Pharmacologic doses of ascorbic acid repress specificity protein (Sp) transcription factors and Sp-regulated genes in colon cancer cells

Abstract

Ascorbic acid (vitamin C) inhibits cancer cell growth, and there is a controversy regarding the cancer chemoprotective effects of pharmacologic doses of this compound that exhibits prooxidant activity. We hypothesized that the anticancer activity of pharmacologic doses of ascorbic acid (<5 mM) is due, in part, to reactive oxygen species-dependent downregulation of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 and Sp-regulated genes. In this study, ascorbic acid (1-3 mM) decreased RKO and SW480 colon cancer cell proliferation and induced apoptosis and necrosis, and this was accompanied by downregulation of Sp1, Sp3, and Sp4 proteins. In addition, ascorbic acid decreased expression of several Sp-regulated genes that are involved in cancer cell proliferation [hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor and cyclin D1], survival (survivin and bcl-2), and angiogenesis [vascular endothelial growth factor (VEGF) and its receptors (VEGFR1 and VEGFR2)]. Other prooxidants such as hydrogen peroxide exhibited similar activities in colon cancer cells, and cotreatment with glutathione inhibited these responses. This study demonstrates for the first time that the anticancer activities of ascorbic acid are due, in part, to ROS-dependent repression of Sp transcription factors.

Fig. 1

Ascorbate is cytotoxic to colon cancer cells. (A) Cell growth inhibition. RKO and SW480 cells were treated with 1 – 3 mM ascorbate for 3 hr and, after 24 hr, cell number was determined as described in the Materials and Methods. Results are expressed as means ± SE for 3 replicate determinations and significant (P<0.05) growth inhibition is indicated (*). Induction of PARP cleavage (B) and necrosis and apoptosis (C) in RKO and SW480 cells. RKO and SW480 cells were treated with 3 mM ascorbate for 3 hr and, after 24 hr, cell lysates were analyzed by western blots for PARP cleavage (B) or were stained with the fluorescent dye kit containing FITC-Annexin V, ethidium homodimer III and Hoechst 3342 and analyzed for apoptosis and necrosis as described in the Materials and Methods.

Fig. 2

Ascorbate-induced effects on protein involved in cell growth and cell death. The effects of ascorbate on expression of EGFR, c-Met and cyclin D1 proteins in RKO (A) and SW480 (B) cells and also on levels of survivin and bcl-2 and cleaved PARP in RKO (C) and SW480 (D) cells were determined on whole cell lysates by western blots as described in the Materials and Methods. Results are typical of duplicate experiments.

Fig. 3

Ascorbate, H₂O₂ and T-BOOH downregulate Sp1, Sp3 and Sp4 proteins. RKO and SW480 cells were treated with ascorbate (A, B), H₂O₂ (C) and T-BOOH (D), and whole cell lysates were analyzed for Sp1, Sp3 and Sp4 proteins or VEGF, VEGFR1 and VEGFR2 proteins by western blots as described in the Materials and Methods. Gels are typical of results obtained in multiple (2 – 3) experiments. Cleaved PARP was also determined in cells treated with H₂O₂ or T-BOOH.

Fig. 4

Antioxidants inhibit ascorbate-induced ROS and growth inhibition. Induction of ROS in RKO (A) and SW480 (B) cells. Cells were treated with DMSO, 3 mM ascorbate alone or in combination with antioxidants and, after 12 hr, ROS was determined as described in the Materials and Methods. Cell proliferation in RKO (C) and SW480 (D) cells. Cells were treated with DMSO, ascorbate alone, or in combination with antioxidants (3 hr) and, after 24 hr, cell numbers were counted as described in the Materials and Methods. Results (A, B) are means ± SE for at least 3 separate experiments per treatment group and significant (P<0.05) responses induced by ascorbate (*) and inhibited after cotreatment with antioxidants (**) are indicated.

Fig. 5

Antioxidants inhibit ascorbate-induced repression of Sp1, Sp3, Sp4 and Sp-regulated gene products. RKO and SW480 cells were treated with 3 mM ascorbate for 3 hr in the presence or absence of thiol antioxidants and, after 24 hr, the expression of Sp1, Sp3 and Sp4 proteins (A), Sp-regulated growth promoting (B), and angiogenic (C) proteins were determined by western blot analysis of whole cell lysates. The blots are representative of replicated (2 – 3) experiments.

Fig. 6

Antioxidants block H₂O₂- and T-BOOH-induced responses in colon cancer cells. (A) Cell proliferation. RKO and SW480 cells were treated with H₂O₂ (24 hr) or T-BOOH (24 hr) alone or in the presence of antioxidants. Cells were then counted as described in the Materials and Methods. Results are expressed as means ± SE for at least 3 replicate experiments for each treatment group and significant (P<0.05) H₂O₂- and T-BOOH-induced responses (*) and inhibition by antioxidant (**) are indicated. Effects of antioxidants on H₂O₂ (B) and T-BOOH (C)-dependent downregulation of Sp transcription factors. Cells were treated as described in (A) and whole cell lysates were analyzed by western blots. Gels are typical of replicate (at least 2) experiments.