Proteomic Analysis For The Molecular Mechanism Of Anti-CDK4 Intrabody In Breast Cancer Cells

Breast cancer is one of the common female malignant tumors.It has become the cancer with the highest incidence rate in China,which is a serious threat to women's health.There is still lacking of effective treatment for breast cancer.Cell cycle dysregulation is one of the main causes of malignant proliferation of tumor cells.In cell cycle progression,CDK4 plays a vital role in regulating cell cycle entry and promoting the G1/S phase transition.Studies have shown that CDK4 is overexpressed and excessively activated in a variety of tumor cells and tissues,and has become an important target for tumor therapy.Intrabody can target to the intracellular specific antigen to knockout the phenotype of important intracellular target protein.Therefore,an anti-CDK4 intrabody with good anti-tumor effect was prepared successfully by using intrabody technique in our previous study.However,the underlying molecular mechanism of anti-CDK4 intrabody has not been addressed.Proteomics is the large-scale and systematic study of structures and functions of all proteins in a cell or biological tissue,and their relationships with other molecules.It plays an important role in cancer biomarkers screening and molecular mechanism of drugs.This study was designed to investigate the underlying molecular mechanisms of anti-CDK4 intrabody in breast cancer cells by using proteomics techniques.The results are as follows:Firstly,we cultured breast cancer cells MDA-MB-231/plpBg(MDA-MB-231 transfected stably with empty vector),and MDA-MB-231 transfected stably with expression plasmids encoding 3 different anti-CDK4 intrabody with a nuclear localization signal(NLS)(MDA-MB-231/pBg-NLS-AKl,MDA-MB-231/pBg-NLS-AK2 and MDA-MB-231/pBg-NLS-AK3).Then total proteins of these cells were extracted successfully.Secondly,the extracted proteins were subjedted to two-dimensional electrophoresis(2-DE).The differential expression protein spots from 2-DE gels stained with silver were analyzed by ImageMasterTM 2D Platinum 6.0 software.The results showed that there were 83 differential expression protein spots.29 protein spots with high repeatbility and distict difference were cut from 2-DE gels stained with Coomassie brilliant blue and analyzed by mass spectrometry,24 of which were successfully identified.The results from Gene Ontology analysis of the identified differentially expressed proteins showed that the identified proteins mainly included molecular chaperones,cytoskeletal protein,enzyme modulator,nucleic acid binding protein.The identified proteins are involved in a variety of functions,such as combination,catalytic activity,structural molecule activity,translation regulator activity,transporter activity and so on.On the other hand,these proteins participate in the process of apoptosis,metabolic process,response to stimulus and many other biological processes.Functional diversity of differential expression proteins suggests that the CDK4 protein targeted by anti-CDK4 intrabody takes part in the regulation of multiple biological functions in breast cancer cells.Moreover,majority of the identified differentially expressed proteins are closely related with the occurrence and development of tumors.Finally,we also analyzed the function of identified proteins and then found some of the more important proteins,such as HINT,RhoGDI,CBX3,KHSRP,EIF3F,LGALS1 and so on.HINT and RhoGDI protein levels increased in response to anti-CDK4 intrabody expression,but CBX3,KHSRP,EIF3F and LGALS1 protein levels decreased.These proteins play an important role in the regulation of cell cycle,protein synthesis and transport,cell apoptosis,cell growth and proliferation,metabolism and migration of tumor cells.In summary,according to all the above results,we hypothesize that the anti-CDK4 intrabody with nuclear localization signal inhibited the growth and proliferation of breast cancer cells by blocking CDK4 activity,which partially through increasing protein levels of HINT1 and other tumor suppressors,and decreasing protein levels of CBX3 and other oncoproteins.The further detailed investigations are warranted to elucidate the underlying molecular mechanisms of anti-CDK4 intrabody.This study provides the experimental evidence and a sound basis for anti-tumor clinical treatment of intrabody targeting CDK4.