Increased Expression of the E3-Ubiquitin Ligase Receptor Subunit βTRCP1 Relates to Constitutive Nuclear Factor-κB Activation and Chemoresistance in Pancreatic Carcinoma Cells

Abstract

The permanent activation of the transcription factor nuclear factor-n nB (NF-n nB) in pancreatic cancer cells is associated with a profound resistance towards chemotherapy. In the present study, we show that chemoresistant pancreatic cancer cell lines exhibiting constitutive NF-n nB activity (i.e., PancTu-1, BxPc3, and Capan-1) express significantly elevated levels of the E3-ubiquitin ligase receptor subunit B BTRCP1, compared with pancreatic carcinoma cell lines lacking constitutive NF-n nB activity and chemoresistance (i.e., PT45-P1 and T3M4). If transfected with B BTRCP1, PT45-P1 cells exhibit an elevated NF-n nB activity and become less sensitive towards anticancer drug treatment (i.e., etoposide). Conversely, blockade of B BTRCP1 expression in PancTu-1 cells by transfection with a vector-expressed small interfering RNA reduces NF-K nB activation and chemoresistance. In PancTu-1 cells, B BTRCP1 expression is inhibited, at least in part, by the interleukin-1 (IL-1) receptor(I) antagonist, whereas stimulation of PT45-P1 cells with IL-1B B resulted in an increased expression of B BTRCP1, and transfection of this cell line with B BTRCP1 induced IL-1B B secretion in a NF-n nB-dependent fashion. Thus, via its close and mutual link to IL-1B B secretion, B BTRCP1 expression might substantially contribute to the persistent, IL-1B B-dependent activation of NF-n nB in pancreatic carcinoma cells. In support of this, B BTRCP1 expression is detectable at considerable levels in a great number of pancreatic ductal adenocarcinoma specimens, along with an intense staining for activated NF-n nB. Altogether, our findings of the elevated B BTRCP1 expression in pancreatic carcinoma cells pinpoint to another important mediator of constitutive NF-n nB activation and thereby of chemoresistance. (Cancer Res 2005; 65(4): 1316-24)

Inflammatory Macrophages Induce Nrf2 Transcription Factor-dependent Proteasome Activity in Colonic NCM460 Cells and Thereby Confer Anti-apoptotic Protection

“…Then the labeled NCM460 cells were incubated in OPTIMEM (PAA Laboratories) for 5 min at 37°C followed by mono-or coculture in OPTIMEM for 1, 3, and 6 h. Western Blotting-Preparation of nuclear and cytoplasmic extracts or total cell lysates was carried out as described before (11,42). After electrophoresis and semi-dry electroblotting onto PVDF membranes, the following primary antibodies were used for immunodetection at a 1000-fold dilution in 5% (w/v) nonfat milk powder, 0.05% Tween 20 in TBS (50 mM Tris/HCl, pH 7.6, and 150 mM NaCl): Nrf2 (Epitomics via Biomol), Hsp90 and Nrf1 (Santa Cruz, Heidelberg, Germany), tubulin (Sigma), S5a and ␣5 (both from Affiniti/Biomol, Hamburg, Germany), and cleaved caspase-3 and PARP1 (Cell Signaling Technology, Frankfurt, Germany).…”

Nuclear factor-κB and inhibitor of κB kinase pathways in oncogenic initiation and progression

“…Relative to other signaling pathways, in a significant number of lung adenocarcinomas, Fas-associated death domain phosphorylation is associated with poor clinical outcome and induces IKK-mediated NF-kB activation (Chen et al, 2005). Interestingly, in pancreatic cancer cells, high levels of expression of the E3-ubiquitin ligase receptor beta-transducin repeat-containing protein 1 (bTRCP1) is proposed to activate NF-kB (Muerkoster et al, 2005; Figure 2). Finally, in breast cancer, CK2 was proposed to induce NF-kB ( Figure 2) and this effect was recently proposed to involve the upregulation of the IKKe subunit (Eddy et al, 2005).…”

Acquired chemoresistance in pancreatic carcinoma cells: induced secretion of IL-1β and NO lead to inactivation of caspases

“…Thus, the innate as well as the acquired chemoresistance in PDAC cells are both characterized by these two cellular mediators. In addition, another common mediator closely related to IL-1b and NO is NF-kB, which is constitutively activated in the course of innate as well as acquired chemoresistance (Mu¨erko¨ster et al, 2004(Mu¨erko¨ster et al, , 2005.…”

“…This autocrine loop obviously evolves from tumor-stroma interactions, as shown for the chemosensitive PDAC cell lines PT45-P1 and T3M4 that, upon coculture with stromal fibroblasts, gained NF-kB-dependent chemoresistance preceeded by elevated IL-1b-secretion (Mu¨erko¨ster et al, 2004). The importance of these factors in the development of innate chemoresistance was further supported by immunohistochemical stainings of human PDAC specimens, which revealed increased expression of the activated NF-kB subunit p65 and IL-1b in a large number of the tested tumor samples but not in normal pancreatic tissue (Mu¨erko¨ster et al, 2004;Mu¨erko¨ster et al, 2005). As we have already described a model for the gain of innate chemoresistance in PT45-P1 cells emerging from tumor-stroma interactions, the aim of this study was to identify mechanisms underlying the acquired chemoresistance of this anti cancer-drug-sensitive cell line.…”

“…ELISA-based NF-kB assay NF-kB activation was analysed by the TransAM NF-kB-kit (Active Motif) using nuclear extracts as described previously (Mu¨erko¨ster et al, 2005). For each sample, 20 ml of nuclear extracts containing 5 mg protein (determined by the D c -Protein assay, BioRad; Mu¨nchen, Germany) were used according to the manufacturer's instructions.…”