Simultaneous proteosome inhibition and heat shock protein induction by bortezomib is beneficial in experimental pancreatitis

Abstract

The proteosome inhibitor bortezomib is used in the treatment of patients with multiple myeloma. Proteosomes are responsible for the degradation of I-κB, the inhibitory protein of transcription factor nuclear factor kappa B (Nf-κB). The heat shock protein (HSP) inducing effect of bortezomib is also documented. The aim of our work was to test the anti-inflammatory effect of bortezomib in cholecystokinin-octapeptide (CCK-8)-induced acute pancreatitis. Male Wistar rats were divided into three groups (n = 8 in each). Group P received an i.p. injection of physiological saline (p.s.) 60 min. before the induction of acute pancreatitis by three hourly s.c. injections of 100 µg/kg CCK-8. Group BP received 1 mg/kg bortezomib dissolved in p.s. 1 h previous to pancreatitis induction. Group C was treated with the vehicle (p.s.). Animals were exsanguinated 4 h after the last injection of CCK-8. Bortezomib pre-treatment significantly reduced the pancreatic weight/body weight ratio, and improved the histology by decreasing the extent of vacuolization and infiltration. Bortezomib pre-treatment inhibited I-κBβ degradation, and induced the synthesis of HSP72. The results confirmed the anti-inflammatory effect of bortezomib in acute experimental pancreatitis. This effect of the drug is presumably mediated by the inhibition of Nf-κB activation and induction of HSP synthesis.

Introduction

Acute pancreatitis is an inflammatory disease caused by the premature activation of pancreatic exocrine enzymes which leads to the injury of the gland and other organs (Figarella et al., 1988). Heat shock protein (HSP) induction and nuclear factor kappa B (NF-κB) inhibition were identified in the past as two effective mechanisms reducing the severity of the disease.

NF-κB is a transcription factor regulating the inflammatory process of acute pancreatitis (Chen et al., 2002, Storz et al., 2004). NF-κB binding elements have been found in the promoter region of many genes encoding inflammatory cytokines and enzymes. In the pancreas, NF-κB exists as a homo or heterodimer of two subunits called p65 and p50 (Gukovsky et al., 1998). Under physiological conditions, NF-κB is located in the cytosol of the cell and is bound to the inhibitory protein I-kappa B (I-κB). During the inflammatory processes, I-κB is phosphorylated by I-κB kinase followed by ubiquitination. This complex is then degraded by the proteosome, a protein complex with multiple proteolytic sites. The unbound NF-κB can translocate to the nuclei. Inhibition of NF-κB activation has been shown to exert anti-inflammatory effects in acute pancreatitis (Letoha et al., 2005a, Letoha et al., 2005b, Rakonczay et al., 2003a). Nf-κB is believed to be a key factor in the development of acute pancreatitis as it regulates the synthesis of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and 6 (IL-6), inducible nitric oxide synthase (iNOS), cyclooxigenase-2 (COX-2) and many other molecules.

HSPs were identified in the past as protective factors during the course of acute pancreatitis (Rakonczay et al., 2003b). These proteins are molecular chaperones responsible for the correct folding, transportation of newly synthesized proteins and for the reconstruction of the three dimensional structure of damaged molecules. HSP synthesis is regulated through transcription factors called heat shock factors (HSF). These factors are bound to HSP70 in the resting cells. If the amount of denaturized intracellular proteins increases, HSP70 dissociates from HSF which allows the transcription factor to bind to its specific responsive element on the DNA. Induction of HSP synthesis in acute pancreatitis reduces the amount of denaturized proteins in the cells which otherwise would lead to the induction of cell death (Garrido et al., 2001, Kubisch et al., 2004, Nollen et al., 1999, Otaka et al., 1994, Parcellier et al., 2003, Rakonczay et al., 2002, Strowski et al., 1997, Wagner et al., 1996).

Bortezomib is a proteosome inhibitor synthesized in 1995 and approved for the treatment of patients with myeloma multiplex or mantle cell lymphoma (Chauhan et al., 2005, Pei et al., 2003, Zhao et al., 2008). Proteosomes are responsible for the degradation of ubiquitinylated proteins and also abnormal or misfolded proteins. One target of proteosomes is I-κB, but proteosomes are also responsible for the degradation of anti- or pro-apoptotic proteins, increasing the sensitivity of immortal tumor cells to chemotherapeutic agents (Grisham et al., 1999). Based on our present knowledge about the molecular mechanism of acute pancreatitis, we hypothesize that bortezomib can have a beneficial effect on the inflammatory process of acute pancreatitis.

The most commonly used experimental model of acute edematous pancreatitis is the cholecystokinin-octapeptide (CCK-8)-induced pancreatitis model in rats. CCK-8 consists of the C-terminal 8 amino acids of CCK and has almost the same biological effects as CCK. In rats, repeated supramaximal doses of CCK-8 lead to the development of a mild edematous pancreatitis. The model is characterized by elevated serum amylase and lipase activities, changes of free-radical-scavenger activities, edema of the pancreas and increased amounts of inflammatory cytokines in the pancreas and serum (Czako et al., 1998, Grady et al., 1996, Laszik et al., 1989, Takacs et al., 1994, Takacs et al., 1996). As CCK-8-induced pancreatitis is a rapidly developing pancreatitis model, it is ideal for the investigation of early events of pancreatitis. These characteristics and the high reproducibility of CCK-8-induced pancreatitis make it the most widely used model for testing the effect of a new compound in acute pancreatitis.