Cross-communication between cancer cells and macrophages within the tumor microenvironment fulfills the critical roles in the progression of cancers, including hepatocellular carcinoma (HCC). Ligustilide exerts anti-inflammation, anti-injury, and anti-tumor pleiotropic pharmacological functions. Nevertheless, its roles in HCC cells and tumor microenvironment remain elusive. In the current study, ligustilide dramatically restrained HCC cell viability and migration but had little cytotoxicity to normal hepatocytes. Importantly, ligustilide antagonized HCC cell co-culture-induced macrophage recruitment and M2 polarization by enhancing the percentage of CD14+CD206+ cells and macrophage M2 markers (CD163, Arg1, CD206, CCL22, IL-10, and TGF-β). Mechanistically, ligustilide repressed yes-associated protein (YAP) activation by reducing nuclear translocation, protein expression, transcriptional regulatory activity of YAP, and increasing p-YAP levels. Noticeably, blocking the YAP offset the suppressive effects of ligustilide on macrophage recruitment and M2 polarization evoked by HCC cells. Moreover, the release of interleukin-6 (IL-6) was mitigated by ligustilide in a YAP-dependent manner in HCC cells, concomitant with inhibition of IL-6R/STAT3 signaling activation. Of interest, interdicting the IL-6 aggravated ligustilide-mediated suppression in HCC-induced macrophage recruitment and M2 polarization; whereas exogenous IL-6 treatment reversed the above effects. Additionally, blockage of IL-6R signaling also overturned IL-6-induced macrophage recruitment and M2 phenotype. Consequently, these findings support a notion that ligustilide not only restrains HCC cell malignancy but also antagonizes HCC cell-evoked macrophage recruitment and M2 polarization by inhibiting YAP/IL-6 release-induced activation of the IL-6 receptor/signal transducer and activator of transcription 3 (IL-6R/STAT3) signaling. Thus, ligustilide may be a promising therapeutic agent to fight HCC by regulating cancer cells and cross-talk between tumor cells and macrophages in tumor microenvironment.

Background: Drug resistance is still a major obstacle during the cisplatin-based chemotherapy of hepatocellular carcinoma (HCC). Recently, studies have indicated that the population of CD133+ cancer cells is partially responsible for the failure of cancer treatment. However, the potential mechanisms are still unclear.Methods: CD133+ HepG2 and Huh7 cells were sorted via flow cytometry. CCK-8 assay was used to detect the cytotoxicity of cisplatin and silibinin against HCC cells. Western blot assay was performed to detect the protein expression, cleavage of caspases and release of cytochrome c from mitochondria into cytosol. Flow cytometry analysis was used to measure the apoptotic rate of CD133+ HepG2 and Huh7 cells.Results: CD133+ HepG2 and Huh7 cells were observed to exhibit obvious resistance against cisplatin. However, co-treatment with silibinin significantly reduced the cisplatin resistance of CD133+ HepG2 and Huh7 cells. Furthermore, although CD133+ HepG2 and Huh7 cells were resistant to cisplatin-induced apoptosis, co-treatment with silibinin enhanced the cisplatin-induced apoptosis through promoting the release of cytochrome c from mitochondria into cytosol. In the mechanism research, we proved that silibinin inhibited the expression of OPA1 in CD133+ HepG2 and Huh7 cells. Under the stress of cisplatin, silibinin promoted the collapse of mitochondria and increased the release of cytochrome c. As a result, caspases-dependent apoptosis was induced in CD133+ HepG2 and Huh7 cells which were co-treated with cisplatin and silibinin.Conclusion: Silibinin sensitizes CD133+ HCC cells to cisplatin-induced apoptosis through suppression of OPA1.