Abstract
This research aims to investigate the effect of gemcitabine (GEM) on various activities and functions of macrophages. Phagocytosis, cell autophagy and reactive oxygen species (ROS) were analysed by laser scanning confocal microscope. The cell cycle status and major histocompatibility complex II (MHC-II) expression were examined by flow cytometry. Inflammatory cytokine secretion such as tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) was detected by Elisa assay. The expression of proteins was analysed by western blot method. The results revealed that GEM-induced immune inhibition of M1-type RAW264.7 macrophages activated by interferon-γ (IFN-γ) and lipopolysaccharide (LPS). We also found that GEM inhibited autophagy, as evidenced by the reduced formation of autophagosome-like vacuoles and autophagosomes. Further study showed that incubation of activated macrophages with the autophagy inhibitor 3-MA induced immune suppression. In contrast, treatment with the autophagy inducer trehalose (Tre) restored phagocytosis, TNF-α and IL-6 secretion, and MHC-II expression in GEM-induced immune-inhibited macrophages. GEM reduced immune effect of M1-type RAW264.7 macrophages via inhibiting TNF-α, IL-6 and MHC-II expression. Furthermore, activation of autophagy by Tre reversed GEM-induced immune inhibition of RAW264.7 macrophages.
Highlights
IFN-γ and LPS treatment alone did not impact the cell cycle of the macrophages; treatment with GEM combined with IFN-γ/LPS led to fewer cells in G 2/M and S phase and more cells in G0/G1 phase (Fig. 1A–B)
The effects of GEM on phagocytosis in activated macrophages were assessed by zymosan particles (Fig. S2) and fluorescently labelled bacteria
The results showed that IFN-γ/LPS-treated macrophages displayed obvious green fluorescence of internalized zymosan (P < 0.01) (Fig. 1C and D) and FITClabelled Escherichia coli DH5α (E.coli DH5α) (Fig. S3), indicating increased phagocytic activity
Summary
Gemcitabine (2′,-2′-difluoro-deoxycytidine, GEM) is a firstline treatment for pancreatic cancer. Its efficacy is limited by the acquisition of drug resistance after long-term use [1]. In addition to its cytotoxic effect, GEM has been shown to exert immunomodulatory activity in different animal tumour models. GEM has been confirmed to selectively deplete regulatory T cells, myeloid-derived suppressor cells and B lymphocytes in tumour-bearing models [2, 3]. Macrophage-based immunotherapies for cancer are more effective and tolerable than radiotherapeutics, chemotherapy or surgery [4].
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