Nitro-NSAIDs are a new class of NSAIDs that have been developed to provide the prototypical anti-inflammatory and analgesic effects of NSAIDs but have minimal toxic gastrointestinal side-effects observed for aspirin and related drugs. The NO moiety enhances gastric mucosal repair and ulcer healing and ameliorates NSAID-induced toxicity. NO-NSAIDs have also been investigated for their anticancer activities and the results of in vitro
cell culture and in vivo
animal studies have been promising (17
). These compounds typically inhibit cell growth and induce apoptosis in various cancer cell lines and this includes inhibition of β-catenin/Tcf complexes and downregulation of cyclin D1 in colon cancer cells (17
). Nitro-aspirin derivatives inhibit growth of tumors in mouse xenograft experiments using HT-29 colon cancer cells and this is accompanied by decreased vascularity in the tumors and downregulation of VEGF (27
). GT-094 also inhibits cancer cell and tumor growth and, in both Caco-2 and HT-29 colon cancer cells, there was no evidence for induction of apoptosis (25
Studies in this laboratory have shown that COX-2 inhibitors such as celecoxib decreased colon cancer cell growth and the NSAID tolfenamic acid decreased pancreatic cancer cell growth, and these effects were due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors (30
). GT-094 inhibited growth of RKO and SW480 colon cancer cells (), induced PARP cleavage (), and TUNEL staining () consistent with induction of apoptosis. Moreover, GT-094 also decreased expression of the antiapoptotic survivin and bcl-2 proteins and this was consistent with induction of apoptosis. GT-094 decreased expression of other proteins that play a role in cell growth (cyclin D1, c-Met and EGFR) and angiogenesis (VEGF and VEGFR1) () and this was consistent with previous studies on NONSAIDs which report downregulation of cyclin D1 and VEGF (24
). Previous studies in this laboratory have demonstrated by RNA interference that knockdown of Sp1, Sp3 and Sp4 individually or in combination decreases expression of the prosurvival, growth promoting, and angiogenic proteins (29
). Results shown in suggest that an underlying mechanism for these responses in RKO and SW480 cells may be related to downregulation of Sp1, Sp3 and Sp4 proteins and this was observed in these cells treated with GT-094 (). The time-dependent decrease in expression of Sp1, Sp3, Sp4 and Sp-regulated genes was also determined in RKO and SW480 cells treated with GT-094 (). Although the rate of decrease in expression of these proteins was similar in each of the cell lines, there were also notable exceptions, particularly for some putative Sp-regulated genes which have previously been characterized by Sp1, Sp3 and Sp4 knockdown in RNA interference studies (29
). For example, expression of cyclin D1 (RKO cells) and bcl-2 (SW480 cells) was rapidly decreased (< 6 h) prior to Sp transcription factors or most other Sp-regulated genes, suggesting that GT-094 also induced other pathways that repress genes independent of Sp downregulation.
At least two different pathways have been linked to drug-dependent downregulation of Sp transcription factors, namely via activation of proteasomes (29
) or through downregulation of miR-27a and the subsequent induction of the Sp repressor ZBTB10 (35
). GT-094-dependent downregulation of Sp1, Sp3 and Sp4 was not reversed by proteasome inhibitors (), whereas the NSAID tolfenamic acid and the triterpenoid betulinic acid induce proteasome-dependent degradation of Sp transcription factors in pancreatic and prostate cancer cells, respectively (34
). In contrast, CDODA-Me-dependent downregulation of Sp1, Sp3 and Sp4 in colon cancer cells was proteasome-independent as observed in this study, and the effects of CDODA-Me were due to decreased miR-27a and the subsequent induction of ZBTB10, a miR-27a-regulated mRNA (35
). Moreover, in SW480 and RKO cells transfected with antisense-miR-27a or ZBTB10 expression plasmid, there is also a decrease in Sp1, Sp3, Sp4 and Sp-regulated genes (35
). This pathway was also relevant for GT-094 which induced ZBTB10 and decreased miR-27a in colon cancer cells ( and Supplemental Figure 1
). ZBTB10 is a member of the POK family of transcriptional repressors (42
) and was first identified as an inhibitor of gastrin gene expression (43
). ZBTB10 binds to the GC-rich gastrin gene promoter and inhibits Sp1-dependent transactivation and presumably inhibits expression of Sp1, Sp3, Sp4 and Sp-regulated genes through similar interactions with their GC-rich promoters.
In ongoing studies with arsenic trioxide (40
) and other pro-oxidants including curcumin and CDDO-Me in pancreatic cancer cells (38
), we have observed that these compounds decreased MMP and increased ROS and ROS-induced downregulation of Sp and Sp-regulated gene products. Results illustrated in and indicate that GT-094 decreased MMP and induced ROS in RKO and SW480 cells and this was reversed in cells cotreated with the thiol antioxidant GSH. The effects of GT-094 on MMP and ROS were similar to the induction of ROS by NO-ASA in colon cancer cell lines (18
). However, induction of ROS by GT-094 in this study was also related to modulation of the miR-27a:ZBTB10-Sp1/Sp3/Sp4 axis since cotreatment with GSH attenuated GT-094-mediated downregulation of miR-27a and induction of ZBTB10 () decreased expression of Sp1, Sp3 and Sp4 (), increased TUNEL staining (), and decreased growth (). It was also apparent from the cotreatment studies (GT-094 + GSH) that there were cell context-dependent differences in the inhibitory effects of GSH. For example, GSH almost totally reversed GT-094-mediated inhibition of RKO cell proliferation but only partially reversed these effects in SW480 cells (), suggesting that other GT-094-induced growth inhibitory responses were important in the latter cell line. In RKO cells, 20 μM GT-094 induced ZBTB10 mRNA but did not downregulate miR-27a (), suggesting that other miRs or other factors contributed to expression of this gene and these are currently being investigated.
In summary, this study shows that like CDODA-Me, GT-094 decreases miR-27a and induces ZBTB10 expression in colon cancer cells and the subsequent downregulation of Sp1, Sp3, Sp4 and Sp-regulated proteins contributes to the anticancer activities of this compound. Moreover, GT-094 disruption of mitochondria and induction of ROS are critical elements for the subsequent ROS-dependent downstream disruption of the miR-27a:ZBTB10-Sp transcription factor axis (). In addition, this study and a previous report in pancreatic cancer cells demonstrates that ROS suppresses miR-27a, and we have also observed similar ROS-dependent effects on the miR-27a promoter. We are currently investigating the specific cis-elements and trans-acting factors responsible for ROS-miR-27a interactions. These results demonstrate a hitherto unknown mechanism of action for GT-094 and other NONSAIDs in cancer cells (data not shown), and current studies are focused on the role of Sp transcription factors as targets for NO-NSAIDs and NSAIDs in cancer cells and tumors.