BommaReddy Raja Reddy, Patel Rekha, Smalley Tracess, Acevedo-Duncan Mildred
Department of Chemistry, University of South Florida, Tampa, FL 33620, USA.
Onco Targets Ther. 2020 Feb 24;13:1661-1676. doi: 10.2147/OTT.S224866. eCollection 2020.
The options for treating lung cancers are limited, as diagnosis typically occurs during the late stages of the disease. There is a dire need to develop aPKC (atypical Protein Kinase C) inhibitors due to aPKC overexpression and contributions to lung cancer malignancies. In this study, we investigate the role of atypical PKCs (aPKCs) in cell proliferation and migration in lung cancer cell lines and the effect of the novel aPKC inhibitor DNDA (3,4-amino-2,7 napthalene disulfonic acid).
The normal and lung cancer cells were treated with various concentrations of DNDA. We used a WST assay to determine lung cell viability, then analyzed cell apoptosis through Annexin V/PI staining and flow cytometry. Immunoprecipitation determined the proteins' associations, and Western blot allowed testing of the expression of interest proteins. We also employed the UbiTest to identify the ubiquitination of the FAK. The scratch and transwell assays measured cell migration and invasion of lung cancer cells.
Our data from cell viability and flow cytometry showed a significant reduction in cell proliferation and induction of apoptosis with DNDA treatment in lung cancer cells, as well as no toxic effect on normal BEAS-2B lung cells. Western blot results showed that the phosphorylation of PKC-iota and phosphorylation of FAK decreased in A549 lung cancer cells upon DNDA treatment. Immunoprecipitation (IP) data revealed an association of PKC-ι with FAK and FAK with Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b). UbiTest results suggest that PKC-ι regulates FAK cleavage through its ubiquitination by Cbl-b, thereby inhibiting A549 lung cancer cells' migration. This was evident from scratch, invasion, and migration assays.
Our study data suggest that DNDA inhibits cell proliferation and induces apoptosis in lung cancer cells. Moreover, DNDA inhibit A549 lung cancer cells' migration by PKC- ι/FAK ubiquitination via Cbl-b.
由于肺癌通常在疾病晚期才被诊断出来，其治疗选择有限。鉴于非典型蛋白激酶C（aPKC）在肺癌中过表达并促进肺癌恶性发展，迫切需要开发aPKC抑制剂。在本研究中，我们调查了非典型蛋白激酶C（aPKC）在肺癌细胞系中的细胞增殖和迁移中的作用，以及新型aPKC抑制剂DNDA（3,4-氨基-2,7-萘二磺酸）的作用。
用不同浓度的DNDA处理正常细胞和肺癌细胞。我们使用WST法测定肺细胞活力，然后通过Annexin V/PI染色和流式细胞术分析细胞凋亡。免疫沉淀确定蛋白质之间的关联，蛋白质印迹法检测目标蛋白的表达。我们还使用泛素化检测来鉴定粘着斑激酶（FAK）的泛素化。划痕试验和Transwell试验检测肺癌细胞的迁移和侵袭能力。
我们从细胞活力和流式细胞术获得的数据显示，DNDA处理可显著降低肺癌细胞的增殖并诱导其凋亡，且对正常BEAS-2B肺细胞无毒性作用。蛋白质印迹结果显示，DNDA处理后，A549肺癌细胞中PKC-ι的磷酸化和FAK的磷酸化水平降低。免疫沉淀（IP）数据显示PKC-ι与FAK以及FAK与Casitas B系淋巴瘤原癌基因b（Cbl-b）之间存在关联。泛素化检测结果表明，PKC-ι通过Cbl-b对其进行泛素化来调节FAK的切割，从而抑制A549肺癌细胞的迁移。这在划痕试验、侵袭试验和迁移试验中得到了证实。
我们的研究数据表明，DNDA可抑制肺癌细胞的增殖并诱导其凋亡。此外，DNDA通过Cbl-b介导的PKC-ι/FAK泛素化抑制A549肺癌细胞的迁移。
