The Intrabody Against Cyclin D1Inhibits Tumor Growth And Metastasis

Tumor is a kind of severe disease that affects human health. Moreover, theinvasion and metastasis of tumor results in the high mortality of patients. Tumor is acell cycle-disease. Cyclin D1, the first synthesized cyclin and regulatory subunit ofcdk4/6, promotes the G1to S transition of the cell cycle and plays a vital role intumorgenesis. The Cyclin D1gene is overexpressed in many human cancers, such asbreast cancer, liver cancer, stomach cancer, and hematopoietic malignancies.Therefore, Cyclin D1gene has been recognized as an oncogene and a potential targetfor cancer therapy.At present, there are several other methods targeting Cyclin D1, such asRNAi, antisense oligonucleotides, but the application of those methods ononcotherapy is limited because they are lack of stability and tumor-specificity.Intracellular antibodies (intrabodies) have high binding specificity and stability andtherefore possesse a great promise and potential in anti-cancer therapy.In our previous study, the eukaryotic expression plasmid with ER-ADκ,coding an endoplasmic reticulum-retained anti-Cyclin D1intrabody was transfectedto MCF-7cells and then the intrabody can be expressed efficiently. Stable expressionof ER-ADκ can significantly inhibit the growth and metastasis of tumor cells and leadto cell cycle arrest and cell apoptosis. Several evidence has confirmed the relationshipbetween Cyclin D1and tumor invision and metastasis. However, the role andmolecular mechanisms of anti-Cyclin D1intrabody mediated tumor invision andmetastasis has not been addressed. This study was designed to explore the role andmechanisms of anti-Cyclin D1intrabody in tumor growth and metastasis by the softagar colony-formation assay, wound healing assay, transwell and western blot. The follows are the results:1. ER-ADκ inhibits the colony formation of MCF-7cellsSoft agar assay was conducted with stable-transfected cell linesMCF-7/pBg-ER-ADκ and the control cells, MCF-7and MCF-7/plpBg. Compared tothe control cells, MCF-7/pBg-ER-ADκ cells show significantly lower number ofcolonies, the rate of colony formation and the size of colonies after Giemsa staining.2. ER-ADκ inhibits the migration of MCF-7cellsWound healing assay was done with stable-transfected cell linesMCF-7/pBg-ER-ADκ and its control cells, MCF-7and MCF-7/plpBg. The width ofthe wound and the number of migrated cells were measured at0h,48h,96h,respectively. The results showed that in contrast to the control cells, the significantlylower cell migrating rate, obviously slower “wound” healing and the reduced woundhealing extent appeared in MCF-7/pBg-ER-ADκ cells. These results demonstrated thatthe expression of ER-ADκ could inhibit the migration of MCF-7cells.3. ER-ADκ inhibits the invasion of MCF-7cells in vitroTranswell assays was carried out with stable-transfected cell linesMCF-7/pBg-ER-ADκ and its control cells, MCF-7and MCF-7/plpBg. As a result,the number of migrated cells of MCF-7/pBg-ER-ADκ had obviously reduced, whichdemonstrated that the expression of ER-ADκ inhibited invasion of MCF-7cell invitro.4. The mechanism of ER-ADκ inhibiting the growth and metastasis ofMCF-7cellsWestern blot was conducted with stable-transfected cell linesMCF-7/pBg-ER-ADκ and its control cells, MCF-7and MCF-7/plpBg to investigatethe change of cell proliferation and metastasis related factors, and western blot forβ-actin was shown as the loading control. The results demonstrate that the level ofβ-actin is uniform, however, due to the expression of ER-ADκ inMCF-7/pBg-ER-ADκ, E-cadherin and p21are up-regulated, COX-2、gp130、CDK4、CDK2and PSTAIR were down-regulated, moreover the phosphorylation of FamilinA (Ser2152) was significantly inhibited. These findings demonstrate that ER-ADκ can inhibit growth and metastasis oftumor cells, moreover, this effect may be related to the up-regulated level ofE-cadherin、p21, the down-regulated level of COX-2，gp130、PSTAIR、CDK4、CDK2and the inhibition of phosphorylation of Familin A.