The anticancer and anti-inflammatory activities ofHalodule uninervisethanolic extract: Pharmaco-molecular mechanism of action and bioactive constituents

Abstract ID 93445 Poster Board 082 Background: Breast cancer is the second leading cause of cancer-related morbidity and mortality in the world. More specifically, triple negative breast cancer (TNBC) is the most aggressive subtype and accounts for 10-20% of all breast cancer cases. TNBC is highly metastatic and lacks the three receptors that represent primary targets of chemotherapy, making it resistant to different treatment regimens, compared with hormone receptor positive breast cancers. Hypothesis/Purpose: Halodule uninervis, a seagrass in the family Cymodoceaceae, has been reported to be rich in bioactive metabolites such as alkaloids, flavonoids, phenols, and steroids. As such, the plant exhibits a plethora of pharmacological activities including anticancer, antioxidant, and antimicrobial. However, its anticancer effects against TNBC and anti-inflammatory potential remain poorly investigated. In the present study, we investigated the anticancer effects of the ethanolic extract of Halodule uninervis (HUE) on TNBC using the MDA-MB-231 breast cancer cell line an as in vitro model. We also explored the anti-inflammatory effects of HUE using the RAW 264.7 cell line as an in vitromodel of macrophages. Methods: LC-MS was used to identify the phytochemical constituents of HUE. The MTT assay was used to evaluate the antiproliferative activity of HUE against MDA-MB-231 cells. This was further validated by studying the effect of the extract on cell cycle progression by flow cytometry and assessing the protein levels of key markers of cell proliferation (p21, Ki67). The induction of apoptosis was examined by bright field microscopy and DAPI staining of the cancer cells. Protein levels of markers of apoptosis were also investigated (Bcl-2, Bax, Capase-3). The effect of HUE on cancer cell phenotypes, such as aggregation, adhesion, migration, invasion in vitro by using the aggregation, scratch, and transwell assays, respectively, while angiogenesis was investigated in ovo with the Chorioallontoic Membrane (CAM) assay. Moreover, the role of the p38 MAPK and STAT3 signaling axis was investigated. The anti-inflammatory potential of HUE was assessed on LPS-induced RAW264.7 cells. And protein levels of inflammatory markers (COX2, iNOS, STAT3, p38, and p65) were determined by immunoblotting. And DCFDA staining was used to quantify intracellular ROS levels. Results: We revealed that HUE decreased MDA-MB-231 cell proliferation and induced G0/G1 cell cycle arrest, which was associated with an increase in the phosphorylation of p38 MAPK, an increase in the levels of tumor suppressor protein p21, and a decrease in the levels of proliferation marker protein Ki67. HUE induced intrinsic apoptosis by reducing protein levels of Bcl-2 and pro-Caspase-3 and promoting Bax protein expression, in correlation with activation of STAT3/p53 pathway. Furthermore, HUE diminished the migration and invasive properties of MDA-MB-231 cells by inducing Occludin and inhibiting Integrin β1. HUE also decreased the production of iNOS and inhibited in ovo angiogenesis. In LPS-activated RAW264.7 macrophages, HUE decreased protein expression of COX-2, iNOS, and p65, in correlation with inhibiting STAT3/p38 pathway. Conclusion: Our findings reveal that HUE exhibits prominent anti-breast cancer and anti-inflammatory activities, suggesting that Halodule uninervis could be used as a source of potential therapeutics against cancer and other inflammatory diseases.