Steroids inversely affect the biosynthesis and secretion of human prostatic acid phosphatase and prostate-specific antigen in the LNCaP cell line

In order to elucidate the mechanism of androgen-regulation of genes expressed only in the prostate gland, the effects of steroid hormones on the biosynthesis and secretion of human prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) were studied in the human prostatic carcinoma cell line, LNCaP. This cell line produces PAP and PSA, both of which were found to be similar to the proteins purified from and located in human prostatic tissue, as shown by Western blot analysis. The synthetic androgen, R1881, regulated the biosynthesis of these two important tumour marker proteins inversely: the amount of PSA released into the medium was increased to 506%±100 of the control levels, while that of PAP was decreased to 26%±3, in 7 days. These effects were dependent on the concentration of the steroid in the growth medium. The androgen-dependent changes observed in the amounts of the secreted proteins were correlated with alterations in their intra-cellular levels. LNCaP cells were found to have very different capacities for secreting PAP and PSA. Whereas the measurable, cellular amounts of PSA and PAP were of similar magnitudes, much larger amounts of PSA than PAP were secreted into the medium. PSA was also found to be more stable than PAP in the culture medium of the LNCaP cells. Other steroids could elicit effects on PAP and PSA biosynthesis similar to those induced by R1881, and the combined effects of effective concentrations of these steroids were undistinguishable from those caused by each one of them separately, suggesting that all these compounds compete for binding to the same modified androgen receptors of the LNCaP cells. Thus, our results confirm the observations of the altered nature of the LNCaP androgen receptors, and demonstrate the ability of these ligands to produce changes in the expression of androgen-dependent prostatic genes. The fact that the changes observed at the protein level were accompanied by increased levels of PSA mRNAs and by decreased levels of PAP mRNA in steroid-treated cells, suggests that one of the targets of androgen and steroid action in the regulation of these genes is at the mRNA level.     

Autologous down-regulation of androgen receptor messenger ribonucleic acid

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.     

Autoregulation of androgen receptor expression in rodent prostate: immunohistochemical and in situ hybridization analysis

Autoregulation of androgen receptor mRNA and protein was investigated by immunohistochemical and in situ hybridization techniques. In both mouse and rat prostate, the epithelial cell nuclei were stained with the monoclonal or polyclonal antibodies raised against human androgen receptor. It was observed that 3 days after castration, nuclear staining of the epithelium was greatly reduced, while androgen treatment restored the staining intensity to a normal level. In situ hybridization using an androgen receptor cDNA fragment as probe demonstrated that the change in androgen receptor mRNA level correlated with the change in antibody staining intensity. These data suggested an up-regulation of androgen receptor expression by androgen.     

The Effects of the Organic Flame‐Retardant 1,2‐Dibromo‐4‐(1,2‐dibromoethyl) Cyclohexane (TBECH) on Androgen Signaling in Human Prostate Cancer Cell Lines

The effects of the organic flame retardant 1,2‐Dibromo‐4‐(1,2‐dibromoethyl) cyclohexane (TBECH) on androgen receptor target gene expression were examined in the human LNCaP prostate cancer cell line. While γ‐/δ‐TBECH alone led to a significant increase in prostate‐specific antigen (PSA) mRNA accumulation, both the α‐/‐TBECH and γ‐/δ‐TBECH mixtures repressed androgen‐inducible PSA mRNA and protein accumulation in human LNCaP cells. Thus, we hypothesize that isomeric mixtures of TBECH may act as partial agonists of the androgen receptor.     

Identification of genes differentially expressed in benign prostatic hyperplasia.

Differences between benign prostatic hyperplasia (BPH) and normal prostate tissue at the level of mRNA expression provide an opportunity to identify candidate genes for this disease. A cDNA subtraction procedure was used to isolate differentially expressed genes in BPH. The subtraction was done by solution hybridization of BPH cDNA against excess normal prostate cDNA. We identified known, EST, and novel genes by sequence and database analysis of the subtracted cDNAs. Several of these cDNAs were used as probes in Northern blotting analysis to confirm over-expression of their corresponding mRNAs in BPH tissues. One highly upregulated sequence of interest shared identity with a known mRNA encoding human NELL2, a protein containing epidermal growth factor-like domains. NELL2 was not previously reported to be expressed in prostate and may code for a novel prostatic growth factor. In situ hybridization analysis of hyperplastic prostate specimens demonstrated that NELL2 mRNA expression is predominantly localized in basal cells of the epithelium. Disease-related changes in the levels of NELL2 may contribute to alterations in epithelial-stromal homeostasis in BPH. (J Histochem Cytochem 49:669-670, 2001)     

Androgen regulation of prostatic binding protein messenger RNA studied by using cloned complementary DNA

The construction of a double-stranded cDNA library using rat prostatic poly(A)RNA and pBR322/kappa 1776 system and the isolation of three prostatic binding protein (PBP) cDNA clones are described. These cDNA clones were characterized and identified by in situ hybridization, mRNA selection-translation and immuno-precipitation as coding for the three subunit components, C1, C2, and C3, of PBP. These clones were used in hybridization experiments with prostatic poly(A)RNA to determine the effect of testosterone on the levels of PBP-mRNA. The results showed that synthesis of these mRNAs varied in response to either androgen withdrawal or replacement. Accumulation of PBP-mRNAs coding for C2 and C3 components occurred 1 hr after androgen administration to castrated rat, whereas the mRNA coding for the C1 component did not appear until 4 hr after androgen replacement. Quantitation of PBP-mRNA sequences in nuclear and polysomal poly(A)RNAs showed that they did not vary coordinately in response to androgen withdrawal. These results indicate differential regulation of PBP genes and suggest possible multiple levels of androgen control of PBP synthesis.     

Differential responsiveness of prostatic acid phosphatase and prostate-specific antigen mRNA to androgen in prostate cancer cells

Androgens regulate the expression of both human prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), two major prostate epithelium-specific differentiation antigens. Due to the important role of these two enzymes as prostate epithelium differentiation markers, we investigated their regulation of expression at the mRNA level in LNCaP human prostate carcinoma cells. Interestingly, phenol red, a pH indicator in the culture medium, promoted cell growth. To eliminate this non-specific effect, a phenol red-free, steroid-reduced medium was utilized. When high-density cells were grown in that medium, 5alpha-dihydrotestosterone (DHT) suppressed PAcP but stimulated PSA. However, tumor promoter phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) functioned as a potent inhibitor of both PAcP and PSA expression. Prolonged treatment with DHT as well as TPA resulted in a similar down-regulation of protein kinase C and cellular PAcP activities. Thus, the levels of PAcP and PSA mRNA are differentially regulated by androgens in LNCaP cells.     

Farnesyl diphosphate synthase is abundantly expressed and regulated by androgen in rat prostatic epithelial cells

Farnesyl diphosphate synthase (FPPS) has been identified as an androgen-response gene in the rat ventral prostate using a highly sensitive PCR-based cDNA subtraction technique. FPPS is an essential enzyme that catalyzes the synthesis of farnesyl diphosphate (FPP), which is required for cholesterol biosynthesis as well as protein prenylation. We have characterized the expression of FPPS in the rat prostate in response to androgen manipulation. Northern blot analysis showed that castration induced a 10-fold down-regulation of FPPS mRNA within 24 h in the ventral prostate and androgen replacement up-regulated FPPS mRNA rapidly in the regressed ventral prostate of a castrated rat. The expression of FPPS was also regulated by androgen in the lateral and dorsal prostate, indicating that FPPS is important to androgen action in all three lobes of the prostate. Western blot analysis showed that FPPS protein level was also regulated by androgen in the prostate. Northern blot analysis of tissue specificity indicated that FPPS was most abundantly expressed in the ventral prostate of a mature rat and was responsive to androgen manipulation in the prostate and seminal vesicles, but not in other tissues. In situ hybridization study showed that FPPS mRNA was localized to the prostatic epithelium. Interestingly, the expression of FPPS was elevated in Dunning rat prostate tumor cell lines. The above findings suggest that FPPS has the potential to play an important role in androgen action and prostate cancer progression.     

Sex steroid hormone metabolism and prostate cancer

The growth and function of the prostate is dependent on androgens. The two predominant androgens are testosterone, which is formed in the testis from androstenedione and 5alpha-dihydrotestosterone, which is formed from testosterone by 5alpha-reductases and is the most active androgen in the prostate. Prostate cancer is one of the most common cancers among men and androgens are involved in controlling the growth of androgen-sensitive malignant prostatic cells. The endocrine therapy used to treat prostate cancer aims to eliminate androgenic activity from the prostatic tissue. Most prostate cancers are initially responsive to androgen withdrawal but become later refractory to the therapy and begin to grow androgen-independently. Using LNCaP prostate cancer cell line we have developed a cell model to study the progression of prostate cancer. In the model androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in the oxidative 17beta-hydroxysteroid dehydrogenase activity was seen whereas the reductive activity seemed to increase. The changes suggest that during transformation estrogen influence is increasing in the cells. This is supported by the cDNA microarray screening results which showed over-expression of several genes up-regulated by estrogens in the LNCaP cells line representing progressive prostate cancer. Since local steroid metabolism controls the bioavailability of active steroid hormones in the prostate, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of the organ.     

Trihydrophobin 1 attenuates androgen signal transduction through promoting androgen receptor degradation

The androgen‐signaling pathway plays critical roles in normal prostate development, benign prostatic hyperplasia, established prostate cancer, and in prostate carcinogenesis. In this study, we report that trihydrophobin 1 (TH1) is a potent negative regulator to attenuate the androgen signal‐transduction cascade through promoting androgen receptor (AR) degradation. TH1 interacts with AR both in vitro and in vivo, decreases the stability of AR, and promotes AR ubiquitination in a ligand‐independent manner. TH1 also associates with AR at the active androgen‐responsive prostate‐specific antigen (PSA) promoter in the nucleus of LNCaP cells. Decrease of endogenous AR protein by TH1 interferes with androgen‐induced luciferase reporter expression and reduces endogenous PSA expression. Taken together, these results indicate that TH1 is a novel regulator to control the duration and magnitude of androgen signal transduction and might be directly involved in androgen‐related developmental, physiological, and pathological processes. J. Cell. Biochem. 109: 1013–1024, 2010. © 2010 Wiley‐Liss, Inc.     

CaT1 expression correlates with tumor grade in prostate cancer

Ca(2+) signaling is important for growth and survival of prostatic carcinoma (PCa) cells. Here we report that the gene for CaT1, a channel protein highly selective for Ca(2+), is expressed at high levels in human PCa and in the LNCaP PCa cell line. CaT1 mRNA levels were elevated in PCa specimens in comparison to benign prostatic hyperplasia (BPH) specimens and positively correlated with Gleason grade in a PCa series. CaT1 mRNA was suppressed by androgen and was induced by a specific androgen receptor antagonist in LNCaP cells, suggesting that the gene is negatively regulated by androgen. These findings are the first to implicate a Ca(2+) channel in PCa progression and suggest that CaT1 may be a novel target for therapy.     

Difference in Protein Expression Profile and Chemotherapy Drugs Response of Different Progression Stages of LNCaP Sublines and Other Human Prostate Cancer Cells

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months after treatment with a median overall survival time of 1-2 years after relapse. LNCaP is a commonly used cell line established from a human lymph node metastatic lesion of prostatic adenocarcinoma. We previously established two relapsed androgen receptor (AR)-rich androgen-independent LNCaP sublines 104-R1 (androgen depleted for 12 months) and 104-R2 cells (androgen depleted for 24 months) from AR-positive androgen-dependent LNCaP 104-S cells. LNCaP 104-R1 and 104-R2 mimics the AR-positive hormone-refractory relapsed tumors in patients receiving androgen ablation therapy. Androgen treatment stimulates proliferation of 104-S cells, but causes growth inhibition and G1 cell cycle arrest in 104-R1 and 104-R2 cells. We investigated the protein expression profile difference between LNCaP 104-S vs. LNCaP 104-R1, 104-R2, PC-3, and DU-145 cells as well as examined the sensitivity of these prostate cancer cells to different chemotherapy drugs and small molecule inhibitors. Compared to 104-S cells, 104-R1 and 104-R2 cells express higher protein levels of AR, PSA, c-Myc, Skp2, BCL-2, P53, p-MDM2 S166, Rb, and p-Rb S807/811. The 104-R1 and 104-R2 cells express higher ratio of p-Akt S473/Akt, p-EGFR/EGFR, and p-Src/Src, but lower ratio of p-ERK/ERK than 104-S cells. PC-3 and DU-145 cells express higher c-Myc, Skp2, Akt, Akt1, and phospho-EGFR but less phospho-Akt and phospho-ERK. Overexpression of Skp2 increased resistance of LNCaP cells to chemotherapy drugs. Paclitaxel, androgen, and inhibitors for PI3K/Akt, EGFR, Src, or Bcl-2 seem to be potential choices for treatment of advanced prostate cancers. Our study provides rationale for targeting Akt, EGFR, Src, Bcl-2, and AR signaling as a treatment for AR-positive relapsed prostate tumors after hormone therapy.     

Overexpression of CXCL10 in human prostate LNCaP cells activates its receptor (CXCR3) expression and inhibits cell proliferation

Chronic or recurrent inflammation plays a role in the development of many types of cancer including prostate cancer. CXCL10 (interferon-gamma inducible protein-10, IP-10) is a small secretory protein of 8.7 kDa. Recently, it has been shown that normal prostate epithelial (PZ-HPV-7) cells produce lower amounts of angiogenic CXC chemokines (GRO-alpha, IL-8) and higher amounts of angiostatic chemokines (CXCL10, CXCL11) as compared to prostate cancer cells (CA-HPV-10 and PC-3). Accordingly, we studied the effects of overexpression of CXCL10 in human prostate cancer LNCaP cells. LNCaP cells were transiently transfected with CXCL10 cDNA in pIRES2-EGFP vector. CXCL10, CXCR3, PSA and G3PDH mRNA levels were determined by semi-quantitative conventional and quantitative real-time RT-PCR and fluorescence-activated cell sorting (FACS). The expression of CXCL10 was markedly enhanced in the transfected cells at mRNA and protein levels in the cells. Overexpression of CXCL10 inhibited cell proliferation of the transfected cells by 30%-40% in serum-limited medium (1% FCS in RPMI1640 medium) and decreased PSA production. CXCR3 expression was significantly induced by the overexpression of CXCL10 as determined by RT-PCR and FACS. These results indicated that CXCL10 inhibited LNCaP cell proliferation and decreased PSA production by up-regulation of CXCR3 receptor. CXCL10 may be potentially useful in the treatment of prostate cancer.