Antioxidant supplements may accelerate the formation and progression of hepatocellular carcinoma in vitro and in vivo

Abstract

Antioxidants are electron donors that can neutralize and therefore inhibit reactive oxygen species (ROS). Some evidence has shown that not only normal cells but cancer cells also require antioxidants, as cancer cells produce even more ROS as a result of their rapid growth. N-acetylcysteine (NAC) and glutathione (GSH) are popular antioxidant supplements for humans. A recent study done by Martin O’ team has revealed that NAC could accelerate lung cancer through reducing the ROS-related DNA damage in mice. Tak Mak’s group has reported that the GSH antioxidant pathway is essential for breast cancer initiation in mice. Liver cancer (hepatocellular carcinoma) is the fifth leading cause of cancer-related death worldwide. Whether NAC and GSH would aggravate HCC remains unknown. In this study, we aimed to investigate the effects of popular antioxidant supplements including NAC and GSH on HCC both in vitro and in vivo. We first screened the cell viability and colony formation effects of NAC and GSH on 5 different HCC cell lines, a human HCC cell line MHCC97L showed the most promoting effects upon treatments. To further evaluate the effects of NAC and GSH on HCC initiation, growth and progression in vitro, we performed functional tests such as sphere formation, cell proliferation, migration and invasion assays in MHCC97L cells. Subcutaneous implantation and orthotopic liver injection mouse models were employed to further investigate the effects of NAC on tumorigenicity and tumor progression in vivo. To reveal the underlying mechanism, we also tested the expression levels of proteins involved in the ROS defence system. From the in vitro experiments, NAC-treated HCC cells promoted colony formation, suggesting that NAC might facilitate tumor cell growth. GSH increased the sphere formation, which is a test for self-renewal ability in vitro, and cell proliferation in MHCC97L cells. Significant enhancement in cell migratory and invasive abilities was observed in both NAC- and GSH-treated HCC cells. For the in vivo tumorigenicity assay, 120 mg/kg/day of NAC (based on conversion from the human equivalent dosage) was given to mice immediately after the tumor cell injection subcutaneously. Toxic side effects were observed in NAC-treated mice such as distended abdomen and weight loss despite a reduction in tumor incidence. In the orthotopic liver injection model, mice were treated with 60 mg/kg/day of NAC after tumor onset. NAC administration significantly increased the tumor masses and incidence of lung metastases when compared to the control group. Furthermore, NAC reduced the expression of p21 by western blotting while GSH showed no significant effect on protein markers involved in ROS defence pathways, such as AMT, P53 and H2AX. To conclude, our data show that antioxidants supplements could exacerbate HCC growth and progression in HCC cells in vitro and mice in vivo. The role of antioxidant supplements such as NAC and GSH in cancer therapy deserves more extensive studies.