Antitumor Effects And The Underlying Molecular Mechanisms Of The Intrabody Against Cyclin D1

Hepatocellular carcinoma(HCC)is one of the most common malignancies and the second leading cause of cancer-related deaths worldwide,which has been a great threat to human health at present.There has been no effective treatment for HCC.Incidence of HCC is increasing in Africa,and South-East Asia,especially in China.Therefore,exploring new comprehensive treatment of HCC will achieve important scientific and social impacts.HCC is mostly caused by chronic hepatitis and hepatocirrhosis,accounting for the chronic hepatitis infection.Accompanying with the development of HCC,cyclin D1 is overexpressed,which promotes the activation of CDK4 and the proliferation of hepatocytes.Abnormally high gene amplification and overexpression of cyclin D1 has been linked to the development and progression of several types of human cancer including HCC.Thus Cyclin D1 has become a potential target for the treatment of cancer.Single-chain fragment variable(scFv)antibody,which is a kind of smaller antibody with unaltered specific affinity to the antigen,take the advantage of small size,low immunogenicity,easy access to cells and easy engineering modification such as intrabody.There is tremendous potential for scFv in cancer prevention and therapy.The intrabody(or intracellular antibody)can be genetically fused with known intracellular protein trafficking signals and thereby be directed to different subcellular compartments,resulting in modulation of the target function by degradation,inhibition,or sequestration.According to our previous study,a single-chain fragment of antibody variable region against cyclin D1(AD9)was prepared by phage antibody library display technique.Then an expression plasmid pBg-ER-ADK habouring an endoplasmic reticulum(ER)-retained scFv gene against human cyclin D1(ER-AD?)was constructed.Our previous studies have shown that the intrabody against cyclin D1 can be express effectively in tumor cells,interact with the endogenous cyclin D1 and inhibit the growth and proliferation of tumor cells while is associated with induction of apoptosis and prevention of metastasis of breast cancer cells.The present study is designed to examine the inhibitory role and the underlying molecular mechanisms of anti-cyclin D1 intrabody in HCC by investigating the interaction model and molecular basis between cyclin D1 and AD9 and the downstream signal transduction mediated by anti-cyclin D1 intrabody.The results as follow:Firstly,to investigate the effects of ER-AD? on the growth and proliferation of hepatocellular carcinoma in vitro and in vivo,cell lines and animal model experiments were conducted.The results showed that ER-AD? can bind effectively to endogenous cyclin D1.The expression of ER-AD? induced cell apoptosis and resulted in cell cycle arrest in G1/S phase in HepG2 cells.Moreover,ER-AD? also significantly suppressed the growth of subcutaneous tumors in nude mice in vivo after the intratumoral injection of pBg-ER-AD?.Secondly,we used the phage peptide library display and computer stimulation to assess the key sites of cyclin D1 interacting with AD9,to reveal the molecular basis of antigen-antibody interaction.Results from phage peptides library display and sequence alignment showed that the epitopes on cyclin D1 were in its N-terminal including the pRB and CDK4 binding motifs.The mimic epitope peptides were homologous to amino acids 29-33,93-96,111-114 of cyclin D1.Truncated mutation assay showed that the epitopes were in its N-terminal before amino acid A120.And these results were verified by computer modeling and docking.Moreover the key amino acids recognized by AD9 were Asn24,Lys33,Lys112,Met113,Lys114 and Glu115.Site-directed mutation assay demonstrated that all mutants were invovled in the interaction between cyclin D1 and AD9 except cyc D E115A.Lys112 was more important for cyclin Dl to bind to AD9.Thus,we supposed that ER-AD? inhibited cell proliferation through blocking the binding between cyclin D1 and CDK4 mediated by Lys112 in cyclin D1.At last,the antigen-antibody interaction analysis in tumor cells verified the key amino acids,and the underlying molecular mechanisms of anti-cyclin Dl intrabody-inhibiting the tumor cell proliferation.The co-immunoprecipitation assay further demonstrated that the expression of ER-AD? in HepG2 cells significantly inhibited binding of cyclin D1 with CDK4 and the recruitment of pRB,and inhibited the phosphorylation of pRB compared with the control group.These data suggest that the ER-AD? may affect cell proliferation by inhibiting the function of CDK4 and pRB.Moreover,expression of ER-AD? decreased mRNA levels of CDK1,CDK4,IL-6,gp130,Bcl-2,c-Met,survivin,but increased mRNA levels of.some anti-proliferation molecules such as p21,p27 and p53 in HepG2 cells and human HCC xenografts in nude mice.In summary,all the above results showed the anti-cyclin D1 intrabody inhibited the growth and proliferation of HCC partially through inhibiting the interaction between cyclin D1 and CDK4,pRB,and further blocking the phosphorylation of pRB to affect the downstream proteins involved in cell growth and proliferation.Our study provides an experimental basis for anti-tumor clinical treatment of intrabody targeting cylin D1.