Extraction of proteins and preliminary characterization of physicochemical properties in Toona sinensis fruit.

We investigated the extraction of Toona sinensis fruit proteins and preliminarily characterized their physicochemical properties. The results showed that optimal extraction occurred under conditions of pH 10.5, a duration of 40 min, a liquid-to-solid ratio of 25:1, and a temperature of 40°C by an orthogonal design using T. sinensis fruit protein as the index and single factor. The total nitrogen content was 13.8 g/100 g and included 17 different amino acids. The glutamate level was highest at 35.37%, followed by arginine at 15.31%. The isoelectric point of T. sinensis fruit protein was between 6.8 and 10.0 with a typical absorption peak by infrared chromatography. Three protein bands were analyzed using SDS-polyacrylamide gel electrophoresis, with relative molecular weights of 55, 51, and 22 kDa. This study provides a theoretical basis for the comprehensive utilization of T. sinensis fruit by further investigating the biological activity of its proteins.

Correlation between Interleukin-17 gene polymorphism and gastric cancer susceptibility in Han Chinese population.

OBJECTIVE: The present study aims to investigate the correlation between Interleukin (IL)-17 gene polymorphism with gastric cancer susceptibility in Han Chinese population. PATIENTS AND METHODS: Between November 2013 and October 2014, 386 patients with gastric cancer who had undergone surgeries at our institution and 374 age- and sex-matched healthy controls were included in this study. Single nucleotide polymorphisms (SNPs) of IL-17 gene (rs2275913, rs3748067, rs4711998 and rs763780) in patients and health controls were studied by using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) and DNA sequencing technology. The correlation between IL-17 polymorphism and gastric cancer susceptibility was analyzed using logistic regression analysis. RESULTS: For IL17 rs2275913, no significant differences were observed in the frequencies of AA genotype and A allele between patients and controls (c2 = 0.870 p > 0.05; c2 = 0.814 p > 0.05). In IL17 rs3748067, the frequencies of TT genotype and T allele were significantly higher in patients than in controls (c2 = 12.82 p < 0.01; c2 = 12.805 p < 0.01). For IL17A rs4711998, no significant differences were observed in the frequencies of AA genotype and A allele between patients and controls (c2 = 2.636, p > 0.05; c2 = 1.462, p > 0.05). As for ILl7F rs763780, the frequencies of GG genotype and G allele in patients were significantly different from those in controls (c2 = 16.534, p < 0.01; c2 = 16.399, p < 0.01). CONCLUSIONS: Polymorphism of IL-17 rs3748067 and rs763780 is closely associated with gastric cancer development. Polymorphism of L-17 rs2275913 and rs4711998 may be correlated with the risk for gastric cancer.

Inhibition of ß-catenin signaling by nongenomic action of orphan nuclear receptor Nur77.

Dysregulation of ß-catenin turnover due to mutations of its regulatory proteins including adenomatous polyposis coli (APC) and p53 is implicated in the pathogenesis of cancer. Thus, intensive effort is being made to search for alternative approaches to reduce abnormally activated ß-catenin in cancer cells. Nur77, an orphan member of the nuclear receptor superfamily, has a role in the growth and apoptosis of cancer cells. Here, we reported that Nur77 could inhibit transcriptional activity of ß-catenin by inducing ß-catenin degradation via proteasomal degradation pathway that is glycogen synthase kinase 3ß and Siah-1 independent. Nur77 induction of ß-catenin degradation required both the N-terminal region of Nur77, which was involved in Nur77 ubiquitination, and the C-terminal region, which was responsible for ß-catenin binding. Nur77/ΔDBD, a Nur77 mutant lacking its DNA-binding domain, resided in the cytoplasm, interacted with ß-catenin, and induced ß-catenin degradation, demonstrating that Nur77-mediated ß-catenin degradation was independent of its DNA binding and transactivation, and might occur in the cytoplasm. In addition, we reported our identification of two digitalis-like compounds (DLCs), H-9 and ATE-i2-b4, which potently induced Nur77 expression and ß-catenin degradation in SW620 colon cancer cells expressing mutant APC protein in vitro and in animals. DLC-induced Nur77 protein was mainly found in the cytoplasm, and inhibition of Nur77 nuclear export by the CRM1-dependent nuclear export inhibitor leptomycin B or Jun N-terminal kinase inhibitor prevented the effect of DLC on inducing ß-catenin degradation. Together, our results demonstrate that ß-catenin can be degraded by cytoplasmic Nur77 through their interaction and identify H-9 and ATE-i2-b4 as potent activators of the Nur77-mediated pathway for ß-catenin degradation.

Discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs.

Finding good drug leads de novo from large chemical libraries, real or virtual, is not an easy task. High-throughput screening is often plagued by low hit rates and many leads that are toxic or exhibit poor bioavailability. Exploiting the secondary activity of marketed drugs, on the other hand, may help in generating drug leads that can be optimized for the observed side-effect target, while maintaining acceptable bioavailability and toxicity profiles. Here, we describe an efficient computational methodology to discover leads to a protein target from safe marketed drugs. We applied an in silico "drug repurposing" procedure for identification of nonsteroidal antagonists against the human androgen receptor (AR), using multiple predicted models of an antagonist-bound receptor. The library of marketed oral drugs was then docked into the best-performing models, and the 11 selected compounds with the highest docking score were tested in vitro for AR binding and antagonism of dihydrotestosterone-induced AR transactivation. The phenothiazine derivatives acetophenazine, fluphenazine, and periciazine, used clinically as antipsychotic drugs, were identified as weak AR antagonists. This in vitro biological activity correlated well with endocrine side effects observed in individuals taking these medications. Further computational optimization of phenothiazines, combined with in vitro screening, led to the identification of a nonsteroidal antiandrogen with improved AR antagonism and marked reduction in affinity for dopaminergic and serotonergic receptors that are the primary target of phenothiazine antipsychotics.

p53 and Nur77/TR3 - transcription factors that directly target mitochondria for cell death induction.

The complex apoptotic functions of the p53 tumor suppressor are central to its antineoplastic activity in vivo. Conversely, p53 function is altered or attenuated in one way or another in the majority of human cancers. Besides its well-understood action as a transcriptional regulator of multiple apoptotic genes, p53 also exerts a direct pro-apoptotic role at the mitochondria by engaging in protein-protein interactions with anti- and pro-apoptotic Bcl2 family members, thereby executing the shortest known circuitry of p53 death signaling. Nur77, also known as TR3 or NGFI-B, is a unique transcription factor belonging to the orphan nuclear receptor superfamily. Even more extreme than p53, Nur77 can exert opposing biological activities of survival and death. Its activities are regulated by subcellular distribution, expression levels, protein modification and heterodimerization with retinoid X receptor. In cancer cells, Nur77 functions in the nucleus as an oncogenic survival factor, but becomes a potent killer when certain death stimuli induce its migration to mitochondria, where it binds to Bcl2 and conformationally converts it to a killer that triggers cytochrome c release and apoptosis. This review focuses on their unexpected transcription-independent pro-death programs at mitochondria and highlights the remarkable mechanistic similarities between them. Moreover, an accumulating body of evidence provides ample rationale to further investigate how these mitochondrial p53 and Nur77 pathways could become exploitable targets for new cancer therapeutics.

Regulation of Nur77 nuclear export by c-Jun N-terminal kinase and Akt.

Proapoptotic nuclear receptor family member Nur77 translocates from the nucleus to the mitochondria, where it interacts with Bcl-2 to trigger apoptosis. Nur77 translocation is induced by certain apoptotic stimuli, including the synthetic retinoid-related 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN)/CD437 class. In this study, we investigated the molecular mechanism by which AHPN/CD437 analog (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces Nur77 nuclear export. Our results demonstrate that 3-Cl-AHPC effectively activated Jun N-terminal kinase (JNK), which phosphorylates Nur77. Inhibition of JNK activation by a JNK inhibitor suppressed 3-Cl-AHPC-induced Nur77 nuclear export and apoptosis. In addition, several JNK upstream activators, including the phorbol ester TPA, anisomycin and MAPK kinase kinase-1 (MEKK1), phosphorylated Nur77 and induced its nuclear export. However, Nur77 phosphorylation by JNK, although essential, was not sufficient for inducing Nur77 nuclear export. Induction of Nur77 nuclear export by MEKK1 required a prolonged MEKK1 activation and was attenuated by Akt activation. Expression of constitutively active Akt prevented MEKK1-induced Nur77 nuclear export. Conversely, transfection of dominant-negative Akt or treatment with a phosphatidylinositol 3-kinase (PI3-K) inhibitor accelerated MEKK1-induced Nur77 nuclear export. Furthermore, mutation of an Akt phosphorylation residue Ser351 in Nur77 abolished the effect of Akt or the PI3-K inhibitor. Together, our results demonstrate that both activation of JNK and inhibition of Akt play a role in translocation of Nur77 from the nucleus to the cytoplasm.

Vitamin A and apoptosis in prostate cancer.

Apoptosis represents an effective way to eliminate cancer cells. Unfortunately, advanced prostate tumors eventually progress to androgen-independent tumors, which are resistant to current therapeutic approaches that act by triggering apoptosis. Vitamin A and its natural and synthetic analogs (retinoids) induce apoptosis in prostate cancer cells in vitro and in animal models, mainly through induction of retinoic acid receptor-beta (RARbeta). Expression levels of RARbeta, however, are significantly reduced in hormone-independent prostate cancer cells. Recently, a new class of synthetic retinoids related to 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) (also called CD437) that effectively induces apoptosis of both hormone-dependent and -independent prostate cancer cells in a retinoid receptor-independent manner was identified and has drawn a lot of attention in the field. The apoptotic effect of AHPN requires expression of orphan receptor TR3 (also called nur77 or NGFI-B). Paradoxically, TR3 expression is also induced by androgen and other mitogenic agents in prostate cancer cells to confer their proliferation. The recent finding that TR3 migrates from the nucleus to mitochondria to trigger apoptosis in response to AHPN suggests that the opposing biological activities of TR3 are regulated by its subcellular localization. Thus, agents that induce translocalization of TR3 from the nucleus to mitochondria will have improved efficacy against prostate cancer. TR3, therefore, represents an unexplored molecule that may be an ideal target for developing new agents for prostate cancer therapy.