Association for Academic Surgery
Inhibition of MIF Leads to Cell Cycle Arrest and Apoptosis in Pancreatic Cancer Cells

https://doi.org/10.1016/j.jss.2009.03.048Get rights and content

Background

Pancreatic ductal adenocarcinoma (PDAC) is the eighth most common cancer with the lowest overall 5-y relative survival rate. Gene expression profiling of PDAC revealed an overexpression of the macrophage migration inhibitory factor (MIF), a lymphokine involved in cell-mediated immunity and inflammation, as well as in the regulation of cellular signal transduction.

Materials and Methods

Endogenous MIF expression was silenced by treatment of pancreatic cancer cell lines using two independent MIF siRNAs. The expression of MIF RNA and protein after siRNA treatment was investigated using quantitative RT-PCR and Western blot. Induction of apoptosis was analyzed using fluorescence activated cell sorting (FACS).

Results

Transfection of MiaPaCa-2 cells with MIF siRNA resulted in a reduction of MIF RNA and protein levels by more than 85%. After treatment, we observed an inhibition of cellular proliferation accompanied by induction of apoptosis. Analysis of the phosphorylation state of Akt showed a markedly increase of the phosphorylation at the Thr308 residue.

Conclusions

Using post-transcriptional silencing with small interfering RNAs, we could show that MIF acts as an autocrine growth factor involved in cell cycle progression. Since MIF is a secreted protein, a therapy directed against MIF or its receptor might lead to a significant growth reduction of PDAC.

Introduction

Pancreatic cancer is one of the most aggressive malignancies in industrialized countries, with an incidence of 6.3/100 000 [1]. It is characterized by early metastasis and aggressive, infiltrative growth along the endothelium basement membrane and neurons [2]. Most patients with pancreatic cancer have a poor outcome due to the absence of early diagnosis and its highly invasive and metastatic features, which leaves more than 85% of the patients inoperable at the time of diagnosis. Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine involved in a wide range of pathophysiologic events in association with inflammation, innate and acquired immune responses, and cell growth [3]. Its first discovery showed an involvement in delayed hypersensitivity and various macrophage and T-cell functions [4]. MIF protein is reported to be constitutively expressed in the human gastrointestinal tract [5], and has been implicated in the pathogenesis of several acute and chronic inflammatory conditions, including septic shock and rheumatoid arthritis [6]. MIF acts as a potent initiator to promote the expression of cytokines as tumor necrosis factor (TNF)-α, IL-1β, IL-6, IL-8, PGE2 in LPS-driven responses, and to induce glucocorticoid-counter regulating activity [7]. Several reports have suggested that MIF plays a critical role in tumorigenesis, angiogenesis, and metastasis [8]. Recently, a functional interdependence between MIF and HIF-1A leading to the stabilization of HIF-1A was demonstrated in breast and pancreas cancer cell lines 9, 10. Overexpression of MIF has been shown in several human neoplasms, including prostate [11], breast [12], colorectal carcinoma, lung cancer, and pancreatic ductal adenocarcinoma (PDAC) [13].

Section snippets

Cell Lines

The cell lines MiaPaCa-2 (CRL-1420), AsPC-1 (CRL-1682), BxPC-3 (CRL-1687), Capan-1 (HTB-79), CFPAC-1 (CRL-1918), HPAF-II (CRL-1997), and PANC-1 (CRL-1469) were purchased from LGC Promochem (Wesel, Germany); the cell line Colo357 (EACC 94072245) from HyperCLDB, PANC-89 (T3M4), PancTuI (PaCa2), PT 45, and PT 64 were kindly made available by Professor H. Kalthoff (Kiel, Germany).

Cell Culture, Transfection, and Cellular Assays

MiaPaCa-2 cells were grown in D-MEM (Gibco, Karlsruhe, Germany), 10% fetal calf serum (PAN Biotech GmbH), 2.5% horse

Gene Profiling in PDAC

A previous search for genetic alterations by gene expression profiling of microdissected PDAC compared with microdissected normal ductal pancreatic tissue, tissue specimen of patients suffering from chronic pancreatitis and stromal tissue of PDAC revealed an overexpression of the macrophage migration inhibitory factor [15] (Fig. 1).

To evaluate expression of MIF in established cell lines we, conducted a quantitative RT–PCR. PT-45, CFPAC-1, PancTuI, and Capan-1 displayed the highest MIF

Discussion

Our findings support that silencing of MIF by siRNA in pancreatic cancer cell lines induces an inhibition of cellular proliferation accompanied by induction of apoptosis emphasizes the central role of MIF in another human neoplastic disease. MIF acts as an autocrine and paracrine growth factor for MiaPaCa-2 cells, and is likely contributing to the development of pancreatic cancer. MIF silencing further entailed reduced protein levels of total Akt and a shifting in phosphorylated residues with a

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