Overexpression of klotho suppresses growth and pulmonary metastasis of osteosarcoma in vivo

Abstract Klotho is originally discovered as an anti-aging gene and knock-out of klotho accelerates aging in mice. Subsequent studies support the anti-carcinogenesis role of klotho in a variety of human malignancies. The present study investigated the role of klotho on growth and metastasis of osteosarcoma cells. The osteosarcoma cells were transduced with lentivirus particles encoding klotho or scramble control. The reconstructed osteosarcoma cells were injected into the femoral medullary cavity of nude mice to establish a xenograft animal model. The anti-tumor properties of klotho were evaluated in terms of tumor growth, apoptosis, glycogen production, and pulmonary metastasis. Lentivirus-mediated overexpression of klotho significantly decreased tumor volume and weight in osteosarcoma mice. Determination of PCNA and Ki67 expression revealed that overexpression of klotho inhibited cell proliferation in tumor tissues obtained from osteosarcoma xenografts. PAS staining also showed that overexpression of klotho significantly decreased the production of glycogen in osteosarcoma. Moreover, TUNEL positive cells were significantly increased after lentivirus-mediated overexpression of klotho. Furthermore, lentivirus-mediated upregulation of klotho reduced the number of pulmonary metastatic lesions in mice compared to control mice. These findings demonstrated that elevated klotho could inhibit osteosarcoma cell growth and pulmonary metastasis in vivo, suggesting that klotho may be a valuable therapeutic target for osteosarcoma.

摘要：克洛托基因（Klotho）最初被鉴定为抗衰老基因，敲除该基因会加速小鼠的衰老进程。后续研究证实，克洛托基因在多种人类恶性肿瘤中发挥抑癌作用。本研究探究了克洛托基因对骨肉瘤细胞生长与转移的调控作用：将骨肉瘤细胞分别转导编码克洛托基因的慢病毒颗粒与乱序对照慢病毒颗粒（scramble control），随后将构建完成的重组骨肉瘤细胞注射至裸鼠股骨骨髓腔，以此建立骨肉瘤异种移植瘤模型。本研究从肿瘤生长、细胞凋亡、糖原生成及肺转移四个维度评估克洛托基因的抗肿瘤活性：慢病毒介导的克洛托基因过表达可显著降低骨肉瘤裸鼠模型的肿瘤体积与重量；对增殖细胞核抗原（PCNA）与Ki67表达的检测结果显示，克洛托基因过表达可抑制异种移植瘤组织中的细胞增殖；过碘酸-雪夫染色（PAS staining）结果同样表明，克洛托基因过表达可显著降低骨肉瘤细胞的糖原生成水平；此外，慢病毒介导的克洛托基因过表达可显著提升TUNEL阳性细胞的数量；进一步实验显示，与对照组小鼠相比，克洛托基因的慢病毒介导上调可减少小鼠肺部转移瘤病灶的数量。上述研究结果证实，克洛托基因高表达可在体内抑制骨肉瘤细胞的生长与肺转移，提示克洛托基因或可成为骨肉瘤治疗的潜在有效靶点。