Mechanisms of Action of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in Colon Cancer

Non-steroidal anti-inflammatory drugs (NSAIDs) and their NO derivatives (NO-NSAIDs), and synthetic analogs are highly effective as anticancer agents that exhibit relatively low toxicity compared to most clinically used drugs. However, the mechanisms of action for NSAIDs and NO-NSAIDs are not well defined and this has restricted their clinical applications and applications for combined therapies. Earlier studies from our laboratory reported that specificity protein (Sp) transcription factors (Sp1, Sp3 and Sp4) are overexpressed in several types of human cancers including colon cancer and many Sp-regulated genes are pro-oncogenic and individual targets for cancer chemotherapy. Based on published results showing that NSAIDs downregulate several putative Sp-regulated genes, we hypothesized that the anticancer properties of NSAIDs may be due, in part, to downregulation of Sp transcription factors. NSAIDs including aspirin and tolfenamic acid (TA) and nitro derivatives of NSAIDs such as GT-094 have been investigated in colon cancer cells and in vivo xenograft models. Aspirin and TA induced apoptosis and decreased colon cancer cell growth and tumor growth in vivo and downregulated genes associated with cell growth, survival, and angiogenesis. Previous RNA interference studies in this laboratory have shown that many of these genes are regulated, in part, by Sp transcription factors Sp1, Sp3 and Sp4 that are overexpressed in colon and other cancer cell lines. Not surprisingly, these NSAIDs also decreased Sp1, Sp3 and Sp4 proteins and Sp-regulated gene products in colon cancer cells and this was due to caspase-dependent proteolysis of Sp1, Sp3 and Sp4 proteins. Aspirin-induced activation of caspases and degradation of Sp1, Sp3 and Sp4 was due to sequestration of zinc and could be reversed by addition of zinc sulphate, whereas TA mediated induction of caspases was independent of zinc ions and is currently being investigated. GT-094 is a novel NO chimera-containing NSAID, which also inhibited colon cancer cell proliferation and induced apoptosis; these effects were accompanied by decreased mitochondrial membrane potential (MMP) and induction of reactive oxygen species (ROS), and were reversed after cotreatment with the antioxidant glutathione. GT-094 also downregulated Sp and Sp-dependent gene products and was due to decreased expression of microRNA-27a (miR-27a) and induction of ZBTB10, an Sp transcriptional 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 ROS-dependent disruption of miR-27a:ZBTB10. The importance of ROS induction in targeting Sp transcription factors was also confirmed using pro-oxidants such as ascorbic acid, hydrogen peroxide and t-butyl hydroperoxide and similar results have been observed in collaborative studies with other ROS inducers in colon cancer cells. Many cancer cell lines and tumors exhibit addiction to non-oncogenes such as Sp1, Sp3 and Sp4 for maintaining the oncogenic phenotype and future research will focus on the mechanisms of ROS-mediated targeting of Sp transcription factors which represents a novel approach for cancer chemotherapy

Sulindac sulfide inhibits colon cancer cell growth and downregulates specificity protein transcription factors

BACKGROUND: Specificity protein (Sp) transcription factors play pivotal roles in maintaining the phenotypes of many cancers. We hypothesized that the antineoplastic effects of sulindac and its metabolites were due, in part, to targeting downregulation of Sp transcription factors. METHODS: The functional effects of sulindac, sulindac sulfone and sulindac sulfide on colon cancer cell proliferation were determined by cell counting. Effects of these compounds on expression of Sp1, Sp3, Sp4 and pro-oncogenic Sp-regulated genes were determined by western blot analysis of whole cell lysates and in transient transfection assays using GC-rich constructs. RESULTS: Sulindac and its metabolites inhibited RKO and SW480 colon cancer cell growth and the order of growth inhibitory potency was sulindac sulfide > > sulindac sulfone > sulindac. Treatment of SW480 and RKO cells with sulindac sulfide downregulated expression of Sp1, Sp3 and Sp4 proteins. Sulindac sulfide also decreased expression of several Sp-regulated genes that are critical for cancer cell survival, proliferation and angiogenesis and these include survivin, bcl-2, epidermal growth factor receptor (EGFR), cyclin D1, p65 subunit of NFκB and vascular endothelial growth factor (VEGF). Sulindac sulfide also induced reactive oxygen species (ROS) and decreased the level of microRNA-27a in colon cancer cells, which resulted in the upregulation of the Sp-repressor ZBTB10 and this resulted in downregulation of Sp proteins. CONCLUSIONS: The results suggest that the cancer chemotherapeutic effects of sulindac in colon cancer cells are due, in part, to its metabolite sulindac sulfide which downregulates Sp transcription factors and Sp-regulated pro-oncogenic gene products

Aspirin Inhibits Colon Cancer Cell and Tumor Growth and Downregulates Specificity Protein (Sp) Transcription Factors

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

Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention

Mechanism of Action of Phenethylisothiocyanate and Other Reactive Oxygen Species-Inducing Anticancer Agents

Reactive oxygen species (ROS)-inducing anticancer agents such as phenethylisothiocyanate (PEITC) activate stress pathways for killing cancer cells. Here we demonstrate that PEITC-induced ROS decreased expression of microRNA 27a (miR-27a)/miR-20a:miR-17-5p and induced miR-regulated ZBTB10/ZBTB4 and ZBTB34 transcriptional repressors, which, in turn, downregulate specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 in pancreatic cancer cells. Decreased expression of miR-27a/miR-20a:miR-17-5p by PEITC-induced ROS is a key step in triggering the miR-ZBTB Sp cascade leading to downregulation of Sp TFs, and this is due to ROS-dependent epigenetic effects associated with genome-wide shifts in repressor complexes, resulting in decreased expression of Myc and the Myc-regulated miRs. Knockdown of Sp1 alone by RNA interference also induced apoptosis and decreased pancreatic cancer cell growth and invasion, indicating that downregulation of Sp transcription factors is an important common mechanism of action for PEITC and other ROS-inducing anticancer agents

Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp) transcription factors

<p>Abstract</p> <p>Background</p> <p>Betulinic acid (BA) inhibits growth of several cancer cell lines and tumors and the effects of BA have been attributed to its mitochondriotoxicity and inhibition of multiple pro-oncogenic factors. Previous studies show that BA induces proteasome-dependent degradation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the mechanism of action of BA in colon cancer cells.</p> <p>Methods</p> <p>The effects of BA on colon cancer cell proliferation and apoptosis and tumor growth <it>in vivo </it>were determined using standardized assays. The effects of BA on Sp proteins and Sp-regulated gene products were analyzed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a) and ZBTB10 mRNA expression.</p> <p>Results</p> <p>BA inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are overexpressed in colon cancer cells and decreased levels of several Sp-regulated genes including survivin, vascular endothelial growth factor, p65 sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary tumor transforming gene-1. The mechanism of action of BA was dependent on cell context, since BA induced proteasome-dependent and proteasome-independent downregulation of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of reactive oxygen species (ROS), ROS-mediated repression of microRNA-27a, and induction of the Sp repressor gene ZBTB10.</p> <p>Conclusions</p> <p>These results suggest that the anticancer activity of BA in colon cancer cells is due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors; however, the mechanism of this response is cell context-dependent.</p

Expression and role of PAICS, a de novo purine biosynthetic gene in prostate cancer

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143605/1/pros23533_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143605/2/pros23533.pd

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

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