Human Hepatoblastoma HepG2 Cells Research Articles

Suppressive effect of isofraxidin on the overexpression of IL-6 and its molecular mechanism in a TPA-treated human hepatocellular carcinoma cell line, HuH-7.

Interleukin-6 (IL-6) is a pleiotropic cytokine that has many biological activities, including inflammation, hematopoiesis, bone metabolism, embryonic development, and other fundamental processes. Recently, IL-6 has been widely recognized as an important pro-inflammatory cytokine involved in cytokine storm pathogenesis during severe inflammatory diseases, such as coronavirus disease 2019 (COVID-19). Therefore, IL-6 is considered to be a therapeutic target for inhibiting cytokine storm. In the present study, we investigated the suppressive effect of isofraxidin, a major coumarin compound of Acanthopanax senticosus, on the overexpression of IL-6 and its molecular mechanism. The expression of IL-6 mRNA was measured using quantitative real-time PCR, and intracellular signaling molecules were detected using western blotting. When the HuH-7 human hepatocellular carcinoma cell line and HepG2 human hepatoblastoma cell line were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), a marked induction of IL-6 mRNA expression was observed in HuH-7 cells compared with HepG2 cells. Isofraxidin significantly suppressed TPA-induced IL-6 mRNA expression in HuH-7 cells in a dose-dependent manner. Furthermore, isofraxidin inhibited TPA-induced phosphorylation of ERK1/2 in a dose-dependent manner. Similarly, the MAPK/ERK inhibitor U0126 suppressed TPA-induced IL-6 mRNA expression. However, isofraxidin had no effects on TPA-induced phosphorylation of SAPK/JNK, Akt (Ser473), and STAT3 (Tyr705), nuclear translocation of NF-κB p65, and degradation of IκB. Taken together, isofraxidin suppresses TPA-induced overexpression of IL-6 mRNA by selectively inhibiting the activation of the MAPK/ERK pathway in HuH-7 cells, indicating that isofraxidin may be an effective anti-inflammatory agent for treating cytokine storm.

3D Bioprinting of Sugar Beet Pectin through Horseradish Peroxidase-Catalyzed Cross-Linking.

Horseradish peroxidase (HRP)-mediated hydrogelation, caused by the cross-linking of phenolic groups in polymers in the presence of hydrogen peroxide (H2O2), is an effective route for bioink solidification in 3D bioprinting. Sugar beet pectin (SBP) naturally has cross-linkable phenols through the enzymatic reaction. Therefore, chemical modifications are not required, unlike the various polymers that have been used in the enzymatic cross-linking system. In this study, we report the application of SBP in extrusion-based bioprinting including HRP-mediated bioink solidification. In this system, H2O2 necessary for the solidification of inks is supplied in the gas phase. Cell-laden liver lobule-like constructs could be fabricated using bioinks consisting of 10 U/mL HRP, 4.0 and 6.0 w/v% SBP, and 6.0 × 106 cells/mL human hepatoblastoma (HepG2) cells exposed to air containing 16 ppm of H2O2 concurrently during printing and 10 min postprinting. The HepG2 cells enclosed in the printed constructs maintained their viability, metabolic activity, and hepatic functions from day 1 to day 7 of the culture, which indicates the cytocompatibility of this system. Taken together, this result demonstrates the potential of SBP and HRP cross-linking systems for 3D bioprinting, which can be applied in tissue engineering applications.

The importance of cell uptake in photothermal treatments mediated by biomimetic magnetic nanoparticles

Biomimetic magnetic nanoparticles (BMNPs) mediated by MamC have proven to be photothermal agents able to allow an optimized cytotoxicity against tumoral cells when used simultaneously as drug nanotransporters and as hyperthermia agents. However, it remains unclear whether BMNPs need to be internalized by the cells and/or if there is a threshold for internal Fe concentration for the photothermal therapy to be effective. In this study, three different situations for photothermal treatments have been simulated to disentangle the effect of BMNPs cell uptake on cell viability after photothermal treatments. Human hepatoblastoma (HepG2) cell line was treated with suspensions of BMNPs, and protocols were developed to have only intracellular BMNPs, only extracellular BMNPs or both, followed by photothermal exposure of the treated cell cultures. Our data demonstrate that: (1) Although the heating efficiency of the photothermal agent is not altered by its location (intra/extracellular), the intracellular location of BMNPs is crucial to ensure the cytotoxic effect of photothermal treatments, especially at low Fe concentration. In fact, the concentration of BMNPs needed to reach the same cytotoxic effect following upon laser irradiation of 0.2 W/cm2 is three times larger if BMNPs are located extracellularly compared to that needed if BMNPs are located intracellularly; (2) For a given location of the BMNPs, cell death increases with BMNPs (or Fe) concentration. When BMNPs are located intracellularly, there is a threshold for Fe concentration (∼ 0.5 mM at laser power intensities of 0.1 W/cm2) needed to affect cell viability following upon cell exposure to photothermia. (3) Bulk temperature rise is not the only factor accounting for cell death. Actually, temperature increases inside the cells cause more damage to cell structures and trigger cell death more efficiently than an increase in the temperature outside the cell.

Horseradish Peroxidase-Mediated Bioprinting via Bioink Gelation by Alternately Extruded Support Material.

Horseradish peroxidase (HRP)-mediated extrusion bioprinting has a significant potential in tissue engineering and regenerative medicine. However, they often face challenges in terms of printing fidelity and structural integrity when using low-viscosity inks. To address this issue, a method that alternately extrudes bioinks and support material was developed in this study. The bioinks consisting of cells, HRP, and phenolated polymers, and the support material contained hydrogen peroxide (H2O2). The support material not only prevented the collapse of the constructs but also supplied H2O2 to facilitate the enzymatic reaction. 3D constructs with tall and complex shapes were successfully printed from a low-viscosity ink containing 10 U/mL HRP and 1.0% w/v phenolated hyaluronic acid (HA-Ph), with a support material containing 10 mM H2O2. Over 90% viability of mouse fibroblasts (10T1/2) was achieved following the printing process, along with a morphology and proliferation rate similar to that of nontreated cells. Furthermore, human hepatoblastoma (HepG2) cells showed an increased spheroid size over 14 days in the printed constructs. The 10T1/2 cells adhered and proliferated on the constructs printed from inks containing both phenolated gelatin and HA-Ph. These results demonstrate the great potential of this HRP-mediated extrusion bioprinting technique for tissue engineering applications.

Microcystins-Loaded Aged Nanoplastics Provoke a Metabolic Shift in Human Liver Cells.

Studies concerning the toxicity of pollutant-loaded nanoplastics (NPs) toward humans are still in their infancy. Here, we evaluated the adsorption of microcystins (MCs) by pristine and aged polystyrene nanoplastics (PSNPs), prepared MCs-loaded aged PSNPS (1, 5, 10, 15, and 19 μg/mg), and systematically mapped the key molecular changes induced by aged and MCs-loaded PSNPs to human hepatoblastoma (HepG2) cells. According to the results, MC-LR adsorption is increased 2.64-fold by aging, and PSNP accumulation is detected in HepG2 cells. The cytotoxicity of the MC-LR-loaded aged PSNPs showed a positive relationship with the MC-LR amount, as the cell viability in the 19 μg/mg loading treatment (aPS-MC19) was 10.84% lower than aged PSNPs; meanwhile, more severe oxidative damage was observed. Primary approaches involved stressing the endoplasmic reticulum and reducing protein synthesis that the aged PSNPs posed for HepG2 cells, while the aggravated cytotoxicity in aPS-MC19 treatment was a combined result of the metabolic energy disorder, oxidative damage, endoplasmic reticulum stress, and downregulation of the MC-LR target protein. Our results confirm that the aged PSNPs could bring more MC-LR into the HepG2 cells, significantly interfere with biological processes, and provide new insight into deciphering the risk of NPs to humans.

Visible light photocrosslinking of sugar beet pectin for 3D bioprinting applications

Herein, we report the hydrogelation of sugar beet pectin (SBP) via visible light-mediated photocrosslinking and its applications in extrusion-based 3D bioprinting. Rapid hydrogelation (<15 s) was achieved by applying 405 nm visible light to an SBP solution in the presence of tris(bipyridine)ruthenium(II) chloride hexahydrate ([Ru(bpy)3]2+) and sodium persulfate (SPS). The mechanical properties of the hydrogel could be tuned by controlling the visible light irradiation time and concentrations of SBP, [Ru(bpy)3]2+, and SPS. High-fidelity 3D hydrogel constructs were fabricated by extruding inks containing 3.0 wt% SBP, 1.0 mM [Ru(bpy)3]2+, and 1.0 mM SPS. Human hepatoblastoma (HepG2) cells encapsulated in SBP hydrogels remained viable and metabolically active after 14 d of culture. Overall, this study demonstrates the feasibility of applying SBP and a visible light-mediated photocrosslinking system to the 3D bioprinting of cell-laden constructs for tissue engineering applications.

Red biocolorant from endophytic Talaromyces minnesotensis: production, properties, and potential applications.

Fungal colorants are gradually entering the global color market, given their advantages of being less harmful to human health, as well as having greater stability and biotechnological potential, compared to other natural sources. The present work concerns the isolation and identification of an endophytic filamentous fungus, together withthe chemical characterization and assessment of the fluorescence, toxicity, stability, and application potential of its synthesized red colorant. The endophytic fungus was isolated from Hymenaea courbaril, a tree from the Brazilian savannah, and was identified as Talaromyces minnesotensis by phenotypic and genotypic characterization. Submerged cultivation of the fungus resulted in the production of approximately 12 AU500 of a red biocolorant which according to LC-DAD-MS analysis is characterized by being a complex mixture of molecules of the azaphilone class. Regarding cytotoxicity assays, activity against human hepatoblastoma (HepG2) cells was only observed at concentrations above 5.0g L-1, while antimicrobial effects against pathogenic bacteria and yeast occurred at concentrations above 50.0g L-1. The biocolorant showed high stability at neutral pH values and low temperatures (10 to 20°C) and high half-life values (t1/2), which indicates potential versatility for application in different matrices, as observed in tests using detergent, gelatin, enamel, paint, and fabrics. The results demonstrated that the biocolorant synthesized by Talaromyces minnesotensis has potential for future biotechnological applications. KEY POINTS: • An endophytic fungus, which was isolated and identified, synthesize a red colorant. • The colorant showed fluorescence property, low toxicity, and application potential. • The red biocolorant was highly stable at pH 8.0 and temperatures below 20°C.

Role of biotransformation in the diazinon-induced toxicity in HepG2 cells and antioxidant protection by tetrahydrocurcumin.

Diazinon (DZN) is an insecticide extensively used to control pests in crops and animals. However, its indicriminated use may lead to liver damage in animals and humans. This study aimed to evaluate the toxicity of DZN (25-150µM) on human hepatoblastoma (HepG2) cells after 24 and 48h of exposure and the role of its biotransformation on the toxicological potential. We also tested the protective effect of tetrahydrocurcumin (THC), an antioxidant agent, in the DZN-induced citotoxicity. DZN caused cytotoxicity in the HepG2 cells, inhibiting cell proliferation and reducing cell viability in a dose- and time-dependent manner. The pre-incubation of HepG2 cells with chemical inducers of cytochrome P450 monooxygenase 3-methylcholanthrene and phenobarbital resulted in a further decrease of cell viability associated with DZN exposure. In addition, the metabolite diazoxon was more toxic than DZN. Our results also revealed that THC alleviated DZN-induced cytotoxicity and reactive oxygen and nitrogen species (RONS) generation in HepG2 cells. In conclusion, our data provide novel insights into the involvement of biotransformation in the mechanisms of DZN-induced cytotoxicity and suggest that amelioration of RONS accumulation might be involved in the protective effect of THC on DZN-induced liver injury.

Poloxamers-based nanomicelles as delivery vehicles of hypericin for hepatic photodynamic therapy

Polymeric micelles in oxyalkylic blocks have been investigated as carriers of hydrophobic photosensitizers for intravenous administration in photodynamic therapy. This study evaluated the uptake of hypericin carried by polymeric micelles formed with Pluronics® P123, F127 and P84 and their photoinduced actions on gluconeogenesis in perfused livers of mice. Uptake and photoinduced action of F127-carried hypericin were also evaluated against cultured human hepatoblastoma HepG2 cells. Respiratory activity was assessed in isolated hepatic mitochondria. Hypericin is also investigated as a promising photosensitizer and it was herein photoinduced with red light. This procedure should activate this agent to a lower extension and prevent damage against healthy tissues. Hypericin carried by P123, P84 and F127 micelles was substantially taken up by perfused livers of healthy mice. However, P123 and P84 inhibited hepatic gluconeogenesis by impairing the aerobic energy supply of the cell, effects probably associated with their more hydrophobic character. Hypericin carried by F127, a more hydrophilic micelle, did not inhibit gluconeogenesis and did not modify the mitochondrial respiration at concentrations lower than 2 μM in the dark or when photoinduced with red light. What is more, F127-carried hypericin was substantially taken up by HepG2 cells and, when photoinduced with red light, considerably decreased the viability of these cells. The results show that F127-carried hypericin could be a suitable photosensitizer system to be used in photodynamic therapy against liver tumors, especially if it is photoinduced with red light, which should activate the molecule to a lower extension and prevent damage to the healthy tissue.

Combined Magnetic Hyperthermia and Photothermia with Polyelectrolyte/Gold-Coated Magnetic Nanorods.

Magnetite nanorods (MNRs) are synthesized based on the use of hematite nanoparticles of the desired geometry and dimensions as templates. The nanorods are shown to be highly monodisperse, with a 5:1 axial ratio, and with a 275 nm long semiaxis. The MNRs are intended to be employed as magnetic hyperthermia and photothermia agents, and as drug vehicles. To achieve a better control of their photothermia response, the particles are coated with a layer of gold, after applying a branched polyethyleneimine (PEI, 2 kDa molecular weight) shell. Magnetic hyperthermia is performed by application of alternating magnetic fields with frequencies in the range 118-210 kHz and amplitudes up to 22 kA/m. Photothermia is carried out by subjecting the particles to a near-infrared (850 nm) laser, and three monochromatic lasers in the visible spectrum with wavelengths 480 nm, 505 nm, and 638 nm. Best results are obtained with the 505 nm laser, because of the proximity between this wavelength and that of the plasmon resonance. A so-called dual therapy is also tested, and the heating of the samples is found to be faster than with either method separately, so the strengths of the individual fields can be reduced. Due to toxicity concerns with PEI coatings, viability of human hepatoblastoma HepG2 cells was tested after contact with nanorod suspensions up to 500 µg/mL in concentration. It was found that the cell viability was indistinguishable from control systems, so the particles can be considered non-cytotoxic in vitro. Finally, the release of the antitumor drug doxorubicin is investigated for the first time in the presence of the two external fields, and of their combination, with a clear improvement in the rate of drug release in the latter case.

A Gedunin-Type Limonoid, 7-Deacetoxy-7-Oxogedunin, from Andiroba (Carapa guianensis Aublet) Reduced Intracellular Triglyceride Content and Enhanced Autophagy in HepG2 Cells.

The seed oil of Carapa guianensis Aublet (Andiroba) has been used in folk medicine for its insect-repelling, anti-inflammatory, and anti-malarial activities. This study aimed to examine the triglyceride (TG) reducing effects of C. guianensis-derived limonoids or other commercially available limonoids in human hepatoblastoma HepG2 cells and evaluate the expression of lipid metabolism or autophagy-related proteins by treatment with 7-deacetoxy-7-oxogedunin (DAOG; 1), a principal limonoid of C. guianensis. The gedunin-type limonoids, such as DAOG (% of control at 20 μM: 70.9 ± 0.9%), gedunin (2, 74.0 ± 1.1%), epoxyazadiradione (4, 73.4 ± 2.0%), 17β-hydroxyazadiradione (5, 79.9 ± 0.6%), 7-deacetoxy-7α-hydroxygedunin (6, 61.0 ± 1.2%), andirolide H (7, 87.4 ± 2.2%), and 6α-hydroxygedunin (8, 84.5 ± 1.1%), were observed to reduce the TG content at lower concentrations than berberine chloride (BBR, a positive control, 84.1 ± 0.3% at 30 μM) in HepG2 cells pretreated with high glucose and oleic acid. Andirobin-, obacunol-, nimbin-, and salannin-type limonoids showed no effect on the intracellular TG content in HepG2 cells. The TG-reducing effect of DAOG was attenuated by the concomitant use of compound C (dorsomorphin), an AMPK inhibitor. Further investigation on the detailed mechanism of action of DAOG at non-cytotoxic concentrations revealed that the expressions of autophagy-related proteins, LC3 and p62, were upregulated by treatment with DAOG. These findings suggested that gedunin-type limonoids from Andiroba could ameliorate fatty liver, and that the action of DAOG in particular is mediated by autophagy.

Design and Characterization of a Bioinspired Polyvinyl Alcohol Matrix with Structural Foam-Wall Microarchitectures for Potential Tissue Engineering Applications.

Traditional medical soft matrix used in a surgical treatment or in wound management was not good enough in both the structural support and interconnectivity to be applied in tissue engineering as a scaffold. Avian skeleton and feather rachises might be good reference objects to mimic in designing a scaffold material with good structural support and high interconnectivity because of its structural foam-wall microarchitectures and structural pneumaticity. In this study, a biomimetic airstream pore-foaming process was built up and the corresponding new medical soft matrix derived from polyvinyl alcohol matrix (PVAM) with air cavities inspired by avian skeleton and feather rachises was prepared. Furthermore, the resulting medical soft matrix and bovine Achilles tendon type I collagen could be employed to prepare a new collagen-containing composite matrix. Characterization, thermal stability and cell morphology of the bioinspired PVA matrix and the corresponding collagen-modified PVA composite matrix with open-cell foam-wall microarchitectures were studied for evaluation of potential tissue engineering applications. TGA, DTG, DSC, SEM and FTIR results of new bioinspired PVA matrix were employed to build up the effective system identification approach for biomimetic structure, stability, purity, and safety of target soft matrix. The bioinspired PVA matrix and the corresponding collagen-modified PVA composite matrix would be conductive to human hepatoblastoma HepG2 cell proliferation, migration, and expression which might serve as a promising liver cell culture carrier to be used in the biological artificial liver reactor.

Antiproliferative and Cytotoxic Activities of Fluorescein-A Diagnostic Angiography Dye.

Fluorescein is a fluorescent dye used as a diagnostic tool in various fields of medicine. Although fluorescein itself possesses low toxicity, after photoactivation, it releases potentially toxic molecules, such as singlet oxygen (1O2) and, as we demonstrate in this work, also carbon monoxide (CO). As both of these molecules can affect physiological processes, the main aim of this study was to explore the potential biological impacts of fluorescein photochemistry. In our in vitro study in a human hepatoblastoma HepG2 cell line, we explored the possible effects on cell viability, cellular energy metabolism, and the cell cycle. We observed markedly lowered cell viability (≈30%, 75–2400 μM) upon irradiation of intracellular fluorescein and proved that this decrease in viability was dependent on the cellular oxygen concentration. We also detected a significantly decreased concentration of Krebs cycle metabolites (lactate and citrate < 30%; 2-hydroxyglutarate and 2-oxoglutarate < 10%) as well as cell cycle arrest (decrease in the G2 phase of 18%). These observations suggest that this photochemical reaction could have important biological consequences and may account for some adverse reactions observed in fluorescein-treated patients. Additionally, the biological activities of both 1O2 and CO might have considerable therapeutic potential, particularly in the treatment of cancer.

Effects of imidacloprid on viability and increase of reactive oxygen and nitrogen species in HepG2 cell line

Imidacloprid (IMD) is a neonicotinoid insecticide used in large quantities worldwide in both veterinary and agronomic applications. Several studies have shown adverse effects of IMD on non-target organisms, with the liver being identified as the main affected organ. This study aimed to evaluate the effects of IMD on human hepatoblastoma (HepG2) cells. HepG2 were exposed to IMD (0.25–2.0 mM) for 24 and 48 h. IMD treatment resulted in cytotoxicity in the HepG2, inhibiting cell proliferation in a dose- and time-dependent manner, starting at concentrations of 0.5 mM (24 h) and 0.25 mM (48 h), and reducing cell viability from 0.5 mM onwards (24 and 48 h). IMD significantly decreased the mitochondrial membrane potential at both time points investigated (2.0 mM), and also induced damage to the cell membrane, demonstrated by significant dose and time-dependent increases in lactate dehydrogenase (LDH) release from concentrations of 1.0 mM (24 h) and 0.5 mM (48 h) upwards. IMD treatment also increased the production of reactive oxygen and nitrogen species (ROS/RNS) at rates above 50% following 0.5 mM (24 h) or 0.25 mM (48 h) concentrations, and caused a significant decrease in reduced/oxidized glutathione ratio (GSH/GSSG), indicating oxidative stress. Furthermore, the antioxidant dithiothreitol, which reacts with ROS/RNS and acts as a thiol reducing agent, inhibited the cytotoxic effect of IMD. In addition, the metabolite IMD-olefin was more toxic than IMD. Our results indicate that IMD induces cytotoxicity in HepG2 cells and that this effect may be associated with an increase in the generation of ROS/RNS.

Long noncoding RNA Gas5 induces cell apoptosis and inhibits tumor growth via activating the CHOP-dependent endoplasmic reticulum stress pathway in human hepatoblastoma HepG2 cells.

In recent years, long noncoding RNAs (lncRNAs) have been demonstrated to be important tumor-associated regulatory factors. LncRNA growth arrest-specific transcript 5 (Gas5) acts as an anti-oncogene in most cancers. Whether Gas5 acts as an oncogene or anti-oncogene in hepatocellular carcinoma (HCC) remains unclear. In the present study, the expression and role of Gas5 in HCC were investigated in vitro and in vivo. Lower expression levels of Gas5 were determined in HCC tissues and cells by quantitative reverse transcription-polymerase chain reaction. Overexpressed Gas 5 lentiviral vectors were constructed to analyze their influence on cell viability, migration, invasion, and apoptosis. Fluorescence in situ hybridization was used to identify the subcellular localization of Gas5. Protein complexes that bound to Gas5 were isolated from HepG2 cells through pull-down experiments and analyzed by mass spectrometry. A series of novel Gas5-interacting proteins were identified and bioinformatics analysis was carried out. These included ribosomal proteins, proteins involved in protein folding, sorting, and transportation in the ER, some nucleases and protein enzymes involved in gene transcription, translation, and other proteins with various functions.78 kDa glucose-regulated protein (GRP78) was identified as a direct target of Gas5 by Rip-qPCR and Western blot analysisassay. Gas5 inhibited HepG2 cell growth and induced cell apoptosis via upregulating CHOP to activate the ER stress signaling pathway. Further studies indicated that the knockdown of CHOP by shRNA partially reversed Gas5-mediated apoptosis in HepG2 cells. Magnetic resonance imagingshowed that the ectopic expression of Gas5 inhibited the growth of HCC in nude mice. These findings suggest that Gas5 functions as a tumor suppressor and induces apoptosis through activation of ER stress by targeting the CHOP signal pathway in HCC.

Effect of fatty acid synthase gene silencing on lipid metabolism and biological behaviors of human hepatoblastoma HepG2 cells

To investigate the effect of fatty acid synthase (FASN) gene silencing on lipid metabolism and biological behaviors of human hepatoblastoma HepG2 cells. Small interfering RNA (siRNA) targeting FASN gene or a negative control siRNA sequence (NC-siRNA) was transfected into HepG2 cells, and the gene silencing efficiency was evaluated with qRT-PCR and Western blotting. Triglyceride level in the cells was detected using enzyme-linked immunosorbent assay, and Oil red O staining was performed to examine intracellular lipid droplets. The proliferation ability of the transfected cells was tested by CCK-8 assay, and cell apoptosis was evaluated using annexin V-FITC/PI apoptosis detection kit. Wound healing assay and Transwell assay were performed to assess the migration ability of the transfected cells. Transfection of the cells with FASN-siRNA, but not NC-siRNA, significantly lowered FASN expression at both the mRNA and protein level (P < 0.001) and decreased the number of lipid droplets (P < 0.001) and triglyceride level (P < 0.01) in the cells. FASN gene silencing significantly inhibited proliferation, increased apoptosis rate and suppressed migration of HepG2 cells (P < 0.001). FASN gene silencing inhibits proliferation and migration and promotes apoptosis of HepG2 cells possibly by suppressing lipid synthesis in the cells.

Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells.

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.

The Effects of Bilirubin and Lumirubin on Metabolic and Oxidative Stress Markers.

For severe unconjugated hyperbilirubinemia the gold standard treatment is phototherapy with blue-green light, producing more polar photo-oxidation products, believed to be non-toxic. The aim of the present study was to compare the effects of bilirubin (BR) and lumirubin (LR), the major BR photo-oxidation product, on metabolic and oxidative stress markers. The biological activities of these pigments were investigated on several human and murine cell lines, with the focus on mitochondrial respiration, substrate metabolism, reactive oxygen species production, and the overall effects on cell viability. Compared to BR, LR was found to be much less toxic, while still maintaining a similar antioxidant capacity in the serum as well as suppressing activity leading to mitochondrial superoxide production. Nevertheless, due to its lower lipophilicity, LR was less efficient in preventing lipoperoxidation. The cytotoxicity of BR was affected by the cellular glycolytic reserve, most compromised in human hepatoblastoma HepG2 cells. The observed effects were correlated with changes in the production of tricarboxylic acid cycle metabolites. Both BR and LR modulated expression of PPARα downstream effectors involved in lipid and glucose metabolism. Proinflammatory effects of BR, evidenced by increased expression of TNFα upon exposure to bacterial lipopolysaccharide, were observed in murine macrophage-like RAW 264.7 cells. Collectively, these data point to the biological effects of BR and its photo-oxidation products, which might have clinical relevance in phototherapy-treated hyperbilirubinemic neonates and adult patients.

Candidone Inhibits Migration and Invasion, and Induces Apoptosis in HepG2 Cells.

The aim of the study was to investigate the inhibitory activity of candidone, the active constituent of Derris (D.) indica, on the proliferation, migration, and invasiveness of human hepatoblastoma (HepG2) cells. Cancer cell death was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis-associated morphological changes were observed by phase contrast microscopy. Additionally, Western blotting was used to study protein expression following treatment with candidone, and transwell migration and invasion assays were used for observing cancer cell migration and invasiveness, respectively. The results suggest that candidone possesses potent inhibitory activity against HepG2 cells (concentration, 100 µM; 24 h treatment). Cancer cells treated with candidone exhibited apoptosis-associated changes, including detachment, cell shrinkage and death. Furthermore, candidone was shown to promote cell death by activating caspase-3 and -9, and decreasing the expression of antiapoptotic proteins, including p65, induced myeloid leukemia cell differentiation protein Mcl-1, B-cell lymphoma 2 (Bcl2), Bcl2-associated agonist of cell death and survivin. Moreover, candidone inhibited the migration and invasion abilities of HepG2 cells and decreased the levels of proteins associated with these processes, including phospho-p38 and active matrix metallopeptidase 9. Collectively, the results of the present study indicate that candidone is able to inhibit the proliferation, migration and invasive potential of HepG2 cells.

Synthesis, Anticancer Evaluation, Computer-Aided Docking Studies, and ADMET Prediction of 1,2,3-Triazolyl-Pyridine Hybrids as Human Aurora B Kinase Inhibitors.

A novel series of 1,2,3-triazolyl-pyridine hybrids were prepared through the reaction of the triazole derivative (1) with the appropriate aldehyde (2a–g) and malononitrile or ethyl cyanoacetate in the presence of ammonium acetate in refluxed acetic acid. The chemical composition of the products was established on the basis of spectral and elemental analyses. Aurora B kinase is a protein with diverse biological actions in controlling tumorigenesis by inhibiting apoptosis and promoting proliferation and metastasis, making it an emerging target for diseases such as hepatocellular carcinoma (HCC). Alteration in the target protein expression causes unequal distribution of genetic information, causing HCC. The new compounds were tested for their antihepatic cancer activity, and some of them had strong efficacy against human hepatoblastoma (HepG2) cell lines.