Exogenous Antioxidants, N-acetylcysteine and Glutathione, Enhance Cancer Initiation and Growth in Hepatocellular Carcinoma

Abstract

ABSTRACT Introduction The controversy over the intake of antioxidants supplements in cancer patients persists for decades without efficient guideline. In tumor cells, increased metabolic activities are accompanied with increased antioxidants production to adapt to oxidative stress and maintain the malignancy. Here we investigated the effects of exogenous antioxidants on tumor initiation and growth, using hepatocellular carcinoma (HCC) as a model. Methods We performed functional assays to investigate the effects of exogenous N-acetylcysteine (NAC) and glutathione (GSH) on tumor initiation and growth in multiple mouse models in vivo including diethylnitrosamine (DEN)-induced and CRISPR-Cas9-mediated Trp53KO/C-MycOE HCC models, and subcutaneous and orthotopic xenograft tumor models, as well as in HCC cell lines. We also evaluated the effect of antioxidants on Sorafenib treatment in vitro and in vivo. RNA-sequencing was performed to identify the pathways affected by antioxidants. Pharmacological inhibitors targeting NAC and GSH metabolism were used to investigate the effects of the antioxidants. Results Exogenous NAC and GSH promoted colony formation, cell proliferation, sphere formation, and migratory and invasive abilities in human HCC cell lines. Both NAC and GSH enhanced tumor initiation in DEN-induced HCC model and CRISPR-Cas9 mediated Trp53KO/C-MycOE spontaneous HCC model in mice, accompanied with significant reduction of intra-tumoral reactive oxygen species (ROS) levels in the tumors. Limiting dilution assay in subcutaneous xenografts in mice also revealed a significantly increased tumor incidence rate of human HCC cells upon NAC and GSH treatment. Using the orthotopic injection mouse model, we found both NAC and GSH significantly boosted tumor growth of mouse hepatoma cells Hepa1-6 and human HCC cells, with significantly reduced intracellular ROS levels in the tumors. Histological analysis further revealed there was more aggressive and invasive growth behavior in NAC- and GSH-treated mice livers; there were significantly more lung metastasis foci in GSH-treated lungs in nude mice. On the other hand, upon testing the effects of these antioxidants on Sorafenib treatment, we observed that the tumor suppressive effect of Sorafenib was abrogated by NAC or GSH, which increased the cell proliferation and reduced the apoptotic cell population and ROS levels in vitro. Moreover, the tumor suppressive effect by Sorafenib was also abrogated by NAC and GSH in vivo when compared to Sorafenib alone. The increase in liver tumor burden was also accompanied by increased lung metastases and reduction of ROS levels. RNA-sequencing and GSEA analysis revealed that both NAC and GSH altered similar molecular pathways to affect metabolism and epigenetic pathways. The NRF2/GCLC-related antioxidant production pathway was down-regulated upon supplementation of NAC and GSH in the presence of ROS inducer. Of note, sulfasalazine (SSA) and buthionine sulphoximine (BSO), which are inhibitors targeting NAC and GSH metabolism respectively, partially abrogated the pro-oncogenic effects induced by NAC and GSH, and inhibited cell proliferation, sphere formation and increasing ROS levels in vitro. In vivo tumor promoting effect by GSH was abolished by addition of BSO, accompanied by significant increase in ROS level. Conclusion Our data implicate that NAC and GSH as exogenous antioxidants promote HCC initiation, enhance tumor growth, and counteract the therapeutic effect of Sorafenib both in vitro and in vivo by reducing the intracellular ROS levels. Our study provides scientific insights regarding the use of exogenous antioxidant supplements in cancer patients.