(+)-Clausenamide protects against drug-induced liver injury by inhibiting hepatocyte ferroptosis.

Drug-induced liver injury is the major cause of acute liver failure. However, the underlying mechanisms seem to be multifaceted and remain poorly understood, resulting in few effective therapies. Here, we report a novel mechanism that contributes to acetaminophen-induced hepatotoxicity through the induction of ferroptosis, a distinctive form of programmed cell death. We subsequently identified therapies protective against acetaminophen-induced liver damage and found that (+)-clausenamide ((+)-CLA), an active alkaloid isolated from the leaves of Clausena lansium (Lour.) Skeels, inhibited acetaminophen-induced hepatocyte ferroptosis both in vivo and in vitro. Consistently, (+)-CLA significantly alleviated acetaminophen-induced or erastin-induced hepatic pathological damages, hepatic dysfunctions and excessive production of lipid peroxidation both in cultured hepatic cell lines and mouse liver. Furthermore, treatment with (+)-CLA reduced the mRNA level of prostaglandin endoperoxide synthase 2 while it increased the protein level of glutathione peroxidase 4 in hepatocytes and mouse liver, confirming that the inhibition of ferroptosis contributes to the protective effect of (+)-CLA on drug-induced liver damage. We further revealed that (+)-CLA specifically reacted with the Cys-151 residue of Keap1, which blocked Nrf2 ubiquitylation and resulted in an increased Nrf2 stability, thereby leading to the activation of the Keap1-Nrf2 pathway to prevent drug-induced hepatocyte ferroptosis. Our studies illustrate the innovative mechanisms of acetaminophen-induced liver damage and present a novel intervention strategy to treat drug overdose by using (+)-CLA.

Fuziline alleviates isoproterenol-induced myocardial injury by inhibiting ROS-triggered endoplasmic reticulum stress via PERK/eIF2α/ATF4/Chop pathway.

Fuziline, an aminoalcohol-diterpenoid alkaloid derived from Aconiti lateralis radix preparata, has been reported to have a cardioprotective activity in vitro. However, the potential mechanism of fuziline on myocardial protection remains unknown. In this study, we aimed to explore the efficacy and mechanism of fuziline on isoproterenol (ISO)-induced myocardial injury in vitro and in vivo. As a result, fuziline effectively increased cell viability and alleviated ISO-induced apoptosis. Meanwhile, fuziline significantly decreased the production of ROS, maintained mitochondrial membrane potential (MMP) and blocked the release of cytochrome C, suggesting that fuziline could play the cardioprotective role through restoring the mitochondrial function. Fuziline also could suppress ISO-induced endoplasmic reticulum (ER) stress via the PERK/eIF2α/ATF4/Chop pathway. In addition, using ROS scavenger NAC could decrease ISO-induced apoptosis and block ISO-induced ER stress, while PERK inhibitor GSK2606414 did not reduce the production of ROS, indicating that excess production of ROS induced by ISO triggered ER stress. And fuziline protected against ISO-induced myocardial injury by inhibiting ROS-triggered ER stress. Furthermore, fuziline effectively improved cardiac function on ISO-induced myocardial injury in rats. Western blot analysis also showed that fuziline reduced ER stress-induced apoptosis in vivo. Above these results demonstrated that fuziline could reduce ISO-induced myocardial injury in vitro and in vivo by inhibiting ROS-triggered ER stress via the PERK/eIF2α/ATF4/Chop pathway.

Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo / 中国药理学与毒理学杂志

OBJECTIVE To discover a small molecule targeting ULK1-modulated cell death of triple negative breast cancer and exploreits potential mechanisms. METHODS ULK1 expression was analyzed by The Cancer Genome Atlas (TCGA) analysis and tissue microarray (TMA) analysis. ULK1 agonist was designed by using in silico screening, as well as modified by chemical synthesis and screened by kinase and anti-proliferative activities. The amino acid residues that key to the activation site of LYN-1604 were determined by site-directed mutagenesis, as well as in vitro kinase assay and ADP-Glo kinase assay. The mechanisms of LYN-1604 induced cell death were investigated by fluores?cence microscope, western blotting, flow cytometry analysis, immunocytochemistry, as well as siRNA and GFP-mRFP-LC3 plasmid transfections. Potential ULK1 interactors were discovered by performing comparative microarray analysis and the therapeutic effect of LYN-1604 was assessed by xenograft breast cancer mouse model. RESULTS We found that ULK1 was remarkably downregulated in breast cancer tissue samples, especially in triple negative breast cancer (TNBC). 32 candidate small molecules were synthesized, and we discovered a small molecule named LYN-1604 as the best candidate ULK1 agonist. Additionally, we identified that three amino acid residues (LYS50, LEU53 and TYR89) were key to the activation site of LYN-1604 and ULK1. Subsequently, we demonstrated that LYN-1604 could induce autophagy-associated cell death via ULK complex (ULK1-mATG13-FIP200-ATG101) in MDA-MB-231 cells. We also found that LYN-1604 induced cell death involved in ATF3, RAD21 and caspase 3, accompanied with autophagy and apoptosis. Moreover, we demonstrated that LYN-1604 had a good therapeutic potential on TNBC by targeting ULK1- modulated cell death in vivo. CONCLUSION We discovered a small molecule (LYN-1604) has therapeutic potential by targeting ULK1-modulated cell death associated with autophagy and apoptosis of TNBC in vitro and in vivo, which could be utilized as a new anti-TNBC drug candidate.

Inhibition of BET bromodomains as a therapeutic strategy for cancer drug discovery.

As a conserved protein interaction module that recognizes and binds to acetylated lysine, bromodomain (BRD) contains a deep, largely hydrophobic acetyl lysine binding site. Proteins that share the feature of containing two BRDs and an extra-terminal domain belong to BET family, including BRD2, BRD3, BRD4 and BRDT. BET family proteins perform transcription regulatory function under normal conditions, while in cancer, they regulate transcription of several oncogenes, such as c-Myc and Bcl-2. Thus, targeting BET proteins may be a promising strategy, and intense interest of BET proteins has fueled the development of structure-based bromodomain inhibitors in cancer. In this review, we focus on summarizing several small-molecule BET inhibitors and their relevant anti-tumor mechanisms, which would provide a clue for exploiting new targeted BET inhibitors in the future cancer therapy.

Design and synthesis of a novel candidate compound NTI-007 targeting sodium taurocholate cotransporting polypeptide [NTCP]-APOA1-HBx-Beclin1-mediated autophagic pathway in HBV therapy.

Sodium taurocholate cotransporting polypeptide (NTCP) is a multiple transmembrane transporter predominantly expressed in the liver, functioning as a functional receptor for HBV. Through our continuous efforts to identify NTCP as a novel HBV target, we designed and synthesized a series of new compounds based on the structure of our previous compound NT-5. Molecular docking and MD simulation validated that a new compound named NTI-007 can tightly bind to NTCP, whose efficacy was also measured in vitro virological examination and cytotoxicity studies. Furthermore, autophagy was observed in NTI-007 incubated HepG2.2.15 cells, and results of q-PCR and Western blotting revealed that NTI-007 induced autophagy through NTCP-APOA1-HBx-Beclin1-mediated pathway. Taken together, considering crucial role of NTCP in HBV infection, NTCP-mediated autophagic pathway may provide a promising strategy of HBV therapy and given efficacy of NTI-007 triggering autophagy. Our study suggests pre-clinical potential of this compound as a novel anti-HBV drug candidate.

Beclin-1: autophagic regulator and therapeutic target in cancer.

Beclin-1 (the mammalian ortholog of yeast ATG6) has been well-characterized to play a pivotal role in autophagy that is a major catabolic pathway in which the cell degrades macromolecules and damaged organelles. Beclin-1 structure has been identified to contain three identifiable domains, including a short Bcl-2-homology-3 (BH3) motif, a central coiled-coil domain (CCD) and a C-terminal half encompassing the evolutionarily conserved domain (ECD). Recent data indicate that Beclin-1 may interact with some co-factors such as Class III phosphatidylinositol 3-kinase (PI3KCIII)/Vps34, Vps15, ATG14L/Barkor, UVRAG, Bif-1, Rubicon, Ambra1, HMGB1, Survivin, Akt and Bcl-2/Bcl-XL to positively or negatively orchestrate the Beclin-1 interactome, thereby co-regulating the autophagy process. Here, we summarize that Beclin-1 serves not only as a key autophagic regulator with its specific interactors, but as a potential therapeutic target in cancer.

Network-based identification of novel connections among apoptotic signaling pathways in cancer.

MicroRNAs (miRNAs), highly conserved, non-coding endogenous RNA and nearly ~22 nucleotides (nt) in length, are well-known to regulate several apoptotic pathways in cancer. In this study, we computationally constructed the initial human apoptotic PPI network by several online databases, and further integrated these high-throughput datasets into a Naïve Bayesian model to predict protein functional connections. Based on the modified apoptotic network, we identified several apoptotic hub proteins such as TP53, SRC, M3K3/5/8, cyclin-dependent kinase2/6, TNFR16/19, and TGF-ß receptor 1/2. Subsequently, we identified some microRNAs that could target the aforementioned apoptotic hub proteins by using TargetScan, PicTar, and Diana-MicroH. In conclusion, these results demonstrate the PPI network-based identification of new connections amongst apoptotic pathways in cancer, which may shed new light on the intricate relationships between core apoptotic pathways and some targeted miRNAs in human cancers.

MicroRNA-modulated autophagic signaling networks in cancer.

MicroRNAs (miRNAs) are small, non-coding endogenous RNAs ∼22 nucleotides (nt) in length that may play the essential roles for regulation of programed cell death, referring to apoptosis and autophagy. Of note, autophagy is an evolutionarily conserved, multi-step lysosomal degradation process in which a cell degrades long-lived proteins and damaged organelles. Accumulating evidence has recently revealed that miRNAs can modulate the autophagic pathways in many pathological processes, most notably cancer. In this review, we focus on highlighting the dual functions of miRNAs as either oncogenes (e.g., miRNA-183, miRNA-376b, miRNA-106a, miRNA-221/222, miRNA-31 and miRNA-34c) or tumor suppressors (e.g., miRNA-30a, miRNA-101 and miRNA-9*) via mediating several autophagic signaling pathways in cancer pathogenesis. Taken together, these findings may uncover the regulatory mechanisms of oncogenic and tumor suppressive miRNAs in autophagy, which would provide a better understanding of miRNA-modulated autophagic signaling networks for future cancer therapeutics.

Plant lectins: targeting programmed cell death pathways as antitumor agents.

Lectins, a group of highly diverse, carbohydrate-binding proteins of non-immune origin that are ubiquitously distributed in plants, animals and fungi, are well-characterized to have numerous links a wide range of pathological processes, most notably cancer. In this review, we present a brief outline of the representative plant lectins including Ricin-B family, proteins with legume lectin domains and GNA family that can induce cancer cell death via targeting programmed cell death pathways. Amongst these above-mentioned lectins, we demonstrate that mistletoe lectins (MLs), Ricin, Concanavalin A (ConA) and Polygonatum cyrtonema lectin (PCL) can lead to cancer cell programmed death via targeting apoptotic pathways. In addition, we show that ConA and PCL can also result in cancer cell programmed death by targeting autophagic pathways. Moreover, we summarize the possible anti-cancer therapeutic implications of plant lectins such as ConA, Phaseolus vulgaris lectin (PHA) and MLs that have been utilized at different stages of preclinical and clinical trials. Together, these findings can provide a comprehensive perspective for further elucidating the roles of plant lectins that may target programmed cell death pathways in cancer pathogenesis and therapeutics. And, this research may, in turn, ultimately help cancer biologists and clinicians to exploit lectins as potential novel antitumor drugs in the future.