Erchen decoction exerts anti-colorectal cancer effects by inhibiting Adcy2 expression and suppressing the cAMP/PKA signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Recent findings have highlighted the potential of Erchen decoction (ECD), a traditional Chinese medicine formula, as a promising therapeutic agent for the management of CRC; however, the fundamental mechanisms are still poorly understood.

PURPOSE: This study was conducted to elucidate the molecular mechanisms of the anti-CRC effects of ECD.

METHODS

The anti-tumor activity of ECD was initially evaluated in multiple CRC cell lines using cell proliferation, Transwell, and colony formation assays. Subsequently, a mouse model harboring subcutaneous tumors was utilized for the assessment of anti-tumor efficacy of ECD in vivo. RNA-seq analysis was conducted for the identification of core proteins and key pathways associated with the anti-CRC effects of ECD. Then, multiple molecular biology approaches, such as Western blotting and immunofluorescence staining, were employed to validate these findings, and core protein was overexpressed to further investigate its specific role in ECD-mediated anti-CRC activity. Finally, the key bioactive constituents of ECD against colorectal cancer (CRC) were identified through molecular docking, cellular thermal shift assay (CETSA), and in vitro experiments.

RESULTS

Notably, ECD dose-dependently inhibited CRC cell proliferation, invasion, and migration. Western blot analysis demonstrated that ECD suppressed EMT pathway in CRC cells, as evidenced by downregulation of N-cadherin and vimentin and upregulation of E-cadherin. RNA-seq analysis identified Adcy2 as a key target regulated by ECD, while KEGG enrichment analysis uncovered that the cAMP signaling pathway played a key part in ECD-mediated anti-CRC effects. Further molecular experiments confirmed that ECD exerted its anti-tumor activity through inhibition of the Adcy2/cAMP/PKA signaling axis. Notably, overexpression of Adcy2 significantly attenuated the inhibitory effects of ECD, whereas Adcy2 knockdown failed to enhance its anti-CRC activity. These findings support the specificity of the Adcy2/cAMP/PKA pathway in the anti-CRC activity of ECD. Furthermore, molecular docking revealed that Rhoifolin binds tightly to Adcy2 and suppresses CRC progression by inhibiting the Adcy2/cAMP/PKA pathway, identifying it as one of the key bioactive constituents of ECD.

CONCLUSION

Erchen Decoction and its key bioactive constituent, Rhoifolin, exert potent anti-tumor effects against CRC by suppressing the Adcy2/cAMP/PKA signaling axis.