Antineoplastic Activity of Chrysin against Human Hepatocellular Carcinoma: New Insight on GPC3/SULF2 Axis and lncRNA-AF085935 Expression

The natural flavonoid chrysin possesses antiproliferative activity against various types of cancers, including hepatocellular carcinoma (HCC), which is a common malignancy. However, the exact mechanism of chrysin antiproliferative activity remains unclear. This research was executed to explore the impact of chrysin on glypican-3 (GPC3)/sulfatase-2 (SULF2) axis and lncRNA-AF085935 expression in HCC using HepG2 cells. Cisplatin (20, 50, 100 μg/mL), chrysin (15, 30, and 60 μg/mL) and the combination of 50 μg/mL cisplatin with different concentrations of chrysin were applied for 24/48 h. Cell viability was determined by MTT assay. Protein levels of GPC3 and SULF2 were measured by ELISA at 24/48 h. GPC3 immunoreactivity was detected by immunocytochemistry. Moreover, GPC3 and SULF2 mRNA expressions in addition to lncRNA-AF085935 expression were assessed by qPCR at 48 h. The GPC3 protein, immunostaining and mRNA levels, SULF2 protein and mRNA levels, as well as lncRNA-AF085935 expression, were decreased significantly with cisplatin and chrysin alone when compared with the control untreated HepG2 cells. However, the combination treatment exhibited a better chemopreventive effect in a dose- and time-dependent manner. This study demonstrated, for the first time, the antiproliferative activity of chrysin against HCC through the suppression of the GPC3/SULF2 axis along with the downregulation of lncRNA-AF085935 expression. Synergistic effect of chrysin with cisplatin could potentiate their antiproliferative action in a dose- and time-dependent manner.

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Bioactivity Screening of Antarctic Sponges Reveals Anticancer Activity and Potential Cell Death via Ferroptosis by Mycalols

Marine Drugs ◽

10.3390/md19080459 ◽

2021 ◽

Vol 19 (8) ◽

pp. 459

Author(s):

Gennaro Riccio ◽

Genoveffa Nuzzo ◽

Gianluca Zazo ◽

Daniela Coppola ◽

Giuseppina Senese ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Lipid Peroxidation ◽

Cell Death ◽

Programmed Cell Death ◽

Hepg2 Cells ◽

Iron Homeostasis ◽

Protein Levels ◽

Antarctic Research ◽

First Time ◽

Antarctic Sponges

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.

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Mycobacterium tuberculosis Requires Cholesterol Oxidase to Disrupt TLR2 Signalling in Human Macrophages

Mediators of Inflammation ◽

10.1155/2019/2373791 ◽

2019 ◽

Vol 2019 ◽

pp. 1-17

Author(s):

Izabela Szulc-Kielbik ◽

Michal Kielbik ◽

Patrycja Przygodzka ◽

Anna Brzostek ◽

Jaroslaw Dziadek ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Cholesterol Oxidase ◽

Interleukin 10 ◽

Signalling Pathway ◽

Mrna Levels ◽

Dependent Manner ◽

Wild Type ◽

Human Macrophages ◽

Protein Levels ◽

The Impact

This study tested the hypothesis that Mycobacterium tuberculosis (Mtb) uses a cholesterol oxidase enzyme (ChoD) to suppress a toll-like receptor type 2- (TLR2-) dependent signalling pathway to modulate macrophages’ immune response. We investigated the impact of Mtb possessing or lacking ChoD as well as TBChoD recombinant protein obtained from Mtb on the expression and activation of two key intracellular proteins involved in TLR2 signalling in human macrophages. Finally, the involvement of TLR2-related signalling proteins in an inflammatory/immunosuppressive response of macrophages to Mtb was evaluated. We demonstrate that wild-type Mtb but not the ∆choD mutant decreased the cytosolic IRAK4 and TRAF6 protein levels while strongly enhancing IRAK4 and TRAF6 mRNA levels in macrophages. Our data show that the TLR2 present on the surface of macrophages are involved in disturbing the signalling pathway by wild-type Mtb. Moreover, recombinant TBChoD effectively decreased the cytosolic level of TRAF6 and lowered the phosphorylation of IRAK4, which strongly confirm an involvement of cholesterol oxidase in affecting the TLR2-related pathway by Mtb. Wild-type Mtb induced an immunosuppressive response of macrophages in an IRAK4- and TRAF6-dependent manner as measured by interleukin 10 production. In conclusion, ChoD is a virulence factor that enables Mtb to disturb the TLR2-related signalling pathway in macrophages and modulate their response.

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Emodin Inhibits the Growth and Invasion and Induces Apoptosis and Autophagy in Hepatocellular Carcinoma via PI3K/AKT/mTOR and Wnt/β-catenin Pathway

10.21203/rs.3.rs-826021/v1 ◽

2021 ◽

Author(s):

binyu qin ◽

Zhili Zeng ◽

Jianliang Xu ◽

Jing Shangwen ◽

Zeng Jie Ye ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

Western Blot ◽

Hepg2 Cells ◽

Cancer Metastasis ◽

Migration And Invasion ◽

Autophagic Flux ◽

Dependent Manner ◽

Mesenchymal Transition ◽

The Impact

Abstract Background: Previous studies reported that emodin extracted from Rheum palmatum L. exerts antiproliferation and antimetastatic effects in a variety of human cancer types. However, the role of emodin in hepatocellular carcinoma (HCC) remain unknown.Methods: EdU and colony formation assays were performed to evaluate the effects of emodin on proliferation. The mobility capacities of HCC treated with emodin were evaluated using wound healing assay. Transwell invasion and migration assays were performed to evaluate anti-migratory and anti-invasive effects of emodin on HCC. Annexin V-FITC/PI was performed to analyze the apoptosis. PI stain was performed to analyze cell cycle. RNA sequencing technology was used to identify the differentially expressed genes (DEGs) induced by emodin in HCC. The impact of emodin on autophagic flux in HepG2 cells was examined by mCherry-GFP-LC3 analysis. Western blot was used to assess the protein expressions of epithelial-mesenchymal transition (EMT), autophagy, PI3K/AKT/mTOR and Wnt/β-catenin signaling pathway.Results: We found that emodin inhibited the growth of HepG2 cells in a dose- and time-dependent manner. In addition, emodin inhibited cell proliferation, induced S and G2/M phases arrest, and promoted apoptosis in HepG2 cells. The migration and invasion of HepG2 cells were also suppressed by emodin. Enrichment analysis revealed that DEGs involved in cell adhesion, cancer metastasis and cell cycle arrest. Moreover, western blot and bioinformatics analysis indicated that emodin induced autophagy and inhibited the EMT in part through suppression of the PI3K/AKT/mTOR and Wnt/β-catenin pathways. Conclusion: Our study indicated that emodin inhibited cell metastasis in HCC via the crosstalk between autophagy and EMT.

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The Effects of Vitamin K1 and Warfarin on Prothrombin Expression in Human Hepatoblastoma (HepG2) Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656315 ◽

1992 ◽

Vol 68 (01) ◽

pp. 040-047 ◽

Cited By ~ 3

Author(s):

C Scott Jamison ◽

Bryan F Burkey ◽

Sandra J Friezner Degen

Keyword(s):

Total Protein ◽

Hepg2 Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Response Analysis ◽

Secreted Protein ◽

Mrna Levels ◽

Vitamin K1 ◽

Maximal Increase ◽

Protein Levels ◽

Warfarin Treatment

SummaryCultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 µg vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 µg/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 µg/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 µg/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240–350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on γ-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

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Negative Correlation Between Serum Levels of Homocysteine and Apolipoprotein M

Current Molecular Medicine ◽

10.2174/1566524019666190308115624 ◽

2019 ◽

Vol 19 (2) ◽

pp. 120-126

Author(s):

J. Wei ◽

Y. Yu ◽

Y. Feng ◽

J. Zhang ◽

Q. Jiang ◽

...

Keyword(s):

Negative Correlation ◽

Hepg2 Cells ◽

Serum Levels ◽

Significant Negative Correlation ◽

Mrna Levels ◽

Rt Pcr ◽

Apolipoprotein M ◽

Protein Mass ◽

Protein Levels ◽

Phosphoinositide 3 Kinase

Background: Homocysteine (Hcy) has been suggested as an independent risk factor for atherosclerosis. Apolipoprotein M (apoM) is a constituent of the HDL particles. The goal of this study was to examine the serum levels of homocysteine and apoM and to determine whether homocysteine influences apoM synthesis. Methods: Serum levels of apoM and Hcy in 17 hyperhomocysteinemia (HHcy) patients and 19 controls were measured and their correlations were analyzed. Different concentrations of homocysteine (Hcy) and LY294002, a specific phosphoinositide 3- kinase (PI3K) inhibitor, were used to treat HepG2 cells. The mRNA levels were determined by RT-PCR and the apoM protein mass was measured by western blot. Results: We found that decreased serum apoM levels corresponded with serum HDL levels in HHcy patients, while the serum apoM levels showed a statistically significant negative correlation with the serum Hcy levels. Moreover, apoM mRNA and protein levels were significantly decreased after the administration of Hcy in HepG2 cells, and this effect could be abolished by addition of LY294002. Conclusions: resent study demonstrates that Hcy downregulates the expression of apoM by mechanisms involving the PI3K signal pathway.

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The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620999200818155814 ◽

2020 ◽

Vol 20 ◽

Author(s):

Hongtao Li ◽

Peng Chen ◽

Lei Chen ◽

Xinning Wang

Keyword(s):

Cell Growth ◽

Western Blot ◽

Wilms Tumor ◽

Critical Role ◽

Time Dependent ◽

Luciferase Assay ◽

Dependent Manner ◽

Tlr4 Expression ◽

Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

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Synthesis and Evaluation of the Antitumor Activity of Novel 1-(4-Substituted phenyl)-2-ethyl Imidazole Apoptosis Inducers In Vitro

Molecules ◽

10.3390/molecules25184293 ◽

2020 ◽

Vol 25 (18) ◽

pp. 4293

Author(s):

Zhen-Wang Li ◽

Chun-Yan Zhong ◽

Xiao-Ran Wang ◽

Shi-Nian Li ◽

Chun-Yuan Pan ◽

...

Keyword(s):

Antitumor Activity ◽

Tumor Cells ◽

Antiproliferative Activity ◽

Antitumor Agent ◽

Positive Control ◽

Selectivity Index ◽

Time Dependent ◽

Potential Candidate ◽

Dependent Manner

Novel imidazole derivatives were designed, prepared, and evaluated in vitro for antitumor activity. The majority of the tested derivatives showed improved antiproliferative activity compared to the positive control drugs 5-FU and MTX. Among them, compound 4f exhibited outstanding antiproliferative activity against three cancer cell lines and was considerably more potent than both 5-FU and MTX. In particular, the selectivity index indicated that the tolerance of normal L-02 cells to 4f was 23–46-fold higher than that of tumor cells. This selectivity was significantly higher than that exhibited by the positive control drugs. Furthermore, compound 4f induced cell apoptosis by increasing the protein expression levels of Bax and decreasing those of Bcl-2 in a time-dependent manner. Therefore, 4f could be a potential candidate for the development of a novel antitumor agent.

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Aggregated Tau-PHF6 (VQIVYK) Potentiates NLRP3 Inflammasome Expression and Autophagy in Human Microglial Cells

Cells ◽

10.3390/cells10071652 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1652

Author(s):

Chinmaya Panda ◽

Clara Voelz ◽

Pardes Habib ◽

Christian Mevissen ◽

Thomas Pufe ◽

...

Keyword(s):

Tau Protein ◽

Nlrp3 Inflammasome ◽

Time Dependent ◽

Microglial Cells ◽

Inflammasome Activation ◽

Dependent Manner ◽

Progressive Dementia ◽

Microglial Polarization ◽

Protein Levels ◽

Pyrin Domain

Intra-neuronal misfolding of monomeric tau protein to toxic β-sheet rich neurofibrillary tangles is a hallmark of Alzheimer’s disease (AD). Tau pathology correlates not only with progressive dementia but also with microglia-mediated inflammation in AD. Amyloid-beta (Aβ), another pathogenic peptide involved in AD, has been shown to activate NLRP3 inflammasome (NOD-like receptor family, pyrin domain containing 3), triggering the secretion of proinflammatory interleukin-1β (IL1β) and interleukin-18 (IL18). However, the effect of tau protein on microglia concerning inflammasome activation, microglial polarization, and autophagy is poorly understood. In this study, human microglial cells (HMC3) were stimulated with the unaggregated and aggregated forms of the tau-derived PHF6 peptide (VQIVYK). Modulation of NLRP3 inflammasome was examined by qRT-PCR, immunocytochemistry, and Western blot. We demonstrate that fibrillar aggregates of VQIVYK upregulated the NLRP3 expression at both mRNA and protein levels in a dose- and time-dependent manner, leading to increased expression of IL1β and IL18 in HMC3 cells. Aggregated PHF6-peptide also activated other related inflammation and microglial polarization markers. Furthermore, we also report a time-dependent effect of the aggregated PHF6 on BECN1 (Beclin-1) expression and autophagy. Overall, the PHF6 model system-based study may help to better understand the complex interconnections between Alzheimer’s PHF6 peptide aggregation and microglial inflammation, polarization, and autophagy.

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Synthesis, Characterization of Nano-β-Tricalcium Phosphate and the Inhibition on Hepatocellular Carcinoma Cells

Journal of Nanomaterials ◽

10.1155/2018/7083416 ◽

2018 ◽

Vol 2018 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Langlang Liu ◽

Yanzeng Wu ◽

Chao Xu ◽

Suchun Yu ◽

Xiaopei Wu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Particle Size ◽

Cell Viability ◽

Tricalcium Phosphate ◽

Hepg2 Cells ◽

Drug Carrier ◽

Average Particle Size ◽

Crystal Habit ◽

Average Particle ◽

Dependent Manner

It is difficult to synthesize nano-β-tricalcium phosphate (nano-β-TCP) owing to special crystal habit. The aim of this work was to synthesize nano-β-TCP using ethanol-water system and characterize it by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Malvern laser particle size analyzer, and transmission electron microscope (TEM). In addition, the inhibitory effect of nano-β-TCP on human hepatocellular carcinoma (HepG2) cells was also investigated using MTT assay, lactate dehydrogenase (LDH) leakage test, and 4′-6-diamidino-2-phenylindole (DAPI) staining. The results showed that negatively charged rod-like nano-β-TCP with about 55 nm in diameter and 120 nm in length was synthesized, and the average particle size of nano-β-TCP was 72.7 nm. The cell viability revealed that nano-β-TCP caused reduced cell viability of HepG2 cells in a time- and dose-dependent manner. These findings presented here may provide valuable reference data to guide the design of nano-β-TCP-based anticancer drug carrier and therapeutic systems in the future.

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Regulation of AMH by oocyte-specific growth factors in human primary cumulus cells

Reproduction ◽

10.1530/rep-17-0421 ◽

2017 ◽

Vol 154 (6) ◽

pp. 745-753 ◽

Cited By ~ 10

Author(s):

Scott Convissar ◽

Marah Armouti ◽

Michelle A Fierro ◽

Nicola J Winston ◽

Humberto Scoccia ◽

...

Keyword(s):

Fold Increase ◽

Cumulus Cells ◽

Maximal Effect ◽

Mrna Levels ◽

Dependent Manner ◽

Protein Levels ◽

Secreted Factors ◽

Morphogenetic Protein ◽

Growth Differentiation Factor 9 ◽

Signaling Inhibitors

The regulation of AMH production by follicular cells is poorly understood. The purpose of this study was to determine the role of the oocyte-secreted factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on AMH production in primary human cumulus cells. Cumulus cells from IVF patients were cultured with a combination of GDF9, BMP15, recombinant FSH and specific signaling inhibitors. Stimulation with GDF9 or BMP15 separately had no significant effect onAMHmRNA levels. In contrast, simultaneous stimulation with GDF9 and BMP15 (G + B) resulted in a significant increase inAMHmRNA expression. Increasing concentration of G + B (0.6, 2.5, 5 and 10 ng/mL) stimulated AMH in a dose-dependent manner, showing a maximal effect at 5 ng/mL. Western blot analyses revealed an average 16-fold increase in AMH protein levels in cells treated with G + B when compared to controls. FSH co-treatment decreased the stimulation of AMH expression by G + B. The stimulatory effect of G + B on the expression of AMH was significantly decreased by inhibitors of the SMAD2/3 signaling pathway. These findings show for the first time that AMH production is regulated by oocyte-secreted factors in primary human cumulus cells. Moreover, our novel findings establish that the combination of GDF9 + BMP15 potently stimulates AMH expression.

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