Effect of culture conditions on androgen sensitivity of the human prostatic cancer cell line LNCaP

Several effects of androgens on LNCaP-FGC prostate tumor cells showed a biphasic pattern. Stimulation of growth and inhibition of secretion of prostatic acid phosphatase (PAP) was observed at low androgen concentrations (below 1 nM of the synthetic androgen R1881), and inhibition of growth and stimulation of PAP secretion was observed at higher concentrations. In contrast, prostate specific antigen (PSA) secretion did not show this biphasic response pattern. Comparable effects were found for two sublines of the LNCaP-FGC cells: an early (passage 20, androgen-dependent) and relatively late (passage 70, androgen-sensitive) passage of the cells. Culturing of both sublines in the presence of a high concentration of androgens (10 nM R1881) resulted initially in a decrease in growth rate, but the cells started to proliferate within 3 weeks. These cells became less sensitive to androgens, lost their biphasic response pattern, and showed reduced androgen receptor levels. Three weeks after removal of the excess of androgens, the passage 70 cells regained a biphasic growth response to androgens. Culture in medium without steroids but with EGF resulted in a decrease of both androgen sensitivity and androgen receptor level. In conclusion, rapid changes of the androgen sensitivity and receptor level of the LNCaP cells occurred under the influence of culture conditions. These changes were partly reversible and, therefore, were most likely due to adaptation of the cells. © 1993 Wiley-Liss, Inc.     

Androgen receptor isoforms in human prostatic cancer tissue and LNCaP cell line

Aim: To investigate the androgen receptor (AR) isoform expressions in human prostatic cancer tissue and LNCaP cell line. Methods: With high resolution isoelectric focusing (IEF) method we demonstrated the different expressions of AR isoforms in human prostatic cancer tissues and LNCaP cell line. Results: Data were obtained from three prostatic cancer specimens and the LNCaP cell line. Three types of AR isoforms were detected with pI values at 6.5, 6.0, and 5.3. For the 3 prostatic cancer specimens, 1 sample showed all the three types of AR isoforms, the second specimen expressed at 6.5 and 6.0, and the third failed to show any type of isoforms. The LNCaP cell line expressed all the three AR isoforms. Binding of 3^H-dihydrotestosterone (^3H-DHT) to these three isoforms was inhibited by the addition of 100-fold excess of DHT or testosterone, while not by progesterone, oestradiol and diethylstilboestrol. Conclusion: The expression of AR isoforms is different in different prostate cancer tissues, which may be related to the difference in the effect of anti-androgen therapy in different patients.     

The effect of oxytocin on cell proliferation in the human prostate is modulated by gonadal steroids: implications for benign prostatic hyperplasia and carcinoma of the prostate

BACKGROUND: Oxytocin (OT) is implicated in regulating prostate growth. OT concentrations are increased in benign, and decreased in malignant prostate disease. This study investigated whether the altered concentrations of OT present in prostate disease affect the proliferation of malignant and non-malignant human prostate cells. METHODS: The effects of varying concentrations of OT and gonadal steroids on cell proliferation of non-malignant prostatic epithelial (PrEC) and stromal (PrSC) cells and androgen dependent (LNCaP) and independent (PC-3) malignant cell lines were assessed. RESULTS: OT (>0.5 nmol . L(-1)) had no effect on PrEC proliferation when cells were cultured alone. When co-cultured with PrSC and gonadal steroids, OT inhibited epithelial cell proliferation. OT inhibited PrSC proliferation, when cells were cultured alone. When PrSC were co-cultured in the presence of estrogen physiological concentrations of OT were inhibitory. No effect on cell proliferation was observed with higher concentrations of OT. OT did not affect the proliferation of malignant cell lines in the absence of androgens but, in the presence of testosterone, low concentrations of OT (<1 nmol . L(-1)) stimulated proliferation of PC-3 cells. Disruption of caveolae in the plasma membrane removed the inhibitory effect of OT on PrSC proliferation but did not affect the stimulatory effect of OT on PC-3 cells cultured in the presence of androgens. CONCLUSIONS: Changes in prostatic concentrations of OT that occur with aging and malignant disease may act to facilitate cell proliferation. The localization of the OT receptor within the plasma membrane modulates OT's proliferative response in the prostate.     

Physiological normal levels of androgen inhibit proliferation of prostate cancer cells in vitro

For more than 70 years, it has been believed that a severe reduction of serum androgen levels caused regression of prostate cancer （PCa） and that increasing androgen levels enhanced growth of PCa. However, numerous recent studies have questioned this traditional belief. In our study, LNCaP and MDA PCa 2b PCa cells were treated with various levels of androgens for 10 or 20 days, and the cell growth was measured with crystal violet mitogenic assay. The results indicated that the effect of androgens on the proliferation of PCa cells occurs in a biphasic pattern, with the androgen levels promoting optimal cell growth at approximately 0.23 ng m1-1 for LNCaP cells and between I and 2 ng m1-1 for MDA PCa 2b cells. Both of the optimal androgen levels are within the adult men＇s physiological low range （〈2.4 ng ml-1）. At lower concentrations than the optimal androgen level, increasing androgen concentration promoted the proliferation of PCa cells. However, at the higher concentrations, increasing androgen concentration resulted in a dose-dependent proliferative inhibition. We conclude that physiologically normal levels of androgen inhibit the proliferation of PCa cells in vitro. However, at very low levels androgens are essential for initial growth of PCa cells.     

Establishment and characterization of androgen-independent human prostate cancer LNCaP cell model

BACKGROUND: The acquisition of an androgen-independent phenotype is the most serious issue of prostate cancer treatment. Although several experimental cell models have been reported for studying androgen independence, they have limited applications related to hormone-refractory prostate cancer. To investigate the molecular mechanism of androgen-independent growth of prostate cancer, we established a useful LNCaP cell model that resembles the clinical scenario of hormone-refractory prostate cancer. METHODS: Androgen-sensitive LNCaP parental cells were continuously maintained in a regular cell-culture medium, that is, phenol red-positive RPMI 1640 medium supplemented with 5% fetal bovine serum and 1% glutamine. Upon passage, the androgen responsiveness of those cells decreased, to a level lower than that of parental cells. We examined the growth properties and androgen responsiveness of these different LNCaP cells in vitro and in vivo. Cytogenetic characteristics and expression of androgen receptors (ARs) and prostate-specific antigen (PSA) were determined. RESULTS: Upon continuous passage, the biological behavior of parental C-33 cells (passage number less than 33) was altered. C-81 cells (passage number higher than 81) clearly exhibited more aggressive growth and lower androgen responsiveness than C-33 and C-51 cells (passage number between 35 and 80) in vitro and in vivo. Nevertheless, all these cells expressed a similar level of functional AR protein as well as a similar genetic profile. Moreover, in a steroid-reduced culture condition, C-81 cells secreted a higher level of PSA than C-33 cells. CONCLUSIONS: Our LNCaP cell model closely recapitulates the progression of human prostate cancer from the androgen-responsive to the hormone-refractory state under the androgen nondeprived condition. This cell model may provide the opportunity to understand the molecular mechanisms associated with the acquisition of androgen independence during human prostate cancer progression.     

C16 ceramide accumulates following androgen ablation in LNCaP prostate cancer cells

BACKGROUND: Adenocarcinoma of the prostate is the most frequently diagnosed non-cutaneous cancer and the second leading cause of cancer-related deaths among men in the United States. The most successful therapies to date for this tumor have involved some form of androgen ablation. However, these therapies become ineffective as the tumor evolves to an androgen-insensitive state. Ceramide is a lipid second messenger that has been shown to mediate growth arrest or cell death when added exogenously to prostate cancer cells. As a first step toward understanding the events that lead to the transition of prostate cancer cells to an androgen-independent state, we considered investigating the effect of androgen ablation on endogenous ceramide levels in androgen-sensitive and androgen-insensitive prostate cancer cells. METHODS: To investigate the mechanisms of growth arrest/apoptosis in androgen-sensitive (LNCaP) and insensitive (DU-145, PC-3) cells, we used various methods including nonyl acridine orange (NAO) staining, propidium iodide (PI) staining/cell-cycle analysis, lipid analysis, and Western blotting assays. RESULTS: In this study, we demonstrate that androgen ablation drives G(0)/G(1)-phase cell-cycle arrest followed by progressive apoptosis in vitro, in LNCaP cells. Lipid analysis indicated an increase in C16 ceramide, which was generated via the de novo pathway as revealed by blockade of ceramide synthase by fumonisin B1. The addition of 5alpha-dihydrotestosterone (DHT) or fumonisin B1 rescued LNCaP cells from apoptosis induced by androgen ablation, and decreased levels of intracellular C16 ceramide. Neither apoptosis nor an increase in C16 ceramide was observed in androgen-independent cell lines following androgen ablation.     

Emodin induces apoptosis in human prostate cancer cell LNCaP

AIM: To elucidate effects and mechanisms of emodin in prostate cancer cells. METHODS: Viability of emodin-treated LNCaP cells and PC-3 cells was measured by MTT assay. Following emodin treatments, DNA fragmentation was assayed by agarose gel electrophoresis. Apoptosis rate and the expression of Fas and FasL were assayed by flow cytometric analysis. The mRNA expression levels of androgen receptor (AR), prostate-specific antigen (PSA), p53, p21, Bcl-2, Bax, caspase-3, -8, -9 and Fas were detected by RT-PCR, and the protein expression levels of AR, p53 and p21 were detected by Western blot analysis. RESULTS: In contrast to PC-3, emodin caused a marked increase in apoptosis and a decrease in cell proliferation in LNCaP cells. The expression of AR and PSA was decreased and the expression of p53 and p21 was increased as the emodin concentrations were increased. In the same time, emodin induced apoptosis of LNCaP cells through the upregulation of caspase-3 and -9, as well as the increase of Bax /Bcl-2 ratio. However, it did not involve modulation of Fas or caspase-8 protein expression. CONCLUSION: In prostate cancer cell line, LNCaP, emodin inhibites the proliferation by AR and p53-p21 pathways, and induces apoptosis via the mitochondrial pathway.     

Androgenic and antiandrogenic control on epidermal growth factor,epidermal growth factor receptor,and androgen receptor expression in human prostate cancer cell line LNCaP

Both androgen and antiandrogen treatments enhance the proliferation rate of the hormone-dependent prostate cancer cell line LNCaP, expressing a mutated androgen receptor (AR). We studied the modification of the expression of epidermal growth factor (EGF), of its receptor (EGF-R), and of androgen receptor (AR) in the LNCaP cell line, under basal conditions and during androgen (R1881) and antiandrogen hydroxy-flutamide (OH-FLU) treatment. After prolonged R1881 administration, a marked increase of EGF release was observed, completely blocked by the addition of OH-FLU. The Scatchard plot analysis of EGF-R binding revealed two classes of binding sites with high and low affinity. The administration of OH-FLU alone or combined with R1881 did not modify the affinity constants, while the low-affinity component disappeared after androgen administration. Both androgen and antiandrogen administration led to a significant increase of the EGF-R high-affinity component. AR mRNA and protein levels were downregulated by R1881 treatment. Following OH-FLU administration, AR mRNA was slightly downregulated, and there was not a strict parallelism between AR mRNA levels and AR binding capacity. When combined with R1881, OH-FLU partially counteracted the androgen-induced AR downregulation. Our data show that EGF-R binding capacity is the only parameter constantly raised in cell proliferation with respect to quiescent cells, and highlights the nonunivocal action of OH-FLU on androgen-induced effects.     

Chimeric molecules facilitate the degradation of androgen receptors and repress the growth of LNCaP cells

Post-translational degradation of protein plays an important role in cell life. We employed chimeric molecules （dihydrotestosterone-based proteolysis-targeting chimeric molecule [DHT-PROTAC]） to facilitate androgen receptor （AR） degradation via the ubiquitin-proteasome pathway （UPP） and to investigate the role of AR in cell proliferation and viability in androgen-sensitive prostate cancer cells. Western blot analysis and immunohistochemistry were applied to analyse AR levels in LNCaP cells after DHT-PROTAC treatment. Cell counting and the 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyl tetrazolium bromide （MTT） cell viability assay were used to evaluate cell proliferation and viability after AR elimination in both LNCaP and PC-3 cells. AR was tagged for elimination via the UPP by DHT-PROTAC, and this could be blocked by proteasome inhibitors. Degradation of AR depended on DHT-PROTAC concentration, and either DHT or an ALAPYIP-（arg）8 peptide could compete with DHT-PROTAC. Inhibition of cell proliferation and decreased viability were observed in LNCaP cells, but not in PC-3 or 786-0 cells after DHT-PROTAC treatment. These data indicate that AR elimination is facilitated via the UPP by DHT-PROTAC, and that the growth of LNCaP cells is repressed after AR degradation.     

环巴胺对人前列腺癌LNCaP细胞增殖和凋亡作用及对PCA3基因表达的影响

目的:探讨环巴胺对人前列腺癌LNCaP细胞增殖和凋亡的作用及对PCA3基因表达的影响。方法:不同浓度环巴胺(1、5、10、15μmol/L)干预LNCaP细胞,以只加入RPMI 1640培养液为空白对照组,分别在不同作用时间(24、48、72h),用MTT法检测其对细胞增殖的抑制、流式细胞术观察细胞凋亡率变化、Hoechst染色观察凋亡细胞形态变化、实时荧光定量逆转录聚合酶链反应(FQ-RT-PCR)观察对PCA3基因表达的影响。结果:5、10、15μmol/L环巴胺对LNCaP细胞增殖均有显著抑制作用,与空白对照组相比差异有显著性(P0.01),10μmol/L组于48h达到半数抑制量(IC50);10、15μmol/L组24、48、72hLNCaP细胞的凋亡率分别为37.21%、57.38%、57.98%和21.16%、71.31%、72.90%,与空白对照组相比差异均有显著性(P0.01)。随着环巴胺浓度增大和作用时间的延长,LNCaP细胞凋亡显著增加。PCA3基因的表达随着环巴胺浓度的上升呈现明显的递减趋势,较空白对照组显著降低(P0.01)。浓度为10μmol/L时,不同作用时间PCA3基因的表达均极低。结论:环巴胺浓度为10、15μmol/L作用48、72h时能够明显抑制LNCaP细胞的增殖,诱导细胞凋亡,并显著下调LNCaP细胞PCA3基因的表达。     

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.     

Effects of galectin-3 expression on growth and tumorigenicity of the prostate cancer cell line LNCaP.

BACKGROUND: Galectin-3 is a beta-galactoside-binding vertebrate lectin. In human prostate cancer, galectin-3 expression has been shown to decrease with progression of disease. In the present study, we further investigated the role of galectin-3 in this malignancy by examining the phenotype of galectin-3-transfected prostate cancer cells. METHODS: Stably transfected galectin-3-expressing cell lines were developed from the prostate cancer cell line LNCaP, which does not constitutively express this molecule. Transfected cells lines were analyzed for alterations in morphology and growth rates, and for ability to form tumors in nude mice. RESULTS: Morphologically, when compared to the parental LNCaP cells, the galectin-3 transfectants had broader, flatter cell bodies, shorter and less finely branched dendritic processes, and large nuclei with pronounced and often multiple nucleoli. The galectin-3 lines were found to proliferate at a slower rate in vitro than either the vector control-transfected lines or parental LNCaP. When injected subcutaneously in nude mice, four of six galectin-3 lines formed tumors at a slower rate than control lines. Twenty-four tumors that formed from the transfected cell lines were examined by immunohistochemistry for galectin-3 expression. Only one tumor was found to express galectin-3, suggesting that the transfected cells which formed tumors were those which successfully down-regulated galectin-3 expression. CONCLUSIONS: In contrast to an apparent stimulatory role in some tumor types, galectin-3 is an inhibitory molecule for prostate cancer.