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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. Results are expressed as means ± SE (3 replicates) and significant (p<0.05) inhibition is indicated (*).</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>.</p

Mechanisms of aspirin-induced Sp protein degradation.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>.</p

Aspirin inhibits colon cancer cell growth and induces apoptosis.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. The expression of apoptotic proteins PARP cleavage was determined by western blot analysis of whole cell lysates as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048208#s3" target="_blank">Experimental Procedures</a>. Results in (A) and (B) are means ± SE for 3 replicate determination for each treatment group, and significant (p<0.05) inhibition is indicated (*).</p

siNR4A1 and NR4A1 antagonist inhibit mTOR in 786-O cells.

<p>Cells were transfected with siNR4A1 (A) and treated with DIMI-C-pPhOH (B) and DIM-C-pPhCO₂Me (C), and whole cell lysates were analyzed by western blots as outlined in the Materials and Methods. (D) Induction of sestrin 2 by western blots was determined in 786-O cells treated with DIM-C-pPhOH and DIM-C-pPhCO₂Me or transfected with siNR4A1 in the presence or absence of 5 mM GSH.</p

NR4A1 plays a role in expression of Sp-regulated growth promoting and survival genes.

<p>(A) Cells were transfected with siNR4A1 and whole cell lysates were analyzed by western blots as outlined in the Materials and Methods. Cells were treated with DIM-C-pPhOH (B) or DIM-C-pPhCO₂Me (C) and after 24 hr, whole cell lysates were analyzed by western blots. (D) Western blot analysis of tumor lysates from athymic nude mice bearing ACHN xenografts and treated with vehicle (control) or DIM-C-pPhOH (30 mg/kg/d) was also determined. Band intensities were quantitated relative to β-actin (means ± SE) and significantly decreased staining intensities are indicated (*; p<0.05).</p

NR4A1 plays a role in RCC proliferation.

<p>(A) Pathways activated by NR4A1 and targeted by NR4A1 antagonists. (B) Cells were transfected with two different oligonucleotides targeting NR4A1 [NR4A1(1) and NR4A1(2)] and after 72 hr, the number of cells were determined as outlined in the Materials and Methods. (C) Cells were treated with DIM-C-pPhOH and DIM-C-pPhCO₂Me and cell numbers were determined after treatment for 24 hr. (D) Cells were transfected with NBRE₃-luc and 40 ng FLAG-NR4A1, treated with DMSO, DIM-C-pPhOH (20 μM) and DIM-C-pPhCO₂Me (15 μM), and luciferase activity was determined as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128308#pone.0128308.ref017" target="_blank">17</a>]. (E) Cells were transfected with siCtl (non-specific) or siNR4A1 and treated with DIM-C-pPhOH and DIM-C-pPhCO₂Me and cell numbers were determined. siNR4A1 alone decreased cell proliferation as indicated in (B) and this value was set at 100% to determine the effects of C-DIMs in cells after loss of NR4A1. (F) Athymic nude mice bearing ACHN cells as xenografts were treated with 30 mg/kg/d DIM-C-pPhOH and tumor volumes were determined. Results (C—E) are means ± SE for at least 3 separate determinations and significantly (p < 0.05) decreased growth/volume is indicated (*). Significant (p < 0.05) attenuation of C-DIM-induced growth or luciferase activity or after transfection with siNR4A1 is also indicated (**).</p