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

3,3'-Diindolylmethane (DIM) and its ring-substituted halogenated analogs (ring-DIMs) induce differential mechanisms of survival and death in androgen-dependent and -independent prostate cancer cells.

International audienceWe recently reported that novel ring-substituted analogs of 3,3'-diindolylmethane (ring-DIMs) induce apoptosis and necrosis in androgen-dependent and -independent prostate cancer cells. In this paper, we have focused on the mechanism(s) associated with ring-DIM-mediated cell death, and on identifying the specific intracellular target(s) of these compounds. The 4,4'- and 7,7'-dichloroDIMs and 4,4'- and 7,7'-dibromoDIMs induced the death of LNCaP, C42B and DU145 prostate cancer cells, but not that of immortalized normal human prostate epithelial (RWPE-1) cells. Ring-DIMs caused the early loss of mitochondrial membrane potential (MMP) and decreased mitochondrial ATP generation in prostate cancer cells. Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore, inhibited ring-DIM-mediated cell death, and salubrinal, an inhibitor of ER stress, inhibited cell death mediated only by 4,4'-dihaloDIMs. We found that although salubrinal did not inhibit the onset of ER stress, it prevented 4,4'-dibromoDIM mediated loss of MMP. Salubrinal potentiated cell death in response to 7,7'-dihaloDIMs and DIM, and this effect concurred with increased loss of MMP. Using in silico 3-D docking affinity analysis, we identified Ca2+/calmodulin-dependent kinase II (CaMKII) as a potential direct target for the most toxic ring-DIM, 4,4'-dibromoDIM. An inhibitor of CaMKII, KN93, but not its inactive analog KN92, abrogated cell death mediated by 4,4'-dibromoDIM. The ring-DIMs induced ER stress and autophagy, but these processes were not necessary for ring-DIM-mediated cell death. Inhibition of autophagy with bafilomycin A1, 3-methyladenine or by LC3B gene silencing sensitized LNCaP and C42B, but not ATG5-deficient DU145 cells to ring-DIM- and DIM-mediated cell death. We propose that autophagy induced by the ring-DIMs and DIM has a cytoprotective function in prostate cancer cells

A chalcone synthase-like bacterial protein catalyzes heterocyclic c-ring cleavage of naringenin to alter bioactivity against nuclear receptors in colonic epithelial cells

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The aryl hydrocarbon receptor ligand omeprazole inhibits breast cancer cell invasion and metastasis

BACKGROUND: Patients with ER-negative breast tumors are among the most difficult to treat and exhibit low survival rates due, in part, to metastasis from the breast to various distal sites. Aryl hydrocarbon receptor (AHR) ligands show promise as antimetastatic drugs for estrogen receptor (ER)-negative breast cancer. METHODS: Triple negative MDA-MB-231 breast cancer cells were treated with eight AHR-active pharmaceuticals including 4-hydroxtamoxifen, flutamide leflunomide, mexiletine, nimodipine, omeprazole, sulindac and tranilast, and the effects of these compounds on cell proliferation (MTT assay) and cell migration (Boyden chamber assay) were examined. The role of the AHR in mediating inhibition of MDA-MB-231 cell invasion was investigated by RNA interference (RNAi) and knockdown of AHR or cotreatment with AHR agonists. Lung metastasis of MDA-MB-231 cells was evaluated in mice administered cells by tail vein injection and prometastatic gene expression was examined by immunohistochemistry. RESULTS: We showed that only the proton pump inhibitor omeprazole decreased MDA-MB-231 breast cancer cell invasion in vitro. Omeprazole also significantly decreased MDA-MB-231 cancer cell metastasis to the lung in a mouse model (tail vein injection), and in vitro studies showed that omeprazole decreased expression of at least two prometastatic genes, namely matrix metalloproteinase-9 (MMP-9) and C-X-C chemokine receptor 4 (CXCR4). Results of RNA interference studies confirmed that omeprazole-mediated downregulation of CXCR4 (but not MMP-9) was AHR-dependent. Chromatin immunoprecipitation assays demonstrated that omeprazole recruited the AHR to regions in the CXCR4 promoter that contain dioxin response elements (DREs) and this was accompanied by the loss of pol II on the promoter and decreased expression of CXCR4. CONCLUSIONS: AHR-active pharmaceuticals such as omeprazole that decrease breast cancer cell invasion and metastasis may have important clinical applications for late stage breast cancer chemotherapy

NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

Overexpression of the nuclear receptor 4A1 (NR4A1) in breast cancer patients is a prognostic factor for decreased survival and increased metastasis, and this has been linked to NR4A1-dependent regulation of transforming growth factor β (TGF-β) signaling. Results of RNA interference studies demonstrate that basal migration of aggressive SKBR3 and MDA-MB-231 breast cancer cells is TGF-β independent and dependent on regulation of β1-integrin gene expression by NR4A1 which can be inhibited by the NR4A1 antagonists 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and a related p-carboxymethylphenyl [1,1-bis(3′-indolyl)-1-(p-carboxymethylphenyl)methane (DIM-C-pPhCO(2)Me)] analog. The NR4A1 antagonists also inhibited TGF-β-induced migration of MDA-MB-231 cells by blocking nuclear export of NR4A1, which is an essential step in TGF-β-induced cell migration. We also observed that NR4A1 regulates expression of both β1- and β3-integrins, and unlike other β1-integrin inhibitors which induce prometastatic β3-integrin, NR4A1 antagonists inhibit expression of both β1- and β3-integrin, demonstrating a novel mechanism-based approach for targeting integrins and integrin-dependent breast cancer metastasis

Piperlongumine is a ligand for the orphan nuclear receptor 4A1 (NR4A1)

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