The Intrabody Against CDK4Inhibits Tumor Cell Growth And Metastasis

Cancer is a significant health problem. Currently, there is no effective treatmentfor cancers because its high invasion and metastasis. A body of research evidenceshows that the cell cycle disorder is closely related to the occurrence of malignanttumors. The activation of cyclin-dependent kinase (CDK) is the control center of cellcycle regulation. As the first activated CDK since cells enter the cell cycle, CDK4promotes cell cycle progression from G1to S phase by forming active kinase complexwith cyclin D1. Overexpression and excessive activation of CDK4has been identifiedin many cancer cells and tissues, which is closely related to tumor occurrence anddevelopment. At present, there are several other methods targeting CDK4, such asRNAi, antisense oligonucleotides. However the application of those methods ononcotherapy is limited because they are lack of stability and tumor-specificity.Intracellular antibodies (intrabodies) have shown a great promise and potential inanti-cancer therapy due to their high binding specificity and stability.A great quantity of investigations have demonstrated that cyclin D1promotestumor invision and metastasis. In our previous study, the eukaryotic expressionplasmid with ER-ADκ coding an endoplasmic reticulum-retained anti-cyclin D1intrabody was transfected to MCF-7cells and then the expression of ER-ADκ caninhibit tumor invision and metastasis. However, the role and molecular mechanismsof CDK4and anti-CDK4intrabody mediating tumor invision and metastasis has notbeen addressed. This study was designed to explore the role of anti-CDK4intrabodyin tumor growth and metastasis.Our previous studies have got an expression plasmid pBg-NLS-AK including anuclear localization signal (NLS) and scFv gene against human CDK4(AK). In order to test the expression and activity of pBg-NLS-AK, triple-negative breast cancerMDA-MB-231cells were transfected with pBg-NLS-AK and plpBg, respectively.Then expression and subcellular distribution of NLS-AK was detected by RT-PCR,Western-blot, and immunofluorescence staining analysis. At the same time, MTTassay was used for cell proliferation analysis. The cell cycle and apoptosis changes ofthe stably transfected cells were detected by Flow Cytometry. Cancer cell migrationand metastasis was detected by wound healing assay and transwell.The results show that stable expression of NLS-AK inMDA-MB-231/pBg-NLS-AK cells induces cell cycle arrest, promotes cellproliferation and inhibits invasion and metastasis of MDA-MB-231/pBg-NLS-AKcells. These data strongly suggested that intracellular anti CDK4scFv may become aneffective approach for triple-negative breast cancer therapy.