Dendrimer-Based Selective Proteostasis-Inhibition Strategy to Control NSCLC Growth and Progression.
Dendrimer-Based Selective Proteostasis-Inhibition Strategy to Control NSCLC Growth and Progression.
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DOI：
10.1371/journal.pone.0158507
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Vij N
中科院分区：
文献类型：
--
作者：
Walworth K;Bodas M;Campbell RJ;Swanson D;Sharma A;Vij N
含Valosin蛋白（VCP/p97）水平升高可促进非小细胞肺癌（NSCLC）的进展。尽管许多VCP抑制剂是可用的，但这些治疗化合物中的大多数对于靶向肿瘤细胞递送具有低特异性。因此，本研究的主要目的是评价树状聚合物包封的强效VCP抑制剂药物在控制非小细胞肺癌（NSCLC）进展中的体外疗效。测试VCP抑制剂（以其纯形式或包封在具有羟基表面的第4代PAMM-树枝状聚合物中）在调节H1299（NSCLC细胞）增殖、迁移、侵袭、凋亡和细胞周期进程中的体外功效。我们的结果表明，与未处理的对照细胞相比，DBeQ对VCP的抑制作用显著强于NMS-873，这通过降低细胞增殖（p<0.0001，MTT-测定）和迁移（p<0.05;划痕-测定）以及增加细胞凋亡（p<0.05;半胱天冬酶-3/7-测定）来证明。接下来，我们发现，树枝状聚合物封装的DBeQ（DDNDBeQ）处理增加了泛素化蛋白的积累，可溶性蛋白质级分（免疫印迹）的H1299细胞相比，DDN-控制，这意味着DBeQ的有效性蛋白质沉积抑制。我们通过免疫染色证实DDNDBeQ处理增加了与ER标记物KDEL共定位的泛素化蛋白的积累。我们观察到，与对照DDN处理相比，DDNDBeQ的蛋白质沉积抑制显著降低了细胞迁移率（划痕测定和transwell侵袭）（p<0.05）。此外，与DDN对照相比，DDNDBeQ处理显示细胞增殖显著降低（p<0.01，MTT测定）和半胱天冬酶-3/7介导的凋亡性细胞死亡增加（p<0.05）。这通过细胞周期分析（碘化丙啶染色）进一步证实，其证明与对照DDN相比，DDNDBeQ处理在G2/M期中显著的细胞周期停滞（p<0.001）。此外，我们通过克隆形成测定证实，与对照/DDN相比，DDNDBeQ处理显著（p<0.001）抑制H1299集落形成。总的来说，将有效的VCP抑制剂DBeQ封装到树枝状聚合物中允许选择性VCP介导的蛋白质沉积抑制，用于控制NSCLC肿瘤生长和进展，以允许肿瘤靶向的持续药物递送。
Elevated valosin containing protein (VCP/p97) levels promote the progression of non-small cell lung carcinoma (NSCLC). Although many VCP inhibitors are available, most of these therapeutic compounds have low specificity for targeted tumor cell delivery. Hence, the primary aim of this study was to evaluate the in vitro efficacy of dendrimer-encapsulated potent VCP-inhibitor drug in controlling non-small cell lung carcinoma (NSCLC) progression. The VCP inhibitor(s) (either in their pure form or encapsulated in generation-4 PAMAM-dendrimer with hydroxyl surface) were tested for their in vitro efficacy in modulating H1299 (NSCLC cells) proliferation, migration, invasion, apoptosis and cell cycle progression. Our results show that VCP inhibition by DBeQ was significantly more potent than NMS-873 as evident by decreased cell proliferation (p<0.0001, MTT-assay) and migration (p<0.05; scratch-assay), and increased apoptosis (p<0.05; caspase-3/7-assay) as compared to untreated control cells. Next, we found that dendrimer-encapsulated DBeQ (DDNDBeQ) treatment increased ubiquitinated-protein accumulation in soluble protein-fraction (immunoblotting) of H1299 cells as compared to DDN-control, implying the effectiveness of DBeQ in proteostasis-inhibition. We verified by immunostaining that DDNDBeQ treatment increases accumulation of ubiquitinated-proteins that co-localizes with an ER-marker, KDEL. We observed that proteostasis-inhibition with DDNDBeQ, significantly decreased cell migration rate (scratch-assay and transwell-invasion) as compared to the control-DDN treatment (p<0.05). Moreover, DDNDBeQ treatment showed a significant decrease in cell proliferation (p<0.01, MTT-assay) and increased caspase-3/7 mediated apoptotic cell death (p<0.05) as compared to DDN-control. This was further verified by cell cycle analysis (propidium-iodide-staining) that demonstrated significant cell cycle arrest in the G2/M-phase (p<0.001) by DDNDBeQ treatment as compared to control-DDN. Moreover, we confirmed by clonogenic-assay that DDNDBeQ treatment significantly (p<0.001) inhibits H1299 colony-formation as compared to control/DDN. Overall, encapsulation of potent VCP-inhibitor DBeQ into a dendrimer allows selective VCP-mediated proteostasis-inhibition for controlling NSCLC-tumor growth and progression to allow tumor-targeted sustained drug delivery.