| Prostate cancer is one of the most prevalent malignant tumors in men worldwide. An estimated 192,280 new cases was diagnosed in 2009 in the US, and prostate cancer is expected to account for 25% of new cases in men in 2009. With the development of prostate cancer early detection technology,intensifying of aging population and changing of people,s living habits in China, the incidence rate of prostate cancer has been increasing year by year and prostate cancer has been one of the most common malignancy in men in China,and is second only to bladder cancer and kidney cancer as a cause of cancer death. Chinese people and Chinese government pay more attention to the research of prostate cancer early detection, prevention and treatment. It is well known that surgery, radiotherapy, chemotherapy, endocrine therapy and biologic therapy are the main strategies of prostate cancer therapy. But all of these methods have their own limitation, So it is increasing urgent and great significant to find a more effective and less side effect therapy strategy for prostate cancer patients.ADEPT is a promising therapy strategy, initially put up by Professor Philpott in mid 1980,s, which may overcome the limitation of chemotherapy. In this strategy, chemotherapeutic agents can only be activated in the tumor cells and it can greatly improve the effect of drugs and dramatically decrease the toxicity of chemotherapeutic agents to the normal cells. It needs three prerequisite in this strategy, firstly, antigens only expressed in the tumors cell are used to target enzymes to the tumor site. Secondly, an enzyme-antibody complex is administered and allowed sufficient time to bind to the tumor cells and to be cleared from the circulation. Lastly, a prodrug is administered and selectively activated extracellularly at the tumor site and effectively kill the tumor cells.In our lab, Our research team has been working on the carboxypeptidase A1 system for ADEPT to treat prostate cancer and has achieved some exciting progresses. We have synthesized two prodrugs called MTX-α-phenylalanine and MTX-α-arginine. Both of them were derived from methothexate-α-peptide. The in vitro cell toxicity assay showed that the MTX-α-phenylalanine cleaved theα-carboxyl phenylalanine residue to yield MTX after hydrolysis by carboxypeptidase A1, and the cytotoxicitic activity of MTX is about 100 times as high as that of the prodrug. And in vivo anti-tumor assay, using the prostate cancer xenograft model, also showed a good prospect. The data indicated that the combination of anti-humanγ-seminoprotein monoclonal antibody (E4B7 McAb)-CPA conjugated with MTX-α–Phe resulted in a tumor inhibition rate of more than 85%. Our research team has prepared every peptide of fused proteins such as hCPA1 holoenzyme and the active center. We found that the hCPA1 gene fused with anti-humanγ-seminoprotein single chain fragments VH monoclonal antibody gene has a lower expression as well as less quantity and less activated fused protein because the molecular weight of the fusion is too big. Although the molecular weight of the active center is short, about 25kd, only to half in comparison with the hCPA1 holoenzyme gene, the activity of it also decreases to as half of that in the hCPA1 holoenzyme gene. We also found that there is aβ-sheet in N-terminal and aα-Helix in C-terminal of the active center, both of them might cover the critical site of the active center and result in that substrate binding site of active center cannot be disclose completely. And we speculated that these two domain might dramatically decrease the activity of the active center.To find a optimum enzyme with high activity but low molecular weight, we designed and cloned a active short peptide without one part of N-terminal (65bp) and one part of C-terminal (67bp) of the active center. Then we did the prokaryotic expression of the active center and the short active peptide and also detected their activity. Two novel anticipated protein bands of 25KD and 20KD were abtained by inducetion, Westernblot confirmed that they are the expected proteins. To detection the activity of proteins by H-L-P, MTT and Apoptotic Assay. Results showed that the activity of the short active peptide is almost the same to that of the active center, not stronger as we expected. It may due to denaturation and renaturation of inclusion body. So we used the eukaryotic expression vector to transfect them to PC-3 cells and examined the expression of this two proteins by RT-PCR and immunofluorescence assay. And we detected the activity of them by MTT assay and cell apoptotic assay in the last. The results showed that both of the active center and short active peptide possessed the activity of hydrolysis, moreover, the activity of the short active peptide was higher than that of the active center. And combination with scFv effected its activity of hydrolysis to some extent. We speculated that combination Vk of scFv with enzyme by overlap-PCR might partly effect the structural collapse of them and further decease its activity. To confirm this speculation needs more experiments.Currently, it is still not clear whether the short active peptide of hCPA1 can largely activate the prodrug MTX-α-Phe because of its enhanced permeability and then possess a more powerful ability of killing tumor cells compared to full-length hCPA1. And it will be our further research direction. The short active peptide of hCPA1 we gained in the experiments lays foundation further research on using the anti-humanγ-seminoprotein monoclonal antibody(E4B7McAb)- hCPA fusion protein to treat prostate cancer. |
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