Corticotropin-releasing hormone receptors mediate apoptosis via cytosolic calcium-dependent phospholipase A₂ and migration in prostate cancer cell RM-1

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   Figure 1

   Knockdown of cPLA₂ inhibited apoptosis with CRH or Ucn2 treatment. (a) Detection of transfection efficiency on three sequences of siRNA against cPLA₂ by real-time PCR (left) and western blotting (middle and right). Vector, negative control; siRNA1, cPLA₂-mus-1186; siRNA2, cPLA₂-mus-379; siRNA3, cPLA₂-mus-296. (b) Annexin V–FITC/PI staining of apoptotic cells by fluorescence microscopy at ×100 magnification (upper) and flow cytometry (lower). Early apoptotic cells showed apple green fluorescence, while necrotic and late apoptotic cells had yellow–red cytoplasm and red nuclear staining. (c) Western blotting analysis showed that cPLA₂ inhibition increased the Bcl-2:Bax ratio. si-cPLA₂, cPLA₂-mus-296. Western blotting results were quantified and counted at the right sides. Experiments were performed three times and results of a representative experiment are shown. Data were expressed as mean±s.e.m. of three independent experiments. *P<0.05 vs control; ^&&P<0.01 vs TG; ^#P<0.05 vs CRH; ^##P<0.01 vs CRH; ^$P<0.05 vs Ucn2; and ^@@P<0.01 vs Ucn2+TG.
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   Figure 2

   CRH upregulated and activated cPLA₂ but Ucn2 decreased cPLA₂ expressions. Concentration- and time-dependent increases in cPLA₂ mRNA (a, left two panels) and protein (b, top row) expression induced by CRH. cPLA₂ mRNA (a, right two panels) and protein (b, bottom row) were decreased by Ucn2 in a concentration- and time-dependent manner. (c) Cells were stimulated with 10⁻⁶ M Anta or Anti-30 followed by incubation with 10⁻⁷ M CRH or Ucn2 for 30 min. After 24 h, expression of cPLA₂ was evaluated by western blotting. (d) Time-dependent cPLA₂ phosphorylation of cells exposed to CRH assessed by western blotting. (e) CRH antagonist, Anta, inhibited CRH-induced phosphorylation of cPLA₂. Experiments were performed five times and results of a representative experiment are shown. Data were expressed as mean±s.e.m. of five independent experiments. *P<0.05 vs control/0 min; **P<0.01 vs control/0 min; ***P<0.001 vs 0 min; ^#P<0.05 vs CRH; ^##P<0.01 vs CRH; and ^$P<0.05 vs Ucn2. Anta, antalarmin; Anti-30, antisauvagine-30.
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   Figure 3

   cPLA₂ expression was induced by CRH but suppressed by Ucn2 in HEK293 cells displaying CRHR1 or CRHR2 overexpression. (a) Detection of transfection efficiency by western blotting assay. (b) Effects of CRHR1 or CRHR2 on RNA (left) and protein (middle and right) expression of cPLA₂. Experiments were performed three times and results of a representative experiment are shown. Data were expressed as mean±s.e.m. of three independent experiments. *P<0.05 vs control; **P<0.01 vs control; ***P<0.001 vs control; ^&P<0.05 vs Vector; ^&&P<0.01 vs Vector; ^#P<0.05 vs CRH; and ^$P<0.05 vs Ucn2. R1, CRHR1-N1; R2, CRHR2-N1; and Vector, empty vector.
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   Figure 4

   CRH increased IL1β protein expression and cPLA₂ mRNA stability, but Ucn2 increased both TNFα and IL1β protein expression. (a) Western blotting analysis of TNFα and IL1β under CRH (upper) or Ucn2 (lower) treatment for times ranging from 0 to 48 h. (b) The cells were treated with siRNA against IL1β or TNFα for 24 h and CRH/Ucn2 for another 24 h. Interference efficiency (left) and cPLA₂ expression (right) by transfection and CRH/Ucn2 treatments were examined by western blot. (c) Degradation curve of cPLA₂ mRNA with CRH or Ucn2. Actinomycin D was used to suppress mRNA production. Experiments were performed five times and results for a representative experiment are shown. Data were expressed as mean±s.e.m. of five independent experiments. *P<0.05 vs 0 h/control (Vector) and **P<0.01 vs 0 h.
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   Figure 5

   CRH and Ucn2 induced migration of RM-1 cells via CRHR1 and CRHR2. (a) Transwell chamber assay of stimulatory effect of CRH and Ucn2 on cell migration. Cells were seeded in transwell chambers and then stimulated with CRH/Ucn2 (10⁻⁷ M) alone or along with Anta/Anti-30 (10⁻⁶ M) for 48 h and the number of migrated cells, which were observed using a microscope at ×200 magnification, was determined. (b) Cells were subjected to a wound-healing assay in the absence or presence of CRH/Ucn2 alone or along with Anta/Anti-30. Cultures were photographed at ×100 magnification, right after the scratch and 24 and 48 h later. Wound healing was measured using Image J Software for percentage of wound closure (right). Experiments were performed three times and results of a representative experiment are shown. Data were expressed as mean±s.e.m. of three independent experiments. *P<0.05 vs control; ^#P<0.05 vs CRH; ^##P<0.01 vs CRH; and ^$P<0.05 vs Ucn2. Anta, antalarmin; Anti-30, antisauvagine-30.

• © 2014 Society for Endocrinology