Apoptosis-inducing potential of tilorone dihydrochloride in triple-negative breast cancer: in silico and in vitro evidences.

Tilorone is an antiviral agent with interferon-inducing properties. The present study focused on evaluating the apoptosis-inducing effects of tilorone in triple-negative breast cancer (TNBC) using in silico and in vitro models. Molecular docking and dynamics were performed to investigate the interactions of cGAMP and tilorone with human STING. Time- and dose-dependent effects of tilorone on the proliferation of TNBC cells were measured by MTT assay. Colony and mammosphere formation assays were performed to test the effects of tilorone on cell proliferation and stemness. Transwell and wound healing assays were performed to test the antimetastatic potential of tilorone. IFNβ levels in cultured supernatants were measured by ELISA; loss of mitochondrial membrane potential (MMP) and nuclear changes were measured by JC-1 and DAPI staining, respectively. Apoptotic proteins were measured by Western blot technique and caspase-3 assay. The in silico molecular docking and dynamics simulation studies revealed that tilorone establishes similar binding with human STING as that of the natural ligand, cGAMP. In vitro experiments further demonstrated that tilorone significantly suppressed TNBC cell proliferation in a time- and dose-dependent manner. Tilorone markedly reduced the ability of TNBC cells to form colonies and mammospheres and cell migration, indicating its inhibitory effects on proliferation, stemness, and metastasis. Mechanistically, treatment with tilorone led to an increase in the level of IFN-β released in culture supernatants of TNBC cell lines. Additionally, tilorone modulated the expression of caspase-3 and PARP proteins with loss of MMP. Thus, tilorone exerts antiproliferative, antistemness, antimetastatic, and pro-apoptotic effects in TNBC, highlighting its potential as a promising therapeutic candidate in TNBC.