Department of Thoracic Medical Oncology, Tianjin Medical University Cancer Institute and Hospital, School of Basic Medical Sciences, International Medical School, School of Pharmacy, Tianjin Medical University, No. 22 Qixiangtai Road, Heping District, Tianjin, 300070, People's Republic of China.
J Exp Clin Cancer Res. 2017 Sep 2;36(1):115. doi: 10.1186/s13046-017-0585-2.
Metastatic malignant melanoma is one of the most aggressive malignancies and its treatment remains challenging. Recent studies demonstrate that the melanoma metastasis has correlations with the heightened activations of protein kinase C ζ (PKCζ) and cyclooxygenase-2 (COX-2) signaling pathways. Targeted inhibitions for PKCζ and COX-2 have been considered as the promising strategies for the treatment of melanoma metastasis. Thus, the PKCζ inhibitor J-4 and COX-2 inhibitor Celecoxib were combined to treat melanoma metastasis in this study.
The Transwell assay, Wound-healing assay and Adhesion assay were used to evaluate the inhibition of combined therapy of J-4 and Celecoxib on melanoma cells invasion, migration and adhesion in vitro, respectively. The impaired actin polymerization was observed by confocal microscope and inactivated signal pathways about PKCζ and COX-2 were confirmed by the Western blotting assay. The B16-F10/C57BL mouse melanoma model was used to test the inhibition of combined therapy of J-4 and Celecoxib on melanoma metastasis in vivo.
The in vitro results showed that the combination of J-4 and Celecoxib exerted synergistic inhibitory effects on the migration, invasion and adhesion of melanoma B16-F10 and A375 cells with combination index less than 1. The actin polymerization and phosphorylation of Cofilin required in cell migration were severely impaired, which is due to the inactivation of PKCζ related signal pathways and the decrease of COX-2. The combined inhibition of PKCζ and COX-2 induced Mesenchymal-Epithelial Transition (MET) in melanoma cells with the expression of E-Cadherin increasing and Vimentin decreasing. The secretion of MMP-2/MMP-9 also significantly decreased after the combination treatment. In C57BL/6 mice intravenously injected with B16-F10 cells (5 × 10 cells/mouse), co-treatment of J-4 and Celecoxib also severely suppressed melanoma lung metastasis. The body weight monitoring and HE staining results indicated the low toxicity of the combination therapy.
This study demonstrates that the combination therapy of PKCζ and COX-2 inhibitors can significantly inhibit melanoma metastasis in vitro and in vivo, which will be an efficient strategy for treatment of melanoma metastasis in clinics.
转移性恶性黑色素瘤是最具侵袭性的恶性肿瘤之一，其治疗仍然具有挑战性。最近的研究表明，黑色素瘤转移与蛋白激酶 C ζ（PKCζ）和环氧化酶-2（COX-2）信号通路的高度激活有关。针对 PKCζ 和 COX-2 的靶向抑制已被认为是治疗黑色素瘤转移的有前途的策略。因此，本研究中使用 PKCζ 抑制剂 J-4 和 COX-2 抑制剂塞来昔布联合治疗黑色素瘤转移。
使用 Transwell 测定法、划痕愈合测定法和黏附测定法分别评估 J-4 和塞来昔布联合治疗对黑色素瘤细胞体外侵袭、迁移和黏附的抑制作用。通过共聚焦显微镜观察到受损的肌动蛋白聚合，并通过 Western 印迹测定法证实 PKCζ 和 COX-2 信号通路失活。使用 B16-F10/C57BL 小鼠黑色素瘤模型测试 J-4 和塞来昔布联合治疗对体内黑色素瘤转移的抑制作用。
体外结果表明，J-4 和塞来昔布联合使用对黑色素瘤 B16-F10 和 A375 细胞的迁移、侵袭和黏附具有协同抑制作用，其联合指数小于 1。肌动蛋白聚合和细胞迁移所需的纽蛋白磷酸化严重受损，这是由于 PKCζ 相关信号通路失活和 COX-2 减少所致。黑色素瘤细胞中 PKCζ 和 COX-2 的联合抑制诱导间充质上皮转化（MET），E-钙黏蛋白表达增加，波形蛋白表达减少。联合治疗后 MMP-2/MMP-9 的分泌也显著减少。在静脉注射 B16-F10 细胞（5×10 个细胞/只小鼠）的 C57BL/6 小鼠中，J-4 和塞来昔布的联合治疗也严重抑制了黑色素瘤肺转移。体重监测和 HE 染色结果表明联合治疗的毒性较低。
本研究表明，PKCζ 和 COX-2 抑制剂的联合治疗可显著抑制黑色素瘤在体外和体内的转移，这将是临床治疗黑色素瘤转移的有效策略。
