also noteworthy that the longevity effect of calycosin is likely not associated with the calorie restriction mechanism. Collectively, our results strongly suggest that calycosin could function as an antioxidant to extend the lifespan of C. elegans by enhancing nucleus translocation of DAF-16 through the insulin-signaling pathway.

Cinchonine induces apoptosis of HeLa and A549 cells through targeting TRAF6.

BACKGROUND: Cancer cells are known to over-express TRAF6 that is critical for both AKT and TAK1 activations. The Really Interesting New Gene (RING) domain of TRAF6 is believed to be responsible for the E3 ligase activity, ZINC fingers of TRAF6 provide critical support for the activity of the RING domain which is critical for both AKT and TAK1 activations. METHODS: We employed computational docking program to identify small molecules that could effectively and competitively bind with the RING domain of TRAF6, which is believed to be responsible for its E3 ligase activity. MTT assay and flow cytometry were employed to analyze apoptosis of cancer cells. Signaling pathways were detected using immunoprecipitation and western blotting, and immunofluorescence was pursued to assess the nature of binding of cinchonine to TRAF6. We also performed animal experiments to test effect of cinchonine in vivo. RESULTS: Cinchonine, a naturally occurring Cinchona alkaloid identified from the docking study, could bind to TRAF6 in HeLa and A549 cells and induce apoptosis of these cancer cells. We found that AKT ubiquitination and phosphorylation as well as phosphorylation of TAK1 were decreased. These activities would lead to subsequent suppression anti-apoptotic protein Bcl-2, while elevating pro-apoptotic protein Bax. Immunofluorescence staining unambiguously demonstrated the binding of cinchonine specifically at the RING domain of TRAF6 in cells, thereby validating the computational modeling. Animal experiments showed that cinchonine could suppress tumor growth in mice without showing significant acute toxicity. CONCLUSION: These investigations suggest that through competitive binding with the RING domain of TRAF6, cinchonine could induce apoptosis via inhibiting AKT and TAK1 signaling pathways.

Therapeutic role of human hepatocyte growth factor (HGF) in treating hair loss.

Hepatocyte growth factor (HGF) is a paracrine hormone that plays an important role in epithelial-mesenchymal transition. HGF secreted by mesenchymal cells affects many properties of epithelial cells, such as proliferation, motility, and morphology. HGF has been reported to promote follicular growth. The purpose of the present study is to investigate the therapeutic role of HGF in hair loss treatment. A recombinant vector containing the human HGF (hHGF) gene (pTARGET-hHGF) was constructed, and the expression of hHGF in vitro was quantitatively and qualitatively evaluated. The effect of hHGF on hair growth was tested in mice, and results demonstrated that pTARGET-hHGF was successfully delivered into fibroblasts in vitro leading to a high expression of hHGF. Local injections of the pTARGET-hHGF recombinant vector into mice resulted in multiple beneficial effects compared to placebo, including faster hair regeneration, improved follicle development, and significantly increased HGF receptor (HGF-R). In conclusion, we have established a nonviral vector of hHGF which could be utilized to manipulate the sheath fibroblasts surrounding hair follicles (HF), thereby stimulating hair regeneration.

Suppression of tumor cell proliferation by quinine via the inhibition of the tumor necrosis factor receptor­associated factor 6­AKT interaction.

Protein kinase B (AKT), is a pivotal component of pathways associated with cell survival, metabolism, invasion and metastasis. AKT mediates anti­apoptotic and proliferative signaling in response to essential cytokines. Tumor necrosis factor receptor­associated factor (TRAF)6, an E3 ubiquitin ligase, has been shown to ubiquitylate, as well as activate AKT. The present study used computational methods to determine the relevant amino acid residues at the binding site of TRAF6 and selected small molecules, which may bind to TRAF6. An ex vivo assay was performed to determine their antitumor activities and the possible mechanism of action. Quinine, a natural alkaloid that is well­known for its therapeutic treatment of malaria, exhibited a distinct antiproliferative and pro­apoptotic effect in HeLa and A549 tumor cell lines via the inhibition of the antiapoptotic protein, B­cell lymphoma (BCL)­2, and activation of the pro­apoptotic factor, BCL­2­associated X protein. Quinine inhibited the lipopolysaccharide (LPS)­induced activation of AKT by inhibiting its phosphorylation at Thr­308 and Ser­473, and reversing LPS­induced proliferation. These results suggested that the inhibition of AKT activation via targeting of TRAF6 with quinine may be a viable anticancer therapeutic approach and a successful example of the alternative use of the original therapeutic properties of this well­known natural product.