Original ArticleSimvastatin attenuates macrophage-mediated gemcitabine resistance of pancreatic ductal adenocarcinoma by regulating the TGF-β1/Gfi-1 axis
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with an estimated 5-year survival rate below 5% [1]. Most patients with PDAC are diagnosed in an advanced stage when the tumors are deemed unresectable and exhibit resistance to chemotherapeutic drugs, including gemcitabine and 5-fluorouracil [2]. Thus, it is urgent to explore the mechanism of this resistance and identify novel therapeutic strategies for treating PDAC.
Increasing evidence has identified a key role of tumor microenvironments (TMEs) in the progression of cancer, especially in the stroma-rich PDAC microenvironment [3], [4], [5]. Current standard-of-care therapies provide few consistent responses in PDAC, largely due to the heterogeneous TMEs [6]. The cellular component of the TME contains pancreatic stellate cells (PSCs), cancer-associated fibroblasts, various immune cells, endothelial cells and pericytes, in addition to cancer cells [6]. The immune cell population is composed of tumor-associated macrophages (TAMs), monocytic myeloid-derived suppressor cells (Mo-MDSCs), and granulocytic MDSCs (G-MDSCs) [7]. TAMs are the most abundant immune cells in the PDAC stroma [8]. Tumor cells secrete specific cytokines that stimulate TAMs, which in turn promote tumor vascularization, accelerate metastases, and confer resistance to chemotherapeutics [9]. Gemcitabine, the most frequently employed chemotherapeutic agent for treating PDAC, is metabolized to active forms by deoxycytidine kinase. Cytidine deaminase serves as a key enzyme that catalyzes the conversion of gemcitabine to inactive metabolites, and TAMs have been found to enhance the chemoresistance of PDAC by up-regulating cytidine deaminase [10]. In accordance with these findings, gemcitabine is more effective in macrophage-depleted mice than in their wild-type counterparts [11]. Thus, it seems that TAMs attenuate the responses of tumors to gemcitabine. However, the exact mechanism by which TAMs contribute to the gemcitabine resistance of PDAC remains largely unknown.
Statins, inhibitors of HMG-CoA, the enzyme that catalyzes cholesterol synthesis, exhibit chemopreventive effects beyond their cholesterol-lowing function. An epidemiological study showed that the use of statins for over 6 months caused a 67% reduction in the risk of pancreatic cancer [12]. A recent clinical case-control study suggested that the reduction in the risk of pancreatic cancer in statin users mainly appears in men and long-term users [13]. A population-based cohort study also implied that statin use significantly reduces the risk of pancreatic cancer in patients with type 2 diabetes [14]. However, two meta-analyses found no significant reduction in pancreatic cancer risks [15], [16]. Although a series of studies focused on the association between statin use and the risk of pancreatic cancer have been performed, little attention has been paid to the effect of statins on the resistance of PDAC to gemcitabine.
In this study, we found that TAMs enhanced the resistance of PDAC cells to gemcitabine and that this effect was attenuated by simvastatin treatment. Further investigation demonstrated that simvastatin treatment reduced TGF-β1 levels in TAM-conditioned medium (TAM-CM), leading to increased Gfi-1 expression in PDAC cells. Gfi-1 negatively regulated the expression of CTGF and HMGB1, contributing to the gemcitabine resistance of PDAC cells. Our data provide a novel mechanism for TAM-mediated gemcitabine resistance and suggest simvastatin as a candidate for overcoming the gemcitabine resistance of PDAC.
Section snippets
Antibodies and reagents
The following monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used: anti-Gfi-1 (pAb, sc-8558) was purchased from Santa Cruz Biotechnology and anti-CTGF (pAb, ab6992), anti-HMGB1 (pAb, ab79823), anti-TGF-β1 (pAb, ab64715) and anti-β-actin (pAb, ab8227) were purchased from Abcam (Cambridge, MA). Cell Counting Kit-8 was purchased from Dojindo Laboratories (Tokyo, Japan). Simvastatin was purchased from Sigma Chemical Co. (USA). Gemcitabine was purchased from Eli Lilly
Simvastatin attenuated TAM-mediated gemcitabine resistance in PDAC cells
We first examined the effect of simvastatin on PDAC cell viability. Treatment with simvastatin for 72 h reduced the viability of Panc-1 and BxPC-3 cells in a dose-dependent manner (Fig. 1A). The antiproliferative effect of simvastatin was most significant at 40 μM, whereas the effect of 5 μM simvastatin was negligible. Since various inflammatory cell types, such as macrophages, play key roles in triggering gemcitabine resistance [10] and simvastatin exhibits an anti-inflammatory effect on
Discussion
Chemotherapy resistance is a huge challenge in improving the survival rate of patients with PDAC. Treatment with the standard drug gemcitabine remains largely ineffective. Thus, clarification of the mechanism underlying chemotherapy resistance in PDAC would contribute to the identification of novel therapeutic strategies for overcoming advanced PDAC. Macrophages have been found to be predominant in the cell population in the microenvironment of various solid tumors [24], [25], [26]. Recent
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant No. 81201778; 81572272), the Promotive Research Fund for Excellent Young and Middle-aged Scientists of Shandong Province (Grant No. BS2011SW036) and the Science and Technology Development Plan Project of Shandong Province (Grant No. 2013GSF11862; 2016GSF201127; 2013G0021810).
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