Discrepancy Of Lung Cancer Cell Growth In Bone Microenvironment And Inhibitory Activity Of Zoledronate On Bone Metastasis In Nude Mice

Discrepancy of lung cancer cell growth in bone microenvironment Background and Purpose:Disseminated cancer cells may initially require local nutrients and growth factors to thrive and survive in bone marrow before developing into overt metastasis.Bone seems a milieu particularly favorable for the growth,vascularization and survival of lung cancer or prostate cancer cells,but not colon or gastric cancer cells.In order to explore the mechanism how bone stromal factors contribute to the bone tropism,we therefore use serum of bone marrow aspiration(BM) and condition medium from bone stromal cells(BSC) to investigate their effects on lung cancer cells.Materials and Methods:Lung cancer cell lines,A549,H460,gastric cancer cell line SGC-7901 were obtained originally from ATCC.SPC-A1 lung cancer cell line,tongue cancer cell line T8113 and primary bone stromal cells were established in our lab before,and reported in many publications.These cells were cultured in phenored -free DMEM / 10%fetal calf serum.Regular medium(RM),condition medium from fibroblast cell(FC) were chosen as controls.ELISA was utilized to compare the factors potentially related to bone metastasis among BM,BSC,RM,and FC.MTT assay was applied to analyze these different treatments on proliferation of lung cancer cells and controls.Flow cytometry analysis of DNA content was performed to assess the cell cycle phase distribution using propidium iodide fluorescence.Double staining of Anexin V-FITC and propidium iodide(PI) was used to examine apoptosis.Results:Both supematants from BM and BSC significantly increased the proliferation of H460 lung cancer cells,which had widespread skeletal metastatic potential.This activity was not shown in A549 and SPC-A1 lung caner cells,which had low bone metastasis potential according to literal reports and our animal study.BM did not improve the proliferation of tongue cancer cells T8113,which had very low bone metastasis potential,and even inhibited the growth of SGC7901 gastric cancer cells compared with regular medium. Bone stromal cells promoted proliferation of H460 cells by reducing bone early and later apoptosis,but not affecting cell cycle.Some factors literally related to bone metastasis were compared.ELISA showed the levels of TGF-b,HGF,IGF-1,IL-6 and PDGF were identical in BM,BSC,RM and FC.Conclusion:Although a supporting role of bone stromal factors on metastatic breast or prostate cancer cells have been repeatedly proposed,it is largely unknown the mechanism of bone tropism for lung cancer.In our study,we found bone stromal factors favored the growth of cancer cells with bone tropism by increasing proliferation,reducing apoptosis,but not affecting cell cycle.Bone stromal cells should secret some growth factors although not identified by ELISA,which promoted the proliferation of high bone metastasis potential cells.The high through-out assay,2D gel followed by mass spectromics,is currently ongoing to recognize the effective factors.Whether bone stromal cells could influence angionenosis and three-dimension growth of cancer cells will also be clear after our further investigation. The effect of adhesion between lung cancer cells and bone stromal cells using microarrayBackground:Bone metastasis occurs in about 20%of patients and causes pain and detrimental complications including spinal cord compression and pathologic fracture. Circulating tumor cells adhere to extro-cellular matrix,and invade the interstitial space of the target organs,which is essential for development of metastasis.Therefore,it is postulated adhesion effect play an important role for the bone tropism of lung cancer.Materials and methods:Adhesion assay was utilized to compare the adhesion capability difference among different lung cancer cell lines,and high throughout microarray technique was used to evaluate the regulated downstream genes by adhestion effect of H 460 cells to bone stromal cells.Results:31 genes were consistently up-regulated for H460 cells after adhering to the plate coated with the conditional medium from bone stromal cell.These data was confirmed by real-time PCR thereafter.Conclusion:Our study showed adhesion of H460 cells to bone stromal cells can activate some signal transduction pathways,and alter the expression of adhesion associated factors,therefore favoring the growth of cancer cells with high bone metastasis potential. Synergistic inhibitory activity of Zoledronate and Paclitaxel on bone metastasis in nude miceBackground and objective:Bone metastases contribute to burden of disease in patients with NSCLC,zoledronic acid is now suggesting the cytostatic effects on anti-tumour effects.The purpose of this study was to establish a human lung adenocarcinoma cell line(SPC-A-1BM) with high metastatic potential in bone of immunodelificient(SCID) mouse model,and to evaluate the efficacy of zoledronate alone and combined with paclitaxel reducing the tumor-induced bone diseaseMaterials and Methods:The human lung adenocarcinoma cancer cells SPC-A-1BM were inoculated into the cardiac ventricle of 40 SCID mice(NIH-BNX).They were randomized into 4 groups,and were treated with zoledronate alone,or combined with paclitaxel,or paclitaxel alone,or normal sodium,respectively for 5 weeks after 7 days of tumor implantation.Bone metastases was assessed,measurement of total-body nuclei bone scan,X-rays,serum NTx,bone lesions for pathologic evaluation,the incidence of other organ metastasis,weight loss and survival time until 8 weeks.Results:The zoledronate combined with paclitaxel was very effective in reducing NSCLC bone metastatic.Bone nuclei scan,radiographic and histological results revealed that there were more bone metastatic lesions in the control group than that in the treatment group.In the treatment groups,bone nuclei scan and Radiographic imagine proveded the combined group significantly reducing bone metastasis(p=0.048,p=0.036).Histological analysis showed there was a marginal difference(p=0.058) between the combined group and the control.The numbers of bone metastases mice in combined group equaled to that in the paclitaxel group,they were less bone metastases than the control's.Moreover,the bone turnover biomarker NTx level was reduced by inhibiting osteoclast activity in the combined group,there was a significant difference between the control and paclitaxel group,respectively(p=0.017,p=0.022).Combined group inhibited lung matastases (p=0.036).Only the zoledronate combined with paclitaxel significantly prolonged the survival time(p=0.022).Conclusion:The results indicated zoledronate inhibited the osteoclast activity,zoledronate enhance the effects of paclitaxel synergistically,reducing the incident of NSCLC bone metastasis,prolonged survival time.