Effect Of Combination Arsenic Trioxide With Ascorbic Acid Inhibited Proliferation And Induced Apoptosis In Hepatocellular Carcinoma

Arsenci trioxide (AS₂O₃) approved for the treatment of a specific type of leukemia known as Acute promyelocytic leukemia (APL) .And preclinical and mechanistic studies suggest that this agent may have broad applicability in haematological and other malignancies. Although it has been widely accepted , many chemotherapeutic drugs function as proapoptotic signals which can trigger apoptosis in tumor cells, the heterogeneity of cellular susceptibility to these cytotoxic agents limited their usage on tumors in clinic. Cancer cells that were intrinsically sensitive to AS₂O₃ contained lower levels of GSH, whereas resistant cancer cells contained higher levels of Glutathione (GSH). The GSH redox systems against oxidative stress, inhibiting GSH production may lead to oxidative stress and induction of apoptosis, through manipulate of the GSH redox system, Arsenci trioxide (AA) can additionally enhance apoptosis cells by AS₂O₃.We choose the less sensitivity hepG₂ cell line. To study the synergistic effect of AS₂O₃ and AA on inhibition of proliferation and induction ofapoptosis in hepatocellular carcinoma cell line and explore the drug's mechanism of action. We set up co-culture system of AS2O3 and AA to hepG2 cells which were treated with AS2O3 alone or in combination with AA. The cell growth curves were detected by cell counting , glutathione(GSH) content were detected by GSH assay. Morphologic evidence for apoptosis was determined by Acridine Orange/ethidium bromide (AO/EB) staining, cell cycle and Sub-Gi and AnnexinV-PI staining were detected by FCM. The release of Cyt c and activation of Caspase-3 and the expression of Bcl-2 were demostrated by immunocytochemistry and western blotting respectively.The major findings of the present study are:1. Effect of combination AS2O3 with AA inhibited proliferation and detected intracellular GSH content in hepG2 cells.We found that AS2O3 can significantly inhibited the proliferation of hepG2 cells in a dose and time-dependent manner. Compared to separate usage of either drug alon, AS2O3 and AA can effectively enhanced growth inhibition with AS2O3 at the dosage of 5umol/L at the third day, while at the dosage of 2umol/L at the fourth day. Cell cycle analysis by flow cytometry showed that AS2O3 inhibited proliferation by arrest at the G2/M and S phase cell, while cell cycle arrest at the G0/G1 after combining AA. With AA treated cells, intracellular GSH content were significant decreased, two agents treated cells decreased GSH content than usage of AS2O3 alone.2. Effect of combination AS2O3 with AA induced apoptosis in hepG2 cells.AS2O3 induced apoptosis of hepG2 cells were confirmed by AO/EB staining, it were obviously seen the early and later apoptosis cell in AS2O3 treated cells. With AS2O3 treated cell at the dosage of 5umol/L alone or...