Paris polyphylla saponins II inhibits invasive, migration and epithelial-mesenchymal transition of melanoma cells through activation of autophagy.

Malignant melanoma is a kind of malignant tumor derived from normal epidermal melanocytes or original nevus cells. It has a high degree of malignancy, rapid progress, dangerous condition, and poor prognosis. In recent years, the innovation of traditional Chinese medicine has broadened the scope and effect of tumor treatment. It is a hotspot and breakthrough to find new anti-tumor invasion and migration drugs from natural plants or traditional Chinese medicine. This study explored the role of PPII in promoting autophagy to inhibit EMT of melanoma cells, the role of the PI3K/Akt signaling pathway in the invasion and migration of melanoma cells induced by PPII. We found that PPII effectively inhibited the proliferation, invasion and migration of melanoma B16 and B16F10 in vitro, and induced autophagy. We also established the xenograft tumor and metastatic tumor model of C57BL/6 mice with B16F10 cells. Results showed that PPII effectively inhibited the growth of transplanted tumors, induced autophagy and inhibited the expression level of EMT related protein; Metastasis experiment showed that PPII inhibited the invasion and migration of B16F10, the effect of inhibiting lung metastasis is the most significant. Further mechanism studies showed that the inhibition of PPII on melanoma invasion and migration is related to its induction of autophagy and then inhibition of EMT.

Phenomenography: An emerging qualitative research design for nursing.

BACKGROUND: Phenomenography emerged from pedagogy to examine the qualitatively different ways that individuals experience and perceive the same phenomenon. Despite its uniqueness, the uptake of phenomenography in nursing research is still limited. Potentially, this may be related to confusion regarding what the design is about, its philosophical underpinnings and how distinct it is from other qualitative designs. OBJECTIVES: To offer a better understanding of phenomenography by comparing it with other established qualitative research designs, examining its theoretical foundations, highlighting some studies that have employed the approach in nursing and offering methodological guidance to improve its uptake in nursing. DESIGN: Discussion paper. FINDINGS: Compared to the traditional qualitative designs employed in nursing, phenomenography has been utilized in fewer studies. The ontological, epistemological and methodological basis of phenomenography highlights it as a distinct design. The strength of phenomenography lies in its emphasis on understanding the collective variations between participants and presenting these holistically as an 'outcome space'. DISCUSSION: Phenomenography is a distinct qualitative research approach that presents a unique opportunity for nursing to further its use. Issues regarding bracketing, the inclusion of phenomenography studies in qualitative meta-synthesis and employing a hermeneutic approach to phenomenography are avenues for further work in nursing. PATIENT AND PUBLIC CONTRIBUTION: No patient or public contribution.

[Effect of electroacupuncture on the expression of autophagy related protein in lung tissue of rats with chronic obstructive pulmonary disease].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli" (ST36) and "Feishu" (BL13) on the expression of autophagy related proteins in the lung tissue of rats with chronic obstructive pulmonary disease (COPD), so as to explore the mechanism of EA underlying improvement of COPD. METHODS: Thirty male SD rats were randomly divided into normal, model and EA groups (n=10 in each group). The COPD model was established by intratracheal infusion of Lipopolysaccharide (LPS, 1 mg/kg) and exposure in cigarette smoke. EA was applied to bilateral ST36 and BL13 for 30 min, once every other day for 2 weeks. The pulmonary function (forced vital capacity ï¼»FVCï¼½, forced expiratory volume in 0.1 s and 0.3 s ï¼»FEV0.1, FEV0.3ï¼½, FEV0.1/FVC and FEV0.3/FVC) was detected by animal pulmonary function analysis system. Histopathological changes of the airway and lung were displayed by H.E. staining. Autophagosomes in the airway and lung tissues were observed by electron microscope. The expression of AMP activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), Unc-51 like autophagy activating kinase 1(ULK1), autophagy related protein ATG6(Beclin1)mRNAs in lung tissue were examined by quantitative real-time PCR. The expression of AMPK, mTOR, ULK1, Beclin1 and microtubule-associated protein 1 light chain 3 (LC3)proteins in lung tissue were examined by Western blot. The contents of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in the broncho alveolar lavage fluid (BALF) were assayed by ELISA. RESULTS: Following modeling, the FVC, FEV0.1, FEV0.3, FEV0.1/FVC and FEV0.3/FVC levels were significantly decreased (P<0.01), the infiltration of inflammatory cells and the increase of autophagosomes were obvious in airway and lung tissue, the mRNA and protein expression of AMPK, ULK1, Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ were increased (P<0.01), while the mRNA and protein expression of mTOR were decreased (P<0.01), the contents of TNF-α and IL-6 in the BALF were increased in the model group compared with the normal group (P<0.01). After EA intervention, all the indexes mentioned above were completely reversed in the EA group relevant to the model group (P<0.01, P<0.05). CONCLUSION: EA at ST36 and BL13 can improve the lung function of COPD rats, which may be related to its effects in inhibiting the autophagy level and reducing the inflammation response in the lung.

Brusatol reverses lipopolysaccharide-induced epithelial-mesenchymal transformation and induces apoptosis through PI3K/Akt/NF-кB pathway in human gastric cancer SGC-7901 cells.

Brusatol is a butyrolactone compound isolated from traditional Chinese medicine Brucea javanica. It has been reported to possess strong cytotoxicity against various cancer cells, thus showing its potential as an anticancer drug. Besides, lipopolysaccharide (LPS) plays a central role in the tumor microenvironment, while epithelial-mesenchymal transformation (EMT), a biological process by which epithelial cells are transformed into mesenchymal phenotypic cells through specific procedures, participates in chronic inflammation and tumor metastasis. This study aimed to investigate the inhibition of LPS-induced tumor cell invasion and metastasis and the molecular mechanism of apoptosis induced by brusatol in human gastric cancer SGC-7901 cells. Cell viability, cell migration and invasion ability, inflammatory factor release, and protein expression were detected using methyl thiazolyl tetrazolium assays, transwell assays, ELISA kit, and Western blot analysis, respectively. The change of EMT marker protein vimentin was assessed using immunofluorescence, while the apoptosis rate was measured using flow cytometry. In summary, brusatol inhibited LPS-induced EMT via the deactivation of the PI3K/Akt/NF-кB signaling pathway. This provides a useful new theoretical basis for the treatment of gastric cancer in the future.

Gambogenic acid induces ferroptosis in melanoma cells undergoing epithelial-to-mesenchymal transition.

Melanoma is characterized by high malignancy and early onset of metastasis. Epithelial-to-mesenchymal transition (EMT) is an early event during tumor metastasis. Tumor cells that develop EMT can escape apoptosis, but they are vulnerable to ferroptosis inducers. Gambogenic acid (GNA), a xanthone found in Gamboge, has cytotoxic effects in highly invasive melanoma cells. This study investigated the anti-melanoma effect and mechanism of action of GNA in TGF-ß1-induced EMT melanoma cells. We found that GNA significantly inhibited the invasion, migration and EMT in melanoma cells, and these cells exhibited small mitochondrial wrinkling (an important feature of ferroptosis). An iron chelator, but not an apoptosis inhibitor or a necrosis inhibitor, abolished the inhibitory effects of GNA on proliferation, invasion and migration of TGF-ß1-stimulated melanoma cells. GNA upregulated the expression of p53, solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in the model cells, contributing to the mechanisms underlying GNA-induced ferroptosis. Collectively, our findings suggest that GNA induces ferroptosis in TGF-ß1-stimulated melanoma cells via the p53/SLC7A11/GPX4 signaling pathway.

Gambogenic acid triggers apoptosis in human nasopharyngeal carcinoma CNE-2Z cells by activating volume-sensitive outwardly rectifying chloride channel.

Gambogenic acid (GNA) is one of the main components of Gamboge, and its anti-cancer effects have been well confirmed by previous researches. Nasopharyngeal carcinoma (NPC) has not been thoroughly studied, and the pathogenesis of NPC is unclear. Scientists have neither discovered effective therapies nor achieved a desirable prognosis. Some studies have found that the regulation of intra- and extracellular ion channels hinges directly on cell apoptosis, and treatment with GNA brings changes to the volume-sensitive outwardly rectifying chloride (VSOR Cl-) current of CNE-2Z cells recorded by the patch clamp method. Nevertheless, rarely have any researchers probed into the relevance between this variation and the anti-tumor mechanism of GNA. This paper is suggested that 2.0 µmol/L GNA activates VSOR Cl- currents on CNE-2Z cell membranes and that the activation of VSOR Cl- currents by GNA in CNE-2Z cells is blocked by the chloride channel blockers DIDS (400 µmol/L) and DCPIB (20 µmol/L). MQAE experiment further proves that GNA leads to the opening of chloride ion channel, which in turn results in the efflux of VSOR Cl- current; GNA induces the downregulation of GRP78 and the upregulation of ATF4 and CHOP proteins. These effects are correlated with endoplasmic reticulum (ER) stress. GNA can activate VSOR Cl- channels, leading to ER stress, inducing apoptosis and inhibiting proliferation in CNE-2Z cells.

[Mechanism of gambogenic acid in resisting angiogenesis of lung cancer in vitro].

The aim of this paper was to observe the effect of gambogenic acid on angiogenesis of lung cancer and its preliminary mechanism. After culturing lung adenocarcinoma A549 cells, the conditioned medium was treated with gambogenic acid and then used to culture human umbilical vein endothelial cells (HUVECs) to establish the indirect contact cell co-culture system. A two-dimensional culture model of HUVEC was established with matrigel to observe the effect of gambogenic acid on angiogenesis. DAPI staining was used to observe the morphological changes in HUVEC cells after treatment with gambogenic acid under the fluorescence microscope. Annexin V-FITC/PI staining and flow cytometry analysis were used to determine gambogenic acid's effect on HUVEC cell apoptosis rate. The protein expressions of PI3K, p-PI3K, Akt, p-Akt were measured by Western blot. PTEN-siRNA was transfected into cells, and RT-PCR was used to detect the expression levels of PI3K and Akt genes. Gambogenic acid can significantly inhibit angiogenesis, and its inhibitory effect was dose-dependent. DAPI staining showed apoptotic morphological features of HUVEC cells under fluorescence microscope. Annexin V-FITC/PI staining showed that gambogenic acid induced apoptosis in HUVECs. The results of Western blot showed that the expressions of p-PI3K and p-Akt protein were down-regulated with gambogenic acid, while the expressions of PI3K and Akt protein was insignificant. The results of RT-PCR indicated that the expressions of PI3K and Akt protein were up-regulated by PTEN siRNA. Gambogenic acid can inhibit angiogenesis in lung cancer in vitro, and the mechanism of inhibiting angiogenesis may be related to the PI3K/Akt signaling pathway.

High-density SNP linkage map construction and QTL mapping for flavonoid-related traits in a tea plant (Camellia sinensis) using 2b-RAD sequencing.

BACKGROUND: Flavonoids are important components that confer upon tea plants a unique flavour and health functions. However, the traditional breeding method for selecting a cultivar with a high or unique flavonoid content is time consuming and labour intensive. High-density genetic map construction associated with quantitative trait locus (QTL) mapping provides an effective way to facilitate trait improvement in plant breeding. In this study, an F1 population (LJ43×BHZ) was genotyped using 2b-restriction site-associated DNA (2b-RAD) sequencing to obtain massive single nucleotide polymorphism (SNP) markers to construct a high-density genetic map for a tea plant. Furthermore, QTLs related to flavonoids were identified using our new genetic map. RESULTS: A total of 13,446 polymorphic SNP markers were developed using 2b-RAD sequencing, and 4,463 of these markers were available for constructing the genetic linkage map. A 1,678.52-cM high-density map at an average interval of 0.40 cM with 4,217 markers, including 427 frameset simple sequence repeats (SSRs) and 3,800 novel SNPs, mapped into 15 linkage groups was successfully constructed. After QTL analysis, a total of 27 QTLs related to flavonoids or caffeine content (CAF) were mapped to 8 different linkage groups, LG01, LG03, LG06, LG08, LG10, LG11, LG12, and LG13, with an LOD from 3.14 to 39.54, constituting 7.5% to 42.8% of the phenotypic variation. CONCLUSIONS: To our knowledge, the highest density genetic map ever reported was constructed since the largest mapping population of tea plants was adopted in present study. Moreover, novel QTLs related to flavonoids and CAF were identified based on the new high-density genetic map. In addition, two markers were located in candidate genes that may be involved in flavonoid metabolism. The present study provides valuable information for gene discovery, marker-assisted selection breeding and map-based cloning for functional genes that are related to flavonoid content in tea plants.

(-)-Epicatechin regulates blood lipids and attenuates hepatic steatosis in rats fed high-fat diet.

SCOPE: (-)-Epicatechin (EC) is a natural flavanol monomer found in cocoa, green tea, and a variety of other plant foods. In this study, effects of EC on blood lipids and hepatic steatosis, and the underlying mechanisms were investigated. METHODS AND RESULTS: A hyperlipidemic rat model was induced by high-fat, high-cholesterol diet. EC was then administrated to the animals by gavage at doses of 10, 20, 40 mg/kg body weight (BW) for 12 weeks. Simvastatin was included as a positive control. The results showed that EC significantly reduced total cholesterol, LDL cholesterol and triglyceride, alleviated liver fat accumulation, while increased HDL cholesterol, in hyperlipidemic rats. EC also reduced lipid peroxidation, inhibited the pro-inflammatory cytokines, and lowered serum AST and ALT. The potential molecular mechanisms of EC underlying these effects were proposed to be associated to regulating Insig-1-SREBP-SCAP pathway, and other lipid metabolic related genes including LXR-α, FAS, and SIRT1. CONCLUSION: EC effectively improved blood lipid profile and protected liver from accumulating excessive fat in hyperlipidemic rats. The results shed a light on the potential role of EC as a promising natural product in preventing hyperlipidemia and nonalcoholic fatty liver disease.

[Study of gambogenic acid-induced apoptosis of melanoma B16 cells through PI3K/Akt/mTOR signaling pathways].

OBJECTIVE: To discuss the mechanism of gambogenic acid (GNA) in inducing the apoptosis of melanoma B16 cells. METHOD: The inhibitory effect of GNA on the proliferation of B16 cells was measured by the methyl thiazolyl tetrazolium (MTT) assay. The effect of GNA on B16 cells was detected by the Hoechst 33258 staining. The transmission electron microscopy was used to observe the ultra-structure changes of B16 cells. The changes in PI3K, p-PI3K, Akt, p-Akt, p-mTOR, PTEN proteins were detected by the Western blotting to discuss the molecular mechanism of GNA in inducing the apoptosis of B16 cells. RESULT: GNA showed a significant inhibitory effect in the growth and proliferation of melanoma B16 cells. The cell viability remarkably decreased with the increase of GNA concentration and the extension of the action time. The results of the Hoechst 33258 staining showed that cells processed with GNA demonstrated apparent apoptotic characteristics. Under the transmission electron microscope, B16 cells, after being treated with GNA, showed obvious morphological changes of apoptosis. The Western blot showed a time-dependent reduction in the p-PI3K and p-Akt protein expressions, with no change in p-PI3K and p-Akt protein expression quantities. The p-mTOR protein expression decreased with the extension of time, where as the PTEN protein expression showed a time-dependent increase. CONCLUSION: GNA could inhibit the proliferation of melanoma B16 cells and induce their apoptosis within certain time and concentration ranges. Its mechanism in inducing the cell apoptosis may be related to PI3K/Akt/mTOR signaling pathways.

(24S)-ergostane-3ß,5α,6ß-triol from Hedyotis chrysotricha with inhibitory activity on migration of SK-HEP-1 human hepatocarcinoma cells.

The methanol extract of the whole plant of Hedyotis chrysotricha demonstrated cytotoxicity against SK-HEP-1 human hepatocarcinoma cells in a primary screening for novel antitumour agents. Bioassay-guided fractionation and purification led to an active principle (24S)-ergostane-3ß,5α,6ß-triol (1) along with four inactive compounds (2-5). The in vitro transwell migration assay showed that compound 1 remarkably reduced the migration of SK-HEP-1 cells by 78.9% at a dose of 30 µM, without any apoptotic effect on this cell line. Moreover, all the isolated compounds were further evaluated for their cytotoxicities against another four human cancer cell lines (MCF-7, NUGC3, SH-SY5Y and PC-3).

Triterpenoids and steroids from the fruits of Melia toosendan and their cytotoxic effects on two human cancer cell lines.

Ten new triterpenoids, named meliasenins I-R (1-10), one new steroid (11), and 11 related known compounds (12-22) were isolated from fruits of Melia toosendan. The structures of the new compounds were established on the basis of spectroscopic methods, including 2D NMR techniques and mass spectrometry. The relative configuration of 1, (20R*,23E)-25-hydroperoxyeupha-7,23-diene-3ß,16ß-diol (meliasenin I), was confirmed by single-crystal X-ray diffraction analysis. All isolated triterpenoids (1-10, 12-15) and two steroids (11, 20) were tested for their cytotoxicity against U20S human osteosarcoma and MCF-7 human breast cancer cells using the MTT assay, and some of them were significantly cytotoxic (IC(50) <10 µg/mL). The insecticidal properties of compounds 1-15 and 20 were also briefly evaluated.

Gambogenic acid inhibits proliferation of A549 cells through apoptosis-inducing and cell cycle arresting.

Although anticancer effect of gambogic acid (GA) and its potential mechanisms were well documented in past decades, limited information is available on the anticancer effect of gambogenic acid (GNA), another major active component of Gamboge. Here we performed a study to determine whether GNA possesses anticancer effect and find its potential mechanisms. The results suggested that GNA significantly inhibited the proliferation of several tumor cell lines in vitro and in vivo. Treatment with GNA dose and time dependently induced A549 cells apoptosis, arrested the cells to G0/G1 phase in vitro and down-regulated the expression of cyclin D1 and cyclooxygenase (COX)-2 in mRNA level. In addition, anticancer effect was further demonstrated by applying xenografts in nude mice coupled with the characteristic of apoptosis in the GNA treated group. Taken together, these observations might suggest that GNA inhibits tumor cell proliferation via apoptosis-induction and cell cycle arrest.