Inhibition of cytohesin-1 by siRNA leads to reduced IGFR signaling in prostate cancer

To explore how cytohesin-1 (CYTH-1) small interfering RNA (siRNA) influences the insulin-like growth factor receptor (IGFR)associated signal transduction in prostate cancer, we transfected human prostate cancer PC-3 cell lines with liposome-encapsulated CYTH-1 siRNA in serum-free medium and exposed the cells to 100 nM IGF-1. The mRNA and protein levels of the signal molecules involved in the IGFR signaling pathways were determined by real-time PCR and detected by Western blotting. The relative mRNA levels of CYTH-1, c-Myc, cyclinD1, and IGF-1R (CYTH-1 siRNA group vs scrambled siRNA group) were 0.26 vs 0.97, 0.34 vs 1.06, 0.10 vs 0.95, and 0.27 vs 0.41 (P < 0.05 for all), respectively. The relative protein levels of CYTH-1, pIGF-1R, pIRS1, pAkt1, pErk1, c-Myc, and cyclinD1 (CYTH-1 siRNA group vs scrambled siRNA group) were 0.10 vs 1.00 (30 min), 0.10 vs 0.98 (30 min), 0.04 vs 0.50 (30 min), 0.10 vs 1.00 (30 min), 0.10 vs 1.00 (30 min), 0.13 vs 0.85 (5 h), and 0.08 vs 0.80 (7 h), respectively. The tyrosine kinase activity of IGF-1R was associated with CYTH-1. The proliferative activity of PC-3 cells transfected with CYTH-1 siRNA was significantly lower than that of cells transfected with scrambled siRNA at 48 h (40.5 vs 87.6%, P < 0.05) and at 72 h (34.5 vs 93.5%, P < 0.05). In conclusion, the interference of siRNA with cytohesin-1 leads to reduced IGFR signaling in prostate cancer; therefore, CYTH-1 might serve as a new molecular target for the treatment of prostate cancer

Cytohesins (CYTHs) are a group of ADP ribosylation factor (ARF) guanosine triphosphatase guanine nucleotide exchange factors that have recently become known to be important regulators of signal transduction in vertebrate and invertebrate biology.They function to regulate the reassembly of the cytoskeleton and the activation of integrin or the integrin signaling system (8).Hafner et al. (9) used a CYTH analogue, SecinH3, to inhibit CYTH gene expres-sion in human liver cells and in a mouse model.The results showed that the insulin receptor complex-associated CYTHs were required by the insulin signaling system in liver cells and that SecinH3 inhibited CYTH-induced insulin resistance in liver cells.
There is an apparent correlation between IGF and insulin and IGFRs and insulin receptors (IR) share the same signaling pathway.Therefore, CYTHs are likely to play an important role in IGF-1R signal transduction.

Screening of cytohesins
PC-3 cells were harvested and total RNA was extracted by the Trizol method according to manufacturer instructions.cDNA was synthesized according to manufacturer instructions.The CYTH-1, CYTH-2 and CYTH-3 genes were amplified and total protein was extracted.The procedures are described in detail below.

Transfection and detection
PC-3 cells were inoculated into each well of 6-well plates at a concentration of 2 x 10 5 cells/mL in 1 mL DMEM containing 10% calf serum and incubated for 24 h.The medium was then refreshed with 200 µL serum-free medium.Simultaneously, in accordance with manufacturer instructions, compounds for transfection were prepared by mixing 5 µL lipofectamin 2000 and 8 µL 10 nM CYTH-1 siRNA together and diluting in 100 µL serum-free DMEM.The compounds were left to stand at room temperature for 20 min.Transfection was performed by adding the compounds to the PC-3 cells and culturing in serum-free medium for 48 h.
Stimulation was performed with IGF-1 (the final concentration was 100 nM) for 10 min or 30 min, followed by Western blotting to detect the protein expression of CYTH-1, tyrosine phospho-IGF-1R (pIGF-1R), tyrosine phospho-IRS1 (pIRS1), threonine phospho-Akt1 (pAkt1), and threonine-tyrosine phospho-Erk1 (pErk1).Simultaneously, stimulation was performed with IGF-1 for 30 min, 1, 3, 5, 7, or 9 h; real-time PCR and Western blotting were performed to detect the mRNA and protein expression of c-Myc and cyclinD1, respectively.Subsequent cell culturing was performed for 24, 48, or 72 h.Cell proliferation was then detected by the MTT assay.Non-transfected PC-3 cells treated with IGF-1 alone and PC-3 cells treated with both IGF-1 and scrambled siRNA were used as positive controls and negative controls, respectively.

Western blotting
The PC-3 cells were harvested and cell lysis was performed using the eukaryotic cell lysis buffer according to manufacturer instructions, followed by extraction of total protein according to manufacturer instructions.Protein quantity was determined by a BCA method.Using 30 μg for each sample, proteins were separated by 12% SDS-PAGE and were blotted with a wet transfer device (BioRad Laboratories, Inc., China) to a nitrocellulose membrane.The membrane was then immersed in blocking solution containing 10% skim milk in PBS Tween-20 (PBST), followed by shaking for 1 h.After washing three times with Tris-buffered saline Tween-20 (TBST) for 5 min each time, the membrane was immersed in primary antibody diluted 1:1000 in the blocking solution at room temperature and then shaken for 1 h.After washing again, the membrane was incubated in HRP-labeled secondary antibody diluted 1:10,000 in blocking solution at room temperature and then shaken for 1 h.After another rinse, the membrane underwent color development by an ECL method, followed by X-film photography.GAPDH protein was used as an inner control.The gray scale values (total raw density) of blots were measured with the VisionWorksLS analysis software available in the UVP EC3 (600) Imaging System (UVP, LLC, USA).

MTT assay
Each well containing cells was supplemented with 20 μL MTT solution (5 mg/mL), followed by incubation in a CO 2 incubator for 4 h.The supernatant was discarded and each well was supplemented with 100 μL dimethylsulfoxide (DMSO, Sigma, USA).When the purple crystals at the bottom of the well were completely dissolved, the absorbance value was measured with a Thermo Multiskan MK3 microplate reader with a wavelength of λ = 490 nm.Cell viability% = experimental absorbance/normal absorbance x 100%.

Statistical analysis
Data are reported as means ± SD.The statistical software SPSS12.0 was used for statistical analysis.Paired comparisons were performed by the Student t-test.P < 0.05 indicated a statistically significant difference.

Discussion
The IGFR signaling system involves complex molecular phosphorylation and includes many molecules for signal transduction, such as IGF-1R, IRS1, Akt1, and Erk1, which can also influence other functional cellular proteins, such as c-Myc and cyclinD1 (10).In the present study, to explore the activity of CYTH-1 and its association with the IGF-1R signaling system in prostate cancer, we used CYTH-1 siRNA to interfere with CYTH-1 expression in PC-3 cells and used qPCR technology and phosphorylated protein-specific antibodies to detect the corresponding changes in the series of IGF-1R signaling molecules mentioned above.
With IGF-1 exposure, PC-3 cells revealed an increase in CYTH-1 mRNA and protein levels.IGF-1 stimulation also increased phosphorylation of IGR-1R at tyrosine 1161 and then pIGF-1R revealed its tyrosine kinase activity to phosphorylate IRS1 at tyrosine 612.Then, the signals were transduced to Akt1 and Erk1 and there was an increase in phosphorylation for both Akt1 (threonine 308 phosphorylation) and Erk1 (threonine 185 and tyrosine 187 phosphorylation).By contrast, the use of CYTH-1 siRNA caused a decrease in CYTH-1 mRNA and protein levels, as well as a decrease in tyrosine 1161 phosphorylation of IGF-1R.The tyrosine kinase activity of IGF-1R also caused a decrease in tyrosine 612 phosphorylation of IRS1 (13).This finding demonstrated that the tyrosine kinase activity of IGF-1R was associated with CYTH-1.Additionally, Akt1 (threonine 308 phosphorylation) and Erk1 (threonine 185 and tyrosine 187 phosphorylation) were decreased, indicating that the signal amplification and transduction pathways were inhibited in an efficient manner (9,13).Hafner et al. (9) reported that phosphorylation of IGF-1R, IRS1, Akt1, and p44MAPK was significantly reduced in liver cells by Secinh3 inhibiting CYTH-1.c-Myc is related to tumor transformation (14) and cy-clinD1 overexpression can shorten the G1 phase of the cell cycle, reduce cell size and reduce dependence on a mitogen (15).In the present study, the qPCR and Western blotting results showed that both c-Myc and cyclinD1 overexpression were apparently inhibited and the MTT assay results showed that the growth and proliferation of tumor cells were significantly inhibited by CYTH-1 siRNA interference.
In addition, the experimental results showed that the IGFR signaling pathway was not completely suppressed and that a certain degree of cell growth and proliferation remained in the prostate cancer cells.Two possible explana-tions are: 1) only CYTH-1 is inhibited in this study, so that CYTH-2 and CYTH-3 may play a role in tumor promotion even though their expression was weak; 2) some inherent bypass signaling pathways may function to rescue the tumor cells, leading to the incomplete suppression of the IGFR signaling pathway.
In conclusion, CYTH-1 plays an important role in the IGFR signaling pathway in prostate cancer cells and suppression of CYTH-1 can reduce the tumor-promoting role of IGFR signaling.Therefore, CYTH-1 might be a new target for treatment of prostate cancer.

Table 1 .
The gray scale values (total raw density) of the blots indicating the total protein levels of detected proteins.