Synergistic apoptosis by combination of metformin and an O-GlcNAcylation inhibitor in colon cancer cells

by Selleckchem | Sep 24 2023

Background: Although autophagy is an important mediator of metformin antitumor activity, the role of metformin in the crosstalk between autophagy and apoptosis remains unclear. The aim was to confirm the anticancer effect by inducing apoptosis by co-treatment with metformin and OSMI-1, an inhibitor of O-GlcNAcylation, in colon cancer cells.

Methods: Cell viability was measured by MTT in colon cancer cell lines HCT116 and SW620 cells. Co-treatment with metformin and OSMI-1 induced autophagy and apoptosis, which was analyzed using western blot, reverse transcription-polymerase chain reaction (RT-PCR) analysis, and fluorescence-activated cell sorting (FACS). Combined treatment with metformin and OSMI-1 synergistically inhibit the growth of HCT116 was confirmed by xenograft tumors.

Results: We showed that metformin inhibited mammalian target of rapamycin (mTOR) activity by inducing high levels of C/EBP homologous protein (CHOP) expression through endoplasmic reticulum (ER) stress and activating adenosine monophosphate-activated protein kinase (AMPK) to induce autophagy in HCT116 cells. Interestingly, metformin increased O-GlcNAcylation and glutamine:fructose-6-phosphate amidotransferase (GFAT) levels in HCT116 cells. Thus, metformin also blocks autophagy by enhancing O-GlcNAcylation, whereas OSMI-1 increases autophagy via ER stress. In contrast, combined metformin and OSMI-1 treatment resulted in continuous induction of autophagy and disruption of O-GlcNAcylation homeostasis, resulting in excessive autophagic flux, which synergistically induced apoptosis. Downregulation of Bcl2 promoted apoptosis via the activation of c-Jun N-terminal kinase (JNK) and CHOP overexpression, synergistically inducing apoptosis. The activation of IRE1α/JNK signaling by OSMI-1 and PERK/CHOP signaling by metformin combined to inhibit Bcl2 activity, ultimately leading to the upregulation of cytochrome c release and activation of caspase-3.

Conclusions: In conclusion, combinatorial treatment of HCT116 cells with metformin and OSMI-1 resulted in more synergistic apoptosis being induced by enhancement of signal activation through ER stress-induced signaling rather than the cell protective autophagy function. These results in HCT116 cells were also confirmed in xenograft models, suggesting that this combination strategy could be utilized for colon cancer treatment.

Comments:

The study aimed to investigate the anticancer effects of co-treatment with metformin and OSMI-1, an inhibitor of O-GlcNAcylation, in colon cancer cells. The researchers performed various experiments to evaluate cell viability, autophagy, apoptosis, and signaling pathways involved in the combined treatment.

They used two colon cancer cell lines, HCT116 and SW620, and assessed cell viability using the MTT assay. The co-treatment of metformin and OSMI-1 led to the induction of autophagy and apoptosis in the HCT116 cells, as confirmed by western blot, RT-PCR analysis, and fluorescence-activated cell sorting (FACS). Furthermore, the researchers performed xenograft tumor experiments to validate the synergistic inhibitory effect of the combined treatment on HCT116 cell growth.

The results demonstrated that metformin inhibited the activity of mammalian target of rapamycin (mTOR), a protein kinase involved in cell growth and proliferation. This inhibition was achieved by inducing high levels of C/EBP homologous protein (CHOP) expression through endoplasmic reticulum (ER) stress and activating adenosine monophosphate-activated protein kinase (AMPK), which triggered autophagy in HCT116 cells. Notably, metformin also increased O-GlcNAcylation and glutamine:fructose-6-phosphate amidotransferase (GFAT) levels in HCT116 cells. This suggested that metformin could block autophagy by enhancing O-GlcNAcylation.

On the other hand, OSMI-1 increased autophagy through ER stress. When metformin and OSMI-1 were combined, they induced continuous autophagy and disrupted the homeostasis of O-GlcNAcylation, resulting in excessive autophagic flux. This excessive autophagy, in conjunction with downregulation of the anti-apoptotic protein Bcl2, synergistically induced apoptosis. The activation of IRE1α/JNK signaling by OSMI-1 and PERK/CHOP signaling by metformin played a role in inhibiting Bcl2 activity. Ultimately, this led to the upregulation of cytochrome c release and activation of caspase-3, key events in the apoptotic pathway.

The researchers concluded that the combination of metformin and OSMI-1 resulted in a synergistic induction of apoptosis in HCT116 cells. This effect was achieved by enhancing signaling activation through ER stress-induced pathways rather than relying on the cell-protective function of autophagy. The findings from the HCT116 cell experiments were further supported by similar results observed in xenograft models, suggesting the potential of this combination strategy for the treatment of colon cancer.