CTRP3 Stimulates Proliferation and Anti-Apoptosis of Prostate Cells through PKC Signaling Pathways

Abstract

C1q/TNF-related protein-3 (CTRP3) is a novel adipokine with roles in multiple cellular processes. However, little is known about its function in prostate cells. This study investigated the effects and mechanisms of CTRP3 in prostate cells. We first generated and purified CTRP3 protein in HEK 293T cells. Proliferation of RWPE-1 prostate cells was evaluated by MTT analyses under treatment with different concentrations of CTRP3 for various exposure times. The results revealed maximum enhancement of proliferation with 10 μg/mL CTRP3 for 72 h. Cell apoptosis and cell cycle were determined by TUNEL staining and flow cytometry analysis. TUNEL assay showed decreased TUNEL-positive cells in RWPE-1 prostate cells treated with CTRP3, and flow cytometry showed significantly decreased apoptotic cells upon CTRP3 treatment (treated cells, 8.34±1.175 vs. controls, 20.163±0.35) (P < 0.01). Moreover, flow cytometry analysis also showed a significant decrease of cells in the G1 phase and an increase of cells in the S and G2 phase upon CTRP3 treatment (treated cells, 42.85±1.40 vs. control, 52.77±0.90; 28.41±0.57 vs. 23.49±1.13; 27.08±1.97 vs. 22.20±1.32, respectively) (all P < 0.05). Two-dimensional gel electrophoresis and mass spectrometry identified differentially expressed proteins, including cytokeratin-19, GLRX3 and DDAH1, which were upregulated in CTRP3 treated cells, and cytokeratin-17 and 14-3-3 sigma, which were downregulated. GLRX3, DDAH1 and 14-3-3 sigma were confirmed using western blot analysis. A PKC inhibitor, staurosporine, was used to inhibit PKC activity in CTRP3 treated RWPE-1 cells. Staurosporine completely abolished the CTRP3-induced increased phosphorylation of intracellular PKC substrates and CTRP3-stimulated effect by RWPE-1 cells. Our results provide the first evidence for a physiological role of the novel adipokine, CTRP3, in prostate cells. Our findings suggest that CTRP3 could improve proliferation and anti-apoptosis of prostate cells through protein kinase C signaling pathways.

Fig 1. Generation and confirmation of CTRP3.

(A) RT-PCR analysis of the pcDNA3.1 expression construct encoding a C-terminal FLAG-tagged CTRP3. (B) Western blot analysis of HEK 293T cells expressing ectopic CTRP3 protein. (C) Western blot analysis of purified CTRP3 protein. CTRP3 protein expressed in prostate cancer tissue was used as a positive control. Lane 1 and Lane 2 were the results from the same sample.

Fig 2. Growth curve demonstrating the effects of CTRP3 on the proliferation of prostate cells.

Cells were treated with various concentrations of CTRP3: (A) 0 μg/mL, (B) 3 μg/mL, (C) 10 μg/mL, (D) 30 μg/mL. The optimum concentration of CTRP3 for the growth of RWPE-1 was 10 μg/mL.

Fig 3. Effect of CTRP3 on the apoptosis of prostate cells.

(A) TUNEL-positive cells among DAPI-positive cells were decreased in cells treated with CTRP3. TUNEL-positive apoptotic nuclei and DAPI-stained nuclei were visualized at ×200 magnification. (B) Flow cytometry analysis demonstrated a significantly decreased ratio of apoptotic cells in cells treated with CTRP3. Data are representative of three independently performed experiments. Mean±SD. *P < 0.01, as compared with the control group.

Fig 4. 2-dimensional polyacrylamide gel electrophoresis maps of prostate cells in CTRP3 treated cells compared with control cells.

Data are representative of three independent performed experiments.

Fig 5. Cropped 2D gel images and western blot analyses of selected proteins.

(A) 14-3-3 sigma, (B) DDAH1, (C) GLRX3.

Fig 6. Effect of CTRP3 on proliferation and apoptosis was mediated through the PKC signaling pathway.

(A) Phosphorylation of intracellular PKC substrates increased in CTRP3 treated RWPE-1 cells and went back to normal when pretreated with PKC inhibitor. (B) RWPE-1 cells were treated with 10 μg/mL of CTRP3 or staurosporine and then analyzed by a MTT assay. PKC inhibitor staurosporine completely abolished the CTRP3-stimulated proliferation in RWPE-1 cells. (C) RWPE-1 cells were treated with 10 μg/mL of CTRP3 or staurosporine and then analyzed by flow cytometry. PKC inhibitor staurosporine completely abolished the CTRP3-stimulated anti-apoptosis effect. Mean±SD. *P < 0.05, as compared with the control group; #P < 0.05, as compared with the CTRP3-treated group.

Fig 7. Effect of CTRP3 on cell cycle was mediated through the PKC signaling pathway.

RWPE-1 cells were treated with 10 μg/mL of CTRP3 or staurosporine and then analyzed by flow cytometry. The percent of RWPE-1 cells in the G1 phase decreased significantly upon CTRP3 treatment whereas the percentage of cells in the S and G2 phase increased. PKC inhibitor staurosporine completely abolished the CTRP3-stimulated effect. Mean±SD. *P < 0.05, as compared with the control group; #P < 0.05, as compared with the CTRP3-treated group.