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The effects against tumors exerted by marine active compounds have been highlighted and investigated. Polymeric nanoparticles made from biodegradable and biocompatible molecules such as hyaluronan (HA) and chitosan (CHI) are able to aggregate the compounds to enhance their activities against tumor cells and reduce the toxicity on normal cells. Here, we extensively examined the antitumor activities and the mechanisms of HA/CHI nanoparticles-aggregated heteronemin (HET) extracted from the sponge Hippospongia sp. The half-maximal inhibitory concentration (IC50) of pure HET toward T24 bladder carcinoma cells is ~0.28 µg/mL. Pure HET from 0.2 to 0.8 µg/mL and HA nanoparticles-aggregated HET at 0.1 and 0.2 µg/mL significantly reduced T24 cell viability. Compared to pure HET, HA nanoparticles/HET aggregates showed much weaker viability-inhibitory effects on L929 normal fibroblasts. HET dose-dependently suppressed cancer cell migration as HA/CHI nanoparticles-aggregated HET displayed stronger migration-inhibitory effects than pure HET. Flow cytometric analysis showed that pure HET increased early/total apoptosis and JC-1 monomer fluorescence, while HA/CHI nanoparticles-aggregated HET induced higher apoptosis and JC-1 monomer rates than pure HET, suggesting that aggregation of HA nanoparticles offers HET stronger apoptosis-inducing capacity through mitochondrial depolarization. Western blot analysis showed that HA nanoparticles-aggregated HET further increased mitochondrial-associated, caspase-dependent and caspase-independent, as well as endoplasmic reticulum stress-related factors in comparison with pure HET. These data indicated that pure HET possesses cytotoxic, antimigratory, and apoptosis-inducing effects on bladder cancer cells in vitro, and its induction of apoptosis in bladder carcinoma cells is mainly caspase dependent. Moreover, HA nanoparticle aggregation reinforced the cytotoxic, antimigratory, and apoptosis-inducing activities against bladder carcinoma cells and attenuated the viability-inhibitory effects on normal fibroblasts. This aggregation reinforces antibladder carcinoma effects of HET via diverse routes, including mitochondrial-related/caspase-dependent, caspase-independent, and endoplasmic reticulum stress-related pathways. The current data also strongly suggested that HA/CHI nanoparticles-aggregated HET would be a potential treatment for urothelial cancer in vivo.

Improvement and enhancement of antibladder carcinoma cell effects of heteronemin by the nanosized hyaluronan aggregation