Synergistic Cytotoxicity of Rapana venosa Hemolymph Fractions with Mitonafide in Lung Cancer Cells

Abstract

Lung cancer remains one of the most prevalent and lethal malignancies worldwide, and new therapeutic strategies are urgently needed. Marine-derived bioactive compounds have recently attracted attention as potential anticancer agents. In this study, we examined the anti-proliferative effects of two hemolymph fractions, with molecular weights below 30 kDa and above 50 kDa, isolated from the marine snail Rapana venosa after the removal of native hemocyanin. These fractions were evaluated in combination with two 1,8-naphthalimides (mitonafide and amonafide) using A549 (p53 wild-type) and H1299 (p53-null) lung cancer cell lines, as well as non-malignant MRC-5 fibroblasts. Both naphthalimides showed intrinsic cytotoxicity, with mitonafide being more potent and selective than amonafide. The hemolymph fractions alone exhibited weak cytotoxicity and low toxicity to normal cells. Remarkably, the fraction with MW>50 kDa strongly potentiated mitonafide, reducing IC50 values and increasing selectivity indices (SI up to 26 in A549 and 13 in H1299), while amonafide combinations provided only modest improvements. Synergy analysis using the Highest Single Agent (HSA) model confirmed that the fraction with MW>50 kDa enhanced mitonafide activity in lung cancer cells, with meaningful synergy observed in A549. Synergy was also detected in MRC-5 fibroblasts. The fraction with  MW<30 kDa similarly potentiated mitonafide and, to a lesser extent, amonafide.