Harmine suppresses hyper-activated Ras-MAPK pathway by selectively targeting oncogenic mutated Ras/Raf in Caenorhabditis elegans.

BACKGROUND: Mutationally activated Ras proteins are closely linked to a wide variety of human cancers. Hence, there has been an intensive search for anti-Ras therapies for cancer treatment. The sole Ras gene, which encodes LET-60, in Caenorhabditis elegans regulates vulval development. While the loss of let-60 function leads to failure of vulva formation, the let-60(n1046gf) allele, which contains a missense mutation mimicking a Ras codon 13 mutation found in human cancers, results in extra vulval tissue, a phenotype named Muv (multiple vulvas). METHODS: By taking advantage of the easy-to-score Muv phenotype of let-60(n1046gf), we used a step-by-step screening approach (from crude extract to active fraction to active natural compound) to search for inhibitors of oncogenic Ras. Mutants of other key components in the Ras-mitogen-activated protein kinase (MAPK) pathway were used to identify other candidate targets. RESULTS: The natural compound harmine, isolated from the plant Peganum harmala, was found to suppress the Muv phenotype of let-60(n1046gf). In addition, harmine targets the hyper-activation of the Ras/MAPK pathway specifically caused by overexpression or mutated forms of LET-60/Ras and its immediate downstream molecule LIN-45/Raf. Finally, harmine can be absorbed into the worm body and probably functions in its native form, rather than requiring metabolic activation. CONCLUSION: In sum, we have revealed for the first time the anti-Ras activity of harmine in a C. elegans model system. Our results revealed the potential anti-cancer mechanism of harmine, which may be useful for the treatment of specific human cancers that are associated with oncogenic Ras mutations.

Simultaneous determination of five N-alkylamides in the root of Anacyclus pyrethrum by HPLC and profiling of components in its methanolic root extract by UPLC/Q-TOF-MS

ABSTRACT The root of Anacyclus pyrethrum (L.) Lag., Asteraceae, is very widely used for treating various diseases in Traditional Uygur Medicine, particularly in the treatment of vitiligo. However, there have been few studies on the quality standards of A. pyrethrum in China. A. pyrethrum contains abundant N-alkylamides, which are considered to be the principal components. Therefore, based on the previous research in our group, six N-alkylamides were obtained by using column chromatography. We used ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry to determine the mass spectrometry cleavage mechanism of these six monomer components and established the mass spectrometry cleavage law of N-alkylamides. Then, we used the ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method to rapidly identify and analyze the N-alkylamide components of the A. pyrethrum methanol extract. Finally, twenty N-alkylamides were identified, including eleven N-isobutylamides, two N-methyl isobutylamides, six 4-hydroxyphenylethyl-amide and one 2-phenylethylamide. Five of these compounds were identified as new compounds that have not been reported to date. Two of these compounds were identified for the first time in this herb. Therefore, this work provides an approach for the quality analysis of N-alkylamides in the root of A. pyrethrum. A search of the literature showed that the content determination in the A. pyrethrum quality standard is still a remaining problem. N-alkylamides are the main components of A. pyrethrum. Even though ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry has the advantages of lower time and higher efficiency compared to high-performance liquid chromatography, considering the ease of repeatability and universality of the quality control method, we chose to use high-performance liquid chromatography for content determination. In this experiment, high-performance liquid chromatography was used for the first time to establish a simple, rapid and accurate method for evaluating the N-alkylamide content in A. pyrethrum with five N-alkylamides used as the standards. Finally, this work provides a qualitative and quantitative method for the analysis of N-alkylamides in A. pyrethrum, improving the quality control standards for A. pyrethrum.

Isorhamnetin suppresses PANC-1 pancreatic cancer cell proliferation through S phase arrest.

Isorhamnetin, a flavonoid ingredient derived from Vernonia anthelmintica (L.) Willd., has shown a spectrum of antitumor activity. However, the chemopreventive potential of isorhamnetin on advanced pancreatic cancer and the underlying molecular mechanism remain unknown. In the current study, treatment of the advanced pancreatic adenocarcinoma cell line PANC-1 with isorhamnetin resulted in robust cell growth arrest. PI-annexin V double staining and Hoechst 33258 staining revealed that isorhamnetin moderately induced early apoptosis without morphological alterations of nuclei. Instead, isorhamnetin caused cell cycle S-phase arrest through downregulation of cyclin A. In addition, isorhamnetin decreased the phosphorylation levels of MEK and ERK in the Ras/MAPK pathway, which is involved in regulating cell proliferation, differentiation and apoptosis. Wound-healing experiments demonstrated isorhamnetin significantly reduced the migratory behavior of PANC-1 cells. Altogether, the present study suggests that isorhamnetin may be a potential agent for prevention of pancreatic carcinoma.

[Study on screening antitumor active fractions and chemical components in active fractions from root of Anaycclus pyrethrum].

Components that systematic separated from the root of Anaycclus pyrethrum were identified, in order to lay a foundation for future study of the root of A. pyrethrum. The CCK-8 assay showed that dichloromethane fraction exhibited the highest degree of cytotoxicity than others. Ten monomeric components were obtained from dichloromethane fraction and ethyl acetate fraction extracted from the root of A. pyrethrum, including 7 N-alkylamides, one coumarin and two flavonoid glycosides. They were identified as tetradeca-2E,4E,8E-trienoic acid 4-hydroxyphenylethylamide(1), deca-2E,4E-dienoicacid isobutylamide(2), undeca-2E,4E-diene-8,10-diynoic acid phenylethylamide(3), tetradeca-2E,4E-dienoic acid 4-hydroxyphenylethylamide(4), tetradeca-2E,4E-diene-8,10-diynoic acid isobutylamide(5), deca-2E,4E- dienoic acid 4-hydroxyphenylethylamide(6), dodeca-2E,4E-dienoic acid 4-hydroxy -phenyl-ethylamide(7), isoscopoletin(8), quercetin-7-O-ß-D-glucopyranoside(9), isorhamnetin-7-O-ß-D-glucopyranoside(10). Among them, compound 1 was identified as a new compound, Compounds 2-4, 8-10 were isolated from this herb for the first time.

[Influential factors associated with 2-week prevalence of fever and diarrhea among infants and young children at the age of 6-23 months in poor rural areas].

OBJECTIVE: To investigate the status and influential factors associated with 2-week prevalence of fever and diarrhea among infants and young children at the age of 6-23 months in poor rural areas.
 Methods: A total of 8 735 rural infants and young children aged 6-23 months in 30 poor counties of Wuling Mountains and Luoxiao Mountains in Hunan Province were selected in August 2015, and the questionnaires were used to collect information on the prevalence of fever and diarrhea, person and families, and feeding status. The data for prevalence of fever and diarrhea in infants and young children were calculated, and multi-non-conditional logistic regression model were used to analyze the influential factors.
 Results: The 2-week prevalence of fever and diarrhea in infants and young children was 20.8% and 12.2% respectively. The ages (OR=0.66, 95%CI 0.58 to 0.75), Dong ethnicity(OR=1.42, 95%CI 1.17 to 1.74) and low body weight (OR=1.31, 95%CI 1.11 to 1.54) were influential factors for fever among infants and young children in poor rural areas; female (OR=0.86, 95%CI 0.76 to 0.98), 12-17 months (OR=0.80, 95%CI 0.69 to 0.93), 18-23 months (OR=0.51, 95%CI 0.43 to 0.60), other ethnic minorities (OR=1.70, 95%CI 1.13 to 2.56), non-complementary feeding (OR=1.65, 95%CI 1.05 to 2.59) and low body weight (OR=1.39, 95%CI 1.14 to 1.70) were the influential factors of diarrhea among infants and young children.
 Conclusion: The 2-week prevalence of fever and diarrhea among infants and young children aged 6-23 months in poor rural areas were quite serious. Low age, Dong ethnicity, and low birth weight are high risk factors for fever. Male, no addition of complementary feeding, and low birth weight are high risk factors for diarrhea.

Sugar Composition Analysis of Fuzi Polysaccharides by HPLC-MSn and Their Protective Effects on Schwann Cells Exposed to High Glucose.

Fuzi has been used to treat diabetic complications for many years in china. In a previous study, we have shown that Fuzi aqueous extract can attenuate Diabetic peripheral neuropathy (DPN) in rats and protect Schwann cells from injury. Thus, the protective effect of Fuzi polysaccharides (FPS) on high glucose-induced SCs and the preliminary mechanism were investigated. Firstly, the FPS were obtained and their monose composition was analyzed by the combination of pre-column derivatization and high performance liquid chromatography coupled with electrospray ionization multi-tandem mass spectrometry (HPLC/ESI-MSn). The results witnessed the efficiency of this method and seven monosaccharides were tentatively identified, among which fucose was first reported. Simultaneously, m/z 215 can be considered as diagnostic ions to confirm the number of monosaccharides. Next, high glucose-induced SC model was applied and divided into model group, treated group of FPS, normal and osmotic control group. After treatment for 48 h, the data showed FPS could significantly decrease the intracellular ROS and apoptosis, which were determined by the corresponding fluorescent probes. Then, the expression of oxidative stress-related proteins in SCs were measured by Western blot. Furthermore, the protein tests found that FPS markedly up-regulated superoxide dismutase (SOD), catalase (CAT) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein level, but down-regulated NADPH oxidase-1 (Nox1) protein level. Moreover, FPS could also increase AMP-activated protein kinase (AMPK) activation significantly. Hence, we preliminary deduced that AMPK-PGC-1α pathway may play an important role in the protective effect of FPS against high glucose-induced cell damage.

An optical-parameters-free method for locating light source in deep tissue.

Bioluminescence of luciferase-expressing cells in live animals is a promising technique for image guided tumor resection. However, requiring exact optical tissue parameters is an underlying problem of diffusion equation when recovering bioluminescent images of luciferase activity within a volume using emission optical signal data from internal light sources. In this paper, an equivalence method based on the equation of radiative transfer (ERT) is proposed to locate bioluminescence source without optical tissue parameters. The solution of equivalence method is satisfied with the unique of localization as well as the complex tissue environment. The reconstructed images of bioluminescence are presented with simulated heterogeneous tissue models, and an 8.7% relative error of the source localization is obtained.