Androgen-responsive Human Prostate Cancer Cell Research Articles

Abstract 1467: Metabolic reprogramming fuels prostate cancer cells towards enzalutamide resistance

Abstract Androgen deprivation therapy is standard care for advanced-stage prostate cancer. Enzalutamide (Xtandi®), a second-generation antiandrogen is the most clinically prescribed drug that has prolonged survival in men. However, this response is temporary and results in the rapid emergence of resistance. The underlying mechanisms of enzalutamide resistance remain unclear. Emerging evidence suggests that metabolic reprogramming mediates resistance to standard-of-care therapies. To identify the molecular underpinnings of enzalutamide resistance, we performed Next-Gen Sequencing (NGS), analyzing global metabolic changes in enzalutamide-resistant clones utilizing two androgen-responsive human prostate cancer cells and comparing them with parental counterparts. Enzalutamide-resistant LNCaP and C4-2B cells were generated from 20µM enzalutamide exposed for 6 months and maintained in media containing 5µM drug, referred to as LNCaP/C42B ENZU cells. Gene set enrichment analysis of differentially expressed genes (DEGs) demonstrated an overlap between genes of metabolic signaling pathways together with fatty acid signaling, fatty acid β oxidation, drug resistance, bile acid biosynthesis, lipid metabolism, and peroxisome signaling. Interestingly, NGS data of LNCaP-ENZU cells showed a higher number of DEGs genes associated with metabolic reprogramming, compared to C42B-ENZU cells. Analysis of LNCaP-ENZU cells exhibited more than 10 fold higher (log2 fold) expression of XIST, AKT3, ZNF655, IRS4, HOXB3, FBN2, FHL1, GSTP1, VCAN, KIAA0408 genes; whereas ZNF544, KLK2, CSMD1, ZG16B, SPDEF, AR, C1R1, FOLH1, HISTIHIB, and TNPR222 were downregulated (-10 to -12 log2 fold), compared to parental counterpart. The majority of these genes belong to the metabolic regulatory network. Furthermore, genes viz. AKT3, HOXB3, and KIA0048 RALYL and KLK2 exhibited interaction with androgen receptor (AR), and may influence the ligand-binding domain of AR resulting in stimulation of AR/AR targeted genes. Q-PCR data were in agreement with NGS data where the signature genes associated with the metabolic synthesis of fatty acid, lipid and glucose were overexpressed in LNCaP-ENZU cells. In particular, fatty acid synthesis genes viz. ACADL, ACSL4, FHL2, and SLC27A6 were high up to 14.7, 15.8, 6.02 and 17.8 log2 fold value. Expression of solute carrier SLC12A5, SLC13A4, SLC24A4, SLC30A3, SLC35D1, and SLC47A1 genes showed 1195, 5, 20.01, 4.6, 9.5, and 4.3 log2 fold higher expression, which function in increasing the nucleotide-sugar transmembrane transporter activity. Expression of lipid-associated genes viz. ABCB4, CYP2D6, CYP4X1, CYP39A1, and SLC25A17 exhibited 30, 25.6, 25.5, 8.4, and 17.5 log2 fold higher expression. These gene expression correlated with higher-end products of TCA cycle, glucose metabolism, citric acid and lactate production in LNCaP ENZU-resistant cells. Taken together, our findings demonstrate molecular alterations after enzalutamide exposure may provide fuel to the cells through metabolic reprogramming. Clinically, targeting the aforementioned genes associated with metabolic reprogramming may lead to control of the enzalutamide-mediated recurrence of prostate cancer. Citation Format: Shiv S. Verma, Eswar Shankar, Rajnee Kanwal, Ricky Chan, Sanjay Gupta. Metabolic reprogramming fuels prostate cancer cells towards enzalutamide resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1467.

Vitamin D3 regulates the formation and degradation of gap junctions in androgen-responsive human prostate cancer cells.

1α-25(OH)2 vitamin D3 (1-25D), an active hormonal form of Vitamin D3, is a well-known chemopreventive and pro-differentiating agent. It has been shown to inhibit the growth of several prostate cancer cell lines. Gap junctions, formed of proteins called connexins (Cx), are ensembles of cell-cell channels, which permit the exchange of small growth regulatory molecules between adjoining cells. Cell-cell communication mediated by gap junctional channels is an important homeostatic control mechanism for regulating cell growth and differentiation. We have investigated the effect of 1-25D on the formation and degradation of gap junctions in an androgen-responsive prostate cancer cell line, LNCaP, which expresses retrovirally-introduced Cx32. Connexin32 is expressed by the luminal and well-differentiated cells of normal prostate and prostate tumors. Our results document that 1-25D enhances the expression of Cx32 and its subsequent assembly into gap junctions. Our results further show that 1-25D prevents androgen-regulated degradation of Cx32, post-translationally, independent of androgen receptor (AR)-mediated signaling. Finally, our findings document that formation of gap junctions sensitizes Cx32-expressing LNCaP cells to the growth inhibitory effects of 1-25D and alters their morphology. These findings suggest that the growth-inhibitory effects of 1-25D in LNCaP cells may be related to its ability to modulate the assembly of Cx32 into gap junctions.

Indole-3-Carbinol and 3′,3′-Diindolylmethane Modulate Androgen's Effect on C-C Chemokine Ligand 2 and Monocyte Attraction to Prostate Cancer Cells

Inflammation has a role in prostate tumorigenesis. Recruitment of inflammatory monocytes to the tumor site is mediated by C-C chemokine ligand 2 (CCL2) through binding to its receptor CCR2. We hypothesized that androgen could modulate CCL2 expression in hormone-responsive prostate cancer cells and thereby promote recruitment of monocytes. Given the inhibitory effect of broccoli-derived compounds indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) on androgen-dependent pathways, we also reasoned that I3C and DIM could modulate the effect of androgen on CCL2-mediated pathways. Dihydrotestosterone was found to induce a time-dependent (0-72 hours) and concentration-dependent (0-1 nmol/L) increase in CCL2 mRNA levels in androgen-responsive human prostate cancer cells (LNCaP). This increase in CCL2 mRNA corresponded with increased secretion of CCL2 protein. The effect of dihydrotestosterone was mediated through an androgen receptor (AR)-dependent pathway as small inhibitor RNA against AR negated the induction of CCL2. Although dihydrotestosterone also induced TWIST1 mRNA, an epithelial-mesenchymal transition-related factor, and purported inducer of CCL2, blocking its expression with small inhibitor RNA did not inhibit dihydrotestosterone induction of CCL2 mRNA. Moreover, conditioned media from androgen-treated cells promoted human monocyte THP-1 cell migration and this effect was blocked by antibody against CCL-2. Both I3C and DIM inhibited promotional effects of dihydrotestosterone on CCL2 and migration. These results show that androgen may regulate CCL2 and promote inflammatory microenvironment in prostate tumors and that this process can be blocked by broccoli-derived compounds.

Retinoids Regulate the Formation and Degradation of Gap Junctions in Androgen-Responsive Human Prostate Cancer Cells

The retinoids, the natural or synthetic derivatives of Vitamin A (retinol), are essential for the normal development of prostate and have been shown to modulate prostate cancer progression in vivo as well as to modulate growth of several prostate cancer cell lines. 9-cis-retinoic acid and all-trans-retinoic acid are the two most important metabolites of retinol. Gap junctions, formed of proteins called connexins, are ensembles of intercellular channels that permit the exchange of small growth regulatory molecules between adjoining cells. Gap junctional communication is instrumental in the control of cell growth. We examined the effect of 9-cis-retinoic acid and all-trans retinoic acid on the formation and degradation of gap junctions as well as on junctional communication in an androgen-responsive prostate cancer cell line, LNCaP, which expressed retrovirally introduced connexin32, a connexin expressed by the luminal cells and well-differentiated cells of prostate tumors. Our results showed that 9-cis-retinoic acid and all-trans retinoic acid enhanced the assembly of connexin32 into gap junctions. Our results further showed that 9-cis-retinoic acid and all-trans-retinoic acid prevented androgen-regulated degradation of gap junctions, post-translationally, independent of androgen receptor mediated signaling. Finally, our findings showed that formation of gap junctions sensitized connexin32-expressing LNCaP cells to the growth modifying effects of 9-cis-retinoic acid, all-trans-retinoic acid and androgens. Thus, the effects of retinoids and androgens on growth and the formation and degradation of gap junctions and their function might be related to their ability to modulate prostate growth and cancer.

Preferential induction of G1 arrest in androgen-responsive human prostate cancer cells by androgen receptor signaling antagonists DL3 and antiandrogen bicalutamide

The purpose of this study was to further characterize cell growth-inhibitory effects of a recently identified androgen receptor (AR) signaling inhibitor 6-amino-2-[2-(4-tert-butyl-pnenoxy)-ethylsulfanyl]-1H-pyrimidin-4-one (DL3) 5 and antiandrogen bicalutamide (Bic). DL3 was more potent than Bic in induction of G1 arrest and reduction of G1-related cell cycle protein expression in AR-positive LNCaP cells. DL3, but not Bic, moderately inhibited growth of AR-negative PC-3 cells independent of G1 arrest. The data indicated that DL3 inhibit cell growth in both AR-dependent and -independent manners and is potentially a potent therapeutic agent for the management of advanced human prostate cancer.

Androgen-regulated Formation and Degradation of Gap Junctions in Androgen-responsive Human Prostate Cancer Cells

The constituent proteins of gap junctions, called connexins (Cxs), have a short half-life. Despite this, the physiological stimuli that control the assembly of Cxs into gap junctions and their degradation have remained poorly understood. We show here that in androgen-responsive human prostate cancer cells, androgens control the expression level of Cx32-and hence the extent of gap junction formation-post-translationally. In the absence of androgens, a major fraction of Cx32 is degraded presumably by endoplasmic reticulum-associated degradation, whereas in their presence, this fraction is rescued from degradation. We also show that Cx32 and Cx43 degrade by a similar mechanism. Thus, androgens regulate the formation and degradation of gap junctions by rerouting the pool of Cxs, which normally would have been degraded from the early secretory compartment, to the cell surface, and enhancing assembly into gap junctions. Androgens had no significant effect on the formation and degradation of adherens and tight junction-associated proteins. The findings that in a cell culture model that mimics the progression of human prostate cancer, degradation of Cxs, as well as formation of gap junctions, are androgen-dependent strongly implicate an important role of junctional communication in the prostate morphogenesis and oncogenesis.

Unraveling androgen receptor interactomes by an array-based method: Discovery of proto-oncoprotein c-Rel as a negative regulator of androgen receptor

The androgen receptor (AR) plays a key role in the development and function of male reproductive organs. Using a high-throughput transcription factor–transcription factor (TF–TF) interaction array method, we captured the AR interactomes in androgen-responsive LNCaP cells. Several known and unknown partners of AR, including AP-2, Pax 3/5 (BSAP), c-Rel, RREB-1, LIII BP, and NPAS2 were identified. We investigated one unreported AR-associated transcription factor, the proto-oncoprotein c-Rel, in detail. C-Rel belongs to the NF-kB/Rel families and is persistently active in a number of diseases, including cancer. The presence of c-Rel transcript, protein, and its in vitro and in vivo association with AR was determined. Co-localization of c-Rel with AR both in cytoplasm and nucleus was confirmed by indirect immunofluorescence analysis. Chromatin immunoprecipitation data indicated that c-Rel, like AR, is a part of the nucleoprotein complex regulating the androgen-responsive prostate-specific antigen (PSA) promoter. Overexpression of c-Rel downregulated the promoter activity of both PSA and GRE4-TATA-Luc plasmids in LNCaP and COS cells. Analysis of AR and c-Rel protein levels indicated that the promoter downregulation was not due to reciprocal decrease in the amounts of AR or c-Rel. In summary, we have identified several new partners of AR by using the TF–TF array method and have provided the first evidence of a functional role for c-Rel in androgen-responsive human prostate cancer cells.

Identification and characterization of PLZF as a prostatic androgen-responsive gene.

Promyelocytic leukemia zinc finger protein (PLZF) was initially identified by virtue of its fusion with RARalpha as a result of a variant t(11;17) chromosomal translocation that occurs in a small subset of acute promyelocytic leukemia (APL) patients. PLZF has been reported to have pro-apoptotic and anti-proliferative activity both in vivo and in vitro. Using a modified subtractive hybridization, we identified PLZF as an androgen-responsive gene in the rat ventral prostate. Northern blot and Western blot were used to characterize the regulation of PLZF by androgens in LNCaP cells. Stable transfections of PLZF in LNCaP cells were performed to assay the effect of PLZF overexpression on LNCaP cell proliferation. PLZF mRNA was transiently up-regulated by androgens in the regressed ventral prostate of castrated adult rat. PLZF was also up-regulated by androgens, at both mRNA and protein levels, in the androgen-responsive human prostate cancer cell line LNCaP. Androgen induction of PLZF mRNA was not inhibited by protein synthesis inhibitor cycloheximide but inhibited by androgen receptor antagonist bicalutamide, indicating that PLZF is a direct androgen-responsive gene. To study the functions of PLZF in androgen action, LNCaP sublines stably overexpressing PLZF were generated. PLZF overexpression inhibited LNCaP proliferation either in the presence or absence of androgen, which is consistent with the reported anti-proliferative activity of PLZF. The above observations indicate that PLZF is an androgen-responsive gene with anti-proliferative activity in prostate cancer cells.

Gadd45γ is androgen-responsive and growth-inhibitory in prostate cancer cells

In our previous microarray analysis searching for genes differentially regulated by androgens in the rat ventral prostate, we identified GADD45γ (growth arrest and DNA damage inducible, gamma) as one of the genes up-regulated by androgens. GADD45γ was initially identified to be a gene involved in negative growth control and its overexpression induced cycle arrest and apoptosis in vitro. In this study, we showed that GADD45γ was transiently up-regulated by androgens in the androgen-responsive human prostate cancer cell line LNCaP. The GADD45γ up-regulation was blocked by an androgen receptor (AR) antagonist, bicalutamide, suggesting the involvement of the androgen receptor. However, this up-regulation was inhibited by cycloheximide, indicating that GADD45γ induction by androgens requires new protein synthesis. Overexpression of GADD45γ inhibited cell growth of LNCaP and PC3 cells and resulted in dramatic morphological changes in both cell lines, arguing that GADD45γ is likely to participate in the differentiation program induced by androgens in the prostate. The above observations provide evidence that GADD45γ is an androgen-responsive gene with growth-inhibitory activity in human prostate cancer cells.

Transcriptional programs activated by exposure of human prostate cancer cells to androgen.

Androgens are required for both normal prostate development and prostate carcinogenesis. We used DNA microarrays, representing approximately 18,000 genes, to examine the temporal program of gene expression following treatment of the human prostate cancer cell line LNCaP with a synthetic androgen. We observed statistically significant changes in levels of transcripts of more than 500 genes. Many of these genes were previously reported androgen targets, but most were not previously known to be regulated by androgens. The androgen-induced expression programs in three additional androgen-responsive human prostate cancer cell lines, and in four androgen-independent subclones derived from LNCaP, shared many features with those observed in LNCaP, but some differences were observed. A remarkable fraction of the genes induced by androgen appeared to be related to production of seminal fluid and these genes included many with roles in protein folding, trafficking, and secretion. Prostate cancer cell lines retain features of androgen responsiveness that reflect normal prostatic physiology. These results provide a broad view of the effect of androgen signaling on the transcriptional program in these cancer cells, and a foundation for further studies of androgen action.

Potential involvement of mt1 receptor and attenuated sex steroid-induced calcium influx in the direct anti-proliferative action of melatonin on androgen-responsive LNCaP human prostate cancer cells.

Melatonin, a pineal secretory product, has been shown to exert a direct anti-proliferative action on the androgen-sensitive LNCaP prostate cancer cell line through hitherto undefined mechanisms. In this communication, expression of mt1 melatonin receptor protein in human prostate cancer tissues and LNCaP cells was demonstrated by immunohisto(cyto)chemistry and western blotting, hence supporting the use of LNCaP cell line as a model for the study of melatonin signaling in prostate cancer cell growth. Using 3H-thymidine incorporation assay, LNCaP cell proliferation was inhibited by 2-iodomelatonin, a high-affinity melatonin receptor agonist. Furthermore, melatonin inhibited 3H-thymidine incorporation into LNCaP cells and attenuated 5alpha-dihydrotestosterone (DHT) or 17beta-estradiol (E2)-induced stimulation of LNCaP cell proliferation at physiological and pharmacological concentrations. Similar concentration-dependent inhibition of sex steroid-induced stimulation of thymidine incorporation into LNCaP cells by 2-iodomelatonin was also observed. Interestingly, attenuation of sex steroid-stimulated calcium influx into LNCaP cells by pharmacological concentrations of melatonin was recorded, whereas 2-iodomelatonin had no effect on cytosolic calcium changes induced by sex steroids. In addition, proliferative and cytosolic calcium changes were associated with inhibition of total prostate-specific antigen (PSA) production by LNCaP cells at high physiological and pharmacological concentrations of melatonin. Our data suggest that activated mt1 receptor and attenuated sex steroid-induced calcium influx are two important mechanisms mediating the direct anti-proliferative action of melatonin on androgen-responsive human prostate cancer cells.

Induction of androgen receptor by 1alpha,25-dihydroxyvitamin D3 and 9-cis retinoic acid in LNCaP human prostate cancer cells.

We have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits proliferation of LNCaP cells, an androgen-responsive human prostate cancer cell line. Also, 1,25-(OH)2D3 increases androgen receptor (AR) abundance and enhances cellular responses to androgen in these cells. In the current study, we have investigated the mechanism by which 1,25-(OH)2D3 regulates AR gene expression and the involvement of AR in the 1,25-(OH)2D3- and 9-cis retinoic acid (RA)-mediated growth inhibition of LNCaP cells. Northern blot analyses demonstrated that the steady-state messenger RNA (mRNA) level of AR was significantly increased by 1,25-(OH)2D3 in a dose-dependent manner. Time-course experiments revealed that the increase of AR mRNA by 1,25-(OH)2D3 exhibited delayed kinetics. In response to 1,25-(OH)2D3, AR mRNA levels were first detected to rise at 8 h and reached a maximal induction of 10-fold over the untreated control at 48 h; the effect was sustained at 72 h. Furthermore, the induction of AR mRNA by 1,25-(OH)2D3 was completely abolished by incubation of cells with cycloheximide, a protein synthesis inhibitor. 1,25-(OH)2D3 was unable to induce expression of an AR promoter-luciferase reporter. Together, these findings indicate that the stimulatory effect of 1,25-(OH)2D3 on AR gene expression is indirect. Western blot analyses showed an increase of AR protein in 1,25-(OH)2D3-treated cells. This increased expression of AR was followed by 1,25-(OH)2D3-induced inhibition of growth in LNCaP cells. Similar to 1,25-(OH)2D3, 9-cis RA also induced AR mRNA expression, and the effect of both hormones was additive. Moreover, 1,25-(OH)2D3 and 9-cis RA acted synergistically to inhibit LNCaP cell growth. These antiproliferative effects of 1,25-(OH)2D3 and 9-cis RA, alone or in combination, were blocked by the pure AR antagonist, Casodex. In conclusion, our results demonstrate that growth inhibition of LNCaP cells by 1,25-(OH)2D3 and 9-cis RA is mediated by an AR-dependent mechanism and preceded by the induction of AR gene expression. This finding, that differentiating agents such as vitamin D and A derivatives are potent inducers of AR, may have clinical implications in the treatment of prostate cancer.

The Conventional Transforming Growth Factor-β (TGF-β) Receptor Type I Is Not Required for TGF-β1 Signaling in a Human Prostate Cancer Cell Line, LNCaP

LNCaP is an androgen-responsive human prostate cancer cell line that has a defective gene for ALK-5, the conventional TGF-β receptor type I. Yet, these cells respond to exogenous TGF-β1 under appropriate concentrations of dihydrotestosterone (DHT). Because a heteromeric complex composed of type I and type II receptor is required for TGF-β signaling, the expression of these receptors was investigated in LNCaP cells at following concentrations of DHT—0, 0.1, and 100 nM. These concentrations were selected because they represent the zero DHT control in which LNCaP cells are not sensitive to TGF-β1, the proliferative dose of DHT in which these cells are sensitive to exogenous TGF-β1, and the growth-arrest dose of DHT in which LNCaP exhibits signs of TGF-β signaling but are insensitive to exogenous TGF-β1, respectively. Results of Western blot analysis showed that LNCaP cells express an increased level of type II receptor at 0.1 nM DHT, the TGF-β1-sensitive dose. However, results of competitive quantitative RT-PCR demonstrated that DHT did not significantly change the level of type II receptor mRNA, suggesting that DHT modulates the level of type II receptor at the posttranscriptional level. In contrast, ALK-5 was not detected in these cells by either Western blot analysis or RT-PCR at all concentrations of DHT used in this study. Subsequently, the expression of ALK-1, -2, and -4 in LNCaP cells was examined because these proteins have been shown to bind TGF-β1in vitro.ALK-1 and -2 were detected in these cells. Further analysis by competitive quantitative RT-PCR and Western blot demonstrated that DHT did not affect the level of expression of ALK-1 and -2 in LNCaP cells. These observations, taken together, demonstrate that ALK-5 is not required for TGF-β1 signaling and that there may be alternative mechanism(s) for TGF-β1 signal transduction in some systems.

1alpha,25-dihydroxyvitamin D3 actions in LNCaP human prostate cancer cells are androgen-dependent.

We and others have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibits cell proliferation and increases secretion of prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive human prostate cancer cell line. The present study was designed to investigate the possible interactions between 1,25-(OH)2D3 and androgens in the regulation of LNCaP cellular function. LNCaP cell growth was dose-dependently inhibited by 1,25-(OH)2D3 (60% inhibition at 10 nM) when cells were cultured in medium supplemented with FBS (FBS medium). 1,25-(OH)2D3-treated cells showed a 5-fold increase in PSA secretion, similar to the increase seen in dihydrotestosterone (DHT)-treated cells. In combination, 1,25-(OH)2D3 and DHT synergistically enhanced PSA secretion 22-fold. This synergistic effect was even greater when cells were cultured in medium supplemented with charcoal-stripped serum (CSS medium), where endogenous steroids are substantially depleted. Under these conditions, 1,25-(OH)2D3 and DHT together stimulated PSA secretion up to 50-fold over the untreated control. Radioligand binding assays and Western blot analyses showed that the androgen receptor (AR) content was increased significantly by 1,25-(OH)2D3 at 48 h. Furthermore, the steady-state mRNA level of AR was up-regulated approximately 2-fold by 1,25-(OH)2D3 at 24 h. When cells were grown in CSS medium, 1,25-(OH)2D3 alone no longer inhibited cell growth or induced PSA secretion. Titration experiments revealed that the addition of DHT at 1 nM to the medium restored the antiproliferative activity of 1,25-(OH)2D3. Conversely, an antiandrogen, Casodex, completely blocked 1,25-(OH)2D3 antiproliferative and PSA stimulation activities when cells were cultured in FBS medium. In conclusion, these results demonstrate that the antiproliferative and PSA induction activities of 1,25-(OH)2D3 in LNCaP cells are dependent upon androgen action and that AR up-regulation by 1,25-(OH)2D3 likely contributes to the synergistic actions of 1,25-(OH)2D3 and DHT in these cells.

Apoptosis: therapeutic significance in the treatment of androgen-dependent and androgen-independent prostate cancer.

To improve survival in men with metastatic prostatic cancer, a therapeutic modality that can effectively eliminate androgen-independent cancer cells is needed desperately. Combination of such an effective modality with androgen ablation could affect all of the heterogeneous populations within prostate tumors of individual patients, thus optimizing the chances of complete cure. Such a therapeutic approach will probably require two types of agents, one with antiproliferative activity affecting the small number of dividing androgen-independent cells and one with the capacity to increase the rate of cell death among the non-proliferating androgen-independent prostatic cancer cells present, i.e. the majority. Androgen-responsive human prostate cancer cells are able to undergo programmed cell death after androgen ablation (even if the cells are not in the proliferative cell cycle). Androgen-independent human prostate cancer cells, however, do not activate this apoptotic pathway of cell death in response to androgen ablation. In contrast, androgen-independent human prostate cancer cells can be induced to undergo apoptosis following such alternative treatment modalities as: (a) non-androgen ablative cytotoxic drugs, such as fluorinated pyrimidines, which result in the "thymine-less state", and (b) ionizing irradiation. The apoptotic effect induced by radiation can be significantly potentiated by post-irradiation treatment of the cells with suramin. In contrast, this radiation induced apoptosis can be substantially inhibited by pretreatment of cells with suramin, probably through suramin's ability to arrest proliferating cells in the GO/Gl phase of the cell cycle. These results suggest that treatment of prostate cancer patients with suramin prior to irradiation is likely to inhibit radiation palliation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aberrant response in vitro of hormone-responsive prostate cancer cells to antiandrogens.

Antiandrogens are in use alone and in combination with other agents as hormonal therapy for prostate cancer. We conducted studies on the androgen-responsive human prostate cancer cell line LNCaP to determine the direct effects of three antiandrogens (hydroxyflutamide, RU23908, and cyproterone acetate) on hormone-responsive human prostate cancer cells in culture. Dihydrotestosterone (DHT) stimulated the growth of LNCaP cells in a dose-dependent fashion. These cells contained approximately 31,000 high-affinity (Kd = 9 x 10(-10) M) androgen binding sites per cell. In the absence of any androgenic stimulation, all three antiandrogens tested showed agonistic properties by increasing the cell number and uptake of [3H]-thymidine. Competitive uptake studies using [3H]-R1881, a nonmetabolized androgen, showed that the three antiandrogens inhibited specific R1881 uptake with IC50s of 0.9 x 10(-7) M for hydroxyflutamide, 2 x 10(-7) M for RU23908, and 1 x 10(-7) M for cyproterone acetate. It is not known whether these unexpected agonistic effects are due to an altered receptor, previously unmasked agonistic properties of the antiandrogens, or emergence of a hypersensitive clone of cells.