Tigecycline exerts an antiproliferative effect in vitro
The antiproliferative effect of tigecycline was evaluated by performing clonogenic and MTS assays in the colon cancer cell lines HCT116 and Caco-2, as well as in the epithelial cell line derived from ​​​​​​​​​​the normal colon mucosa NCM356. Treatment with tigecycline reduced proliferation (Figure 1A) and clonogenic capacity (Figure 1B) of all the cell lines tested in a dose-dependent manner, similarly to 5-FU. However, 5-FU showed more cytotoxic effect as observed in the MTS assay (Figure 1A). These results were confirmed in HCT116 cells by immunofluorescence assay using the antibody anti-MKI67 (Figure 1C).
Then, we studied the molecular mechanisms involved in the anti-proliferative effects of tigecycline, focusing on the role of β-catenin (CTNNB1), a mediator in the Wnt signaling pathway, involved in the development of several types of cancer, including CRC (Zhanget al. , 2018).
Tigecycline and 5-FU reduced nuclear CTNNB1 as well as increased cytoplasmic CTNNB1, thus lowering nuclear/cytoplasmic ratio in comparison with non-treated cells (Figure 2A). Consequently, tigecycline hinders CTNNB1 nuclear translocation, maybe increasing cytoplasmic CTNNB1 phosphorylation (Figure 2A). Moreover, they also reduced MYC levels (Figure 2A), downstream of the CTNNB1 pathway. In addition, tigecycline also inhibited STAT3 activation (Figure 2A), which promotes nuclear accumulation of CTNNB1 (Kawada et al. , 2006).
To confirm these results the Wnt/β-catenin pathway was stimulated with Wnt3a in HCT116 cells, which caused a reduction of CTNNB1 phosphorylation and increased nuclear CTNNB1 (Figure 2B), thus resulting in upregulated AXIN2 and MYC transcription (Figure 2C). However, tigecycline and 5-FU enhanced CTNNB1 phosphorylation and lowered its nuclear translocation, evidenced by the lower nuclear CTNNB1 levels in treated cells (Figure 2B). Downregulated expression of CTNNB1 target genes, MYC and AXIN2, was also observed with tigecycline and 5-FU (Figure 2C). Therefore, we confirmed that tigecycline and 5-FU act as inhibitors of the Wnt/β-catenin pathway in tumor cells, both in basal and Wnt3a-stimulated proliferation.