School of Life Sciences, Liaoning Normal University, Dalian 116081, China.
Department of Pharmacology, Dalian Medical University, Dalian 116044, China.
Mar Drugs. 2019 Jan 23;17(2):75. doi: 10.3390/md17020075.
Lj-RGD3, which contains three Arg⁻Gly⁻Asp (RGD) motifs, was first identified from the buccal glands of and has been shown to suppress the tumor progression in the previous studies. Apart from the three RGD motifs, Lj-RGD3 is also characterized by its high content of histidine in its amino acid sequence. In order to clarify whether the histidine-rich characterization of Lj-RGD3 is also associated with its anti-tumor activity, mutants were designed in which the three RGD motifs (Lj-112), or all histidines (Lj-27) or both (Lj-26) were deleted. Furthermore, a mutant (Lj-42) in which all histidines and three RGD motifs were respectively substituted with alanines and three Ala⁻Gly⁻Asp (AGD) motifs, as well as a mutant (Lj-41) in which all histidines were substituted with alanines was synthesized to avoid alterations in structure which might further cause changes in the peptides' functions. After recombination and purification, recombinant Lj-112 (rLj-112), recombinant Lj-27 (rLj-27), recombinant Lj-41 (rLj-41), and recombinant Lj-RGD3 (rLj-RGD3) exhibited anti-proliferative activity in B16 cells, respectively; while recombinant Lj-26 (rLj-26) and recombinant Lj-42 (rLj-42) did not affect the proliferation of B16 cells significantly. In addition, the anti-proliferative activity of rLj-112 in B16 cells was due to apoptosis. Typical apoptosis features were observed, including chromatin condensation, fragmented DNA, and increased levels of cleaved caspase 3/caspase 7/nuclear enzyme poly (ADP-ribose) polymerase (PARP) in B16 cells. Similar to rLj-RGD3, rLj-112 was also capable of suppressing the migration and invasion of B16 cells by disturbing the F-actin arrangement. After labeling with FITC, rLj-112 was found localized in the cytoplasm of B16 cells, which induced the internalization of epidermal growth factor receptor (EGFR), suggesting that rLj-112 might block the EGFR mediated signaling pathway. Actually, the phosphorylation level of EGFR and its downstream signal molecules including Akt, PI3K, p38, and ERK1/2 was reduced in the rLj-112 treated B16 cells. In vivo, rLj-112 also inhibited the growth, weight, and volume of the tumors in B16 xenografted C57BL/6 mice without reducing their body weight, indicating that rLj-112 might be safe and might be used as an effective anti-tumor drug in the near future.
Lj-RGD3 最初从 的颊腺中被鉴定出来，含有三个 Arg⁻Gly⁻Asp（RGD）基序，之前的研究表明它能够抑制肿瘤的进展。除了三个 RGD 基序外，Lj-RGD3 的氨基酸序列还具有高组氨酸含量的特点。为了阐明 Lj-RGD3 的组氨酸丰富特征是否也与其抗肿瘤活性有关，设计了突变体，其中三个 RGD 基序（Lj-112）或所有组氨酸（Lj-27）或两者（Lj-26）被删除。此外，还合成了一个突变体（Lj-42），其中所有组氨酸和三个 RGD 基序分别被丙氨酸和三个 Ala⁻Gly⁻Asp（AGD）基序取代，以及一个突变体（Lj-41），其中所有组氨酸被丙氨酸取代，以避免结构的改变，这可能进一步导致肽功能的改变。重组 Lj-112（rLj-112）、重组 Lj-27（rLj-27）、重组 Lj-41（rLj-41）和重组 Lj-RGD3（rLj-RGD3）在 B16 细胞中分别表现出抗增殖活性，而重组 Lj-26（rLj-26）和重组 Lj-42（rLj-42）对 B16 细胞的增殖没有显著影响。此外，rLj-112 在 B16 细胞中的抗增殖活性是由于细胞凋亡。在 B16 细胞中观察到典型的凋亡特征，包括染色质浓缩、DNA 片段化以及切割的 caspase 3/半胱天冬酶 7/核酶多聚（ADP-核糖）聚合酶（PARP）水平升高。与 rLj-RGD3 相似，rLj-112 还能够通过干扰 F-肌动蛋白排列来抑制 B16 细胞的迁移和侵袭。用 FITC 标记后，发现 rLj-112 位于 B16 细胞的细胞质中，这诱导了表皮生长因子受体（EGFR）的内化，表明 rLj-112 可能阻断 EGFR 介导的信号通路。实际上，在 rLj-112 处理的 B16 细胞中，EGFR 及其下游信号分子如 Akt、PI3K、p38 和 ERK1/2 的磷酸化水平降低。在体内，rLj-112 也抑制了 B16 异种移植 C57BL/6 小鼠肿瘤的生长、重量和体积，而不降低其体重，表明 rLj-112 可能是安全的，并且可能在不久的将来作为一种有效的抗肿瘤药物使用。
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