Screening potential antagonists of epidermal growth factor receptor from Marsdenia tenacissima via cell membrane chromatography model assisted by HPLC-ESI-IT-TOF-MS.

Marsdenia tenacissima, or Tongguanteng in Chinese, is a traditional Chinese herb and has a broad application in clinical practice for its pharmacological effects of treating asthma, pneumonia, tonsillitis, pharyngitis tumors, etc. However, few studies have reported the screening of the active components of this medicine for tumor therapy. In this work, a two-dimensional analytical system was developed to screen antagonists of epidermal growth factor receptor (EGFR) from M. tenacissima. A fraction was retained on the EGFR cell membrane chromatography (CMC) column, separated and identified as tenacissoside G (TG), tenacissoside H (TH) and tenacissoside I (TI) by two-dimensional HPLC-IT-TOF-MS. Molecular docking and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay were carried out to assess the activity of TS (including TG, TH and TI). Molecular docking results showed that the binding mode of TS on EGFR is similar to that of gefitinib. The MTT assay demonstrated that gefitinib and TS (especially TI) could inhibit the growth of EGFR highly expressed cell lines in a dose-dependent manner in the range of 5-50 µmol/L. In conclusion, the two-dimensional EGFR/CMC-HPLC-IT-TOF-MS system could be a useful approach in drug discovery from traditional Chinese medicines for searching for potential antitumor candidates.

HMQ-T-F5 (1-(4-(2-aminoquinazolin-7-yl)phenyl)-3-(2­bromo­5- (trifluoromethoxy)phenyl) thiourea) suppress proliferation and migration of human cervical HeLa cells via inhibiting Wnt/ß-catenin signaling pathway.

BACKGROUND: Drug therapy plays an important role in the treatment of cervical cancer, which is one of the most common solid tumors in women. Therefore, it is important to seek more effective and less toxic therapies. PURPOSE: The aim of this study is to investigate the therapeutic potential of HMQ-T-F5 (1-(4-(2-aminoquinazolin-7-yl)phenyl)-3-(2­bromo­5-(trifluoromethoxy) phenyl)thiourea) (F5) for cervical cancer and explore the related mechanism. METHODS: By performing MTT assay, colony formation assay, flow cytometry, wound-healing assay, transwell assay, immunofluorescent staining and siRNA assay, we study the effect of F5 on human cervical HeLa cells. The mechanism of F5 was also investigated. RESULTS: We found that F5 significantly inhibited HeLa cell proliferation, led to accumulation of cells in the S phase, and induced apoptosis and inhibited migration. Mechanistically, F5 inhibited HeLa cell growth and migration through repressing the expression and nuclear translocation of ß-catenin, enhancing Axin expression, inhibiting the phosphorylation of LRP5/6 and GSK3ß, as well as downregulating the Wnt downstream targeted proteins. Knockdown of a checkpoint ß-catenin by siRNA significantly attenuated HeLa cell proliferation. Furthermore, XAV939, an inhibitor of ß-catenin, was used to treat HeLa cells and the results demonstrated that F5 inhibited proliferation and migration via the inhibition of the Wnt/ß-catenin pathway. CONCLUSION: Our findings demonstrated that F5 can target ß-catenin potentially and is useful in the treatment of cervical cancer.

Wnt/ß-catenin signaling pathway is involved in regulating the migration by an effective natural compound brucine in LoVo cells.

BACKGROUND: Colorectal cancer remains the third most common malignancies and migration is one of the main factors for its high mortality rate. Brucine, a natural plant alkaloid, has been proved to possess a variety of pharmacological functions including anti-tumor activities. PURPOSE: The aim of this study was to investigate the inhibitory effect of brucine on the colorectal cancer and the underlying mechanism. METHODS: In this study, colony formation assay and transwell assay were used to investigate the effect of brucine on LoVo cells viability and migration. Immunofluorescence assay, western blot assay and Gelatin zymography assay were used to study the mechanism of brucine. Xenograft model in nude mice was induced to investigate the in vivo effect of brucine on LoVo cells. RESULTS: Brucine could significantly decrease the viability, inhibit the colony formation and induce the apoptosis of LoVo cells. Brucine could also suppress the migration of LoVo cells in a dose-dependent manner. Western blot analysis elucidated that the inhibition of migration was associated with the decreasing expression of matrix metalloproteinases including MMP2, MMP3 and MMP9. Moreover, we found that treatment of brucine could downregulate the expression of Frizzled-8, Wnt5a, APC and GSNK1A1, and increase the expression of AXIN1. Meanwhile, brucine also decreased the phosphorylation level of LRP5/6 and GSK3ß, and increased the level of p-ß-catenin. Xenografted model in nude mice study also revealed that oral administration of brucine could inhibit the growth and migration of LoVo cells by activating the expression of AXIN1 and p-ß-catenin. CONCLUSION: Brucine could suppress the migration of the colorectal cancer in vitro and in vivo and the effect was associated with the inhibition of the Wnt/ß-catenin signaling pathway.

Screening allergic components of Yejuhua injection using LAD2 cell membrane chromatography model online with high performance liquid chromatography-ion trap-time of flight-mass spectrum system.

Yejuhua (YJH) injection is a traditional Chinese medicine (TCM) injection and has a widely application in clinical practice. However, adverse drug reactions (ADRs) caused by YJH injection, majorly manifested as allergic reactions, have been reported. Hence, Effective and practical method for allergen screening and identification is needed. In this work, a LAD2/CMC model coupled online with HPLC-IT-TOF-MS system was developed to screen, analyze, and identify the allergenic components of YJH injection. A fraction was retained on the LAD2/CMC column, and identified as linarin (LN). Histamine release assay was performed by the multiple reaction monitoring (MRM) method of UPLC-ESI-MS/MS. Results showed that YJH injection and LN were in accord with their allergic effects by increasing histamine release. In conclusion, the LAD2/CMC-HPLC-IT-TOF-MS system developed in this study could be used to screen allergenic components in complex systems.