Experimental Study In Construction And Bioassay Of The Specific Dual-Function MR Molecular Probe Against HER2 Positive Prostate Cancer Cells

Objective 1. To culture human prostate cancer PC-3 cells and LNCAP cells; to detect the expression of human epidermal growth factor receptor-2(HER2) in the two prostate cancer cells. 2. To construct the recombinant plasmid p QE30-t BID and further express and purify human proapoptosis protein t BID. 3. To construct a novel specific dual-function MR molecular probe t BID-Gold Mag-Anti HER2 with human proapoptosis protein t BID and HER2 antigen combining with Gold Mag nanoparticles; to evaluate the biological activities of the molecular probe targeting and killing prostate cancer cells with HER2 antigen highly expressed and further assess the possibilities of the molecular probe imaging in MRI.Methods 1. Human prostate cancer PC-3 cells and LNCAP cells were cultured in culture-flasks and put in a cell incubator with a temperature of 37 degrees and 5% carbon dioxide. The growth and morphology of the two cells were observed at regular intervals. When in good conditions, cells were handled with molecular probes for flow cytometry to detect the expression of HER2 in the cells membranes. 2. According to the gene sequences of t BID, upstream primer and downstream primer were designed and synthesized and t BID gene fragment was obtained via polymerase chain reaction. By restriction enzyme digestion, ligation and transformation, the new recombinant plasmid p QE30-t BID was finally constructed, which would be transformed into the expression vector E. coli BL21(DE3). IPTG, an inducer, could derive the expression of t BID protein. SDS-PAGE and Western Blot(WB) analysis were used to detect the actual expression of t BID. Finally, the t BID protein was purified via Ni-NTA affinity chromatography and verified by WB analysis. 3. Protein t BID and anti-HER2 antibody were respectively conjugated with Gold Mag nanoparticles based on the characterizations of colloid gold particles to construct a molecular probe t BID-Gold Mag-Anti HER2. 4. The condition of PC-3 cells and LNCAP cells treated with molecular probes was analyzed respectively via flow cytometry and scanning electron microscopy to evaluate the ability of the molecular probes targeting to prostate cancer cells with HER2 antigen highly expressed. In addition, Annexin-V-FITC staining by flow cytometry was used to detect if the molecular probes could induce apoptosis of the HER2 positive prostate cancer cells. 5. The two prostate cancer cells handled with molecular probes were placed in the head coil and prepared to undergo MR examination. The signal intensity of cells in each group was measured and recorded. The measurement data were analyzed by ANOVA to compare the signal intensity between each two groups, P < 0.05 was considered as statistical significance.Results 1. Prostate cancer PC-3 and LNCAP cells were all anchorage-dependent cells. Under a light microscope, the PC-3 cells were observed to be ovoid shaped, while the LNCAP cells were fusiform to rod shaped. In addition, the growth rate of the former was faster than the latter. 2. The t BID gene fragment of about 411 bp was amplified by PCR and recombinant plasmid p QE30-t BID was finally proved to be constructed correctly via bacteria liquid PCR and enzyme digestion. Transformed into the expression vector E. coli BL21(DE3), the p QE30-t BID was induced to express soluble protein t BID in the supernatant and showed the protein band with 15 k D in SDS-PAGE. After the Ni-NTA affinity chromatography, the protein t BID was successfully purified and verified to be correct with a specific protein band of 15 k D. 3. Flow cytometry was used to detect the expression of HER2 in the prostate cancer cells membranes, which showed that PC-3 cells were HER2 positive, while LNCAP cells were HER2 negative. 4. The novel molecular probe t BID-Gold Mag-Anti HER2 was generated with Gold Mag nanoparticles binding with proapoptosis t BID protein and anti-HER2 antibody respectively. The flow cytometry and scanning electron microscopy were used to evaluate the ability of the molecular probes targeting to prostate cancer cells and showed that the molecular probes could specifically target PC-3 cells with HER2 antigen highly expressed, but could not bind with HER2 negative LNCAP cells. In addition, Annexin-V-FITC staining of the two cells showed that the molecular probes could induce apoptosis in HER2 positive PC-3 cells, while had no effect on HER2 negative LNCAP cells. 5. MR images and statistical results showed that the signal intensity of the group with PC-3 cells treated with molecular probes was significantly lower than that of the four remaining groups in T2 WI respectively, and in the four remaining groups, there was no significant statistical difference between each two groups.Conclusion In our study, we have constructed a novel specific molecular probe t BID-Gold Mag-Anti HER2 against HER2 positive prostate cancer cells successfully with human proapoptosis t BID protein and anti-HER2 antibody, respectively. Through a series of experiments, we have verified the function of the molecular probe in killing prostate cancer HER2 positive cells and imaging in MR in vitro, which is promising in specific targeting therapy of malignant tumors including prostate cancer in the future.