Disulfiram inhibits LPS-induced TLR4/NF-κB signaling in spheroids derived from human colorectal cancer cells

Bacterial lipopolysaccharide (LPS) is a major cause of inflammation-associated colorectal cancer (CRC) after surgery, and recurrence of CRC is often due to the formation of highly resistant cancer stem cells (CSCs). Here, TLR4 antagonistic effects of disulfiram (DSF), an antabuse compound, were analyzed using MTT assay, colony formation assay, RT-PCR, targeted gene silencing studies, and western blotting in spheroids derived from monolayer HT-29 and HCT-116 cells.

We attempted to understand the mechanism of LPS-associated stemness by targeting the colon CSCs with DSF in spheroid models representing tumor microenvironment.

Initially, we optimized LPS concentration as 1 µg/mL and IC₅₀ of DSF at 48 h as 50 µM and 10 µM in HT-29 and HCT-116 cells, respectively. We demonstrated increased colony formation by LPS and antagonistic effect of DSF against LPS-mediated tumorigenicity by reducing the number of colonies. Furthermore, we generated the spheroid models from monolayer HT-29 and HCT-116 cells and their stemness properties were confirmed by over-expression of CSC markers (Lgr5, Oct4, Dclk1, Aldh1, CD44, and CD133). Moreover, targeted gene silencing studies revealed that TLR4 is necessary for LPS-induced effects in spheroids. DSF inhibited the CSC markers, TLR4/NF-κB, ERK1/2, and p38 MAPK, whereas LPS promoted these pathways in spheroids.

Our results unraveled the mechanism of CRC stemness and anti-CSC effect of DSF in LPS-induced CRC using spheroids. Further work may be required to study the effect of DSF on CRC progression using spheroids from CRC tissues or animal models.