Antiproliferative effect of Filopaludina bengalensis fluid inducing apoptosis-necroptosis synergy with immunogenic remodelling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents a highly aggressive phenotype of breast carcinoma, distinguished by unique metastatic patterns and poor prognosis driven by its immune microenvironment. Recent advances integrating anticancer agents with personalized immunotherapies are emerging as promising avenues for TNBC management. Freshwater molluscs, though primarily consumed as nutraceuticals and less explored scientifically, hold promising therapeutic potential as immunomodulatory, natural anticancer agents. In the current investigation the anticancer potential of the extrapallial fluid of Filopaludina bengalensis (FBF), a widespread mollusca in south-eastern India, was evaluated against TNBC, exploring its underlying molecular mechanism. Dose-dependent antiproliferative effect in fluid-treated MDA-MB-231 cells ranged from 89.28% at 3.125 µg/ml to 28.4% at 200 µg/ml (p < 0.0001), with an IC50 at 49 ± 1.6 µg/ml. Cytotoxicity driven by reactive oxygen species generation results in membrane damage, mitochondrial depolarization, and apoptosis (confirmed by DAPI, AO/EtBr). Annexin V/PI staining further endorsed late apoptosis and necrosis at 25 and 50 µg/ml and inhibited cell cycle progression at G2/M checkpoint, comparable to that of doxorubicin. Gene expression analysis revealed p53 activation, Bax upregulation, Bcl-2 suppression, caspase cascade initiation, and necroptosis with inflammatory mediator modulation, highlighting an immunogenic tumor microenvironment. Furthermore, high-resolution mass spectrometric analysis (HRMS) profiling of FBF predominantly revealed peptide fragments or related components, largely correlating with its profound anti-proliferative activity. Thus, F. bengalensis can be a potential breakthrough in oncotherapy by averting apoptotic resistance while eliciting complementary immunogenic necroptosis in human triple-negative breast carcinoma.