Huang Han Hsiang, Kuo Shyh Ming, Wu Yi-Jhen, Su Jui-Hsin
Department of Veterinary Medicine, National Chiayi University, Chiayi City, Taiwan.
Department of Biomedical Engineering, I-Shou University, Kaohsiung City, Taiwan.
Int J Nanomedicine. 2016 Mar 29;11:1237-51. doi: 10.2147/IJN.S99911. eCollection 2016.
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.
海洋活性化合物对肿瘤的作用已得到重点关注和研究。由透明质酸（HA）和壳聚糖（CHI）等可生物降解且具有生物相容性的分子制成的聚合物纳米颗粒，能够聚集这些化合物，以增强其对肿瘤细胞的活性，并降低对正常细胞的毒性。在此，我们广泛研究了从海绵属 Hippospongia sp. 中提取的 HA/CHI 纳米颗粒聚集的异名海绵素（HET）的抗肿瘤活性及其作用机制。纯 HET 对 T24 膀胱癌细胞的半数抑制浓度（IC50）约为 0.28 µg/mL。0.2 至 0.8 µg/mL 的纯 HET 以及 0.1 和 0.2 µg/mL 的 HA 纳米颗粒聚集的 HET 均显著降低了 T24 细胞活力。与纯 HET 相比，HA 纳米颗粒/HET 聚集体对 L929 正常成纤维细胞的活力抑制作用要弱得多。HET 呈剂量依赖性地抑制癌细胞迁移，因为 HA/CHI 纳米颗粒聚集的 HET 显示出比纯 HET 更强的迁移抑制作用。流式细胞术分析表明，纯 HET 增加了早期/总凋亡率以及 JC-1 单体荧光，而 HA/CHI 纳米颗粒聚集的 HET 诱导的凋亡率和 JC-1 单体率高于纯 HET，这表明 HA 纳米颗粒的聚集通过线粒体去极化赋予 HET 更强的凋亡诱导能力。蛋白质印迹分析表明，与纯 HET 相比，HA 纳米颗粒聚集的 HET 进一步增加了与线粒体相关的、半胱天冬酶依赖性和非依赖性以及内质网应激相关因子。这些数据表明，纯 HET 在体外对膀胱癌细胞具有细胞毒性、抗迁移和诱导凋亡的作用，并且其在膀胱癌细胞中诱导凋亡主要依赖半胱天冬酶。此外，HA 纳米颗粒聚集增强了对膀胱癌细胞的细胞毒性、抗迁移和诱导凋亡活性，并减弱了对正常成纤维细胞的活力抑制作用。这种聚集通过多种途径增强了 HET 的抗膀胱癌作用，包括线粒体相关/半胱天冬酶依赖性、非依赖性以及内质网应激相关途径。目前的数据还强烈表明，HA/CHI 纳米颗粒聚集的 HET 在体内可能是一种治疗尿路上皮癌的潜在药物。
