Jinfeng Cui

Ochratoxin A induces oxidative DNA damage and G1 phase arrest in human peripheral blood mononuclear cells in vitro

Ochratoxin A is one of the most abundant food-contaminating mycotoxins worldwide, and its immunosuppressive effects in human caused more and more concern in biomedical field. In the present study, the toxicity of OTA on human peripheral blood mononuclear cells (hPBMC) was explored by analyzing the involvement of oxidative pathway. It was found that OTA treatment led to the release of reactive oxygen species (ROS) and the increase of 8-hydroxydeoxyguanosine (8-OHdG), an important biomarker of oxidative DNA stress. Moreover, we found that OTA treatment induced DNA strand breaks in hPBMC as evidenced by DNA comet tails formation and increased γ-H2AX expression. In addition, OTA could induce cell cycle arrest at G1 phase by down-regulating the expression of CDK4 and cyclinD1 protein, as well as apoptosis in hPBMC in vitro. Pre-treatment of hPBMC with antioxidant, N-acetyl-L-cysteine (NAC), could reduce OTA-induced ROS release and DNA damage, thus confirming the involvement of oxidative DNA damage in the OTA genotoxicity in hPBMC. NAC pre-treatment could also significantly prevent OTA-induced down-regulation of CDK4 and cyclinD1 expression in hPBMC. All the results demonstrated the involvement of oxidative pathway in OTA mediated cytotoxicity in human immune cells, which including the ROS accumulation-oxidative DNA damage-G1 arrest and apoptosis. Our results provide new insights into the molecular mechanisms by which OTA might promote immunotoxicity.

Ochratoxin A induces G2 phase arrest in human gastric epithelium GES-1 cells in vitro.

Ochratoxin A (OTA) is a mycotoxin commonly found in several food commodities worldwide. OTA exposure was involved in the nephrotoxicity, hepatotoxicity as well as immunotoxicity in experimental model. Our previous study showed that the high level of OTA contamination in wheat might be related to the high incidence of gastric carcinoma in rural area of China. However, there were no available data regarding the gastric toxicity of OTA up to now. In the present study, we explored the toxicity of OTA in human gastric epithelium immortalized cells (GES-1) by analyzing the regulation of the cell cycle, apoptosis and its molecular mechanism. We found that OTA could induce GES-1 cells arrested in G(2)/M phase. Among these cycle-arrested cells, the proportion of cells in M phase was down-regulated after OTA treatment by the mitotic index and the level of phospho-histone H3. Thus, it was clear that OTA exerted a major influence on G(2) phase arrest instead of M phase. We further detected the expression of the key factors which are critical to the G(2)/M phase transmission such as Cdc25C, Cdc2 and cyclinB1. The cyclinB1-Cdc2 complex was reduced and the expression of Cdc25C, Cdc2 and cyclinB1 were significantly decreased by OTA treatment both at protein and mRNA level, respectively. Considering that the cells may undergo apoptosis or death due to the cell cycle arrest, so we next detected the apoptosis of cells by OTA treatment. The results confirmed that OTA did induce apoptosis of GES-1 cells and activate the cleavage of capase-3. In conclusion, cell apoptosis and G(2) phase arrest mediated by Cdc25C, Cdc2 and cyclinB1 may be the initiating event in the gastric toxicity of OTA.

Low serum pepsinogen I and pepsinogen I/II ratio and Helicobacter pylori infection are associated with increased risk of gastric cancer: 14-year follow up result in a rural Chinese community

The correlation between low serum PG level and H. pylori infection with the development of gastric cancer has caused considerable concerns all over the world. Some authors exclaimed that gastric cancer developed only in patients infected with H. pylori, whereas the other had different findings. In this study, 1,501 adult local residents with determined serum PG levels and anti H. pylori IgG status were followed for 14 years for the development of gastric cancer in a rural community with high risk of gastric cancer in Hebei Province, China. The results showed the accumulated gastric cancer incidence in the subjects with abnormal PG level and those with H. pylori infection were all significantly higher than that in the corresponding normal controls (53.9‰ vs. 12.7‰, p < 0.05 and 23.1‰ vs. 5.93‰, p < 0.05). The highest gastric cancer incidence was seen in the subjects with both abnormal serum PG and positive H. pylori (56.0‰), and followed by the subjects with abnormal PG and negative H. pylori (47.6‰) and those with normal serum PG and positive H. pylori (18.4‰). The abnormal serum PG level (OR 3.029) and H. pylori infection (OR 4.345) were all risk factors for the development of gastric cancer. The results suggested that the subjects with abnormal serum PG level and/or positive H. pylori infection in the rural area of China were all high risk population for gastric carcinoma and the subjects with both abnormal serum PG and positive H. pylori infection were at especially high risk for the development of gastric carcinoma.

ERK and p38 MAPK signaling pathways are involved in ochratoxin A-induced G2 phase arrest in human gastric epithelium cells

Ochratoxin A (OTA) is a ubiquitous mycotoxin with potential nephrotoxic, hepatotoxic, and immunotoxic effects. Recent work from our laboratory found that OTA evoked G2 phase arrest in GES-1 cells in vitro by modulating the key factors Cdc25C, Cdc2 and cyclinB1, which were critical to the G2/M phase transmission, suggested that OTA-induced G2 arrest mediate at least in part OTA toxicity effect. However, the molecular mechanism of this effect is currently unclear. In the present study, we showed that treatment of GES-1 cells with OTA could induce the activation of MAPK family members ERK and p38. ERK inhibitor PD98059 and p38 inhibitor SB203580 significantly reversed the depression of Cdc25C/p-Cdc25C, Cdc2/p-Cdc2, cyclinB1 as well as the cyclinB1-Cdc2 complex, thereby, abolished the delay in G2 phase. In addition, silencing ERK and p38 expression with siRNA significantly inhibited OTA-induced G2 arrest in GES-1 cells as well. Collectively, these data suggest that the ERK and p38 MAPK signaling pathways play important roles in the regulation of OTA-induced G2 arrest in GES-1 cells.

Oxidative DNA damage is involved in ochratoxin A-induced G2 arrest through ataxia telangiectasia-mutated (ATM) pathways in human gastric epithelium GES-1 cells in vitro

Ochratoxin A (OTA), one of the most abundant mycotoxin food contaminants, is classified as “possibly carcinogenic to humans.” Our previous study showed that OTA could induce a G2 arrest in immortalized human gastric epithelium cells (GES-1). To explore the putative roles of oxidative DNA damage and the ataxia telangiectasia-mutated (ATM) pathways on the OTA-induced G2 arrest, the current study systematically evaluated the roles of reactive oxygen species (ROS) production, DNA damage, and ATM-dependent pathway activation on the OTA-induced G2 phase arrest in GES-1 cells. The results showed that OTA exposure elevated intracellular ROS production, which directly induced DNA damage and increased the levels of 8-OHdG and DNA double-strand breaks (DSBs). In addition, it was found that OTA treatment induced the phosphorylation of the ATM protein, as well as its downstream molecules Chk2 and p53, in response to DNA DSBs. Inhibition of ATM by the pharmacological inhibitor caffeine or siRNA effectively prevented the activation of ATM-dependent pathways and rescued the G2 arrest elicited by OTA. Finally, pretreatment with the antioxidant N-acetyl-l-cysteine (NAC) reduced the OTA-induced DNA DSBs, ATM phosphorylation, and G2 arrest. In conclusion, the results of this study suggested that OTA-induced oxidative DNA damage triggered the ATM-dependent pathways, which ultimately elicited a G2 arrest in GES-1 cells.

Assessment on pollution of ochratoxin a in grain in China and its apoptosis effect on vitro-cultured human tubular kidney cells

Ochratoxin A (OTA) is nephrotoxic, immunosuppressive, and teratogenic in many species and is a possible human carcinogen. In this study, we investigated the OTA pollution situations of grains in northern China and the signaling pathway that mediated OTA‐induced apoptosis in human tubular kidney cells (HKCs). Samples of grains collected from three representative areas were determined by using high‐performance liquid chromatography fluorescence method. The effects of OTA on cell apoptosis, caspase‐3, Bax, and Bcl‐2 expression, and phosphorylation of c‐Jun NH2 terminal kinase (JNK) were detected in cultured HKCs via flow cytometry (FCM), Hoechst 33258 staining, and Western blot. It showed that OTA pollution of edible grains was very common in north China. OTA could affect caspase‐3, Bax, and Bcl‐2 expression and increased cell apoptosis in cultured HKCs. The JNK signalling pathway might play an important role during these cellular events.

Sterigmatocystin-Induced DNA Damage Triggers G2 Arrest via an ATM/p53-Related Pathway in Human Gastric Epithelium GES-1 Cells In Vitro

Sterigmatocystin (ST), which is commonly detected in food and feed commodities, is a mutagenic and carcinogenic mycotoxin that has been recognized as a possible human carcinogen. Our previous study showed that ST can induce G2 phase arrest in GES-1 cells in vitro and that the MAPK and PI3K signaling pathways are involved in the ST-induced G2 arrest. It is now widely accepted that DNA damage plays a critical role in the regulation of cell cycle arrest and apoptosis. In response to DNA damage, a complex signaling network is activated in eukaryotic cells to trigger cell cycle arrest and facilitate DNA repair. To further explore the molecular mechanism through which ST induces G2 arrest, the current study was designed to precisely dissect the role of DNA damage and the DNA damage sensor ataxia telangiectasia-mutated (ATM)/p53-dependent pathway in the ST-induced G2 arrest in GES-1 cells. Using the comet assay, we determined that ST induces DNA damage, as evidenced by the formation of DNA comet tails, in GES-1 cells. We also found that ST induces the activation of ATM and its downstream molecules, Chk2 and p53, in GES-1 cells. The ATM pharmacological inhibitor caffeine was found to effectively inhibit the activation of the ATM-dependent pathways and to rescue the ST-induced G2 arrest in GES-1 cells, which indicating its ATM-dependent characteristic. Moreover, the silencing of the p53 expression with siRNA effectively attenuated the ST-induced G2 arrest in GES-1 cells. We also found that ST induces apoptosis in GES-1 cells. Thus, our results show that the ST-induced DNA damage activates the ATM/53-dependent signaling pathway, which contributes to the induction of G2 arrest in GES-1 cells.

Sterigmatocystin induces G1 arrest in primary human esophageal epithelial cells but induces G2 arrest in immortalized cells: key mechanistic differences in these two models.

Sterigmatocystin (ST), a mycotoxin commonly found in food and feed commodities, has been classified as a “possible human carcinogen.” Our previous studies suggested that ST exposure might be a risk factor for esophageal cancer and that ST may induce DNA damage and G2 phase arrest in immortalized human esophageal epithelial cells (Het-1A). To further confirm and explore the cellular responses of ST in human esophageal epithelia, we comparatively evaluated DNA damage, cell cycle distribution and the relative mechanisms in primary cultured human esophageal epithelial cells (EPC), which represent a more representative model of the in vivo state, and Het-1A cells. In this study, we found that ST could induce DNA damage in both EPC and Het-1A cells but led to G1 phase arrest in EPC cells and G2 phase arrest in Het-1A cells. Furthermore, our results indicated that the activation of the ATM–Chk2 pathway was involved in ST-induced G1 phase arrest in EPC cells, whereas the p53–p21 pathway activation in ST-induced G2 phase arrest in Het-1A cells. Studies have demonstrated that SV40 large T-antigen (SV40LT) may disturb cell cycle progression by inactivating some of the proteins involved in the G1/S checkpoint. Het-1A is a non-cancerous epithelial cell line immortalized by SV40LT. To evaluate the possible perturbation effect of SV40LT on ST-induced cell cycle disturbance in Het-1A cells, we knocked down SV40LT of Het-1A cells with siRNA and found that under this condition, ST-induced G2 arrest was significantly attenuated, whereas the proportion of cells in the G1 phase was significantly increased. Furthermore, SV40LT-siRNA also inhibited the activation of the p53–p21 signaling pathway induced by ST. In conclusion, our data indicated that ST could induce DNA damage in both primary cultured and immortalized esophageal epithelial cells. In primary human esophageal epithelial cells, ST induced DNA damage and then triggered the ATM–Chk2 pathway, resulting in G1 phase arrest, whereas in SV40LT-immortalized human esophageal epithelial cells, SV40LT-mediated G1 checkpoint inactivation occurred, and ST-DNA damage activated p53–p21 signaling pathway, up-regulating G2/M phase regulatory proteins and finally leading to a G2 phase arrest. Thus, the SV40LT-mediated G1 checkpoint inactivation is responsible for the difference in the cell cycle arrest by ST between immortalized and primary cultured human esophageal epithelial cells.

Ochratoxin A induces DNA damage and G2 phase arrest in human esophageal epithelium Het-1A cells in vitro.

Ochratoxin A (OTA), a toxin produced by several species of Aspergillus and Penicillium, is one of the most abundant food-contaminating mycotoxins. The International Agency for Research on Cancer (IARC) has classified OTA as a possible human carcinogen. Our previous study showed that there were high levels of OTA contaminations in wheat in the areas with high incidence of esophageal cancer in north China. This finding suggests that exposure to low levels of OTA may be a critical etiological factor for esophageal cancer in these areas. However, up to now, the potential biological effects of OTA on human esophageal epithelial cells have not been fully elucidated. In the present study, we explored the cytotoxicity of OTA in human esophageal epithelium immortalized cells (Het-1A). We found that OTA could induce DNA strand breaks and chromosome aberrations in Het-1A cells. OTA-induced DNA damage was followed by G2 cell cycle arrest, and down-regulation of Cdc2 and cyclinB1 contributed to the OTA-induced G2 arrest in Het-1A cells. Additionally, OTA induced apoptosis in Het-1A cells by activating caspase-3. In conclusion, our results indicated that OTA could induce DNA damage, G2 arrest and apoptosis in Het-1A cells, which may be involved in the esophageal toxicity of OTA.

Differential expression of Pim-3, c-Myc, and p-p27 proteins in adenocarcinomas of the gastric cardia and distal stomach

Gastric cardia adenocarcinoma (GCA) is distinct from adenocarcinoma of the distal stomach because of its different etiological factors, tumor characteristics, and biological behavior. However, its pathogenesis is not fully understood. The purpose of this study is to characterize the role of Pim-3, c-Myc, and p-p27 in the tumorigenesis and progression of different sites of gastric adenocarcinoma by determining its pathogenetic significance. The expression of Pim-3, c-Myc, and p-p27 proteins was evaluated by immunohistochemistry in 140 resection specimens of gastric adenocarcinomas (78 GCAs , 62 DGAs and 20 normal gastric tissues). The level of expression of Pim-3, c-Myc, and p-p27 and the co-expression of all three markers (Pim-3+/c-Myc+/p-p27+) in GCA were significantly lower than that in DGA tumors (P < 0.05). Detailed analysis of the immunoreactivity patterns showed that in DGA, Pim-3 immunoreactivity was associated significantly with poor tumor differentiation, advanced tumor stage, and presence of lymph node metastasis. In addition, c-Myc overexpression correlated with tumor stage and lymph node metastasis, and positive p-p27 expression correlated with poor differentiation and tumor stage. The phenotype of Pim-3+/c-Myc+/p-p27+ co-expression was closely correlated with tumor stage and lymph node metastasis (P < 0.05). In contrast, GCA only demonstrated a close correlation of Pim-3 overexpression with poor tumor differentiation and tumor stage (P < 0.05). Our results demonstrate the presence of different expression patterns of Pim-3, c-Myc, p-p27, and Pim-3+/c-Myc+/p-p27+ and their clinicopathologic significance in GCA and DGA tumors. Our results add support to the notion that distinct molecular mechanisms may be involved in the development and progression of adenocarcinomas from the gastric cardia and distal portion of stomach.