Mechanisms of Action of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in Colon Cancer

Non-steroidal anti-inflammatory drugs (NSAIDs) and their NO derivatives (NO-NSAIDs), and synthetic analogs are highly effective as anticancer agents that exhibit relatively low toxicity compared to most clinically used drugs. However, the mechanisms of action for NSAIDs and NO-NSAIDs are not well defined and this has restricted their clinical applications and applications for combined therapies. Earlier studies from our laboratory reported that specificity protein (Sp) transcription factors (Sp1, Sp3 and Sp4) are overexpressed in several types of human cancers including colon cancer and many Sp-regulated genes are pro-oncogenic and individual targets for cancer chemotherapy. Based on published results showing that NSAIDs downregulate several putative Sp-regulated genes, we hypothesized that the anticancer properties of NSAIDs may be due, in part, to downregulation of Sp transcription factors. NSAIDs including aspirin and tolfenamic acid (TA) and nitro derivatives of NSAIDs such as GT-094 have been investigated in colon cancer cells and in vivo xenograft models. Aspirin and TA induced apoptosis and decreased colon cancer cell growth and tumor growth in vivo and downregulated genes associated with cell growth, survival, and angiogenesis. Previous RNA interference studies in this laboratory have shown that many of these genes are regulated, in part, by Sp transcription factors Sp1, Sp3 and Sp4 that are overexpressed in colon and other cancer cell lines. Not surprisingly, these NSAIDs also decreased Sp1, Sp3 and Sp4 proteins and Sp-regulated gene products in colon cancer cells and this was due to caspase-dependent proteolysis of Sp1, Sp3 and Sp4 proteins. Aspirin-induced activation of caspases and degradation of Sp1, Sp3 and Sp4 was due to sequestration of zinc and could be reversed by addition of zinc sulphate, whereas TA mediated induction of caspases was independent of zinc ions and is currently being investigated. GT-094 is a novel NO chimera-containing NSAID, which also inhibited colon cancer cell proliferation and induced apoptosis; these effects were accompanied by decreased mitochondrial membrane potential (MMP) and induction of reactive oxygen species (ROS), and were reversed after cotreatment with the antioxidant glutathione. GT-094 also downregulated Sp and Sp-dependent gene products and was due to decreased expression of microRNA-27a (miR-27a) and induction of ZBTB10, an Sp transcriptional repressor that is regulated by miR-27a in colon cancer cells. Moreover, the effects of GT-094 on Sp1, Sp3, Sp4, miR-27a and ZBTB10 were also inhibited by glutathione suggesting that the anticancer activity of GT-094 in colon cancer cells is due, in part, to ROS-dependent disruption of miR-27a:ZBTB10. The importance of ROS induction in targeting Sp transcription factors was also confirmed using pro-oxidants such as ascorbic acid, hydrogen peroxide and t-butyl hydroperoxide and similar results have been observed in collaborative studies with other ROS inducers in colon cancer cells. Many cancer cell lines and tumors exhibit addiction to non-oncogenes such as Sp1, Sp3 and Sp4 for maintaining the oncogenic phenotype and future research will focus on the mechanisms of ROS-mediated targeting of Sp transcription factors which represents a novel approach for cancer chemotherapy.