| Prostate cancer, the most common cancer in the male urinary and reproductive system, is difficult to treat after the occurrence of local invasion and distal metastasis. Therefore, elucidation of the novel mechanisms of prostate cancer metastasis and identification of potential targets for the diagnosis and treatment of highly malignant prostate cancers are needed. Moreover, no specific protein associated with metastasis of prostate cancer has been identified. Therefore, in this study, we aimed to identify prostate cancer metastasis-associated membrane proteins. In our previous study, we developed a phage-displayed 7-mer peptide library to screen the target peptides that were specifically bound to PC-3M cells with subtractive panning from normal prostate cells and PC-3 prostate cancer cells. A novel short peptide(B04) was found to have high affinity to highly metastatic PC-3M cells. And we found that B04 can inhibit the proliferation and metastasis of highly metastatic PC-3M cells. Then, ATP synthase beta subunit(ATP5B) was then identified as a binding partner of B04 on the PC-3M cell surface by methods of proteomics. In order to further confirm whether ATP5 B expressed on the cell membrane of the PC-3M cells, and whether B04 play the role of inhibiting invasion of PC-3M cells through specific inhibition of ATP5 B that expression on the membrane of PC-3M cells. And further test whether B04 has effect on the tubule formation of HUVEC.Results: 1. ATP5 B was expressed on the membrane of highly malignant human prostate cancer specimens.Using the method of organization immunofluorescence double stain to test the expression of ATP5 B and E-Cadherin in high malignant prostate cancer tissue(clinical stage IV), E-Cadherin was expression on cellular membrane in epithelial tissue, which helps detect the expression position of ATP5 B. The results showed that ATP5 B was expressed on the membrane and cytoplasm of highly malignant human prostate cancer specimens. 2. The expression and subcellular localization of ATP5 B in high metastasis of prostate cancer cell line PC-3M cells and low metastasis of prostate cancer cell line PC-3.Using the method of cell immunofluorescence technique to test the expression of ATP5 B in PC-3M and PC-3 cells. The results showed that ATP5 B was expressed in the cytoplasm of PC-3M and PC-3 cells, but ATP5 B was only expressed on the membrane of PC-3M cells, which was consistent with the results of immune electron microscopy. Then, we used flow cytometry and Western blot to further confirm the presence of extracellular ATP5 B on PC-3M and PC-3 cells with anti-ATP5 B antibodies. ATP5 B was present on the surface of PC-3M cells, and the percentage of ATP5B-positive cells and the fluorescence intensity of ATP5 B were significantly higher in PC-3M cells than in PC-3 cells(61.68%±3.94% versus 20.10%±1.79%, respectively, for the percentage of ATP5B-positive cells; 53.87±5.14 versus17.46±0.31, respectively, for the fluorescence intensity.), and the difference was statistically significant(P<0.05). The results of Western blot showed that gray relative value of membrane protein ATP5 B and GAPDH in PC-3M and PC-3 cells were 0.428± 0.075 and 0.161 ± 0.065 respectively, and the difference was statistically significant(P<0.05). From the above results, we concluded that ATP5 B was ectopically expressed on the cellular membrane of PC-3M cells, and the expression quantity was obviously higher than PC-3 cells. 3. B04 bound to ATP5 B on PC-3M cells.Using the methods of flow cytometry, real time PCR and Western blot to further study on the combination of short peptide B04 and ectopic ATP5 B. The results showed that incubation of B04 with PC-3M cells reduced the detecting signal of ATP5B(from 61.25%±2.56% to 17.23%±1.13%) by flow cytometry and increased the detecting signal of ATP5 B m RNA(from 1.00±0.03 to 1.34±0.06) by real time PCR, and the difference was statistically significant(P<0.05). Finally, we confirmed the binding of B04 and ATP5 B using competitive binding assays after SDS-PAGE and transblotting of proteins onto PVDF membranes. The results showed that ATP5 B on PC-3M cells were specifically blocked by B04. 4. Reduction of membrane-bound ATP5 B levels inhibited the invasion of PC-3M cells.In transwell assays, we found that the invasive ability of PC-3M cells incubated with B04(7.83±1.31) was significantly decreased compared with that of PC-3M cells alone(45.67±4.32), and the difference was statistically significant(P< 0.05). 5. B04 inhibit ability of HUVEC tubule formation.Using HUVEC tubule formation experiment to test the ability of HUVEC tubule formation after incubating with B04. The result showed that the short peptide B04 could inhibit ability of HUVEC tubule formation.In summary, our results showed that ATP5 B was highly expressed on the membrane of highly metastatic PC-3M cells. High metastasis associated peptide B04 inhibited the invasion of PC-3M cells and HUVEC tubule formation by specific inhibition of ATP5 B that expression on the cell membrane. Therefore, we further indicated that ATP5 B that was highly expressed on the cell membrane of PC-3M cells may promote the high metastasis of prostate cancer. And ATP5 B may be a potential biomarker or therapeutic target in highly metastatic tumors. |
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