Abstract
Unmethylated CpG dinucleotides, present in bacterial DNA, are recognized in vertebrates via the Toll-like receptor 9 (TLR9) and are known to act as an anticancer agent by stimulating immune cells to induce a proinflammatory response. Although the effects of CpG-oligodeoxynucleotides (CpG-ODNs) in immune cells have been widely studied, little is known regarding their molecular effects in TLR9-positive tumor cells. To better understand the role of these bacterial motifs in cancer cells, we analyzed proteome modifications induced in TLR9-positive tumor cells in vitro and in vivo after CpG-ODN treatment in a rat colon carcinoma model. Proteomics analysis of tumor cells by two-dimensional gel electrophoresis followed by mass spectrometry identified several proteins modulated by bacterial CpG motifs. Among them, several are related to autophagy including potential autophagic substrates. In addition, we observed an increased glyceraldehyde-3-phosphate dehydrogenase expression, which has been shown to be sufficient to trigger an autophagic process. Autophagy is a self-digestion pathway whereby cytoplasmic material is sequestered by a structure termed the autophagosome for subsequent degradation and recycling. As bacteria are known to trigger autophagy, we assessed whether bacterial CpG motifs might induce autophagy in TLR9-positive tumor cells. We showed that CpG-ODN can induce autophagy in rodent and human tumor cell lines and was TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after CpG motif intratumoral injection. Our findings bring new insights on the effect of bacterial CpG motifs in tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues.
Highlights
Unmethylated CpG dinucleotides, present in bacterial DNA, are recognized in vertebrates via the Toll-like receptor 9 (TLR9) and are known to act as an anticancer agent by stimulating immune cells to induce a proinflammatory response
These differences lead the immune system to recognize unmethylated CpG sequences as a “danger signal” indicating a potential pathogen infection [2]. Recognition of these pathogen-associated molecular patterns (PAMPs)1 is mediated by the Toll-like receptor 9 (TLR9) [3] and followed by downstream signaling events leading to the activation of target genes [4]
CpG-ODN-induced Effects on TLR9-positive Colon Cancer Cells in Vitro and in Vivo—To investigate the molecular events underlying the various effects of CpG-ODN reported in tumor cells (14 –21), we analyzed proteome modifications induced in the PROb TLR9-positive rat colon carcinoma cell line after CpG-ODN treatment
Summary
Oligonucleotides and Plasmids—The following sequences of the phosphorothioate-modified ODNs (Eurogentec, Seraing, Belgium) were used: for experiments in rodent cells (or human embryonal kidney (HEK) 293 cells expressing the murine TLR9): CpG-ODN 1826, 5Ј-TCCATGACGTTCCTGACGTT-3Ј; and control ODN, 5Ј-TCCATGACCTTCCTGACCTT-3Ј; for experiments in human cells: CpG-ODN 2006, 5Ј-TCGTCGTTTTGTCGTTTTGTCGTT-3Ј; and control ODN, 5ЈTGCTGCTTTTGTGCTTTTGTGCTT-3Ј. Cells were resuspended in the electroporation V-buffer at a final concentration of 1.5 ϫ 107 (PROb and HEK 293) or 2 ϫ 107 (MCF-7 and PC-3) cells/ml and transfected with 2 g of pLC3-GFP plasmid in the absence or in the presence of 1 g of CpG-ODN or 1 g of control ODN. The MS/MS parameters were as follows: isolation width, 3; collision energy, 35%; micro-HPLC Surveyor system (Thermo Fisher); 60-min gradient; and BioBasic C18 (Thermo Scientific) column (100 ϫ 0.32 mm) Such high mass accuracy on the precursor ion allowed the elimination of virtually any false positive peptide identifications, suggesting that peptides that do not match the specificity of the protease used in the digestion should not automatically be considered as false positives. The statistics were computed with MINITABTM V12.2 software (Minitab, Inc., State College, PA). p values were considered to be statistically significant when less than 0.05 for one comparison and less than 0.0125 for four comparisons
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