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.