| Background and purposeTumstatin, a 244 amino acids domain fragment of typeⅣcollagenα3 chain, possess two active regions. The one consisting of amino acids 74-98 is associated with anti-angiogenic properties and the other one consisting of amino acids 197-215 is associated with anti-tumor activity. However, tumstatin does not show a direct anti-tumor effect because its anti-tumor effect region (197-215aa) is masked with another peptide. In previous study, we have constructed a fusion protein based on the above two tumstatin-derived sequences linked with the human IgG3 upper hinge region, named recombined vascular basement membrane derived multifunctional peptide (rVBMDMP), and preliminarily confirmed its double inhibition effects on human umbilical vein endothelial cells and colon cancer cells. Based on our previous work, the present study will be further systematicly observe inhibition effect of rVBMDMP on the growth of lung cancer cell and analysis possible mechanism so that provide a theoretical basis for the development of new anti-tumor protein drugs.Methods(1) MTS assay was peformed to measure the effect of rVBMDMP on the proliferation of human lung adenocarcinoma (A549) cell lines, large cell lung cancer (H460) cell lines, small cell lung cancer (H446) cell lines, lung squamous cell carcinoma (H520) and human diploid fibroblasts (KMB-17) cell line. Plate cloning method was used to detect inhibitory effect of rVBMDMP on A549 cell. AO and EB duel stain under fluorescence microscope and DNA agarose gel electrophoresis were employed to detect apoptosis induction of rVBMDMP on A549 cells. Scratching assay and transwell method were used to measure the effect of rVBMDMP on the migration and invasion/metastasis abilities of A549 cells in vitro respectively. (2) Functional classification using genechips-cancer pathway finder microarray was employed to identify gene expression change induced by rVBMDMP at the transcriptional level. Then Real-time PCR and weatern bloting technology were used to confirm the results of cDNA microarray. (3) Immunoprecipitation was applied to detect interaction between rVBMDMP and integrinαⅤβ3. The changes of FAK/PI3K signal transduction and the Fas/caspase-8 signaling pathway related molecules expression after rVBMDMP treatment in A549 cells were surveyed by Western blotting, and within nude mice tumor were surveyed by immunohistochemistry. TUNEL assay was adopted to detect apoptosis of tumor tissue in nude mice. Western blotting was used to detect phosphorylation of PI3K/Akt, drug resistance associated protein MRP, anti-apoptotic protein bcl-2 and caspase-3 expression change in A549/DDP cells after rVBMDMP exposure.Results(1) rVBMDMP can inhibit proliferative activity of NSCLC cells in different degrees, in which the strongest inhibition was on the A549. (2) rVBMDMP can promote DNA non-random cut in a time-dependent manner and morphological changes of apoptosis in a time-dependent manner in A549 cells. (3) 10.0μM rVBMDMP can lead to IC50 values of cisplatin reduction in A549 cells from 4.614μg/mL to 1.320μg/mL and that in A549/DDP cells from 31.19μg/mL to 11.82μg/mL. (4) After treatment with10.0μM rVBMDMP, cDNA microarray showed that expression of total of 16 genes including p27 gene, granzyme A, metastasis suppressor gene KISS-1, signal transduction and transcription factor(RAF1, RASA1, SRC), cyclin D1, IntegrinαⅤ,α1,α2,α6 were up-regulated; expression of total of 21 genes including Signal transduction and transcription factors(CTNNB1, JUN, MYC, MAP2K1), apoptosis protease activating factor(APAF1), angiogenesis gene regulation(FGF2, IGF1, THBS1), invasion and metastasis related gene(SERPINB2, SERPINE1, SERPINB5, uPA, uPAR) were down-regulated. Among these genes, the test results of CDC25A, uPA, uPAR, IntegrinαV and KISS1 were consistent with microarray results. (5) rVBMDMP can interact with integrinαⅤ/β3, then inhibits phosphorylation of integrinαV/β3 and downstream of FAK, PI3K and Akt in the A549 cell. Immunohistochemical test results in nude mice xenograft tumor were consistent with the cell detection. (6) In early stage (12h), rVBMDMP can up-regulate the expressions of Fas and activate caspase-8, and down-regulate the expressions of bcl-2 and MRP and activate caspase-3 24h later. Immunohistochemical test and TUNEL results in nude mice xenograft tumor were consistent with the cell detection too.Conclusion(1) rVBMDMP can inhibit the proliferation and abilities of migration and invasion/metastasis in vitro of lung adenocarcinoma A549 cell and directly induce apoptosis. (2) The effects of growth inhibition and apoptosis induction of rVBMDMP on A549 cells may be related to binding to integrinαVβ3 and then inhibition of protein phosphorylation of integrinαVβ3/FAK/PI3K/Akt. (3) Induction of Apoptosis by rVBMDMP may be associated with upregulation of Fas and inhibition of bcl-2. (4) rVBMDMP suppressed invasion and metastasis of human lung cancer cell by upregulating the expression of KISS-1 and downregulating the expression of uPA and uPAR. (5) rVBMDMP can increase the sensitivity of A549 and A549/DDP cells to DDP, which may be related to the above-mentioned mechanism and inhibition of MRP expression. (6) Established a gene expression profiles of rVBMDMP acting on A549 cell, and obtained 37 differentially expressed genes.
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