Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Lepu Biopharma Co., Ltd., Beijing 101204, China.
Biochem Pharmacol. 2022 Jul;201:115057. doi: 10.1016/j.bcp.2022.115057. Epub 2022 Apr 28.
KRAS mutation and NF-κB both play crucial role in pancreatic cancer; in addition, defensin, the peptide mediator in innate immunity, can inhibit NF-κB. Assuming a strategy that targets both NF-κB and concomitantly the mutated KRAS indirectly via intensive macropinocytosis, we designed and generated a recombinant protein DF-HSA which consists of two molecules of human beta-defensin 2 (HBD2) and a moiety of human serum albumin (HSA). As shown, the recombinant protein DF-HSA markedly down-regulated NF-κB in both KRAS mutant MIA PaCa-2 cells and wild type BxPC-3 cells. Determined by confocal microscopy, the uptake of DF-HSA in MIA PaCa-2 cells was more intense than that in BxPC-3 cells. The uptake was blocked by the specific inhibitor EIPA, indicating that DF-HSA internalized via macropinocytosis. DF-HSA displayed more potent cytotoxicity to cancer cells than HBD2. DF-HSA induced apoptosis in cancer cells. Notably, DF-HSA inhibited tumor cell spheroid formation, an effect comparable to that of salinomycin. DF-HSA inhibited tumor cell migration and invasion. As detected with scanning electron microscopy, DF-HSA strongly depleted filopodia on cell surface; and salinomycin induced similar changes. By in vivo imaging, DF-HSA displayed intense tumor-site accumulation and lasting retention for over 14 days; however, HBD2 showed much less tumor-site accumulation and a shorter retention time for only 24 h. DF-HSA suppressed the growth of pancreatic cancer MIA PaCa-2 xenograft in athymic mice; and its combination with gemcitabine achieved higher antitumor efficacy. In summary, the recombinant defensin/HSA fusion protein that inhibits NF-κb associated with intensive macropinocytosis is highly effective against pancreatic cancer.
KRAS 突变和 NF-κB 在胰腺癌中均发挥关键作用；此外，防御素作为先天免疫中的肽介质，可抑制 NF-κB。假设一种通过密集的巨胞饮作用间接靶向 NF-κB 及其同时突变的 KRAS 的策略，我们设计并生成了一种重组蛋白 DF-HSA，它由两种人β防御素 2（HBD2）分子和一部分人血清白蛋白（HSA）组成。结果表明，重组蛋白 DF-HSA 可显著下调 KRAS 突变的 MIA PaCa-2 细胞和野生型 BxPC-3 细胞中的 NF-κB。通过共聚焦显微镜确定，DF-HSA 在 MIA PaCa-2 细胞中的摄取量强于 BxPC-3 细胞。该摄取被特异性抑制剂 EIPA 阻断，表明 DF-HSA 通过巨胞饮作用内化。DF-HSA 对癌细胞的细胞毒性强于 HBD2。DF-HSA 诱导癌细胞凋亡。值得注意的是，DF-HSA 抑制肿瘤细胞球体形成，其效果与盐霉素相当。DF-HSA 抑制肿瘤细胞迁移和侵袭。通过扫描电子显微镜检测，DF-HSA 强烈耗尽细胞表面的丝状伪足；盐霉素诱导了类似的变化。通过体内成像，DF-HSA 在 14 天以上时间内显示出强烈的肿瘤部位聚集和持续保留；然而，HBD2 显示出较少的肿瘤部位聚集和仅 24 小时的较短保留时间。DF-HSA 抑制了裸鼠中胰腺癌 MIA PaCa-2 异种移植瘤的生长；并且与吉西他滨联合使用可实现更高的抗肿瘤疗效。总之，抑制与密集巨胞饮作用相关的 NF-κB 的重组防御素/HSA 融合蛋白对胰腺癌具有高度疗效。
