Department of Chemistry, University of South Florida, Tampa, FL 33620, USA.
Department of Pathology, Immunology and Laboratory Medicine, University of Florida, College of Medicine, Gainesville, FL 32610, USA.
Int J Oncol. 2017 Nov;51(5):1370-1382. doi: 10.3892/ijo.2017.4131. Epub 2017 Sep 22.
Atypical protein kinase Cs (aPKC) are involved in cell cycle progression, tumorigenesis, cell survival and migration in many cancers. We believe that aPKCs play an important role in cell motility of melanoma by regulating cell signaling pathways and inducing epithelial to mesenchymal transition (EMT). We have investigated the effects of two novel aPKC inhibitors; 2-acetyl-1,3-cyclopentanedione (ACPD) and 3,4-diaminonaphthalene-2,7-disulfonic acid (DNDA) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular docking data suggested that both inhibitors specifically bind to protein kinase C-zeta (PKC-ζ) and PKC-iota (PKC-ι) and kinase activity assays were carried out to confirm these observations. Both inhibitors decreased the levels of total and phosphorylated PKC-ζ and PKC-ι. Increased levels of E-cadherin, RhoA, PTEN and decreased levels of phosphorylated vimentin, total vimentin, CD44, β-catenin and phosphorylated AKT in inhibitor treated cells. This suggests that inhibition of both PKC-ζ and PKC-ι using ACPD and DNDA downregulates EMT and induces apoptosis in melanoma cells. We also carried out PKC-ι and PKC-ζ directed siRNA treatments to prove the above observations. Immunoprecipitation data suggested an association between PKC-ι and vimentin and PKC-ι siRNA treatments confirmed that PKC-ι activates vimentin by phosphorylation. These results further suggested that PKC-ι is involved in signaling pathways which upregulate EMT and which can be effectively suppressed using ACPD and DNDA. Our results summarize that melanoma cells proliferate via aPKC/AKT/NF-κB mediated pathway while inducing the EMT via PKC-ι/Par6/RhoA pathway. Overall, results show that aPKCs are essential for melanoma progression and metastasis, suggesting that ACPD and DNDA can be effectively used as potential therapeutic drugs for melanoma by inhibiting aPKCs.
非典型蛋白激酶 C（aPKC）参与许多癌症中的细胞周期进程、肿瘤发生、细胞存活和迁移。我们认为，aPKC 通过调节细胞信号通路和诱导上皮间质转化（EMT），在黑色素瘤细胞的运动中发挥重要作用。我们研究了两种新型 aPKC 抑制剂；2-乙酰-1,3-环戊二酮（ACPD）和 3,4-二氨基萘-2,7-二磺酸（DNDA）对两种恶性黑色素瘤细胞系与正常黑素细胞相比的细胞增殖、凋亡、迁移和侵袭的影响。分子对接数据表明，这两种抑制剂特异性结合蛋白激酶 C-ζ（PKC-ζ）和蛋白激酶 C-ι（PKC-ι），并进行激酶活性测定以证实这些观察结果。两种抑制剂均降低了总 PKC-ζ 和 PKC-ι 及其磷酸化水平。抑制剂处理细胞中 E-钙粘蛋白、RhoA、PTEN 水平升高，磷酸化波形蛋白、总波形蛋白、CD44、β-连环蛋白和磷酸化 AKT 水平降低。这表明使用 ACPD 和 DNDA 抑制 PKC-ζ 和 PKC-ι 可下调 EMT 并诱导黑色素瘤细胞凋亡。我们还进行了 PKC-ι 和 PKC-ζ 靶向 siRNA 处理以证明上述观察结果。免疫沉淀数据表明 PKC-ι 与波形蛋白之间存在关联，而 PKC-ι siRNA 处理证实 PKC-ι 通过磷酸化激活波形蛋白。这些结果进一步表明，PKC-ι 参与信号通路，上调 EMT，而 ACPD 和 DNDA 可通过抑制 aPKC 有效抑制。我们的研究结果总结为黑色素瘤细胞通过 aPKC/AKT/NF-κB 介导的途径增殖，同时通过 PKC-ι/Par6/RhoA 途径诱导 EMT。总的来说，结果表明 aPKC 对于黑色素瘤的进展和转移至关重要，这表明 ACPD 和 DNDA 可以通过抑制 aPKC 有效用作黑色素瘤的潜在治疗药物。
