Effects In HepG2 Cells Research Articles

FGF21 affects the glycolysis process via mTOR-HIF1α axis in hepatocellular carcinoma.

Metabolic reprogramming, particularly glycolysis, is essential in processes like cancer and immune response. While FGF21's role in hepatocyte glucose metabolism has been linked to glucose transporters and its impact on aerobic glycolysis and cellular growth in HCC remain unclear. In this study, we investigated FGF21-mediated modulation of glucose metabolism in HCC through mTOR and HIF1α axis in HCC. The study evaluated the dysregulation of FGF21 and its prognostic impact in HCC using various datasets. The literature review was done to identify glycolysis related genes to find significant interaction with FGF21 using stringdb and their correlation in datasets. The regulation of FGF21 was validated in HepG2 cell lines by transfecting FGF21 and measuring its effects on glycolysis, including glucose uptake, lactate levels, and key glycolytic enzymes using rt-PCR. Additionally, the effect of FGF21 transfection on mTOR and HIF1α was also evaluated using rt-PCR. The insilico analysis indicates that the FGF21-mTOR-HIF1α signaling axis regulates glucose metabolism, with mTOR as a central integrator of signals from FGF21 and HIF1α. Invitro experiments showed that silencing FGF21 expression via siRNA reduced glycolytic enzyme expression, glucose uptake, lactate levels, and cell proliferation in HepG2 cells. Conversely, recombinant FGF21 treatment has a reverse effect in HepG2 cells. Additionally, FGF21 treatment also affected mTOR and HIF1α expression, highlighting its role in metabolic regulation and disease through the mTOR-HIF1α axis. The regulation of FGF21 influences glycolysis via the mTOR-HIF1α axis, highlighting its critical role in glucose metabolism and metabolic adaptation in response to energy availability.

Pro-oxidant Tetrapleura tetraptera polyphenol extract parallels quercetin as an in vitro cytotoxic agent in human liver cancer cells

Tetrapleura tetraptera (TT) fruits, also called aidan fruits, are traditionally used in Africa and other parts of the world as a spice in foods and in treating diseases such as arthritis, diabetes, infections and cardiovascular diseases but have remained largely underutilized. To find new ways of utilizing TT dry fruits, this study produced pro-oxidant polyphenols from it by applying the combined effects of high-power, low frequency ultrasound and the Fenton-like mechanism. The efficacy of the pro-oxidant polyphenols as cytotoxic agent was tested in human liver cancer cells (HepG2), evaluating its potential use as an anti-cancer remedy. The Folin Ciocalteau, DPPH, ABTS and cellular antioxidant activity (CAA) assays were used to determine total phenolic content (TPC) and antioxidant property of extracts. Pro-oxidant property of extracts was confirmed using lipid oxidation assays that measured malondialdehyde (MDA) levels and peroxide value (PV) in samples. It was found that TT polyphenol (TTP) extract treated with FeCl3 (TTP-FeCl3) showed poor antioxidant properties evident in its ability to promote lipid oxidation (i.e. increasing MDA levels from 0.17 to 2.53 µmol g−1 and PV from 0.40 to 1.16 meq O2 kg−1 fat). It also induced cytotoxic effects in HepG2 cells. From LC-ESI-MS analysis, the major phenolic compounds and their concentrations in the TTP-FeCl3 extract were kaempferol-3-O-rutinoside (23.75 µg g−1), 4-O-methylgallic acid (19.05 µg g−1), chlorogenic acid (16.22 µg g−1), rutin (16.15 µg g−1), quercetin-3-O-glucuronide (13.19 µg g−1), cyanidin-3-O-galactoside (12.58 µg g−1), orientin (10.99 µg g−1), isorhamnetin-3-O-glucoside (10.11 µg g−1) and gallic acid (10.47 µg g−1). This study showed that prolonged high-power, low-frequency ultrasonication in combination with FeCl3 treatment can be used to produce pro-oxidant polyphenols capable of eliciting cytotoxic effects in HepG2 cells in vitro. Exploring targeted delivery of TTP pro-oxidants with cytotoxic properties has great potential in disease management.

Molecular insights into acetyl triethyl citrate (ATEC) induced toxic effect in HepG2 cells based on multi‐omics integrative analysis

AbstractCitrate esters have become the main alternatives to traditional plasticizers in food packaging materials. However, there is a lack of understanding of their toxic effects, particularly the combined effects and inner mechanisms has not been well studied. Our group pioneered the study on combined toxicity of tributyl citrate (TBC) and acetyl triethyl citrate (ATEC), two commonly co‐used citrate esters in food packaging materials. The results showed that exposure to TBC and ATEC can decrease the viability of HepG2 cells in a dose dependent manner. When the mixtures of ATEC and TBC exposed to HepG2 cells, they exhibited antagonism effect. Therefore, ATEC was selected to investigate the molecular mechanisms with multi‐omics techniques at its 1/4 EC50 concentration. A total of 31 metabolites with significant changes were found as potential biomarkers. The LIPG (Lipase G, endothelial type) and GCLM (glutamate‐cysteine ligase modifier subunit) were identified as differentially expressed genes based on transcriptomic analysis. Moreover, downregulated l‐glutamate and l‐glutamine which participate in TCA cycle, resulting in the collapse of energy production and cytotoxicity. These findings on major metabolic pathways will provide insight into the mechanism of cytotoxicity of HepG2 cells after ATEC exposure.

Exploring Nanocarriers for Boosting Entacapone Bioavailability: A Journey through System Characterization and Assessment of Toxicity and Pharmacological and 2D Permeability Paybacks.

Catechol-O-methyltransferase inhibitors (iCOMT), such as entacapone, have been successfully employed to treat tremor-related symptoms of Parkinson's disease. However, iCOMT has been associated with a short half-life and poor oral bioavailability. Nanobased drug delivery systems have often been used to overcome this type of setbacks. Therefore, entacapone was encapsulated in PEGylated poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) via a nanoprecipitation process, as well as in PEGylated nanostructured lipid carriers (NLCs) using a solvent emulsification/evaporation method. Both nanoformulations presented sub-200 nm populations, with zeta-potential (ZP) values close to -30 mV, and showed stability at different pHs, while maintaining their physicochemical properties mostly intact, presenting only a change in their superficial charge (ZP values), indicating their interaction. Both nanoformulations presented interaction with mucins, which anticipates good permeation and bioavailability for oral and topical administration. No cytotoxic effects were observed for lyophilized PLGA NPs encapsulating entacapone, in which 2-hydroxypropyl-ß-cyclodextrin (HPβCD) was used as a cryoprotectant at 3% concentration (HP-PLGA@Ent), in human hepatocellular carcinoma (HepG2), human neuroblastoma (SH-SY5Y), or human epithelial colorectal adenocarcinoma (Caco-2) cell lines. Conversely, NLCs encapsulating entacapone (W-NLCs@Ent) presented cytotoxic effects on the HepG2 cell line, likely due to intracellular lipid accumulation or storage. Both nanoformulations maintained a COMT inhibition effect in HepG2 cells, using 3-BTD as the COMT probe. An increase of entacapone permeability in both monolayer and coculture models (Caco-2 and Caco-2/HT29-MTX, respectively) was observed for the developed nanoformulations. Overall, this work shows that encapsulated entacapone in different nanocarriers could be a stimulating alternative to solve entacapone setbacks, since they improve its physicochemical properties and permeability while still maintaining the COMT inhibitory activity.

A novel fluorescent probe for viscosity and polarity detection in real tobacco root cells and biological imaging.

The disruption of lipid droplet function is associated with the pathogenesis of various diseases. Clarifying the response behavior of lipid droplets to the microenvironment at the cellular level is of great significance. Plant lipids not only exist in phospholipids in cell membranes, but also in aromatic essential oils. Monitoring the level of lipid droplets in plant cells using fluorescent probes provides a simple method for screening lipid-rich varieties. We synthesized a polarity-viscosity responsive coumarin fluorescent probe, Cou-CN, which achieved sensitive detection of polarity and viscosity in dilute solution environments by constructing this simple probe with ICT and TICT properties and verifying it using Gaussian computational simulation. Cou-CN exhibited good lipid droplet illumination effects in HepG2 cells with a correlation coefficient of 0.92 compared to the commercial lipid droplet dye BODIPY. Additionally, co-staining the probe with the lipophilic commercial dye Nile Red in tobacco root stem seedling cells resulted in a high correlation coefficient of 0.9.

The metabolic response of HepG2 cells to extracellular vesicles derived from Raphanus sativus L. var. caudatus Alef microgreens probed by chemometrics-assisted LC-MS/MS analysis

Extracellular vesicles (EVs) derived from Thai rat-tailed radish (Raphanus sativus L. var. caudatus Alef) microgreens were previously reported as novel bioactive bioparticles against cancer. This study aimed to investigate the metabolic disruption associated with the antiproliferative effect against HepG2 liver cancer cells, a representative of metabolizing cells and tissue. In this study, the neutral red uptake assay was performed to screen for the antiproliferative effect and determine the cytotoxic concentrations of EVs against HepG2 cells. An untargeted approach to cellular metabolomics was conducted using liquid chromatography coupled with the high-resolution mass spectrometry system with multivariate and univariate analyses to determine the metabolic changes of HepG2 liver cancer cells after EV treatment. EVs showed an antiproliferative effect in HepG2 cells with a half-maximal inhibitory concentration (IC50) of 685.5 ± 26.4 and 139.7 ± 4.2 μg/ml at 24 and 48 h, respectively. In the metabolomics study, 163 metabolites were annotated, with 61 significantly altered metabolites. Among these significant metabolites, 18 were related to glycerophospholipid metabolism. Phosphatidylcholine—the important lipid building blocks for cell membranes, lipid mediators for cell proliferation, and immunosuppressive signaling—was mainly decreased by EV treatment. The alteration of cellular phospholipids in cancer was discussed. This finding suggested the possible mechanism of anticancer action of EVs by disrupting phospholipid metabolism and survival signaling in cancer cells. Further studies should be made to confirm EVs' potential as single and combination therapy in vivo to reduce cancer resistance. This may close the gap between in vitro study and clinical setting.

Direct and macrophage stimulation mediated effects of active, inactive, and cell-free supernatant forms of Akkermansia muciniphila and Faecalibacterium duncaniae on hepcidin gene expression in HepG2 cells.

Hepcidin is a crucial regulator of iron homeostasis with protective effects on liver fibrosis. Additionally, gut microbiota can also affect liver fibrosis and iron metabolism. Although the hepatoprotective potential of Akkermansia muciniphila and Faecalibacterium duncaniae, formerly known as F. prausnitzii, has been reported, however, their effects on hepcidin expression remain unknown. We investigated the direct and macrophage stimulation-mediated effects of active, heat-inactivated, and cell-free supernatant (CFS) forms of A. muciniphila and F. duncaniae on hepcidin expression in HepG2 cells by RT-qPCR analysis. Following stimulation of phorbol-12-myristate-13-acetate (PMA) -differentiated THP-1 cells with A. muciniphila and F. duncaniae, IL-6 concentration was assessed via ELISA. Additionally, the resulting supernatant was treated with HepG2 cells to evaluate the effect of macrophage stimulation on hepcidin gene expression. The expression of genes mediating iron absorption and export was also examined in HepG2 and Caco-2 cells via RT-qPCR. All forms of F. duncaniae increased hepcidin expression while active and heat-inactivated/CFS forms of A. muciniphila upregulated and downregulated its expression, respectively. Active, heat-inactivated, and CFS forms of A. muciniphila and F. duncaniae upregulated hepcidin expression, consistent with the elevation of IL-6 released from THP-1-stimulated cells as a macrophage stimulation effect in HepG2 cells. A. muciniphila and F. duncaniae in active, inactive, and CFS forms altered the expression of hepatocyte and intestinal iron-mediated absorption /exporter genes, namely dcytb and dmt1, and fpn in HepG2 and Caco-2 cells, respectively. In conclusion, A. muciniphila and F. duncaniae affect not only directly but also through macrophage stimulation the expression of hepcidin gene in HepG2 cells. These findings underscore the potential of A. muciniphila and F. duncaniae as a potential therapeutic target for liver fibrosis by modulating hepcidin and intestinal and hepatocyte iron metabolism mediated gene expression.

Anticancer Activity and Molecular Mechanisms of Acetylated and Methylated Quercetin in Human Breast Cancer Cells

Quercetin, a flavonoid polyphenol found in many plants, has garnered significant attention due to its potential cancer chemoprevention. Our previous studies have shown that acetyl modification of the hydroxyl group of quercetin altered its antitumor effects in HepG2 cells. However, the antitumor effect in other cancer cells with different gene mutants remains unknown. In this study, we investigated the antitumor effect of quercetin and its methylated derivative 3,3′,4′,7-O-tetramethylquercetin (4Me-Q) and acetylated derivative 3,3′,4′,7-O-tetraacetylquercetin (4Ac-Q) on two human breast cancer cells, MCF-7 (wt-p53, caspase-3-ve) and MDA-MB-231 (mt-p53, caspase-3+ve). The results demonstrated that 4Ac-Q exhibited significant cell proliferation inhibition and apoptosis induction in both MCF-7 and MDA-MB-231 cells. Conversely, methylation of quercetin was found to lose the activity. The human apoptosis antibody array revealed that 4Ac-Q might induce apoptosis in MCF-7 cells via a p53-dependent pathway, while in MDA-MB-231 cells, it was induced via a caspase-3-dependent pathway. Furthermore, an evaluation using a superoxide inhibitor, MnTBAP, revealed 4Ac-Q-induced apoptosis in MCF-7 cells in a superoxide-independent manner. These findings provide valuable insights into the potential of acetylated quercetin as a new approach in cancer chemoprevention and offer new avenues for health product development.

Therapeutic Potential of Sol-Gel ZnO Nanocrystals: Anticancer, Antioxidant, and Antimicrobial Tri-Action.

Zinc oxide nanocrystals (ZnO NCs) hold great promise in nanomedicine with fascinating multifunctional properties. We investigated the therapeutic potential of sol-gel synthesized ZnO NCs with crystal sizes of 52.65 and 25.11 nm, focusing on their anticancer effects on HepG2 and HT29 cells, antioxidant properties, and antimicrobial activity. Both samples displayed a hexagonal wurtzite ZnO structure, wherein the crystal sizes diminished with lower calcination temperatures according to X-ray diffraction. The scanning electron microscopy analysis revealed that lowering the calcination temperature resulted in a decrease in the grain size of the ZnO NCs, as expected. This reduction in grain size combined with a decrease in crystal size resulted in a significant 40% reduction in the reflectance of the ZnO NCs in UV-vis-NIR spectroscopy. It was also observed that the ZnO NCs calcined at higher temperatures exhibited larger particle sizes with a reduced surface area mean of 69.30 μm and a stable negative zeta potential of -11.2 mV. In contrast, the ZnO NCs calcined at lower temperatures exhibited a larger surface area mean of 34.56 μm and a positive zeta potential of +10 mV. In both cell lines, the cytotoxic potential was found to be higher in HepG2 cells. Specifically, when ZnO nanocrystals (NCs) with a crystal size of 52.65 nm were used, the lowest cell viability was observed at a concentration of 5.74 μg/mL. Based on oxidative stress index values, a lower crystal size of ZnO NCs displayed greater effectiveness in HT29 cells, while a higher crystal size of ZnO NCs had pronounced effects in HepG2 cells. Moreover, both ZnO NCs exhibited significant antimicrobial activity against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and Candida parapsilopsis fungus. These findings emphasize sol-gel ZnO NCs' potential as versatile agents in nanomedicine, spurring research on targeted cancer therapies and antimicrobial innovations.

Safety assessment of high fructose corn syrup and fructose used as sweeteners in foods

High Fructose Corn Syrup (HFCS) and Fructose (FR) are widely used sweeteners in many foods and beverages. This study aimed at investigating the cytotoxic effects of HFCS (5%–30%) and FR (62.5–2000 μg/mL) using MTT assay in Human Hepatocellular Carcinoma (HepG2) cells, and genotoxic effects of using Chromosome Aberrations (CAs), Sister Chromatid Exchanges (SCEs), Micronuclei (MN) and comet assays in human lymphocytes. HFCS significantly reduced the cell viability in HepG2 cells at between 7.5% and 30% for 24 and 48 h. 30% HFCS caused a very significant toxic effect. FR had a cytotoxic effect in HepG2 cells at all treatments. However, as fructose concentration decreased, the cell viability decreased. HFCS (10%–20%) and FR (250–2000 μg/mL) decreased the mitotic index at higher concentrations. IC50 value was found to be a 15% for 48 h. IC50 value of FR was detected as 62.5 μg/mL for 24 h and 48 h. HFCS significantly increased CAs frequency at 15% and 20%. FR significantly increased the frequency of CAs at 250, 1000, and 2000 μg/mL for 48 h. Both sweeteners increased the frequency of SCEs at all concentrations. HFCS (15% and 20%) and FR (250, 1000, and 2000 μg/mL) induced MN frequency at higher concentrations. HFCS caused DNA damage in comet assay at 10% −30%. FR increased tail intensity and moment at 125-2000 μg/mL and tail length at 62.5, 250 and 500 μg/mL. Therefore, HFCS and FR are clearly seen to be cytotoxic and genotoxic, especially at higher concentrations.

Assessing the mitochondrial safety profile of the molnupiravir active metabolite, β-d-N4-hydroxycytidine (NHC), in the physiologically relevant HepaRG model.

β-d-N4-Hydroxycytidine (NHC) is the active metabolite of molnupiravir, a broad-spectrum antiviral approved by the MHRA for COVID-19 treatment. NHC induces lethal mutagenesis of the SARS-CoV-2 virus, undergoing incorporation into the viral genome and arresting viral replication. It has previously been reported that several nucleoside analogues elicit off-target inhibition of mitochondrial DNA (mtDNA) or RNA replication. Although NHC does not exert these effects in HepG2 cells, HepaRG are proven to be advantageous over HepG2 for modelling nucleoside analogue-induced mitochondrial dysfunction. Therefore, the objective of this work was to assess the mitotoxic potential of NHC in HepaRG cells, a model more closely resembling physiological human liver. Differentiated HepaRG cells were exposed to 1-60 μM NHC for 3-14 days to investigate effects of sub-, supra-, and clinically-relevant exposures (in the UK, molnupiravir for COVID-19 is indicated for 5 days and reported Cmax is 16 μM). Following drug incubation, cell viability, mtDNA copy number, mitochondrial protein expression, and mitochondrial respiration were assessed. NHC induced minor decreases in cell viability at clinically relevant exposures, but did not decrease mitochondrial protein expression. The effects on mtDNA were variable, but typically copy number was increased. At supra-clinical concentrations (60 μM), NHC reduced mitochondrial respiration, but did not appear to induce direct electron transport chain dysfunction. Overall, NHC does not cause direct mitochondrial toxicity in HepaRG cells at clinically relevant concentrations, but may induce minor cellular perturbations. As HepaRG cells have increased physiological relevance, these findings provide additional assurance of the mitochondrial safety profile of NHC.

Determination of the cyto-genotoxic effects of sodium acetate and sodium sulfite by MTT and comet assays

Sodium acetate (NaA) and sodium sulfite (NaS) are two food additives in the class of preservatives. In this study, 3-(4,5-dimethyl thiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay was established to detect the cytotoxicity, and comet assay was used to determine the genotoxicity of NaA and NaS. For the MTT assay, human hepatocellular carcinoma (HepG2) cells were treated with different concentrations of each preservative (15.63, 31.25, 62.50, 125, 250, 500, 1000 and 2000 µg/mL for NaA; 3.91, 7.81, 15.62, 31.25, 62.50, 125, 250 and 500 µg/mL for NaS, respectively) for 24-h. non-treated wells used as control (only medium) were included. Comet assay was performed on lymphocytes isolated from healthy donors with multiple concentrations of NaA (15.63, 31.25, 62.50, 125, 250 µg/mL) and NaS (3.91, 7.81, 15.62, 31.25, 62.50 µg/mL) for 1 h. A negative (distilled water) and a positive control (100 µM H2O2) were also included. Significant cytotoxic activity was detected for NaA and NaS only at the highest concentration. Besides, both substances significantly increased DNA damage compared to the control at almost all concentrations (except at low concentrations). In general, both food preservatives exhibited weak cytotoxic effects in HepG2 cells. These food preservatives showed genotoxic activity, especially at higher concentrations.

Phytochemical Screening and In Vitro Hepatoprotective Activity of Japanese Glorybower (Clerodendrum japonicum) Leaves

Objective The objective of this study was to assess the in vitro hepatoprotective activity of Clerodendrum japonicum leaves. Methods Clerodendrum japonicum leaves were subjected to extraction process followed by phytochemical studies. The obtained extracts were studied to assess the cytotoxicity and in vitro hepatoprotective activity using HepG2 cell line culture. Results The phytochemical analysis of Clerodendrum japonicum leaves signaled the presence of phytosterols terpenoids phenolics tannins flavonoids carbohydrates and saponins. The alcoholic extract showed 38.50 cell growth inhibition indicating lower cytotoxicity of alcoholic extract compared to the other extracts and also 35.99 of protective effect in HepG2 cells against paracetamol induced toxicity in comparison to Silymarin as the standard drug. Conclusion The present study suggests that the ethanolic extract of Clerodendrum japonicum leaves can be useful as a hepatoprotective agent.nbsp

Comparative toxicity of coal and coal ash: Assessing biological impacts and potential mechanisms through in vitro and in vivo testing

BackgroundCoal and coal ash present inorganic elements associated with negative impacts on environment and human health. The objective of this study was to compare the toxicity of coal and coal ash from a power plant, assess their inorganic components, and investigate the biological impacts and potential mechanisms through in vitro and in vivo testing. MethodsParticle-Induced X-ray Emission method was used to quantify inorganic elements and the toxicity was evaluated in Caenorhabditis elegans and Daphnia magna in acute and chronic procedures. The genotoxic potential was assessed using alkaline and FPG-modified Comet assay in HepG2 cells and mutagenicity was evaluated using Salmonella/microsome assay in TA97a, TA100, and TA102 strains. ResultsInorganic elements such as aluminum (Al) and chromium (Cr) were detected at higher concentrations in coal ash compared to coal. These elements were found to be associated with increased toxicity of coal ash in both Caenorhabditis elegans and Daphnia magna. Coal and coal ash did not induce gene mutations, but showed genotoxic effects in HepG2 cells, which were increased using the FPG enzyme, indicating DNA oxidative damage. ConclusionsThe combined findings from bioassays using C. elegans and D. magna support the higher toxicity of coal ash, which can be attributed to its elevated levels of inorganic elements. The genotoxicity observed in HepG2 cells confirms these results. This study highlights the need for continuous monitoring in areas affected by environmental degradation caused by coal power plants. Additionally, the analysis reveals significantly higher concentrations of various inorganic elements in coal ash compared to coal, providing insight into the specific elemental composition contributing to its increased toxicity.

Comparative bioactivity analysis of hydro-methanolic extracts of root, leaf, and inflorescence of Tupistra nutans Wall. ex Lindl: An in vitro study on antioxidant and anti-hepatocellular carcinoma activity

Tupistra nutans, native to the Eastern Himalayas, is traditionally used as vegetables and in manaand pressure. Despite being a medicinal agricultural plant, no in-depth antioxidant and anticancer study was done. Therefore, in this study, we investigated antioxidant and anti-hepatocellular carcinoma activities of hydro-methanolic extracts of its root (ETNRt), leaf (ETNLf), and inflorescence (ETNIn). DPPH, ABTS, FRAP, and total antioxidant capacity (TAC) assays revealed that ETNLf had the highest antioxidant potential. Also, extracts showed antioxidant activity in human hepatocellular carcinoma (HepG2) cells by reducing H2O2-induced ROS generation at lower concentrations. However, at higher concentrations, ETNRt, ETNLf, and ETNIn had anticancer effects in HepG2 cells with IC50 (12 h) values of 92.62, 34.60, and 85.55 μg/ml respectively, which was further confirmed by cell migration and clonogenicity reduction. Spectrofluorometric and fluorescence microscopy revealed that ETNRt, ETNLf, and ETNIn increased intracellular ROS, superoxide, and calcium levels and reduced mitochondrial membrane potential and mass. ETNRt, ETNLf, and ETNIn also depleted reduced glutathione levels in HepG2. Acridine orange/propidium iodide and scanning electron microscopy analysis confirmed the membrane damage and apoptotic changes, whereas confocal microscopic analysis with DAPI staining indicated cytoplasmic shrinkage and DNA damage. GC-MS analysis of ETNRt, ETNLf, and ETNIn revealed the differential distribution of anticancer phytochemicals such as beta-citronellol, piperidine, diosgenin, beta-sitosterol, and stigmasterol in Tupistra nutans. Our study suggested that Tupistra nutans had a promising antioxidant and in-vitro anti-hepatocellular carcinoma activity. However, the anticancer potential of inflorescence, used as a vegetable, decreased when it was cooked compared to its hydro-methanolic extract.

İZOİMPERATORİN ARACILIKLI ANTİKANSER AKTİVİTE: MİTOKONDRİYAL DİSFONKSİYONUN HEPG2 HÜCRELERİNDEKİ ROLÜ

Objective: The first goal of the present study is to investigate the role of mitochondria due to the Crabtree effect in HepG2 cells exposed to ISO in either glucose- or galactose-conditioned media. The second aim is to predict the interactions between electron transport chain (ETC) complexes and ISO, which might be the possible reason for mitochondrial dysfunction. Material and Method: Cell viability and membrane damage for HepG2 cells exposed to ISO (12.5, 25, 50, 100, and 250 µM) were assessed by MTT and LDH leakage assays in either glucose- or galactose-conditioned media. The affinity of ISO to ETC complexes was also determined by a molecular docking study. Result and Discussion: MTT assay showed that 250 µM ISO leads to cytotoxic activity in glucose-conditioned media, while 25 µM and higher concentrations of ISO decrease cell viability in galactose-conditioned media. A membrane damage assay conducted in a glucose-conditioned media assay revealed that 250 µM ISO disrupts the cell membrane. 100 and 250 µM ISO increased membrane damage in galactose-conditioned media. According to docking simulations, binding affinities of ISO to ETC complexes are in descending order: Complex IV > Complex I > Complex III > Complex II. Inhibition of complex IV by ISO inhibits the transfer of electrons from cytochrome c to oxygen, and the proton gradient collapses. The present study proposed that ISO leads to mitochondrial dysfunction via inhibition of the ETC.

Induction of Apoptosis and Cell Cycle Arrest by Ethyl Acetate Extract of Salsola kali and Flavonoid Constituents Analysis

In our previous study on Salsola kali, the ethyl acetate extract revealed an in vitro cytotoxic effect against the tumor cell lines (MCF-7, HepG2, and A549). Therefore, this study was conducted to identify phenolic and flavonoid components in this extract by LC-ESI-TOF/MS technique and explore the mechanism of the cytotoxic effect on the progression of the cell cycle and the induction of apoptosis by flow cytometry. Further, protein markers were evaluated as cytotoxicity mechanisms to confirm apoptosis initiation. LC-ESI-TOF/MS analysis identified eleven phenolics and forty-five flavonoid compounds from the ethyl acetate extract. Ethyl acetate extract induced G1 cell population arrest in HepG2 cells after 24 hours of exposure. The flow cytometry data confirmed that ethyl acetate primarily initiates cell death by starting early and late apoptotic manners. Induction of apoptosis by ethyl acetate extract was proved by a significant increase in cytochrome C, caspase 3, and Bax levels, while the level of antiapoptotic BCl2 was significantly decreased. Moreover, the Bax/BCl2 ratio was increased after treatment in a dose-dependent manner of ethyl acetate extract compared to that of the untreated group. These findings suggest that ethyl acetate extract may have a remarkable anticancer effect in HepG2 cells through cell cycle regulation and apoptosis.

Lipid metabolic disturbance induced by triphenyl phosphate and hydroxy metabolite in HepG2 cells

Triphenyl phosphate (TPHP) has been widely used as flame retardants and been detected with increasing frequency in environment. TPHP can transform into mono-hydroxylated phosphate (OH-TPHP) and diester diphenyl phosphate (DPHP) through biotransformation. So far, information on the cytotoxicity and molecular regulatory mechanisms of TPHP metabolites are still limit. This study investigated the adverse effects of TPHP, OH-TPHP, and DPHP in HepG2 cells in terms of cell proliferation, lactate dehydrogenase release, reactive oxygen species generation, and mitochondrial membrane potential. The transcriptomic changes were measured using RNA sequencing, and bioinformatics characteristics including biological functions, signal pathways and protein-protein interaction were analyzed to explore the potential molecular mechanisms. Results displayed that the order of cytotoxicity was OH-TPHP> TPHP> DPHP. The prioritized biological functions changes induced by TPHP and OH-TPHP were correlated with lipid metabolism. Significant lipid accumulation was observed as confirmed by increased total cholesterol and triglycerides contents, and enhanced oil red O staining. Enrichment of PPARα/γ and down-stream genes suggested the participation of PPARs signal pathway in lipid metabolism disorder. In addition, TPHP and OH-TPHP induced endoplasmic reticulum stress (ERS), which was further confirmed by the ERS inhibitor experiment. In general, TPHP and OH-TPHP had obvious cytotoxic effects in HepG2 cells. PPARs signal pathway and endoplasmic reticulum stress may be involved in the lipid metabolism disorder induced by TPHP and OH-TPHP.

Synthesis and Anticancer Activity of Ergosterol Peroxide Hybrids With Paclitaxel Side Chain Inducing Apoptosis in Human Hepatoma Carcinoma Cells

The antitumor activities of natural paclitaxel (PTX), semisynthetic docetaxel, and cabazitaxel are highly dependent on their C-13 side chains. Therefore, using natural ergosterol peroxide (EP, 1) as the lead compound, two EP-PTX hybrids (EP-A2 and EP-B2) were prepared and their antitumor activities were evaluated against 4 kinds of human MCF-7, HepG2, HCT-116, and A549 cell lines in vitro. The results showed that both EP-A2 and EP-B2 inhibited the growth of all four kinds of tested tumor cell lines. For paclitaxel-resistant MCF-7 cells, both EP-A2 and EP-B2 showed significant inhibitory activity with relatively low IC50 values (9.39 μM and 8.60 μM, respectively). In addition, EP-B2 inhibited the growth of the HepG2 cells (IC50 = 7.82 μM) more successfully than EP. Preliminary studies of the mechanism suggest that EP-B2 could arrest the G1 phase transition in HepG2 cells. In addition, EP-B2 showed an obvious apoptosis-inducing effect in HepG2 cells, as detected by the Annexin V/PI binding assay and the Western blot assay. Hybrid EP-B2 has the potential to become a novel antitumor drug through further study of the mechanism of action and its structural modifications.

Induction of clastogenesis and gene mutations by carbamazepine (at its therapeutically effective serum levels) in mammalian cells and the dependence on human CYP2B6 enzyme activity

Carbamazepine (CBZ, an antiepileptic) is metabolized by multiple CYP enzymes to its epoxide and hydroxides; however, whether it is genotoxic remains unclear. In this study, molecular docking (CBZ to CYPs) and cytogenotoxic toxicity assays were employed to investigate the activation of CBZ for mutagenic effects, in various mammalian cell models. Docking results indicated that CBZ was valid as a substrate of human CYP2B6 and 2E1, while not for CYP1A1, 1A2, 1B1 or 3A4. In the Chinese hamster (V79) cell line and its derivatives genetically engineered for the expression of human CYP1A1, 1A2, 1B1, 2E1 or 3A4 CBZ (2.5 ~ 40μM) did not induce micronucleus, while in human CYP2B6-expressing cells CBZ significantly induced micronucleus formation. In a human hepatoma C3A cell line, which endogenously expressed CYP2B6 twofold higher than in HepG2 cells, CBZ induced micronucleus potently, which was blocked by 1-aminobenzotriazole (inhibitor of CYPs) and ticlopidine (specific CYP2B6 inhibitor). In HepG2 cells CBZ did not induce micronucleus; however, pretreatment of the cells with CICTO (CYP2B6 inducer) led to micronucleus formation by CBZ, while rifampicin (CYP3A4 inducer) or PCB126 (CYP1A inducer) did not change the negative results. Immunofluorescent assay showed that CBZ selectively induced centromere-free micronucleus. Moreover, CBZ induced double-strand DNA breaks (γ-H2AX elevation, by Western blot) and PIG-A gene mutations (by flowcytometry) in C3A (threshold being 5μM, lower than its therapeutic serum concentrations, 17 ~ 51μM), with no effects in HepG2 cells. Clearly, CBZ may induce clastogenesis and gene mutations at its therapeutic concentrations, human CYP2B6 being a major activating enzyme.