Kinoshita Rie, Watanabe Masami, Huang Peng, Li Shun-Ai, Sakaguchi Masakiyo, Kumon Hiromi, Futami Junichiro
Department of Biotechnology, Division of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan.
Center for Innovative Clinical Medicine, Okayama University Hospital, Okayama, Japan.
Oncol Rep. 2015 Jun;33(6):2908-14. doi: 10.3892/or.2015.3885. Epub 2015 Mar 30.
Reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3 is a tumor-suppressor gene and has been studied as a promising therapeutic gene for cancer gene therapy. Intratumoral injection of an adenovirus vector carrying the human REIC/Dkk-3 gene (Ad-REIC) elicits cancer cell-specific apoptosis and anticancer immune responses. The cytokine-like effect of secretory REIC/Dkk-3 on the induction of dendritic cell (DC)-like cell differentiation from monocytes plays a role in systemic anticancer immunity. In the present study, we generated recombinant full-length and N-terminally truncated REIC/Dkk-3 to characterize the biological activity of the protein. During the purification procedure, we identified a 17 kDa cysteine-rich stable product (C17-REIC) showing limited degradation. Further analysis showed that the C17-REIC domain was sufficient for the induction of DC-like cell differentiation from monocytes. Concomitant with the differentiation of DCs, the REIC/Dkk-3 protein induced the phosphorylation of glycogen synthase kinase 3β (GSK-3β) and signal transducers and activators of transcription (STAT) at a level comparable to that of granulocyte/macrophage colony-stimulating factor. In a mouse model of subcutaneous renal adenocarcinoma, intraperitoneal injection of full-length and C17-REIC proteins exerted anticancer effects in parallel with the activation of immunocompetent cells such as DCs and cytotoxic T lymphocytes in peripheral blood. Taken together, our results indicate that the stable cysteine-rich core region of REIC/Dkk-3 is responsible for the induction of anticancer immune responses. Because REIC/Dkk-3 is a naturally circulating serum protein, the upregulation REIC/Dkk-3 protein expression could be a promising option for cancer therapy.
永生细胞中表达降低（REIC）/Dickkopf（Dkk）-3是一种肿瘤抑制基因，已作为癌症基因治疗中有前景的治疗基因进行研究。瘤内注射携带人REIC/Dkk-3基因的腺病毒载体（Ad-REIC）可引发癌细胞特异性凋亡和抗癌免疫反应。分泌型REIC/Dkk-3对单核细胞诱导树突状细胞（DC）样细胞分化的细胞因子样作用在全身抗癌免疫中发挥作用。在本研究中，我们构建了重组全长和N端截短的REIC/Dkk-3以表征该蛋白的生物学活性。在纯化过程中，我们鉴定出一种17 kDa富含半胱氨酸的稳定产物（C17-REIC），其降解有限。进一步分析表明，C17-REIC结构域足以诱导单核细胞分化为DC样细胞。与DC分化同时，REIC/Dkk-3蛋白诱导糖原合酶激酶3β（GSK-3β）和信号转导及转录激活因子（STAT）磷酸化，其水平与粒细胞/巨噬细胞集落刺激因子相当。在皮下肾腺癌小鼠模型中，腹腔注射全长和C17-REIC蛋白可发挥抗癌作用，同时激活外周血中的免疫活性细胞，如DC和细胞毒性T淋巴细胞。综上所述，我们的结果表明，REIC/Dkk-3富含半胱氨酸的稳定核心区域负责诱导抗癌免疫反应。由于REIC/Dkk-3是一种天然循环的血清蛋白，上调REIC/Dkk-3蛋白表达可能是癌症治疗的一个有前景的选择。
