摘要
【研究背景及目的】肺癌是呼吸系统最常见的恶性肿瘤，其发病率和死亡率在世界范围内居高不下，特别是在我国大中城市呈现逐年上升趋势。临床上所采用的手术、放疗、化疗、微波局部灭活以及分子靶向治疗等综合治疗模式使得肺癌患者的生活质量及生存期得到一定程度的改善，但5年总生存率只有16%，控制肺癌的转移和复发仍面临严峻挑战。唑来膦酸（zoledronic acids）对恶性肿瘤骨转移的治疗效果已经在临床实践中得到证实，并作为一线药物广泛应用。最新的研究结果表明，唑来膦酸不但可以抑制破骨细胞的破坏与吸收，还可以直接作用于肿瘤细胞而发挥抗肿瘤作用。其机制涉及唑来膦酸可以诱导肿瘤细胞的凋亡与自噬、抑制肿瘤细胞的增殖、预防肿瘤细胞对骨的侵袭与粘附以及降低肿瘤组织中的血管生成。但目前关于唑来膦酸在肺癌的发生及转移过程中如何发挥作用，作用的靶点及其信号通路还不清楚。已知无论是心肌或者平滑肌等可兴奋细胞，还是肿瘤细胞等非兴奋性细胞，其细胞膜表面都存在大量离子通道。离子通道的主要作用是调控细胞膜内外K+、Na~+、Ca~(2+)与Cl~-等离子的稳态平衡，对细胞的病理及生理过程起着关键的调控作用。
T型Ca~(2+)通道在许多细胞膜上均有表达，其可受细胞膜电位与细胞浆内Ca~(2+)的双重调控。T型Ca~(2+)通道的抑制可显著诱导血管平滑肌细胞（VSMCs）的凋亡。近年的研究已表明，在肿瘤细胞膜上的T型Ca~(2+)通道也高度表达，并且T型Ca~(2+)通道的异常可能与血管内皮生长因子（VEGF）等肿瘤分子标志物高度相关，籍此推测T型Ca~(2+)通道也可能参与了肺癌的发生与发展过程。已有实验提示唑来膦酸可显著抑制肺癌细胞系A549的增殖及血管生成，那么是否像VSMCs一样，唑来膦酸是通过抑制T型Ca~(2+)通道诱导了肺癌细胞的凋亡抑制细胞增殖，并且唑来膦酸所引起的T型Ca~(2+)通道抑制是否也调控了肺癌细胞的迁移、侵袭与血管形成等过程。为此，我们在细胞和动物实验两个层面设计了本实验来验证唑来膦酸对肺癌A549细胞的影响及其作用机制。
【方法】以体外培养的肺腺癌A549细胞系进行系统的系列实验，观察唑来膦酸对肺腺癌A549细胞系的凋亡、增殖、迁移、侵袭以及血管形成能力的影响。通过免疫细胞化学、流式细胞仪检测细胞凋亡，采用MTT与细胞增殖核抗原（Proliferatingcell nuclear antigen, PCNA）免疫组化染色法检测A549细胞的增殖能力；通过划痕实验与transwell小室实验检测A549细胞的侵袭与转移能力；以人脐静脉内皮细胞（Human umbilical vein endothelial cells, HUVECs）的体外成环实验以及鸡胚尿膜囊（chicken embryo allantoids membrane, CAM）实验来检测血管的生成能力。
通过加入T型Ca~(2+)通道抑制剂米贝拉地尔（Mibefradil）来探讨唑来膦酸对肺癌A549细胞影响的可能机制。选择肺腺癌A549细胞系与转染了T型Ca~(2+)通道α亚基(CaV3.1)的人胚胎肾细胞293（human embryonic kidney293, HEK293），利用全细胞膜片钳技术记录唑来膦酸对T型Ca~(2+)通道电流的影响；并检测了A549细胞内Ca~(2+)的变化以探讨诱导凋亡发生的可能机制。在细胞实验基础上，我们还建立了A549细胞的裸鼠种植瘤动物模型，观察唑来膦酸对肺癌A549细胞种植瘤的生长抑制作用，且采用免疫荧光染色法对裸鼠种植瘤组织的凋亡相关基因caspase-3、粘附相关基因E-cadherin与血管内皮生长因子（Vascular endothelial growth factor, VEGF）进行了观察。
【结果】1、唑来膦酸可通过抑制T型Ca~(2+)通道来改变细胞内环境，诱导A549肺癌细胞的凋亡，抑制其增殖：通过膜片钳技术我们证实肺腺癌A549细胞系上有T型Ca~(2+)通道的存在。唑来膦酸可显著抑制T型Ca~(2+)通道的活性，而受抑制的T型Ca~(2+)通道可诱导A549细胞凋亡，且凋亡具有剂量依赖性与时间依赖性，其机制涉及胞内Ca~(2+)浓度的升高。我们又把T型Ca~(2+)通道α亚基转染到HEK293细胞上，证实唑来膦酸抑制T型Ca~(2+)通道的作用靶点就在α亚基上。而T型Ca~(2+)通道阻断剂Mibefradil可协同增强唑来膦酸的这种作用。本实验结果表明唑来膦酸可能通过抑制T型Ca~(2+)通道来促进A549细胞的凋亡，同时抑制A549细胞的增殖。
2、唑来膦酸通过抑制T型Ca~(2+)通道的作用，降低细胞的迁移、侵袭以及血管生成能力：我们发现唑来膦酸可以显著抑制A549细胞的迁移与侵袭能力，100μM唑来膦酸作用24h后肺癌细胞迁移生长的距离明显短于对照组，其迁移率也显著低于对照组细胞（17.3±3.6%vs.75.9±4.7%）；A549肺癌细胞穿过基底膜的数目显著减少（17.1±6.6%vs.63.8±8.2%）；唑来膦酸处理组的HUVEC生长缓慢，贴壁后几乎无移动，小管形成的数目明显减少。唑来膦酸可抑制体外与体内血管形成，其中的机制涉及T型Ca~(2+)通道信号的抑制，通过实验证实其具有明显降低HUVECs的体外成环与CAM的体内血管生成的作用。
3、唑来膦酸通过抑制T型Ca~(2+)通道抑制A549细胞裸鼠皮下种植瘤的生长：我们观察到注射唑来膦酸可抑制A549肺癌细胞种植瘤的生长，唑来膦酸注射组中裸鼠肿瘤生长显著受到抑制，肿瘤组织的体积明显小于对照组，且种植瘤组织的凋亡相关基因caspase-3、粘附相关基因E-cadherin与血管生成促进因子-血管内皮生长因子（Vascular endothelial growth factor, VEGF）均发生显著改变。
【结论】通过细胞与动物实验均证实唑来膦酸可通过调控T型Ca~(2+)通道发挥抗肺癌的作用，这可能是唑来膦酸抑制肺腺癌A549细胞生长的一条新的信号通路，可为肺癌的治疗提供新的靶点与思路。
Background: Lung cancer is the most common respiratory disease, which has a strongpropensity to metastasize. Most patients with advanced lung cancer always develop bonemetastasis. More recently, multiple clinical studies have be reported that bisphosphonatesare successful drugs to reduce the hypercalcaemia and skeletal morbidity in the treatmentof lung cancer and bone metastasis. Zoledronic acid has been regarded as one of the mostclinical potent nitrogen-containing biphosphonates in the adjuvant and neoadjuvantsettings of treatment for metastatic lung cancer, which has been reported not only toinhibit osteoclast-mediated bone resorption, but also have direct anti-tumor andanti-metastatic properties in lung cancer in vitro and in vivo. The primary mechanismsresponsible for the anti-tumor properties of zoledronic acid may involve the induction ofapoptosis, inhibition of proliferation, prevention of invasion and adhesion, the reduction ofangiogenesis, and the stimulation of innate anti-cancer immunity. However, the precisemechanisms remain to be determined.
T-type voltage-dependent calcium channels (CaT) are ubiquitously present in most human cells and play an essential role in the regulation of cell growth. The basic functionalsubunit of CaTchannel is the pore forming α-subunit encoded by a single gene,CACNA1G or CACNA1H. CaTchannels are activated by membrane potential andphosphorylation. In excitable cells, activation of CaTchannel may increase theintracellular Ca~(2+)concentration and then contribute to the regulation of muscle contraction.In contrast, activation of CaTchannel has been considered to promote cell-proliferation byincreasing intracellular Ca~(2+)concentration in non-excitable cells. Previous studies haveimplicated that CaTchannel has been related to the progression of several malignanttumors. However, the role of CaTchannel in cancerous phenomenon is still controversial.For example, activation of CaTchannel has been reported to be involved in theproliferation, migration, and invasion of glioblastoma, human mesangial cell, and PC-3human prostate carcinoma cells. On the contrary, some work also suggested that activationof CaTchannel inhibited the proliferation of human breast cancer cells. In particular, it hasbeen demonstrated that CaTchannels are highly expressed in various established humanlung cancer cell lines, such as A549,95D NSCLC cells, and normal mammary epithelialcells. However, there have been no studies addressing the possibility of CaTchannel in thetreatment of human lung cancer with zoledronic acid to date. In the present study, weinvestigated the specific role of CaTchannel in the treatment of lung cancer cells withzoledronic acid.
Methodology: The A549cell lines were chosen for the experiment. The action ofzoledronic acid on CaTchannel was investigated by whole-cell patch clamp techniquesand Western blotting. Cell apoptosis was assessed with immunocytochemistry, analysis offragmented DNA by agarose gel electrophoresis, and flow cytometry assays. Cellproliferation was investigated by MTT test and immunocytochemistry. In addition, suchfindings were further confirmed from human embryonic kidney293(HEK293) cellswhich were transfected with functional CaTα1G-subunit. Finally, intracellular Ca~(2+)inA549cells was examined to investigate the possible mechanisms. The migration and theinvasive ability of A549cells were investigated by wound healing assays and Trans-wellmigration assays respectively. The tube formation of human umbilical vein endothelialcells (HUVECs）and neovascularization in chicken embryo allantoids membrane (CAM) were also assessed. At last, we established the nude mice xenograft with A549cells andassessed the expression of caspase-3, vascular endothelial growth factor (VEGF), andE-cadherine.
Principal Findings: Our results clearly indicate that zoledronic acid directly decreased theactivities of CaT channels, and then inhibition of CaT channel by zoledronic acidcontributed to induce apoptosis and suppress proliferation in A549cells. The possiblemechanisms were associated with the elevated level of intracellular Ca~(2+) in A549cells.
Zoledronic acid inhibited the migration and the invasive ability of A549cells. In addition,zoledronic acid also inhibited the vascular angiogenesis in the tube formation of HUVECsand neovascularization in CAM. In addition, the inhibitors of CaTchannel (Mibefradil)could imitate the effects of zoledronic acid in the migration and invasive ability of A549cells and vascular angiogenesis. These results suggested that inhibition of CaTchannelsmay participate in the treatment of lung cancer with zoledronic acid.
Zoledronic acid inhibited the growth of nude mice xenograft with A549cells. In addition,zoledronic acid also influenced apoptosis and the expression of caspase-3, VEGF, andE-cadherine. Furthermore, Mibefradil, the inhibitor of CaTchannel could imitate theeffects of zoledronic acid in nude mice xenograft with A549cells.
Conclusions: Inhibition of CaTchannel may be a novel molecular pathway, which isinvolved in the treatment of lung cancer with zoledronic acid in vivo and in vitro.
引文
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