Human Hepatoma G2 Research Articles

Locusta migratoria hydrolysates attenuate lipopolysaccharide (LPS)/D-Galactosamine (D-Gal)-induced cytotoxicity and inflammation in Hep G2 cells via NF-κB signaling suppression

The prolonged state of hepatic inflammation can lead to liver damage, a critical driving force in the progression of liver-related diseases. Locusta migratoria (LM), an edible insect, is recognized for its protein richness and potential to produce a range of bioactive polypeptides, presenting a novel solution for liver disease. This study investigated the hepatoprotective effects of LM hydrolysates in human hepatoma G2 (Hep G2) cells challenged with lipopolysaccharide (LPS)/D-Galactosamine (D-Gal), a model of liver injury. Remarkably, LM hydrolysates significantly ameliorated cell damage, as evidenced by the inhibition of the LPS/D-Gal-induced decrease in cell viability and reduction in lactate dehydrogenase (LDH) release. Furthermore, LM hydrolysates alleviated the release of aspartate aminotransferase (AST) from cells exposed to LPS/D-Gal and lowered the secretion of inflammatory cytokines while suppressing the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a key pathway in inflammation. In particular, LM-N hydrolysate mitigated hepatotoxicity by attenuation of inflammatory responses to reduce interleukin 6 (IL-6) levels, and NF-κB nuclear translocation. These findings suggest that LM hydrolysates could potentially offer hepatoprotective effects by mitigating the inflammatory responses induced by LPS/D-Gal.

Marein reduces lipid levels via modulating the PI3K/AKT/mTOR pathway to induce lipophagy

Ethnopharmacological relevanceThe capitulum of Coreopsis tinctoria Nutt. (CT, Xue-Ju in Chinese) is a precious medicine in Xinjiang Uygur Autonomous region of China. The Coreopsis tinctoria Nutt. is used to prevent and treat dyslipidemia, coronary heart disease, etc. Recent studies have shown that its extract has a pharmacological effect on hyperlipidemia and hyperglycemia. Aim of the studyThe study aimed to systematically evaluate the lipid-lowering activity of CT through a mice model of hyperlipidemia and a human hepatoma G2 (HepG2) cells model of lipid accumulation, and to investigate its main active components and mechanism. Materials and methodsBiochemical analysis of blood/liver lipids and liver histopathology were used to evaluate the effect of the aqueous extract of Coreopsis tinctoria Nutt. (AECT) on hyperlipidemia mice. High-performance liquid chromatography (HPLC) analysis was used to identify the main components in the AECT. Oil red O staining, immunofluorescence, western blotting, and determination of the total cholesterol (TC), total triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were used to further study the effect and potential mechanism of the AECT main components on sodium oleate-induced lipid accumulation in HepG2 cells. ResultsWe confirmed the lipid-lowering activity of the aqueous extract and further identified flavonoids as its main components. Among them, five Coreopsis tinctoria Nutt. flavonoids mixture (FM) significantly reduced lipid droplet area, lipid content, TC, TG, and LDL-C levels, and elevated HDL-C levels in HepG2 cells induced by sodium oleate. Furthermore, they increased lipophagy in HepG2 lipid-accumulating cells, while decreasing the ratio of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR. Most importantly, marein may be a key component. ConclusionsOur study demonstrated that AECT, with flavonoids as the main component, can improve diet-induced hyperlipidemia in obese mice. Among the main five flavonoids, marein plays a key role in promoting lipophagy by regulating the PI3K/AKT/mTOR pathway, resulting in a lipid-lowering effect.

Molecular mechanism of Ganji Fang in the treatment of hepatocellular carcinoma based on network pharmacology, molecular docking and experimental verification technology.

Background: Hepatocellular carcinoma (HCC) is a malignant tumor harmful to human health. Ganji Fang (GJF) has good clinical efficacy in the treatment of HCC, but its mechanism is still unclear. Objective: The aim of this study was to investigate the mechanism of action of GJF in the treatment of HCC through network pharmacology, molecular docking and in vitro experiments. Methods: A series of network pharmacology methods were used to identify the potential targets and key pathways of GJF in the treatment of HCC. Then, molecular docking technology was used to explore the binding ability of key active ingredients and targets in GJF. Multiple external databases were used to validate the key targets. In in vitro experiments, we performed MTT assays, wound-healing assays, cell cycle assays, apoptosis assays and RT‒qPCR to verify the inhibitory effect of GJF on the Human hepatoma G2 (HepG2) cells. Result: A total of 162 bioactive components and 826 protein targets of GJF were screened, and 611 potential targets of HCC were identified. Finally, 63 possible targets of GJF acting on HCC were obtained. KEGG enrichment analyses showed that the top five pathways were the cell cycle, cellular senescence, p53 signaling pathway, PI3K/Akt signaling pathway, and progesterone-mediated oocyte maturation. Among them, we verified the PI3K/Akt signaling pathway. CCNE1, PKN1, CCND2, CDK4, EPHA2, FGFR3, CDK6, CDK2 and HSP90AAI were enriched in the PI3K/Akt pathway. The molecular docking results showed that the docking scores of eight active components of GJF with the two targets were all less than -5.0, indicating that they had certain binding activity. In vitro cell experiments showed that GJF could inhibit the proliferation and migration of HepG2 cells, block the cell cycle and induce apoptosis of HepG2 cells, which may be related to the PI3K/Akt signaling pathway. In summary, EPHA2 may be an important target of GJF in HCC, and pachymic acid may be an important critical active compound of GJF that exerts anticancer activity. Conclusion: In general, we demonstrated, for the first time, that the molecular mechanism of GJF in HCC may involve induction of G0/G1 phase cycle arrest through inhibition of the PI3K/Akt signaling pathway and promote apoptosis of hepatoma cell lines. This study provides a scientific basis for the subsequent clinical application of GJF and the in-depth study of its mechanism.

Metformin Protects Against Sunitinib-induced Cardiotoxicity: Investigating the Role of AMPK.

Sunitinib is associated with cardiotoxicity through inhibition of AMP-protein kinase (AMPK) signaling. By contrast, the common antidiabetic agent metformin has demonstrated cardioprotection through indirect AMPK activation. In this study, we investigate the effects of metformin during sunitinib-induced cytotoxicity. Left ventricular developed pressure, coronary flow, heart rate, and infarct size were measured in Langendorff-perfused rat hearts treated with 1 µM sunitinib ±50 µM metformin ±1 µM human equilibrative nucleoside transporter inhibitor S-(4-Nitrobenzyl)-6-thioinosine (NBTI). Western blot analysis was performed for p-AMPKα levels. Primary isolated cardiac myocytes from the left ventricular tissue were used to measure live cell population levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess adjunctive treatment of and metformin in human hepatoma G2 and promyelocytic leukemia (HL-60) cells treated with 0.1-100 µM sunitinib ±50 µM metformin. In the perfused hearts, coadministration of metformin attenuated the sunitinib-induced changes to left ventricular developed pressure, infarct size, and cardiac myocyte population. Western blot analysis revealed a significant decrease in p-AMPKα during sunitinib treatment, which was attenuated after coadministration with metformin. All metformin-induced effects were attenuated, and NBTI was coadministered. The MTT assay demonstrated an increase in the EC50 value during coadministration of metformin with sunitinib compared with sunitinib monotherapy in hepatoma G2 and HL-60 cell lines, demonstrating the impact and complexity of metformin coadministration and the possible role of AMPK signaling. This study highlights the novel cardioprotective properties of metformin and AMPK activation during sunitinib-induced cardiotoxicity when administered together in the Langendorff heart model.

Rational deuteration of dronedarone attenuates its toxicity in human hepatic HepG2 cells.

Deuteration is a chemical modification strategy that has recently gained traction in drug development. The replacement of one or more hydrogen atom(s) in a drug molecule with its heavier stable isotope deuterium can enhance its metabolic stability and pharmacokinetic properties. However, it remains uninterrogated if rational deuteration at bioactivation "hot-spots" could attenuate its associated toxicological consequences. Here, our preliminary screening with benzofuran antiarrhythmic agents first revealed that dronedarone and its major metabolite N-desbutyldronedarone elicited a greater loss of viability and cytotoxicity in human hepatoma G2 (HepG2) cells as compared with amiodarone and its corresponding metabolite N-desethylamiodarone. A comparison of dronedarone and its in-house synthesized deuterated analogue (termed poyendarone) demonstrated that deuteration could attenuate its in vitro toxicity in HepG2 cells by modulating the extent of mitochondrial dysfunction, reducing the dissipation of mitochondrial membrane potential, and evoking a distinct apoptotic kinetic signature. Furthermore, although pretreatment with the CYP3A inducer rifampicin or the substitution of glucose with galactose in the growth media significantly augmented the loss of cell viability elicited by dronedarone and poyendarone, a lower loss of cell viability was consistently observed in poyendarone across all concentrations. Taken together, our preliminary investigations suggested that the rational deuteration of dronedarone at its benzofuran ring reduces aberrant cytochrome P450 3A4/5-mediated bioactivation, which attenuated its mitochondrial toxicity in human hepatic HepG2 cells.

Gryllus bimaculatus De Geer hydrolysates alleviate lipid accumulation, inflammation, and endoplasmic reticulum stress in palmitic acid-treated human hepatoma G2 cells

Ethnopharmacological relevanceNonalcoholic fatty liver disease (NAFLD) is one of the most common hepatic diseases closely intertwined with saturated fatty acids intake. Therefore, various studies are being conducted to find natural substances to prevent either the onset or progression of NAFLD. According to traditional medicinal literature, it has been reported that Gryllus bimaculatus De Geer (GB) has systemic detoxifying activity; however, the preventive effects of GB on NAFLD have not been elucidated to date. Aim of studyTo evaluate the potential of GB as a material for the mitigation of NAFLD, we investigated the effects of GB hydrolysates on the hepatic lipid accumulation, inflammation, and endoplasmic reticulum (ER) stress in human hepatoma G2 (Hep G2) cells treated with palmitic acid (PA). MethodsSteamed and dried GB was defatted, pulverized, and then lyophilized following hydrolyzation using Neutrase® (GB-N) or Flavourzyme® (GB-F). Hep G2 cells were incubated with GB-N or GB-F at various concentrations (0, 0.25, 0.5, and 1 mg/mL) for 24 h, and then PA was treated for another 24 h. ResultsThe GB-N and GB-F significantly prevented the PA-induced intracellular lipid accumulation in the human liver cells (p < 0.05). Moreover, the GB-N and GB-F increased the hepatic cellular viability against the PA-treatment (p < 0.05). In addition, the GB-N and GB-F significantly ameliorated the PA-inducible proinflammatory cytokines mRNA expression, such as tumor necrosis factor-α and interleukin-1β, compared to the PA-treated hepatic cells (p < 0.05). Furthermore, the GB-N and GB-F inhibited the PA-inducible lipogenic mRNA expression, such as fatty acid synthase, sterol regulatory element-binding protein 1c, and peroxisome proliferator-activated receptor-γ (p < 0.05). Moreover, the GB-N and GB-F alleviated the ER stress-related mRNA expression, such as glucose regulatory protein 78 and X-box binding protein increased in PA-treated cells (p < 0.05). ConclusionsThese results indicate that GB-N and GB-F could be used as materials to prevent the NAFLD onset or progression with alleviating hepatic lipid accumulation, inflammation, and ER stress.

Simultaneous Tests of Theaflavin-3,3'-digallate as an Anti-Diabetic Drug in Human Hepatoma G2 Cells and Zebrafish (Danio rerio).

Theaflavin-3,3′-digallate (TF3) is the most important theaflavin monomer in black tea. TF3 was proved to reduce blood glucose level in mice and rats. However, the elaborate anti-diabetic mechanism was not well elucidated. In this work, human hepatoma G2 (HepG2) cells and zebrafish (Danio rerio) were used simultaneously to reveal anti-diabetic effect of TF3. The results showed that TF3 could effectively rise glucose absorption capacity in insulin-resistant HepG2 cells and regulate glucose level in diabetic zebrafish. The hypoglycemic effect was mediated through down-regulating phosphoenolpyruvate carboxykinase and up-regulating glucokinase. More importantly, TF3 could significantly improve β cells regeneration in diabetic zebrafish at low concentrations (5 μg/mL and 10 μg/mL), which meant TF3 had a strong anti-diabetic effect. Obviously, this work provided the potential benefit of TF3 on hypoglycemic effect, regulating glucose metabolism enzymes, and protecting β cells. TF3 might be a promising agent for combating diabetes.

Fumonisin B1 Epigenetically Regulates PTEN Expression and Modulates DNA Damage Checkpoint Regulation in HepG2 Liver Cells.

Fumonisin B1 (FB1), a Fusarium-produced mycotoxin, is found in various foods and feeds. It is a well-known liver carcinogen in experimental animals; however, its role in genotoxicity is controversial. The current study investigated FB1-triggered changes in the epigenetic regulation of PTEN and determined its effect on DNA damage checkpoint regulation in human liver hepatoma G2 (HepG2) cells. Following treatment with FB1 (IC50: 200 µM; 24 h), the expression of miR-30c, KDM5B, PTEN, H3K4me3, PI3K, AKT, p-ser473-AKT, CHK1, and p-ser280-CHK1 was measured using qPCR and/or Western blot. H3K4me3 enrichment at the PTEN promoter region was assayed via a ChIP assay and DNA damage was determined using an ELISA. FB1 induced oxidative DNA damage. Total KDM5B expression was reduced, which subsequently increased the total H3K4me3 and the enrichment of H3K4me3 at PTEN promoters. Increased H3K4me3 induced an increase in PTEN transcript levels. However, miR-30c inhibited PTEN translation. Thus, PI3K/AKT signaling was activated, inhibiting CHK1 activity via phosphorylation of its serine 280 residue preventing the repair of damaged DNA. In conclusion, FB1 epigenetically modulates the PTEN/PI3K/AKT signaling cascade, preventing DNA damage checkpoint regulation, and induces significant DNA damage.

Human-on-Leaf-Chip: A Biomimetic Vascular System Integrated with Chamber-Specific Organs.

The vascular network is a central component of the organ-on-a-chip system to build a 3D physiological microenvironment with controlled physical and biochemical variables. Inspired by ubiquitous biological systems such as leaf venation and circulatory systems, a fabrication strategy is devised to develop a biomimetic vascular system integrated with freely designed chambers, which function as niches for chamber-specific vascularized organs. As a proof of concept, a human-on-leaf-chip system with biomimetic multiscale vasculature systems connecting the self-assembled 3D vasculatures in chambers is fabricated, mimicking the in vivo complex architectures of the human cardiovascular system connecting vascularized organs. Besides, two types of vascularized organs are built independently within the two halves of the system to verify its feasibility for conducting comparative experiments for organ-specific metastasis studies in a single chip. Successful culturing of human hepatoma G2 cells (HepG2s) and mesenchymal stem cells (MSCs) with human umbilical vein endothelial cells (HUVECs) shows good vasculature formation, and organ-specific metastasis is simulated through perfusion of pancreatic cancer cells and shows distinct cancer encapsulation by MSCs, which is absent in HepG2s. Given good culture efficacy, study design flexibility, and ease of modification, these results show that the bioinspired human-on-leaf-chip possesses great potential in comparative and metastasis studies while retaining organ-to-organ crosstalk.

Subcritical water extraction, identification, antioxidant and antiproliferative activity of polyphenols from lotus seedpod

In this study, polyphenols from lotus seedpod were extracted by subcritical water. Based on single factor experiments, the extraction conditions were optimized by response surface methodology (RSM). Under optimal conditions [temperature of 140 °C, time of 20 min, liquid-solid ratio of 70 mL/g and 4‰ (W/V) NaHSO3], the maximal yeild of lotus seedpod polyphenols (LSPP) was 178.32 mg of gallic acid equivalents (mg GAE) per gram dry weight (g DW), significantly higher than that of hot water extraction (HWE). This study further investigated antioxidant activity of LSPP. The results showed that, compared with HWE, LSPP extracted by SWE had better reducing power and ability to scavenge DPPH, ABTS*+ and NO2−, (P < 0.05). In addition, the antiproliferative ability on human hepatoma G2 (HepG2) cells of LSPP extracted by SWE was also evaluated to be significant (P < 0.05). Moreover, the antioxidant and antiproliferative activity of LSPP were found to be positively correlated with the polyphenols concentration. Detailed HPLC-ESI-MSn analyses with literatures and contrasted with authentic standards allowed the identification of 8 compounds (proanthocyanidin dimer 1, proanthocyanidin dimer 2, catechin, cyanidin-3-O-glucoside, quercetin-3-O-glucuronoside, isoquercetin, kaempferol-3-O-glucuronide and isorhamnetin) in LSPP (SWE). This work can provide a reference for the utilization of subcritical water extraction in the field of natural product extraction and the development and utilization of lotus seedpod as bioactive materials.

Subcritical water extraction, identification and antiproliferation ability on HepG2 of polyphenols from lotus seed epicarp

In this study, polyphenols were extracted with subcritical water (SWE) from lotus seed epicarp. Based on single factor experiments, the extraction parameters were optimized by response surface methodology (RSM). Lotus seed epicarp polyphenols (LSEP) were purified by column chromatography (AB-8 macroporous resin) and identified by HPLC-ESI-MS. The antiproliferation ability of LSEP was also investigated. The optimal extraction parameters for the polyphenols were the temperature of 160℃, the time of 15 min, the liquid-solid ratio of 60 mL/g and the sodium bisulfite (NaHSO3) addition amount of 2‰ (W/V). Under these extraction conditions, the maximal yeild of LSEP was 88.72 mg of gallic acid equivalents (GAE) per 1 g dry weight (DW), significantly higher than that of hot water extraction (HWE). Ten polyphenols were identified and quantified. Total content of ten polyphenols was 896.604 mg/g in LSEP. This study also found that LSEP extracted by SWE has better antiproliferation ability on human hepatoma G2 (HepG2) cells than that of HWE.

Design, synthesis and anticancer activity evaluation of aziridine-1,2,3-triazole hybrid derivatives

Abstract New 1-aryl-4-[(aziridine-1-yl)diarylmethyl]-5-methyl-1H-1,2,3-triazole derivatives 7a–i were synthesized by a one-pot reaction of diaryl-(1-aryl-5-methyl-1H-1,2,3-triazol-4-yl)methanols 6a–i derived from 1-aryl-5-methyl-1H-1,2,3-triazole-4-carboxylic acids 4a–c. Structures of compounds 7a–i were confirmed by analysis of proton nuclear magnetic resonance (1H NMR) and carbon-13 nuclear magnetic resonance (13C NMR), mass spectrometry (MS) and infrared (IR) data. The structure of compound 7f was studied by X-ray diffraction analysis. The anticancer activities of compounds 7a–i against human leukemia HL-60 cells and human hepatoma G2 cells were evaluated. Some of the compounds are highly active.

Patulin induced ROS-dependent autophagic cell death in Human Hepatoma G2 cells

Patulin (PAT) is a secondary metabolite produced by certain species of Penicillium, Byssochlamys and Aspergillus. It has been shown to induce liver toxicity, but the possible molecular mechanisms are not completely elucidated. In our study, we treated Human Hepatoma G2 (HepG2) cells by 3-methyladenine (3-MA), an autophagosome formation inhibitor, and rapamycin, an autophagosome formation stimulator. The results showed that 3-MA protected the HepG2 cells against PAT cytotoxicity, while rapamycin decreased the cell viability. Thus, autophagy may play an important role in PAT-induced toxicity. To uncover the mechanism by which cells decrease proliferation and activation of autophagy, we found that collapses of mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) level were increased under treatment with PAT. Further, we elucidated that the expression of p-Akt1 and p-MTOR was inhibited during this process. N-acetyl-l-cysteine (NAC), a ROS inhibitor, protected against PAT-induced cytotoxicity, decreased the protein expression of LC3-II, and up-regulated the level of p-Akt1 and p-MTOR. These findings suggested that PAT-induced autophagic cell death was ROS-dependent in HepG2 cells. In conclusion, it is possible that PAT elicited autophagy through ROS-Akt1-MTOR pathway in the HepG2 cells.

Effects of 2-amino-9H-pyrido[2,3-b]indole (AαC) metabolic bio-activation on oxidative DNA damage in human hepatoma G2 (HepG2) cells

2-Amino-9H-pyrido[2,3-b]indole (AαC), which is a hazardous compound present in cigarette smoke, has been listed as probable human carcinogens (Group 2B). The carcinogenicity and genotoxicity of AαC were activated by the process of metabolic bio-activation. Whereas, few studies about genotoxicity induced by AαC have been reported. In this study, we took HepG2 cells as the model to investigate the relationship between oxidative DNA damage induced by AαC and metabolic bio-activation of AαC, which is of importance to unveil the mechanism of AαC genotoxicity. Firstly, the HepG2 cells were treated with 10 and 20 μg/mL AαC, respectively. Then different concentrations of protein ranging from 0 to 1 mg/mL in S9 mixture solution were utilized to make cells have different capacities for metabolic activation. Intracellular AαC hydroxylated metabolites and 8-OHdG were estimated by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that, at the same concentration of AαC, with the increment of concentration of protein in S9 mixture solution, the levels of hydroxylated metabolites and 8-OHdG/106dG increased. And at the same concentration of protein in S9 mixture solution, with the increment of concentration of AαC, the levels of hydroxylated metabolites and 8-OHdG/106dG increased. The hydroxylated metabolites and 8-OHdG were positively related by correlation analysis. In addition, the correlation coefficients of N-OH-AαC and 8-OHdG were maximum (R2 = 0.73 and 0.66). Taken together, these results indicated that the metabolic bio-activation of AαC might result in oxidative DNA damage.

Design, synthesis, and anticancer activity evaluation of novel aziridine-1,2,3-triazole hybrid derivatives

ABSTRACTSome new 1-aryl-4-[(aziridine-1-yl)diaryl-methyl]-5-methyl-1H-1,2,3-triazole derivatives 7j–s were synthesized by the one-pot reaction of diaryl-(1-aryl-5-methyl-1H-1,2,3-triazol-4-yl)methanol compounds 6j–s formed from 1-aryl-5-methyl-1H-1,2,3-triazole-4-carboxylic acid derivatives. The new compounds 7j–s and 6j–s are investigated by 1H and 13C NMR, MS, and IR. The anticancer activity of the synthesis target compounds was evaluated against human leukemia (HL-60) cells and human hepatoma G2 cells. Some of the compounds were highly efficient. The 1H-NMR signals of the aziridine-ring cis-H/trans-H protons were found to be two group peaks at 1.800–1.884 and 1.183–1.327 ppm.

A rapid forming, injectable and photo-cleavable hydrogel for controlled drug release

The autophagy of human hepatoma G2 (HepG2) cells induced by procyanidins from chestnut (Castanea mollissima Bl.) shell (CSPCs) was investigated, and the inherent relationship between autophagic levels and reactive oxygen species (ROS) generation was studied. The results showed that CSPCs induced HepG2 cell death in a time- and concentration-dependent manner, increased the accumulation of autophagolysosomes and microtubule-associated proteins light chain 3-II (LC3-II, a marker of autophagy). However, these phenomena were not observed in the group pretreated with the autophagy inhibitor 3-MA, suggesting that CSPCs induced HepG2 cell autophagy. Furthermore, we found that CSPCs triggered ROS generation in cells, while the levels of ROS decreased in the N-acetylcysteine (Nac) co-treatment, revealing that CSPCs-mediated autophagy was partly blocked by Nac. In addition, treatment with CSPCs decreased the mitochondrial membrane potential of HepG2 cells. These results suggested CSPCs could trigger autophagy via ROS generation, which may be associated with the mitochondria-dependent signaling way.

Critical role of p21 on olaquindox-induced mitochondrial apoptosis and S-phase arrest involves activation of PI3K/AKT and inhibition of Nrf2/HO-1pathway

Olaquindox, a quinoxaline 1,4-di-N-oxide, is known as an antibacterial agent and feed additive to treat bacterial infections and promote animal growth. However, the potential mechanism of toxicity is still unknown. The present study aims to explore the molecular mechanism of p21 on olaquindox-induced mitochondrial apoptosis and S-phase arrest in human hepatoma G2 cells (HepG2). As a result, olaquindox promoted production of ROS, suppressed the protein expression p21 in p53-independent way and phosphorylated p21. Meanwhile, olaquindox activated AKT and Nrf2/HO-1 pathway, up-regulated Bax/Bcl-2 ratio, disrupted mitochondrial membrane potential (MMP) and subsequently caused cytochrome c release and a cascade activation of caspase, eventually induced apoptosis. Olaquindox could induce S-phase arrest in HepG2 cells involved with the increase of Cyclin A, Cyclin E and CDK 2. Furthermore, knockdown of p21 decreased cell viability, enhanced oxidative stress, aggravated olaquindox-induced mitochondrial apoptosis and S-phase arrest involvement of activating PI3K/AKT and inhibiting Nrf2/HO-1 pathway. PI3K/AKT inhibitor (LY294002) and HO-1inhibitor (ZnPP-IX) both increased olaquindox-induced apoptosis and S-phase arrest. In conclusion, knockdown of p21 increased olaquindox-induced mitochondrial apoptosis and S-phase arrest through further activating PI3K/AKT and inhibiting Nrf2/HO-1pathway. Our study provided new insights into the molecular mechanism of olaquindox and shed light on the role of p21.

GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways.

Olaquindox, a quinoxaline 1,4-dioxide derivative, is widely used as a feed additive in many countries. The potential genotoxicity of olaquindox, hence, is of concern. However, the proper mechanism of toxicity was unclear. The aim of the present study was to investigate the effect of growth arrest and DNA damage 45 alpha (GADD45a) on olaquindox-induced DNA damage and cell cycle arrest in HepG2 cells. The results showed that olaquindox could induce reactive oxygen species (ROS)-mediated DNA damage and S-phase arrest, where increases of GADD45a, cyclin A, Cdk 2, p21 and p53 protein expression, decrease of cyclin D1 and the activation of phosphorylation-c-Jun N-terminal kinases (p-JNK), phosphorylation-p38 (p-p38) and phosphorylation-extracellular signal-regulated kinases (p-ERK) were involved. However, GADD45a knockdown cells treated with olaquindox could significantly decrease cell viability, exacerbate DNA damage and increase S-phase arrest, associated with the marked activation of p-JNK, p-p38, but not p-ERK. Furthermore, SP600125 and SB203580 aggravated olaquindox-induced DNA damage and S-phase arrest, suppressed the expression of GADD45a. Taken together, these findings revealed that GADD45a played a protective role in olaquindox treatment and JNK/p38 pathways may partly contribute to GADD45a regulated olaquindox-induced DNA damage and S-phase arrest. Our findings increase the understanding on the molecular mechanisms of olaquindox.

Effect of GADD45a on olaquindox-induced apoptosis in human hepatoma G2 cells: Involvement of mitochondrial dysfunction.

Olaquindox, a quinoxaline 1, 4-dioxide derivative, has been widely used as a feed additive for promoting animal growth in China. The aim of present study was to investigate the effect of grow arrest and DNA damage 45 alpha (GADD45a) on olaquindox-induced apoptosis in HepG2 cells. The result showed that olaquindox induced the decrease of cell viability in a dose dependent manner. Compared to the control group, olaquindox treatment at 400 and 800μg/mL increased the expression level of GADD45a protein and reactive oxygen species (ROS) production, decreased mitochondrial membrane potential (MMP), and subsequently increased the expression of Bax while decreased the expression of Bcl-2, leading to the release of cytochrome c (Cyt c). However, knockdown of GADD45a enhanced olaquindox-induced ROS production, disrupted MMP and subsequently caused Cyt c release, then further increased olaquindox- induced cell apoptosis by increasing the activities of caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP). In conclusion, the results revealed that GADD45a played a critical role in olaquindox-induced apoptosis in HepG2 cells, which may embrace the regulatory ability on the mitochondrial apoptosis pathway.

Procyanidins from Nelumbo nucifera Gaertn. Seedpod induce autophagy mediated by reactive oxygen species generation in human hepatoma G2 cells.

In this study, autophagic effect of procyanidins from lotus (Nelumbo nucifera Gaertn.) seedpod (LSPCs) on human hepatoma G2 (HepG2) cells, and the inherent correlation between autophagic levels and reactive oxygen species (ROS) generation were investigated. The results showed that LSPCs increased monodansylcadaverine (MDC) fluorescence intensity and LC3-I/LC3-II conversion in HepG2 cells. In addition, the typically autophagic characteristics (autophagosomes and autolysosomes) were observed in LSPCs-treated cells, but not found in the cells treated with autophagy inhibitor 3-methyladenine (3-MA). Furthermore, the elevated ROS level was in line with the increasing of autophagy activation caused by LSPCs, however, both 3-MA and the ROS scavenger N-acetylcyteine (NAC) inhibitors effectively suppressed the autophagy and ROS generation triggered by LSPCs. As a result, these results indicated that LSPCs induced HepG2 cell autophagy in a time- and dose-dependent manner, and promoted reactive oxygen species (ROS) generation on HepG2 cells. Moreover, we found that LSPCs caused DNA damage, S phase arrest and the decrement of mitochondria membrane potential (MMP) which were associated with ROS generation. In summary, our findings demonstrated that the LSPCs-induced autophagy and autophagic cell death were triggered by the ROS generation in HepG2 cells, which might be associated with ROS generation through the mitochondria-dependent signaling way.