Rhein-induced apoptosis in colorectal cancer cell lines: A mechanistic study of the myeloid differentiation primary response gene 88/toll-like receptor 4/nuclear factor kappa-B signaling pathway.

Objective: Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide, and targeted therapies for CRC are urgently needed. This study aimed to investigate the mechanisms through which rhein induces apoptosis in CRC cells, focusing on its influence on the myeloid differentiation primary response gene 88 (MYD88)/toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway.

Material and methods: Cell Counting Kit-8 assay was conducted, with three non-cytotoxic concentrations of rhein selected for further analysis. Cells were allocated into four groups: control, 10 μM rhein, 20 μM rhein, and 50 μM rhein. Migration ability was evaluated through wound healing assay, and invasive potential was assessed using Transwell invasion assay. Apoptotic rates were determined through terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. The expression levels of apoptosis-related proteins and the key components of the MYD88/TLR4/NF-κB pathway were analyzed by quantitative reverse-transcription polymerase chain reaction and Western blotting after rhein treatment.

Results: The CRC HT-29 and SW480 cells' capacity to migrate and invade was markedly reduced by rhein treatment. (P < 0.05) while markedly enhancing the apoptotic rates (P < 0.05). This finding was marked by a reduction in the expression levels of B-cell lymphoma 2 (BCL-2) protein and messenger RNA (mRNA, P < 0.05), along with a notable increase in the levels of Bcl-2-associated X and cysteinyl aspartate-specific protease 3 proteins and mRNAs (P < 0.05). The expression levels of MYD88, TLR4, and NF-κB proteins and mRNAs were substantially downregulated (P < 0.05). Adding the TLR4 agonist lipopolysaccharide partially reversed the inhibitory effects of rhein on this signaling pathway, thereby restoring some cellular functional behavior.

Conclusion: Rhein appears to promote apoptosis in CRC cells through the MYD88/TLR4/NF-κB signaling pathway, thus inhibiting tumor initiation and progression.