Exploring the mechanism of daphne-type diterpenes against gastric cancer cells.

Gastric cancer is one of the common malignant tumors. It is reported that daphne-type diterpenes have inhibitory effects on gastric cancer cells, but the mechanism is still unknown. To explore the detailed mechanism of the anticancer effect of daphne-type diterpenes, we carried out an integrated network pharmacology prediction study and selected an effective component (yuanhuacine, YHC) for the following validation in silico and in vitro. The result showed that daphne-type diterpenes exerted an anti-tumor effect by targeting proto-oncogene tyrosine-protein kinase SRC as well as regulating the Ras/MAPK signaling pathway, which caused the apoptosis and mitochondrial damage in gastric cancer cells.

Potential Targets and Mechanisms of Bitter Almond-Licorice for COVID-19 Treatment Based on Network Pharmacology and Molecular Docking.

BACKGROUND: The outbreak of Corona Virus Disease 2019 (COVID-19) has resulted in millions of infections and raised global attention. Bitter almonds and licorice are both Traditional Chinese Medicines (TCM), often used in combination to treat lung diseases. Several prescriptions in the guidelines for the diagnosis and treatment of coronavirus disease 2019 (trial version ninth) contained bitter almond-licorice, which was effective in the treatment of COVID-19. However, the active ingredients, drug targets and therapeutic mechanisms of bitter almonds-licorice for the treatment of COVID-19 remain to be elucidated. METHODS: The active ingredients and targets were derived from the Traditional Chinese Medicine Systems Pharmacology (TCMSP). Meanwhile, targets associated with COVID-19 were obtained from the GeneCards database, PharmGkb database and DrugBank database. Then, the potential targets of bitter almond-licorice against COVID-19 were screened out. Protein-protein interaction (PPI) networks and core targets were analyzed through the String database and Cytoscape software. In addition, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed based on potential targets using R statistical software. Finally, molecular docking was used to validate the binding of the active ingredients to the core targets. RESULTS: The results of the TCMSP database showed that the bitter almond-licorice had 89 active components against COVID-19, involving 102 targets. PPI network and core target analysis indicated that IL-6, TNF, MAPK1, and IL1B were the key targets against COVID-19. In addition, GO and KEGG enrichment analysis showed that the bitter almond-licorice were involved in various biological processes through inflammation-related pathways such as TNF signaling pathway and IL-17 signaling pathway. Finally, molecular docking approaches confirmed the affinity between the active components of the bitter almond-licorice and the therapeutic targets. CONCLUSION: The bitter almond-licorice could be used to treat COVID-19 by inhibiting inflammatory responses and regulating cellular stress. This work is based on data mining and molecular docking, and the findings need to be interpreted with caution.

Screening and analysis of key genes in the biological behavior of bone mesenchymal stem cells seeded on gradient nanostructured titanium compared with native pure Ti.

Titanium (Ti) and Ti-based alloy materials are ideal brackets that restore bone defect, and the mechanism of related genes inducing bone mesenchymal stem cells (BMSCs) to osteogenic differentiation is currently a hot research topic. In order to screen key genes of BMSCs during the osteogenic expression process, we acquired data sets (GSE37237 and GSE84500) which were in the database Gene Expression Omnibus (GEO). Investigations on differentially expressed genes (DEGs) and their enrichment of functions were conducted. We constructed relative protein-protein interaction (PPI) network by using Search Tool for the Retrieval of Interacting Genes (STRING) and visualized the expression of DEGs with Cytoscape. A total of 279 DEGs were discerned, which could be divided into 177 down regulated genes and 102 up regulated genes. In addition, the DEGs' enrichment and pathways included regulation of actin cytoskeleton, inflammatory mediator regulation of transient receptor potential (TRP) channels, peroxisome proliferator-activated receptors (PPAR) pathway, cell cycle, Rheumatoid arthritis, mitogen-activated protein kinases (MAPK) signaling pathway and Ras signaling pathway ect. It showed that 10 notable up regulated genes were mainly in AMP-activated protein kinase (AMPK) pathway. Then we used a technology named surface mechanical attrition treatment (SMAT) to prepare gradient nanostructured (GNS) surface Ti and seeded well-growing BMSCs on the surface of SMAT Ti and native pure Ti. Cell Counting Kits-8 (CCK-8), apoptosis experiment, immunofluorescence technology and staining experiments for alka-line phosphatase (ALP) and alizarin red staining (ARS) were used to research the proliferation, adhesion and differentiation ability of BMSCs seeded on SMAT Ti compared with native pure Ti. We used quantitative real-time PCR (qRT-PCR) technology so as to verify the expression of the most significant 5 genes. In summary, these results indicated novel point of views into candidate genes and potential mechanism for the further study of BMSCs' behaviors seeded on SMAT Ti.

A simple method for preparation of lignin/TiO2 nanocomposites by sulfonation degree regulation and their application in polyurethane films.

The waterborne polyurethane (WPU) exposed to outdoor environment for a long time are more likely to reduce their mechanical performance and service life. This work describes a simple and effective method to obtain the homogeneous lignin/TiO2 nanocomposite as the anti-UV additive to improve the applicability of WPU. The SKLs/TiO2 were prepared by the gradient sulfonation kraft lignin (SKLs) and tetrabutyl titanate. The particle morphology and hybrid structure of SKLs/TiO2 are characterized by FT-IR, zeta potential analysis, XPS, TG and SEM. Interestingly, it was found that the change of π-π interactions and electrostatic repulsion between SKL molecules effected the forming of SKLs/TiO2 nanocomposite. The lignin content and morphology of SKLs/TiO2 nanocomposite could be controlled by regulating the sulfonate group content on lignin molecular. Furthermore, the SKLs/TiO2 nanocomposites was successfully applied on water polyurethane film as the additive, when SKLs/TiO2 content increased from 0 wt% to 5.0 wt%, the tensile strength increased 43%, the elongation at break increased from 240.0% to 352.0% and the UV transmittance reduced from 87% to 1.7% below 400 nm, which greatly improved the UV resistance and mechanical properties. The results of this study are of significant and practical importance to the high-value-added utilization of lignin.

Daphnegiravone D from Daphne giraldii induces cell death by targeting ATR in Hep3B cells.

Ataxia telangiectasia and Rad3-related protein (ATR) plays a crucial role in cancer and has become a promising target for cancer therapy. Daphnegiravone D (DGD), which could induce apoptosis and oxidative stress in hepatocellular carcinoma (HCC) cells, but the detailed target protein was still unclear. The study provided that the possible target of DGD against HCC cells was determined by isobaric labels for relative and absolute quantification (iTRAQ) assay. In all changed proteins the fold change of ATR was particularly significant. The results from GO, KEGG and PPI analysis showed that DNA damage, cell cycle, apoptosis, DNA repair related pathways changed and ATR was exactly related to them. Moreover, the mRNA and protein of ATR were both decreased in a concentration-dependent manner, and the results of molecular docking also verified the binding. Additionally, cellular thermal shift assay (CETSA) suggested that DGD could directly target at ATR protein. Furthermore, the knockdown of ATR could increase apoptosis and reactive oxygen species (ROS) which induced by DGD. Since ATR inhibitors were generally used in combination with chemotherapy drugs (especially DNA damage drugs) in clinical trials, we investigated the combined application of DGD and oxaliplatin. The results showed that DGD combined with OXA also increased the apoptosis and ROS production of Hep3B cells over either drug alone. Taken together, this study revealed that DGD targeting ATR could be a promising therapeutic strategy for the treatment of liver cancer.

Application of gradient acid fractionation protocol to improve decolorization technology by lignin-based adsorbent.

Wastewater contaminated with dyes is discharged by huge amount daily, and involved many hazardous materials. Thus, this study focused on introducing low cost, ecofriendly and available removal agent (lignin-based adsorbent). Three adsorbents, APKL-4, APKL-5 and APKL-6 were obtained using gradient acid precipitation technology and used for methylene blue (MB) removal. The samples were characterized by SEM, FT-IR and zeta potential analyzer. The results indicated that the three adsorbents exhibit significantly different adsorption behavior due to the structural differences caused by fractionation. The APKL-5 and APKL-6 have fewer hydrophilic groups in their molecules and thus have more adsorption active sites to load MB molecules. A pore structure inside of APKL-5 molecules is form in acid fractionation, which allows it to carry more MB molecules. The adsorption capacity of APKL-5 increased 3.8 times (from 345 to 1310 mg g-1) in the alkaline solution which showing excellent pH responsiveness. This paper presents a new promising approach for preparing high efficiency, low cost and eco-friendly adsorbents and builds a foundation for developing further applications of lignin-based adsorbents.

Network pharmacology predicted HDAC6 as a potential target of flavones from Daphne giraldii on hepatocellular carcinoma.

Daphne giraldii Nitsche, belongs to Daphne genus, has been reported to exert anti-tumor activities. Our previous study suggested that flavones from Daphne giraldii have significant inhibitory effects on hepatocellular carcinoma (HCC) cells. However, the potential target of this type flavone was still unknown. In this study, 74 flavonoids compounds of Daphne giraldii and 41 potential targets of HCC were analyzed by the network, the most potential target was histone deacetylase 6 (HDAC6). Considering the cytotoxicity, compound 70 (Daphnegiravone D, DGD) was chosen for further confirmation. Molecular docking study revealed that DGD formed high binding affinity with HDAC6. Concomitantly, pharmacological studies indicated that DGD could inhibit the expression of HDAC6 in vitro and in vivo. In this study, network pharmacology along with experimental validation predicted and verified HDAC6 as one of potential targets of flavones, these investigations provide a new insight for further study of Daphne giraldii on HCC treatment.

Enantiomeric 8-O-4'-type neolignans from Crataegus pinnatifida exhibit cytotoxic effect via apoptosis and autophagy in Hep3B cells.

Crataegus pinnatifida has been famous for its nutritional purpose. However, systematic investigation on the bioactive constituents is still lacking, although this fruit has been reported for its cytotoxic effect before. In this study, two pairs of new lignan enantiomers (1a/1b, 2a/2b), which isolated using chiral chromatographic column from the fruits of C. pinnatifida were studied. The absolute configurations of enantiomers were determined by comparison between the experimental electronic circular dichroism (ECD) and calculated ECD spectra. Among them, 1a/1b exhibited a better cytotoxic effect in hepatocellular carcinoma Hep3B cells with an IC50 value of 34.97 ± 2.74 and 17.42 ± 0.71 µM, respectively. In addition, 1b induced much more apoptotic, autophagic cells than 1a in Hep3B cells. Furthermore, the underlying mechanism was demonstrated that p38 activation could promote 1b-induced apoptosis and autophagy. Moreover, 1b-induced apoptosis was significantly decreased in the presence of autophagic inhibitor Bafilomycin A1 (Baf A1), suggesting that the induction of autophagy enhanced apoptotic cell death in 1b-treated cells. In general, these findings provide a valuable basis for further understanding the effect of 8-O-4' lignans in C. pinnatifida on cytotoxic effect.

Brusatol: A potential anti-tumor quassinoid from Brucea javanica.

Brusatol, a triterpene lactone compound mainly from Brucea javanica, sensitizes a broad spectrum of cancer cells. It is known as a specific inhibitor of nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway. In this review, we provide a comprehensive overview on the antitumor effect and molecular mechanisms of brusatol in vitro and in vivo. This review also covers pharmacokinetics studies, modification of dosages forms of brusatol. Increasing evidences have validated the value of brusatol as a chemotherapeutic agent in cancers, which may contribute to drug development and clinical application.

Yuanhuatine from Daphne genkwa selectively induces mitochondrial apoptosis in estrogen receptor α-positive breast cancer cells in vitro.

Breast cancer is one of the most common cancers diagnosed among women worldwide. Estrogen receptor alpha (ERα) is a transcriptional factor that plays an important role in the development and progression of breast cancer. Yuanhuatine, a natural daphnane-type diterpenoid extracted from Daphne genkwa, was reported to exhibit significant cytotoxicity against breast cancer cells. However, the underlying mechanism is still unclear. In this study, we evaluated the cytotoxicity of yuanhuatine on two breast cancer cell lines that are ERα-positive and -negative. The results show that yuanhuatine inhibits the growth of ERα-positive cells (MCF-7) with much stronger inhibitory activity (IC50 = 0.62 µM) compared with positive control tamoxifen (IC50 = 14.43 µM). However, no obvious cytotoxicity was observed in ERα-negative cells (MDA-MB-231). Subsequent experiment also indicated that yuanhuatine markedly induced mitochondrial dysfunction, leading to apoptosis in MCF-7 cells. Molecular docking studies suggest the potential interactions between yuanhuatine and ERα. Immunofluorescence staining and Western blot analysis indicated that yuanhuatine down-regulated the expression of ERα in MCF-7 cells. MPP, a specific ERα inhibitor, significantly enhanced yuanhuatine-induced mitochondrial dysfunction and apoptosis in MCF-7 cells. On the contrary, the treatment with yuanhuatine causes no apoptosis in MM231 cells. Altogether, in vitro and in silico results suggested that ERα down-regulation was involved in yuanhuatine-induced mitochondrial dysfunction and apoptosis in ERα-positive breast cancer cells. Thus, yuanhuatine could be a potential candidate for treating ERα-positive breast cancer.

Racemic neolignans from Crataegus pinnatifida: Chiral resolution, configurational assignment, and cytotoxic activities against human hepatoma cells.

Phytochemical investigation of the fruit of Crataegus pinnatifida led to the isolation of four pairs of dihydrobenzofuran neolignan enantiomers (1a/1b-4a/4b) including six new compounds (1a/1b, 2a/2b, 3a and 4a). The enantioseparations of the racemates were achieved successfully by chiral chromatographic column. Their structures were established by comprehensive spectroscopic analyses and the absolute configurations were determined by quantum mechanical calculation of electronic circular dichroism (ECD) spectra. All compounds were evaluated in vitro for their cytotoxicity using human hepatocellular carcinoma Hep3B and HepG2 cells. Among them, it was found that 2a had a selective cytotoxicity against Hep3B cells with IC50 value of 25.47 µM, while the IC50 value of its enantiomer 2b on Hep3B cells was 59.37 µM. These results implied that the absolute configurations of 2a and 2b possessed remarkable influences on their cytotoxicity. Further flow cytometry analysis indicated that 2a performed more significant effect on cell apoptosis compared with its enantiomer 2b.

Eclalbasaponin I causes mitophagy to repress oxidative stress-induced apoptosis via activation of p38 and ERK in SH-SY5Y cells.

Oxidative stress accompanying excessive accumulation of reactive oxygen species (ROS) and mitochondrial dysfunction leads to the occurrence of neurodegenerative diseases. Our previous study showed that Eclalbasaponin I (EcI), a triterpene saponin isolated from Aralia elata (Miq.) Seem. (A. elata), repressed oxidative stress in human neuroblastoma SH-SY5Y cells. However, the detailed mechanism remains unclear. In this study, pretreatment with EcI in SH-SY5Y cells significantly activated the p38-mitogenactivated protein kinase (p38), the extracellular regulated protein kinase (ERK), whereas it did not affect the c-jun NH2 terminal kinases (JNK). In accordance with the initial findings, EcI-induced neuroprotective effect was attenuated by SB203580 (SB, a p38 inhibitor) or FR180204 (FR, an ERK inhibitor), being further confirmed by specific small interfering RNA (siRNA). Inhibition of either p38 or ERK up-regulated the apoptosis induction in EcI- and H2O2-cotreated cells. Furthermore, p38 or ERK suppression enhanced intracellular and mitochondrial ROS generation, decreased the activities of endogenous antioxidant defences as well as the mitochondrial membrane potential (MMP), resulting in dysfunction of mitochondria. In addition, EcI-induced autophagy and mitophagy were obviously down-regulated when p38 or ERK activation was blocked. Cumulatively, these findings supported that EcI-caused mitophagy contributed to the neuroprotective effect through p38 or ERK activation. Mitophagy induction might be an effective therapeutic intervention in neurodegenerative diseases.

Phenylpropanoid derivatives from the fruit of Crataegus pinnatifida Bunge and their distinctive effects on human hepatoma cells.

Ten undescribed phenylpropanoid derivatives including four pairs of enantiomers and two 8-9' linked neolignans, together with fifteen known ones were isolated from the fruit of Crataegus pinnatifida Bunge. Their structures were established by comprehensive spectroscopic analyses. Enantiomers were separated successfully by chiral chromatographic column and their absolute configurations were determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. The in vitro cytotoxicity of the isolates were evaluated against two human hepatocellular carcinoma, HepG2 and Hep3B cells. Among them, (±)-crataegusanoid A, (±)-crataegusanoid B and crataegusanoid F exhibited moderate cytotoxicity. Interestingly, the different absolute configurations of (±)-crataegusanoid A and B demonstrated enantioselective cytotoxicity in HepG2 cells. Further flow cytometry analysis indicated that both (-)-crataegusanoid A and (-)-crataegusanoid B performed more significant effects on cell apoptosis, autophagy, and cell cycle progression compared with their enantiomers (+)-crataegusanoid A and (+)-crataegusanoid B. In addition, the results revealed that these two pairs of enantiomers induced protective autophagy in HepG2 cells.

Sesquiterpenoids from the roots of Croton crassifolius.

Phytochemical investigation of Croton crassifolius roots afforded five sesquiterpenes (1-5), including two new sesquiterpenes 6S-hydroxy-cyperenoic acid (1) and crassifterpenoid A (5), together with three known compounds (2-4). The structures of the new compounds were determined by comprehensive spectroscopic methods, and their absolute configurations were determined by quantum chemical ECD calculation. Crassifterpenoid A (5) is the first germacrane-type sesquiterpene isolated from C. crassifolius, which enriched the diversity of chemical constituents in Croton crassifolius. In addition, the cytotoxicities of all compounds against human liver cancer lines HepG2 and Hep3B were determined, but none showed significant activity.

Triterpenes from the fruit of Camptotheca acuminata suppress human hepatocellular carcinoma cell proliferation through apoptosis induction.

The fruit of Camptotheca acuminata, a kind of mainly medicinal plant, possesses good antitumor properties. In order to explore the bioactive compounds for the treatment of hepatocellular carcinoma, the study focused on the isolation of cytotoxic compounds from the fruit of Camptotheca acuminata, which led to the discovery of fourteen compounds, including one new triterpene, 3ß,20-dihydroxy-30α-methyl,17(29)-ß-epoxy-28-norlupane (1), together with thirteen known compounds (2-14). The structures of isolated compounds were demonstrated by spectroscopic methods including 1D and 2D NMR spectroscopy. Moreover, all triterpenes were evaluated for antiproliferative activities against two human hepatocellular carcinoma cell lines, HepG2 and Hep3B. Compound 3 showed the strongest cytotoxic activity against the HepG2 with IC50 value at 29.6 µM. Further study demonstrated that compound 3 exhibited cytotoxic activity through the induction of apoptosis.

Prenylated flavans from Daphne giraldii and their cytotoxic activities.

Nine new prenylated flavan compounds with multi-chiral centers including two pairs of epimers were isolated from the stem and root bark of Daphne giraldii. Their structures were established by extensive NMR and HR-ESIMS spectroscopic data analyses. The in vitro cytotoxicity experiments indicated that compound 6 showed the most significant cytotoxicity against Hep3B cells, with an IC50 value of 9.83 µM. Hoechst 33258 and Annexin V-FITC/PI staining suggested that 6 could induce apoptosis of Hep3B cells in a concentration-dependent manner. Further mechanism study indicated that the apoptosis was associated with the up-regulations of Bax, cl-PARP and a decrease in Bcl-2 expression.

Daphnegiravone D from Daphne giraldii Nitsche induces p38-dependent apoptosis via oxidative and nitrosative stress in hepatocellular carcinoma cells.

Daphnegiravone D (DGD), a prenylated flavonoid from Daphne giraldii Nitsche, significantly inhibited cell growth of several cancer cell lines without cytotoxicity on human normal cells. Our previous study showed that DGD could induce apoptosis in hepatocellular carcinoma Hep3B and HepG2 cells, but the detailed mechanism was still unclear. The present study provides that DGD-induced oxidative and nitrosative stress contribute to apoptotic cell death in Hep3B and HepG2 cells. Furthermore, there is a positive loop between oxidative stress and p38 activation, similar result is observed between nitrosative stress and p38. N-Acetylcysteine (NAC), a reactive oxygen species scavenger, could relieve DGD-induced oxidative stress, but exerts little effect on nitrosative stress. In addition, carboxy-PTIO (PTIO, a well-known scavenger of reactive nitrogen species) down-regulates the induction of nitrosative stress without obvious effect on oxidative stress in DGD-treated cells. In conclusion, the induction of oxidative and nitrosative stress could enhance p38-mediated apoptosis in DGD-treated Hep3B and HepG2 cells. Moreover, we speculated that OS and NS could not ultimately affect each other in DGD-treated HCC cells. This study gives a new insight on the mechanism of DGD-induced apoptotic cell death via oxidative and nitrosative stress in HCC cells.

Bioactivity-guided isolation of ß-Carboline alkaloids with potential anti-hepatoma effect from Picrasma quassioides (D. Don) Benn.

ß-Carboline alkaloids in Picrasma quassioides (D. Don) Benn. have been proven to possess inhibitory activity against various cancer cells. However, their effect on hepatocellular carcinoma and structure-activity relationships (SAR) have not been systematically reported. In this work, bioactivity-directed fractionation of P. quassioides led to the separation of active fraction A2-2. A total of 39 ß-carbolines, including 4 new ones (1-4), were obtained from the active fraction. Moreover, all the isolated compounds were identified in the active fraction A2-2 by LC-MS. The cytotoxicity on HepG2 and Hep3B cells of all compounds was screened by MTT assay, and the SAR were established. The SAR were also supported by the apoptosis ratio of HepG2 cells using flow cytometry analysis after treatment with potential compounds 1, 2, 9, 10, 12, 29, 36 and 38. It suggested that these active compounds caused death of hepatoma cells through apoptosis induction. In addition, further study revealed that compounds 12, 29, 36 significantly activated caspase-3 in HepG2 cells.