Autophagy plays an important role in response to intracellular and extracellular stress to sustain cell survival. However, dysregulated or excessive autophagy may lead to cell death, known as “type II programmed cell death,” and it is closely associated with apoptosis. In our previous study, we proposed that olaquindox induced apoptosis of HepG2 cells through a caspase-9 dependent mitochondrial pathway. In this study, we investigated autophagy induced by olaquindox and explored the crosstalk between apoptosis and autophagy in olaquindox-treated HepG2 cells. Olaquindox-induced autophagy was demonstrated by the accumulation of monodansylcadervarine, as well as elevated expression of autophagy-related MAP-LC3 and Beclin 1 proteins. The autophagy inhibitor 3-methyladenine significantly increased the apoptotic rate induced by olaquindox, which was correlated with increased ratio of Bax/Bcl-2. The further studies showed that olaquindox increased the levels of reactive oxygen species (ROS), and antioxidant N-acetyl-L-cysteine (NAC) effectively blocked the accumulation of ROS but failed to block autophagy. Moreover, olaquindox induced the activation of c-Jun N-terminal protein kinase (JNK), and JNK inhibitor SP600125 failed to block autophagy. Instead, olaquindox-induced autophagy was enhanced by NAC or SP600125. Meanwhile, JNK activation was remarkably blocked by NAC, indicating that ROS may be the upstream signaling molecules of JNK activation and involved in the negative regulation of olaquindox-induced autophagy. These results suggest that olaquindox induces autophagy in HepG2 cells and that olaquindox-induced apoptosis can be enhanced by 3-methyladenine. Olaquindox-induced autophagy in HepG2 cells is upregulated by Beclin 1 but downregulated by ROS-dependent JNK. Copyright © 2014 John Wiley & Sons, Ltd.

Given the previously described essential role for the p38 mitogen-activation protein kinase (p38 MAPK) signaling pathway in human hepatoma G2 cells (HepG2), we undertook the present study to investigate the role of the p38 MAPK signaling pathway in cell-cycle arrest induced by Furazolidone (FZD). The aim of this study was to determine the effects of FZD on HepG2 cells by activating and inhibiting the p38 MAPK signaling pathway. The cell cycle and proliferation of HepG2 cells treated with FZD were detected by flow cytometry and MTT assay in the presence or absence of p38 MAPK inhibitors (SB203580), respectively. Cyclin D1, cyclin D3 and CDK6 were detected by quantitative real-time PCR and western blot analysis. Our data showed that p38 MAPK became phosphorylated after stimulation with FZD. Activation of p38 MAPK could arise S-phase cell-cycle arrest and suppress cell proliferation. Simultaneously, inhibition of the p38 MAPK signaling pathway significantly prevented S-phase cell-cycle arrest, increased the percentage of cell viability and decreased the expression of cyclin D1, cyclin D3 and CDK6. These results demonstrated that FZD arose S-phase cell-cycle arrest via activating the p38 MAPK signaling pathway in HepG2 cells. Cyclin D1, cyclin D3 and CDK6 are target genes functioning at the downstream of p38 MAPK in HepG2 cells induced by FZD. Copyright © 2012 John Wiley & Sons, Ltd.

A sensitive and robust HPLC method was developed for the determination of kanamycin (Kan) in swine tissues. Kan was extracted from tissue with 10% TCA (w/v) solution and cleaned up with MCX SPE column. The purified extract was derivatized with 9-fluorenylmethyl chloroformate, and then separated on a C18 column and detected by a fluorescence detector. The linear range of the calibration curve was 0.025–1 μg/mL with R² of 0.9998. The limits of quantification were determined to be 0.1 mg/kg in muscle, 0.2 mg/kg in liver, and 0.6 mg/kg in kidney, and the average recoveries of Kan from swine tissues at different fortification levels (0.1–0.4 mg/kg for muscle, 0.2–1.2 mg/kg for liver, and 0.6–5.0 mg/kg for kidney) ranged from 80.7 to 91.3% with intraday and interday coefficient of variations less than 12.1%. The validated method was successfully applied to determine Kan in incurred samples, indicating that it can be used as a routine tool for the surveillance of Kan residue in swine tissues.