[Effect of Cell Autophagy on the Apoptosis of Hepatocellular Carcinoma Cells Induced by Arsenic Trioxide].

OBJECTIVES: To determine the effect of autophagy on the apoptosis of hepatocellular carcinoma cells induced by arsenic trioxide (ATO). METHODS: Hepatocellular carcinoma HepG2 cells were exposed to ATO. The cell viability was detected by MTT after adjustments for autophagy agonist (Rap) and autophagy inhibitor (3-MA). The autophagosome was observed under electronic microscope. The autophagy related proteins (LC3 and Beclin1) were detected by immunofluorescence. The cell apoptosis was measured by flow cytometry. RESULTS: With 5-20 µmol/Lof ATO, HepG2 cells exposed to 3-MA had significantly lower viability (P <0.05) and higher early apoptosis (P <0.05) than those without exposure to 3-MA. Exposure to 3-MA was also associated with lower expressions of LC3 and Beclin1, with HepG2 cells showing typical apoptotic characteristics. By contrast, with 5-20 µmol/Lof ATO, the cells exposed to Rap showed significantly higher viability (P <0.05) and lower early apoptosis (P<0.05) than those without exposure to Rap. A large number of autophagosome appeared in the cells exposed to Rap. Exposure to Rap was associated with increased expressions of LC3 and Beclin1, but with no statistical significance (P >0.05). CONCLUSION: Targeted autophagy inhibition can significantly increase the sensitivity of HepG2 to ATO. The underlining mechanism is associated with enhanced apoptosis of hepatocellular carcinoma cells.

[miR-155/BACH1 Signaling Pathway in Human Lung Adenocarcinoma Cell Death Induced by Arsenic Trioxide].

OBJECTIVE: To explore the changes of micro RNA 155 (miR-155),BTB and CNC homologous protein 1 (BACH1),quinone oxidoreductase 1 (NQO1) and heme-oxygenase-1 (HO-1) in the process of arsenic trioxide-induced cell death,and to clarify the relationship between miR-155 and BACH1,providing experimental basis for the sensitivity of arsenic trioxide (ATO) treatment. METHODS: Human lung adenocarcinoma cell line A549 cells were treated with different concentrations of ATO. MTT assay and total antioxidant capacity detection kit were used to determine cell viability and total antioxidant capacity,respectively. BACH1,NQO1 and HO-1 protein expression were probed by Western blot and real-time fluorescence quantitative (qRT-PCR) was utilized to test the miR-155 level. A549 cells were transfected with miR-155 mimic and its negative control,then the expression level of miR-155 was detected by qRT-PCR,and these cells were treated with 20 µmol/L for 24 h followed by MTT and Western blot detection. RESULTS: 10 µmol/L ATO significantly reduced the cell viability in A549 cells. 10 µmol/L and 20 µmol/L ATO treatment activated BACH1 expression and inhibited miR-155,NQO1 and HO-1 expression,leading to decreased total antioxidant capacity. Importantly,the cell death induced by 20 µmol/L ATO was significantly decreased in miR-155 mimic transfection cells in comparison with non-transfected cells and miR-155 mimic negative control transfected cells. Moreover,high expression of miR-155 reduced BACH1 activation and increased NQO1 and HO-1 expression in cells treated with 20 µmol/L ATO ( P<0.05). CONCLUSION: Restraining total antioxidant capacity contributes to ATO induced cell death,the underlying mechanisms may be that ATO can activate BACH1 expression through inhibition of the miR-155 level,leading to subsequent inhibition of NQO1 and HO-1 expression. Taken together,these data suggest that miR-155 and BACH1 could be used as sensitivity targets for ATO treatment in lung cancer.

[The Experiment Study and Mechanism of Aspirin Enhances Cellular Sensitivity of Hepatocellular Carcinoma Cell Line to Arsenic Trioxide].

OBJECTIVE: To explore whether aspirin could sensitize arsenic trioxide on human hepatocelluar carcinoma cell line and understanding the combination mechanisms underlying co-treatment. METHODS: Cell viability was detected by MTT assay, cell apoptosis rate and reactive oxygen species (ROS) level were measured by flow cytometry, and Western blot assay was used to estimated the protein expression of heme oxygenase-1 (HO-1) in total protein and NF-E2-related factor 2 (Nrf2) in nuclear protein. RESULTS: 10 µmol/L arsenic trioxide can decreased the cell viability, while cell apoptosis rate, ROS level, HO-1 and Nrf2 protein expression was increased (P < 0.05). When compared with arsenic trioxide alone, co-treatment of arsenic trioxide with aspirin in different concentration (0, 0.1, 1.0, 2.5, 5.0 mmol/L) exhibited dual effects in intracellular ROS level, HO-1 and Nrf2 expression. Specifically, with the increasing of aspirin concentrations, the level of ROS induced by arsenic trioxide showed a rising trend after the first reduction, whereas, HO-1 and Nrf2 protein expression were decreased at first and then increased. CONCLUSION: Low concentration, less than 2.5 mmol/L, of aspirin may reduce the ROS accumulation through activating of Nrf2-HO-1 pathway, therefore decreasing the apoptotic cell death induced by arsenic trioxide. On the contrary, 5 mmol/L aspirin could increase the sensitivity of HepG2 to arsenic trioxide through enhancing the arsenic trioxide-induced apoptosis by ROS accumulation resulting in inhibiting the Nrf2-HO-1 pathway.