具有增强的体外和体内抗脉络膜黑色素瘤细胞活性的色素上皮衍生因子磷酸模拟突变体。

Phosphomimetic mutants of pigment epithelium-derived factor with enhanced anti-choroidal melanoma cell activity in vitro and in vivo.

机构信息

Experimental Research Center, First People's Hospital, School of Medicine, Shanghai Jiaotong University, China.

出版信息

Invest Ophthalmol Vis Sci. 2012 Oct 3;53(11):6793-802. doi: 10.1167/iovs.12-10326.

DOI:10.1167/iovs.12-10326

PMID:22952124

Abstract

PURPOSE

Currently, choroidal melanoma is chemoresistant and there is no routine adjuvant chemotherapy for it. We investigated whether pigment epithelium-derived factor (PEDF) and its triple phosphomimetic mutants could more efficiently suppress melanoma tumor growth and metastasis, as well as how the triple phosphomimetic mutants act as antitumor agents.

METHODS

Phosphomimetic mutants of PEDF were constructed by site mutagenesis. Lentiviruses carrying wild type (WT) PEDF, S24E114E227A (EEA)-PEDF, and S24E114E227E (EEE)-PEDF were produced in 293 fast-growing, highly transfectable (FT) cells and used to infect human choroidal melanoma cell line (OCM-1). The growth, migration, invasion and metastasis abilities of OCM-1 cells expressing WT-PEDF, EEA-PEDF or EEE-PEDF were investigated in vitro and in vivo, while the underlying mechanism of PEDF phosphomimetic mutants were investigated via Western blotting.

RESULTS

OCM-1 cells infected with lentiviruses carrying WT-PEDF, EEA-PEDF, and EEE-PEDF displayed reduced proliferation, migration and invasion abilities, and were more prone to apoptosis. Cell media containing WT-PEDF, EEA-PEDF, or EEE-PEDF protein inhibited the tube forming capacity of human umbilical vein endothelial cells (HUVEC) in vitro. OCM-1 cells expressing WT-PEDF, EEA-PEDF, or EEE-PEDF displayed significantly reduced tumor growth and metastasis in the melanoma xenograft of nude mice models, with the PEDF mutants displaying much stronger effects than the wild type. The antitumor effects of PEDF are associated with the inhibition of VEGF and nuclear factor kappa-B (NF-κB) expression, as well as further inhibition of Akt phosphorylation.

CONCLUSIONS

The phosphomimetic mutants of PEDF showed enhanced anti-melanoma activity by directly affecting tumor cells and indirectly affecting angiogenesis. These findings encourage the development of PEDF mutants as innovative anticancer agents.

摘要

目的

目前，脉络膜黑色素瘤对化疗具有耐药性，且尚无常规辅助化疗方法。本研究旨在探讨色素上皮衍生因子（PEDF）及其三重磷酸化模拟突变体是否能更有效地抑制黑色素瘤肿瘤生长和转移，并探讨三重磷酸化模拟突变体作为抗肿瘤药物的作用机制。

方法

通过定点突变构建 PEDF 的磷酸化模拟突变体。在 293 快速生长、高转染（FT）细胞中产生携带野生型（WT）PEDF、S24E114E227A（EEA）-PEDF 和 S24E114E227E（EEE）-PEDF 的慢病毒，并用于感染人脉络膜黑色素瘤细胞系（OCM-1）。体外和体内研究 OCM-1 细胞中表达 WT-PEDF、EEA-PEDF 或 EEE-PEDF 的生长、迁移、侵袭和转移能力，同时通过 Western blot 研究 PEDF 磷酸化模拟突变体的作用机制。

结果

携带 WT-PEDF、EEA-PEDF 和 EEE-PEDF 的慢病毒感染的 OCM-1 细胞显示出增殖、迁移和侵袭能力降低，并且更易发生凋亡。细胞培养基中含有 WT-PEDF、EEA-PEDF 或 EEE-PEDF 蛋白，可抑制人脐静脉内皮细胞（HUVEC）的管形成能力。在裸鼠黑色素瘤异种移植模型中，表达 WT-PEDF、EEA-PEDF 或 EEE-PEDF 的 OCM-1 细胞显示出明显的肿瘤生长和转移减少，而 PEDF 突变体的作用明显强于野生型。PEDF 的抗肿瘤作用与抑制血管内皮生长因子（VEGF）和核因子κB（NF-κB）表达以及进一步抑制 Akt 磷酸化有关。