Research On Mechanisms Of Apoptosis Of Hepatocellular Carcinoma Induced By Combination Of Arsenic Trioxide And Ascorbic Acid

Recently, the antineoplastic properties of As₂O₃ aroused wide interest. Studies in vitro showed that As₂O₃ induced apoptosis of acute promyelocytic leukemia (APL) cell lines at micromolar concentration, and these preclinical studies were confirmed by controlled clinical trials showing that treatment with As₂O₃ led to complete remission in APL patients. More recently, studies revealed that apoptosis-inducing properties of As₂O₃ were not restricted to APL, and different types of tumor cell's viability, including hepatocellular carcinoma cell lines, were shown to be altered in vitro by exposed to As₂O₃. Key mediators of sensitivity to As₂O₃-induced apoptosis included intracellular GSH and ROS. The loss of inner mitochondrial membrane potential was also an important step in As₂O₃-mediated cell apoptosis.Ascorbic acid (AA), which was regarded as an anti-oxidant, was found to have pro-oxidative properties which could enhance the sensitivity of myeloid and lymphoid malignant cells to As₂O₃ in vitro. In our previous study, it was found that AA could promote As₂O₃-induced apoptosis of human hepatocellular carcinoma cell line HepG2 in vitro. Since ROS played a key role in AA combined with As₂O₃-induced apoptosis in other cell lines, we investigated ROS and downstream activated by ROS in the mechanisms of apoptosis induced by AA+As₂O₃ combination in HepG2 cells.1. Effects of combination of arsenic trioxide and ascorbic acid on intracellular ROS and antioxidant level in HepG2 cells§1 Effects of combination of arsenic trioxide and ascorbic acid on intracellular ROS level in HepG2 cells Intracellular ROS was detected by means of an oxidation-sensitive fluorescent probe (DCFH-DA).Exposure of HepG2 cells to 2.0μmol/L As₂O₃ plus 100μmol/L AA led to a remarkable increase in intracellular ROS compared with As₂O₃ alone did, with increased ROS level represented by the DCF intensity from 127.61±5.12 to152.60±5.88(P<0.05).§2 Effects of combination of arsenic trioxide and ascorbic acid on intracellular antioxidant level in HepG2 cellsThe GSH activity was measured by reacting on DTNB to form a yellow substance, which can be determined by colorimetry. The GPx activity was detected by the oxidizing speed of the GSH, which can be expressed by the GSH reduction in a certain time. The SOD activity was determined by hydroxylamine assay-developed from xanthine oxidase assay.A remarkable depletion in cellular GSH was found after AA or As₂O₃ exposure(P<0.01). And a higher level of decrease in GSH was observed in combinative treatment than As₂O₃ treatment(P<0.01).Significant reductions in GPx and SOD activity were observed after As₂O₃+AA exposure, but SOD and GPx depletion were found equal to As₂O₃ treated alone (P>0.05).2. Effects of combination of arsenic trioxide and ascorbic acid on mitochondria membrane potential in HepG2 cells The mitochondrial membrane potential was measured by flow cytometry analysis with rhodamine 123 staining As₂O₃ treatment caused depolarization of the inner mitochondrial membrane while AA alone had no significant effect on mitochondrial membrane potential(P<0.01). The combination of As₂O₃ and AA increased the depolarization of the inner mitochondrial membrane compared to As₂O₃ treatment alone(P<0.01).3. Effects of combination of arsenic trioxide and ascorbic acid on apoptosis proteins in HepG2 cellsAnti-apoptotic Bcl-2 protein, pro-apoptotic Bax protein and p17 subunit of caspase-3 were analysed by Western blot method.The results showed that there was an approximately 9-fold decrease in the ratio of Bcl-2/Bax expression in As₂O₃ treatment and combinative treatment compared with control(P<0.01), but there was no significant alteration between As₂O₃ treatment and the combinative treatment(P>0.05). And a marked increase of caspase-3 p17– activated caspase-3 subunit– in As₂O₃ or combinative treatment was observed(P<0.01), further more, a significant increase in combinative treatment group compared with As₂O₃ treatment group was found(P<0.01).4. NAC attenuates As₂O₃ and AA mediated ROS increase and cell apoptosis§1 NAC attenuates As₂O₃ and AA mediated ROS increase Intracellular ROS was detected by means of an oxidation-sensitive fluorescent probe (DCFH-DA). 10mmol/L antioxidant NAC attenuated combinative treatment mediated ROS elevation(P<0.05).§2 NAC attenuates As₂O₃ and AA induced apoptosis 10mmol/L NAC abolished the apoptosis effect of As₂O₃ and AA combinative treatment, and the apoptosis rate decreased from 15.60 %±1.14% to 9.48%±0.67% after10mmol/L NAC was added to combinative treatment(P<0.05). ConclusionAA potentiated As₂O₃-induced apoptosis through oxidative pathway by increasing ROS level which may be the result of delpeting intracellular GSH. It may influence the cascade downstream following ROS, such as, mitochondria depolarization and caspase-3 activation, but SOD and GPx depletion and the ratio of Bcl-2 to Bax influenced by As₂O₃ was not found to be potentiated by AA.