Differential expression of FAAH mRNA and protein in prostate carcinoma cells
To determine the relative amounts of FAAH mRNA, quantitative RT-PCR was performed on various cell lines to determine the cycle thresholds (bold line, ). Cycle thresholds (Ct) of the prostate carcinoma cell lines were normalized to the housekeeping gene, GADPH (data not shown). Normalized FAAH mRNA expression levels in prostate carcinoma cells were compared to the expression in normal prostate epithelial cells (PrEC) cells (). In comparison to PrEC cells, FAAH mRNA expression was 20-fold higher in LNCaP cells, 1.52-fold higher in DU-145 cells, and 40-fold lower in PC-3 cells ().
Figure 1 Expression of FAAH in normal and prostate carcinoma cells. A) Real time PCR profiles for the amplification of FAAH cDNAs in normal and prostate carcinoma cells. B) Quantitative RT-PCR of FAAH mRNA (normalized to GAPDH) in prostate carcinoma cells expression (more ...)
FAAH protein expression in membrane fractions of PrEC, LNCaP, DU-145, and PC-3 cells was determined by Western immunoblot analysis with a polyclonal anti-FAAH antibody (). PrEC cells expressed relatively low level of FAAH protein (band intensity ratio to β-actin = 0.06). In prostate carcinoma cells, LNCaP cells expressed high level of FAAH protein (ratio = 2.42) while DU-145 and PC-3 cells expressed low level of FAAH protein (ratio = 0.08 and 0.01, respectively). FAAH protein expression correlated well with FAAH mRNA in these cells. FAAH protein was not detected in cytosolic fractions of these cells indicating that FAAH is present only in the membrane fractions. These results indicated differential FAAH expression in normal prostate epithelial cells and the three prostate carcinoma cell lines.
Specific hydrolysis activities in prostate carcinoma cell membranes
Since AEA is known to be hydrolyzed by FAAH only, it was used to demonstrate the enzymatic activity of FAAH. The specific hydrolysis activities for AEA were 32.19, 7.73, 30.70 and 104.88 pmol/min/mg protein by PrEC, PC-3, DU-145, and LNCaP cells, respectively (). These specific FAAH hydrolysis activities correlated with the FAAH protein expression levels (). The specific hydrolysis activities for 2-OG were 96.52, 86.53, 159.20, and 166.02 pmol/min/mg protein by PrEC, PC-3, DU-145, and LNCaP cells, respectively ().
The high specific hydrolysis activities for both AEA and 2-OG in LNCaP cells indicates that FAAH in most likely responsible for the hydrolysis, which was supported by the high levels of FAAH gene and protein expression in LNCaP cells. High specific hydrolysis activity for 2-OG suggests other 2-OG (2-AG) hydrolyzing enzyme(s) are present, particularly in DU-145 cells.
Effects of FAAH siRNA on endocannabinoid hydrolysis and invasion of LNCaP cells
Since LNCaP cells have very high levels of FAAH mRNA and protein expression (), the effects of FAAH siRNA on endocannabinoid hydrolysis and cell invasion were investigated. LNCaP cells were treated with two separated siRNA sequences; siRNA #1-GUGCAGAAGUUACACAGUAUU or siRNA #2-GAAGGGCUGUGUCUAUGGAUU, which both effectively inhibited FAAH protein expression. However, siRNA #1 (40 nM) is presented due to the lower effective concentration needed compared to siRNA #2 (80 nM). LNCaP cells transfected with a FAAH-specific siRNA exhibited an 80% reduction in FAAH protein expression (band intensity ratio to β-actin = 0.29) as compared to non-transfected control or siControl transfected cells (ratio = 1.49 and 1.38, respectively) (). There was approximately a 70% reduction in AEA hydrolysis in FAAH siRNA-transfected LNCaP cell membranes as compared to control and siControl-transfected membranes (). A 30% reduction in 2-OG hydrolysis was observed in these same membrane fractions (), suggesting the presence of other enzyme(s) that contribute to 2-OG hydrolysis. These results demonstrated that FAAH contributes to the hydrolysis of AEA and 2-OG (2-AG) in LNCaP cell membranes.
Figure 2 Effects of siRNA on FAAH expression, hydrolysis of AEA and 2-OG, and invasion of LNCaP cells. A) Western blot depicting immunoreactive bands corresponding to FAAH in control, siControl, and FAAH siRNA in membrane fractions of LNCaP cells. The intensity (more ...)
FAAH siRNA transfection significantly reduced invasion of LNCaP cells (74.9 ± 4.3%) as compared to control (100.0 ± 6.0%) and siControl (94.0 ± 7.7%) (). Exogenously added 2-AG (10 μM) to FAAH siRNA-transfected LNCaP cells further reduced cell invasion to 53.0 ± 2.3% (). This reduction in cell invasion was blocked by SR141716A, a selective CB1 receptor antagonist (500 nM) ().
Interestingly, exogenous AEA (1 μM) reduced invasion of FAAH siRNA-transfected LNCaP cells to 50.8 ± 1.6% (). Again, SR141716A (500 nM) blocked the reduction of cell invasion by AEA (). These results demonstrated that blocking endocannabinoid hydrolysis by FAAH siRNA enhanced the inhibition of cell invasion by AEA and 2-AG and the inhibition was abolished by the selective CB1 receptor antagonist.
Effects of CAY10401, a specific FAAH inhibitor, and FAAH siRNA on AEA hydrolysis and invasion of LNCaP cells
To further demonstrate the role of FAAH in cell invasion, FAAH activity in LNCaP cells was inhibited with both pharmacological inhibitor and FAAH siRNA transfection. LNCaP cells treated with CAY10401, a specific FAAH inhibitor, at 0.1, 1.0, and 10.0 μM reduced cell invasion to 68.4 ± 2.9%, 56.6 ± 1.5%, and 59.6 ± 3.0%, respectively (). CAY10401 (100 nM) inhibited specific AEA hydrolysis activity in LNCaP membranes (71% reduction) compared to vector transfected membranes (). Furthermore, CAY10401 (100 nM) augmented the inhibition of specific AEA hydrolysis activity in FAAH siRNA membranes (90% reduction) of LNCaP cells ().
Figure 3 Effects of a specific FAAH inhibitor and FAAH siRNA on invasion of LNCaP cells. A) Effect of CAY10401 on invasion of LNCaP cells. Values are mean ± S.E.M. (n = 6–12). *, significantly lower than control, p < 0.05. B) Specific hydrolysis (more ...)
FAAH siRNA transfection reduced invasion of LNCaP cells (74.9 ± 4.3%) as compared with vector transfected cells (control). CAY10401 (100 nM) reduced invasion of vector transfected cells to 68.37 ± 1.71%. CAY10401 (100 nM) further reduced invasion of FAAH siRNA transfected LNCaP cells to 50.7 ± 2.86% (). These studies demonstrated that a combined inhibition of FAAH by a specific FAAH inhibitor and FAAH siRNA further reduced endocannabinoid hydrolysis and cell invasion.
Effects of overexpression of FAAH on endocannabinoid hydrolysis and invasion of PC-3 cells
PC-3 cells have extremely low levels of FAAH protein expression; therefore, they were transiently transfected with pCMV6-XL5 vector containing FAAH cDNA to overexpress FAAH. Overexpression of FAAH markedly increased FAAH protein in PC-3 cells (). AEA hydrolysis in the membrane fractions of PC-3 cells overexpressing FAAH increased 5-fold compared to control cells (); however, the hydrolysis of 2-OG increased by about 1.8-fold compared to control PC-3 cell membranes ().
Figure 4 Effects of FAAH overexpression on FAAH protein, hydrolysis of AEA and 2-OG, and cell motility of PC-3 cells. A) Western blot depicting immunoreactive bands corresponding to FAAH and β-actin in PC-3 cell membrane fractions of control, vector control, (more ...)
Overexpression of FAAH in PC-3 cells led to an increase of cell invasion (118.5 ± 4.0%) compared to the vector control (100.0 ± 3.2%) (). The addition of exogenous 2-AG (10 μM) further enhanced cell invasion of PC-3 cells in both vector control (122.1 ± 1.8%) and FAAH overexpressing cells (131.3 ± 3.54%). A similar increase in cell invasion was observed when PC-3 cells overexpressing FAAH were treated with AEA (1 μM). However, AEA (1 μM) reduced invasion of vector transfected PC-3 cells (). The inhibition of cell invasion by AEA is due to the low FAAH expression and AEA hydrolysis activity () in PC-3 cells.
Endogenous AEA concentrations were 166 ± 13, 164 ± 14, and 160 ± 18 (pg/mg protein) in PC-3, DU-145, and LNCaP cells, respectively. These concentrations were relatively low as compared with 2-AG concentrations. Since LNCaP cells have high FAAH expression and AEA hydrolysis, these results may suggest that LNCaP cells have relatively higher AEA synthesis than other cells.
In addition, PC-3 cells overexpressing FAAH exhibited a significant increase in cell migration at 24 hr compared to vector-transfected cells (). These results further support the role of FAAH in the regulation of 2-AG and AEA hydrolysis and motility of prostate carcinoma cells.
FAAH expression in human prostate tissues
FAAH protein expression was determined in commerically available prostate cancer TMAs with different Gleason scores. In the normal stroma and prostate glands, a weak FAAH staining (level of staining of 1) was detected in one of 8 cores. Within 4 of 12 cores of benign prostatic hyperplasia (BPH), cells of the basal layer showed moderate granular cytoplasmic positivity while the luminal cells had no staining (). Well-differentiated prostate cancer tissues (Gleason scores 2–4), FAAH protein staining was detected in 18 of 19 cores with the averaged staining level of 1.94 ± 0.17. In moderately differentiated prostate cancer tissues (Gleason score 5–6), FAAH protein staining was detected in 6 of 8 cores with averaged staining level of 1.50 ± 0.22 (). For poorly differentiated or undifferentiated prostate cancer tissues (Gleason score = 7–10), FAAH protein staining was detected in 85 of 130 cores with an average staining of 1.94 ± 0.09 (). These TMA staining patterns of a limited number of tissue samples suggested that FAAH is expressed in 69% (109 of 157) of prostate cancer tissues while it has a low incidence (12% or 1 of 8 cores) in normal tissues ().
Figure 5 Immunohistochemical analysis of FAAH protein expression. A) A representative photograph of FAAH protein staining in benign prostatic hyperplasia (BPH). B) A representative photograph of FAAH protein staining in moderately differentiated prostate cancer (more ...)