Aspirin inhibits colon cancer cell and tumor growth and downregulates specificity protein (Sp) transcription factors

Abstract

Acetylsalicylic acid (aspirin) is highly effective for treating colon cancer patients postdiagnosis; however, the mechanisms of action of aspirin in colon cancer are not well defined. Aspirin and its major metabolite sodium salicylate induced apoptosis and decreased colon cancer cell growth and the sodium salt of aspirin also inhibited tumor growth in an athymic nude mouse xenograft model. Colon cancer cell growth inhibition was accompanied by downregulation of Sp1, Sp3 and Sp4 proteins and decreased expression of Sp-regulated gene products including bcl-2, survivin, VEGF, VEGFR1, cyclin D1, c-MET and p65 (NFκB). Moreover, we also showed by RNA interference that β-catenin, an important target of aspirin in some studies, is an Sp-regulated gene. Aspirin induced nuclear caspase-dependent cleavage of Sp1, Sp3 and Sp4 proteins and this response was related to sequestration of zinc ions since addition of zinc sulfate blocked aspirin-mediated apoptosis and repression of Sp proteins. The results demonstrate an important underlying mechanism of action of aspirin as an anticancer agent and, based on the rapid metabolism of aspirin to salicylate in humans and the high salicylate/aspirin ratios in serum, it is likely that the anticancer activity of aspirin is also due to the salicylate metabolite.

Figure 1. Aspirin inhibits colon cancer cell growth and induces apoptosis.

Inhibition of SW480 and RKO (A) and HT29 and HCT116 (B) cell proliferation. Cells were treated with DMSO or 2.5–10 mM aspirin for 3 days, and cell numbers were determined as described in the Experimental Procedures. Induction of Annexin V staining (C) and apoptotic responses in RKO and SW480 (D) and HT29 and HCT116 (E) cells. Annexin V staining was determined as described in the Experimental Procedures. The expression of apoptotic proteins PARP cleavage was determined by western blot analysis of whole cell lysates as described in the Experimental Procedures. Results in (A) and (B) are means ± SE for 3 replicate determination for each treatment group, and significant (p<0.05) inhibition is indicated (*).

Figure 2. Aspirin decreases expressions of Sp1, Sp3, Sp4 and Sp-regulated gene products in colon cancer cells.

Downregulation of Sp proteins in RKO and SW480 (A) and HT29 and HCT116 (B) and Sp-regulated gene products in RKO and SW480 (C) and HT29 and HCT116 (D) cells. Cells were treated with 5 or 10 mM aspirin for 24 or 48 hr, and whole cell lysates were analyzed by western blot analysis as described in the Experimental Procedures. Results are typical of duplicate experiments. (E) Aspirin decreases reporter gene activity. Cells were transfected with pSp1For4, pSp3For5, pVEGF and pSurvivin and treated with DMSO or aspirin (5 or 10 mM). Luciferase activity was determined as described in the Experimental Procedures. Results are expressed as means ± SE (3 replicates) and significant (p<0.05) inhibition is indicated (*).

Figure 3. Salicylate inhibits colon cancer cell growth and downregulates Sp1, Sp3, Sp4 and Sp-regulated genes.

Inhibition of RKO and SW480 (A) and HCT116 and HT29 (B) cell growth. Cells were treated with 2.5–10 mM sodium salicylate for up to 3 days, and cell numbers were determined as described in the Experimental Procedures. Protein downregulation in RKO and SW480 (C) and HCT116 and HT29 (D) cells. Cells were treated with 5 or 10 mM salicylate for 24 or 48 hr, and whole cell lysates were analyzed by western blots as described in the Experimental Procedures.

Figure 4. Aspirin decreases expression of NFκB and β-catenin in colon cancer cells.

Decreased p65/p50 in RKO (A) and SW480 (B) cells. Cells were treated for 48 hr with 5 or 10 mM, and whole cell, nuclear and cytosolic extracts were analyzed by western blot analysis as described in the Experimental Procedures. Levels of p65 and p50 proteins (relative to β-actin) in whole cell lysates were quantitated from 3 replicate experiments and were significantly decreased by aspirin. (C) Aspirin decreases NFκB-luc. The construct was transfected into RKO and SW480 cells treated with DMSO or aspirin, and luciferase activity determined as described in the Experimental Procedures. Results are means ± SE (3 replicates) and significant (p<0.05) inhibition is indicated (*). (D) Downregulation of β-catenin. Cells were treated with 5 or 10 mM aspirin for 48 hr, and whole cell lysates were analyzed by western blots as described in the Experimental Procedures.

Figure 5. Knockdown of Sp1, Sp3 and Sp4 (alone and combined) by RNA interference.

Knockdown of Sp1, Sp3, Sp4 and Sp1/3/4 (A) and p65/p50 (B) in colon cancer cells. Cells were transfected with various oligonucleotides, and whole cell lysates were analyzed by western blot analysis as outlined in the Experimental Procedures. (C) Knockdown of Sp1/3/4 (combined) inhibits NFκB-luc. Cells were transfected with iLamin (control) and iSp1/3/4 (combined oligonucleotides) and NFκB-luc, and luciferase activity was determined as described in the Experimental Procedures. Results are expressed as means ± SE (3 replicates), and significantly (p<0.05) decreased activity is indicated (*). Sp knockdown decreases β-catenin (D) and induces PARP cleavage (E). Cells were transfected with various oligonucleotides, and whole cell lysates were analyzed by western blots as described in the Experimental Procedures.

Figure 6. Mechanisms of aspirin-induced Sp protein degradation.

(A) Effects of leptomycin B. Cells were treated with 10 mM aspirin in the presence or absence of leptomycin B for 48 hr, and whole cell lysates were analyzed by western blot analysis as described in the Experimental Procedures. Effects of antioxidants (B) and caspase inhibitors (C, D) on aspirin-induced Sp protein downregulation. Cells were treated with DMSO, aspirin alone or in combination with antioxidants or caspase inhibitors, and after 48 hr, whole cell lysates were analyzed by western blots as described in the Experimental Procedures.

Figure 7. Zinc depletion results in induction of caspases and downregulation of Sp proteins.

(A) TPEN induces activation (cleavage) of caspases. Cells were treated with 25 or 50 µM TPEN for 18 hr, and whole cell lysates were analyzed by western blots as described in the Experimental Procedures. (B) TPEN decreases Sp protein expression and zinc sulfate blocks the effects of TPEN on Sp downregulation. Cells were treated with 50 µM ZnSO₄ for 18 hr, and whole cell lysates were analyzed by western blots. (C) Aspirin activates caspases. Cells were treated with aspirin for 48 hr, and whole cell lysates were analyzed by western blots as outlined in the Experimental Procedures. (D) Aspirin-induced PARP cleavage is blocked by ZnSO₄. Cells were treated for 48 hr with aspirin alone or in combination with ZnSO₄ and PARP cleavage was analyzed by western blots of whole cell lysates. (E) Effects of ZnSO₄ on aspirin-induced downregulation of Sp proteins. RKO or SW480 cells were treated with 10 mM aspirin alone or in combination with 50 µM ZnSO₄ for 48 hr, and whole cell lysates were analyzed by western blots as outlined in the Experimental Procedures.

Figure 8. Aspirin inhibits colon tumor growth in athymic nude mice (xenografts).

Inhibition of tumor weight (A) and volume (growth) (B) in athymic nude mice administered the sodium salt of aspirin. Athymic nude mice bearing RKO cells as xenografts were treated with the sodium salt of aspirin, and tumor volumes and weights were determined after sacrifice as described in the Experimental Procedures. (C) Expression of Sp1, Sp3 and Sp4 in colon tumors. Tumor lysates from solvent (control) and aspirin-treated mice were analyzed by western blot analysis as described in the Experimental Procedures. Expression of Sp1, Sp3 and Sp4 in aspirin-treated tumors compared to solvent (control)-treated tumors (set at 100%) was determined by densitometry, and β-actin was used to normalize protein expression. Results are means ± SE (6 replicates) and significant (p<0.05) inhibition of Sp1, Sp3 and Sp4 protein levels by aspirin is indicated (*). (D) Induction of apoptosis. Fixed tumor tissue from control and aspirin-treated mice were analyzed for TUNEL staining as outlined in the Experimental Procedures.