Fig. 1. Docetaxel-induced NO levels were stabilized by sildenafil in prostate cancer (PCa).

A. RT-PCR analyses to determine the relative PDE5 expression levels in human PCa samples (n=41) compared to non-tumor (n=9) samples. The results presented are normalized to GAPDH mRNA levels. B. PDE5 expression levels were determined in the human PCa cell lines used in this study by qRT-PCR and western blot analyses. C. Quantitative RT-PCR and western blot analyses of PDE5 expression in Pten knockout mouse prostate tumor tissue-derived mRNA in comparison with age-matched wild type mouse prostate tissue. D. Quantitative RT-PCR and western blot analyses of PDE5 were performed in Pten knockout mouse prostate-derived cell lines. Western blot analyses of PDE5 expression in mouse cell lines were shown in the upper panel. E. An ELISA was performed to determine the basal cGMP levels in PCa cells. F. For the ELISA, 22Rv1 PCa cells were plated in a 6-well format at a concentration of 5x10⁴ cells/cm², allowed to grow and then treated with increasing concentrations of sildenafil for 30 min. The resulting cells were lysed and analyzed for cGMP levels. For the western blot, a duplicate set of experiments was performed to determine PDE5 expression levels. G. 22Rv1 PCa cells were treated with various concentrations of docetaxel and combination with sildenafil, and cGMP levels were measured. As compared to untreated control (first column), cGMP levels are significantly higher in docetaxel and docetaxel sildenafil combination treatment. H. 22Rv1 PCa cells were treated with docetaxel and the relative NO levels were determined using DAF-FM diacetate. The relative NO levels (fluorescent benzotriazole derivative) in live cells were quantified by a fluorescent plate reader (Left panel). A representative fluorescent image is shown in the right panel. I. qRT-PCR for NOS2 mRNA levels in 22Rv1 PCa cells treated with different concentrations of docetaxel for 72hr. J. 22Rv1 PCa cells were treated with docetaxel and 1400W (NOS2-specific inhibitor) alone or in the combination and analyzed for NO production by a fluorescent plate reader (Left panel) and fluorescent microscope ( Right panel). K. 22Rv1 PCa cells were treated with docetaxel and 1400W (NOS2 inhibitor) alone or in the combination and analyzed for cGMP levels by ELISA. Scale bar: 200 μm. cGMP – cyclic guanosine monophosphate, DAF-FM diacetate – 4-amino-5-methylamino-2’,7’-difluorescein diacetate, DEA – diethylamine NONOate (NO donor, positive control), Doce – docetaxel, GC – guanylate cyclase, NO – nitric oxide, NOS2 – nitric oxide synthase 2. Doce=docetaxel and Sild=sildenafil, and 0.5, 1, and 2 denote the concentration in nM and μM of docetaxel and sildenafil, respectively. Data are expressed as mean±SEM.