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（Klotho）最初被发现是一种抗衰老基因，其基因敲除会加速小鼠的衰老进程。后续研究证实，克洛托（Klotho）在多种人类恶性肿瘤中发挥抑癌作用。本研究探讨了克洛托（Klotho）对骨肉瘤细胞增殖与转移的调控作用。研究人员将编码克洛托的慢病毒颗粒与阴性对照（scramble）慢病毒颗粒分别转导至骨肉瘤细胞中，将重组后的骨肉瘤细胞注射至裸鼠股骨骨髓腔内，以此构建骨肉瘤异种移植动物模型。本研究从肿瘤生长、细胞凋亡、糖原生成及肺转移四个方面评估了克洛托的抗肿瘤特性：慢病毒介导的克洛托过表达可显著降低骨肉瘤裸鼠的肿瘤体积与瘤重；通过检测增殖细胞核抗原（PCNA）与Ki-67的表达水平，发现克洛托过表达可抑制异种移植瘤组织中的细胞增殖；过碘酸雪夫（PAS）染色结果显示，克洛托过表达可显著降低骨肉瘤细胞的糖原生成量。此外，慢病毒介导的克洛托过表达可显著升高脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）阳性细胞的数量；进一步研究发现，与对照组小鼠相比，克洛托上调表达可减少小鼠体内的肺转移灶数量。上述实验结果证实，克洛托表达上调可在体内抑制骨肉瘤细胞的增殖与肺转移，提示克洛托或可成为骨肉瘤治疗的潜在有效靶点。