Antitumor Effects Of Tumor-Targeting Intrabody Against CDK4

The encoding genes of cell cycle regulators are often mutated in tumor cells.The mutation will lead to the abnormal expression of cell cycle regulators and induce the production of tumor Because the cell cycle is out of control. Previous study suggested that cyclin D1 and CDK4 play a key role in cancer development among these regulators. Over-amplification and of cyclin D1 and CDK4 overexpression often appear in a variety of malignant tumors. These show that CDK4 is a vital target in cancer research and clinical treatment. Therefore, it is a trend to inhibit the activity of CDK4 protein in breast cancer clinical treatment.Breast cancer is the most common disease in the female population. Recently, the numbers of patients are rising both in developed countries and developing countries. Recent research has shown that CDK4 is over expression in human breast cancer. So CDK4 maybe a helpful target for breast cancer clinical therapy.Intracellular antibodies can specifically be localized in subcellular region and specifically bind with intracellular molecules with introducting subcellular localization signals sequences to the N terminal or C terminal of intrabody gene sequences. Intracellular antibodies have a long half-life and directly combine with the target protein for a long time. These make intracellular antibody attract more people's attention now. In the present study, anti-CDK4 human single-chain antibody was screened from the phage antibody library. Anti-CDK4 intrabody was make by the introduction of nuclear localization signal sequence and the E-tag peptide tags sequence. Then the modified cDNA was cloned into the tumor-specific eukaryotic expression vector plpBg to construct pBg-NLS-AK. And the anti-tumor effects was conducted in vitro and in vivo.1,Expression of NLS-AK in transiently transfected MCF-7 cells RT-PCR results showed that only the MCF-7/pBg-NLS-AK group had the specific band at the weight of 460 bp. Western-blot showed that only the MCF-7/pBg-NLS-AK group had a protein band at the weight of 31 kD.The results suggested that the anti-CDK4 antibody gene NLS-AK was expresseed in MCF-7 cells on mRNA level and protein level. Immunoprecipitation assay showed that NLS-AK was expresseed efficiently in MCF-7 cells and the protein production of NLS-AK could specifically bind with CDK4 molecule in tumor cells. Immunofluorescence assay showed that only the nuclear region of the experiment group glowed bright yellow green fluorescence .The results suggested that the NLS-AK gene was expressed effiently in MCF-7 cells, and the NLS-AK protein could locate in nuclear region. 2,Anti-tumor effects of NLS-AK in vitroRT-PCR and western blot results showed that the stable cell lines were obtained. Immunoprecipitation assay showed that NLS-AK expressed stably in MCF-7 cells could specifically bind with CDK4 molecule in tumor cells. Immunofluorescence assay showed that only the nuclear region of the experiment group glowed bright yellow green fluorescence.The results suggested that the NLS-AK gene was efficiently expressed in MCF-7 cells, and the NLS-AK protein could locate in nuclear region. The stably transfected cells were detected by Flow Cytometry. The cell cycle G1 phase of MCF-7/pBg-NLS-AK was arrested compared with MCF-7 cell. G0-G1 phase cells proportion was up from 40.10±0.72 % to 72.54±2.55 %,while S phase cells proportion reduced from 38.50±2.51 % to 23.60±5.05 %. The apoptosis rate of MCF-7 cell is 4.00±1.19 %, while the apoptosis rate of MCF-7/pBg-NLS-AK is 22.34±2.17 %. There was no significant difference between MCF-7 cells and MCF-7/plpBg cells (P>0.05). MCF-7/pBg-NLS-AK cells showed much more G0-G1 phase cells and apoptosis than the control group (P<0.05).3,The inhibitory effect of NLS-AK in vivoIn order to verify the anti-tumor effects of NLS-AK, the breast tumor mouse model was established. While the volume of tumor reached around 50mm3 at the tenth day, the mice were divided randomly into three groups including of 5 % glucose control group , plpBg control group and pBg-NLS-AK treatment group and were performed treatment. The statistical analysis showed that the mouse tumor volume has no significant difference at the tenth day (P>0.05). The long diameter and short diameter of tumor were measured every two days. After three times injection (the 19th days), the tumor growth of treatment group was significantly inhibited ( P<0.05). After the end of 5 times injection (the 31th days), the mice were sacrificed. The growth rate of tumor from the pBg-NLS-AK treatment group significantly lower than the control groups (P<0.05). Although 5 % glucose control group growed slightly faster than the empty vector control group, there was no significant difference (p> 0.05). And the tumor weight inhibition rate is 74.92 %. The histological assay demonstrated that the treatment group showed large areas of necrosis, dissolved nucleus, nuclear condensation,nuclear fragmentation and hyalinization. These results suggested that anti-CDK4 intracellular antibody NLS-AK can inhibit the growth of breast tumor in vitro and in vivo. It may become an effective approach for tumor gene therapy.