Incarvine C and its analogues inhibit the formation of cell cytoskeleton by targeting Rac1.

Ras-related C3 botulinum toxin substrate 1 (Rac1) has emerged as a key regulator in the treatment of cancer metastasis because of its involvement in the formation of cell plate pseudopods and effects on cell migration. In this study, we found that incarvine C, a natural product isolated from Incarvillea sinensis, and its seven analogues exhibited antitumour activity by inhibiting cell cytoskeleton formation, with moderate cytotoxicity. Accordingly, these compounds inhibited the cytoskeleton-mediated migration and invasion of MDA-MB-231 cells, with inhibition rates ranging from 37.30 % to 69.72 % and 51.27 % to 70.90 % in vitro, respectively. Moreover, they induced G2/M phase cell cycle arrest in MDA-MB-231 cells. A pull-down assay revealed that the interaction between Rac1 and its downstream effector protein PAK1 was inhibited by these compounds and that the compound Ano-6 exhibited substantial activity, with an inhibition rate of more than 90 %. Molecular docking showed that incarvine C and its analogues could bind to the nucleotide-binding pocket of Rac1, maintaining high levels of inactivated Rac1. As Ano-6 exhibited significant activity in vitro, its anti-cancer activity was tested in vivo. Four weeks of oral treatment with Ano-6 was well-tolerated in mice, and it induced a potential anti-tumour response in xenografts of MDA-MB-231 cells. Further studies demonstrated that Ano-6 was enriched in tumour tissues after 2 h of administration and induced an increase in the number of dead tumour cells. In summary, these findings not only reveal the mechanism of incarvine C but also provide a new molecular template for Rac1 inhibitors and identify a promising candidate for breast cancer treatment.

A New Monoterpene Alkaloid from Incarvillea sinensis with Migration Inhibitory Activity on Cancer Cell.

A novel monoterpene alkaloid, named incarvine G, was isolated from the Incarvillea sinensis Lam. Its chemical structure was elucidated using comprehensive spectroscopic methods. Incarvine G is an ester compound comprised of a monoterpene alkaloid and glucose. This compound showed evident inhibition on cell migration, invasion, and cytoskeleton formation of human MDA-MB-231 with low cytotoxicity.

Corrigendum: Dual-specificity phosphatase CDC25B was inhibited by natural product HB-21 through covalently binding to the active site.

[This corrects the article DOI: 10.3389/fchem.2018.00531.].

A network-based pharmacological study on the mechanism of action of muscone in breast cancer.

Background: The purpose of this study was to investigate the mechanism of action of muscone on breast cancer using network pharmacology and molecular docking techniques. Methods: Targets of muscone acid action were collected using the PubChem and SwissTargetPrediction databases. Relevant target sets of breast cancer were collected using the GeneCards database, and the intersection of the drug-disease targets was used as the potential target of muscone action in breast cancer. The STRING database was used to construct a target protein-protein interaction (PPI) network, and the data were imported into Cytoscape 3.7.1 for topological network analysis to obtain the core target genes of muscone in breast cancer. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the DAVID database. The correlation of core gene expression with breast cancer survival was analyzed using the online Kaplan-Meier plotter tool. Molecular docking of core target genes to muscone was performed using AutoDock Vina. Results: A total of 18 common targets of muscone and breast cancer were obtained through target intersection. The PPI map and topology analysis revealed that androgen receptor (AR), progesterone receptor (PGR), matrix metalloproteinase 9 (MMP9), prostaglandin-endoperoxide synthase 2 (PTGS2), heat shock protein 90 alpha family class A member 1 (HSP90AA1), mitogen-activated protein kinase 14 (MAPK14), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) might be the key targets of muscone acting on breast cancer. The GO enrichment analysis identified 60 terms, while the KEGG pathway enrichment analysis identified 7 signaling pathways, including steroid hormone biosynthesis, ovarian steroidogenesis, cancer pathways, and the tumor necrosis factor (TNF) signaling pathway. The results of survival stage analysis showed that the binding activity between muskone and key targets was better than other targets. The molecular docking results showed that muscone had the highest docking affinity for the key target CYP19A1 gene at -7.0 kJ/moL. Conclusions: Muscone might exert anti-breast cancer effects through cancer pathways, ovarian steroidogenesis, and TNF signaling pathways and has the potential to be developed as a clinical agent.

Comprehensive analysis reveals COPB2 and RYK associated with tumor stages of larynx squamous cell carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is one of the highly aggressive malignancy types of head and neck squamous cell carcinomas; genes involved in the development of LSCC still need exploration. METHODS: We downloaded expression profiles of 96 (85 in advanced stage and 11 in early stage) LSCC patients from TCGA-HNSC. Function enrichment and protein-protein interactions of genes in significant modules were conducted. Univariate and multivariate Cox regression analyses were performed to explore potential prognostic biomarkers for LSCC. The expression levels of genes at different stages were compared and visualized via boxplots. Immune infiltration was examined by the CIBERSORTx web-based tool and depicted with ggplot2. Gene set enrichment analysis (GSEA) was utilized to analyze functional enrichment terms and pathways. Immunohistochemical staining (IHC) was used to verify the expression of genes in the LSCC samples. RESULTS: We identified 25 modules, including 3 modules significantly related to tumor stages of LSCC via weighted gene co-expression network analysis (WGCNA). UIMC1, NPM1, and DCTN4 in the module 'cyan', TARS in the module 'darkorange', and COPB2 and RYK in the module 'lightyellow' showed statistically significant relation to overall survival. The expression of COPB2, DCTN4, RYK, TARS, and UIMC1 indicated association with the change of fraction of immune cells in LSCC patients; two genes, COPB2 and RYK, indicated different expression in various tumor stages of LSCC. Finally, COPB2 and RYK showed high-expression in tumor tissues of advanced LSCC patients. CONCLUSIONS: Our study provided a potential perceptive in analyzing progression of LSCC cells and exploring prognostic genes.

Effect of carrier materials on the properties of the andrographolide solid dispersion

Abstract In the work the andrographolide (AG)-solid dispersions (SDs) were prepared by the spray-drying method, using polyethylene glycol 8000 (PEG8000), Poloxamer188, polyvinylpyrrolidone K30 (PVPK30), Soluplus® as carrier materials. The effect of different polymers as carrier materials on the properties of the AG-SDs were studied. The results showed obvious differences in intermolecular interaction, thermal stability, drug state, powder properties, dissolution behavior, and so on of AG-SDs prepared using different polymers as carrier materials. AG-PEG8000-SD was a partial-crystalline and partial-amorphous powder with smaller surface area and pore volume, but it was easy to wetting and did not swell in contact with dissolved medium. AG-Soluplus®-SD was completely amorphous powder with larger specific surface area and pore volume, but it swelled in contact with water. Therefore, the dissolution profile of AG in AG-PEG8000-SD was similar to that in AG-Soluplus®-SD. Soluplus® and PEG8000 were suitable polymers to design AG-SDs, considering both physicochemical properties and dissolution behaviors. The results of this reseach showed that when selecting carrier materials for SD, we should not only consider the state of drugs in SD and the powder properties of SD, but also consider whether there is swelling when the carrier materials are in contact with the dissolution medium.

Blood sampling from peripherally inserted central catheter is effective and safe for patients with head and neck cancers.

OBJECTIVE: To evaluate the validity of laboratory tests for blood sampling from a peripherally inserted central catheter. METHODS: A total of 22 patients diagnosed with head and neck cancers were enrolled. In total, 101 paired blood samples were taken both via venipuncture and peripherally inserted central catheter for hematology and biochemistry testing. Paired t tests and linear correlation analysis were used to evaluate the results. Blood sampling-related pain was recorded by visual analogue scales and numerical rating scales. Infusion occlusion, hemolysis, and catheter-related blood stream infection were also recorded. RESULTS: The peripherally inserted central catheter-associated test results were slightly lower than those with venipuncture. Some parameters differed more than others. However, the degree of difference was less than 5% for every pair. There was a high correlation between the test results with two methods of blood sampling with the representative equation approximately being "y = x." According to visual analogue scales and numerical rating scale analysis, the pain degree with peripherally inserted central catheter was significantly lower than that of the venipuncture (p < 0.001). No case of infusion occlusion, catheter-related blood stream infection was reported with both methods. Hemolysis rate in blood samples from peripherally inserted central catheter (1/101) was much lower than that seen with venipuncture (11/101) with significant difference (p = 0.0056). CONCLUSION: Blood sampling via peripherally inserted central catheter and venipuncture showed equivalent reliability in laboratory testing. Compared with venipuncture, blood sampling via peripherally inserted central catheter causes less pain and is safer. Blood sampling via peripherally inserted central catheter is strongly recommended for clinical use.

A Comparative Study on Polyphenolic Composition of Berries from the Tibetan Plateau by UPLC-Q-Orbitrap MS System.

Five traditional medicinal food from the Tibetan plateau including Nitraria tangutorum Bobrov (NT), Hippophae rhamnoides L. (HR), Lycium ruthenicum Murray (LR), Lycium barbarum L. (LB) and Rubus corchorifolius L.f. (RC) are rich in phenolic compounds. However, the detailed studies about the phenolic compounds remain scarce. Therefore, we established a rapid method for the simultaneous identification and quantification of the phenolic compounds from berries via Ultra Performance Liquid Chromatography-Quadruple-Orbitrap MS system (UPLC-Q-Orbitrap MS). This method was verified from many aspects including detection limit, quantification limit, precision, repeatability, stability, average recovery rate and recovery range, and then was used to analyze the phenolic compounds in these five species of berries. Finally, a total of 21 phenolic compounds were directly identified by comparing the retention time and exact mass, of which 14 compounds were identified by us for the first time in berries from the Tibetan plateau, including one flavonoid aglycone (myricetin), 11 phenolic acids (gallic acid, protocatechuate, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, 2-hydroxybenzeneacetic acid and ellagic acid), one flavanol (catechin) and one dihydrochalcone flavonoid (phloretin). Quantitative results showed that rutin, myricetin, quercetin and kaempferol were the main flavonoids. Moreover, a variety of phenolic acid compounds were also detected in most of the berries from the Tibetan plateau. Among these compounds, the contents of protocatechuate and chlorogenic acid were high, and high levels of catechin and phloretin were also detected in these plateau berries.

Isoborneol Attenuates Low-Density Lipoprotein Accumulation and Foam Cell Formation in Macrophages.

PURPOSE: Isoborneol has been used in the treatment of cardiovascular disease for several years in China. However, the mechanism is still unclear. The aim of this study was to identify the novel mechanism of isoborneol for its application in atherosclerotic disease. MATERIALS AND METHODS: The whole-genome gene expression profiles of MCF-7 cells treated with/or without isoborneol were detected by mRNA microarray analysis. The degree of similarity between the gene expression profiles was compared with the Connectivity Map (CMAP) database. An MTT assay was used to assess the toxicity of isoborneol on RAW 264.7 cells. Oil red O staining and a Dil-ox-LDL uptake assay in RAW 264.7 cells were also used to detect the accumulation of lipids in the macrophages and the uptake of oxidized low-density lipoprotein (ox-LDL). RESULTS: Isoborneol was proved to have mRNA expression profiles similar to that of ikarugamycin which can inhibit the uptake of ox-LDL. This process has proved to be an important cause of foam cell formation and early atherosclerotic lesions. It is speculated, therefore, that isoborneol may show similar activity to that shown by ikarugamycin. Subsequently, it was shown that RAW 264.7 cells reduced the absorption of ox-LDL and the accumulation of intracellular lipids after treatment with different concentrations of isoborneol. CONCLUSION: The results indicate that isoborneol inhibits macrophage consumption of ox-LDL, thereby preventing the accumulation of lipids in the macrophages. These results provide evidence for the application of isoborneol in atherosclerotic disease.

Discovery of a natural fluorescent probe targeting the Plasmodium falciparum cysteine protease falcipain-2.

The Plasmodium falciparum cysteine protease falcipain-2 (FP-2) is an attractive antimalarial target. Here, we discovered that the natural compound NP1024 is a nonpeptidic inhibitor of FP-2 with an IC50 value of 0.44 µmol L-1. The most exciting finding is that both in vitro and in vivo, NP1024 directly targets FP-2 in malaria parasite-infected erythrocytes as a natural fluorescent probe, thereby paving the way for an integration of malaria diagnosis and treatment.

Quantitative Analyses of Nine Phenolic Compounds and Their Antioxidant Activities from Thirty-Seven Varieties of Raspberry Grown in the Qinghai-Tibetan Plateau Region.

In this work, an efficient method for the rapid extraction and separation of antioxidant phenols was developed and optimized. The method was then applied to extract and separate nine phenols from 37 varieties of raspberry, in which their antioxidant activities were further investigated. First, the extraction was conducted using ultra-sonication, which was then further separated using reversed-phase high-performance liquid chromatography/ultraviolet (RP-HPLC/UV) analysis. In this step, several key parameters (volume of the extraction reagent, time of extraction, and the temperature of extraction) affecting its efficiency were investigated and optimized using the response surface methodology (RSM) combined with the Box-Behnken design (BBD) so that the optimal conditions were obtained. According to the overall results of the optimization study, the optimal conditions were chosen as follows: volume of extraction reagent = 2.0 mL, time of extraction = 50.0 min, and temperature of extraction = 50 °C. The optimal conditions were then applied to extract nine phenols, including gallic acid, catechin, chlorogenic acid, vanillic acid, syringic acid, cumaric acid, ferulic acid, rosemary acid, and quercetin from 37 raspberry varieties. The extracted phenols were characterized and their antioxidant activities, including DPPH- and ABTS- free radical scavenging and intracellular reactive oxygen species (ROS) activity, using HepG2 cells as the model, were subsequently studied. The findings suggested that although their contents varied among most raspberry varieties, these phenols significantly contributed toward their antioxidant capacity and scavenging intracellular ROS activities. This study provides a scientific and theoretical basis for the selection of raspberry varieties and product development in Qinghai province.

Discovery of SCY45, a Natural Small-Molecule MDM2-p53 Interaction Inhibitor.

The disruption of the MDM2-p53 interaction has been regarded as an attractive strategy for anticancer drug discovery. Here, the natural small-molecule SCY45 was identified as a potent MDM2-p53 interaction inhibitor based on fluorescence polarization and molecular modeling. SCY45 inhibited the MDM2-p53 interaction with an IC50 value of 4.93±0.08 µm. The structural modeling results showed that SCY45 not only had high structural similarity with nutlin-3a, a well-reported MDM2-P53 interaction inhibitor, but also bound to the p53 binding pocket of MDM2 with a binding mode similar to that of nutlin-3a. Moreover, SCY45 reduced the cell viability in cancer cells with MDM2 gene amplification. SCY45 showed the highest inhibition for SJSA-1 cells, which exhibit excessive MDM2 gene amplification, with an IC50 value of 7.54±0.29 µm, whereas SCY45 showed a weaker inhibition for 22Rv1 cells and A549 cells, which have a single copy of the MDM2 gene, with IC50 values of 18.47±0.75 µm and 31.62±1.96 µm, respectively.

Shikonin inhibits cancer cell cycling by targeting Cdc25s.

BACKGROUND: Shikonin, a natural naphthoquinone, is abundant in Chinese herb medicine Zicao (purple gromwell) and has a wide range of biological activities, especially for cancer. Shikonin and its analogues have been reported to induce cell-cycle arrest, but target information is still unclear. We hypothesized that shikonin, with a structure similar to that of quinone-type compounds, which are inhibitors of cell division cycle 25 (Cdc25) phosphatases, will have similar effects on Cdc25s. To test this hypothesis, the effects of shikonin on Cdc25s and cell-cycle progression were determined in this paper. METHODS: The in vitro effects of shikonin and its analogues on Cdc25s were detected by fluorometric assay kit. The binding mode between shikonin and Cdc25B was modelled by molecular docking. The dephosphorylating level of cyclin-dependent kinase 1 (CDK1), a natural substrate of Cdc25B, was tested by Western blotting. The effect of shikonin on cell cycle progression was investigated by flow cytometry analysis. We also tested the anti-proliferation activity of shikonin on cancer cell lines by MTT assay. Moreover, in vivo anti-proliferation activity was tested in a mouse xenograft tumour model. RESULTS: Shikonin and its analogues inhibited recombinant human Cdc25 A, B, and C phosphatase with IC50 values ranging from 2.14 ± 0.21 to 13.45 ± 1.45 µM irreversibly. The molecular modelling results showed that shikonin bound to the inhibitor binding pocket of Cdc25B with a favourable binding mode through hydrophobic interactions and hydrogen bonds. In addition, an accumulation of the tyrosine 15-phosphorylated form of CDK1 was induced by shikonin in a concentration-dependent manner in vitro and in vivo. We also confirmed that shikonin showed an anti-proliferation effect on three cancer cell lines with IC50 values ranging from 6.15 ± 0.46 to 9.56 ± 1.03 µM. Furthermore, shikonin showed a promising anti-proliferation effect on a K562 mouse xenograph tumour model. CONCLUSION: In this study, we provide evidence for how shikonin induces cell cycle arrest and functions as a Cdc25s inhibitor. It shows an anti-proliferation effect both in vitro and in vivo by mediating Cdc25s.

The construction and characterization of a novel adenovirus vector of artificial microRNA targeting EGFR.

OBJECTIVE: To construct a recombinant adenovirus with artificial microRNA targeting the epidermal growth factor receptor (EGFR) to inhibit the proliferation and induce the apoptosis of tumor cells. METHODS: An artificial microRNA (amiR) targeting EGFR was designed and cloned into an adenovirus shuttle plasmid to obtain pDC312-SLPI (secretory leukoprotease inhibitor)-EGFR amiR-pA. Then it was co-transfected into 293 cells with the adenovirus backbone plasmid pBHGloxΔE1, 3Cre to pack the recombinant adenovirus Ad-SLPI-EGFR amiR. After the recombinant adenovirus infected Hep-2 cells by Ad-SLPI-EGFR amiR, a morphological change was observed, and an MTT assay was performed to assess the proliferation of the tumor cells. The expression of EGFR was determined by western blotting analyses to assess the effect of Ad-SLPI-EGFR amiR. RESULTS: The sequence of artificial microRNA targeting EGFR was confirmed by DNA sequencing. Ad-SLPI-EGFR amiR significantly downregulated EGFR expression at the protein level and exerted an inhibitory effect on proliferation in Hep-2 cells. The transfected cells became round, shrunken, partly grape-like and floating under a lighted microscope. CONCLUSION: The recombinant adenovirus of Ad-SLPI-EGFR amiR was successfully constructed, and it was able to sufficiently decrease the expression of EGFR and inhibit the proliferation of Hep-2 cells.

Dual-Specificity Phosphatase CDC25B Was Inhibited by Natural Product HB-21 Through Covalently Binding to the Active Site.

Cysteine 473, within the active site of the enzyme, Cdc25B, is catalytically essential for substrate activation. The most well-reported inhibitors of Cdc25 phosphatases, especially quinone-type inhibitors, function by inducing irreversible oxidation at this active site of cysteine. Here, we identified a natural product, HB-21, having a sesquiterpene lactone skeleton that could irreversibly bind to cys473 through the formation of a covalent bond. This compound inhibited recombinant human Cdc25B phosphatase with an IC50 value of 24.25 µM. Molecular modeling predicted that HB-21 not only covalently binds to cys473 of Cdc25B but also forms six hydrogen bonds with residues at the active site. Moreover, HB-21 can dephosphorylate cyclin-dependent kinase (CDK1), the natural substrate of Cdc25b, and inhibit cell cycle progression. In summary, HB-21 is a new type of Cdc25B inhibitor with a novel molecular mechanism.

Dual androgen receptor (AR) and STAT3 inhibition by a compound targeting the AR amino-terminal domain.

Prostate cancer (PCa) often recurs as incurable castration-resistant prostate cancer (CRPC) after the failure of androgen deprivation therapy. CRPC development relies on androgen receptor (AR) signaling. The IL6/STAT3 pathway is also a key driver of CRPC. The crosstalk between IL6/STAT3 and the AR pathways provides opportunities to explore next-generation agents to treat PCa. Through screening of around 600 natural compounds in our newly established prostate tumorigenesis model, potential STAT3 signaling inhibitors were found and additionally examined for effects on AR signaling. The small molecular compound 154 exhibited dual effects on IL6/STAT3 and AR pathways. We show here that compound 154 inhibits AR and STAT3 transcriptional activity, reduces the expression of phosphorylation of STAT3 (Y705) and downregulates the mRNA levels of AR target genes. Compound 154 also inhibits protein expression of AR and AR splice variants (ARv567es and AR-V7) without altering AR mRNA levels. Compound 154 binds to AR directly, but not to STAT3 and is identified as an antagonist of the AR amino-terminal domain (NTD) by disrupting protein-protein interactions between STAT3 and the AR NTD. Moreover, compound 154 does not reduce AR nuclear translocation. Compound 154 possesses the potential to become a leading compound in novel therapies against CRPC.

Veratramine modulates AP-1-dependent gene transcription by directly binding to programmable DNA.

Because the transcription factor activator protein-1 (AP-1) regulates a variety of protein-encoding genes, it is a participant in many cellular functions, including proliferation, transformation, epithelial mesenchymal transition (EMT), and apoptosis. Inhibitors targeting AP-1 have potential use in the treatment of cancer and other inflammatory diseases. Here, we identify veratramine as a potent natural modulator of AP-1, which selectively binds to a specific site (TRE 5'-TGACTCA-3') of the AP-1 target DNA sequence and regulates AP-1-dependent gene transcription without interfering with cystosolic signaling cascades that might lead to AP-1 activation. Moreover, RNA-seq experiments demonstrate that veratramine does not act on the Hedgehog signaling pathway in contrast to its analogue, cyclopamine, and likely does not harbor the same teratogenicity and toxicity. Additionally, veratramine effectively suppresses EGF-induced AP-1 transactivation and transformation of JB6 P+ cells. Finally, we demonstrate that veratramine inhibits solar-ultraviolet-induced AP-1 activation in mice. The identification of veratramine and new findings in its specific regulation of AP-1 down stream genes pave ways to discovering and designing regulators to regulate transcription factor.

A Natural CCR2 Antagonist Relieves Tumor-associated Macrophage-mediated Immunosuppression to Produce a Therapeutic Effect for Liver Cancer.

Hepatocellular carcinoma (HCC) is a common malignant tumor in the digestive tract with limited therapeutic choices. Although sorafenib, an orally administered multikinase inhibitor, has produced survival benefits for patients with advanced HCC, favorable clinical outcomes are limited due to individual differences and resistance. The application of immunotherapy, a promising approach for HCC is urgently needed. Macrophage infiltration, mediated by the CCL2/CCR2 axis, is a potential immunotherapeutic target. Here, we report that a natural product from Abies georgei, named 747 and related in structure to kaempferol, exhibits sensitivity and selectivity as a CCR2 antagonist. The specificity of 747 on CCR2 was demonstrated via calcium flux, the binding domain of CCR2 was identified in an extracellular loop by chimera binding assay, and in vivo antagonistic activity of 747 was confirmed through a thioglycollate-induced peritonitis model. In animals, 747 elevated the number of CD8+ T cells in tumors via blocking tumor-infiltrating macrophage-mediated immunosuppression and inhibited orthotopic and subcutaneous tumor growth in a CD8+ T cell-dependent manner. Further, 747 enhanced the therapeutic efficacy of low-dose sorafenib without obvious toxicity, through elevating the numbers of intra-tumoral CD8+ T cells and increasing death of tumor cells. Thus, we have discovered a natural CCR2 antagonist and have provided a new perspective on development of this antagonist for treatment of HCC. In mouse models of HCC, 747 enhanced the tumor immunosuppressive microenvironment and potentiated the therapeutic effect of sorafenib, indicating that the combination of an immunomodulator with a chemotherapeutic drug could be a new approach for treating HCC.