TRPM8通道抑制剂AMTB对骨肉瘤的治疗作用及机制研究

Osteosarcoma is the most common primary bone tumor. Due to lacking of noval therapies and chemotherapeutic drugs’ severe side effects, there is great limit for drugs use. The survival rate of osteosarcoma patients remains very low in last three decades which is only about 50%. Oxaliplatin is an important chemotherapeutic drug for osteosarcoma. However, Oxaliplatin’s usage is limited because of the cause of cold allodynia. According to our previous study, we found AMTB, a synthesized drug, can greatly inhibit TRPM8 in both vitro and vivo. Besides, we found TRPM8 was highly expressed in osteosarcoma cell lines and patients’ specimen through RTQ-PCR and immunohistochemistry. We also found that AMTB could prohibit osteosarcoma growth both in vitro and in vivo. AMTB also can enhance the sensitivity of osteosarcoma cells to Oxaliplatin. Through comparision of mRNA expression differences between osteosarcoma cells before and after exposing to AMTB, we found some genes associated with Wnt signaling pathway is suppressed, such as β-catenin, TCF7, LEF1 ect. Based on these, we are attempting to study the anti-tumor effect of AMTB against osteosarcoma at molecular, cell and animal level. At the same time we also want to study the synergistic effect against osteosarcoma of AMTB and Oxaliplatin as well as whether AMTB can alleviate Oxaliplatin’s cold allodynia side effect. Beside of presenting a candidate for osteosarcoma chemotherapy, we are also hoping to further explore the mechanism under the high expression of TRPM8 in osteosarcoma to give a new point of view for osteosarcoma study.

骨肉瘤是骨最常见的原发性恶性肿瘤，因缺乏新的治疗方法及现有化疗药物的副作用限制，近30年来骨肉瘤患者生存期未能再有效提高，五年生存率仅约50%。奥沙利铂是骨肉瘤重要的化疗药物，但因冷过敏等副作用限制了其临床使用。课题组前期研究证实化学合成药物AMTB在小鼠体内外对TRPM8通道均有良好的抑制作用。并且TRPM8通道在骨肉瘤临床样本和细胞系中均呈高表达，而使用AMTB在体外和体内均可抑制骨肉瘤的生长。进一步研究表明AMTB可增强骨肉瘤细胞对奥沙利铂的敏感性。通过表达谱测序，课题组发现AMTB可抑制多个Wnt通路相关基因的表达，包括β-catenin、TCF7、LEF1等。以此为基础，本研究拟在分子、细胞、动物水平阐明AMTB的抗骨肉瘤效应，并探索其与奥沙利铂的协同作用和减轻奥沙利铂冷过敏副作用的效应，通过深入研究TRPM8通道在骨肉瘤发展中的生物学机制，为骨肉瘤的治疗提供新的靶点和理论基础。

瞬时受体电位通道（TRP通道）是一类位于动物细胞膜上的非选择性阳离子通道，其可以形成通过钙、镁等阳离子的孔道。钙离子可以通过调节细胞信号转导通路和钙调节蛋白从而影响包括细胞生长、凋亡以及肿瘤发生发展在内的多种细胞事件。钙调节通道在肿瘤中表达的改变可能与肿瘤的发生发展密切相关，成为未来肿瘤治疗的靶点。本研究发现TRPM8通道蛋白在骨肉瘤患者石蜡切片和骨肉瘤多种细胞系中表达明显升高，且其表达与患者预后相关。通过MTT、克隆形成、划痕试验等多种细胞学试验以及裸鼠皮下荷瘤模型证明TRPM8通道特异性抑制剂AMTB可以通过抑制TRPM8通道活性在动物体内及体外产生良好的抗骨肉瘤活性，且该作用具有时间及剂量依赖性。此外，AMTB可以协同骨肉瘤化疗药物奥沙利铂产生抗肿瘤效果。流式细胞学试验发现AMTB可以明显诱导骨肉瘤细胞系发生凋亡，进一步研究发现可能与其抑制TGFβ通路有关。本研究很好的解释了瞬时受体电位通道M8在骨肉瘤发生发展中的作用，为进一步理解骨肉瘤发生发展机制提供了理论依据，也为下一步改进优化骨肉瘤化疗方案提供了新的靶点和苗头药物，具有良好的市场应用前景。