Melatonin synergizes BRAF-targeting agent vemurafenib in melanoma treatment by inhibiting iNOS/hTERT signaling and cancer-stem cell traits
Melatonin synergizes BRAF-targeting agent vemurafenib in melanoma treatment by inhibiting iNOS/hTERT signaling and cancer-stem cell traits
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褪黑素通过抑制 iNOS/hTERT 信号传导和癌症干细胞特征，协同 BRAF 靶向药物维莫非尼治疗黑色素瘤
DOI：
10.1186/s13046-019-1036-z
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发表时间：
2019-02-04
影响因子：
11.3
通讯作者：
Deng, Wuguo
中科院分区：
文献类型：
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作者：
Hao, Jiaojiao;Fan, Wenhua;Deng, Wuguo
背景作为BRAF激酶的选择性抑制剂，vemurafenib对V600 BRAF突变型黑色素瘤患者具有有效的抗肿瘤活性。方法采用免疫组织化学方法检测小鼠移植性黑色素瘤组织中iNOS、hTERT、p-p65、Epcam、CD 44、PCNA的表达。采用MTT法、体外肿瘤球培养、细胞周期、细胞凋亡、AO/EB法和集落形成法、transwell法和划痕法检测黑色素瘤细胞的增殖和迁移能力，并观察裸鼠移植瘤的生长差异。Western blot检测iNOS/hTERT信号通路中关键分子的表达变化。采用细胞核-细胞质分离和免疫荧光技术检测p50/p65在黑色素瘤细胞系中的定位。流式细胞术检测CD 44的表达。Pull down法和ChIP法检测p65与iNOS和hTERT启动子的结合能力。此外，hTERT启动子驱动的荧光素酶质粒转染到黑色素瘤细胞与指定的治疗，以确定荧光素酶活性hTERT.ResultsMelatonin显着和协同增强vemurafenib介导的抑制增殖，集落形成，迁移和入侵，促进vemurafenib诱导的细胞凋亡，细胞周期停滞和干细胞在黑色素瘤细胞减弱。进一步的机制研究表明，褪黑素通过抑制NF-κB B p50/p65的核转位及与iNOS和hTERT启动子的结合，从而抑制iNOS和hTERT的表达，增强维罗非尼B的抗肿瘤作用。还在黑色素瘤异种移植小鼠中评估并证实了维罗菲尼与褪黑激素联合治疗后的抗肿瘤能力增强。结论总的来说，我们的结果表明褪黑激素通过抑制细胞增殖和癌症干细胞特性来协同维罗菲尼在人黑色素瘤中的抗肿瘤作用。通过靶向NF-κB/iNOS/hTERT信号通路，并提示褪黑激素在拮抗维罗非尼毒性和增强其在黑色素瘤治疗中的敏感性方面的潜力。
BackgroundAs the selective inhibitor of BRAF kinase, vemurafenib exhibits effective antitumor activities in patients with V600 BRAF mutant melanomas. However, acquired drug resistance invariably develops after its initial treatment.MethodsImmunohistochemical staining was performed to detect the expression of iNOS and hTERT, p-p65, Epcam, CD44, PCNA in mice with melanoma xenografts. The proliferation and migration of melanoma cells were detected by MTT, tumorsphere culture, cell cycle, cell apoptosis, AO/EB assay and colony formation, transwell assay and scratch assay in vitro, and tumor growth differences were observed in xenograft nude mice. Changes in the expression of key molecules in the iNOS/hTERT signaling pathways were detected by western blot. Nucleus-cytoplasm separation, and immunofluorescence analyses were conducted to explore the location of p50/p65 in melanoma cell lines. Flow cytometry assay were performed to determine the expression of CD44. Pull down assay and ChIP assay were performed to detect the binding ability of p65 at iNOS and hTERT promoters. Additionally, hTERT promoter-driven luciferase plasmids were transfected in to melanoma cells with indicated treatment to determine luciferase activity of hTERT.ResultsMelatonin significantly and synergistically enhanced vemurafenib-mediated inhibitions of proliferation, colony formation, migration and invasion and promoted vemurafenib-induced apoptosis, cell cycle arresting and stemness weakening in melanoma cells. Further mechanism study revealed that melatonin enhanced the antitumor effect of vemurafenib by abrogating nucleus translocation of NF-κB p50/p65 and their binding at iNOS and hTERT promoters, thereby suppressing the expression of iNOS and hTERT. The elevated anti-tumor capacity of vemurafenib upon co-treatment with melatonin was also evaluated and confirmed in mice with melanoma xenografts.ConclusionsCollectively, our results demonstrate melatonin synergizes the antitumor effect of vemurafenib in human melanoma by inhibiting cell proliferation and cancer-stem cell traits via targeting NF-κB/iNOS/hTERT signaling pathway, and suggest the potential of melatonin in antagonizing the toxicity of vemurafenib and augmenting its sensitivities in melanoma treatment.