The optimization and application of cell-based screening model for IL-17-mediated signaling pathway / 药学学报

We explore and verify the optimized condition for HEK-Blue IL-17 screening model, and screen the compounds that inhibits IL-17-medited signaling pathway. HEK-Blue IL-17 cells (5×104 cells per well) were seeded into the 96 plates followed by different concentrations of IL-17A or IL-17F alone, or in combination with tested compounds for 16 h. Then, the supernatant medium was incubated with QUANTI-Blue for 1 or 3 h to detect the OD value at λ655nm. The secreted alkaline phosphatase (SEAP) production was an index of IL-17-mediated signaling activation in HEK-Blue IL-17 cells. We found that both IL-17A and IL-17F can significantly activate the IL-17 signaling pathway in HEK-Blue IL-17 cells. The available dosage of IL-17A and IL-17F were 10 and 100 ng·mL-1, respectively. The reaction time of SEAP and QUANTI-Blue was 1 h. In this model, arctigenin and epigallocatechin gallate (EGCG) could inhibit the IL-17A and IL-17F-mediated signaling pathway. This established and optimized screening model of HEK-Blue IL-17 cells was suitable for screening inhibitors of IL-17-mediated signaling pathway.

Establishment and optimization of an enzyme-cell based high-throughput screening platform for IDO1 inhibitors / 药学学报

In this study, we used the tumor immunotherapy protein indoleamine 2,3-dioxygenase 1 (IDO1) as the target, and proposed an enzyme-cell-based tertiary IDO1 inhibitor high throughput screening platform. Firstly, the recombinant human IDO1 protein was expressed by genetic engineering and efficient IDO enzymatic screening system was established. Secondly, A172 cells stimulated with interferon-γ (IFNγ) or constructed plasmid which could highly express human IDO1 protein in HEK293 cells with transient transfection were used to construct the specific IDO1 cell based screening system. Finally, the effect of the compound on kynurenine and tryptophan in mouse plasma was determined by LC/MS/MS method on C57 mice, which could further verify the inhibitory effect of the selected compounds on IDO1 in vivo. The established and optimized enzyme-cell based screening model in this study can efficiently and effectively obtain IDO1 inhibitors in vitro, which lays a good foundation for the rapid development of clinical drugs. Procedures for animal study were performed with approval of the Animal Care and Use Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College.

The effects of different stimulators on the activation of human lymphocytes / 药学学报

The research aims to study the effects of different stimulants on the activation of human lymphocytes. Human peripheral blood mononuclear cells were prepared by density centrifugation. The blood's sample was provided by National Institutes for Food and Drug Control and approved by its Ethics Committee. The expressions of CD69 in CD3+CD4+ and CD3+CD8+ human T cells were detected by flow cytometry after administrated with CD3/CD28 antibody, phytohaemagglutinin (PHA), Staphylococcus auresus enterooxin B (SEB), interleukin (IL27) and PMA plus ionomycin for 24 h. The proliferation of lymphocyte was detected by CellTiter-Glo kit. The secreted IFNγ in supernatant of medium was examined by ELISA kit. The proliferation of lymphocytes had no change after exposed of CD3/CD28 antibody, SEB, IL27 and PMA plus ionomycin for 24 h. However, the CD69 expressions in CD3+CD4+ and CD3+CD8+ T cells and IFNγ productions were significantly increased by CD3/CD28 antibody, SEB, IL27 and PMA plus ionomycin at 24 h, indicating that CD3+CD4+ and CD3+CD8+ T cells were activated under above-mentioned stimulated condition. CD3/CD28 antibody, SEB, IL27 and PMA plus ionomycin were valid stimulants for T cell activation.

The antitumor activity of STAT3 target inhibitor Bt354 andmolecular mechanism of anti-prostate cancer / 药学学报

Signal transducer and activator of transcription 3 (STAT3) was found in an abnormal constitutively active status in certain cancer tissues, and under these circumstances the interruption of STAT3 signaling pathway was proposed with the potential anti-cancer efficacy. In this study, our previous reported STAT3 inhibitor Bt354 can inhibit tumor growth in DU145 xenograft mice without affecting body weight. In groups treated with Bt354, the inhibition rate of tumor weight was 58.8%, 62.7% and 73.5% in 10, 20, 40 mg·kg-1 group, respectively. Particularly, the number of Ki 67 positive cells in the tumor sections was significantly decreased in Bt354 groups. Furthermore, Bt354 inhibited the nuclear translocation of STAT3 and consequently induced cell growth inhibition, apoptosis in DU145 cells. These findings suggest that Bt354 may be a potent anticancer agent for STAT3 activated prostate cancer cells. Procedures for animal study were performed with approval of the Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College.

Antitumor activity of a novel PARP1/2 inhibitor YHP-743 / 药学学报

Poly(ADP-ribose) polymerase (PARP)-1 and PARP2 function as ADP-ribosylases involved in DNA repair. PARP1/2 is highly expressed in cancers and emerged as an attractive target for antitumor drug. In this study, we investigated the antitumor activity of a novel PARP1/2 inhibitor YHP-743 in vitro and in vivo. The results showed that YHP-743 had potent enzymatic inhibitory activity against PARP1 and PARP2 to down-regulate the PAR level. YHP-743 not only inhibited breast cancer cells with genes deficiency of homologous recombination repair, but also potentiated chemotherapy agent's cytotoxicity, such as temozolomide, topotecan, cisplatin and doxorubicin. YHP-743 elicited good antitumor activity in combination with temo-zolomide in vivo.

Validation and application a cell-based screening model for IL-6/JAK/STAT3 inhibitor / 药学学报

Interleukin-6 (IL-6)/janus kinase (JAK)/signal transducers and activators of transcription 3 (STAT3) is a pivotal signaling pathway in the regulation of cell proliferation, survival, differentiation and T cell activation. Aberration of this pathway is involved in multiple autoimmunity diseases and cancers, therefore the pathway is considered as a hot target for drug development. In our study, we validated a cell-based model of IL-6/JAK/STAT3 and used it in screening of its inhibitors. HEK-Blue IL-6 cells of Invivogen Inc. were used to stably express IL-6 receptor and STAT3-induced secreted embryonic alkaline phosphatase (SEAP) report gene. After stimulation by IL-6, SEAP was secreted from cells and reacted with QUANTI-Blue. The product can be detected at 655 nm. The inhibitory effect of compounds on STAT3 signaling showed as IC50 was calculated by OD value. The results shown that IL-6 specifically activated the cells, which could be applied to screen the inhibitors for IL-6/JAK/STAT3 signaling pathway. The optimized screening conditions were described as below:50 000 cells/well, 1 ng·mL-1 IL-6 incubation for 20 h and reaction with QUANTI-Blue for 1 h. Based on this condition, we screened 14 natural products based on this cell model and arctigenin, cryptotanshinone and curcumin showed potential inhibitory activities on STAT3 signaling pathway with IC50 of 1.28, 2.96 and 6.61 μmol·L-1. Our study suggests that HEK-Blue IL-6 cells were suitable for screening inhibitors for the IL-6/JAK/STAT3 signaling pathway.

Co-chaperones: regulated action in conformational functions of HSP90 and their actions in cancer / 药学学报

Heat shock protein 90(HSP90), as an essential molecular chaperone, regulates the folding, assembly and maturation of a wide range of oncogenic client proteins. The process of adenosine triphosphate(ATP)binding and adenosine diphosphate(ADP)/ATP exchange act as a conformational switch to regulate the chaperone function of HSP90. Furthermore, this process is controlled by a range of accessory proteins(as referred to co-chaperones), such as Hop, CDC37, p23, AHA1, PP5, etc. This article describes the structure and function of several co-chaperones, and their roles in tumor progress.