Integration of In Vitro and In-Silico Analysis of Gracilaria edulis on Anti-Cancer Potential and Apoptotic Signaling Pathway Activity

Abstract

Breast cancer, the most common malignancy in females, and rhabdomyosarcoma (RMS), the most prevalent soft tissue sarcoma in children, remain significant clinical challenges. This study evaluated the anticancer potential and apoptotic signaling pathways of Gracilaria edulis extracts and identified their mechanisms of action against RMS and breast adenocarcinoma (MCF-7) cell lines. Cytotoxicity was assessed using MTT assays, while apoptotic potential was evaluated through phase contrast and fluorescence microscopy, caspase 3/7 activity, DNA fragmentation, and gene expression analysis of apoptosis regulatory genes. In silico analysis was also performed to examine the molecular interactions of bioactive compounds present in Gracilaria edulis with cancer-related proteins involved in apoptotic signaling. The methanol extract was fractionated into hexane, chloroform, and ethyl acetate, with the hexane fraction demonstrating the strongest cytotoxicity (IC₅₀RMS: 32.52 ± 2.15 μg/mL; IC₅₀MCF-7:29.84 ± 0.65 μg/mL) in MTT assays. Apoptotic features, including chromatin condensation, membrane blebbing, cellular shrinkage, and DNA fragmentation, were observed, particularly in RMS cells. The hexane fraction significantly activated caspase 3/7 in RMS cells, while lower activation was noted in MCF-7 cells, possibly due to the partial deletion of the CASP-3 gene. Real-time PCR analysis revealed differential gene expression, with p21 showing dominant upregulation in RMS cells and p53 being more prominently expressed in MCF-7 cells. These findings reflect their distinct roles in apoptotic signaling pathways. A significant increase in the Bax/Bcl-2 ratio in RMS cells (8.45) and MCF-7 cells (29.69) indicated a pro-apoptotic shift. GC-MS analysis identified key bioactive compounds, including 9-octadecenoic acid methyl ester, hexadecenoic acid methyl ester, and 1,2-benzenedicarboxylic acid mono(2-ethylhexyl) ester. In silico docking revealed that 1,2-benzenedicarboxylic acid mono(2-ethylhexyl) ester demonstrated the most promising binding interactions, particularly with BCL-2, while 9-octadecenoic acid methyl ester exhibited weaker binding affinities across all targets (p53, p21, and BCL-2), suggesting limited therapeutic relevance without structural optimization. However, the hexane fraction of G. edulis and its bioactive compounds remain promising as potential anticancer agents, warranting further in vitro and in vivo validation and molecular optimization.

Graphical Abstract