The Role Of Calcineurin Involved In Bone Metastasis Of Lung Cancer

Lung cancer is the leading morbidity and mortality of malignance. Distant metastasis of lung cancer cells is the main cause of patient death. Bone is common site in lung cancer patients, and more than 30% patients will happen bone metastasis. Bone metastasis usually leads to pain, fracture, spinal compression, paraplegia and hypercalcinemia. These complications make life quality of patients very poor. Now, clinical treatment effect of bone metastasis still didn't please patients.Therefore, effective therapeutic methods for bone metastasis of lung cancer are urgently needed. Understanding the mechanism of bone metastases is therefore critical for the design of effective strategies to prevent and treat bone metastasis.Calcineurin(CaN) is multifunctional signal enzyme which involves in regulating function of various cells. A novel role for calcineurin (CaN) has been reported recently regarding the oncogenic potential in pancreatic and colorectal cancer. Recently, an emerging role for CaN is provided with bone metastasis potential: it affects tumor cells, osteoclast and osteoblast. It is the interaction of these three kinds of cells leading to bone metastasis. So, CaN may play important roles in bone metastasis of different human malignancies. Our study previously demonstrated that the distribution of CaN was significantly different between SBC-5 cells which can lead to bone metastasis and SBC-3 cells which can not lead to bone metastasis. The SBC-5 cells and SBC-3 cells have similar genetic background but different potential of bone metastasis. Thus, we speculated that CaN might be responsible for bone metastasis in small cell lung cancer.The aim of this study was to investigate the putative causal role CaN could play in the development of lung cancer with bone metastases. This study is a part of subject financed by National Natural Science Foundation of China.The objectives and results of the study are as follows:Part one: CnAαexpression in lung cancer tissues.1. CnAαwas overexpressed in small cell lung cancer tissue with bone metastasis as compared to small cell lung cancer with non-bone metastasis control samples by RT-PCR.2. We performed immunostaining on small cell lung cancer tissue sections. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasm staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis.Part two: CnAαexpression in lung cancer cells and its effects on biological behavior of lung cancer cell in vitro.1. CnAαwas expressed in small cell lung cancer with bone metastasis cell line (SBC-5), but not in small cell lung cancer with non-bone metastasis cell line (SBC-3) by RT-PCR and Western blot.2. In order to further illustrate the function and mechanism of CnAαon bone metastasis, we established two clones, SBC-3/CnAαwhich expressed exogenous CnAαand SBC-3/neo as empty vector (control) by stable transfection.3. MTT assay and soft agar colony formation assay indicate that CnAαenhanced proliferation ability of lung cancer cells.4. Flow cytometry indicated SBC-3/CnAαcells had a relative lower proportion in the G1 phase and a higher proportion in S phase, as compared to SBC-3/neo cells. Results indicated that CnAαincrease cell proliferation ability. Cell apoptosis was detected by flow cytometry. The number of apoptotic cells of SBC-3/CnAαwas significantly lower compared to SBC-3/neo cells5. Cell migration assay indicated that CnAα-transfected cells increased migration compared with control-transfected cells. Cell invasion assay indicated CnAαtransfectants increased invasion compared with control transfectants.Part three: CnAαeffect on lung cancer with bone metastasis was examined by NOD/SCID mice.1. NOD/SCID mice were inoculated with SBC-3/CnAαand SBC-3/neo, 2.0×106 mouse into the tail vein. Four weeks after tumor cell inoculation, the mice were anesthetized by i.p. injection of pentobarbital and X-ray photographs of the mice were taken to determine bone metastasis. Bone metastasis was discovered.In conclusion, CnAαwas overexpressed in lung cancer tissues with bone metastasis as compared to tumors with non-bone metastases. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasmic staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis. In vitro it was demonstrated that the CnAαgene obviously promoted cell proliferation and inhibited cell apoptosis. Transfection with the CnAαgene promoted cell migration and invasion. Animal experiment found bone metastasis by intravenous injection of SBC-3/CnAαcells in a mouse model. The study indicated that CnAαpromoted generation of bone metastasis.