Evaluation on the Potential for Hepatotoxic Components from Herba Epimedii to Induce Apoptosis in HepG2 Cells and the Analysis of the Influence of Metabolism in Liver Microsomes.

The potential hepatotoxicity of Herba Epimedii is a focal point in traditional Chinese medicine security applications. As determined in our previous study, the flavonoid constituents of Herba Epimedii, sagittatoside A, icariside I, baohuoside I and icaritin, are related to the hepatotoxicity of this herb. However, the hepatotoxic mechanism of these components needs to be clarified further, and whether these components can maintain their injury action following liver metabolism needs to be confirmed. Herein, the effects of sagittatoside A, icariside I, baohuoside I and icaritin on the apoptosis of HepG2 cells and the expression of key proteins, including Bax, Bcl-2, Caspase-3 and Caspase-9, were evaluated. Moreover, with liver microsome incubation, the influences of metabolism on the apoptotic activities of these components were investigated. Then, by HPLC-MS/MS analyses, the in vitro metabolic stability of these components was determined after incubation with different kinds of liver microsomes to explain the reason for the influence. The results suggested that sagittatoside A, baohuoside I and icaritin could induce apoptosis, which is likely to be closely related to the induction of the intrinsic apoptosis pathway. After metabolic incubation, the sagittatoside A and icaritin metabolism mixture could still induce apoptosis due to less metabolic elimination, while the icariside I and baohuoside I metabolism mixtures respectively got and lost the ability to induce apoptosis, probably due to quick metabolism and metabolic transformation. The findings of this study may provide important references to explore the material basis and mechanism of the hepatotoxicity of Herba Epimedii.

Targeting NEDD8-activating enzyme for cancer therapy: developments, clinical trials, challenges and future research directions.

NEDDylation, a post-translational modification through three-step enzymatic cascades, plays crucial roles in the regulation of diverse biological processes. NEDD8-activating enzyme (NAE) as the only activation enzyme in the NEDDylation modification has become an attractive target to develop anticancer drugs. To date, numerous inhibitors or agonists targeting NAE have been developed. Among them, covalent NAE inhibitors such as MLN4924 and TAS4464 currently entered into clinical trials for cancer therapy, particularly for hematological tumors. This review explains the relationships between NEDDylation and cancers, structural characteristics of NAE and multistep mechanisms of NEDD8 activation by NAE. In addition, the potential approaches to discover NAE inhibitors and detailed pharmacological mechanisms of NAE inhibitors in the clinical stage are explored in depth. Importantly, we reasonably investigate the challenges of NAE inhibitors for cancer therapy and possible development directions of NAE-targeting drugs in the future.

Red blood cell membrane-coated functionalized Au nanocage as a biomimetic platform for improved MicroRNA delivery in hepatocellular carcinoma.

Dysregulation of microRNAs (miRNAs) expression is closely related to cancers and managing miRNA expression holds great promise for cancer therapy. However, their wide clinical application has been hampered by their poor stability, short half-life and non-specific biodistribution in vivo. Herein, a novel biomimetic platform designated as RHAuNCs-miRNA for improved miRNA delivery was prepared through wrapping miRNA-loaded functionalized Au nanocages (AuNCs) with red blood cell (RBC) membrane. RHAuNCs-miRNA not only successfully loaded miRNAs but also effectively protected them from enzymatic degradation. With good stability, RHAuNCs-miRNA had the characteristics of photothermal conversion and sustained release. Cellular uptake of RHAuNCs-miRNA by SMMC-7721 cells was in a time-dependent manner via clathrin- and caveolin-mediated endocytosis. The uptake of RHAuNCs-miRNAs was affected by cell types and improved by mild near infrared (NIR) laser irradiation. More importantly, RHAuNCs-miRNA exhibited a prolonged circulation time without the occurrence of accelerated blood clearance (ABC) in vivo, resulting in efficient delivery to tumor tissues. This study may demonstrate the great potential of RHAuNCs-miRNA for improved miRNAs delivery.

Synthesis of S-2-phenylchromane Derivatives and Evaluation of the Antiproliferative Properties as Apoptosis Inducers in Cancer Cell Lines.

BACKGROUND: Cancer remains one of the major health issues globally, where chemotherapy forms the main treatment mode for different types of cancers. Due to cancer cell ability to develop resistance, decreased clinical effectiveness of anticancer drugs can occur. Therefore, the need to synthesize novel antitumor drugs remains important. OBJECTIVE: The aim of our work consisted of synthesizing S-2-phenylchromane derivatives containing the tertiary amide or 1,2,3-triazole fragments with promising anticancer activity. METHODS: A series of S-2-phenylchromane derivatives were synthesized and evaluated for cytotoxic activity against three selected cancer cell lines (HGC-27 human gastric carcinoma cell line, Huh-7 epithelial-like tumorigenic cells, and A549 adenocarcinomic human alveolar basal epithelial cells) using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Hoechst staining was used to detect the effects of S-2-phenylchromane derivatives on apoptosis. The apoptosis percentages were detected by annexin V-fluoresceine isothiocyanate/propidium iodide (Annexin V-FITC/PI) double staining assay with flow cytometry. Expression levels of apoptosis-related proteins were detected by western blot. RESULTS: Cell line A549, consisting of adenocarcinomic human alveolar basal epithelial cells, displayed the highest sensitivity to the S-2-phenylchromane derivatives. Among these compounds, E2 showed the most potent antiproliferative activity against A549 cells with an IC50 value of 5.60 µM. Hoechst staining and flow cytometry analysis revealed apoptosis in A549 cells by compound E2. In addition, activation of the expression levels of caspase-3, caspase-7, and their substrate poly (ADP-ribose) polymerase (PARP) by E2 was detected by western blot. CONCLUSION: In summary, results point towards compound E2, an S-2-phenylchromane derivative, as a potential lead molecule in anticancer agents for human adenocarcinomic alveolar basal cells based on the induction of apoptosis.

Discovery of dual tubulin-NEDDylation inhibitors with antiproliferative activity.

Although various dual-target tubulin inhibitors have been designed and synthesised, no dual tubulin-NEDDylation inhibitors as antiproliferative agents were reported so far. In this work, a series of trimethoxyphenyl analogues as potential dual tubulin-NEDDylation inhibitors were synthesised and evaluated for their antiproliferative activity. Among them, compound C11 exhibited the most potent inhibitory activity with IC50 values of 1.17, 2.48, and 1.47 µM against HepG2, PC3, and MCF7 cells, respectively. In addition, it displayed the potent inhibitory activity against tubulin with an IC50 value of 2.40 µM and obviously inhibited tubulin polymerisation in HepG2 cells. Furthermore, C11 inhibited NEDDylation by a ATP-dependent manner. Molecular docking studies revealed that the methoxy group and dithiocarbamate group of C11 could form hydrogen bonds with residues of tubulin and E1 NEDD8-activating enzyme (NAE). These results suggested that compound C11 was a dual tubulin-NEDDylation inhibitor with antiproliferative activity.

Flavokawain A Reduces Tumor-Initiating Properties and Stemness of Prostate Cancer.

We have previously demonstrated the in vivo chemopreventive efficacy of flavokawain A (FKA), a novel chalcone from the kava plant, in prostate carcinogenesis models. However, the mechanisms of the anticarcinogenic effects of FKA remain largely unknown. We evaluated the effect of FKA on prostate tumor spheroid formation by prostate cancer stem cells, which were sorted out from CD44+/CD133+ prostate cancer cells 22Rv1 and DU145. FKA treatment significantly decreased both the size and numbers of the tumor spheroids over different generations of spheroid passages. In addition, the dietary feeding of FKA-formulated food to Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice bearing CD44+/CD133+ 22Rv1 xenograft tumors resulted in a significant reduction of tumor growth compared to those fed with vehicle control food-fed mice. Furthermore, the expression of stem cell markers, such as Nanog, Oct4, and CD44, were markedly downregulated in both tumor spheroids and tumor tissues. We also observed that FKA inhibits Ubc12 neddylation, c-Myc, and keratin-8 expression in both CD44+/CD133+ prostate tumor spheroids and xenograft tumors. Our results suggest that FKA can reduce the tumor-initiating properties and stemness of prostate cancer, which provides a new mechanism for the chemoprevention efficacy of FKA.

Annual review of LSD1/KDM1A inhibitors in 2020.

Lysine-specific demethylase 1 (LSD1/KDM1A) has emerged as a promising target for the discovery of specific inhibitors as antitumor drugs. Based on the source of compounds, all LSD1 inhibitors in this review are divided into two categories: natural LSD1 inhibitors and synthetic LSD1 inhibitors. This review highlights the research progress of LSD1 inhibitors with the potential to treat cancer covering articles published in 2020. Design strategies, structure-activity relationships, co-crystal structure analysis and action mechanisms are also highlighted.

Discovery of novel indole derivatives that inhibit NEDDylation and MAPK pathways against gastric cancer MGC803 cells.

A series of novel indole derivatives were synthesized and evaluated for their antiproliferative activity against three selected cancer cell lines (MGC803, EC-109 and PC-3). Among these analogues, 2-(5-methoxy-1H-indol-1-yl)-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (V7) showed the best inhibitory activity against MGC803 cells with an IC50 value of 1.59 µM. Cellular mechanisms elucidated that V7 inhibited colony formation, induced apoptosis and arrested cell cycle at G2/M phase. Importantly, indole analogue V7 inhibited NEDDylation pathway and MAPK pathway against MGC803 cells.

Ras/Raf/MEK/ERK pathway axis mediated neurotoxicity induced by high-risk pesticide residue-Avermectin.

Pesticide residues have become a healthy threaten of human beings. Among the pesticides, many of them have neurotoxicity. Extracellular Regulated Protein Kinases (ERK) pathway is an important signaling pathway that regulates a variety of downstream progress. In this work, peach (PRUNUS persica) and cherry (PRUNUS cerasus) were sampled from over 300 plantations in China and assessed for the residue risk. In mechanism studies, high-risk pesticide Avermectin showed a high activity inhibiting three neurotoxicity models, SH-SY5Y, PC-12 and SK-N-SH cells. At protein levels, ERK pathway proteins and their downstream proteins were obviously down-regulated. Moreover, the effects of low-dose Avermectin can be accumulated at protein levels in the low-dose long-term chronic toxicology detection.

Discovery of tertiary amide derivatives incorporating benzothiazole moiety as anti-gastric cancer agents in vitro via inhibiting tubulin polymerization and activating the Hippo signaling pathway.

On the basis and continuation of our previous studies on anti-tubulin and anti-gastric cancer agents, novel tertiary amide derivatives incorporating benzothiazole moiety were synthesized and the antiproliferative activity was studied in vitro. Preliminary structure activity relationships (SARs) were explored according to the in vitro antiproliferative activity results. Some of compounds could significantly inhibit the proliferation of three cancer cells (HCT-116, MGC-803 and PC-3 cells) and compound F10 exhibited excellent antiproliferative activity against HCT-116 cells (IC50 = 0.182 µM), MGC-803 cells (IC50 = 0.035 µM), PC-3 cells(IC50 = 2.11 µM) and SGC-7901 cells (IC50 = 0.049 µM). Compound F10 effectively inhibited tubulin polymerization (IC50 = 1.9 µM) and bound to colchicine binding site of tubulin. Molecular docking results suggested compound F10 could bind tightly into the colchicine binding site of ß-tubulin. Moreover, compound F10 could regulate the Hippo/YAP signaling pathway. Compound F10 activated Hippo signaling pathway from its very beginning MST1/2, as the result of Hippo cascade activation YAP were inhibited. And then it led to a decrease of c-Myc and Bcl-2 expression. Further molecular experiments showed that compound F10 arrested at G2/M phase, inhibited cell colony formatting and induced extrinsic and intrinsic apoptosis in MGC-803 and SGC-7901 cells. Collectively, compound F10 was the first to be reported as a new anticancer agent in vitro via inhibiting tubulin polymerization and activating the Hippo signaling pathway.

ß-Lactams as promising anticancer agents: Molecular hybrids, structure activity relationships and potential targets.

ß-Lactam, commonly referred as azetidin-2-one, is a multifunctional building block for synthesizing ß-amino ketones, γ-amino alcohols, and other compounds. Besides its well known antibiotic activity, this ring system exhibits a wide range of activities, attracting the attention of researchers. However, the structurally diverse ß-lactam analogues as anticancer agents and their different molecular targets are poorly discussed. The purpose of this review is 3-fold: (1) to explore the molecular hybridization approach to design ß-lactams hybrids as anticancer agents; (2) the structure activity relationship of the most active anticancer ß-lactams and (3) to summarize their antitumor mechanisms.

Novel piperidine derivatives as colchicine binding site inhibitors induce apoptosis and inhibit epithelial-mesenchymal transition against prostate cancer PC3 cells.

Tubulin polymerisation inhibitors that target colchicine binding site were powerful anticancer agents. Although along the years many colchicine binding site inhibitors (CBSIs) have been reported, few piperidine derivatives were identified as CBSIs. In this regard, we focussed efforts on the piperidine as a promising chemotype to develop potent CBSIs. Herein, novel piperidine derivatives were synthesised and evaluated for their antiproliferative activities. Among them, compound 17a displayed powerful anticancer activity with the IC50 value of 0.81 µM against PC3 cells, which was significantly better than 5-fluorouracil. It could inhibit tubulin polymerisation binding at the colchicine site and inhibit the tumour growth in vitro and in vivo. Further biological studies depicted that 17a suppressed the colony formation, induced apoptosis, and inhibited epithelial-mesenchymal transition against PC3 cells. These results revealed that compound 17a is a promising colchicine binding site inhibitor for the treatment of cancer and it is worthy of further exploitation.

Sigma-1 receptor is involved in diminished ovarian reserve possibly by influencing endoplasmic reticulum stress-mediated granulosa cells apoptosis.

Sigma non-opioid intracellular receptor 1 (sigma-1 receptor), a non-opioid transmembrane protein, is located on cellular mitochondrial membranes and endoplasmic reticulum. Current research has demonstrated that sigma-1 receptor is related to human degenerative diseases. This study is focused on the effects of sigma-1 receptor on the pathophysiological process of diminished ovarian reserve (DOR) and granulosa cells (GCs) apoptosis. Sigma-1 receptor concentration in follicular fluid (FF) and serum were negatively correlated with basal follicle-stimulating hormone (FSH) and positively correlated with anti-mullerian hormone (AMH), antral follicle count (AFC). Sigma-1 receptor reduction in GCs was accompanied by endoplasmic reticulum stress (ERS)-mediated apoptosis in women with DOR. Plasmid transfection was used to establish SIGMAR1-overexpressed and SIGMAR1-knockdown human granulosa-like tumor (KGN) cell and thapsigargin (TG) was used to induce ERS KGN cells. We found that KGN cells treated with endogenous sigma-1 receptor ligand dehydroepiandrosterone (DHEA) and sigma-1 receptor agonist PRE-084 showed similar biological effects to SIGMAR1-overexpressed KGN cells and opposite effects to SIGMAR1-knockdown KGN cells. DHEA may improve DOR patients' pregnancy outcomes by upregulating sigma-1 receptor and downregulating ERS-mediated apoptotic genes in GCs. Thus, sigma-1 receptor may be a potential ovarian reserve biomarker, and ligand-mediated sigma-1 receptor activation could be a future approach for DOR therapy.

Antiproliferative benzothiazoles incorporating a trimethoxyphenyl scaffold as novel colchicine site tubulin polymerisation inhibitors.

Tubulin polymerisation inhibitors exhibited an important role in the treatment of patients with prostate cancer. Herein, we reported the medicinal chemistry efforts leading to a new series of benzothiazoles by a bioisosterism approach. Biological testing revealed that compound 12a could significantly inhibit in vitro tubulin polymerisation of a concentration dependent manner, with an IC50 value of 2.87 µM. Immunofluorescence and EBI competition assay investigated that compound 12a effectively inhibited tubulin polymerisation and directly bound to the colchicine-binding site of ß-tubulin in PC3 cells. Docking analysis showed that 12a formed hydrogen bonds with residues Tyr357, Ala247 and Val353 of tubulin. Importantly, it displayed the promising antiproliferative ability against C42B, LNCAP, 22RV1 and PC3 cells with IC50 values of 2.81 µM, 4.31 µM, 2.13 µM and 2.04 µM, respectively. In summary, compound 12a was a novel colchicine site tubulin polymerisation inhibitor with potential to treat prostate cancer.

Discovery of novel tertiary amide derivatives as NEDDylation pathway activators to inhibit the tumor progression in vitro and in vivo.

NEDDylation pathway regulates multiple physiological process, unlike inhibitors, NEDDylation activators are rarely studied. Novel amide derivatives were synthesized and evaluated for antiproliferative activity against MGC803, MCF-7 and PC-3 cells. Among them, Ⅶ-31 displayed the most potent activity with an IC50 value of 94 nmol/L against MGC803 cells. Cellular mechanisms elucidated that Ⅶ-31 inhibited the cell viability, arrested cell cycle at G2/M phase and induced apoptosis via intrinsic and extrinsic pathways against MGC803 cells. In addition, Ⅶ-31 activated NAE1-Ubc12-Cullin1 NEDDylation via interacting with NAE1 directly. Furthermore, the activation of NEDDylation resulted in the degradation of inhibitor of apoptosis proteins (IAPs). Importantly, Ⅶ-31 inhibited tumor growth in xenograft models in vivo without the apparent toxicity. In summary, it is the first time to reveal that Ⅶ-31 deserves consideration for cancer therapy as a NEDDylation activator.

Gartanin is a novel NEDDylation inhibitor for induction of Skp2 degradation, FBXW2 expression, and autophagy.

Gartanin, a 4-prenylated xanthone, has been identified from the purple mangosteen fruit as a potent growth inhibitor of various cancer cell lines, including prostate cancer. However, much of Gartanin's anticancer mechanism remains unknown. We have discovered that Gartanin docked onto the regulatory subunit of the precursor cell-expressed developmentally downregulated 8 (NEDD8)-activating enzyme (NAE) complex and next to the NEDD8 binding complex, which leads to inhibit NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 NEDDylation in an in vitro assay. The S phase kinase-associated protein (Skp2) and F-box and WD-repeat domain-containing 2 (FBXW2), the NEDD8 family members of E3 ubiqutin ligases, were also downregulated and upregulated by Gartainin, respectively. Knock-down of NEDD8 expression by short harpin (Sh) RNAs blocked or attenuated these effects of Gartainin. Finally, Gartanin demonstrated its ability to inhibit growth of prostate cancer lines via autophagy initiation. Our data support that Gartanin is a naturally occurring NEDDylation inhibitor and deserves further investigation for prostate cancer prevention and treatment.

Discovery of indoline derivatives that inhibit esophageal squamous cell carcinoma growth by Noxa mediated apoptosis.

A series of novel indoline derivatives were synthesized and evaluated for antiproliferative activity against four selected cancer cell lines (Hela, A549, HepG2 and KYSE30). Among them, compound 20 displayed the potent inhibition activity against esophageal cancer cells (Kyse30, Kyse450, Kyse510 and EC109). Cellular mechanism studies in esophageal squamous cell carcinoma (ESCC) cells elucidated compound 20 inhibited cell growths in vitro and in vivo, reduced colony formation, arrested cell cycle at M phase, and induced Noxa-dependent apoptosis in ESCC. Importantly, compound 20 was identified as a novel Noxa mediated apoptosis inducer. These results suggested that compound 20 might be a promising anticancer agent with potential for development of further clinical applications.

Inulin with a low degree of polymerization protects human umbilical vein endothelial cells from hypoxia/reoxygenation-induced injury.

Here, we identified inulin-type oligosaccharides with 3-13 degrees of polymerization from Morinda officinalis. Radical-scavenging assays showed that Inulins 4-7 had modest anti-oxidative effects. Inulins 4 and 5 dose-dependently increased human umbilical vein endothelial cell survival during hypoxia/re-oxygenation (H/R)-induced injury, and Inulin 5 promoted angiogenesis. Triplicate assays with the Affymetrix Human Transcriptome Array 2.0 showed that Inulin 5 exposure up-regulated genes associated with cell cycle progression, apoptosis, DNA replication and repair, ubiquitin-mediated proteolysis, the mitogen-activated protein kinase pathway, and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-signaling pathway. Flow cytometry, reverse transcription-quantitative polymerase chain reaction, and western blot experiments verified the microarray results and demonstrated that Inulin 5 influenced cell cycle progression and the PI3K-protein kinase B (PKB)-endothelial nitric oxide synthase (eNOS) pathway. Thus, inulin-type oligosaccharides from M. officinalis roots may protect against H/R-induced injury, primarily through an anti-oxidative effect, and promote angiogenesis by activating the PI3K-PKB-eNOS-signaling pathway.

Anti-PD1 up-regulates PD-L1 expression and inhibits T-cell lymphoma progression: possible involvement of an IFN-γ-associated JAK-STAT pathway.

PURPOSE: NK/T-cell neoplasms are rare, highly aggressive, and insensitive to chemotherapy. These lymphomas have a poor prognosis, with patients being vulnerable to relapse. Hence, there is a need for alternative treatments. The purpose of this study is to investigate whether anti-PD1 takes effect on NK/T cell lymphoma. METHODS: The expression of PD-L1 in NK/T cell lines was investigated by flow cytometry and by Western blot. In vivo, overall survival and median survival time of mice bearing an NK/T cell line tumor was assessed. Tumor-infiltrating T cells and monocyte-derived suppressor cells were evaluated by flow cytometry. Levels of PD-L1 and components of the JAK-STAT pathway were assessed in tumor tissues by immunohistochemistry. RESULTS: NK/T cell lines had greater expression of PD-L1 than normal peripheral blood human NK cells. In vivo, anti-PD1 treatment improved overall survival and median survival time of mice bearing an NK/T cell line. Furthermore, anti-PD1 treatment increased levels of PD-L1. Cultured tumor-infiltrating lymphocytes from mice treated with anti-PD1 had greater levels of IFN-γ than cultured lymphocytes from untreated animals. Further, levels of JAK2 and STAT1 were greater in mice treated with anti-PD1. CONCLUSION: In vivo, anti-PD1 inhibited the progression of an NK/T-cell lymphoma and up-regulated PD-L1 expression. This up-regulation may be through the IFN-γ-associated JAK-STAT pathway.