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Backgrounds: Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver diseases. The leaves of Broussonetia papyrifera contain a large number of flavonoids, which have a variety of biological functions., Methods: In vitro experiments, free fatty acids were used to stimulate HepG2 cells. NAFLD model was established in vivo in mice fed with high fat diet (HFD) or intraperitoneally injected with Tyloxapol (Ty). At the same time, Total flavonoids of Broussonetia papyrifera (TFBP) was used to interfere with HepG2 cells or mice., Results: The results showed that TFBP significantly decreased the lipid accumulation induced by oil acid (OA) with palmitic acid (PA) in HepG2 cells. TFBP decreased the total cholesterol (TC), the triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and increased high-density lipoprotein cholesterol (HDLC) in serum. TFBP could also effectively inhibit the generation of reactive oxygen species (ROS) and restrained the level of myeloperoxidase (MPO), and enhance the activity of superoxide dismutase (SOD) to alleviate the injury from oxidative stress in the liver. Additionally, TFBP activated nuclear factor erythroid-2-related factor 2 (Nrf2) pathway to increasing the phosphorylation of AMP-activated protein kinase (AMPK). Meanwhile, protein levels of mTORC signaling pathway were evidently restrained with the treatment of TFBP., Conclusion: Our experiments proved that TFBP has the therapeutic effect in NAFLD, and the activation of Nrf2 and AMPK signaling pathways should make sense., Competing Interests: Declaration of competing interest Qi Wang, Yunfei Wei, Yeling Wang, Ziyang Yu, Haiyan Qin, Lilei Zhao, Jiaqi Cheng, Bingyu Sheng, Meiyu Jing, Haihua Feng declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

- Seven previously undescribed ent-eudesmane sesquiterpenoids (1-7), as well as seven known analogs (8-14), were isolated from the Chinese liverwort Chiloscyphus polyanthus var. rivularis. Their structures were established based on comprehensive spectroscopy analysis, electronic circular dichroism calculations, as well as biosynthetic considerations. The cytotoxicity against HepG2 (Human hepatocellular carcinomas) cancer cell line, and antifungal activity against Candida albicans SC5314 of all isolated ent-eudesmane sesquiterpenoids were preliminarily tested, results showed that the tested compounds did not display obvious cytotoxicity and antifungal activities under the tested concentration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The prevalence of nonalcoholic fatty liver disease (NAFLD) is estimated to be approximately about 25.24% of the population worldwide. NAFLD is a complex syndrome and is characterized by a simple benign hepatocyte steatosis to more severe steatohepatitis in the liver pathology. Phellinus linteus (PL) is traditionally used as a hepatoprotective supplement. Styrylpyrone-enriched extract (SPEE) obtained from the PL mycelia has been shown to have potential inhibition effects on high-fat- and high-fructose-diet-induced NAFLD. In the continuous study, we aimed to explore the inhibitory effects of SPEE on free fatty acid mixture O/P [oleic acid (OA): palmitic acid (PA); 2:1, molar ratio]-induced lipid accumulation in HepG2 cells. Results showed that SPEE presented the highest free radical scavenging ability on DPPH and ABTS, and reducing power on ferric ions, better than that of partitions obtained from n-hexane, n-butanol and distilled water. In free-fatty-acid-induced lipid accumulation in HepG2 cells, SPEE showed an inhibition effect on O/P-induced lipid accumulation of 27% at a dosage of 500 μg/mL. As compared to the O/P induction group, the antioxidant activities of superoxide dismutase, glutathione peroxidase and catalase were enhanced by 73%, 67% and 35%, respectively, in the SPEE group. In addition, the inflammatory factors (TNF-α, IL-6 and IL-1β) were significantly down-regulated by the SPEE treatment. The expressions of anti-adipogenic genes involved in hepatic lipid metabolism of 5' adenosine monophosphate (AMP)-activated protein kinase ( AMPK ), sirtuin 1 ( SIRT1 ) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha ( PGC-1α ) were enhanced in the SPEE supplemented HepG2 cells. In the protein expression study, p-AMPK, SIRT1 and PGC1-α were significantly increased to 121, 72 and 62%, respectively, after the treatment of SPEE. Conclusively, the styrylpyrone-enriched extract SPEE can ameliorate lipid accumulation and decrease inflammation and oxidative stress through the activation of SIRT1/AMPK/PGC1-α pathways.

Hepatocellular carcinoma (HCC) is a major subtype of primary liver cancer with a high mortality rate. Pyroptosis and autophagy are crucial processes in the pathophysiology of HCC. Searching for efficient drugs targeting pyroptosis and autophagy with lower toxicity is useful for HCC treatment. Mallotucin D (MLD), a clerodane diterpenoid from Croton crassifolius, has not been previously reported for its anticancer effects in HCC. This study aims to evaluate the inhibitory effects of MLD in HCC and explore the underlying mechanism. We found that the cell proliferation, DNA synthesis, and colony formation of HepG2 cells and the angiogenesis of HUVECs were all greatly inhibited by MLD. MLD caused mitochondrial damage and decreased the TOM20 expression and mitochondrial membrane potential, inducing ROS overproduction. Moreover, MLD promoted the cytochrome C from mitochondria into cytoplasm, leading to cleavage of caspase-9 and caspase-3 inducing GSDMD-related pyroptosis. In addition, we revealed that MLD activated mitophagy by inhibiting the PI3K/AKT/mTOR pathway. Using the ROS-scavenging reagent NAC, the activation effects of MLD on pyroptosis- and autophagy-related pathways were all inhibited. In the HepG2 xenograft model, MLD effectively inhibited tumor growth without detectable toxicities in normal tissue. In conclusion, MLD could be developed as a candidate drug for HCC treatment by inducing mitophagy and pyroptosis via promoting mitochondrial-related ROS production.

Mycotoxins can impart different types of combined toxicity to humans and animals, therefore, it is critical to understand the underlying mechanisms to eliminate the harm. Herein a combination of zearalenone (ZEA) at 2 μM and deoxynivalenol (DON) at 0.1 μM decreased cell viability and increased ROS level in HepG2 cells, suggesting synergistic toxicity exerted by ZEA and DON even at their low toxic concentrations. Moreover, apoptosis and inflammatory response were promoted after the co-exposure of ZEA and DON, indicated by the increased expression of BAX, Caspase-3, IL-1β and IL-6 genes. Such synergistic toxicity was closely associated with miR-221-mediated PTEN/PI3K/AKT signal pathway, with a negative regulatory relationship between PTEN and PI3K/AKT signaling. MiR-221 could influence cell viability and ROS level to counter the combined toxicity of ZEA and DON through targeting directly PTEN gene. This study demonstrated the toxicological impact of mycotoxin interactions on cells, and critical role of the interplay between miRNAs and PTEN in monitoring the synergistic toxicity of mycotoxin mixture., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Food contaminants of bacterial or fungal origin frequently contaminate staple foods to various extents. Among others, the bacterial toxin cereulide (CER) and the mycotoxin deoxynivalenol (DON) co-occur in a mixed diet and are absorbed by the human body. Both toxins exert dis-tinctive mitotoxic potential. As damaged mitochondria are removed via autophagy, mitochondrial and lysosomal toxicity were assessed by applying low doses of single and combined toxins (CER 0.1-50 ng/mL; DON 0.01-5 µg/mL) to HepG2 liver cells. In addition to cytotoxicity assays, RT-qPCR was performed to investigate genes involved in lysosomal biogenesis and autophagy. CER and DON caused significant cytotoxicity on HepG2 cells after 5 and 24 h over a broad concentration range. CER, alone and in combination with DON, increased the transcription of the autophagy related genes coding for the microtubule associated protein 1A/1B light chain 3 ( LC3) and sequestome 1 ( SQSTM1) as well as LC3 protein expression which was determined using immunocytochemistry. DON increased LC3 protein expression without induction of gene transcription, hence it seems plausible that CER and DON act on different pathways. The results support the hypothesis that CER induces autophagy via the LC3 pathway and damaged mitochondria are therefore eliminated.

Mitochondria are involved in the regulation of apoptosis, making them a promising target for the development of new anticancer drugs. Doxorubicin (DOX), a chemotherapeutic drug, can induce reactive oxygen species (ROS)-mediated apoptosis, improving its anticancer effects. Herein, Rhein, an active ingredient in rhubarb, with the capability of self-assembly and mitochondrial targeting, was used in conjunction with DOX to form efficient nanomaterials (Rhein-DOX nanogel) capable of sustained drug release. It was self-assembled with a hydrogen bond, π-π stacking interactions, and hydrophobic interactions as the main driving force, and its loading efficiency was up to 100%. Based on its self-assembly characteristics, we evaluated the mechanism of this material to target mitochondria, induce ROS production, and promote apoptosis. The IC 50 of the Rhein-DOX nanogel (3.74 μM) was only 46.3% of that of DOX (11.89 μM), and the tumor inhibition rate of the Rhein-DOX nanogel was 79.4% in vivo, 2.3 times that of DOX. This study not only addresses the disadvantages of high toxicity of DOX and low bioavailability of Rhein, when DOX and Rhein are combined for the treatment of hepatoma, but it also significantly improved the synergistic antihepatoma efficacy of Rhein and DOX, which provides a new idea for the development of long-term antihepatoma agents with low toxicity.

<b>Background and Objective:</b> Pineapple (<i>Ananas comosus</i>) is a popular fruit worldwide with natural antioxidant properties. This study examined how pineapple modified the expression of drug-metabolizing enzymes (CYP1A2, CYP2C9, CYP3A4, UGT1A6, NAT2 and SULT1A1) and a drug transporter (OATP1B1) in human hepatocarcinoma (HepG2) cells. <b>Materials and Methods:</b> HepG2 cells (2.5×10<sup>5</sup> cells/well in a 24-well plate) were incubated with pineapple juice extract (125-1,000 μg mL<sup>1</sup>) for 48 hrs in phenol red-free medium. Resazurin reduction, ROS, AST and ALT assays were performed. The mRNA expression of target genes was determined by RT/qPCR. <b>Results:</b> Pineapple juice slightly reduced HepG2 cell viability to 80% of the control, while ROS, AST and ALT levels were not changed. Pineapple juice did not alter the expression of CYP1A2, CYP2C9 and UGT1A6 mRNA. All tested concentrations of pineapple juice suppressed CYP3A4, NAT2 and OATP1B1 expression, while SULT1A1 expression was induced. <b>Conclusion:</b> Though pineapple juice slightly decreased the viability of HepG2 cells, cell morphology and cell function remained normal. Pineapple juice disturbed the expression of phase I (CYP3A4) and phase II (NAT2 and SULT1A1) metabolizing genes and the drug transporter OATP1B1. Therefore, the consumption of excessive amounts of pineapple juice poses a risk for drug interactions.

Microalgae are known as a producer of proteins and lipids, but also of valuable compounds for human health benefits (e.g., polyunsaturated fatty acids (PUFAs); minerals, vitamins, or other compounds). The overall objective of this research was to prospect novel products, such as nutraceuticals from microalgae, for application in human health, particularly for metabolic diseases. Chlorella vulgaris and Chlorococcum amblystomatis were grown autotrophically, and C. vulgaris was additionally grown heterotrophically. Microalgae biomass was extracted using organic solvents (dichloromethane, ethanol, ethanol with ultrasound-assisted extraction). Those extracts were evaluated for their bioactivities, toxicity, and metabolite profile. Some of the extracts reduced the neutral lipid content using the zebrafish larvae fat metabolism assay, reduced lipid accumulation in fatty-acid-overloaded HepG2 liver cells, or decreased the LPS-induced inflammation reaction in RAW264.7 macrophages. Toxicity was not observed in the MTT assay in vitro or by the appearance of lethality or malformations in zebrafish larvae in vivo. Differences in metabolite profiles of microalgae extracts obtained by UPLC-LC-MS/MS and GNPS analyses revealed unique compounds in the active extracts, whose majority did not have a match in mass spectrometry databases and could be potentially novel compounds. In conclusion, microalgae extracts demonstrated anti-obesity, anti-steatosis, and anti-inflammatory activities and could be valuable resources for developing future nutraceuticals. In particular, the ultrasound-assisted ethanolic extract of the heterotrophic C. vulgaris significantly enhanced the anti-obesity activity and demonstrated that the alteration of culture conditions is a valuable approach to increase the production of high-value compounds.

Objective: To investigate evodiamine (EVO)-induced hepatotoxicity and the underlying mechanism., Methods: HepG2 cells were treated with EVO (0.04-25 μmol/L) for different time intervals, and the cell survival rate was examined by cell counting kit-8 (CCK-8) method. After HepG2 cells were treated with EVO (0.2, 1 and 5 μmol/L) for 48 h, the alanine transaminase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) activities and total bilirubin (TBIL) content of supernatant were detected. A multifunctional microplate reader was used to detect the intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in HepG2 cells to evaluate the level of cell lipid peroxidation damage. The interactions between EVO and apoptosis, autophagy or ferroptosis-associated proteins were simulated by molecular docking. The HepG2 cells were stained by mitochondrial membrane potential (MMP) fluorescent probe (JC-10) and annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI), and MMP and apoptosis in HepG2 cells were detected by flow cytometry. The protein expression levels of caspase-9, caspase-3, bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2) were detected by Western blot., Results: The cell survival rate was significantly reduced after the HepG2 cells were exposed to EVO (0.04-25 μmol/L) in a time- and dose-dependent manner. The half maximal inhibitory concentration (IC50) of the HepG2 cells treated with EVO for 24, 48 and 72 h were 85.3, 6.6 and 4.7 μmol/L, respectively. After exposure to EVO (0.2, 1 and 5 μmol/L) for 48 h, the ALT, AST, LDH, ALP activities and TBIL content in the HepG2 cell culture supernatant, and the MDA content in the cells were increased, and SOD enzyme activity was decreased. Molecular docking results showed that EVO interacted with apoptosis-associated proteins (caspase-9 and caspase-3) better. JC-10 and Annexin V-FITC/PI staining assays demonstrated that EVO could decrease MMP and promote apoptosis in the HepG2 cells. Western blot results indicated that the protein expressions of cleaved caspase-9 and cleaved caspase-3 were upregulated in the HepG2 cell treated with EVO for 48 h. In contrast, the protein expressions of pro-caspase-3, BSEP and MRP2 were downregulated., Conclusion: These results suggested that 0.2, 1 and 5 μmol/L EVO had the potential hepatotoxicity, and the possible mechanism involved lipid peroxidation damage, cell apoptosis, and cholestasis.

Phloretin, a dihydrochalcone, widely exists in the fruits of apple trees and crabapple trees ( Malus prunifolia ) with multiple biological activities. Presently, we studied the function of phloretin on the attenuation of hepatic steatosis and further explored the underlying mechanisms both in vitro and in vivo . Male C57BL/6J mice were fed a normal diet or high fat diet (HFD) with or without phloretin (100 mg kg -1 ) for 12 weeks. HepG2 cells were induced by 200 μM palmitic acid (PA) and co-incubated with phloretin (50 μM) for 24 h. The results showed that phloretin treatment significantly decreased the accumulation of lipids in the liver of the HFD-fed C57BL/6J mice and PA-induced HepG2 cells. Also, phloretin effectively ameliorated hepatic steatosis via promoting fatty acid β-oxidation (FAO). This biological activity of phloretin was closely related to its capacity to improve mitochondrial dysfunction, including the promotion of mitochondrial biosynthesis and inhibition of mitochondrial swelling through the AMPK-dependent SIRT1/PGC-1α and SIRT3/CypD signaling pathways, respectively. These results demonstrate that phloretin effectively improves mitochondrial function and ameliorates HFD-induced hepatic steatosis through an AMPK-dependent signaling pathway.

Background/aim: Epirubicin (EPI), an epimer of doxorubicin (DOX), and DOX are anthracycline agents with broad-spectrum antitumor activity. The aim of the present study was to elucidate the transport characteristics of EPI and DOX in human hepatocellular carcinoma HepG2 cells and human non-small cell lung cancer A549 cells, and to examine the relationship of intracellular drug accumulation with their cytotoxic effects., Materials and Methods: Intracellular concentrations of EPI and DOX were measured using high-performance liquid chromatography (HPLC). Expression level of targeted genes was analyzed by real-time quantitative PCR. Cell viability was evaluated using the MTT assay., Results: Similar to DOX, EPI was taken up into HepG2 and A549 cells by organic cation transporter 6 and passive diffusion; however, the efficiency of saturable and non-saturable uptake of EPI was greater than that of DOX in both cell types. EPI served as a substrate of P-glycoprotein and multidrug associated protein (MRP) 1 and MRP2 similarly to DOX, but the efflux efficiency of each transporter was markedly different between EPI and DOX. The intracellular accumulation of EPI was significantly greater than that of DOX in all cells, and the accumulated level reflected the cytotoxic effects of these drugs. However, the intracellular drug amount did not correspond to the degree of cytotoxicity when compared between HepG2 and A549 cells, which can be explained by the higher expression of Bcl-xl in A549 cells., Conclusion: This study suggested that the transport characteristics are markedly different between EPI and DOX in HepG2 and A549 cells, and that intracellular accumulation is the predominant factor affecting the cytotoxicity of EPI and DOX in individual cells., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Although the medical application of betulin has been presented in previous studies, the potential mechanism of the anti-inflammatory action of betulin should be further investigated. This work aims to confirm the hypothesis that betulin has dexamethasone-like anti-inflammatory action through glucocorticoid receptor (GR)-mediated pathway. Firstly, the binding ability of betulin with GR was measured by a fluorescence polarization-based competitive binding assay, with the IC 50 value of 79.18 ± 0.30 mM. Betulin could bind to GR and then induced GR nuclear translocation, but lacked GR transcriptional activity in HeLa cells. Hence, betulin exhibited the potential to be a dissociated modulator for GR, with the loss of glucocorticoid response element (GRE)-associated side effects. In addition, betulin downregulated GRE-driven protein expression of G6P involved in gluconeogenesis, namely side effect. The results of pro-inflammatory cytokines analysis showed that betulin exerted anti-inflammatory action in vitro. Both of the hydrophobic and hydrogen-bonding interactions stabilized the binding between betulin and GR during the simulation process. In conclusion, betulin might be a potential dissociated GR modulator with a reduced side effect profile yet keeping its anti-inflammatory action., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Perfluorooctane sulfonate (PFOS) is one kind of persistent organic pollutants. In previous study, we found that PFOS induced autophagy-dependent lysosomal membrane permeabilization (LMP) in hepatocytes, and siRNA against lysosomal permease spinster 1 (SPNS1) relieved PFOS-induced LMP. However, whether and how SPNS1 functioned as the link between autophagy and LMP was still not defined. In this study, we constructed a stable cell line expressing high levels of SPNS1. We found that SPNS1 interacted specifically with α-tubulin of tyrosinated isotype by pull-down assay. After treatment with PFOS, the level of tyrosinated α-tubulin was autophagy-dependently decreased. SPNS1-tyrosinated α-tubulin interaction was disrupted subsequently, which led to LMP eventually. We also found that stable high-expression of SPNS1 in hepatocytes accelerated lysosomal acidification, and deteriorated PFOS-induced LMP. This study pointed out that SPNS1-tyrosinated α-tubulin interaction mediated the cross-talk between autophagy and LMP induced by PFOS, shedding new light on the mechanism of PFOS hepatotoxicity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Our study investigated the effects of food contaminant 1,3-dichloro-2-propanol (1,3-DCP) on hepatic lipid metabolism and its mechanism. We found that triglyceride (TG), total cholesterol (TC) and the number of lipid droplets (LDs) were increased in the liver of C57BL/6 mice given intragastric administration of 1,3-DCP for 30 days. Meanwhile, 1,3-DCP inhibited autophagosomes and lysosomes formation, reflected by decreased LC3-II, LAMP1, LAMP2, CTSD, CTSB expression, increased p62 expression and decreased LC3 fluorescence. Subsequently, we detected the changes of hepatic lipid accumulation caused by 1,3-DCP using an autophagy inducer or inhibitor. In vivo, Hepatic lipid accumulation caused by 1,3-DCP was mitigated by the autophagy inducer Rapa. On the contrary, the autophagy inhibitor (chloroquine or 3-methyladenine) further exacerbated hepatic lipid accumulation caused by 1,3-DCP. 1,3-DCP reduced the number of autophagosomes encapsulated LDs, assessed by colocalization of LD and LC3. These data demonstrated that 1,3-DCP induced lipid accumulation by inhibiting autophagy. We further investigated the mechanism of 1,3-DCP-inhibited autophagy and found 1,3-DCP increased the ratios of p-AKT/AKT, p-mTOR/mTOR, p-FOXO1/FOXO1, decreased FOXO1 nuclear localization in vivo. These proteins may be involved in the regulation of 1,3-DCP-mediated autophagy. We detected the changes in autophagy marker protein LC3-II and lipid accumulation using an AKT inhibitor ARQ-092 or a mTOR inhibitor rapamycin in HepG2 cells. Compared with 1,3-DCP group, lipid accumulation was decreased, LC3-II and FOXO1 nuclear localization were increased, p-FOXO1 levels were decreased in HepG2 cells pretreated with ARQ-092 or rapamycin. Taken together, these data revealed that the effects of 1,3-DCP on lipid accumulation by inhibiting autophagy were dependent on AKT/mTOR/FOXO1 signaling pathway. Our study not only supplied the mechanism of 1,3-DCP toxicity, but also provided experimental basis for effective intervention measures of 1,3-DCP toxicity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The present study investigated the protective effects and mechanism of action of cyanidin-3-O-glucoside (C3G) and its major metabolite protocatechuic acid (PCA) against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced cytotoxicity in HepG2 cells. The results demonstrated that C3G and PCA dose-dependently suppressed PhIP-induced mutation in Salmonella typhimurium TA98, and inhibited PhIP-induced cytotoxicity and apoptosis in HepG2 cells. Western blot analysis indicated that C3G and PCA minimized PhIP-induced cell damage by reversing the abnormal expression of Bax/Bcl-2, Cytochrome c, cleaved Caspase-3, XIAP, Nrf2, HO-1, LC3 and p62 involved in intrinsic apoptotic and Nrf2/p62 pathways. Molecular docking results revealed that C3G and PCA were able to interfere with Nrf2 signaling and apoptotic cascade through binding to Keap1 and Bcl-2. Moreover, the protective effect of C3G was stronger than that of PCA. These findings suggested that dietary consumption of food sources rich in C3G can fight against the health risks of heterocyclic aromatic amines., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Turbo cornutus , the horned turban sea snail, is found along the intertidal and basaltic shorelines of Jeju Island, Korea. T . cornutus feeds on seaweeds (e.g., Undaria sp., and Ecklonia sp.) composed of diverse antioxidants. This study identified potential antioxidant properties from T. cornutus viscera tissues. Diverse extracts were evaluated for their hydrogen peroxide (H 2 O 2 ) scavenging activities. T . cornutus viscera protamex-assisted extracts (TVP) were purified by gel filtration chromatography (GFC), and potential antioxidant properties were analyzed for their amino acid sequences and its peroxidase inhibition effects by in silico molecular docking and in vitro analysis. According to the results, T. cornutus viscera tissues are composed of many protein contents with each over 50%. Among the extracts, TVP possessed the highest H 2 O 2 scavenging activity. In addition, TVP-GFC-3 significantly decreased intracellular reactive oxygen species (ROS) levels and increased cell viability in H 2 O 2 -treated HepG2 cells without cytotoxicity. TVP-GFC-3 comprises nine low molecular bioactive peptides (ELR, VGPQ, TDY, ALPHA, PAH, VDY, WSDK, VFSP, and FAPQY). Notably, the peptides dock to the active site of the myeloperoxidase (MPO), especially TDY and FAPQY showed the MPO inhibition effects with IC 50 values of 646.0 ± 45.0 µM and 57.1 ± 17.7 µM, respectively. Altogether, our findings demonstrated that T. cornutus viscera have potential antioxidant properties that can be used as high value-added ingredients.

RG7388 (Idasanutlin) is a potent inhibitor of oncoprotein murine double minute 2 (MDM2). Herein we investigated the feasibility of developing 18 F-labeled RG7388 as a radiotracer for imaging MDM2 expression in tumors with positron emission tomography (PET). Two fluorinated analogues of RG7388, 6 and 7 , were synthesized by attaching a fluoronicotinyl moiety to RG7388 via a polyethylene glycol (PEG 3 ) or a propyl linker. The inhibitory potency (IC 50 ) of 6 and 7 against MDM2 was determined by a fluorescence polarization (FP)-based assay. Next, compound 6 was labeled with 18 F using a trimethylammonium triflate precursor to obtain [ 18 F]FN-PEG 3 -RG7388 ([ 18 F] 6 ), and its properties were evaluated in MDM2 expressing wild-type p53 tumor cell lines (SJSA-1 and HepG2) in vitro and in tumor xenografts in vivo. The FP assays revealed an IC 50 against MDM2 of 119 nM and 160 nM for 6 and 7 , respectively. 18 F-labeling of 6 was achieved in 50.3 ± 7.5% radiochemical yield. [ 18 F] 6 exhibited a high uptake (∼70% of input dose) and specificity in SJSA-1 and HepG2 cell lines. Saturation binding assays revealed a binding affinity ( K d ) of 128 nM for [ 18 F] 6 on SJSA-1 cells. In mice, [ 18 F] 6 showed fast clearance from blood with a maximum tumor uptake of 3.80 ± 0.85% injected dose per gram (ID/g) in HepG2 xenografts at 30 min postinjection (p.i.) and 1.32 ± 0.32% ID/g in SJSA-1 xenografts at 1 h p.i. Specificity of [ 18 F] 6 uptake in tumors was demonstrated by pretreatment of mice with SJSA-xenografts with a blocking dose of RG7388 (35 mg/kg body weight, i.p.). In vivo stability studies in mice using HPLC showed ∼60% and ∼30% intact [ 18 F] 6 remaining in plasma at 30 min and 1 h p.i., respectively, with the remaining activity attributed to polar peaks. Our results suggest that RG7388 is a promising molecular scaffold for 18 F-labeled probe development for MDM2. Additional labeling strategies and functionalizing locations on RG7388 are under development to improve binding affinity and in vivo stability of the 18 F-labeled compound to make it more amenable for PET imaging of MDM2 in vivo.

Background: Cancer is a significant health problem around the world and one of the leading causes of human death. The need for novel, selective and non-toxic anti-cancer agents is still urging., Aim of the Work: to investigate the anti-proliferative and pro-apoptotic effects of the synthesized ciprofloxacin 3,4,5 tri-methoxy chalcone hybrid (CCH) on the HepG2 hepatocellular carcinoma and MCF7 breast carcinoma cell lines., Materials and Methods: HepG2 and MCF7cell lines were treated with CCH. Cell viability and cell cycle analysis were performed. Protein and mRNA expression levels of P53, COX-2 and TNF-α were analyzed by western blotting and RT-PCR respectively., Results: CCH caused concentration and time-dependent reduction in the viability of human HepG2 and MCF7 cells, pre-G1 apoptosis and cell cycle arrest at G2/M stage, significantly higher P53 and TNF-α mRNA and protein expression levels but significantly lower COX2 mRNA and protein expression levels., Conclusion: CCH showed obvious anti-proliferative and apoptosis-inducing activities in both cell lines.

An in-depth study on the inhibitory mechanism on protein tyrosine phosphatase 1B (PTP1B) and aldose reductase (AR) enzymes, including analysis of the insulin signalling pathway, of phosphoeleganin, a marine-derived phosphorylated polyketide, was achieved. Phosphoeleganin was demonstrated to inhibit both enzymes, acting respectively as a pure non-competitive inhibitor of PTP1B and a mixed-type inhibitor of AR. In addition, in silico docking analyses to evaluate the interaction mode of phosphoeleganin with both enzymes were performed. Interestingly, this study showed that phosphoeleganin is the first example of a dual inhibitor polyketide extracted from a marine invertebrate, and it could be used as a versatile scaffold structure for the synthesis of new designed multiple ligands.

Sponges are known to produce a series of compounds with bioactivities useful for human health. This study was conducted on four sponges collected in the framework of the XXXIV Italian National Antarctic Research Program (PNRA) in November-December 2018, i.e., Mycale (Oxymycale) acerata , Haliclona (Rhizoniera) dancoi , Hemimycale topsenti , and Hemigellius pilosus . Sponge extracts were fractioned and tested against hepatocellular carcinoma (HepG2), lung carcinoma (A549), and melanoma cells (A2058), in order to screen for antiproliferative or cytotoxic activity. Two different chemical classes of compounds, belonging to mycalols and suberitenones, were identified in the active fractions. Mycalols were the most active compounds, and their mechanism of action was also investigated at the gene and protein levels in HepG2 cells. Of the differentially expressed genes, ULK1 and GALNT5 were the most down-regulated genes, while MAPK8 was one of the most up-regulated genes. These genes were previously associated with ferroptosis, a programmed cell death triggered by iron-dependent lipid peroxidation, confirmed at the protein level by the down-regulation of GPX4, a key regulator of ferroptosis, and the up-regulation of NCOA4, involved in iron homeostasis. These data suggest, for the first time, that mycalols act by triggering ferroptosis in HepG2 cells.

Fluoxetine is a first-line selective serotonin reuptake inhibitor widely applied for the treatment of depression; however, it induces abnormal hepatic lipid metabolism. Considering decreased expression or function of glucose-6-phosphatase (G6Pase), a key enzyme in gluconeogenesis, or the upregulation of fatty acid uptake, causes hepatic lipid accumulation. The aim of this study was to elucidate whether G6Pase regulation and fatty acid uptake alteration contribute to fluoxetine-induced abnormal hepatic lipid metabolism. Our study revealed that 8-week oral administration of fluoxetine dose-dependently increased hepatic triglyceride, causing hepatic steatosis. Concomitantly, the expression of G6Pase in mouse livers and primary mouse hepatocytes (PMHs) was downregulated in a concentration-dependent manner. Furthermore, fluoxetine increased the concentrations of glucose-6-phosphate (G6Pase substrate) and acetyl CoA (the substrate for de novo lipogenesis) in mouse livers. Additionally, fluoxetine also induced lipid accumulation and downregulated G6Pase expression in HepG2 cells. However, the uptake of green fluorescent fatty acid (BODIPY™ FL C16) in PMHs was not changed after fluoxetine treatment, indicating that fluoxetine-induced hepatic steatosis was not associated with fatty acid uptake alteration. In conclusion, fluoxetine downregulated hepatic G6Pase expression, subsequently enhanced the transformation of glucose to lipid, and ultimately resulted in hepatic steatosis, but with no impact on fatty acid uptake., (© 2020 John Wiley & Sons, Ltd.)

α- β unsaturated carboxylic acids containing a heterocyclic moiety is one of the most potent class of bioactive compounds whose speedy generation through novel synthetic techniques has become an enigma for the synthetic chemists. This research project demonstrates a novel method for the synthesis of these compounds using polymer-supported microwave-assisted methodology carried out through one-pot multicomponent reaction. Both soluble and insoluble polymers have been used and their results are comprehensively analyzed. Moreover, the compounds are characterized through spectral analysis like FTIR, GC-MASS, 1 HNMR Spectroscopy. The cytotoxicity of synthesized compounds is evaluated through MTT assay using HEPG 2 cells.

Non-alcoholic steatohepatitis (NASH), one of the deleterious stages of non-alcoholic fatty liver disease, remains a significant cause of liver-related morbidity and mortality worldwide. In the current work, we used an exploratory data analysis to investigate time-dependent cellular and mitochondrial effects of different supra-physiological fatty acids (FA) overload strategies, in the presence or absence of fructose (F), on human hepatoma-derived HepG2 cells. We measured intracellular neutral lipid content and reactive oxygen species (ROS) levels, mitochondrial respiration and morphology, and caspases activity and cell death. FA-treatments induced a time-dependent increase in neutral lipid content, which was paralleled by an increase in ROS. Fructose, by itself, did not increase intracellular lipid content nor aggravated the effects of palmitic acid (PA) or free fatty acids mixture (FFA), although it led to an up-expression of hepatic fructokinase. Instead, F decreased mitochondrial phospholipid content, as well as OXPHOS subunits levels. Increased lipid accumulation and ROS in FA-treatments preceded mitochondrial dysfunction, comprising altered mitochondrial membrane potential (ΔΨm) and morphology, and decreased oxygen consumption rates, especially with PA. Consequently, supra-physiological PA alone or combined with F prompted the activation of caspase pathways leading to a time-dependent decrease in cell viability. Exploratory data analysis methods support this conclusion by clearly identifying the effects of FA treatments. In fact, unsupervised learning algorithms created homogeneous and cohesive clusters, with a clear separation between PA and FFA treated samples to identify a minimal subset of critical mitochondrial markers in order to attain a feasible model to predict cell death in NAFLD or for high throughput screening of possible therapeutic agents, with particular focus in measuring mitochondrial function.

The organochlorine insecticide dichlorodiphenyltrichloroethane (DDT) and heavy metal cadmium (Cd) are widespread environmental pollutants. They are persistent in the environment and can accumulate in organisms. Although the individual toxicity of DDT and Cd has been well documented, their combined toxicity is still not clear. Since liver is their common target, in this study, the individual and combined toxicity of DDT and Cd in human liver carcinoma HepG2 and human normal liver THLE-3 cell lines were investigated. The results showed that DDT and Cd inhibited the viability of HepG2 and THLE-3 cells dose-dependently and altered lysosomal morphology and function. Intracellular reactive oxygen species and lipid peroxidation levels were induced by DDT and Cd treatment. The combined cytotoxicity of DDT and Cd was greater than their individual cytotoxicity, and the interaction between Cd and DDT was additive on the inhibition of cell viability and lysosomal function of HepG2 cells. The interaction was antagonistic on the inhibition of cell viability of THLE-3 cells. These results may facilitate the evaluation of the cumulative risk of pesticides and heavy metal residues in the environment.

Silybum marianum (Milk thistle) has been proven to possess anticancer, lactogenic, neuroprotective, immunomodulatory, hepatoprotective and anti-inflammatory properties. The current study was designed to evaluate the antiproliferative potential of aqueous and various organic fractions (ethanolic, petroleum ether, ethyl acetate, chloroform, n-butanol) of S. marianum against cancerous [HeLa, HepG2] and noncancerous [BHK] cell lines. The MTT assay was performed to access the cytotoxicity of all these fractions and IC50 values were calculated. The cytotoxicity of these fractions was also confirmed through crystal violet and Trypan blue assays. All the tested fractions of S. marianum possessed significant antiproliferative potential. Interestingly, ethyl acetate fraction of S. marianum exhibited the highest antiproliferative activity amongst all the other tested fractions with an IC50 of 13.07 µg/ml, 18.92 µg/ml and 76.15µg/ml against HeLa, HepG2 and BHK cell lines respectively. So it is concluded that S. marianum possess strong anticancer activity against both cervical and liver cancer and low cytotoxicity against normal cell line so it could be used as a source of potent anticancer compounds having high efficacy and minimal side effects.

Current study was intended to isolate bioactive compounds from ethyl acetate fraction of Saussurea hypoleuca root extract and evaluation of their antioxidant, antimicrobial and anti-cancerous activities which might be helpful for their chemo preventive potential against selected bacterial strains. Column chromatography was done for isolation of compounds which were characterized on the basis of extensive spectroscopic analysis; Infra-red (IR), Electron Ionization (EI-Positive), Proton Nuclear Magnetic Resonance ( 1 H-NMR) and Carbon Nuclear Magnetic Resonance ( 13 C-NMR). Two compounds were identified, as sesquiterpenes (40mg) and linoleic acid (33mg) from 10 grams of ethyl acetate fraction. Both compounds have shown in vitro antioxidant activity which in regard; 2, 2- diphenyl-1-picrylhydrazyl (DPPH) scavenging potential was high in sesquiterpenes (261.81) as compared to linoleic acid (90.89). The minimum inhibitory concentrations (MIC) of both compounds were evaluated in various bacterial and fungal strains against respective controls. However, in human hepatocellular carcinoma (Hep G2 cell lines) sesquiterpenes exhibited strong anticancer potential than linolenic acid which might be its potential free radical inactivator in MTT assay. This paper directs the ethano medicinal worth of plant root as it possesses bioactive compounds which in our best knowledge these compounds isolated and reported first time from this plant root specie.

Endogenous cytokinins in mycelia of medicinal mushrooms Hericium coralloides and Fomitopsis officinalis grown in vitro were identified using high-performance liquid chromatography coupled with mass spectrometry. High amounts of zeatin-type cytokinins and isopentenyladenine were found. The qualitative composition and quantitative content of cytokinins were species-specific traits of mushrooms. Optical microscopy was used to perform a comparison analysis of the influence of crude extracts and purified cytokinin fractions from both species' mycelial biomass on HepG2 tumor cell growth in vitro and morphology. The results showed that purified cytokinin fractions from H. coralloides and F. officinalis mycelia demonstrated a cytotoxic effect on HepG2 cells, unlike crude extracts. Under the influence of all mushroom extracts, similar patterns of changes in HepG2 cell morphology were observed, but they were more pronounced for H. coralloides compared with F. officinalis. Purified fractions of both mushroom species caused an increased level of apoptosis compared to crude extracts. Some increase in glucose uptake by cultured cells was found in all investigated samples wherein the influence of H. coralloides extracts was approximately twice the effect of the corresponding F. officinalis extracts. The data obtained confirm the assumption that cytokinins are involved in the expression of therapeutic effects of medicinal mushrooms and indicate the need to take into consideration the methods of cytokinin extraction when preparing pharmacologically active drugs based on fungal raw materials. Thus, extracts from H. coralloides and F. officinalis mycelial biomass are promising in the search for anticancer agents.

<b>Background and Objective:</b> The medicinal herb <i>Plumbago indica</i> (PI) and its major constituent plumbagin have reported pharmacological properties but there is a lack of information about their herb-drug interactions. The effects of methanolic (PI-MeOH) and ethanolic (PI-EtOH) crude extracts of PI and plumbagin on the expression of cytochrome P450s (<i>CYP1A2</i>, <i>CYP2E1</i> and <i>CYP3A4</i>) and transporters (<i>ABCC1</i>, <i>ABCG2</i> and <i>SLC22A11</i>) were investigated in BeWo and HepG2 cells. <b>Materials and Methods:</b> BeWo or HepG2 cells were treated with 0.5-5 μM plumbagin or 25-500 μg mL<sup>1</sup> of PI-MeOH or PI-EtOH for 24 hrs. Total RNA was extracted and mRNA expression of CYPs and transporters were determined using RT-qPCR. <b>Results:</b> PI and plumbagin affected mRNA expression differently in the two tested cell types. In BeWo cells, all concentrations of PI-MeOH induced <i>CYP2E1</i>, 100 and 500 μg Ml<sup>1</sup> PI-MeOH and PI-EtOH up-regulated <i>CYP1A2</i>, <i>CYP3A4 </i>and <i>ABCG2 </i>and 500 μg mL<sup>1</sup> PI-EtOH induced <i>ABCG2</i> expression. Plumbagin suppressed <i>CYP1A2</i> and induced <i>SLC22A11 </i>expression at the highest concentration, 5 μM. In HepG2 cells, 5 μM plumbagin and 500 μg Ml<sup>1</sup> PI-EtOH suppressed <i>CYP3A4 </i>expression and 500 μg mL<sup>1</sup> PI-MeOH and PI-EtOH up-regulated <i>CYP1A2</i> and <i>CYP2E1 </i>expression. <i>ABCC1</i> expression was induced by all treatments while <i>ABCG2</i> and <i>SLC22A11 </i>were induced only by 500 μg mL<sup>1</sup> PI-MeOH and PI-EtOH. <b>Conclusion:</b> The use of PI or plumbagin supplements in large quantities or for long periods should be carefully considered due to the risk of herbal drug interactions via modulated expression of CYPs and transporters.

Bioactive constituents of numerous marine organisms have been investigated recently for their preclinical and clinical anticancer activity. Three marine organisms: black-spotted sea cucumber: Pearsonothuria graeffei ( Pg ), lollyfish: Holothuria atra ( Ha ), and sea hare: Aplysia dactylomela ( Ad ), were collected during winter 2019 from Gulf of Aqaba, Red Sea, Egypt, and macerated with ethanol into three different extracts: Pg E , Ha E , and Ad E , where each was in vitro assessed for its antiproliferative and proapoptotic properties on HepG2 , HCT-116 , and MCF-7 cancer cells. Pg E dose-dependently inhibited the growth of HepG2 , HCT-116 , and MCF-7 cells within IC 50 values 16.22, 13.34, and 18.09  μ g/mL, respectively, while the IC 50 values for the antiproliferative activity of Ha E were 12.48, 10.45, and 10.36  μ g/mL, respectively, and the IC 50 values of Ad E were 6.51, 5.33, and 6.87  μ g/mL, respectively. All extracts were found to induce G 0 /G 1 cell cycle arrest for HepG2 cells side by side with their inhibition of CDK2 on all three cell lines while all extracts were also showed to induce apoptosis in HepG2 cell line at pre- G 1 phase supplemented by their anticancer activity via proapoptotic protein Bax , caspase-3 , and cleavage PARP increase, and antiapoptotic protein Bcl-2 downturn. Moreover, necrosis has been relatively noticed in HepG2 cell line as an additional anticancer activity for each extract. Our data introduced three ethanolic marine extracts as natural chemotherapeutic agents to be further developed for cancer control., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Hong Cui et al.)

In the search for new antibiotic compounds, fractionation of Pseudomonas protegens UP46 culture extracts afforded several known Pseudomonas compounds, including 2,4-diacetylphloroglucinol (DAPG), as well as two new antibacterial alkaloids, 6-(pyrrolidin-2-yl)DAPG (1) and 6-(piperidin-2-yl)DAPG (2). The structures of 1 and 2 were determined by nuclear magnetic resonance spectroscopy and mass spectrometry. Compounds 1 and 2 were found to have antibacterial activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus, with minimal inhibitory concentration (MIC) 2 and 4 μg ml -1 , respectively, for 1, and 2 μg ml -1 for both pathogens for 2. The MICs for 1 and 2, against all tested Gram-negative bacteria, were >32 μg ml -1 . The half maximal inhibitory concentrations against HepG2 cells for compounds 1 and 2 were 11 and 18 μg ml -1 , respectively, which suggested 1 and 2 be too toxic for further evaluation as possible new antibacterial drugs. Stable isotope labelling experiments showed the pyrrolidinyl group of 1 to originate from ornithine and the piperidinyl group of 2 to originate from lysine. The P. protegens acetyl transferase (PpATase) is involved in the biosynthesis of monoacetylphloroglucinol and DAPG. No optical rotation was detected for 1 or 2, and a possible reason for this was investigated by studying if the PpATase may catalyse a stereo-non-specific introduction of the pyrrolidinyl/piperidinyl group in 1 and 2, but unless the PpATase can be subjected to major conformational changes, the enzyme cannot be involved in this reaction. The PpATase is, however, likely to catalyse the formation of 2,4,6-triacetylphloroglucinol from DAPG.

Aims: Recent findings have instituted the role of hyperglycemia-related AGE/RAGE and NF-κB in instigating reactive oxygen species (ROS) mediated mitochondrial dysfunction and apoptosis of hepatocyte, which leads to steatohepatitis. Naringin, a flavanone glycoside found to possess myriads of pharmacological benefits along with its antioxidant and anti-inflammatory properties. Consequently, we aimed to decipher the effect of naringin on RAGE/NF-κB mediated mitochondrial apoptosis in type 2 diabetes mellitus (T2DM)-induced steatohepatitis., Main Methods: Hepatic HepG2 cells were cultured in palmitic acid medium with and without naringin. Lipid content was examined by Oil Red O and Nile Red staining. Cellular apoptosis was determined by Annexin V-FITC/PI staining. An experimental T2DM-induced steatohepatitis was developed in Sprague Dawley rats by high-fat diet (HFD) for 12 weeks. The naringin was administrated orally at a dose of 100 mg/kg, daily for eight weeks. Glucose and insulin tolerance test was performed. Liver sections were stained by hematoxylin-eosin and picrosirius red. The mRNA and protein expression of RAGE and NF-κB were determined by qPCR, Immunofluorescence, and Immunoblotting. Mitochondrial membrane potential (MMP), cellular and mitochondrial ROS were measured by FACS., Key Findings: Palmitic acid encountered HepG2 cells and HFD fed rats exhibited hyperlipidemia, insulin resistance, abnormal aminotransferases, steatosis, and fibrosis. Besides, the level of AGEs, RAGE, NF-κB, and oxidative stress were exacerbated. Moreover, MMP, cellular and mitochondrial ROS were altered in diabetic rats. Nevertheless, the naringin treatment ameliorated the steatohepatitis by improving the levels of aforementioned parameters., Significance: Collectively, these findings suggested anti-steatohepatitis potential of naringin in diabetics., Competing Interests: Declaration of competing interest All authors declared that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

BACKGROUND Oncolytic viruses (OVs) can specifically infect and kill tumor cells. Adeno-associated virus (AAV) is a widely-studied OV. This study aimed to construct a tumor-targeted recombinant AAV using genetic engineering technology. MATERIAL AND METHODS The transgene plasmid pAAV-HE1B19K-TE1A was constructed with 4 genes (hTERT, E1A, HKII, and E1B19K) and co-transfected with pAAV-RC and pHelper to tumor cells (HepG2, A549, BGC-803) and normal cells (HUVEC). rAAV was verified with fluorescence microscopy. Quantitative PCR (qPCR) assay was used to test the titer of rAAV in each cell line. Apoptosis was analyzed using qPCR and Western blot assay. MTT was used to detect the effect of rAAV on cell viability. RESULTS The pAAV-HE1B19K-TE1A transgene plasmid was successfully structured. pAAV-HE1B19K-TE1A was highly expressed in all tumor cells. The titers of pAAV-HE1B19K-TE1A in HepG2, A549, and BGC-803 were 7.4×10⁷, 1.4×10⁸, and 1.1×10⁸ gc/μl, respectively. pAAV-HE1B19K-TE1A significantly decreased cell viability of tumor cells compared to that in HUVEC (p<0.05). pAAV-HE1B19K-TE1A remarkably triggered cleaved caspase 3 (C-caspase 3) activity in tumor cells compared to that in untransfected tumor cells (p<0.05). pAAV-HE1B19K-TE1A significantly induced release of cytochrome C (Cyto C) in tumor cells compared to that in untransfected tumor cells (p<0.05). pAAV-HE1B19K-TE1A demonstrated no toxicity to vital tissues of animals. CONCLUSIONS Tumor-targeted rAAV was successfully produced using the Helper-free system with recombinant plasmid, demonstrating high efficacy in decreasing viability of tumor cells without adverse effects on normal cells.

Sono-photodynamic therapy (SPDT) activates the same photo-/sonosensitizer and exerts more marked antitumor effects than sonodynamic therapy or photodynamic therapy. We aimed to explore the utilization of curcumin (CUR)-loaded poly(L-lactide-co-glycolide) microbubble (MB)-mediated SPDT (CUR-PLGA-MB-SPDT) in HepG2 liver cancer cells. The cytotoxicity and intracellular accumulation of CUR were determined. We used 40 µM CUR as the photo-/sonosensitizer for 3 h. In a comparison of CUR-SDT or CUR-PDT, HepG2 cell viability decreased and apoptotic rate increased in CUR-SPDT. The CUR-PLGA MBs had round spheres with smooth surfaces and an average size of 3.7 µm. In CUR-PLGA MBs, drug entrapment efficiency and drug-loading capacity were 74.29 ± 2.60% and 17.14 ± 0.60%, respectively. CUR-loaded PLGA MBs (CUR-PLGA MBs) had good biocompatibility with normal L02 cells and were almost non-cytotoxic to HepG2 cells. Among CUR-SDT, CUR-PDT, CUR-SPDT or CUR-PLGA-MB-SDT, the cell CUR-PLGA-MB-SPDT had the lowest viability. Transmission electron microscopy revealed pyroptosis and apoptosis in the CUR-PLGA-MB-SPDT group; the potential mechanism was related to the mitochondrial membrane potential loss and increased production of intracellular reactive oxygen species. These findings suggested that CUR-PLGA-MB-SPDT may be a promising treatment for liver cancer., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Although non‑alcoholic fatty liver disease (NAFLD) is considered a benign disorder, hepatic steatosis has been proposed to be involved in the tumorigenesis of liver cancer. However, the underlying mechanism for carcinogenesis in fatty liver diseases remains unclear. Cancer stem cells (CSCs) have been hypothesized to serve a key role in tumorigenesis. Tumor formation begins with a subset of heterogeneous cells that share properties with stem cells, such as self‑renewal and undifferentiated properties. Our previous study reported that the saturated fatty acid palmitate (PA) significantly enhanced the CSC properties of the HepG2 human liver cancer cell line; however, its underlying mechanisms are unknown. In the present study, a proteomic approach was used to investigate the palmitoylation of proteins in HepG2 CSCs. CSC behavior was induced in HepG2 cells via 200 µM PA. Proteomic analysis was performed to identify post‑transcriptional modifications of proteins in HepG2 CSCs in response to PA treatment. The present study identified proteins modified by palmitoylation in HepG2 CSC spheres formed following PA treatment. It was therefore hypothesized that palmitoylation may be crucial for CSC sphere formation. Furthermore, the present study demonstrated that two palmitoylation inhibitors, tunicamycin (5, 10 and 25 µg/ml) and 2‑bromohexadecanoic acid (25, 50 and 150 µM), significantly decreased CSC sphere formation without affecting cell viability. An association was identified between sphere formation capacity and tumor‑initiating capacity of CSCs. The results of the present study demonstrated that protein palmitoylation may influence the PA‑induced CSC tumor‑initiating capacity, and that the inhibition of palmitoylation may be a suitable chemopreventive strategy for treating patients with NAFLD.

The liver plays a pivotal role in maintaining euglycemia. Biogenesis and function of mitochondria within hepatocytes are often the first to be damaged in a diabetic population, and restoring its function is recently believed to be a promising strategy on inhibiting the progression of diabetes. Previously, we demonstrated that the gut microbiota metabolite butyrate could reduce hyperglycemia and modulate the metabolism of glycogen in both db/db mice and HepG2 cells. To further explore the mechanism of butyrate in controlling energy metabolism, we investigated its influence and underlying mechanism on the biogenesis and function of mitochondria within high insulin-induced hepatocytes in this study. We found that butyrate significantly modulated the expression of 54 genes participating in mitochondrial energy metabolism by a PCR array kit, both the content of mitochondrial DNA and production of ATP were enhanced, expressions of histone deacetylases 3 and 4 were inhibited, beta-oxidation of fatty acids was increased, and oxidative stress damage was ameliorated at the same time. A mechanism study showed that expression of GPR43 and its downstream protein beta-arrestin2 was increased on butyrate administration and that activation of Akt was inhibited, while the AMPK-PGC-1alpha signaling pathway and expression of p-GSK3 were enhanced. In conclusion, we found in the present study that butyrate could significantly promote biogenesis and function of mitochondria under high insulin circumstances, and the GPR43- β -arrestin2-AMPK-PGC1-alpha signaling pathway contributed to these effects. Our present findings will bring new insight on the pivotal role of metabolites from microbiota on maintaining euglycemia in diabetic population., Competing Interests: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Tingting Zhao et al.)

Humanity faces great challenges, such as the rise of bacterial antibiotic resistance and cancer incidence. Thus, the discovery of novel therapeutics from underexplored environments, such as marine habitats, is fundamental. In this study, twelve strains from the phylum Firmicutes and thirty-four strains from the phylum Proteobacteria, isolated from marine sponges of the Erylus genus, collected in Portuguese waters, were tested for bioactivities and the secondary metabolites were characterised. Bioactivity screenings comprised antimicrobial, anti-fungal, anti-parasitic and anti-cancer assays. Selected bioactive extracts were further analysed for already described molecules through high performance liquid chromatography and mass spectrometry. Several bioactivities were observed against the fungus Aspergillusfumigatus, the bacteria (methicillin-resistant Staphylococcus aureus and Escherichia coli), the human liver cancer cell line HepG2 and the parasite Trypanosoma cruzi. Medium scale-up volume extracts confirmed anti-fungal activity by strains Proteus mirabilis #118&#95;13 and Proteus sp. (JX006497) strain #118&#95;20. Anti-parasitic activity was also confirmed in Enterococcus faecalis strain #118&#95;3. Moreover, P. mirabilis #118&#95;13 showed bioactivity in human melanoma cell line A2058 and the human hepatocellular carcinoma cell line HepG2. The dereplication of bioactive extracts showed the existence of a variety of secondary metabolites, with some unidentifiable molecules. This work shows that bacterial communities of sponges are indeed good candidates for drug discovery and, as far as we know, we describe anti-parasitic activity of a strain of E. faecalis and the presence of diketopiperazines in Proteus genus for the first time.

Six new compounds, including three terpenoids ( 1 - 3 ) and three lignans ( 4 - 6 ), were isolated from the 95% EtOH extract of the twigs of Tripterygium hypoglaucum . Their structures were determined on the basis of extensive spectroscopic analysis. 9'- O -benzoyl-lariciresinol ( 4 ) showed weak cytotoxicity against HepG2/Adr cells, with an IC 50 value of 30.1  μ M in vitro .

To discover novel structural compounds which are producted by endophytic fungi, a primary chemical profiling of Camptotheca acuminata Decne derived endophytic fungus Penicillum polonicum had been taken. Two new compounds β-lactone polonicin A ( 1 ) and enoic acid polonicin B ( 2 ) together with seven known compounds 3 - 9 were isolated from Penicillum polonicum obtained from C. acuminata . The structures of the new compounds 1 and 2 were identified by modern spectrum technology including detailed 1D, 2D NMR and MS data analyses. When tested against HepG2 hepatocellular carcinoma (HCC) cell lines, compounds 4 - 8 showed moderate anti-HCC activity. In addition, compound 1 - 3 have effects on increasing GLUT4 translocation and glucose uptake in vitro . Compound 1 showed the strongest glucose uptake and GLUT4 translocation activities in rat skeleton (L6) myoblast cell line with enhancements of 1.8 and 2.1 folds respectively compared to the control.

Nonalcoholic fatty liver disease (NAFLD) has become one of the most common medical problems. Inhibition of lipogenesis and promotion of lipolysis are two ways to prevent NAFLD. In this study, oleic acid-induced HepG2 cells are used as a NAFLD cell model to test whether s-petasin exerts inhibition of lipogenesis and promotion of the lipolysis effect. The results showed that s-petasin significantly inhibited the lipid level in oleic acid-induced HepG2 cells. Moreover, results showed that the triacylglycerol level was reduced by s-petasin in oleic acid-induced HepG2 cells. Western blot assay revealed that s-petasin stimulated phosphorylation of AMPKα and ACCα. The results also demonstrated that s-petasin can inhibit lipogenesis and enhance triacylglycerol turnover by down-regulation of FAS and SCD-1 and up-regulation of ATGL and HSL through the AMPK signaling-dependent regulation of transcriptional factors, FKHR and SREBP-1. This in vitro study indicates that s-petasin has potential as a candidate compound for NAFLD therapy.

Zearalenone, produced by various Fusarium species, is a non-steroidal estrogenic mycotoxin that contaminates cereals, resulting in adverse effects on human health. We investigated the effects of zearalenone and its metabolite alpha zearalenol on epigenetic modifications and its relationship with metabolic pathways in human hepatocellular carcinoma cells following 24 h of exposure. Zearalenone and alpha zearalenol at the concentrations of 1, 10 and 50 μM significantly increased global levels of DNA methylation and global histone modifications (H3K27me3, H3K9me3, H3K9ac). Expression levels of the chromatin modifying enzymes EHMT2, ESCO1, HAT1, KAT2B, PRMT6 and SETD8 were upregulated by 50 μM of zearalenone exposure using PCR arrays, consistent with the results of global histone modifications. Zearalenone and alpha zearalenol also changed expression levels of the AhR, LXRα, PPARα, PPARɣ, L-fabp, LDLR, Glut2, Akt1 and HK2 genes, which are related to nuclear receptors and metabolic pathways. PPARɣ, a key regulator of lipid metabolism, was selected from among these genes for further analysis. The PPARɣ promoter reduced methylation significantly following zearalenone exposure. Taken together, the epigenetic mechanisms of DNA methylation and histone modifications may be key mechanisms in zearalenone toxicity. Furthermore, effects of zearalenone in metabolic pathways could be mediated by epigenetic modifications., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A new pentaketide derivative, penilactonol A ( 1 ), and two new hydroxyphenylacetic acid derivatives, (2'R)-stachyline B (2) and (2'R)-westerdijkin A (3), together with five known metabolites, bisabolane-type sesquiterpenoids 4-6 and meroterpenoids 7 and 8, were isolated from the solid culture of a marine alga-associated fungus Penicillium chrysogenum LD-201810. Their structures were elucidated based on extensive spectroscopic analyses, including 1D/2D NMR and high resolution electrospray ionization mass spectra (HRESIMS). The absolute configurations of the stereogenic carbons in 1 were determined by the (Mo 2 (OAc) 4 )-induced circular dichroism (CD) and comparison of the calculated and experimental electronic circular dichroism (ECD) spectra, while the absolute configuration of the stereogenic carbon in 2 was established using single-crystal X-ray diffraction analysis. Compounds 2 and 3 adapt the 2'R-configuration as compared to known hydroxyphenylacetic acid-derived and O-prenylated natural products. The cytotoxicity of 1 -8 against human carcinoma cell lines (A549, BT-549, HeLa, HepG2, MCF-7, and THP-1) was evaluated. Compound 3 exhibited cytotoxicity to the HepG2 cell line with an IC 50 value of 22.0 μM. Furthermore, 5 showed considerable activities against A549 and THP-1 cell lines with IC 50 values of 21.2 and 18.2 μM, respectively., Competing Interests: The authors declare no conflict of interest.

Twelve new sesterterpenes along with eight known sesterterpenes were isolated from the marine sponge Hyrtios erectus collected off the coast of Chuuk Island, the Federated State of Micronesia. Based upon a combination of spectroscopic and computational analyses, these compounds were determined to be eight glycine-bearing scalaranes ( 1 - 8 ), a 3-keto scalarane ( 9 ), two oxidized-furan-bearing scalaranes ( 10 and 11 ), and a salmahyrtisane ( 12 ). Several of these compounds exhibited weak antiproliferation against diverse cancer cell lines as well as moderate anti-angiogenesis activities. The antiproliferative activity of new compound 4 was found to be associated with G0/G1 arrest in the cell cycle., Competing Interests: The authors declare no conflict of interest.

Polygonatum sibiricum is a medicinal and homologous plant grown in China, which is commercially available without the provision of medical prescription. Saponin is one of the biologically active components of Polygonatum sibiricum. This study aimed to extract saponin from Polygonatum sibiricum (PSS) and to investigate its hypoglycemic effect and effect on gut micorbiota in diabetic mice. Through single factor and orthogonal experiments, the extraction conditions of saponin were optimized, and the content of saponin in the extract was 3.07 ± 0.02 mg g -1 . During the experiment evaluating the hypoglycemic ability, it was found that PSS could inhibit the activity of α-amylase and α-glucosidase at first. Then, HepG2 was used to observe the effect of the saponin on insulin resistance. The results showed that PSS could significantly improve the state of insulin resistance in IR-HepG2 cells, increase the glucose consumption and intracellular glycogen content of cells, and the activity of hexokinase (0.455 ± 0.007 μmol min -1 g -1 ) and pyruvate kinase (0.174 ± 0.038 U per g prot). Finally, it was found that PSS could alleviate the symptoms of polyphagia and polydipsia in diabetic mice, regulate the gut microbiota of diabetic mice, increase the number of probiotics, and reduce the number of harmful bacteria. In summary, PSS may play an important role in regulating diabetes and can be developed as a promising natural material for diabetes prevention and treatment.

The fresh leaves of Metapanax delavayi (Araliaceae) have been used as a common wild vegetable for salad and soup, and also herbal tea by the local people living in its growing areas of Yunnan province, China. Detailed chemical investigation led to the identification of a new triterpenoid saponin, 3- O - α -L-arabinopyranosyl-28- O - β -D-glucopyranosyl-(1→6)- O - β -D-glucopyranosyl-3 β -hydroxyolean-12-ene-28,29-dioic acid ( 1 ) from the fresh leaves, together with 11 known compounds, including six triterpenoid saponins ( 2 - 7 ), two caffeoylquinic acid derivatives ( 8 - 9 ), and three flavonoid glycosides ( 10 - 12 ). Their structures were determined on the basis of spectroscopic analysis and acidic hydrolysis. Compounds 3 - 5 and 8 - 12 were isolated from M. delavayi for the first time. Moreover, the known saponins 3- O - β -D-xylopyranosyl-3 β -hydroxyolean-12-ene-28,29-dioic acid ( 3 ) and yiyeliangwanoside IV ( 5 ) exhibited protective effects on HepG2 cells damaged by the alcohol intakes, at a concentration of 1.0 µg/mL. The results indicated M. delavayi is an ideal dietary vegetable and herbal tea with potential hepatoprotective activity.[Formula: see text].

The expression of drug-metabolizing enzymes is deeply involved in chemical-induced cancer progression and prevention. The aryl hydrocarbon receptor (AhR) induces phase I, and certain phase II drug-metabolizing enzymes after the binding of ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We have previously demonstrated that luteolin inhibited TCDD-induced AhR transformation, and modulated the expression of drug-metabolizing enzymes through not only the AhR, but also the nuclear factor-erythroid-2-related factor 2 (Nrf2). We have examined the effect of kaempferol on the expression of drug-metabolizing enzymes through modulation of the AhR- and Nrf2-pathways, and the effect of co-treatment with kaempferol and luteolin. Kaempferol dose-dependently inhibited not only the TCDD-induced expression of phase I and phase II drug-metabolizing enzymes, but also the tertiary butylhydroquinone (t-BHQ)-induced expression of phase II drug-metabolizing enzymes, by modulating the AhR- and Nrf2-pathways. Co-treatment with kaempferol and luteolin enhanced the inhibitory effect on the expression of drug-metabolizing enzymes, compared with either kaempferol or luteolin alone. Moreover, co-treatment with kaempferol and luteolin increased the cellular levels of kaempferol without affecting the levels of luteolin. An in vivo study was also performed and the results demonstrated that co-treatment with kaempferol and luteolin enhanced the inhibition of benzo[a]pyrene-induced drug-metabolizing enzymes compared with either kaempferol or luteolin alone, in the liver of ICR mice. These results suggest that luteolin promoted the incorporation of kaempferol into hepatocytes and enhanced the inhibitory effect of kaempferol on chemical-induced drug-metabolizing enzymes. Thus, luteolin enhances the kaempferol-inhibited expression of drug-metabolizing enzymes.

Previous studies have shown that crude polysaccharides from the Lycium barbarum fruit could inhibit cancer cell growth, but the major effective constituents are yet to be identified. In this study, we compared the effects of L. barbarum fruit polysaccharide fractions on the growth of hepatoma cells (SMMC-7721 and HepG2), cervical cancer cells (HeLa), gastric carcinoma cells (SGC-7901), and human breast cancer cells (MCF-7). LBGP-I-3 showed stronger inhibitory effects on MCF-7 cells (cell viability of 48.96%) than SMMC-7721 (cell viability of 78.91%) and HeLa cells (cell viability of 55.94%), and had no effect on HepG2 and SGC-7901 cells. In addition, LBGP-I-3 had no inhibitory effect on normal liver cells (L02, cell viability of 115.58%). Investigation of the underlying mechanism suggested that LBGP-I-3 inhibited the growth of cancer cells by cell cycle arrest and apoptosis. LBGP-I-3 arrested the cell cycle at the G0/G1 phase, altered mitochondrial function, activated oxidative stress, and regulated the MAPK signaling pathway to induce apoptosis. Thus, LBGP-I-3 may be a potential functional food ingredient for the prevention of cancer without toxicity to normal cells in vitro. These results could help further elucidate the structure-activity relationship of L. barbarum fruit polysaccharides and functional food development., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Phytochemical investigation on the leaves and twigs of Toona ciliata has led to the isolation of four new polyynes ( 1 - 4 ) and two knowns ( 5 and 6 ). Their structures were determined by extensive spectroscopic analysis (MS, UV, IR, and NMR) and Mosher's method. All compounds were evaluated for their inhibitory activities against HepG2 human tumor cell line but were inactive.

Severely burned patients suffer from a hypermetabolic syndrome that can last for years after the injury has resolved. The underlying cause of these metabolic alterations most likely involves the persistent elevated catecholamine levels that follow the surge induced by thermal injury. At the cellular level, endoplasmic reticulum (ER) stress in metabolic tissues is a hallmark observed in patients following burn injury and is associated with several detrimental effects. Therefore, ER stress could be the underlying cellular mechanism of persistent hypermetabolism in burned patients. Here, we show that catecholamines induce ER stress and that adreno-receptor blockers reduce stress responses in the HepG2 hepatocyte cell line. Our results also indicate that norepinephrine (NE) significantly induces ER stress in HepG2 cells and 3T3L1 mouse adipocytes. Furthermore, we demonstrate that the alpha-1 blocker, prazosin, and beta blocker, propranolol, block ER stress induced by NE. We also show that the effects of catecholamines in inducing ER stress are cell type-specific, as NE treatment failed to evoke ER stress in human fibroblasts. Thus, these findings reveal the mechanisms used by catecholamines to alter metabolism and suggest inhibition of the receptors utilized by these agents should be further explored as a potential target for the treatment of ER stress-mediated disease.

A number of liver diseases are known to be caused by oxidative stress. Petroselinum sativum (P. sativum; parsley) is popular for its anti-inflammatory, antimicrobial, anticancer, antioxidant and antidiabetic activities. However, till date the hepatoprotective potential of chloroform extract of P. sativum (PSA) on hydrogen peroxide (H 2 O 2 ) induced cytotoxicity and oxidative stress in human liver (HepG2) cells have not been studied. Therefore, this study was framed to evaluate whether the levels of hydrogen peroxide (H 2 O 2 ) induced cytotoxicity and oxidative stress in HepG2 cells could be diminished by pretreating the cells with PSA. MTT assay, NRU assay, morphological alterations, glutathione (GSH) depletion, lipid peroxidation (LPO), ROS generation and loss of mitochondrial membrane potential (MMP) were assessed by using non-cytotoxic concentrations (5, 10 and 25 μg/mL) of PSA against H 2 O 2 (0.25 mM) induced damage in HepG2 cells. The results demonstrated that pretreatment of HepG2 cells with PSA offered protective properties by lowering the LPO and ROS generation and elevating the cell viability, GSH and MMP levels. Together, these results suggest that PSA has the hepatoprotective effect on H 2 O 2 induced cell death in HepG2 cells.