A Novel Mechanism In The Treatment Of Breast Cancer With Zoledronic Acid: The Activation Of Bk_Ca Channel

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 Ca²⁺-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 Ca²⁺, and phosphorylation. Activation of BKCachannel hyperpolarizes the membrane potential and deactivates thevoltage-dependent Ca²⁺channels （VDCCs）, which leads to a reduction inintracellular Ca²⁺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 Ca²⁺-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 Ca²⁺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 Ca²⁺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.