bay-11-7082 and Colorectal-Neoplasms

Cytokines play important roles in tumorigenesis and progression of cancer cells, while their functions in drug resistance remain to be illustrated. We successfully generated doxorubicin (Dox)-resistant CRC HCT-116 and SW480 cells (namely HCT-116/Dox and SW480/Dox, respectively). Cytokine expression analysis revealed that IL-8, while not FGF-2, EGF, TGF-β, IL-6, or IL-10, was significantly increased in Dox-resistant CRC cells as compared with their corresponding parental cells. Targeted inhibition of IL-8 via siRNAs or its inhibitor reparixin can increase the Dox sensitivity of HCT-116/Dox and SW480/Dox cells. The si-IL-8 can decrease the mRNA and protein expression of multidrug resistance 1 (MDR1, encoded by ABCB1), while has no effect on the expression of multidrug resistance-associated protein 1 (ABCC1), in CRC Dox-resistant cells. IL-8 can increase the phosphorylation of p65 and then upregulate the binding between p65 and promoter of ABCB1. BAY 11-7082, the inhibitor of NF-κB, suppressed the recombination IL-8 (rIL-8) induced upregulation of ABCB1. It confirmed that NF-κB is involved in IL-8-induced upregulation of ABCB1. rIL-8 also increased the phosphorylation of IKK-β, which can further activate NF-κB, while specific inhibitor of IKK-β (ACHP) can reverse rIL-8-induced phosphorylation of p65 and upregulation of MDR1. These results suggested that IL-8 regulates the Dox resistance of CRC cells via modulation of MDR1 through IKK-β/p65 signals. The targeted inhibition of IL-8 might be an important potential approach to overcome the clinical Dox resistance in CRC patients.

Topics: Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B; Cell Line, Tumor; Colorectal Neoplasms; Cytokines; Doxorubicin; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; I-kappa B Kinase; Interleukin-8; NF-kappa B; Nitriles; Phosphorylation; RNA, Small Interfering; Sulfonamides; Sulfones; Transcription Factor RelA; Up-Regulation

Colorectal cancer (CRC) remains a common and deadly cancer due to metastatic disease. Activin and TGFB (TGFβ) signaling are growth suppressive pathways that exert non-canonical pro-metastatic effects late in CRC carcinogenesis. We have recently shown that activin downregulates p21 via ubiquitination and degradation associated with enhanced cellular migration independent of SMADs. To investigate the mechanism of metastatic activin signaling, we examined activated NFkB signaling and activin ligand expression in CRC patient samples and found a strong correlation. We hypothesize that activation of the E3 ubiquitin ligase MDM2 by NFkB leads to p21 degradation in response to activin treatment. To dissect the link between activin and pro-carcinogenic NFkB signaling and downstream targets, we found that activin but not TGFB induced activation of NFkB leading to increased MDM2 ubiquitin ligase via PI3K. Further, overexpression of wild type p65 NFkB increased MDM2 expression while the NFkB inhibitors NEMO-binding domain (NBD) and Bay11-7082 blocked the activin-induced increase in MDM2. In conclusion, in colon cancer cell migration, activin utilizes NFkB to induce MDM2 activity leading to the degradation of p21 in a PI3K dependent mechanism. This provides new mechanistic knowledge linking activin and NFkB signaling in advanced colon cancer which is applicable to targeted therapeutic interventions.

Topics: Activins; Carcinogenesis; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; NF-kappa B; Nitriles; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Sulfones; Ubiquitin-Protein Ligases

Silymarin from milk thistle (Silybum marianum) plant has been reported to show anti-cancer, anti-inflammatory, antioxidant and hepatoprotective effects. For anti-cancer activity, silymarin is known to regulate cell cycle progression through cyclin D1 downregulation. However, the mechanism of silymarin-mediated cyclin D1 downregulation still remains unanswered. The current study was performed to elucidate the molecular mechanism of cyclin D1 downregulation by silymarin in human colorectal cancer cells. The treatment of silymarin suppressed the cell proliferation in HCT116 and SW480 cells and decreased cellular accumulation of exogenously-induced cyclin D1 protein. However, silymarin did not change the level of cyclin D1 mRNA. Inhibition of proteasomal degradation by MG132 attenuated silymarin-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with silymarin. In addition, silymarin increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated silymarin-mediated cyclin D1 downregulation. Inhibition of NF-κB by a selective inhibitor, BAY 11-7082 suppressed cyclin D1 phosphorylation and downregulation by silymarin. From these results, we suggest that silymarin-mediated cyclin D1 downregulation may result from proteasomal degradation through its threonine-286 phosphorylation via NF-κB activation. The current study provides new mechanistic link between silymarin, cyclin D1 downregulation and cell growth in human colorectal cancer cells.

Topics: Antineoplastic Agents; Cell Cycle; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; HCT116 Cells; Humans; Leupeptins; NF-kappa B; Nitriles; Phosphorylation; Point Mutation; Proteasome Endopeptidase Complex; Proteolysis; Silybum marianum; Silymarin; Sulfones; Threonine

A proliferation-inducing ligand (APRIL) which stimulates the cell proliferation is abundantly expressed in colorectal cancer (CRC) tumors. In this report, the promoter region of the APRIL gene was determined and the major transcription factor was investigated for the first time. Deletion analysis of 5'-flanking region of the human APRIL gene and transient transfection revealed that a 538 bp region (from -1539 to -1001) was essential for promoter activation of the APRIL gene. The data from electrophoretic mobility shift assays (EMSA) indicated that the 538 bp promoter region was responsive to the specificity protein 1 (Sp1) and nuclear factor kappa B (NF-kB). Overexpression of Sp1 or NF-kB increased the activity of the APRIL promoter. Mithramycin A (inhibitor of Sp1) and Bay11-7082 (inhibitor of NF-kB) exhibited an inhibitory activity to APRIL promoter. Our results will benefit to the APRIL gene regulation investigation and contribute to discover new drug target for the APRIL gene therapy of CRC.

Topics: Base Sequence; Binding Sites; Colorectal Neoplasms; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Neoplastic; Humans; Luciferases; Molecular Sequence Data; NF-kappa B; Nitriles; Plicamycin; Promoter Regions, Genetic; Protein Binding; Response Elements; Sequence Deletion; Sp1 Transcription Factor; Sulfones; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor Ligand Superfamily Member 13

Nuclear functions for IkappaB kinase (IKK), including phosphorylation of histone H3 and nuclear corepressors, have been recently described. Here, we show that IKK is activated in colorectal tumors concomitant with the presence of phosphorylated SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor that is aberrantly localized in the cytoplasm. In these tumors, IKKalpha associates to the chromatin of specific Notch targets, leading to the release of SMRT. Abrogation of IKK activity by BAY11-7082 or by expressing dominant negative IKKalpha restores the association of SMRT with Notch target genes, resulting in specific gene repression. Finally, BAY11-7082 significantly reduces tumor size in colorectal cancer xenografts (CRC-Xs) implanted in nude mice.

Topics: Animals; Cell Line; Cell Nucleus; Colorectal Neoplasms; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; Male; Mice; NF-kappa B; Nitriles; Phosphorylation; Receptors, Notch; Repressor Proteins; Sulfones