Fig. 5

Activation of eNOS-NO signaling and its downstream effectors in PCSCs and antiandrogen-resistant prostate cancer cells. a Cell viability assay. DU145-sh-eNOS cells grew at the same proliferation rate as DU145-sh-scramble cells under adherent 2D culture condition, suggesting that knockdown of eNOS induced no significant impact on proliferation of DU145 cells. b–d 3D culture spheroid formation assay performed on DU145 and LNCaP cells. b Knockdown of eNOS by sh-eNOS could significantly suppress the spheroid formation capacity of DU145 and LNCaP cells. c Schematic diagram illustrates the actions of different used selective activator and inhibitors of regulators of the eNOS-NO-sGC-cGMP-PKG signaling pathway. d Treatment with eNOS inhibitors, L-NAME and L-NIO (100 μM, respectively), could significantly suppress the spheroid formation capacity of DU145 cells. e Treatment with an NOS3 enhancer AVE3085 (5 μM) could significantly enhance the spheroid formation capacity of DU145 cells. However, treatments with selective sGC inhibitor ODQ (20 μM) and PKG inhibitor KT5823 (10 μM) could significantly suppress the spheroid formation capacity of DU145 cells. f Measurement of cGMP levels in DU145-eNOS and LNCaP-eNOS-transduced cells by direct immunoassay. Results showed that both DU145-eNOS and LNCaP-eNOS cells contained significant higher cGMP levels than their corresponding parental cells, grown as either adherent 2D culture cells or 3D culture spheroids, and with such increased cGMP levels being suppressed by L-NIO treatment (100 μM). g Cell viability assay performed in LNCaP-BC32 cells. Analysis showed that treatments with inhibitors targeting to eNOS (L-NAME, 100 μM; L-NIO, 200 μM) and sGC (ODQ, 20 μM) could induce more growth inhibition on LNCaP-BC32 cells than LNCaP cells. Results repeated at least three times are expressed as mean ± SD, *P < 0.05, **P < 0.01