A Study On The Effect And Mechanism Of Lung Cancer A549Cells With Zoledronic Acid

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 Ca²⁺concentration and then contribute to the regulation of muscle contraction.In contrast, activation of CaTchannel has been considered to promote cell-proliferation byincreasing intracellular Ca²⁺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 Ca²⁺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 Ca²⁺ 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.