GT-094, a NO-NSAID, inhibits colon cancer cell growth by activation of a reactive oxygen species-microRNA-27a: ZBTB10-specificity protein pathway

Abstract

Ethyl 2-((2,3-bis(nitrooxy)propyl)disulfanyl)benzoate (GT-094) is a novel nitric oxide (NO) chimera containing an nonsteroidal anti-inflammatory drug (NSAID) and NO moieties and also a disulfide pharmacophore that in itself exhibits cancer chemopreventive activity. In this study, the effects and mechanism of action of GT-094 were investigated in RKO and SW480 colon cancer cells. GT-094 inhibited cell proliferation and induced apoptosis in both cell lines and this was accompanied by decreased mitochondrial membrane potential (MMP) and induction of reactive oxygen species (ROS), and these responses were reversed after cotreatment with the antioxidant glutathione. GT-094 also downregulated genes associated with cell growth [cyclin D1, hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor (EGFR)], survival (bcl-2, survivin), and angiogenesis [VEGF and its receptors (VEGFR1 and VEGFR2)]. Results of previous RNA interference studies in this laboratory has shown that these genes are regulated, in part, by specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 that are overexpressed in colon and other cancer cell lines and not surprisingly, GT-094 also decreased Sp1, Sp3, and Sp4 in colon cancer cells. GT-094-mediated repression of Sp and Sp-regulated gene products was due to downregulation of microRNA-27a (miR-27a) and induction of ZBTB10, an Sp 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 activation of an ROS-miR-27a:ZBTB10-Sp transcription factor pathway.

Figure 1

GT-094 inhibits growth and induces apoptosis in colon cancer cells. (A) Inhibition of RKO and SW480 cell growth. Cells were treated with different concentrations of GT-094, and cell numbers were determined on days 1, 2 and 3 as described in the Materials and Methods. Western blot analysis of Sp-regulated survival (B), proliferative (C), and angiogenic (D) gene products in RKO and SW480 cells treated with GT-094. Cells were treated with GT-094 (20 and 50 μmol/L) for 24 h, and whole cell lysates were analyzed by western blots as described in the Materials and Methods.

Figure 2

GT-094-mediated effects on Sp protein and Sp-regulated gene products. (A) GT-094 decreases Sp1, Sp3 and Sp4 protein expression in colon cancer cells. RKO and SW480 cells were treated with 20 or 50 μmol/L GT-094 for 24 h, and whole cell lysates were analyzed by western blots as described in the Materials and Methods. (B) Effects of proteasome inhibitors on Sp downregulation. Cells were treated with 50 μmol/L GT-094, MG132 or lactacystin alone or in combination, and whole cell lysates were analyzed by western blots as described in the Materials and Methods. Results in Figure 2 are typical of replicate (at least 2) experiments. Time-course effects of GT-094 (50 μM) in RKO (C) and SW480 (D) cells. Cells were treated with DMSO (0 time) or GT-094 for 6, 12, 18, 24 and 36 h, and whole cell lysates were analyzed by western blots as outlined in the Materials and Methods.

Figure 3

GT-094 decreases MMP in colon cancer cells. Effects of GT-094 in RKO (A) and SW480 (B) cells. Cells were treated with 50 μmol/L GT-094, 25 μmol/L CCCP, and GT-094 plus GST for 12 h and analyzed for changes in MMP as outlined in the Materials and Methods. Quantitative changes in MMP in RKO (C) and SW480 (D) cells. Changes in MMP were determined as described in the Materials and Methods. Significant (p<0.05) decreases by GT-094 or CCCP (*) and reversal of these effects by GSH (**) are indicated.

Figure 4

Role of ROS in GT-094-mediated downregulation of Sp1, Sp3 and Sp4 proteins. Induction of ROS in RKO (A) and SW480 (B) cells. Cells were treated with 50 μmol/L GT-094, 1 mmol/L DTT, and 5 mmol/L GSH alone or in combination for 12 h, and ROS was determined as described in the Materials and Methods. Significant (p<0.05) induction by GT-094 (*) and inhibition of this response by GSH or DTT (**) are indicated. Effects of GSH or DTT on GT-094-mediaited Sp downregulation in RKO (C) and SW480 (D) cells. Cells were treated with different concentrations as indicated for 24 h, and whole cell lysates were analyzed by western blots as outlined in the Materials and Methods. Results are typical of replicate (2) experiments.

Figure 5

GSH blocks GT-094-induced growth inhibition and apoptosis. Inhibition of RKO (A) and SW480 (B) cell growth by GT-094 blocked by GSH. Cells were treated with 50 μmol/L GT-094 or 5 mmol/L GSH alone or in combination for 24 h, and cell numbers were determined as described in the Materials and Methods. Significant (p<0.05) inhibition by GT-094 (*) or reversal of this effect by GSH (**) are indicated. Induction of apoptosis by GT-094 ± GSH in RKO (C) and SW480 (D) cells. Cells were treated with GT-094 ± GSH for 24 h, and apoptosis was determined by TUNEL staining as described in the Materials and Methods. Significant (p<0.05) induction of apoptosis by GT-094 (*) and inhibition after cotreatment with GSH (**) are indicated.

Figure 6

GT-094 modulates miR-27a and ZBTB10 expression. (A) GT-094 decreases miR-27a expression in RKO and SW480 cells. Cells were treated with 20 or 50 μmol/L GT-094 and 5 mmol/L GSH alone or in combination for 24 h, and miR-27a was determined by real time PCR as outlined in the Materials and Methods. (B) GT-094 induces ZBTB10 in RKO and SW480 cells. Cells were treated with 20 or 50 μmol/L GT-094 and 5 mmol/L GSH alone or in combination for 24 h, and ZBTB10 mRNA levels were determined by real time PCR as outlined in the Materials and Methods. Significant (p<0.05) inhibition of miR-27a and induction of ZBTB10 (*) and inhibition of these responses by GSH (**) are indicated. (C) Proposed molecular mechanism for GT-094-induced ROS and ROS-dependent disruption of the miR-27a:ZBTB10-Sp/Sp-regulated gene axis.