Altered staining patterns and expression level of Engrailed-2 in Benign prostatic hyperplasia and Prostate Cancer predict Prostatic disease progression
Abstract Background: Prostate cancer (PC) as a kind of malignant tumor, causes the most death of cancer among males. Successful curing of PC greatly relies on its diagnose in the early stage. Engrailed-2 (EN2), which has been confirmed being existed in the high level in the urine of PC patients, has not been reported as a histochemical diagnostic biomarker of PC. In this study, we analyzed the EN2 expression level and staining patterns in PC and benign prostatic hyperplasia (BPH) samples for seeing the change of EN2 in PC early stage. Methods: EN2 monoclonal antibody was generated and the specificity of this antibody was validated with different maneuvers. Endogenic and exogenous of EN2 in three PC cell lines (LNCap, PC3, and DU145) was detected by immunofluorescence. The expression level and staining patterns of EN2 in 25 of PC and 25 of BPH tissues were detected by immunohistochemistry. RT-PCR was done for further conforming whether EN2 is overexpressed in PC and BPH tissues. Finally, a relationship of EN2 expression and PC occurrence was obtained by case analysis of PC. Results: The results of WB and immunofluorescence showed the monoclonal antibody of EN2 we made could specifically bind endogenic and ectogenic EN2 protein in three different PC cell lines. Results of immunofluorescence showed the endogenic EN2 was generally expressed in the cytoplasm and ectogenic EN2 has mostly existed in the nucleus. Immunohistochemical staining of EN2 in PC was extremely higher than in BPH confirmed by RT-PCR. The staining areas were mostly nucleus and cytoplasm in BPH tissues but cytomembrane in PC tissues. The expression level of EN2 was positively correlated with the PC clinical stage. Conclusion: The EN2 monoclonal antibody we made could distinguish BPH and PC by immunohistochemistry. The expression level and tissue distribution pattern of EN2 can help with the determination of PC’s progression.