L02 Cells Research Articles

Design, synthesis, cell imaging, and bioactivity assessment of novel Rhodamine-Pyrimidine nido-carborane derivatives as fluorescent anticancer agents

Because of the advantages of fluorescent drugs consisting of fluorescent dyes and compounds with anticancer activity for therapeutic diagnostics and personalized medicine. A series of fluorescent antitumor drugs rhodamine pyrimidine derivatives (RP1, RP2, RP3 and RP4) were synthesized by linking rhodamine B and pyrimidine derivatives through ethylenediamine bridge and then introduced into nido-carborane to form fluorescent salt derivatives (RPB1, RPB2, RPB3, and RPB4). It improves the tumor targeting and imaging ability of pyrimidine derivatives. By CCK8, it was found that the inhibition rates of RP4 and RPB4 at 10 μg/mL on HeLa, PC-3 and L02 cells were 70.4 %, 68.7 %, 86.1 % and 72.5 %, 76.4 %, 81.0 %, respectively. The compound has strong inhibitory effect on cancer cells. The logP values of compounds RP4 and RPB4 were between 0 and 3, which were easily absorbed and transported through the cell membrane. Further cell imaging experiments showed that RP4 could enter tumor cells through cell membrane and had good biocompatibility. The experimental results show that compounds RP4 has good imaging capability and tumor targeting, and is a potential fluorescent anticancer agents.

Influence of Five Drying Methods on Active Compound Contents and Bioactivities of Fresh Flowers from Syringa pubescens Turcz

The flower of Syringa pubescens Turcz. is used in Chinese folk medicine and also as a flower tea for healthcare. The effects of five drying methods on the active compound contents, the antioxidant abilities, anti-inflammatory properties and enzyme inhibitory activities were evaluated. The plant materials were treated using shade-drying, microwave-drying, sun-drying, infrared-drying and oven-drying. The seven active compounds were simultaneously determined using an HPLC method. Furthermore, the chemical profile was assessed using scanning electron microscopy, ultraviolet spectroscopy and infrared spectroscopy. The antioxidant capacities and protective effects on L02 cells induced with hydrogen peroxide were measured. The anti-inflammatory effects on lipopolysaccharide-induced RAW264.7 cells were investigated. The enzyme inhibitory activities were determined against α-amylase, α-glucosidase cholinesterases and tyrosinase. The results indicated that drying methods had significant influences on the active compound contents and biological properties. Compared with other samples, the OD samples possessed low IC50 values with 0.118 ± 0.004 mg/mL for DPPH radical, 1.538 ± 0.0972 for hydroxyl radical and 0.886 ± 0.199 mg/mL for superoxide radical, while the SHD samples had stronger reducing power compared with other samples. The SHD samples could be effective against H2O2-induced injury on L02 cells by the promoting of T-AOC, GSH-PX, SOD and CAT activities and the reducing of MDA content compared with other samples. Furthermore, SPF samples, especially the SHD sample, could evidently ameliorate inflammation through the inhibition of IL-6, IL-1β and TNF-α expression. All the studied SPF samples exhibited evidently inhibitory effects on the four enzymes. The IC50 values of inhibitory activity on α-glucosidase and α-amylase from SHD sample were 2.516 ± 0.024 and 0.734 ± 0.034 mg/mL, respectively. SD samples had potential inhibitory effects on cholinesterases and tyrosinase with IC50 values of 3.443 ± 0.060 and 1.732 ± 0.058 mg/mL. In consideration of active compound contents and biological activities, it was recommended that SHD and SD be applied for drying SPF at an industrial scale.

Exploring the underlying mechanisms of fisetin in the treatment of hepatic insulin resistance via network pharmacology and in vitro validation

ObjectiveTo characterize potential mechanisms of fisetin on hepatic insulin resistance (IR) using network pharmacology and in vitro validation.MethodsPutative targets of fisetin were retrieved from the Traditional Chinese Medicine Systems Pharmacology database, whereas the potential genes of hepatic IR were obtained from GeneCards database. A protein–protein interaction (PPI) network was constructed according to the intersection targets of fisetin and hepatic IR using the Venn diagram. The biological functions and potential pathways related to genes were determined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Cell experiments were also conducted to further verify the mechanism of fisetin on hepatic IR.ResultsA total of 118 potential targets from fisetin were associated with hepatic IR. The areas of nodes and corresponding degree values of TP53, AKT1, TNF, IL6, CASP3, CTNNB1, JUN, SRC, epidermal growth factor receptor (EGFR), and HSP90AA1 were larger and could be easily found in the PPI network. Furthermore, GO analysis revealed that these key targets were significantly involved in multiple biological processes that participated in oxidative stress and serine/threonine kinase activity. KEGG enrichment analysis showed that the PI3K/AKT signaling pathway was a significant pathway involved in hepatic IR. Our in vitro results demonstrated that fisetin treatment increased the expressions of EGFR and IRS in HepG2 and L02 cells under normal or IR conditions. Western blot results revealed that p-AKT/AKT levels were significantly up-regulated, suggesting that fisetin was involved in the PI3K/AKT signaling pathway to regulate insulin signaling.ConclusionWe explored the pharmacological actions and the potential molecular mechanism of fisetin in treating hepatic IR from a holistic perspective. Our study lays a theoretical foundation for the development of fisetin for type 2 diabetes.

Lusianthridin ameliorates high fat diet-induced metabolic dysfunction-associated fatty liver disease via activation of FXR signaling pathway.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a common chronic liver disease, but there are few specific medications for it. Lusianthridin, a major phenanthrene component that originates from Dendrobium Sonia, has various in vitro biological functions. In this study, we aimed to evaluate the therapeutic effects of lusianthridin on high-fat diet (HFD)-induced MAFLD as well as to examine the mechanism of its effects. We fed male mice high-fat-diet for 12 weeks to induce MAFLD and then continued to feed them, either with or without lusianthridin, for another six weeks. We found that lusianthridin decreased serum triacylglycerol, hepatic triacylglycerol, and serum low density lipoprotein cholesterol. It also reduced hepatic lipid accumulation based on the results of morphology analysis. Besides, it improved hepatic inflammation as well, including a decrease in serum alanine aminotransferase and a reduction in macrophage and neutrophil infiltration. Mechanistically, surface plasmon resonance, cell thermal shift assay and dual-luciferase report system results suggested that lusianthridin combined with farnesoid X receptor (FXR) ligand binding region and activated its transcriptional activity. Lusianthridin also decreased de no lipogenesis though inhibiting Srebp1c and downstream Scd-1, Lpin1 and Dgat2 expression in a FXR-dependent manner in oleic acid treated L02 cells. Correspondingly, lusianthridin inhibited Srebp1c and downstream lipogenesis in MAFLD liver tissues of mice at both of genetic and protein levels. Finally, the protective effects of lusianthridin on hepatic steaotosis were abolished in Fxr−/− mice. Taken together, our results suggested that lusianthridin attenuated high-fat-diet induced MAFLD via activation the FXR signaling pathway.

A assembled plate column bioreactor for adherent cell culture

The ideal bioreactor should be highly efficient, cost-effective and able to promote cells adhesion growth. In this study, we constructed a plate-tower bioreactor with S-loop in each layer based on multilayer plate bioreactor and plate column theory. Human hepatocellular carcinomas(HepG2) and human normal liver cell L02 (L02) cells were employed evaluation of bioreactor performance. The results show that cells were able to adhere to the plates and the density reached to 1～1.2 × 105/cm2. When circulating flow rate of the reactor is 8 ml/min and the oxygen supply concentration is 40 %, the sufficient material and gas exchange in bioreactor, the amount of albumin, urea and cytochrome P450(CYP450) content in the hyperoxia group were 3.31, 2.58 and 2.6 times higher than that of the control group, there was no obvious cell damage. This condition up the regulation of genes in more metabolic pathways and improved the metabolic capacity of hepatocytes. The single-layer area and the number of bioreactors can be flexibly adjusted, and the manufacturing cost of single-layer is close to that of ordinary plastic cell culture dishes. Either batch or continuous mode can be used in the bioreactor. In view of the above characteristics, the bioreactor is more conducive to practical application.

Novel Platinum(IV) complexes intervene oxaliplatin resistance in colon cancer via inducing ferroptosis and apoptosis

Platinum-based chemotherapeutics are widely used for cancer treatment but are frequently limited because of dosage-dependent side effects and drug resistance. To attenuate these drawbacks, a series of novel platinum(IV) prodrugs (15a–18c) were synthesized and evaluated for anti-cancer activity. Among them, 17a demonstrated superior anti-proliferative activity compared with oxaliplatin (OXA) in the cisplatin-resistant lung cancer cell line A549/CDDP and OXA-resistant colon cancer cell line HCT-116/OXA but showed a lower cytotoxic effect toward human normal cell lines HUVEC and L02. Mechanistic investigations suggested that 17a efficiently enhanced intracellular platinum accumulation, induced DNA damage, disturbed the homeostasis of intracellular reactive oxygen molecules and mitochondrial membrane potential, and thereby activated the mitochondrion-dependent apoptosis pathway. Moreover, 17a significantly induced ferroptosis in HCT-116/OXA via triggering the accumulation of lipid peroxides, disrupting iron homeostasis, and inhibiting solute carrier family 7 member 11 and glutathione peroxidase 4 axial pathway transduction by inhibiting the expression of the phosphorylated signal transducer and activator of transcription 3 and nuclear factor erythroid 2‐related factor 2. Moreover, 17a exerted remarkable in vivo antitumor efficacy in the HCT-116/OXA xenograft models but showed attenuated toxicity. These results indicated that these novel platinum(IV) complexes provided an alternative strategy to develop novel platinum-based antineoplastic agents for cancer treatment.

Catapol attenuates the aseptic inflammatory response to hepatic I/R injury in vivo and in vitro by inhibiting the HMGB1/TLR-4/NF-κB signaling pathway via the microRNA-410–3p

BackgroundHepatic ischemia-reperfusion (I/R) injury involves inflammatory necrosis of liver cells as a significant pathological mechanism. Catapol possesses anti-inflammatory activity that is extracted from the traditional Chinese medicine, Rehmannia glutinosa. MethodsThe liver function and histopathology, Oxidative stress, and aseptic inflammatory responses were assessed in vivo, and the strongest dose group was selected. For mechanism, the expression of miR-410–3p, HMGB1, and TLR-4/NF-κB signaling pathways was detected. The dual luciferase assay can verify the targeting relationship between miR-410–3p and HMGB1. Knockdown of miR-410–3p in L02 cells is applied in interference experiments. ResultsCAT pre-treatment significantly decreased the liver function markers alanine and aspartate aminotransferases and reduced the areas of hemorrhage and necrosis induced by hepatic I/R injury. Additionally, it reduced the aseptic inflammatory response and oxidative stress, with the strongest protective effect observed in the high-dose CAT group. Mechanistically, CAT downregulates HMGB1, inhibits TLR-4/NF-κB signaling pathway activation, and reduces inflammatory cytokines TNF-α, and IL-1β. In addition, the I/R-induced downregulation of microRNA-410–3p was inhibited by CAT pre-treatment in vivo and in vitro. HMGB1 was identified as a potential target of microRNA-410–3p using a dual-luciferase reporter assay. Knockdown of microRNA-410–3p abolished the inhibitory effect of CAT on HMGB1, p-NF-κB, and p-IκB-α protein expression. ConclusionsOur study showed that CAT pre-treatment has a protective effect against hepatic I/R injury in rats. Specifically, CAT attenuates the aseptic inflammatory response to hepatic I/R injury in vivo and in vitro by inhibiting the HMGB1/TLR-4/NF-κB signaling pathway via the microRNA-410–3p.

Norcantharidin-Encapsulated C60-Modified Nanomicelles: A Potential Approach to Mitigate Cytotoxicity in Renal Cells and Simultaneously Enhance Anti-Tumor Activity in Hepatocellular Carcinoma Cells.

The objective of this study was to examine the preparation process of DSPE-PEG-C60/NCTD micelles and assess the impact of fullerenol (C60)-modified micelles on the nephrotoxicity and antitumor activity of NCTD. The micelles containing NCTD were prepared using the ultrasonic method and subsequently optimized and characterized. The cytotoxicity of micelles loaded with NCTD was assessed using the CCK-8 method on human hepatoma cell lines HepG2 and BEL-7402, as well as normal cell lines HK-2 and L02. Acridine orange/ethidium bromide (AO/EB) double staining and flow cytometry were employed to assess the impact of NCTD-loaded micelles on the apoptosis of the HK-2 cells and the HepG2 cells. Additionally, JC-1 fluorescence was utilized to quantify the alterations in mitochondrial membrane potential. The generation of reactive oxygen species (ROS) following micelle treatment was determined through 2',7'-dichlorofluorescein diacetate (DCFDA) staining. The particle size distribution of the DSPE-PEG-C60/NCTD micelles was determined to be 91.57 nm (PDI = 0.231). The zeta potential of the micelles was found to be -13.8 mV. The encapsulation efficiency was measured to be 91.9%. The in vitro release behavior of the micelles followed the Higuchi equation. Cellular experiments demonstrated a notable decrease in the toxicity of the C60-modified micelles against the HK-2 cells, accompanied by an augmented inhibitory effect on cancer cells. Compared to the free NCTD group, the DSPE-PEG-C60 micelles exhibited a decreased apoptosis rate (12%) for the HK-2 cell line, lower than the apoptosis rate observed in the NCTD group (36%) at an NCTD concentration of 75 μM. The rate of apoptosis in the HepG2 cells exhibited a significant increase (49%), surpassing the apoptosis rate observed in the NCTD group (24%) at a concentration of 150 μM NCTD. The HK-2 cells exhibited a reduction in intracellular ROS and an increase in mitochondrial membrane potential (ΔψM) upon exposure to C60-modified micelles compared to the NCTD group. The DSPE-PEG-C60/NCTD micelles, as prepared in this study, demonstrated the ability to decrease cytotoxicity and ROS levels in normal renal cells (HK-2) in vitro. Additionally, these micelles showed an enhanced antitumor activity against human hepatocellular carcinoma cells (HepG2, BEL-7402).

Core-Shell Reactor Partitioning Enzyme and Prodrug by ZIF-8 for NADPH-Sensitive In Situ Prodrug Activation.

Enzyme-prodrug therapies have shown unique advantages in efficiency, selectivity, and specificity of in vivo prodrug activation. However, precise spatiotemporal control of both the enzyme and its substrate at the target site, preservation of enzyme activity, and in situ substrate depletion due to low prodrug delivery efficiency continue to be great challenges. Here, we propose a novel core-shell reactor partitioning enzyme and prodrug by ZIF-8, which integrates an enzyme with its substrate and increases the drug loading capacity (DLC) using a prodrug as the building ligand to form a Zn-prodrug shell. Cytochrome P450 (CYP450) is immobilized in ZIF-8, and the antitumor drug dacarbazine (DTIC) is coordinated and deposited in its outer layer with a high DLC of 43.6±0.8 %. With this configuration, a much higher prodrug conversion efficiency of CYP450 (36.5±1.5 %) and lower IC50 value (26.3±2.6 μg/mL) are measured for B16-F10 cells with a higher NADPH concentration than those of L02 cells and HUVECs. With the tumor targeting ability of hyaluronic acid, this core-shell enzyme reactor shows a high tumor suppression rate of 96.6±1.9 % and provides a simple and versatile strategy for enabling in vivo biocatalysis to be more efficient, selective, and safer.

Core‐Shell Reactor Partitioning Enzyme and Prodrug by ZIF‐8 for NADPH‐Sensitive In Situ Prodrug Activation

AbstractEnzyme‐prodrug therapies have shown unique advantages in efficiency, selectivity, and specificity of in vivo prodrug activation. However, precise spatiotemporal control of both the enzyme and its substrate at the target site, preservation of enzyme activity, and in situ substrate depletion due to low prodrug delivery efficiency continue to be great challenges. Here, we propose a novel core–shell reactor partitioning enzyme and prodrug by ZIF‐8, which integrates an enzyme with its substrate and increases the drug loading capacity (DLC) using a prodrug as the building ligand to form a Zn‐prodrug shell. Cytochrome P450 (CYP450) is immobilized in ZIF‐8, and the antitumor drug dacarbazine (DTIC) is coordinated and deposited in its outer layer with a high DLC of 43.6±0.8 %. With this configuration, a much higher prodrug conversion efficiency of CYP450 (36.5±1.5 %) and lower IC50 value (26.3±2.6 μg/mL) are measured for B16‐F10 cells with a higher NADPH concentration than those of L02 cells and HUVECs. With the tumor targeting ability of hyaluronic acid, this core–shell enzyme reactor shows a high tumor suppression rate of 96.6±1.9 % and provides a simple and versatile strategy for enabling in vivo biocatalysis to be more efficient, selective, and safer.

Developing a novel benzothiazole-based red-emitting probe for intravital imaging of superoxide anion

Superoxide anion (O2•−), the first generated reactive oxygen species (ROS), is a critical player in cellular signaling network and redox homeostasis. Imaging of O2•−, particularly in vivo, is of concern for further understanding its roles in pathophysiological and pharmacological events. Herein, we designed a novel probe, (E)-4-(5-(2-(benzo[d]thiazol-2-yl)-2-cyanovinyl)furan-2-yl)phenyl trifluoromethane-sulfonate (BFTF), by modifying hydroxyphenyl benzothiazole (a widely used dye scaffold) which includes insertion of both an acrylonitrile unit and a furan ring to extend the total π-conjugation system and to enhance push-pull intramolecular charge transfer process, and utilization of trifluoromethanesulfonate as the response unit. Toward O2•−, the probe features near-infrared fluorescent emission (685 nm), large Stokes shift (135 nm), and deep tissue penetration (300 μm). With its help, we successfully mapped preferential generation of O2•− in HepG2 cells over L02 cells, as well as in A549 over BEAS-2B cells by β-lapachone (an anticancer agent that generates O2•−), and more importantly, visualized overproduction of O2•− in living mice with liver injury induced by acetaminophen (a well-known analgesic and antipyretic drug).

Berberine protects hepatocyte from hypoxia/reoxygenation-induced injury through inhibiting circDNTTIP2.

During hepatic ischemia-reperfusion injury, the excessive release of inflammatory cytokines can activate the intracellular signal transduction cascade to induce hepatocyte injury. Apoptosis is an important way of cell death after I/R injury. Berberine, a common quaternary ammonium alkaloid, has anti-inflammatory, anti-oxidative stress, and anti-apoptotic effects. An increasing number of studies have revealed the importance of non-coding RNAs, including microRNA, long non-coding RNAs and circular RNAs (circRNAs), as regulators of the effects of berberine. In this study, we investigated the mechanism of berberine against liver ischemia-reperfusion injury in vitro. In this study, hypoxia-reoxygenation (H/R)-treated L02 cells were pretreated with berberine to study the role and mechanism of berberine in resisting hepatic ischemia-reperfusion injury. The results show that berberine pre-treatment increased the cell viability of H/R-challenged cells, reduced H/R-induced apoptosis and ROS production, reversed H/R-increased on IL-6, IL-1β, TNF-α, and H/R-decreased IL-10 expression. Mechanically, berberine protect hepatocyte from H/R injury, at least partially, through circDNTTIP2. In addition, circDNTTIP2 can bind to the TATA box of caspase3 promoter, thereby promoting caspase 3-related cell apoptosis and the release of inflammatory cytokines. This study found that berberine has a protective effect on H/R-induced hepatocyte damage by inhibiting a novel circRNA, circDNTTIP2. This study provides potential treatment strategies and treatment targets for liver ischemia-reperfusion injury.

Folic Acid Protects against Ethanol-Induced Hepatic Mitophagy Imbalance by ROS Scavenging and Attenuating the Elevated Hcy Levels.

Ample evidence indicates that ethanol-induced oxidative stress and mitochondrial dysfunction are central to the pathogenesis of alcoholic liver disease (ALD). As an adaptive quality control mechanism, mitophagy removes dysfunctional mitochondria to avert hepatic lesions in ALD. Folic acid exhibits potential radical scavenging properties and has been proven to ameliorate mitochondrial disorder in oxidative stress-related diseases. In this study, we aimed to uncover the mitophagy regulatory effects of folic acid in a 10w alcohol C57BL/6J mice feeding model (56% v/v) and L02 cells model cultured with ethanol (2.5% v/v). The results showed that folic acid alleviates ethanol-induced liver injury, decreasing oxidative stress and restoring liver enzyme. Furthermore, folic acid improved the mitochondrial function and inhibited ethanol-activated mitophagy through decreasing PINK1-Parkin and Drp1 expression, which inhibited the release of mitochondrial cytochrome C to the cytoplasm, preventing hepatocyte apoptosis. Intriguingly, folic acid attenuates the elevated hepatic homocysteine (Hcy) level. Additionally, the pretreatment of L02 cells with folic acid also ameliorated Hcy-induced oxidative damage, mitochondrial fission, and mitophagy. In summary, these results suggest that folic acid has beneficial effects in mitophagy remodeling by ROS scavenging and facilitating Hcy metabolism and could be developed as a potential therapeutic agent against ALD.

Cdh1 plays a protective role in nonalcoholic fatty liver disease by regulating PPAR/PGC-1α signaling pathway

BackgroundNonalcoholic fatty liver disease (NAFLD) is a significant etiological factor in liver-related diseases, which can lead to severe consequences such as steatohepatitis, cirrhosis and death. Cdh1 is considered as a crucial protein involved in cell cycle regulation. The purpose of this study is to explore the biological role of Cdh1 in NAFLD. Materials and methodsNAFLD cell model was established, and L02 cells and AML12 cells were infected by shRNA lentivirus with Cdh1 knockdown in vitro, and the effect of Cdh1 deletion on cell lipid deposition was evaluated. The effects of Cdh1 deletion on Akt phosphorylation and PPAR/PGC-1α signaling pathway in L02 cells were examined. In addition, the NAFLD mouse model was constructed, and the conditional knockout mice of Cdh1 were selected to verify the results. ResultsIn vitro experiments showed that the Cdh1 deletion enhanced cell lipid deposition. In vivo experiments showed that conditional knockdown of Cdh1 aggravated fatty degeneration and damage of liver in mice. Cdh1 deletion promotes Akt phosphorylation and inhibits PPAR/PGC-1α signaling pathway in L02 cells. Conditional knockout of Cdh1 down-regulates PPAR/PGC-1α signaling pathway in NAFLD mouse model. ConclusionThe deletion of Cdh1 may promote Akt phosphorylation by up-regulating Skp2 and inhibit the PPAR/PGC-1α signaling pathway. Cdh1 serves a protective function in the occurrence and progression of NAFLD.

In Vitro Investigation of the Anticancer Activity of Peucedanum praeruptorum Dunn Extract on HepG2 Human Hepatoma Cells

The anticancer activity of Peucedanum praeruptorum Dunn extract (PPDE) was investigated in vitro in the HepG2 human hepatoma cell line and compared to normal human liver cells (L02 cells). The effect of the PPDE on the proliferation of the cells was measured by MTT assays, and the levels of enzymes and small molecules implicated in oxidative stress regulation were measured using specific reagent kits. The expression levels of genes implicated in apoptosis (Bax, Bcl-2, caspase-3, caspase-8, and caspase-9) and oxidative stress (SOD1 and SOD2) were quantified by RT-qPCR. Lastly, HPLC was employed to analyze the composition of the PPDE. PPDE was found to significantly inhibit the proliferation of HepG2 cells but had little effect on the proliferation of normal liver cells. PPDE increased the levels of reactive oxygen species and malonaldehyde, a lipid peroxidation product, in HepG2 cells, and it reduced the activities of antioxidant enzymes, as well as the levels of γ-GCS and reduced glutathione (GSH), suggesting that it inhibited the ability of cancer cells to regulate intracellular oxidative stress. PPDE also increased the expression of the genes encoding Bax, caspase-3, caspase-8, and caspase-9 and decreased the expression of Bcl-2, SOD1, and SOD2 in HepG2 cells, suggesting that PPDE induced the apoptosis of the liver cancer cells. HPLC analysis identified that the components of PPDE included caffeic acid, isochlorogenic acid C, myricetin, baicalin, luteolin, and kaempferol, all of which have demonstrated antioxidant properties.

Matrine disrupts Nrf2/GPX4 antioxidant system and promotes hepatocyte ferroptosis

Matrine (MT) is an alkaloid isolated from Sophora flavescens with various bioactivities and is widely used clinically. However, the broader its clinical use, the greater its toxicity concerns. We investigate the role of ferroptosis in MT-induced liver injury caused by an imbalance in the antioxidant pathway. Our results showed that MT could cause pathological changes in liver tissues and lead to a significant reduction in L02 cell viability. MT also reduced superoxide dismutase (SOD) and glutathione (GSH), increased malondialdehyde (MDA), reactive oxygen species (ROS), and lipid peroxidation levels, and disrupted iron homeostasis, leading to ferroptosis. In addition, MT decreased the protein levels of FTH, Nrf2, xCT, GPX4, HO-1 and ferroptosis suppressor protein 1 (FSP1) and increased the protein levels of TRF1 and DMT1, characteristic indicators of ferroptosis. Interestingly, the cytotoxic effects of MT were alleviated by ferroptosis inhibitor, Nrf2 agonist, or selenium supplementation. These results revealed that MT triggers hepatocyte ferroptosis by inhibiting the Nrf2/GPX4 antioxidant system.

Metabolic disturbance and transcriptomic changes induced by methyl triclosan in human hepatocyte L02 cells.

Methyl triclosan (MTCS) is one of the biomethylated by-products of triclosan (TCS). With the increasing use of TCS, the adverse effects of MTCS have attracted extensive attention in recent years. The purpose of this study was to investigate the cytotoxicity of MTCS and to explore the underlining mechanism using human hepatocyte L02 cells as in vitro model. The cytotoxicity results revealed that MTCS could inhibit cell viability, disturb the ratio of reduced glutathione (GSH) and oxidized glutathione (GSSG), and reduce the mitochondrial membrane potential (MMP) in a dose-dependent manner. In addition, MTCS exposure significantly promoted the cellular metabolic process, including enhanced conversion of glucose to lactic acid, and elevated content of intracellular triglyceride (TG) and total cholesterol (TC). RNA-sequencing and bioinformatics analysis indicated disorder of glucose and lipid metabolism was significantly induced after MTCS exposure. Protein-protein interaction network analysis and node identification suggested that Serine hydroxy methyltransferase 2 (SHMT2), Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), Asparagine synthetase (ASNS) and Phosphoglycerate dehydrogenase (PHGDH) are potential molecular markers of metabolism imbalance induced by MTCS. These results demonstrated that oxidative stress and metabolism dysregulation might be involved in the cytotoxicity of MTCS in L02 cells.

Integration approach of transcriptomics and metabolomics reveals the toxicity of Anthracene and its chlorinated derivatives on human hepatic cells

Polycyclic aromatic hydrocarbons (PAHs) and Chlorinated PAHs (Cl-PAHs) are ubiquitous environmental contaminants. The toxicological information of anthracene (Ant) and its chlorinated derivatives is quite limited. In this study, an integrated metabolomic and transcriptomic analysis approach was adopted to assess the toxic effects triggered by Ant and its chlorinated derivatives, 2-chloroanthracene (2-ClAnt) and 9,10-dichloroanthracen (9,10-Cl2Ant), at human-relevant levels on human normal hepatocyte L02 cells. The cell viability test showed no significant effects on the viability of L02 cells exposed to Ant, 2-ClAnt and 9,10-Cl2Ant at doses of 5–500 nM for 24 h. However, based on transcriptomic analysis, Ant, 2-ClAnt and 9,10-Cl2Ant exposure at human-relevant levels obviously perturbed global gene expression in L02 cells and induced the differential expression of several genes related to cancer development. As the number of genes related to cancer development altered by 9,10-Cl2Ant is the largest, 9,10-Cl2Ant posed greater risks of tumor development than Ant and 2-ClAnt did. Metabolomics analysis demonstrated that Ant, 2-ClAnt and 9,10-Cl2Ant caused significant metabolic perturbation in L02 cells. Pathway enrichment analysis indicated that Ant, 2-ClAnt and 9,10-Cl2Ant mainly perturbed the lipid metabolism and nucleotide metabolism pathway. However, 9,10-Cl2Ant caused a wider perturbation to metabolic pathways than Ant and 2-ClAnt did. In addition, dysregulation of nucleotide metabolism perturbed by Ant, 2-ClAnt and 9,10-Cl2Ant may be associated with the genomic instability and further carcinogenesis.

Discovery of Triazolyl Derivatives of Cucurbitacin B Targeting IGF2BP1 against Non-Small Cell Lung Cancer.

Cucurbitacin B (CuB) is a potent but toxic anticancer natural product. Herein, we designed and synthesized 2-OH- and 16-OH-modified CuB derivatives to improve their antitumor efficacy and reduce toxicity. Among them, derivative A11 had the most potent antiproliferative activity against A549 lung cancer cells (IC50 = 0.009 μM) and was approximately 10-fold more potent than CuB, while the cytotoxicity of A11 toward normal L02 cells was about 10-fold less potent, indicating a much wider therapeutic window than CuB. Derivative A11 directly binds to the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) protein with a KD value of 2.88 nM, which is about 23-fold more potent than CuB, leading to the decreased expression of downstream apoptosis- and cell cycle-related proteins. More importantly, A11 exhibited much more potent anticancer efficacy in an A549 xenograft mouse model with a TGI rate of 80% and a superior in vivo safety profile than that of CuB.

Bavachin induces liver injury and cell apoptosis by targeting Wnt/β-catenin/DRP1 signaling pathway mediated mitochondrial dysfunction

Psraleae Fructus (PF) is a well-known traditional Chinese medicine in China. While numerous liver injury reports caused by PF limits its clinical application. Bavachin, a flavonoid compound isolated from the fruits of Psoralea corylifolia L., has been validated to induce direct apoptosis in hepatocytes and liver tissues in our previous studies. However, the subcellular mechanisms of bavachin induced liver injury is still elusive. Here, utilizing 6-week-old C57BL/6 J mice and human embryonic hepatocytes (L02 cells), we report that bavachin activates dynamic-related protein 1 (DRP1) mediated excess mitochondrial fission and endoplasmic reticulum (ER) stress related apoptosis via Wnt/β-catenin signaling pathway. Notably, DRP1 knockdown or XAV-939 induced Wnt/β-catenin inhibition decreased bavachin-induced ER stress and cell apoptosis in L02 cells. In addition, bavachin impaired mitochondrial structural and function in the mice liver tissues. Mdivi-1, a mitochondrial fission inhibitor targeting DRP1, prevented bavachin-induced mitochondrial and ER structural damage, ER stress, and liver injury. Our results demonstrated that bavachin induced mitochondrial fission plays a crucial role in bavachin induced ER stress related liver injury, via the mechanism that involved activation of Wnt/β-catenin signaling pathway.