摘要
乳腺癌是女性最常见的恶性实体肿瘤，其发病率在世界范围内呈现逐年上升的趋势。目前，临床上所采用的早期诊断、手术、放疗、化疗、内分泌以及分子靶向治疗等综合治疗模式使得乳腺癌的死亡率呈现逐渐下降的趋势，但其复发率与转移率仍然很高。唑来膦酸（zoledronic acids）是近年来发展起来的一类新药物，其对乳腺癌骨性转移的治疗效果已经在临床实践中获得证实。最新的研究亦表明，唑来膦酸不但可以抑制破骨细胞的骨破坏与骨吸收，还可以直接作用于乳腺癌细胞而发挥抗肿瘤作用，其机制涉及唑来膦酸诱导乳腺癌细胞的凋亡与自噬、抑制乳腺癌细胞的增殖、预防乳腺癌细胞对骨的侵袭与粘附以及降低乳腺癌组织的血管生成。但目前关于唑来膦酸发挥抗肿瘤作用的分子靶点与信号通路还不清楚，有待更深入的研究。
无论是心肌或平滑肌等可兴奋细胞，还是肿瘤细胞等非兴奋性细胞，其细胞膜表面都有大量离子通道的表达。离子通道的主要作用是调控细胞膜内外K+、Ca~(2+)、Na~+与Cl-等离子的稳态平衡，因而对细胞的病理与生理过程起着关键的调控作用。大电导钙激活钾通道（Big conductance calcium-activated K~+channel,BKCa）在许多细胞膜上均有高度表达，其可受细胞膜电位与细胞浆内Ca~(2+)的双重调控。心血管系统中的BKCa通道对血管的收缩-舒张起着重要的调控作用，而且最新的研究证实BKCa通道的激活可显著诱导血管平滑肌细胞（vascularsmooth muscle cell, VSMCs）的凋亡。近年的研究亦表明，人乳腺癌细胞膜上的BKCa通道也高度表达，并且BKCa通道的异常可能与雌激素受体（estrogenreceptor, ER）、人表皮生长因子受体2（human epidermal growth factor receptor2,HER-2）等经典肿瘤分子标志物高度相关，因而推测BKCa通道也可能参与了乳腺癌的发生与发展过程，但其发挥了何种作用还不清楚。我们在前期实验中发现唑来膦酸可显著增强乳腺癌细胞系MDA-MB-231与MCF-7的BKCa通道电流，那么是否如VSMCs一样，唑来膦酸是否可通过激活BKCa通道诱导了乳腺癌细胞凋亡？并且唑来膦酸所引起的BKCa通道激活是否也调控了乳腺癌细胞的增殖、迁移、侵袭与血管形成等过程？
为了验证以上的假说，我们分别在体外培养的乳腺癌细胞系与裸鼠种植瘤2个水平进行了实验。我们选择了ER阴性的MDA-MB-231乳腺癌细胞系、ER阳性的MCF-7乳腺癌细胞系与转染了BKCa通道α亚基基因(hSloα)的人胚胎肾细胞293（human embryonic kidney293, HEK293），利用全细胞与单通道膜片钳技术记录唑来膦酸对BKCa通道电流的影响；通过免疫细胞化学、DNA片段琼脂糖凝胶电泳分析与流式细胞仪检测细胞凋亡，并检测了MDA-MB-231细胞内Ca~(2+)与线粒体膜电位(Δψm)的变化以探讨凋亡可能的机制；以MTT与细胞增殖核抗原（Proliferating cell nuclear antigen, PCNA）免疫组化染色来检测MDA-MB-231细胞的增殖；以划痕实验与transwell小室检测MDA-MB-231细胞的侵袭与转移能力；以人脐静脉内皮细胞（Human umbilical vein endothelialcells, HUVECs）的体外成环实验以及鸡胚尿膜囊（chicken embryo allantoidsmembrane, CAM）来检测血管的形成能力。最后，我们建立了MDA-MB-231细胞的裸鼠种植瘤模型，观察了BKCa通道抑制剂（Iberiotoxin, IBTX）在唑来膦酸治疗乳腺癌组织中的作用。我们的主要实验结果如下：
1、唑来膦酸通过激活BKCa通道诱导MDA-MB-231乳腺癌细胞的凋亡：
通过膜片钳技术我们证实MDA-MB-231与MCF-7乳腺癌细胞系上均有BKCa通道的存在。对MDA-MB-231细胞而言，唑来膦酸可显著增加BKCa通道的活性，而激活的BKCa通道可诱导MDA-MB-231细胞凋亡，其机制涉及胞内Ca~(~(2+))浓度的升高与线粒体膜电位(Δψm)的去极化。我们还把BKCa通道α亚基转染到HEK293细胞上，证实唑来膦酸激活BKCa通道的作用靶点就在α亚基上。对MCF-7而言，唑来膦酸也可激活MCF-7细胞的BKCa通道，但BKCa通道的激活却不能诱导细胞凋亡。如果以ER拮抗剂ICI182780阻断MCF-7细胞ER的功能，则唑来膦酸则可通过激活BKCa通道诱导MCF-7细胞凋亡，因此，我们推测ER的存在可能干扰了BKCa通道的作用。
2、唑来膦酸通过激活BKCa通道抑制MDA-MB-231细胞的增殖、侵袭以及血管生成：
由于MCF-7细胞膜上BKCa通道不参与凋亡的调控，因此我们的研究重点集中在MDA-MB-231细胞上。我们发现唑来膦酸可显著抑制MDA-MB-231细胞的增殖、迁移与侵袭能力，也可明显降低HUVECs的体外成环与CAM的体内血管生成，但BKCa通道阻断剂IBTX或TEA则可部分逆转唑来膦酸的这种作用。本实验结果表明唑来膦酸激活的BKCa通道不但可促进MDA-MB-231细胞的凋亡，还可以抑制MDA-MB-231细胞的增殖、侵袭、以及血管的形成。
3、唑来膦酸通过调控BKCa通道抑制MDA-MB-231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MDA-MB-231乳腺癌细胞种植瘤的生长，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，而BKCa通道阻断剂IBTX可部分逆转唑来膦酸的这种作用。本实验结果通过裸鼠体内实验表明唑来膦酸可通过激活BKCa通道调控MDA-MB-231细胞裸鼠皮下种植瘤的生长。
总之，我们通过体内与体外实验均证实唑来膦酸可通过调控BKCa通道发挥抗乳腺癌的作用，这可能是唑来膦酸治疗ER阴性MDA-MB-231乳腺癌的一条新的信号通路，可为乳腺癌的治疗提供新的思路与靶点。
Background: Breast cancer is the most common neoplasm in women and has astrong propensity to metastasize to bone. Most patients with advanced breast cancerfrequently develop bone metastases characterized with the increased osteoclasticbone resorption, and at this stage, the disease associated with pain, fractures, andhypercalcemia is considered incurable. More recently, multiple preclinical and earlyclinical studies have demonstrated that bisphosphonates are successfully establisheddrugs to reduce the incidence of hypercalcaemia and skeletal morbidity in thetreatment of breast cancer and bone metastasis. The clinical potential of zoledronicacid, one of the most potent nitrogen-containing biphosphonates, is widelyconfirmed in the adjuvant and neoadjuvant settings of treatment for metastaticbreast cancer. Zoledronic acid has been reported not only to inhibitosteoclast-mediated bone resorption, but also have direct anti-tumor andanti-metastatic properties in breast cancer in vitro and in vivo. The primarymechanisms responsible for the direct anti-tumor activity of zoledronic acid may involve the inhibition of tumor-cell proliferation, the induction of tumor-cellapoptosis and autophagy, the prevention of tumor-cell invasion and adhesion inbone, the reduction of angiogenesis, and the stimulation of innate anti-cancerimmunity. However, the precise mechanisms remain to be determined by whichzoledronic acid directly affects breast cancer cells.
Large conductance Ca~(2+)-activated K~+(BKCa) channels are ubiquitously presentin most human cells and play an essential role in the regulation of basic cellularprocesses. The basic functional unit of BKCachannel is the pore forming α-subunitencoded by a single gene, Sloα or KCNMA1. BKCachannels are activated bymembrane potential, intracellular Ca~(2+), and phosphorylation. Activation of BKCachannel hyperpolarizes the membrane potential and deactivates thevoltage-dependent Ca~(2+)channels (VDCCs), which leads to a reduction inintracellular Ca~(2+)concentration. In excitable cells, such as vascular smooth musclecells (VSMCs), it is well known that BKCachannels contribute to the regulation ofvascular tone in a negative feedback manner which limits VSMCs depolarizationand prevents vasospasm. Recently, activation of BKCachannel has also beenreported to be involved in the regulation of apoptosis besides itselectrophysiological function in vascular relaxation. In contrast, functions of BKCachannel in non-excitable cells are somewhat enigmatic. Previous studies haveimplicated that BKCachannel has been related to the progression of severalmalignant tumor. In particular, it has been demonstrated that BKCachannels arehighly expressed in various established human breast cancer cell lines, such asMCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-435s, MDA-MB-361, andnormal mammary epithelial cells. However, the role of BKCachannel in breastcancerous phenomenon is still controversial. For example, activation of BKCachannel has been described to be involved in the proliferation, migration, andinvasion of breast cancer cells. On the contrary, some work also suggested thatBKCachannel might have no roles in the controlling growth of breast cancer cells because the specific BKCachannel blockers [charybdotoxin or iberiotoxin (IBTX)]did not have any effect on cell proliferation. However, to date there have been nostudies addressing the possibility of BKCachannel in the treatment of human breastcancer with oledronic acid. In the present study, we investigated the specific role oflarge conductance Ca~(2+)-activated potassium (BKCa) channel in the treatment ofbreast cancer cells with zoledronic acid.
Methodology/Principal Findings: The estrogen receptor (ER)-negativeMDA-MB-231and ER-positive MCF-7cell lines were chosen for the experiment.The action of zoledronic acid on BKCachannel was investigated by whole-cell andcell-attached patch clamp techniques. Cell apoptosis was assessed withimmunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis,and flow cytometry assays. Cell proliferation was investigated by MTT test andimmunocytochemistry. In addition, such findings were further confirmed fromhuman embryonic kidney293(HEK293) cells which were transfected withfunctional BKCaα-subunit (hSloα). Finally, intracellular Ca~(2+)and mitochondrialmembrane potential (Δψm) in MDA-MB-231cells were also examined toinvestigate the possible mechanisms. The migration and the invasive ability ofMDA-MB-231cells were investigated by wound healing assays and Trans-wellmigration assays respectively. The tube formation of Human umbilical veinendothelial cells (HUVECs）and neovascularization in chicken embryo allantoidsmembrane (CAM) were also accessed. At last, we established the nude micexenograft with MDA-MB-231cells and assessed the expression of caspase-3,Vascular endothelial growth factor (VEGF), and E-cadherine.
1) Our results clearly indicate that zoledronic acid directly increased theactivities of BKCachannels, and then activation of BKCachannel by zoledronic acid contributed to induce apoptosis in MDA-MB-231cells. The possible mechanismswere associated with the elevated level of intracellular Ca~(2+)and a concomitantdepolarization of mitochondrial membrane potential (Δψm) in MDA-MB-231cells.However, activation of BKCachannels by zoledronic acid was found to be relativelyresistant to apoptosis in ER-positive MCF-7cells. The presence of a functional ERseemed to interfere with the effects of BKCachannel in zoledronic acidinduced-apoptosis of MCF-7cell lines in vitro as demonstrated by the use of the ERantagonist ICI182780and ER-negative MDA-MB-231cells.
2) Zoledronic acid inhibited the migration and the invasive ability ofMDA-MB-231cells. In addition, zoledronic acid also inhibited the vascularangiogenesis in the tube formation of HUVECs and neovascularization in CAM.However, the inhibitors of BKCachannel (IBTX and TEA) could partly reverse theeffects of zoledronic acid in the migration and invasive ability of MDA-MB-231cells and vascular angiogenesis. These results suggested that activation of BKCachannels paly an important role in the treatment of breast cancer with zoledronicacid.
3) Zoledronic acid inhibited the growth of nude mice xenograft withMDA-MB-231cells. In addition, zoledronic acid also influenced apoptosis and theexpression of caspase-3, VEGF, and E-cadherine. However, IBTX, the inhibitor ofBKCachannel could partly reverse the effects of zoledronic acid in nude micexenograft with MDA-MB-231cells.
Conclusions: Activation of BKCachannel may be a novel molecular pathway, which is involved in the treatment of ER-negative breast cancer with zoledronicacid in vivo and in vitro.
引文
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