Lu Zhang

BACKGROUND: Triglycerides with varying chain length and unsaturation degree are now in high demand in the food and pharmaceutical industries. Enzymatic catalyzed synthesis methods are favored over chemical‐based processes in the commercial production of triglycerides. This study found that the application of vacuum can improve the efficiency and productivity of enzymatic synthesis of various triglycerides using Novozym 435 (EC 3.1.1.3), an immobilized lipase B from Candida antarctica. The system was used for esterification of fatty acids varying in chain length, unsaturation degree, and oxidation degree.RESULTS: The vacuum condition was not applicable to short chain fatty acids (C₄–C₆) due to loss of fatty acids in the reaction. The conversions of C₄–C₆ saturated fatty acids were low (15–35%) under atmospheric pressure. Under vacuum, high conversions (>95%) and triglyceride yields (approximately 90%) were achieved in the esterification of glycerol with several medium‐chain and long‐chain (C₈–C₁₈) saturated fatty acids. Moreover, unsaturation degree of fatty acids also affected the triglyceride yield. The triglyceride contents synthesized from C18:2n‐6 (76.17 ± 0.45%) was significantly lower (P < 0.05) than those from C18:0 (91.59 ± 1.58%) and C18:1n‐9 (90.77 ± 0.85%). Furthermore, fatty acids with a high oxidation degree had negative effect on triglyceride synthesis. This vacuum solvent free system allowed the synthesis enzyme to be reused 11 times without significant decrease in the lipase activity for esterification of fatty acids.CONCLUSION: The studied vacuum facilitated triglyceride synthesis process provides an economically attractive yet sustainable solution for the production of a wide range of triglyceride products. © 2020 Society of Chemical Industry

Currently, particular focus is placed on the implication of autophagy in a variety of human diseases, including cancer. Discovery of small-molecule modulators of autophagy as well as their potential use as anti-cancer therapeutic agents would be of great significance. To this end, a series of curcumin analogs previously synthesized in our laboratory were screened. Among these compounds, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one (CA-5f) was identified as a potent late-stage macroautophagy/autophagy inhibitor via inhibiting autophagosome-lysosome fusion. We found that CA-5f neither impaired the hydrolytic function nor the quantity of lysosomes. Use of an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic screen in combination with bioinformatics analysis suggested that treatment of human umbilical vein endothelial cells (HUVECs) with CA-5f for 1 h suppressed the levels of cytoskeletal proteins and membrane traffic proteins. Subsequent studies showed that CA-5f exhibited strong cytotoxicity against A549 non-small cell lung cancer (NSCLC) cells, but low cytotoxicity to normal human umbilical vein endothelial cells (HUVECs), by increasing mitochondrial-derived reactive oxygen species (ROS) production. Moreover, CA-5f effectively suppressed the growth of A549 lung cancer xenograft as a single agent with an excellent tolerance in vivo. Results from western blot, immunofluorescence, and TdT-mediated dUTP nick end labeling (TUNEL) assays showed that CA-5f inhibited autophagic flux, induced apoptosis, and did not affect the level of CTSB (cathepsin B) and CTSD (cathepsin D) in vivo, which were consistent with the in vitro data. Collectively, these results demonstrated that CA-5f is a novel late-stage autophagy inhibitor with potential clinical application for NSCLC therapy. Abbreviations: 3-MA, 3-methyladenine; ANXA5, annexin A5; ATG, autophagy related; CA-5f, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; DMSO, dimethyl sulfoxide; DNM2, dynamin 2; EBSS, Earle’s balanced salt solution; GFP, green fluorescent protein; HCQ, hydroxyl CQ; HEK293, human embryonic kidney 293; HUVEC, human umbilical vein endothelial cells; LAMP1, lysosomal associated membrane protein 1; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LDH, lactic acid dehydrogenase; LMO7, LIM domain 7; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; NAC, N-acetyl cysteine; MYO1E, myosin IE; NSCLC, non-small cell lung cancer; PARP1, poly(ADP-ribose) polymerase 1; PI, propidium iodide; RFP, red fluorescent protein; ROS, reactive oxygen species; SQSTM1, sequestosome 1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Natural polysaccharides are the most widely used biopolymers for green synthesis of eco-friendly silver nanoparticles (AgNPs). In a previous study, a high molecular weight (MW) fraction of exopolysaccharides (EPS) produced by a medicinal fungus Cs-HK1 has been shown useful for green and facile synthesis of AgNPs in water. This study was to further evaluate the effects of molecular properties of EPS on the formation, stability and properties of AgNPs with different EPS fractions at various pH conditions. Three EPS fractions (P_0.5, P_2.0 and P_5.0: MW high to low and protein content low to high) were reacted with silver nitrate at various pH 3.0–8.0 in water. The most favorable pH range was 5.5–8.0 for the formation and stable dispersion of AgNPs. At a given pH, the maximum amount of AgNPs was produced with P_5.0, and the minimum with P_0.5. The shape, size and physiochemical properties of AgNPs were strongly affected by the molecular characteristics of EPS (MW and conformation). The results may be helpful for understanding the factors and mechanisms for formation of stable AgNPs with natural polysaccharides and the interactions between AgNPs and the polysaccharide hydrocolloids in water.

To investigate the effects of 3,4,4'-trihydroxy-trans-stilbene (3,4,4'-THS), an analogue of resveratrol, on human non-small-cell lung cancer (NSCLC) cells in vitro. Cell viability of NSCLC A549 cells was determined by MTT assay. Cell apoptosis was evaluated using flow cytometry and TUNEL assay. Cell necrosis was evaluated with LDH assay. The expression of apoptosis- or autophagy-associated proteins was measured using Western blotting. The formation of acidic compartments was detected using AO staining, neutral red staining and Lysotracker-Red staining. LC3 punctae were analyzed with fluorescence microscopy. Treatment with 3,4,4'-THS (10-80 μmol/L) concentration-dependently inhibited the cell viability. It did not cause cell necrosis, but induced apoptosis accompanied by up-regulation of cleavaged PARP, caspase3/9 and Bax, and by down-regulation of Bcl-2 and surviving. It also increased the formation of acidic compartments, LC3-II accumulation and GFP-LC3 labeled autophagosomes in the cells. It inhibited the mTOR-dependent pathway, but did not impair autophagic flux. 3,4,4'-THS-induced cell death was enhanced by the autophagy inhibitors 3-MA (5 mmol/L) or Wortmannin (2 μmol/L). Moreover, 3,4,4'-THS treatment elevated the ROS levels in the cells, and co-treatment with 3-MA further elevated the ROS levels. 3,4,4'-THS-induced apoptosis and autophagy in the cells was attenuated by NAC (10 mmol/L)Conclusion:3,4,4'-THS induces both apoptosis and autophagy in NSCLC A549 cells in vitro. Autophagy inhibitors promote 3,4,4'-THS-induced apoptosis of A549 cells, thus combination of 3,4,4'-THS and autophagy inhibitor provides a promising strategy for NSCLC treatment.

Angiogenesis plays an important role in many pathological processes. Identification of novel anti‐angiogenic agents will provide new insights into the mechanisms for angiogenesis as well as potential lead compounds for developing new drugs. In the present study, a series of resveratrol methylated derivatives have been synthesized and screened. We found trans‐3,4‐dimethoxystilbene (3,4‐DMS) with the fullest potential to develop as an anti‐angiogenic agent. In vitro and in vivo analyses suggested that 3,4‐DMS could effectively inhibit endothelial cell proliferation, migration, tube formation, and endogenous neovascularization. Our results showed that 3,4‐DMS exerted its anti‐angiogenic effect likely through induction of endothelial cell apoptosis via a pathway involving p53, Bax, cytochrome c, and caspase proteases. Moreover, 3,4‐DMS also induced macroautophagy in endothelial cells through activation of AMPK and the downstream inhibition of mTOR signaling pathway. Further studies indicated that intracellular calcium ([Ca²⁺]_i) might bridge the 3,4‐DMS‐induced apoptosis and macroautophagy through modulating reactive oxygen species (ROS) levels in endothelial cells. Combination of 3,4‐DMS with inhibitor of autophagy, such as 3‐methyladenine (3‐MA) and autophagy‐related gene (ATG) 5 small interfering RNA (siRNA), potentiated the pro‐apoptotic and anti‐angiogenic effects of 3,4‐DMS. Our study provides a novel angiogenic inhibitor and a useful tool in exploring the molecular mechanisms for the crosstalk between apoptosis and macroautophagy in endothelial cells. 3,4‐DMS could be served as a potential lead compound for developing a class of new drugs targeting angiogenesis‐related diseases. J. Cell. Biochem. 114: 697–707, 2013.