This study is the first report describing the promising antitumor activity of biologically active compounds isolated from the hemolymph of marine snail Rapana venosa—a fraction with Mw between 50 and 100 kDa and two structural subunits (RvH1 and RvH2), tested on a panel of human breast cell lines—six lines of different molecular subtypes of breast cancer MDA-MB-231, MDA-MB-468, BT-474, BT-549, SK-BR-3, and MCF-7 and the non-cancerous MCF-10A. The fraction with Mw 50–100 kDa (HRv 50–100) showed good antitumor activity manifested by a significant decrease in cell viability, altered morphology, autophagy, and p53 activation in treated cancer cells. An apparent synergistic effect was observed for the combination of HRv 50–100 with cis-platin for all tested cell lines. The combination of HRv 50–100 with cisplatin and/or tamoxifen is three times more effective compared to treatment with classical chemotherapeutics alone. The main proteins in the active fraction, with Mw at ~50 kDa, ~65 kDa, ~100 kDa, were identified by MALDI-MS, MS/MS analyses, and bioinformatics. Homology was established with known proteins with antitumor potential detected in different mollusc species: peroxidase-like protein, glycoproteins Aplysianin A, L-amino acid oxidase (LAAO), and the functional unit with Mw 50 kDa of RvH. Our study reveals new perspectives for application of HRv 50–100 as an antitumor agent used alone or as a booster in combination with different chemotherapies
