RESUMO
Histone deacetylase inhibitors (HDACi) show high antineoplastic potential in preclinical studies in various solid tumors, including gastric carcinoma; however, their use in clinical studies has not yet yielded convincing efficacies. Thus, further studies on cellular/molecular effects of HDACi are needed, for improving clinical efficacy and identifying suitable combination partners. Here, we investigated the role of oxidative stress in gastric cancer cells upon treatment with HDACi. A particular focus was laid on the role of the Nrf2 pathway, which can mediate resistance to cell-inhibitory effects of reactive oxidative species (ROS). Using fluorescence-based ROS sensors, oxidative stress was measured in human gastric cancer cell lines. Activation of the Nrf2 pathway was monitored in luciferase reporter assays as well as by mRNA and proteomic expression analyses of Nrf2 regulators and Nrf2-induced genes. Furthermore, the effects of ROS scavenger N-acetyl-L-cysteine (NAC) and Nrf2-knockdown on HDACi-dependent antiproliferative effects were investigated in colorimetric formazan-based and clonogenic survival assays. HDACi treatment led to increased oxidative stress levels and consequently, treatment with NAC reduced cytotoxicity of HDACi. In addition, vorinostat treatment stimulated expression of a luciferase reporter under the control of an antioxidative response element, indicating activation of the Nrf2 system. This Nrf2 activation was only partially reversible by treatment with NAC, suggesting ROS independent pathways to contribute to HDACi-promoted Nrf2 activation. In line with its cytoprotective role, Nrf2 knockdown led to a sensitization against HDACi. Accordingly, the expression of antioxidant and detoxifying Nrf2 target genes was upregulated upon HDACi treatment. In conclusion, oxidative stress induction upon HDAC inhibition contributes to the antitumor effects of HDAC inhibitors, and activation of Nrf2 represents a potentially important adaptive response of gastric cancer cells in this context.
RESUMO
Gastric cancer remains among the deadliest neoplasms worldwide, with limited therapeutic options. Since efficacies of targeted therapies are unsatisfactory, drugs with broader mechanisms of action rather than a single oncogene inhibition are needed. Preclinical studies have identified histone deacetylases (HDAC) as potential therapeutic targets in gastric cancer. However, the mechanism(s) of action of HDAC inhibitors (HDACi) are only partially understood. This is particularly true with regard to ferroptosis as an emerging concept of cell death. In a panel of gastric cancer cell lines with different molecular characteristics, tumor cell inhibitory effects of different HDACi were studied. Lipid peroxidation levels were measured and proteome analysis was performed for the in-depth characterization of molecular alterations upon HDAC inhibition. HDACi effects on important ferroptosis genes were validated on the mRNA and protein level. Upon HDACi treatment, lipid peroxidation was found increased in all cell lines. Class I HDACi (VK1, entinostat) showed the same toxicity profile as the pan-HDACi vorinostat. Proteome analysis revealed significant and concordant alterations in the expression of proteins related to ferroptosis induction. Key enzymes like ACSL4, POR or SLC7A11 showed distinct alterations in their expression patterns, providing an explanation for the increased lipid peroxidation. Results were also confirmed in primary human gastric cancer tissue cultures as a relevant ex vivo model. We identify the induction of ferroptosis as new mechanism of action of class I HDACi in gastric cancer. Notably, these findings were independent of the genetic background of the cell lines, thus introducing HDAC inhibition as a more general therapeutic principle.