JDF promotes the apoptosis of M2 macrophages and reduces epithelial-mesenchymal transition and migration of liver cancer cells by inhibiting CSF-1/PI3K/AKT signaling pathway.

ABSTRACT

Background: The interaction between cancer cells and the tumor microenvironment is of critical importance in liver cancer. Jiedu Granule formula (JDF) has been shown to minimize the risk of recurrence and metastasis following liver cancer resection. Investigating the mechanism underlying the therapeutic effects of JDF can extend its field of application and develop novel treatment approaches. Methods: We established a rat liver orthotopic transplantation tumor model, and recorded the prognostic effects of JDF adjuvant therapy on the recurrence and metastasis of liver cancer. Liver and lung tissues were collected for immunofluorescence staining and H&E staining, respectively. In addition, THP-1 cells were incubated with PMA and IL-4 to induce them to differentiate into M2 macrophages. CSF-1 expression was knocked down using lentivirus to determine the function of CSF-1. Liver cancer cells were cultured with a conditioned medium (CM) or co-cultured with macrophages. Cell viability was determined using the MTT assay. The levels of CSF-1, CSF-1R, E-cadherin, N-cadherin, PI3K, AKT, and cleaved caspase-3 were detected using ELISA, Western blotting and qPCR. The ability of cells to migrate was assessed using cell scratch and transwell assays. Apoptosis was evaluated using flow cytometry. Results: The JDF treatment decreased the risk of liver cancer metastasis after surgery and the infiltration of CD206/CD68 cells in liver cancer tissue. In cell experiments, JDF showed effects in suppressing M2 macrophages activity and downregulating the expression of CSF-1 and CSF-1R. The concentration of CSF-1 in the supernatant was also lower in the JDF-treated group. Futhermore, M2-CM was found to promote cancer cell migration and epithelial-mesenchymal transition (EMT); however, these effects were weakened after administering JDF. Knocking down endogenous CSF-1 in M2 macrophages resulted in a comparable suppression of cancer cell migration and EMT. Additionally, JDF treatment inhibited activation of the PI3K/AKT pathway, thus promoting the apoptosis of M2 macrophages. Conclusions: Treatment with JDF reduced the EMT and migratory capacity of liver cancer cells, which might be attributed to the inhibition of M2 macrophage infiltration and interruption of the CSF-1/PI3K/AKT signaling pathway. This mechanism may hold significant implications for mitigating the risk of metastatic spread in the aftermath of hepatic surgery.