Hyperglycemia induced cathepsin L maturation linked to diabetic comorbidities and COVID-19 mortality.

Diabetes, a prevalent chronic condition, significantly increases the risk of mortality from COVID-19, yet the underlying mechanisms remain elusive. Emerging evidence implicates Cathepsin L (CTSL) in diabetic complications, including nephropathy and retinopathy. Our previous research identified CTSL as a pivotal protease promoting SARS-CoV-2 infection. Here, we demonstrate elevated blood CTSL levels in individuals with diabetes, facilitating SARS-CoV-2 infection. Chronic hyperglycemia correlates positively with CTSL concentration and activity in diabetic patients, while acute hyperglycemia augments CTSL activity in healthy individuals. In vitro studies reveal high glucose, but not insulin, promotes SARS-CoV-2 infection in wild-type cells, with CTSL knockout cells displaying reduced susceptibility. Utilizing lung tissue samples from diabetic and non-diabetic patients, alongside Leprdb/dbmice and Leprdb/+mice, we illustrate increased CTSL activity in both humans and mice under diabetic conditions. Mechanistically, high glucose levels promote CTSL maturation and translocation from the endoplasmic reticulum (ER) to the lysosome via the ER-Golgi-lysosome axis. Our findings underscore the pivotal role of hyperglycemia-induced CTSL maturation in diabetic comorbidities and complications.

People with diabetes are at greater risk of developing severe COVID-19 and dying from the illness, which is caused by a virus known as SARS-CoV-2. The high blood sugar levels associated with diabetes appear to be a contributing factor to this heightened risk. However, diabetes is a complex condition encompassing a range of metabolic disorders, and it is therefore likely that other factors may contribute. Previous research identified a link between an enzyme called cathepsin L and more severe COVID-19 in people with diabetes. Elevated cathepsin L levels are known to contribute to diabetes complications, such as kidney damage and vision loss. It has also been shown that cathepsin L helps SARS-CoV-2 to enter and infect cells. This raised the question of whether elevated cathepsin L is responsible for the increased COVID-19 vulnerability in patients with diabetes. To investigate, He, Zhao et al. monitored disease severity and cathepsin L levels in patients with COVID-19. This confirmed that people with diabetes had more severe COVID-19 and that higher levels of cathepsin L are linked to more severe disease. Analysis also revealed that cathepsin L activity increases as blood glucose levels increase. In laboratory experiments, cells exposed to glucose or fluid from the blood of people with diabetes were more easily infected with SARS-CoV-2, with cells genetically modified to lack cathepsin L being more resistant to infection. Further experiments revealed this was due to glucose promoting maturation and migration of cathepsin L in the cells. The findings of He, Zhao et al. help to explain why people with diabetes are more likely to develop severe or fatal COVID-19. Therefore, controlling blood glucose levels in people with diabetes may help to prevent or reduce the severity of the disease. Additionally, therapies targeting cathepsin L could also potentially help to treat COVID-19, especially in patients with diabetes, although more research is needed to develop and test these treatments.

Molecularly generated rat hepatitis E virus strains from human and rat show efficient replication in a human hepatoma cell line.

The hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. Whereas HEV genotypes 1-4 of species Paslahepevirus balayani are commonly found in humans, infections with ratHEV (species Rocahepevirus ratti) were previously considered to be restricted to rats. However, several cases of human ratHEV infections have been described recently. To investigate the zoonotic potential of this virus, a genomic clone was constructed here based on sequence data of ratHEV strain pt2, originally identified in a human patient with acute hepatitis from Hongkong. For comparison, genomic clones of ratHEV strain R63 from a rat and of HEV genotype 3 strain 47832mc from a human patient were used. After transfection of in vitro-transcribed RNA from the genomic clones into the human hepatoma cell line HuH-7-Lunet BLR, virus replication was shown for all strains by increasing genome copy numbers in cell culture supernatants. These cells developed persistent virus infections, and virus particles in the culture supernatant as well as viral antigen within the cells were demonstrated. All three generated virus strains successfully infected fresh HuH-7-Lunet BLR cells. In contrast, the human hepatoma cell lines HuH-7 and PLC/PRF/5 could only be infected with the genotype 3 strain and to a lesser extent with ratHEV strain R63. Infection of the rat-derived hepatoma cell lines clone 9, MH1C1 and H-4-II-E did not result in efficient virus replication for either strain. The results indicate that ratHEV strains from rats and humans can infect human hepatoma cells. The replication efficiency is strongly dependent on the cell line and virus strain. The investigated rat hepatoma cell lines could not be infected and other rat-derived cells should be tested in future to identify permissive cell lines from rats. The developed genomic clone can represent a useful tool for future research investigating pathogenicity and zoonotic potential of ratHEV.

Shell engineering in soft alginate-based capsules for culturing liver spheroids.

Functional interaction between cancer cells and the surrounding microenvironment is still not sufficiently understood, which motivates the tremendous interest for the development of numerous in vitro tumor models. Diverse parameters, for example, transport of nutrients and metabolites, availability of space in the confinement, etc. make an impact on the size, shape, and metabolism of the tumoroids. We demonstrate the fluidics-based low-cost methodology to reproducibly generate the alginate and alginate-chitosan microcapsules and apply it to grow human hepatoma (HepG2) spheroids of different dimensions and geometries. Focusing specifically on the composition and thickness of the hydrogel shell, permeability of the microcapsules was selectively tuned. The diffusion of the selected benchmark molecules through the shell has been systematically investigated using both, experiments and simulations, which is essential to ensure efficient mass transfer and/or filtering of the biochemical species. Metabolic activity of spheroids in microcapsules was confirmed by tracking the turnover of testosterone to androstenedione with chromatography studies in a metabolic assay. Depending on available space, phenotypically different 3D cell assemblies have been observed inside the capsules, varying in the tightness of cell aggregations and their shapes. Conclusively, we believe that our system with the facile tuning of the shell thickness and permeability, represents a promising platform for studying the formation of cancer spheroids and their functional interaction with the surrounding microenvironment.

The 2,3-epoxy naphthoquinol produced by endophyte Arthrinium marii M-211.

A novel 2,3-epoxy naphthoquinol, named (6R,7R,8R)-theissenone A (1), possessing an oxatricyclo[5.4.0.03,5]undeca-trien-2-one skeleton, together with two known compounds, (6S,7R,8R)-theissenone (2) and arthrinone (3), were produced by an endophytic fungus, Arthrinium marii M-211, which was isolated from mangrove plants. The structure of 1, including the absolute stereochemistry, was elucidated by analysis of nuclear magnetic resonance (NMR) and mass spectrometry (MS) data and time-dependent density functional theory (TDDFT) calculations of electronic circular dichroism (ECD) spectra. Additionally, the absolute structure of 2 was deduced as a diastereomer of 1 using ECD spectral data analysis. Compounds 1, 2 and 3 exhibited cytotoxic activity against the H4IIE rat hepatoma cells, with IC50 values of 67.5, 46.6 and 13.4 µM, respectively.

Possible Glutathione Peroxidase 4-Independent Reduction of Phosphatidylcholine Hydroperoxide: Its Relevance to Ferroptosis.

Ferroptosis is mainly caused by iron-mediated peroxidation of phospholipids and has recently attracted attention due to its involvement in various diseases. At the center of it is supposedly the inability of glutathione peroxidase 4 (GPX4) to reduce excess peroxidized phospholipids (e.g., phosphatidylcholine hydroperoxide (PCOOH)) that trigger ferroptosis. However, the involvement of enzymes other than GPX4 in ferroptosis is scarcely known. To elucidate this matter, we evaluated the uptake of PCOOH in a GPX4 knockout (KO) human hepatoma cell line HepG2 generated using CRISPR-Cas9. After confirming that GPX4 expression in the KO cells was below the detection limit, we cultured both wild-type (WT) and GPX4 KO HepG2 cells in a medium containing 50 µM PCOOH for 1-8 hours. By analyzing the level of PCOOH and its reduction product (phosphatidylcholine hydroxide, PCOH) in cells using liquid chromatography-tandem mass spectrometry, we detected the cellular uptake of PCOOH. On top of this, we detected a large amount of PCOH not only in WT HepG2 but also in GPX4 KO HepG2, thus indicating the notable involvement of enzymes other than GPX4 (e.g., other GPX family, glutathione S-transferase, thioredoxin, or peroxiredoxin) in reducing PCOOH. Further corroboration of these findings hopefully leads to the development of novel methods to prevent ferroptosis-related diseases by targeting enzymes other than GPX4.

Microfluidic Chip as a Tool for Effective In Vitro Evaluation of Cyclophosphamide Prodrug Toxicity.

Most drugs are metabolized in the liver, which can lead to their activation or inactivation with a change in the parent compound pharmacology, as well as liver damage by active metabolites. Preclinical animal studies of drug safety do not always predict its effect on humans due to species specificity. Thus, for the rapid drug screening, and especially prodrugs, an in vitro system is required that allows predicting xenobiotic cytotoxicity with consideration of their metabolism in liver cells. The use of a microfluidic chip (BioClinicum) made it possible to cultivate a 2D culture of human HaCaT keratinocytes with spheroids of human hepatoma HepaRG cells. After incubation in a specially selected universal serum-free medium containing 3.8 mM cyclophosphamide, pronounced death of HaCaT cells was observed in comparison with culturing in the absence of liver cells.

Triterpene saponins from the roots of Acacia senegal (L.) Willd.

Six new triterpenoid saponins, named senegalosides A-F (1-6) were isolated from the seedpods and roots of Acacia senegal (Mimosaceae). Their structures were elucidated using 1D and 2D-NMR spectroscopic analysis and mass spectrometry. Compound 1 possesses an unusual sapogenin, 3ß-hydroxy-21-oxo-olean-12-en-28-oic acid (machaeric acid), and was reported here in its natural form for the first time within the genus Acacia. Senegaloside A is the first example of a machaeric-type triterpenoid glycoside in the plant kingdom. The cytotoxic effect of isolated saponins was evaluated on the H4IIE rat hepatoma cell line. As a result, compounds 1, 3-6 were not significantly cytotoxic to H4IIE cells even at 200 µM. Compound 2 was suppressed cell viability at 50-200 µM.

Effect of melittin on programmed necrosis of human hepatocellular carcinoma cell line SMMC￣7721 / 解剖学报

Objective To investigate the lethal effect of melittin on human hepatocellular carcinoma cell line SMMC-7721 and its possible mechanism. Methods MTT assay was used to investigate the killing effect of different concentrations melittin on human hepatocellular carcinoma cells SMMC-7721. Meanwhile, the inhibitory effect of specific programmed necrosis inhibitors necrostain-1(Nec-1) on melittin killing SMMC-7721 cells was detected. Programmed cell necrosis observed by Hoechst 33342 and PI double staining. The necrotosis rate was detected by flow cytometry. Ultrastructural changes of cell was detected by transmission electron microscopy. The expression of receptor interacting protein 1(RIP1) was detected by Western blotting. Results Compared with the control group, the proliferation activity of SMMC-7721 cells was significantly decreased after treatment with different concentrations of melittin for 24 hours (P < 0. 05) . Cells stained in dark blue and red. Cell membrane integrity was destroyed, organelle swelling, organelle membrane was destroy, that demonstrates cell was necrosis. Westen blotting result showed an increased proportion of RIP1 expression in SMMC-7721 cells. Compared with the melittin group, cell proliferation activity was significantly increased, cell necrotosis rate was decreased, and intracellular RIP1 expression was down-regulated in the Nec-1 pretreatment group. Conclusion Melittin induces cell death in SMMC-7721 cells through the RIP1-mediated programmed necrosis pathway.

A bioartificial liver device based on three-dimensional culture of genetically engineered hepatoma cells using hollow fibers.

The bioartificial liver (BAL) device is an extracorporeal liver support system incorporating living hepatocytes. A major problem in BAL device development is to obtain a high number of functional cells. In this study, we focused on a genetically engineered mouse hepatoma cell line, Hepa/8F5, in which elevated liver functions are induced via overexpression of liver-enriched transcription factors activated by doxycycline (Dox) addition. We applied a three-dimensional culture technique using hollow fibers (HFs) to Hepa/8F5 cells. Hepa/8F5 cells responded to Dox addition by reducing their proliferative activity and performing liver-specific functions of ammonia removal and albumin secretion. The functional activities of cells depended on the timing of Dox addition. We also found that Hepa/8F5 cells in the HF culture were highly functional in a low rather than high cell density environment. We further fabricated an HF-type bioreactor with immobilized Hepa/8F5 cells as a BAL device. Although ammonia removal activity of this BAL device was lower than that of the small-scale HF bundle, albumin secretion activity was slightly higher. These results indicated that the BAL device with immobilized Hepa/8F5 cells was highly functional with potential to show curative effects in liver failure treatment.

Tumor-specific inhibitory action of decorin on different hepatoma cell lines.

BACKGROUND: In spite of therapeutic approaches, liver cancer is still one of the deadliest type of tumor in which tumor microenvironment may play an active role in the outcome of the disease. Decorin, a small leucine-rich proteoglycan is not only responsible for assembly and maintenance of the integrity of the extracellular matrix, but a natural inhibitor of cell surface receptors, thus it exerts antitumorigenic effects. Here we addressed the question whether this effect of decorin is independent of the tumor phenotypes including differentiation, proliferation and invasion. METHOD: Four hepatoma cell lines HepG2, Hep3B, HuH7 and HLE, possessing different molecular backgrounds, were selected to investigate. After proliferation tests, pRTK arrays, WB analyses, and immunofluorescent examinations were performed on decorin treated and control cells for comparison. RESULTS: Significant growth inhibitory potential of decorin on three out of four hepatoma cell lines was proven, however the mode of its action was different. Induction of p21WAF1/CIP1, increased inactivation of c-myc and ß-catenin, and decrease of EGFR, GSK3ß and ERK1/2 phosphorylation levels were observed in HepG2 cells, pathways already well-described in literature. However, in the p53 deficient Hep3B and HuH7, InsR and IGF-1R were the main receptors transmitting signals. In harmony with its receptor status, Hep3B cells displayed high level of activated AKT. As the cell line is retinoblastoma mutant, ATR/Chk1/Wee1 system might hinder the cell cycle in G2/M phase via phosphorylation of CDK1. In Huh7 cells, all RTKs were inhibited by decorin followed by downregulation of AKT. Furthermore, HuH7 cell line responded with concentration-dependent ERK activation and increased phospho-c-myc level. Decorin had only a non-significant effect on the proliferation rate of HLE cell line. However, it responded with a significant decrease of pAKT, c-myc and ß-catenin activity. In this special cell line, the inhibition of TGFß may be the first step of the protective effect of decorin. CONCLUSIONS: Based on our results decorin may be a candidate therapeutic agent in the battle against liver cancer, but several questions need to be answered. It is certain that decorin is capable to exert its suppressor effect in hepatoma cells without respect to their phenotype and molecular background.

Killing Effects of IFN R-/- Mouse NK Cells Activated by HN Protein of NDV on Mouse Hepatoma Cells and Possible Mechanism with Syk and NF-κB.

The objective of this article is to evaluate whether the tumoricidal activity of mouse IFN R-/- nature killer (NK) cells is induced by Newcastle disease virus hemagglutinin-neuraminidase (NDV-HN) stimulation, and to investigate what is the mechanism of the HN-stimulated NK cells to kill mouse hepatoma cell line in vitro. The mouse IFN R-/- NK cells were stimulated for 16 hr with 500 ng/mL NDV-HN in 1640 medium. Quantify the cytotoxic activities of NK cells against mouse hepatoma cells (Hepa1-6) by flow cytometry. Granzymes B (GrB) and Fas/FasL concentrations in the supernatants of IFN R-/- NK cells medium were determined by specific ELISA assay. The expression of cell surface GrB and Fas was determined by Western blot. NDV-HN stimulation enhanced tumoricidal activity of IFN R-/- NK cells toward Hepa1-6 in vitro. Treating with anti-HN neutralizing mAb induced significant decline in the cytotoxicity of IFN R-/- NK cells toward Hepa1-6 cell line (P < 0.05). After treating with anti-HN protein (1 µL/mL), Syk-specific inhibitor Herbimycin A(250 ng/mL) and NF-κB inhibitor PDTC (500 ng/mL) downregulated the tumoricidal activity of HN-stimulated IFN R-/- NK cells (P < 0.05). Moreover, significant suppressions in the production of GrB and Fas/FasL were observed in HN-stimulated IFN R-/- NK cells (P < 0.05). Thus, we concluded that killer activation receptors pathway is involved in the IFN-γ-independent GrB and Fas/FasL expression of NDV-HN-stimulated IFN R-/- NK cells, and these are activated by Syk and NF-κB. Anat Rec, 302:1718-1725, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association for Anatomy.

The effects of coadministration of tilorone dihydrochloride and culture supernatants from Lactobacillus reuteri on the mouse hepatoma cell line.

CONTEXT: Tilorone dihydrochloride is a therapeutic agent with a different mechanism in cancer. The species of Lactobacillus have an important role in cytotoxic effect. AIMS: Because of unknown effects of tilorone and culture supernatants from Lactobacillus reuteri on hepatoma, the aim of this study is to evaluate apoptotic, cytotoxic, and therapeutic effects of tilorone on mouse hepatoma cell line with and without culture supernatants from L. reuteri. MATERIALS AND METHODS: To do so, after cell line culture, cells were divided into different groups such as negative control, treatment with four doses of tilorone, positive control of supernatant (single dose), and combination therapy groups of different doses of tilorone with supernatant (constant doses), for 48 h. All groups were studied with pathologic tests, biochemical study, tetrazolium dye (3-(4, 5- dimethylthiazol -2-yl)-2, 5-diphenyltetrazolium bromide [MTT]) assay, and absolute real-time-polymerase chain reaction (RT-PCR) were done to assess Bax and Bcl-2 genes expression, as molecular studies. RESULTS: MTT assay results revealed that the tilorone tissue culture IC50 (TCIC50) on the Hepa1-6 cell line was 50 µg/ml. RT-PCR analysis showed that tilorone dihydrochloride induced upregulation and downregulation in expression of Bax and Bcl-2, respectively. Simultaneous, antioxidant effect has also seen in a way that prevented necrosis, in biochemical analysis. These results were dose dependent and statistically significant compared to the control group. CONCLUSIONS: Based on these results, it appeared that this agent could be a good candidate for further evaluation as effective chemotherapy acting through the induction of apoptosis in hepatoma. The cell death caused through bacterial supernatant was rather necrosis than apoptosis.

In Vitro and In Vivo Protective Effects of Flavonoid-Enriched Lotus Seedpod Extract on Lipopolysaccharide-Induced Hepatic Inflammation.

Endotoxin lipopolysaccharide (LPS) plays an important role in the acceleration of hepatic inflammation. Natural medicinal plants that can prevent inflammation by targeting LPS have potential therapeutic clinical application. The aim of the study is to examine the anti-inflammatory effects of lotus seedpod extract (LSE), used as a traditional Chinese herbal medicine with hemostasis function and for eliminating bruise, on the LPS-induced hepatic inflammation and its underlying molecular mechanisms in vitro and in vivo. In vitro, LSE and its purified compound (-)-epigallocatechin (EGC) dose-dependently inhibited the expressions of pro-inflammatory cytokines and mediators, including tumor necrosis factor (TNF)- α , interleukin (IL)-6, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), without affecting cell viability in LPS-stimulated human hepatoma cell line HepG2. Molecular studies showed the anti-LPS effect of HLP or EGC might be mediated via downregulation of Toll-like receptor 4. (TLR4)-mediated both NF- κ B and p38 signaling, as demonstrated by the usage of pyrrolidine dithiocarbamate (PDTC), a specific NF- κ B inhibitor. In vivo, LPS-induced hepatic inflammation was significantly ameliorated in LSE-fed mice as gauged by dose-dependent inhibition of serum levels of biochemical markers of liver damage, the changes of hepatic lobular architecture and the secretion of pro-inflammatory mediators, as well as induction of anti-oxidant enzymes. As a result, our data presented the first evidence of EGC-enriched LSE as an anti-inflammatory agent in LPS-stimulated HepG2 cells and mice, and these findings may open interesting perspectives to the strategy in treatment for hepatic inflammation.

miR-375 affects the proliferation and invasion of hepatoma cells via targetingYAP / 中国肿瘤生物治疗杂志

@# Objective: To investigate the mechanisms of miR-375 affecting the proliferation and invasion of hepatoma cells via targeting YAP (Yes-associated protein). Methods: The cancerous tissues and corresponding para-cancerous tissues of 70 patients with hepatocellular carcinoma (HCC) who underwent surgical resection at the Second Affiliated Hospital of Henan University of Traditional Chinese Medicine from January 2015 to December 2016, as well as the hepatoma cell lines SMMC-7721, Hb611, HepG2 and BEL-7405 were collected for this study. qPCR method was used to detect the expression level of miR-375 in collected HCC tissues and different hepatoma cell lines; Dual luciferase reporter gene assay was used to verify the interaction between miR-375 and YAP; The relationship between miR-375 and clinicopathological features of HCC patients was also analyzed; MTT assay was used to detect the effect of miR375 on the proliferation of hepatoma cells; Transwell invasion assay was used to detect the invasive ability of hepatoma cells after inhibiting the expression of miR-375; Western blotting was used to detect the expression of YAP in HepG2 cells. The nude mouse model of subcutaneously transplanted xenograft was established, and the tumor volume and mass of transplanted hepatoma cells were detected after inhibiting the expression of miR-375. The expression of YAP in xenograft of nude mice was detected by immunohistochemistry and Western blotting. Results: The expression of miR-375 and YAP in HCC tissues was significantly higher than that in para-cancerous tissues (all P<0.05). The expression of miR-375 in HepG2 cells was the highest (P<0.05). miR-375 could specifically bind to the 3' UTR of YAP and regulate the expression activity of YAP. After inhibiting the expression of miR-375, the proliferation and invasion abilities of HepG2 cells were reduced (all P<0.05); The tumor volume and mass of transplanted xenografts were significantly reduced (both P<0.05); The expression of YAP protein in the transplanted xenografts was down-regulated (P<0.05). Conclusion: miR-375 plays an important role in the occurrence and development of liver cancer, and can influence the malignant biological behaviors of hepatoma cells by targeting and regulating the expression ofYAP.

Study on Synthesis and Anti-tumor Activity of 6 Kinds of Tectorigenin Mannich Base Derivatives / 中国药房

OBJECTIVE： To conduct structural modification of tectorigenin to search for new compounds with anti-tumor activity. METHODS： Tectorigenin was used as a lead compound， and then added into amine reagents as ethanolamine， methylamine， ethylamine， dimethylamine， diethylamine， n-propylamine and formaldehyde solution. Tectorigenin Mannich base derivatives were synthesized by mannich reaction with as the lead compound. The structures of the derivatives were identified according to IR， UV， MS and NMR data. Solubility of tectorigenin and its derivatives were investigated by solubility test method. MTT assay was used to investigate the inhibitory effects of tectorigenin and its derivatives on the proliferation of human colon cancer cell line HCT116， human lung cancer cell line A549 and human hepatoma cell line HepG2， and half inhibitory concentration （IC50） was calculated. The inhibition rate of tectorigenin and its derivatives （100 mg/kg） on H22 hepatoma-bearing mice in vivo was studied. RESULTS： Totally of 6 kinds of tectorigenin mannich base derivatives were synthesized， such as 8-（N-hydroxyethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-methyl）-methyleneamino-5，7，4′-trihydroxy-6- methoxyisoflavone， 8-（N， N-diethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N， N-dimethyl）-methyleneamino- 5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-ethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-propyl）- methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone （compounds 1-6 in turn）. Compared with tectorigenin， the water solubility of six derivatives was significantly improved， and the solubility was 5-20 times higher than that of tectorigenin. IC50 of compounds 1， 3 and 5 to HCT116 cells were （34.82±3.27）， （16.21±4.13）， （33.12±3.25） μmol/L， which were stronger than that of tectorigenin [（45.23±5.74） μmol/L]； IC50 of compounds 1， 3 and 5 to A549 cells were （37.05±5.74）， （26.88±4.52）， （30.13±6.23） μmol/L， which were stronger than that of tectorigenin [（53.24±6.34） μmol/L]； IC50 of compounds 1， 3 and 5 to HepG2 cells were （23.74±1.45）， （18.96±2.34）， （30.95±2.87） μmol/L， which were stronger than that of tectorigenin [（48.98±2.58） μmol/L]. Compounds 1， 3 and 5 showed higher inhibition rates （55.51％， 57.20％ and 49.15％） than tectorigenin （33.05％） on H22 hepatoma-bearing mice， respectively. The other three compounds had no obvious advantage over tectorigenin in anti-tumor activity. CONCLUSIONS： In this study， compounds 1， 3 and 5 of six tectorigenin mannich base derivatives synthesized in this study have stronger antitumor activity than tectorigenin.

On the prediction of cytotoxicity of diverse chemicals for topminnow (Poeciliopsis lucida) hepatoma cell line, PLHC-1$.

Two data sets on the cytotoxicity of diverse chemicals to topminnow (Poeciliopsis lucida) hepatoma cell line (PLHC-1) were modelled with quantitative structure-toxicity relationship (QSTR). The data sets are based on 3-amino-7-dimethylamino-2-methylphenazine hydrochloride (NR) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays representing lysosomal damage and metabolic impairment, respectively. The descriptors were calculated with DRAGON 6 and SPARTAN 10 software packages. Descriptor selection was made by 'all subset' and genetic algorithm-based features implemented in QSARINS software. The proposed QSTR models were validated both internally and externally. For both endpoints, statistically satisfactory QSTR models were generated with nTr = 39; r2Tr = 0.782; RMSETr = 0.466; nTest = 18; r2Test = 0.799; RMSETest = 0.360 for NR-based model and nTr = 32; r2Tr = 0.775; RMSETr = 0.460; nTest = 10; r2Test = 0.864; RMSETest = 0.290 for MTT-based model. Additionally, the QSTR models generated for NR and MTT endpoints were used to predict the cytotoxicity of an external set of 657 and 652 diverse chemicals with structural coverage of 98.6% and 98.3%, respectively. A moderate correlation was observed between the experimental in vivo and predicted in vitro values for external set chemicals. The QSTR models may provide an initial, rapid screening and prioritization of these diverse chemicals for the acute fish toxicity assessment and reduce the need for extensive in vivo toxicity testing.

Synergistic cytotoxicity and mechanism of caffeine and lysozyme on hepatoma cell line HepG2.

The influences of caffeine, lysozyme and the joint application of them on the hepatoma cell line HepG2 proliferation inhibition and cell apoptosis were observed by 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and Hoechst 33342, which showed the proliferation inhibition rate of the joint application on HepG2 cells was 47.21%, significantly higher than caffeine or lysozyme, and the joint application promoted the apoptosis of HepG2 cells obviously. Van't Hoff classical thermodynamics formula, the Föster theory of non-radiation energy transfer and fluorescence phase diagram were used to manifest that the process of lysozyme binding to caffeine followed a two-state model, which was spontaneous at low temperature driven by enthalpy change, and the predominant intermolecular force was hydrogen bonding or Van der Waals force to stabilize caffeine-lysozyme complex with the distance 5.86nm. The attenuated total reflection-Fourier transform infrared spectra indicated that caffeine decreased the relative contents of α-helix and ß-turn, which inferred the structure of lysozyme tended to be "loose". Synchronous fluorescence spectra and ultraviolet spectra supported the above conclusion. The amino acid residues in the cleft of lysozyme were exposed and electropositivity was increased attributing to the loose structure, which were conducive to increasing caffeine concentration on the HepG2 cell surface by electrostatic interaction to show synergistic effect.

Polarity based characterization of biologically active extracts of Ajuga bracteosa Wall. ex Benth. and RP-HPLC analysis.

BACKGROUND: The concept of botanical therapeutics has revitalized due to wide importance of plant derived pharmaceuticals. Therefore, the ameliorative characteristics of Ajuga bracteosa were studied. METHODS: Total phenolic content, flavonoid content, antioxidant capacity, reducing power and free-radical scavenging activity were determined colorimetrically. Specific polyphenols were quantified by RP-HPLC analysis. Preliminary cytotoxicity was tested using brine shrimp lethality assay while antiproliferative activity against THP-1 and Hep-G2 cell lines was determined by MTT and SRB protocols respectively. Antileishmanial potential was assessed via MTT colorimetric method. To investigate antidiabetic prospect, α-amylase inhibition assay was adopted whereas disc diffusion method was used to detect likely protein kinase inhibitory, antibacterial and antifungal activities. RESULTS: Among fifteen different extracts, maximum total phenolic content (10.75 ± 0.70 µg GAE/mg DW), total reducing power (23.90 ± 0.70 µg AAE/mg DW) and total antioxidant capacity (11.30 ± 0.80 µg AAE/mg DW) were exhibited by methanol extract with superlative percent extract recovery (17.50 ± 0.80% w/w). Chloroform-methanol extract demonstrated maximum flavonoid content (4.10 ± 0.40 µg QE/mg DW) and ethanol extract exhibited greatest radical scavenging activity (IC50 14.40 ± 0.20 µg/ml). RP-HPLC based quantification confirmed polyphenols such as pyrocatechol, gallic acid, resorcinol, catechin, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, vanillic acid, coumarin, sinapinic acid, trans-cinnamic acid, rutin, quercetin and kaempferol. The brine shrimp lethality assay ranked 78.60% extracts as cytotoxic (LC50 ≤ 250 µg/ml) whereas significant THP-1 inhibition was shown by methanol-acetone extract (IC50 4.70 ± 0.43 µg/ml). The antiproliferative activity against Hep-G2 hepatoma cancer cell line was demonstrated by n-hexane, ethylacetate and methanol-distilled water (IC50 8.65-8.95 µg/ml) extracts. Methanol extract displayed prominent protein kinase inhibitory activity (MIC 12.5 µg/disc) while n-hexane extract revealed remarkable antileishmanial activity (IC50 4.69 ± 0.01 µg/ml). The antidiabetic potential was confirmed by n-hexane extract (44.70 ± 0.30% α-amylase inhibition at 200 µg/ml concentration) while a moderate antibacterial and antifungal activities were unveiled. CONCLUSION: The variation in biological spectrum resulted due to use of multiple solvent systems for extraction. We also deduce that the valuable information gathered can be utilized for discovery of anticancer, antileishmanial, antioxidant and antidiabetic bioactive lead candidates.

The apoptotic inducible effects of salicylic acid on hepatoma cell line: relationship with nitric oxide signaling.

Clinical and experimental data suggest that salicylic acid (SA) is tumor preventive and NO has a multitude of effects on tumor biology. Therefore, firstly, the aim of our study is to explore the important role of SA in apoptotic induction of liver cancer cells. Secondly, we investigate whether SA mediates the anti-tumor effects by NO signaling pathway. The liver cancer cell line was treated with different concentrations of SA. Cell proliferation was tested using MTS assay and cell apoptosis was assessed by flow cytometry. NO content and NOS activities were measured by biochemical assay. The anti- or pro-apoptotic regulator gene expressions were analyzed by real-time PCR. Our data illustrated that high concentration of SA significantly inhibited liver cancer cell proliferation accompanied by apoptosis induction. In addition, SA led to the release of NO and the increase of NOS activities in above process. Importantly, SA up-regulated a series of apoptosis-related gene expression and reduced the mRNA level of HMGB1. Meanwhile, we also found that NOS inhibitor L-NAME and NO scavenger cPTIO attenuated the above SA-induced effects. Thus, we provided the evidence that SA exerted anti-tumor effects in liver cancer cell in part mediated by the NO pathway.

Analysis of miRNA expression patterns in human and mouse hepatocellular carcinoma cells.

AIM: Hepatocellular carcinoma (HCC), one of the most common malignancies in adults displays aberrant miRNA expression during its pathogenesis. We assessed expression of miRNA in surgically resected human HCC of an early stage and murine HCC with a high malignancy in order to find miRNA overexpressed in HCC regardless of tumor stage and underlying etiology. Further, the role of the deregulated miRNA in HCC pathogenesis was investigated. METHODS: miRNA were isolated from HCC tissues and surrounding non-tumorous tissues from HCC patients and a murine transgenic model of HCC. A quantitative reverse transcription polymerase chain reaction was performed to determine expression levels of miRNA. Human HCC cell lines stably expressing individual miRNA were generated to investigate the biological function of overexpressed miRNA. RESULTS: We found that levels of miR-221, -181b-1, -155-5p, -25 and -17-5p were significantly upregulated in both human and murine HCC regardless of tumor stage, underlying etiology or the presence of fibrosis. Using HCC cell lines stably expressing respective miRNA, we found that miR-221 increased the proliferation of hepatoma cells, while miR-17-5p induced cell migration. CONCLUSION: We identified miRNA that are consistently upregulated in HCC. The overexpressed miRNA could potentially be used as a bona fide biomarker for HCC.