Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing Normal University, Beijing, P. R. China.
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China.
Cancer Med. 2019 Aug;8(9):4441-4453. doi: 10.1002/cam4.2158. Epub 2019 Jun 20.
Recombinant human calcineurin B subunit (rhCNB) has been shown to be an immune-stimulatory protein promoting cytokine production and inducing phenotypic maturation of Dendritic cells (DCs). In vivo, it has good antitumor efficacy, and has potential as an antitumor drug. Exogenous rhCNB was found to be internalized into tumor cells via the Toll-like receptor 4 (TLR4) complex, but it was not known whether its immuno-modulatory and antitumor functions involved entry by this same route.
The production and secretion of the cytokines and chemokines in innate immune cells induced by rhCNB were determined by ELISA, and the expression of CD40, CD80, CD86, and MHCII was analyzed by FACs. Experimental Lewis lung cancer (LLC) model was prepared in C57 BL/6 wild-type (WT) mice, TLR4 mice or their littermates by the inoculation of LLCs in their right armpit, and then administrated daily intraperitoneal injections (0.2 mL) of normal saline, rhCNB 20 mg/kg, and rhCNB 40 mg/kg, respectively.
Recombinant human calcineurin B subunit promoted the production of antitumor cytokines by innate immune cells, and culture supernatants of rhCNB-stimulated immune cells induced apoptosis of LLCs. In addition, rhCNB up-regulated CD40, CD80, CD86, and MHCII expression in macrophages and DCs in TLR4 cells but failed to do so in TLR4 deficient cells. rhCNB also induced the formation of CD4 and CD8 T cells in splenocytes from WT mice, but not from TLR4-deficient littermates. Intraperitoneal administration of WT C57BL/6 mice with rhCNB resulted in a 50% reduction in LLC tumor growth, but failed to inhibit tumor growth in TLR4 littermates.
The in vivo antitumor and immunomodulatory effects of rhCNB are mediated by the TLR4. This conclusion is important for the further understanding and development of rhCNB as an antitumor drug.
重组人钙调神经磷酸酶 B 亚基（rhCNB）已被证明是一种免疫刺激性蛋白，可促进细胞因子的产生，并诱导树突状细胞（DC）的表型成熟。在体内，rhCNB 具有良好的抗肿瘤疗效，具有作为抗肿瘤药物的潜力。外源性 rhCNB 被发现通过 Toll 样受体 4（TLR4）复合物被内化到肿瘤细胞中，但尚不清楚其免疫调节和抗肿瘤功能是否涉及通过相同途径进入。
通过 ELISA 测定 rhCNB 诱导的固有免疫细胞中细胞因子和趋化因子的产生，并通过 FACs 分析 CD40、CD80、CD86 和 MHCII 的表达。通过在右腋窝接种 LLC 细胞，在 C57BL/6 野生型（WT）小鼠、TLR4 小鼠或其同窝仔鼠中制备实验性 Lewis 肺癌（LLC）模型，然后分别每天腹腔内注射（0.2 mL）生理盐水、rhCNB 20 mg/kg 和 rhCNB 40 mg/kg。
重组人钙调神经磷酸酶 B 亚基促进固有免疫细胞产生抗肿瘤细胞因子，rhCNB 刺激的免疫细胞培养上清液诱导 LLC 细胞凋亡。此外，rhCNB 在 TLR4 细胞中上调巨噬细胞和 DC 中的 CD40、CD80、CD86 和 MHCII 表达，但在 TLR4 缺陷细胞中不能。rhCNB 还诱导 WT 小鼠脾细胞中 CD4 和 CD8 T 细胞的形成，但不能诱导 TLR4 缺陷小鼠的形成。腹腔内给予 rhCNB 的 WT C57BL/6 小鼠 LLC 肿瘤生长减少 50%，但不能抑制 TLR4 同窝仔鼠的肿瘤生长。
rhCNB 的体内抗肿瘤和免疫调节作用是由 TLR4 介导的。这一结论对进一步理解和开发 rhCNB 作为抗肿瘤药物具有重要意义。
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