Although transforming growth factor-beta (TGF-beta) has been identified to mainly inhibit cell growth, the correlation of elevated TGF-beta with increasing serum prostate-specific antigen (PSA) ...levels in metastatic stages of prostate cancer has also been well documented.
12-O-tetradecanoylphorbolacetate (TPA) influences proliferation, differentiation, and apoptosis in a variety of cells including prostate cancer cells. Here, we show that androgen treatment ...potentiates TPA-induced apoptosis in androgen-sensitive prostate cancer LNCaP cells but not in androgen-independent prostate cancer cell lines DU145 and PC-3. The use of the antiandrogen bicalutamide (Casodex) rescued LNCaP cells from 5-alpha-dihydrotestosterone (DHT)/TPA-induced apoptosis, suggesting that DHT/TPA-induced apoptosis is mediated by androgen/androgen receptor (AR). In addition, a caspase-3 inhibitor (Ac-DEVD-CHO) reduced the level of apoptosis, suggesting that DHT/TPA-mediated apoptosis occurs through a caspase-3-dependent pathway. A functional reporter assay using nuclear factor (NF) kappaB-luciferase and an electromobility gel shift assay showed that DHT suppressed NFkappaB activity. In addition, apoptosis mediated by combined DHT/TPA treatment was abrogated by overexpression of the NFkappaB subunit p65 in LNCaP-p65 cells, suggesting that NFkappaB may play an important role in regulating the effects of androgen/AR and TPA on apoptosis. Furthermore, use of the c-Jun N-terminal kinase (JNK) inhibitor SB202190 showed that the combination of DHT/TPA increased JNK activation in LNCaP cells but not in LNCaP-p65 cells, demonstrating that NFkappaB may be able to suppress JNK activity. These results indicate that androgen/AR facilitates TPA-induced apoptosis by interruption of the NFkappaB signaling pathway, leading to activation of JNK in LNCaP cells. These data describe a signaling pathway that could potentially be useful in proposed therapeutic treatment strategies exploiting combinations of different agents that control apoptosis in prostate tumors.
The androgen receptor (AR) is a member of the steroid receptor superfamily that binds to the androgen response element to
regulate target gene transcription. AR may need to interact with some ...selected coregulators for maximal or proper androgen
function. Here we report the isolation of a new AR coregulator with a calculated molecular mass of 267 kDa named the androgen
receptor-associated protein 267-α (ARA267-α). ARA267-α contains 2427 amino acids, including one S u(var)3-9, E nhancer-of-zeste, and T rithorax (SET) domain, two L XX LL motifs, three nuclear translocation signal (NLS) sequences, and four plant homodomain (PHD) finger domains. Northern blot
analyses reveal that ARA267-α is expressed predominantly in the lymph node as 13- and 10-kilobase transcripts. HepG2 is the
only cell line tested that does not express ARA267-α. Yeast two-hybrid and glutathione S -transferase pull-down assays show that both the N and C terminus of ARA267-α interact with the AR DNA- and ligand-binding
domains. Unlike other coregulators, such as CBP, which enhance the interaction between the N and C terminus of AR, we found
that ARA267-α had little influence on the interaction between the N and C terminus of AR. Luciferase and chloramphenicol acetyltransferase
assays show that ARA267-α can enhance AR transactivation in a dihydrotestosterone-dependent manner in PC-3 and H1299 cells.
ARA267-α can also enhance AR transactivation with other coregulators, such as ARA24 or PCAF, a histone acetylase, in an additive
manner. Together, our data demonstrate that ARA267-α is a new AR coregulator containing the SET domain with an exceptionally
large molecular mass that can enhance AR transactivation in prostate cancer cells.
Immunocytochemical staining of cultured cells using specific antibodies is a powerful technique to study the expression and subcellular localization of proteins. However, this technique is associated ...with sample-to-sample variations because samples are handled individually and manually. Cell permeation is needed when intracytoplasmic or nuclear proteins are studied. Storage of cultured cells is difficult, and experiments must be repeated if additional studies are desired later, which introduces more variations. We developed a cell culture block array technique that converts cultured cells into a permanently fixed form identical to tissue sections prepared for pathologic examination. Cells from different cultures can be embedded in a single block. Many identical sections, each containing cells from multiple cultures, may be stained with different antibodies using an automated stainer. As a result, sample-to-sample variation is eliminated. Because cells in these blocks are sectioned by knives, all cellular proteins come into direct contact with antibodies, and cell permeation is not needed. Such blocks can be conveniently stored for years without loss of antigens, providing a constant source for future studies. We demonstrated the utility of this technique by studying the proliferation and neuroendocrine differentiation of prostate cancer-derived LNCaP cells cultured in vitro.
It is known that androst-5-ene-3β,17β-diol (Adiol), a precursor of testosterone (T), can activate estrogen target genes. The androgenic activity of Adiol itself, however, is poorly understood. Using ...a transient transfection assay, we here demonstrate in human prostate cancer cells that Adiol can activate androgen receptor (AR) target genes in the presence of AR, and that AR coactivator ARA70 can further enhance this Adiol-induced AR transcriptional activity. In contrast to this finding, an active metabolite of dehydroepiandrosterone, 7-oxo-dehydroepiandrosterone, does not activate AR target gene in the absence or presence of ARA70. Thin layer chromatography analysis reveals that T, dihydrotestosterone, and 17β-estradiol are undetectable in human prostate cancer DU145 cells after treatment with Adiol. Additionally, a proteolysis assay shows that a distinct ligand-receptor conformational difference exists between T-AR and Adiol-AR. Together, the above findings and the fact that T, but not Adiol, can induce transcriptional activity in a mutant AR (mtAR708), suggest that, without being metabolized into T, Adiol itself may represent a natural hormone with androgenic activity in human prostate cancer cells. Because two potent antiandrogens, hydroxyflutamide (Eulexin), and bicalutamide (casodex), that are widely used for the treatment of prostate cancer, fail to block Adiol-mediated induction of AR transcriptional activity in prostate cancer cells, the effectiveness of so-called “total androgen blockage,” a standard treatment for prostate cancer, may need to be reevaluated.
Our earlier report suggested that androst-5-ene-3β,7β-diol (Δ
5
-androstenediol or Adiol) is a natural hormone with androgenic activity and that two potent antiandrogens, hydroxyflutamide (Eulexin) ...and bicalutamide (Casodex), fail to block completely the Adiol-induced androgen receptor (AR) transactivation in prostate cancer cells. Here, we report the development of a reporter assay to screen several selective steroids with anti-Adiol activity. Among 22 derivatives/metabolites of dehydroepiandrosterone, we found 4 steroids no. 4, 1,3,5(10)-estratriene-17α-ethynyl-3,17β-diol; no. 6, 17α-ethynyl-androstene-diol; no. 8, 3β,17β-dihydroxy-androst-5-ene-16-one; and no. 10, 3β-methylcarbonate-androst-5-ene-7,17-dione that have no androgenic activity and could also block the Adiol-induced AR transactivation in prostate cancer PC-3 cells. Interestingly, these compounds, in combination with hydroxyflutamide, further suppressed the Adiol-induced AR transactivation. Reporter assays further showed that these four anti-Adiol steroids have relatively lower glucocorticoid, progesterone, and estrogenic activity. Together, these data suggest some selective steroids might have anti-Adiol activity, which may have potential clinical application in the battle against the androgen-dependent prostate cancer growth.
It is known that androst-5-ene-3beta,17beta-diol (Adiol), a precursor of testosterone (T), can activate estrogen target genes. The androgenic activity of Adiol itself, however, is poorly understood. ...Using a transient transfection assay, we here demonstrate in human prostate cancer cells that Adiol can activate androgen receptor (AR) target genes in the presence of AR, and that AR coactivator ARA70 can further enhance this Adiol-induced AR transcriptional activity. In contrast to this finding, an active metabolite of dehydroepiandrosterone, 7-oxo-dehydroepiandrosterone, does not activate AR target gene in the absence or presence of ARA70. Thin layer chromatography analysis reveals that T, dihydrotestosterone, and 17beta-estradiol are undetectable in human prostate cancer DU145 cells after treatment with Adiol. Additionally, a proteolysis assay shows that a distinct ligand-receptor conformational difference exists between T-AR and Adiol-AR. Together, the above findings and the fact that T, but not Adiol, can induce transcriptional activity in a mutant AR (mtAR708), suggest that, without being metabolized into T, Adiol itself may represent a natural hormone with androgenic activity in human prostate cancer cells. Because two potent antiandrogens, hydroxyflutamide (Eulexin), and bicalutamide (casodex), that are widely used for the treatment of prostate cancer, fail to block Adiol-mediated induction of AR transcriptional activity in prostate cancer cells, the effectiveness of so-called "total androgen blockage," a standard treatment for prostate cancer, may need to be reevaluated.
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