Figure 1

PEc is associated with the induction of proliferation in PC-3 cells. Immunohistochemical analysis of IGF-1Ec expression in human prostate biopsies: (A) benign columnar prostate epithelium and prostate cancer (arrows), 100×; (B) benign columnar prostate epithelium and prostate cancer (arrows), 200×; (C) prostate adenocarcinoma and high grade prostatic intraepithelial neoplasia (PIN) 200×; (D) prostate adenocarcinoma and PIN, 400×. Actions of PEc in prostate cancer. (E) Detection of IGF-1Ec expression levels by qRT-PCR in mPC-3 cells (white column) and in PC-3 cells overexpressing the PEc (PC-3PEc) cells (black column). An at least six-fold increase of IGF-1Ec mRNA expression was detected in the PC-3PEc cells compared with the mock PC-3 (mPC-3) cells using GAPDH as a reference gene. (Student t test, p < 0.05, n = 6. Error bars refer to SD). (F) Immunofluorescence analysis of the PEc expression in mPC-3 and the PC-3PEc cells, using a polyclonal rabbit anti-human IGF-1Ec antibody. (G) Cell proliferation analysis using MTT assay. Exogenous PEc administration (17 nmol/L) induced the growth rate of wtPC-3 cells at 24 and 48 h. Increased cellular proliferation also was observed in PC-3PEc cell lines compared with mPC-3 cells, as measured at 24 and 48 h. (Student t test, p < 0.0001, n = 5. Error bars refer to SD). (H) Cell cycle analysis of mPC-3 and PC-3PEc cells using flow cytometry. PC-3PEc cells presented significantly lower distribution in the G1/G0 phase and higher distribution in the S phase as compared with mPC-3 cells (Student t test, p < 0.001, n = 3). RN2: range in gating; FL5: fluorescent light detector 5. (I) Western analysis of ERK1 and ERK2 (ERK1/2) and Akt phosphorylation in mPC-3 and PC-3PEc cells. Exogenous administration of synthetic PEc (17 nmol/L) induced ERK1/2 but not Akt phosphorylation in mPC-3 cells (0, 15, 30 and 60 min). PC-3PEc possessed constantly constitutive ERK1/2 activation without Akt being affected. (J) Exogenous IGF-1 (17 nmol/L) induced Akt and ERK1/2 phosphorylation in mPC-3 cells at 15, 30 and 60 min (Western blot).