Effect of psychological stress on aging and its implication on the oral cavity : A mini-review

departmental bulletin pape

Omeprazole Inhibits Pancreatic Cancer Cell Invasion through a Nongenomic Aryl Hydrocarbon Receptor Pathway

Omeprazole and 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin (TCDD) are aryl hydrocarbon receptor (AhR) agonists that inhibit the invasion of breast cancer cells through inhibition of CXCR4 transcription. Treatment of highly invasive Panc1 pancreatic cancer cells with TCDD, omeprazole, and seven other AhR-active pharmaceuticals showed that only omeprazole and tranilast, but not TCDD, inhibited invasion in a Boyden chamber assay. Similar results were observed in MiaPaCa2 cells, another quasimensenchymal pancreatic ductal adenocarcinoma (QM-PDA) pancreatic cancer cell line, whereas invasion was not observed with BxPC3 or L3.6pL cells, which are classified as classical (less invasive) pancreatic cancer cells. It was also observed in QM-PDA cells that TCDD, omeprazole, and tranilast did not induce CYP1A1 or CXCR4 and that treatment with these compounds did not result in nuclear uptake of AhR. In contrast, treatment of BxPC3 and L3.6pL cells with these AhR ligands resulted in induction of CYP1A1 (by TCDD) and nuclear uptake of AhR, which was similar to that observed for Ah-responsive MDA-MB-468 breast and HepG2 liver cancer cell lines. Results of AhR and AhR nuclear translocator (Arnt) knockdown experiments in Panc1 and MiaPaCa2 cells demonstrated that omeprazole- and tranilast-mediated inhibition of invasion was AhR-dependent but Arnt-independent. These results demonstrate that in the most highly invasive subtype of pancreatic cancer cells (QM-PDA) the selective AhR modulators omeprazole and tranilast inhibit invasion through a nongenomic AhR pathway

DataSheet1_Piperlongumine is a ligand for the orphan nuclear receptor 4A1 (NR4A1).DOCX

Piperlongumine and derivatives are being developed as anticancer agents which act primarily as inducers of reactive oxygen species (ROS) in cancer cell lines. Many of the anticancer activities of piperlongumine resemble those observed for bis-indole derived compounds that bind the orphan nuclear receptor 4A1 (NR4A1) and act as inverse receptor agonists to inhibit NR4A1-regulated pro-oncogenic pathways and genes. In this study we show that like other NR4A1 inverse agonists piperlongumine inhibited RKO, SW480 and HCT116 colon cancer cell growth migration and invasion and induced apoptosis. Piperlongumine also downregulated the pro-reductant isocitrate dehydrogenase 1 (IDH1) and thioredoxin domain-containing 5 (TXNDC5) gene products resulting in the induction of ROS as previously observed for other inverse NR4A1 agonists. ROS also induced sestrin2 and this resulted in activation of AMPK phosphorylation and inhibition of mTOR pathway signaling. It has previously been reported that these pathways/genes are also regulated by inverse NR4A1 agonists or by knockdown of NR4A1. We also observed that piperlongumine directly bound NR4A1, inhibited NR4A1-dependent transactivation and interactions of the NR4A1/Sp1 complex bound to the GC-rich promoter of the NR4A1-regulated G9a gene.</p

DataSheet2_Piperlongumine is a ligand for the orphan nuclear receptor 4A1 (NR4A1).PDF

Piperlongumine and derivatives are being developed as anticancer agents which act primarily as inducers of reactive oxygen species (ROS) in cancer cell lines. Many of the anticancer activities of piperlongumine resemble those observed for bis-indole derived compounds that bind the orphan nuclear receptor 4A1 (NR4A1) and act as inverse receptor agonists to inhibit NR4A1-regulated pro-oncogenic pathways and genes. In this study we show that like other NR4A1 inverse agonists piperlongumine inhibited RKO, SW480 and HCT116 colon cancer cell growth migration and invasion and induced apoptosis. Piperlongumine also downregulated the pro-reductant isocitrate dehydrogenase 1 (IDH1) and thioredoxin domain-containing 5 (TXNDC5) gene products resulting in the induction of ROS as previously observed for other inverse NR4A1 agonists. ROS also induced sestrin2 and this resulted in activation of AMPK phosphorylation and inhibition of mTOR pathway signaling. It has previously been reported that these pathways/genes are also regulated by inverse NR4A1 agonists or by knockdown of NR4A1. We also observed that piperlongumine directly bound NR4A1, inhibited NR4A1-dependent transactivation and interactions of the NR4A1/Sp1 complex bound to the GC-rich promoter of the NR4A1-regulated G9a gene.</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

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

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

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

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