Deciphering the role of Clinacanthus nutans extract in reshaping the tumor microenvironment and its effect on triple negative breast cancer

Abstract

Triple negative breast cancer (TNBC) is a clinically heterogenous and highly aggressive tumors. Chemotherapy remains as the mainstream therapeutic approach due to limited targeted therapy options. Growing evidence indicates that modulating the tumor microenvironment (TME) with immunotherapy is a promising approach for TNBC. The extracts of Clinacanthus nutans (CN) has demonstrated potent anti-cancer properties. However, the underlying immunomodulatory mechanism of CN remains unclear. Therefore, this study aimed to investigate the potential of CN in reshaping TNBC TME by regulating tumor-associated macrophages and its outcome on treating TNBC. The major bioactive compounds in CN ethanolic extract were saponin, alkaloid, phenol, tannin, flavonoid, terpenoid, glycoside, and steroid. HPLC profiling confirmed the presence of schaftoside, isoorientin, orientin, isovitexin and vitexin. Based on Sulforhodamine B (SRB) cytotoxicity assay, CN extract exerted significant cytotoxicity against MCF-7 (luminal A subtype) at 48 and 72 h with IC50 value of 0.89 mg/ml and 0.81 mg/ml respectively but was less effective against both TNBC cell lines (MDA-MB 231 and MDA-MB 468) without exerting any IC50 value. RNA sequencing analysis revealed an upregulation of Annexin A1 (AnxA1) in TNBC cell line, which was associated with treatment responsiveness, conferring drug resistance to CN. In silico analysis revealed AnxA1 as a key player in TNBC interactome which coordinates aggressive phenotypes of the tumors. Following knockdown of AnxA1, a significant dose-dependent cytotoxicity was observed in MDA-MB 231 challenged with CN, with IC50 values of 1.22 mg/ml and 0.96 mg/ml at 48 and 72 h respectively. Interestingly, using a macrophage-MDA-MB 231 co-culture system, results showed that CN treatment substantially promoted the anti-tumor M1 macrophage phenotype (p<0.05) and reprogrammed the IL-4/IL-13 induced M2 macrophages (pro-tumor) towards M1 phenotype (p<0.0001). MDA-MB 231 treated with CN-induced M1 macrophages conditioned medium were more prone to apoptotic cell death (p<0.0001). CN also significantly reduced the inflammatory cytokines such as IL-6 (p<0.01), TNF-α (p<0.001), IL-8 (p<0.05), IFN-γ (p<0.001) and IL-10 (p<0.01), and pro-migratory molecules like matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) (p<0.001) in the supernatant of the co-culture medium. As most of the pro-inflammatory cytokines and MMPs are target genes of NF-kB, we investigated the inhibitory activity of CN’s active compounds against the key protein of NF-kB activation, IKKβ protein by molecular docking. The docking analysis revealed that all the active compounds had strong binding affinity to the ligand-binding sites of IKKβ, suggesting that an inhibitory effect on NF-kB signaling via competitive inhibition. Taking together, our findings suggested that reshaping the TME by regulating the macrophage polarization through NF-kB signalling may become a novel mechanism underlying the anti-tumor effects of CN in high AnxA1 expressing TNBC.