Tigecycline shows pro-apoptotic effects in colon cancer cell lines.
To evaluate the pro-apoptotic capacity of tigecycline, we used the Annexin V / PI assay in HCT116 cells. We observed a dose-response effect in the percentage of ANXV+IP- cells (Figure 3A), thus revealing its ability to promote early apoptosis after 48 h of treatment. No significant differences in the number of late apoptotic or necrotic cells (ANXV+IP+) were found. These results were confirmed by the TUNEL assay performed in HCT116 cells, since the treatment with tigecycline significantly increased the staining of brown (apoptotic) nuclei (Figure 3B), as did the control treatment (Figure 3B).
Three different pathways of apoptosis have been described: the extrinsic or death receptor pathway, the intrinsic or mitochondrial pathway, and a third less well-known that involves the endoplasmic reticulum (ER). We observed that the cellular pathways involved in 5-FU-mediated apoptosis were slightly different from those engaged by tigecycline (Figure 3C-E). When considering the extrinsic pathway, tigecycline (25 µM) increased the levels of cleaved CASP8, as well as the ratio between the cleaved and the full-length form (Figure S1A), with a trend to reduce full-length CASP8, although not significant (Figure 3C). Subsequently, we evaluated the last steps of the extrinsic pathway. Tigecycline significantly increased CASP9 and the cleaved form of the executioner CASP7 (Figure 3C). Levels of CASP9 and cleaved CASP7 were also increased by 5-FU, which also upregulated the levels of CASP3 and their active forms (Figure 3C). Furthermore, the treatment of HCT116 with tigecycline significantly increased the levels of cleaved PARP1 and reduced the levels of full length PARP1 leading to a significant increment of the ratio (Figure 3C and S1A). This action was even more intense with the 5-FU treatment (Figure 3C and S1A). These results support that treatment with tigecycline exerts a pro-apoptotic effect, as shown by 5-FU.
Besides, tigecycline increased the levels of both BID and tBID, even though this increase was not statistically significant (Figure S1A). Truncation of BID (tBID) by cleaved CASP8 on the mitochondrial membrane is required for the formation of pores and the release of cytochrome C oxidase (COX) to the cytosol, and it has been reported that BID acts as a link between the extrinsic pathway and the intrinsic pathway of apoptosis (Li et al. , 1998). Interestingly, the incubation of the cells with either tigecycline or 5-FU significantly increased the levels of Bcl-2-associated X protein (BAX) and its oligomer (Figure 3D and S1A). BAX is a member of the Bcl-2 family proteins that oligomerizes and promotes a mitochondrial outer membrane permeabilization (MOMP) (Kalkavan et al. , 2018). BCL2 inactivates BAX and its pore-forming function, so the ratio BAX/BCL2 an indicator of the cell apoptosis state. Tigecycline or 5-FU+ did not significantly modify BCL2 levels but increased BAX/BCl2 ratio (Figure 3D), which would promote the pore formation in the mitochondrial membrane and the release of COX. In fact, COX levels were increased in those cells incubated with tigecycline (Figure 3D). In addition, tumor protein 53 (TP53), involved in COX release through BAX stimulation and BCL2 inhibition and, thus, favoring the formation of the apoptosome was up-regulated by 5-FU, but not by tigecycline (Figure 3D). However, tigecycline acted downstream increasing BCL2L11 levels (Figure S1B).
The pro-apoptotic effects of both tigecycline and 5-FU also involve the ER pathway, although in different ways (Figure 3D and S1C). Tigecycline increased the levels of the ER-stress marker immunoglobulin heavy chain-binding protein (HSPA5) (Figure 3E). Similarly,tigecycline upregulated the activating transcription factor 6 (ATF6) levels, both the full length and the cleaved (active) form. This resulted in an increase of the levels of C/EBP homologous protein (DDIT3) (Figure 3E), which links ER and mitochondrial pathways. On the contrary, 5-FU did not significantly modify the levels of ATF6 or DDIT3 (Figure 3E) but increased the levels of the phosphorylated and active form of Jun-N-terminal kinase (JNK) (Figure 3E), which justifies its ability to promote apoptosis through ER pathway. Although tigecycline also increased the levels of phospho-JNK, this was only observed when the p54 isoform was considered, and with a lower efficacy in comparison with 5-FU (Figure 3E). Moreover, both, tigecycline and 5-FU, significantly increased the ratio between phospho-JUN and JUN (Figure 3E), thus favoring the activation of this proapoptotic ligand.