Anti-colorectal cancer effects and mechanisms of action of the Chinese medicine formula Huai-Hua-San

Colorectal cancer（CRC）is one of the common tumors of the digestive system. Current CRC therapies have limitations. Huai-Hua-San（HHS）is a traditional Chinese medicine formula comprising two edible herbs，Sophorae Flos and Gardeniae Fructus. HHS was traditionally used to manage TCM symptoms that resemble CRC. However，pharmacological basis of the formula's application in treating CRC is not known. In the present study，we found that an extract of HHS（HHSE）reduced the viability of，and induced apoptosis in，HCT116，HCT8 and HT-29 CRC cells，while exhibited less cytotoxicity in normal colon cells. HHSE dose-dependently and significantly restrained tumor growth without overt toxicity in an HT-29 xenograft mouse model. Network pharmacology analysis highlighted the involvement of PI3K/AKT signaling pathway in the anti-CRC effects of HHSE. Western blot results demonstrated that HHSE significantly lowered the protein levels of phospho-AKT（Ser 473）and its downstream anti-apoptosis proteins Mcl-1 and Bcl-xL in CRC cells and in mouse tumors. An AKT activator（SC79）significantly diminished the anti-proliferative effects of HHSE，indicating that inhibition of the PI3K/AKT pathway contributes to the anti-CRC effects of the extract. Combined analyses of RNA-Seq and miRNA-Seq data from mouse tumors suggested that the miR-142-3p/FAM98A pathway is another signaling pathway involved in the anti-CRC effects of HHSE. RT-qPCR results showed that HHSE upregulated miR-142-3p micro-RNA levels and downregulated FAM98A mRNA levels in CRC cells. Immunoblotting showed that HHSE significantly downregulated protein levels of FAM98A signaling molecules，including PRMT1，β-catenin，AKT，c-Myc and Bcl-xL. Inhibition of miR-142-3p significantly reduced the anti-proliferative effects of HHSE. Moreover，FAM98A silencing decreased the proliferative and colony-formation abilities of HT-29 cells and enhanced the anti-proliferation effects of HHSE. In summary，HHSE exerts anti-CRC effects in cell and mouse models; inhibition of PI3K/AKT signaling pathway and regulation of miR-142-3p/FAM98A pathway contribute to anti-CRC mechanisms of the extract. Our findings provide pharmacological justifications for the use of HHS in treating CRC and suggest that HHSE could be developed into an effective and safe anti-CRC drug. Additionally，and importantly，this work suggests that targeting miR-142-3p/FAM98A axis is a new strategy for treating CRC.