Selenium nanoparticles coupling with Astragalus Polysaccharides exert their cytotoxicities in MCF-7 cells by inhibiting autophagy and promoting apoptosis.

BACKGROUND: Astragalus Polysaccharides (APS) had been reported to exhibit antitumor activities. Given that nanoparticles possessed unique advantages in cancer treatment, APS was used as the modifier to prepare gold, silver and selenium nanoparticles (APS-Au, APS-Ag and APS-Se NPs) in the present study. METHODS: The three nanoparticles were synthesized via a green approach and characterized by DLS, TEM, XRD, FT-IR and UV-Vis. The inhibitory effects of these nanoparticles on various tumor cells proliferation were examined by MTT assay in vitro. Reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the expression of apoptosis and autophagy-related proteins were also detected. RESULTS: Among these, APS-Se NPs displayed the most potent antitumor activities against MCF-7 cells in vitro. Flow cytometric analysis suggested that after cells were exposed to elevated concentrations of APS-Se NPs (10, 20 and 40 µmol/L), the rate of apoptosis was increasing (16.63 ± 0.89, 38.60 ± 3.46 and 44.38 ± 2.62%, respectively). Further analysis by immunofluorescence revealed an increase in intracellular ROS and a loss of MMP. This was accompanied by increased LC3-I to LC3-II conversion. Also, western blot analysis demonstrated that the ratios of Bax/Bcl-2 and cleaved caspase9/caspase 9 rose, and LC3-II and p62 protein levels increased. The addition of chloroquine, an inhibitor of autophagy, further enhanced protein expression of p62 and LC3-II. CONCLUSION: APS-Se NPs exerted their cytotoxic activity in MCF-7 cells by blocking autophagy and facilitating mitochondrial pathway-mediated apoptosis.

Astragalus membranaceus Injection Suppresses Production of Interleukin-6 by Activating Autophagy through the AMPK-mTOR Pathway in Lipopolysaccharide-Stimulated Macrophages.

Astragalus membranaceus (AM), used in traditional Chinese medicine, has been shown to enhance immune functions, and recently, its anti-inflammatory effects were identified. However, the mechanisms of action remain unclear. Most studies have shown that autophagy might be involved in the immune response of the body, including inflammation. Here, we developed an inflammatory model by stimulating macrophages with lipopolysaccharides (LPS) to explore the anti-inflammatory effect and mechanisms of AM injection from the perspective of the regulation of autophagy. Immunoblot, immunofluorescence, and ELISA were used to determine the effects of AM injection on the production of interleukin-6 (IL-6) and alterations of autophagy markers. It was found that AM injection reduced the expression of IL-6 in LPS-stimulated macrophages and reversed the LPS-induced inhibition of cellular autophagy. After treatment with inhibitors of signaling pathways, it was shown that LPS downregulated autophagy and upregulated the production of IL-6 in macrophages via the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. AM injection reversed the effects of LPS by activating the AMP-activated protein kinase (AMPK) instead of inhibiting Akt. These results were further confirmed by testing activators and siRNA silencing of AMPK. Hence, these 2 distinct signaling molecules appear to exert opposite effects on mTOR, which integrates information from multiple upstream signaling pathways, negatively regulating autophagy. In addition, we demonstrated that autophagy might play a key role in regulating the production of IL-6 by testing activators and inhibitors of autophagy and siRNA silencing of ATG5. These findings showed that AM injection might enhance autophagy by activating AMPK and might further play a repressive effect on the LPS-stimulated expression of IL-6. This study explored the relationship between autophagy, signaling pathways, and the production of inflammatory factors in a model of endotoxin infection and treatment with AM injection.

Farrerol Directly Targets GSK-3ß to Activate Nrf2-ARE Pathway and Protect EA.hy926 Cells against Oxidative Stress-Induced Injuries.

Oxidative stress-mediated endothelial injury is considered to be involved in the pathogenesis of various cardiovascular diseases. Farrerol, a typical natural flavanone from the medicinal plant Rhododendron dauricum L., has been reported to show protective effects against oxidative stress-induced endothelial injuries in our previous study. However, its action molecular mechanisms and targets are still unclear. In the present study, we determined whether farrerol can interact with glycogen synthase kinase 3ß- (GSK-3ß-) nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling, which is critical in defense against oxidative stress. Our results demonstrated that farrerol could specifically target Nrf2 negative regulator GSK-3ß and inhibit its kinase activity. Mechanistic studies proved that farrerol could induce an inhibitory phosphorylation of GSK-3ß at Ser9 without affecting the expression level of total GSK-3ß protein and promote the nuclear translocation of Nrf2 as well as the mRNA and protein expression of its downstream target genes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in EA.hy926 cells. Further studies performed with GSK-3ß siRNA and specific inhibitor lithium chloride (LiCl) confirmed that GSK-3ß inhibition was involved in farrerol-mediated endothelial protection and Nrf2 signaling activation. Moreover, molecular docking and molecular dynamics studies revealed that farrerol could bind to the ATP pocket of GSK-3ß, which is consistent with the ATP-competitive kinetic behavior. Collectively, our results firstly demonstrate that farrerol could attenuate endothelial oxidative stress by specifically targeting GSK-3ß and further activating the Nrf2-ARE signaling pathway.

Selenium nanoparticles fabricated in laminarin polysaccharides solutions exert their cytotoxicities in HepG2 cells by inhibiting autophagy and promoting apoptosis.

Selenium nanoparticles (SeNPs) have been attracting increasing attention as potential cancer therapeutic agents. In the present study, laminarin polysaccharides (LP) decorated selenium nanoparticles (LP-SeNPs) with an average diameter of ca. 60 nm were synthesized. Transmission electron microscope (TEM), laser particle analyzer, UV-visible spectrometer and Energy dispersive X-ray (EDX) spectrometer were applied to characterize the prepared SeNPs. The cytotoxicity, apoptosis, and autophagy were examined using a series of cellular assays. The results revealed that LP-SeNPs exhibited cytotoxicity against HepG2 cells with IC50 value was 23.4 ±â€¯2.7 µM. After cells were treated with various concentrations of LP-SeNPs (10, 20 and 40 µM) for 24h, the total apoptosis rate increased to 17.4 ±â€¯1.6, 20.9 ±â€¯1.3 and 30.9 ±â€¯1.2%, respectively. Additionally, treatment of LP-SeNPs increased the expression of Bax and cleaved caspase-9 but decreased the level of Bcl-2. This suggested that LP-SeNPs induced mitochondria-mediated apoptosis. Further, exposure of cells to LP-SeNPs for 12 h induced the upregulation of LC3-II and p62. Treatment of chloroquine (CQ), the inhibitors of the autophagosome, resulted in further accumulation of p62 and LC3-II. These results demonstrated that LP-SeNPs induced the activation of early autophagy, but blocked the late phase of autophagy. Inhibition of late phase of autophagy resulted in the damaged organelles cannot be cleared and aggravating apoptosis. In conclusion, these results indicated that LP-SeNPs exerted its cytotoxicity in HepG2 cells by inhibiting autophagy and inducing apoptosis.

Green synthesis of selenium nanoparticles with extract of hawthorn fruit induced HepG2 cells apoptosis.

CONTEXT: Selenium nanoparticles (SeNPs) have attracted worldwide attention due to their unique properties and potential bioactivities. Considering that hawthorn is both a traditional medicine and a common edible food, hawthorn fruit extract (HE) was chosen as a reductant to prepare SeNPs. OBJECTIVE: SeNPs were synthesized by using an aqueous HE as a reductant and stabilizer. The antitumor activities and potential mechanisms of SeNPs were explored by using a series of cellular assays. MATERIALS AND METHODS: The HE mediated SeNPs (HE-SeNPs) were examined using various characterisation methods. The cytotoxicity was measured against HepG2 cells after treated with 0, 5, 10 and 20 µg/mL of HE-SeNPs for 24 h. Annexin V-FITC/PI staining analysis was performed to observe the apoptosis of HepG2 cells. Additionally, mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS) levels were evaluated. Finally, the protein expression levels of caspase-9 and Bcl-2 were identified by Western blot. RESULTS: The mono-dispersed and stable SeNPs were prepared with an average size of 113 nm. HE-SeNPs showed obvious antitumor activities towards HepG2 cells with an IC50 of 19.22 ± 5.3 µg/mL. Results from flow cytometry revealed that both early and total apoptosis rates increased after treating with HE-SeNPs. After cells were treated with various concentrations of HE-SeNPs (5, 10 and 20 µg/mL) for 24 h, the total rate increased to 7.3 ± 0.5, 9.7 ± 1.7 and 19.2 ± 1.6%, respectively. Meanwhile, treatment of HE-SeNPs up-regulated intracellular ROS levels and reduced the MMP. In addition, HE-SeNPs induced the up-regulation of caspase-9 and down-regulation of Bcl-2. DISCUSSION AND CONCLUSIONS: HE-SeNPs induced intracellular oxidative stress and mitochondrial dysfunction to initiate HepG2 cell apoptosis through the mitochondrial pathway. Therefore, HE-SeNPs may be a candidate for further evaluation as a chemotherapeutic agent for human liver cancer.

Synthesis, characterization and antitumor properties of selenium nanoparticles coupling with ferulic acid.

Selenium nanoparticles (Se NPs) attract a lot of attention as potential cancer therapeutic agents. However, the antitumor activities of pure Se NPs are poor, and some modifiers are needed to enhance the activities. In the present study, we prepared Ferulic Acid (FA)-modified selenium nanoparticles in a facile synthetic approach. The obtained FA-Se NPs were characterized using transmission electron microscope (TEM), dynamic light scattering (DLS), ultraviolet-visible spectrophotometer (UV-VIS), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and Energy dispersive X-ray (EDX) spectroscopy. In vitro antitumor effects of FA, Se NPs and FA-Se NPs in HepG-2 cells were examined by methyl thiazolyl tetrazolium (MTT) assay. It showed that FA-Se NPs effectively inhibited the growth of HepG-2 cells with IC50 value of 11.57 ±â€¯3.6 µg/ml, while the value of Se NPs was >100 µg/ml. In addition, FA behaves no obvious antitumor effects at high concentrations up to 100 µg/ml. In order to investigate the antitumor mechanism of FA-Se NPs, fluorescence morphological examination and Annexin V-FITC/PI staining analysis were performed to observe the apoptosis of HepG-2 cells induced by FA-Se NPs. Meanwhile, mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS) levels and caspase-3 and -9 activities were detected. The results revealed that FA-Se NPs induced intracellular ROS generation and MMP disruption by finally activating caspase-3/9 to trigger HepG-2 cells apoptosis through mitochondrial pathway. Further investigation on the interactions of FA-Se NPs with calf thymus DNA (ctDNA) indicated that the antitumor activities may be associated with the DNA-binding properties of FA-Se NPs.

Apoptosis induced by farrerol in human gastric cancer SGC-7901 cells through the mitochondrial-mediated pathway.

Farrerol, a typical flavanone isolated from the Chinese medicinal plant Rhododendron dauricum L., has been found to show various biological activities. However, to the best of our knowledge, its inhibitory actions against cancer cells have not been reported as yet. Therefore, the present study aimed to investigate the cytotoxic and apoptotic effects of farrerol on human gastric cancer SGC-7901 cells. Farrerol showed a 50% inhibition of SGC-7901 cell growth at a concentration of 40.4 µmol/l for 24 h according to MTT assays. The cell morphology results indicated that SGC-7901 cells treated with farrerol showed several features of apoptotic cell death, which was also confirmed by the Annexin-V FITC/PI double-staining assay. Further studies showed that farrerol treatment induced the attenuation of mitochondrial membrane potential, accompanied by the release of Cyt-c and the activation of caspase-9 and caspase-3. Furthermore, farrerol decreased the gene expression of Bcl-2, whereas the gene expression level of Bax was found to increase after farrerol treatment. These combined results indicated that farrerol can induce apoptosis through a mitochondrial-mediated pathway.

Comparison of the phenolic content and antioxidant activities of Apocynum venetum L. (Luo-Bu-Ma) and two of its alternative species.

The leaves of Apocynum venetum L. (AV), a native Chinese plant, have been used as folk medicine in China and Japan. This study evaluated the content of the active antioxidant component and antioxidant activities of AV, and its two alternative species, Poacynum pictum (Schrenk) Baill. (PP) and Poacynum hendersonii (Hook.f.) Woodson (PH). The total phenolic and total flavonoid contents were determined. In addition, the quantitative analysis of two major flavonoid compounds (hyperoside and isoquercitrin) was carried out by HPLC. The antioxidant activities were investigated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity method, the reducing power test and the chelating ability of ferrous ions. The highest total phenolic and flavonoid contents were observed in the AV methanolic extract, followed by the PP and PH methanolic extracts. HPLC analysis indicated that isoquercitrin was one of the major components in all three species, however, hyperoside was only detected in AV at high levels. All the antioxidant assays we performed demonstrated that the AV extract was markedly superior to those of the other two species.

Chemopreventive effects of Peucedanum praeruptorum DUNN and its major constituents on SGC7901 gastric cancer cells.

In this study, the effects of Peucedanum praeruptorum DUNN methanolic extract (PPME) and its major constituents on SGC7901 human gastric cancer cells were evaluated. Two pyranocoumarins, namely, (±) praeruptorin A (PA) and (±) praeruptorin B (PB), were isolated from PPME. A high performance liquid chromatographic (HPLC) method was developed to determine the contents of PA and PB in PPME. The anti-proliferative and cytotoxic actions of PPME were observed using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and release of lactate dehydrogenase (LDH) assays. At 300 µg/mL, PPME inhibited cell growth by 51.2% (P < 0.01), probably linked to the high concentration of PA and PB. Both PA and PB exhibited antiproliferative and cytotoxic activities on the SGC7901 cells. Furthermore, the active principle compound, PA, also enhanced the actions of doxorubincin (DOX) on SGC7901 cells. Cell growth decreased higher with the combined treatment of PA and DOX than that with the chemotherapy agent applied alone, suggesting that PA could reduce the dose of DOX for the desired effects.

[Simultaneous determination of four flavonoids in Malus prunifolia from Shanxi province by RP-HPLC].

OBJECTIVE: To develop an HPLC method for simultaneous determination of rutin, hyperoside, quercetin and kaempferol in Malus prunifolia from Shanxi province in China. METHOD: The separation was performed on a Hypersil C18 column (4.6 mm x 250 mm, 5 microm), using a gradient elution with methanol-water containing 0.2% phosphoric acid as the mobile phase. The flow rate was 1.0 mL x min(-1), the detection wavelength was 360 nm and the temperature of column was 25 degrees T. RESULT: The linear ranges of rutin, hyperoside, quercetin and kaempferol were 1.87-46.67 mg x L(-1), 6.40-160.0 mg x L(-1), 3.33-83.33 mg x L(-1), 0.80-20.00 mg x L(-1), respectively. The average recoveries (n=6) of the four constituents were 99.2% (RSD 2.9%), 98.2% (RSD 1.9%), 97.4% (RSD 2.3%), 97.2% (RSD 1.3%), respectively. CONCLUSION: The method was simple, accurate and can be used to evaluate medicinal value of Malus prunifolia.