Dutasteride, the dual 5alpha-reductase inhibitor, inhibits androgen action and promotes cell death in the LNCaP prostate cancer cell line.

BACKGROUND: Reduction of T to DHT by 5alphaR in the prostate enhances androgenic activity for most targets. Inhibition of 5alphaR activity with finasteride attenuates androgen action in men and animal models. The objective of this study was to compare and contrast the effects of a potent new 5alphaR inhibitor, dutasteride, with finasteride in the LNCaP prostate cancer cell line. METHODS: LNCaP cells were incubated for varying times with T or DHT in steroid-free medium in the absence or presence of increasing doses of dutasteride or finasteride and the effects on 5alphaR activity, PSA accumulation in the medium, and on cell proliferation were determined. Drug effects on apoptosis were investigated using Annexin V staining and a cell death ELISA assay. Effects of the drugs on AR ligand-binding activity and on AR protein levels were determined. RESULTS: Dutasteride inhibited (3)H-T conversion to (3)H-DHT and, as anticipated, inhibited T-induced secretion of PSA and proliferation. However the drug also inhibited DHT-induced PSA secretion and cell proliferation (IC(50) approximately 1 microM). Finasteride also inhibited DHT action but was less potent than dutasteride. Dutasteride competed for binding the LNCaP cell AR with an IC(50) approximately 1.5 microM. High concentrations of dutasteride (10-50 microM), but not finasteride, in steroid-free medium, resulted in enhanced cell death, possibly by apoptosis. This was accompanied by loss of AR protein and decreased AR ligand-binding activity. Occupation of AR by R1881 partly protected against cell death and loss of AR protein. PC-3 prostate cancer cells, which do not contain AR, also were killed by high concentrations of dutasteride, as well as by 50 microM finasteride. CONCLUSIONS: Dutasteride exhibited some inhibitory actions in LNCaP cells possibly related to 5alphaR inhibition but also had antiandrogenic effects at relatively low concentrations and cell death-promoting effects at higher concentrations. Finasteride also was antiandrogenic, but less than dutasteride. The antiandrogenic effects may be mediated by the mutant LNCaP cell AR. Promotion of cell death by dutasteride can be blocked, but only in part, by androgens.

5alpha-reductase type 1 immunostaining is enhanced in some prostate cancers compared with benign prostatic hyperplasia epithelium.

PURPOSE: In the prostate testosterone is converted to the more potent androgen dihydrotestosterone by the enzymes 5alpha-reductase (5alphaR) types 1 (5alphaR1) and 2 (5alphaR2). Since 5alphaR2 is the dominant prostatic enzyme, the 5alphaR2 selective inhibitor finasteride has been widely used to treat benign prostatic hyperplasia (BPH). However, inhibition of both 5alphaR enzymes provides a greater decrease in serum dihydrotestosterone. We developed a specific antibody to 5alphaR1 and assessed expression in BPH and prostate cancer (pCa) tissue. The presence of this isoenzyme in localized prostate cancer would provide a rationale for assessing the efficacy of dual inhibition for prostate cancer prevention. MATERIALS AND METHODS: A polyclonal antibody to 5alphaR1 was developed and validated using 5alphaR1 and 5alphaR2 transfected COS-1 cells. A total of 26 BPH and 53 pCa specimens were assessed for 5alphaR1 protein expression using immunocytochemical methods. Also, 29 BPH and 37 pCa specimens were assayed for 5alphaR1 and 5alphaR2 enzyme activity. RESULTS: Specificity of the 5alphaR1 antibody was confirmed using transfected COS-1 cells. Cells transfected with 5alphaR1 showed specific staining in immunocytochemistry experiments and on Western blotting of cell lysates the expected 24 kDa band was observed. High intensity immunoreactivity for 5alphaR1 was observed in the tumor epithelium of 28% of pCa specimens. No high intensity epithelial staining was observed in BPH specimens. In 19% of pCa and 7% of BPH specimens 5alphaR1 enzyme activity was detected. CONCLUSIONS: The presence of increased 5alphaR1 in some prostatic malignancies suggests that it is worthwhile to investigate the use of a dual 5alphaR inhibitor to prevent or treat early stage prostate cancer.

The utility of tissue transglutaminase as a marker of apoptosis during treatment and progression of prostate cancer.

PURPOSE: To determine the extent of cell proliferation and apoptosis during treatment and progression of prostate cancer and to determine whether staining for tissue transglutaminase is a better histological marker than TUNEL for neoadjuvant androgen ablation treatment of localized prostate cancer. MATERIALS AND METHODS: Immunocytochemistry techniques were used on archival prostate tissue from four groups of men: 14 men with BPH, 18 men with untreated, localized prostate cancer, 21 men with localized prostate cancer who received neoadjuvant hormone therapy prior to prostatectomy and 18 men with metastatic androgen-independent prostate cancer. Cell proliferation was evaluated by staining for the Ki67 nuclear antigen, and apoptosis was evaluated by staining for DNA fragmentation (TUNEL technique) and tissue transglutaminase (tTG). Image analysis was used to quantitate the results. RESULTS: TUNEL staining increased by 37% in localized prostate cancer compared with BPH, with a further increase of 43% seen after neoadjuvant therapy, although variation was such that neither was statistically significant. In androgen-independent cancer, TUNEL staining was decreased compared with neoadjuvant hormone treated cancer (p = 0.02). Staining for tTG was not increased in untreated prostate cancer compared with BPH; however, staining more than doubled after neoadjuvant therapy, compared with untreated prostate cancer (p = 0.04). Staining for tTG was markedly decreased in androgen-independent cancer (p = 0.07 compared with BPH and p = 0.0004 compared with neoadjuvant hormone treated cancer). Ki67 immunoreactivity did not significantly change in localized prostate cancer, either before or after neoadjuvant therapy, compared with BPH, but it more than doubled in androgen-independent prostate cancer (p = 0.07 compared with BPH and p = 0.05 compared with untreated prostate cancer). CONCLUSIONS: This study shows that cell proliferation increases and apoptosis decreases as prostate cancer progresses to androgen independence, and, that of the markers used in this study, tissue transglutaminase most accurately reflects the anticipated effect of neoadjuvant hormone therapy on localized prostate cancer. An assessment of these parameters provides a valuable tool for appraising new prostate cancer therapies.

Androgen-induced regrowth in the castrated rat ventral prostate: role of 5alpha-reductase.

Testosterone (T), the major circulating androgen, must be converted to dihydrotestosterone (DHT) by the enzyme 5alpha-reductase (5alpha-R) to be maximally active in the prostate. The present study was designed to determine the relative potency of T and DHT on regrowth of the involuted prostate and to elucidate the role of 5alpha-R in the growing prostate. To create dose-response curves for intraprostatic T or DHT, rats were castrated for 2 weeks to allow their prostates to fully regress and then given T implants of various sizes in the presence or absence of the 5alpha-R inhibitor, finasteride. Markers for androgen effects on regrowth of the prostate were prostate weight, duct mass (a measure of secretory activity) and DNA content (a measure of cell number). To assess the relative uptake of T and DHT by the prostate, a comparison was made of intraprostatic DHT levels resulting from T and DHT implants. In the prostate, 1.6-1.9 times more T than DHT was required to achieve a half-maximal response for each of the three markers of prostate regrowth. The dose-response curves revealed that thresholds for intraprostatic T and DHT had to be attained before significant growth was observed. The threshold for T was 2- to 3-fold greater than that for DHT. However, at high intraprostatic concentrations, the effects of T mimicked those of DHT. When the relationship between serum T levels and prostate regrowth was considered, 13 times more serum T was required for half-maximal prostate regrowth when its conversion to DHT was blocked by finasteride. This is partly due to decreased androgen accumulation in the prostate when T was the major intraprostatic androgen. Finally, T or DHT implants in the absence of finasteride resulted in similar intraprostatic DHT levels, indicating that uptake of each serum androgen into the prostate was similar. However, to achieve similar levels of DHT or T in serum, much larger DHT pellets were needed, suggesting more rapid metabolism of DHT in tissues other than the prostate. We conclude that the role of 5alpha-R is 2-fold: it converts testosterone into a modestly more potent androgen and enhances prostatic accumulation of androgen. DHT, in principle, could serve equally well as T as the circulating androgen, although the rate of DHT production would have to be considerably higher to counter the apparent rapid clearance from serum. In addition, we hypothesize that T has arisen as the major circulating androgen instead of DHT because it can be aromatized to estradiol, which itself has important roles in male reproductive function and bone physiology.

Insulin-like growth factor binding protein 5 is associated with involution of the ventral prostate in castrated and finasteride-treated rats.

BACKGROUND: Insulin-like growth factor binding protein (IGFBP)-5 has been proposed as a signal for apoptosis in the ovary. To determine the relationship between IGFBP-5 and apoptosis during regression of the androgen-deprived prostate, rats were castrated or treated with the 5alpha-reductase inhibitor finasteride for 4, 9, 14, 21, and 28 days. METHODS: Ventral prostate tissue was immunostained for IGFBP-5, and apoptotic cells were identified by in situ end-labeling of fragmented DNA (TUNEL). To compare the distribution of IGFBP-5 with the distribution of apoptotic cells, mirror-image serial sections of prostate tissues from normal and day 4 finasteride-treated rats were examined. RESULTS: In normal rats, 4+/-1% of prostate epithelial cells stained positively for IGFBP-5, and 0.1+/-0.03% demonstrated DNA fragmentation. IGFBP-5 staining peaked at day 9 with 93 +/-2% and 64+/-13% of epithelial cells staining positively in castrated and finasteride-treated rats, respectively. In contrast, DNA fragmentation peaked at day 4 in tissues from both castrated and finasteride-treated rats with 7+/-1% and 0.7+/-0.3% of epithelial cells, respectively, staining. In the serial sections, TUNEL and IGFBP-5 staining were not usually expressed in the same cells. CONCLUSIONS: Prostatic involution involves both programmed cell death and inhibition of cell growth. Because of the distribution of staining and the delayed expression of IGFBP-5 relative to initiation of apoptosis, we postulate that IGFBP-5 functions as an inhibitor of cell proliferation rather than as a signal for apoptosis.

Relative potency of testosterone and dihydrotestosterone in preventing atrophy and apoptosis in the prostate of the castrated rat.

Although dihydrotestosterone (DHT) is the principal androgen in the prostate, testosterone can also act as an androgen in this tissue. To determine the relative potencies of testosterone and DHT in preventing prostate regression, castrated rats were implanted for 4 d with varying doses of testosterone in the presence or absence of the 5alpha-reductase inhibitor finasteride. In the absence of finasteride, testosterone in the prostate is converted to DHT, creating an intraprostatic DHT dose response. In the presence of finasteride, this conversion is blocked, and an intraprostatic testosterone dose response is achieved. DHT was 2.4 times more potent than testosterone at maintaining normal prostate weight and duct lumen mass, a measure of epithelial cell function. The two androgens were equipotent at preventing DNA fragementation and expression of testosterone-repressed prostate message, two measures of apoptosis (cell death). The intraprostatic testosterone concentration that results from finasteride treatment in rats is sufficient to inhibit apoptosis but will not maintain normal epithelial cell activity. In conclusion, whereas DHT is more potent than testosterone at stimulating prostate epithelial cell function as measured by ductal mass, the two androgens are equipotent at preventing prostate cell death after castration. These results explain why finasteride causes prostate involution in the rat with minimal evidence of prostate cell death.