MK Involved In Immune Escape Of Gastric By Regulating MICA/B Expression And Evaluation Of Efficacy Of MK-targeted-cancer.

Midkine (MK), a heparin-binding growth factor, is most strongly expressed in midgestation, with the progress of embryonic development, the expression of tissue gradually limitations, and is weakly expressed in the tissues of the lung, colon, stomach, kidney, and spleen, and not at all in the liver. Recently research found that MK expression in tumor tissues was higher than the corresponding adjacent normal tissues. Our previous research by RT-PCR and immunohistochemical analysis showed that compared with the healthy tissues, MK is highly expressed in gastric carcinoma tissues in Chinese patients. Which is related with the clicical stage and the far metastasis and mainly locate in the matrix of tumor cells. The research of MK expression characteristics and mechanism in tumor tissue of cancer patients, have provide new ways for tumor diagnosis and treatment to us.To explore the possibility of MK as a tumor marker, we have immunized Balb/c mice three times with the MK recombinant protein, and then, taked the mice's spleen cells to fuse with the SP2/0 cells. We got three anti-human-MK hybridoma cell lines by screening with HAT and HT, three kinds of monoclonal antibody and those labeled with HRP by these cells. By using hybrid matching method with those antibodies and the HRP labeled's, we established the ELISA detection system to detect the MK concentrations in the 24 cancer patient's serum and in the health's serum. Conclusion showed that this ELISA kits can be used to detect the level of MK in the patient serum and urine of patient in clinical. The current study showed that the roles of MK during the tumor formation, mainly were related to the occurrence of induced tumor cells, promote fibrinolysis and a chemotactic role in promoting tumor angiogenesis, increased drug resistance of tumor cells, have not MK and tumor immunity relevant aspects of the reported escape. In order to explore and study the occurrence of MK in the role of tumor immunity, using DNA microarray analysis of genes associated with the MK expression analysis found that high expression of the MK, the tumor killer cells (mainly NK cells and CD8+T cells) activation ligand MIC A/B high expression, and after RT-PCR, Q-PCR and FCM and ELISA tests to prove that MK expression of tumor cells, the cell surface and soluble MIC A/B expression were increased. After further study found, MK can increased the expression of transcription factor CHOP through the P38 signaling pathway, and then CHOP was combined with AP-1 together to promote the MIC A /B expression. We first found the new regulation mechanism of MIC A/B expression. We concluded that MK promot the MIC A/B expression in the tumor cells through the P38 signaling pathway to make tumor cells to escape from the immunal killing effect of NK cell and T cell killing effect.MK-SCFV-DOX, which is an immunoconjugate of doxorubicin (DOX) and a single-chain Fv fragment against human midkine (MK-SCFV), was used to target chemotherapy to a mouse tumor model expressing high levels of the human MK antigen. The recombinant MK-SCFV expressed in bacteria was purified by metal affinity chromatography and then conjugated to the DOX by using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was MK-SCFV(Dex)1.3(DOX)20. Apoptosis assay showed that the MK-SCFV-DOX had higher cytotoxicity against MK-over-expressed BGC823 cells (BGC823-MK cells) as compared to BGC823-3.1 cells. When given intravenously to tumor-bearing mice implanted with BGC823-MK cells, MK-SCFV-DOX suppressed tumor growth more effectively than free DOX (tumor growth inhibition rate of 51.83% as compared to 40.81%). Histologic analysis of the tumor tissue revealed that the MK-SCFV-DOX promoted more DOX accumulation in tumors and more tumor cell death than did free DOX. These results showed that MK-SCFV-DOX is more effective in the targeted inhibition of tumor growth and that MK-SCFV may be a competent drug-carrier.