Daowen Li

Curcumin attenuates quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells

Abstract Quinocetone (QCT), a new quinoxaline 1,4-dioxides, has been used as antimicrobial feed additive in China. Potential genotoxicity of QCT was concerned as a public health problem. This study aimed to investigate the protective effect of curcumin on QCT-induced oxidative stress and genotoxicity in human hepatocyte L02 cells. Cell viability and intracellular reactive oxygen species (ROS), biomarkers of oxidative stress including superoxide dismutase (SOD) activity and glutathione (GSH) level were measured. Meanwhile, comet assay and micronucleus assay were carried out to evaluate genotoxicity. The results showed that, compared to the control group, QCT at the concentration ranges of 2–16 μg/mL significantly decreased L02 cell viability, which was significantly attenuated with curcumin pretreatment (2.5 and 5 μM). In addition, QCT significantly increased cell oxidative stress, characterized by increases of intracellular ROS level, while decreased endogenous antioxidant biomarkers GSH level and SOD activity (all p < 0.05 or 0.01). Curcumin pretreatment significantly attenuated ROS formation, inhibited the decreases of SOD activity and GSH level. Furthermore, curcumin significantly reduced QCT-induced DNA fragments and micronuclei formation. These data suggest that curcumin could attenuate QCT-induced cytotoxicity and genotoxicity in L02 cells, which may be attributed to ROS scavenging and anti-oxidative ability of curcumin. Importantly, consumption of curcumin may be a plausible way to prevent quinoxaline 1,4-dioxides-mediated oxidative stress and genotoxicity in human or animals.

Curcumin Ameliorates Furazolidone-Induced DNA Damage and Apoptosis in Human Hepatocyte L02 Cells by Inhibiting ROS Production and Mitochondrial Pathway

Furazolidone (FZD), a synthetic nitrofuran derivative, has been widely used as an antibacterial and antiprotozoal agent. Recently, the potential toxicity of FZD has raised concerns, but its mechanism is still unclear. This study aimed to investigate the protective effect of curcumin on FZD-induced cytotoxicity and the underlying mechanism in human hepatocyte L02 cells. The results showed that curcumin pre-treatment significantly ameliorated FZD-induced oxidative stress, characterized by decreased reactive oxygen species (ROS) and malondialdehyde formation, and increased superoxide dismutase, catalase activities and glutathione contents. In addition, curcumin pre-treatment significantly ameliorated the loss of mitochondrial membrane potential, the activations of caspase-9 and -3, and apoptosis caused by FZD. Alkaline comet assay showed that curcumin markedly reduced FZD-induced DNA damage in a dose-dependent manner. Curcumin pre-treatment consistently and markedly down-regulated the mRNA expression levels of p53, Bax, caspase-9 and -3 and up-regulated the mRNA expression level of Bcl-2. Taken together, these results reveal that curcumin protects against FZD-induced DNA damage and apoptosis by inhibiting oxidative stress and mitochondrial pathway. Our study indicated that curcumin may be a promising combiner with FZD to reduce FZD-related toxicity in clinical applications.

Quinocetone triggered ER stress-induced autophagy via ATF6/DAPK1-modulated mAtg9a trafficking

The present study is undertaken to explore quinocetone-induced autophagy and its possible mechanism. Western blotting and green fluorescence protein (GFP)-LC3 vector transfection were performed to determine the ratio of LC3 conversion and its subcellular localization. Results revealed that the quinocetone induced autophagy in time- and dose-dependent manners. Besides, we tested the expressions of immunoglobulin heavy chain binding protein (BiP) and C/EBP homologous protein (CHOP) and the transcription of BiP, HerpUD, and sec24D by western blotting and RT-PCR, respectively. Results showed that quinocetone also induced endoplasmic reticulum (ER) stress during quinocetone-induced autophagy. Furthermore, we observed the cleavage of ATF6, the phosphorylation of MRLC, and the expression of death-associated protein kinase (DAPK1) by western blotting; the transcription of DAPK1 by RT-PCR; and the subcellular localization of ATF6 and mAtg9 by immunofluorescence. These results suggest that quinocetone stimulates the MRLC-mediated mAtg9 trafficking, which is critical for autophagosome formation, via the ATF6 upregulated expression of DAPK1. Last, we generated ATF6 and DAPK1 stable knockdown HepG2 cell lines and found that the conversion ratios of LC3 were decreased upon the treatment of quinocetone. Together, we propose that quinocetone induces autophagy through ER stress signaling pathway-induced cytoskeleton activation.

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.

ML-7 amplifies the quinocetone-induced cell death through akt and MAPK-mediated apoptosis on HepG2 cell line

Abstract The study aims at evaluating the combination of the quinocetone and the ML-7 in preclinical hepatocellular carcinoma models. To this end, the effect of quinocetone and ML-7 on apoptosis induction and signaling pathways was analyzed on HepG2 cell lines. Here, we report that ML-7, in a nontoxic concentration, sensitized the HepG2 cells to quinocetone-induced cytotoxicity. Also, ML-7 profoundly enhances quinocetone-induced apoptosis in HepG2 cell line. Mechanistic investigations revealed that ML-7 and quinocetone act in concert to trigger the cleavage of caspase-8 as well as Bax/Bcl-2 ratio up-regulation and subsequent cleavage of Bid, capsases-9 and -3. Importantly, ML-7 weakened the quinocetone-induced Akt pathway activation, but strengthened the phosphorylation of p-38, ERK and JNK. Further treatment of Akt activator and p-38 inhibitor almost completely abolished the ML-7/quinocetone-induced apoptosis. In contrast, the ERK and JNK inhibitor aggravated the ML-7/quinocetone-induced apoptosis, indicating that the synergism critically depended on p-38 phosphorylation and HepG2 cells provoke Akt, ERK and JNK signaling pathways to against apoptosis. In conclusion, the rational combination of quinocetone and ML-7 presents a promising approach to trigger apoptosis in hepatocellular carcinoma, which warrants further investigation.

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.

DIDS inhibits overexpression BAK1-induced mitochondrial apoptosis through GSK3β/β-catenin signaling pathway

Bcl‐2 homologous antagonist/killer (BAK1) is a critical regulator of mitochondrial apoptosis. Although upregulation of BAK1 induces apoptosis has been established, the underlying molecular mechanism is far from clear. 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS), an organic anion used as a blocker of anion exchangers and chloride channels, has been proved to rescue cell apoptosis both in vitro and in vivo. However, whether DIDS can inhibit BAK1‐induced mitochondrial apoptosis remains undefined. Thus, this study aimed to explore whether DIDS could protect BAK1‐induced apoptosis through GSK3β/β‐catenin signaling pathway. The results showed overexpression BAK1 in 293T cells induced mitochondrial apoptosis accompanied by increasing the expression levels of cleaved caspase‐9, ‐3, poly (ADP‐ribose) polymerase (PARP) and reducing the MMP. Furthermore, overexpression BAK1 decreased the expression levels of Ser9‐GSK3β and β‐catenin. In addition, lithium chloride (LiCl), an activator of Wnt/β‐catenin signaling pathway, markedly attenuated overexpression BAK1‐induced mitochondrial apoptosis by restoring the expression levels of Ser9‐GSK3β and β‐catenin. Finally, DIDS absolutely abolished overexpression BAK1‐mediated mitochondrial apoptosis through recovering the expression levels of Ser9‐GSK3β and β‐catenin. Taken together, our results reveal that DIDS blocks overexpression BAK1‐induced mitochondrial apoptosis through GSK3β/β‐catenin pathway.

TCS2 Increases Olaquindox-Induced Apoptosis by Upregulation of ROS Production and Downregulation of Autophagy in HEK293 Cells

Olaquindox, a feed additive, has drawn public attention due to its potential mutagenicity, genotoxicity, hepatoxicity and nephrotoxicity. The purpose of this study was to investigate the role of tuberous sclerosis complex (TSC2) pathways in olaquindox-induced autophagy in human embryonic kidney 293 (HEK293) cells. The results revealed that olaquindox treatment reduced the cell viability of HEK293 cells and downregulated the expression of TSC2 in a dose- and time-dependent manner. Meanwhile, olaquindox treatment markedly induced the production of reactive oxygen species (ROS), cascaded to autophagy, oxidative stress, and apoptotic cell death, which was effectively eliminated by the antioxidant N-acetylcysteine (NAC). Furthermore, overexpression of TSC2 attenuated olaquindox-induced autophagy in contrast to inducing the production of ROS, oxidative stress and apoptosis. Consistently, knockdown of TSC2 upregulated autophagy, and decreased olaquindox-induced cell apoptosis. In conclusion, our findings indicate that TSC2 partly participates in olaquindox-induced autophagy, oxidative stress and apoptosis, and demonstrate that TSC2 has a negative regulation role in olaquindox-induced autophagy in HEK293 cells.