Hormone-insensitive Prostate Cancer Cell Research Articles

Abstract 1824: Hexadecanoic acid mediated cell cycle arrest involving inhibition of cell migration

Abstract The Caribbean region accounts for the highest incidence and mortality rates of prostate cancer in the world. This warrants the need for targeted therapy as currently the most prevalent and effective option include chemotherapeutic drugs. Previous studies showed that an enriched sample extracted from the Jamaican Round Leaf yellow yam exhibited antiproliferative effects on DU145 prostate cancer cells. Based on the results obtained, this present study aims at exploring the underlying mechanism of action as well as the antimetastatic activities that the yam derived hexadecanoic acid may exhibit. Flow cytometry analysis was used to assess cell cycle arrest exhibited by hexadecanoic acid on the hormone insensitive DU145 prostate cancer cells. The western blot analysis was then utilized to determine the effects of the hexadecanoic acid enriched sample on cyclins in the cells. Transwell migration assays were then used to evaluate cell metastatic capabilities. Hexadecanoic acid enriched sample induced G1 S cell cycle arrest in the hormone insensitive prostate cancer cell line. Western blot assays also indicated that cell death was possibly induced through the cell cycle regulatory protein, cyclin D1 as its expression was downregulated in the treatment groups. Additionally, the observed metastatic inhibition of prostate cancer cells due to the action of the hexadecanoic acid in the wound healing assay may be attributed to a mechanism involving cyclin D1 as studies have indicated that abnormal Ccnd1 Cdk4 expression promotes tumor growth and metastasis. These results are substantial as it indicates a mechanistic pathway through which hexadecanoic acid is able to cause antiproliferative effects through a cell cycle arrest of hormone insensitive prostate cancer cell lines. Citation Format: Sasha-gay A. Wright, William Aiken, Helen Asemota, Kimberly Foster, Rupika Delgoda. Hexadecanoic acid mediated cell cycle arrest involving inhibition of cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1824.

The influence of hypoxia on the prostate cancer proteome.

Prostate cancer accounts for around 15% of male deaths in Western Europe and is the second leading cause of cancer death in men after lung cancer. Mounting evidence suggests that prostate cancer deposits exist within a hypoxic environment and this contributes to radio-resistance thus hampering one of the major therapies for this cancer. Recent reports have shown that nitric oxide (NO) donating non-steroidal anti-inflammatory drugs (NSAIDs) reduced tumour hypoxia as well as maintaining a radio-sensitising/therapeutic effect on prostate cancer cells. The aim of this study was to evaluate the impact of hypoxia on the proteome of the prostate and to establish whether NO-NSAID treatment reverted the protein profiles back to their normoxic status. To this end an established hormone insensitive prostate cancer cell line, PC-3, was cultured under hypoxic and normoxic conditions before and following exposure to NO-NSAID in combination with selected other common prostate cancer treatment types. The extracted proteins were analysed by ion mobility-assisted data independent acquisition mass spectrometry (MS), combined with multivariate statistical analyses, to measure hypoxia-induced alterations in the proteome of these cells. The analyses demonstrated that under hypoxic conditions there were well-defined, significantly regulated/differentially expressed proteins primarily involved with structural and binding processes including, for example, TUBB4A, CIRP and PLOD1. Additionally, the exposure of hypoxic cells to NSAID and NO-NSAID agents, resulted in some of these proteins being differentially expressed; for example, both PCNA and HNRNPA1L were down-regulated, corresponding with disruption in the nucleocytoplasmic shuttling process.

Enhanced radiosensitization of enzalutamide via schedule dependent administration to androgen-sensitive prostate cancer cells.

Prostate cancer (PCa) is a progressive disease and the most diagnosed cancer in men. The current standard of care for high-risk localized PCa is a combination of androgen deprivation therapy (ADT) and radiation (XRT). The majority of these patients however become resistant due to incomplete responses to ADT as a result of selective cells maintaining androgen receptor (AR) activity. Improvement can be made if increasing radiosensitivity is realized. Therefore, the aim of this study is to investigate the efficacy of the next-generation PCa drug Enzalutamide (ENZA), as a radiosensitizer in XRT therapy. Using a number of androgen-dependent (LNCaP, PC3-T877A) and androgen-independent (C4-2, 22RV1, PC3, PC3-AR V7) cell lines, the effect of ENZA as a radiosensitizer was studied alone or in combination with ADT and/or XRT. Cell viability and cell survival were assessed, along with determination of cell cycle arrest, DNA damage response and repair, apoptosis and senescence. Our results indicated that either ENZA alone (in AR positive, androgen-dependent PCa cells) or in combination with ADT (in AR positive, hormone-insensitive PCa cells) potentiates radiation response [Dose enhancement factor (DEF) of 1.75 in LNCAP and 1.35 in C4-2] stronger than ADT + XRT conditions. Additionally, ENZA sensitized androgen dependent PCa cells to XRT in a schedule-dependent manner, where concurrent administration of ENZA and radiation lead to a maximal radiosensitization when compared to either drug administration prior or after XRT. In LNCaP cells, ENZA treatment significantly prolonged the presence of XRT-induced phospho-γH2AX up to 24 h after treatment; suggesting enhanced DNA damage. It also significantly increased XRT-induced apoptosis and senescence. Our data indicates that ENZA acts as a much stronger radiosensitizer compared to ADT. We have also observed that its efficacy is schedule dependent and related to increased levels of DNA damage and a delay of DNA repair processes. Finally, the initial abrogation of DNA-PKcs activity by AR inhibition and its subsequent recovery might represent an important mechanism by which PCa cells acquire resistance to combined anti-androgen and XRT treatment. This work suggests a new use of ENZA in combination with XRT that could be applicable in clinical trial settings for patients with early and intermediate hormone responsive disease.

Autophagy activated by the c-Jun N-terminal kinase-mediated pathway protects human prostate cancer PC3 cells from celecoxib-induced apoptosis.

The aim of the present study was to investigate the role of autophagy in celecoxib-induced apoptosis in human hormone-insensitive prostate cancer cell line PC3 cells and to explore the underlying molecular mechanism leading to autophagic activation. A cell viability assay was applied to investigate the effect of various concentrations of celecoxib (0, 40, 60, 80, 100 and 120 µmol/l) on PC3 cells for 24 and 48 h, respectively. The 50% inhibitory concentration of celecoxib for 24 h was chosen for subsequent experiments. Annexin V-fluorescein isothiocyanate/propidium iodide double staining flow cytometry, as well as caspase 3 and poly (ADP-ribose) polymerase proteins detected by western blotting, were applied to analyze cellular apoptosis induced by celecoxib. Ultrastructural cellular changes observed by transmission electron microscopy and the level of LC-3 II and P62 detected by western blotting were used to determine the activation of autophagy. It was demonstrated that celecoxib induced apoptosis and activated autophagy in PC3 cells in a dose- and time-dependent manner. Furthermore, flow cytometry and western blotting were applied to elucidate whether the role of autophagy in celecoxib-induced apoptosis is protective or destructive. Blockade of autophagy markedly increased apoptosis, suggesting that celecoxib-activated autophagy was cytoprotective. Additionally, c-jun-N-terminal kinase (JNK) was demonstrated to have a role in autophagic activation, and suppression of JNK was able to reduce autophagy and increase apoptosis. In conclusion, the results of the present study indicate that celecoxib induces apoptosis in PC3 cells; however, celecoxib also activates JNK-mediated autophagy, which exerts cytoprotective effects in prostate cancer PC3 cells. Blockade of autophagy via the JNK-mediated pathway may provide a promising strategy for prostate cancer therapy.

Androgen Receptor-dependent Expression of Low-density Lipoprotein Receptor-related Protein 6 is Necessary for Prostate Cancer Cell Proliferation.

Androgen receptor (AR) signaling is important for prostate cancer (PCa) cell proliferation. Here, we showed that proliferation of hormone-sensitive prostate cancer cells such as LNCaP was significantly enhanced by testosterone stimulation whereas hormone-insensitive prostate cancer cells such as PC3 and VCaP did not respond to testosterone stimulation. Blocking of AR using bicalutamide abolished testosterone-induced proliferation of LNCaP cells. In addition, knockdown of AR blocked testosterone-induced proliferation of LNCaP cells. Basal expression of low-density lipoprotein receptor-related protein 6 (LRP6) was elevated in VCaP cells whereas stimulation of testosterone did not affect the expression of LRP6. However, expression of LRP6 in LNCaP cells was increased by testosterone stimulation. In addition, knockdown of LRP6 abrogated testosterone-induced proliferation of LNCaP cells. Given these results, we suggest that androgen-dependent expression of LRP6 plays a crucial role in hormone-sensitive prostate cancer cell proliferation.

Antiandrogen Gold Nanoparticles Dual-Target and Overcome Treatment Resistance in Hormone-Insensitive Prostate Cancer Cells

Prostate cancer is the most commonly diagnosed cancer among men in the developed countries.(1) One in six males in the U.S.(2) and one in nine males in the U.K.(3) will develop the disease at some point during their lifetime. Despite advances in prostate cancer screening, more than a quarter of a million men die from the disease every year(1) due primarily to treatment-resistance and metastasis. Colloidal nanotechnologies can provide tremendous enhancements to existing targeting/treatment strategies for prostate cancer to which malignant cells are less sensitive. Here, we show that antiandrogen gold nanoparticles--multivalent analogues of antiandrogens currently used in clinical therapy for prostate cancer--selectively engage two distinct receptors, androgen receptor (AR), a target for the treatment of prostate cancer, as well as a novel G-protein coupled receptor, GPRC6A, that is also upregulated in prostate cancer. These nanoparticles selectively accumulated in hormone-insensitive and chemotherapy-resistant prostate cancer cells, bound androgen receptor with multivalent affinity, and exhibited greatly enhanced drug potency versus monovalent antiandrogens currently in clinical use. Further, antiandrogen gold nanoparticles selectively stimulated GPRC6A with multivalent affinity, demonstrating that the delivery of nanoscale antiandrogens can also be facilitated by the transmembrane receptor in order to realize increasingly selective, increasingly potent therapy for treatment-resistant prostate cancers.

MicroRNA-133 inhibits cell proliferation, migration and invasion in prostate cancer cells by targeting the epidermal growth factor receptor

It has been shown that regulation of EGFR expression in prostate cancer cells is mostly at the transcriptional level. microRNA-133 (miR-133) has long been recognized as a muscle-specific miRNA which may regulate myoblast differentiation and participate in many myogenic diseases. Recently, it has been reported that miR-133 is also involved in other tumors, such as bladder cancer, esophageal cancer and may regulate cell motility in these cancer cells. In the present study, we examined the expression and effects of miR-133 in two hormone-insensitive prostate cancer cell lines. The expression of miR-133a and miR-133b were analyzed by quantitative RT-PCR. After transfection of miR-133a and miR-133b, cell viability assay, luciferase assay, western blot analysis, cell migration and invasion assay were conducted in Du145 and PC3 cells. In this study, we showed that miR‑133a and miR-133b are expressed at the detection limit in two hormone-insensitive prostate cancer cell lines, PC3 and DU145. Ectopic expression of miR-133 inhibited cell proliferation, migration and invasion in these cells. We also provide the first evidence that miR-133 may target EGFR. Our study provided the first glimpse of the functional role of miR-133 in two hormone-independent prostate cancer cell lines. These results may add to our knowledge on the molecular basis of prostate cancer progression.

Expression of resistin in the prostate and its stimulatory effect on prostate cancer cell proliferation

To determine whether resistin, a novel adipokine, induces prostate cancer cell proliferation. To identify the mechanisms underlying the activation of prostate cancer cells by resistin. Semi-quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical staining were performed to investigate the intensity of prostate epithelial resistin expression. Human full-length resistin gene (RETN) was transfected into the PC-3 cells using the pEGFP-N1 vector to assess the effect of overexpression of resistin in prostate cancer cell line PC-3. Various concentrations of human recombinant protein resistin were added to the hormone-insensitive prostate cancer cell lines PC-3 and DU-145 for 48 h, and cell proliferation was assessed by a water-soluble tetrazolium salt assay. Human prostate cancer cell lines PC-3 and DU-145 were found to express the human resistin mRNA. Resistin protein was strongly detected in high-grade prostate cancer tissue, whereas BPH or low-grade prostate cancer tissue revealed fainter expression of resistin. Cell proliferation was stimulated by both the full-length resistin gene overexpression and resistin treatment. Akt phosphorylation occurred after addition of resistin to PC-3 and DU-145 cells. LY294002, a pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K), significantly inhibited PC-3 and DU-145 cell proliferation after resistin treatment. Resistin is expressed in human prostate cancers. Resistin induces prostate cancer cell proliferation through PI3K/Akt signalling pathways. The proliferative effect of resistin on prostate cancer cells may account in part for prostate cancer progression.

Compensatory Upregulation of Tyrosine Kinase Etk/BMX in Response to Androgen Deprivation Promotes Castration-Resistant Growth of Prostate Cancer Cells

We previously showed that targeted expression of non-receptor tyrosine kinase Etk/BMX in mouse prostate induces prostate intraepithelial neoplasia, implying a possible causal role of Etk in prostate cancer development and progression. Here, we report that Etk is upregulated in both human and mouse prostates in response to androgen ablation. Etk expression seems to be differentially regulated by androgen and interleukin 6 (IL-6), which is possibly mediated by the androgen receptor (AR) in prostate cancer cells. Our immunohistochemical analysis of tissue microarrays containing 112 human prostate tumor samples revealed that Etk expression is elevated in hormone-resistant prostate cancer and positively correlated with tyrosine phosphorylation of AR (Pearson correlation coefficient rho = 0.71, P < 0.0001). AR tyrosine phosphorylation is increased in Etk-overexpressing cells, suggesting that Etk may be another tyrosine kinase, in addition to Src and Ack-1, which can phosphorylate AR. We also showed that Etk can directly interact with AR through its Src homology 2 domain, and such interaction may prevent the association of AR with Mdm2, leading to stabilization of AR under androgen-depleted conditions. Overexpression of Etk in androgen-sensitive LNCaP cells promotes tumor growth while knocking down Etk expression in hormone-insensitive prostate cancer cells by a specific shRNA that inhibits tumor growth under androgen-depleted conditions. Taken together, our data suggest that Etk may be a component of the adaptive compensatory mechanism activated by androgen ablation in prostate and may play a role in hormone resistance, at least in part, through direct modulation of the AR signaling pathway.

Effects of sorafenib on proliferation of hormone-sensitive and hormone-insensitive prostate cancer cells

e16092 Background: Sorafenib is a multi-targeted kinase inhibitor approved for treatment of renal and hepatocellular cancer. In patients with castration therapy-resistant prostate cancer sorafenib treatment is associated with discordant prostate-specific antigen (PSA) and clinical responses, possibly due to an effect on PSA expression. The mechanisms of its action in advanced prostate tumors are not investigated so far. The aim of the present study was to evaluate the effects of sorafenib in androgen-sensitive (LNCaP) and -insensitive (PC 3) prostate cancer cell lines. Methods: Proliferation was evaluated by measurement of cell number and protein. Expression of cell cycle and apoptosis regulatory proteins of the Bcl-2 family was determined by Western blot. Results: Treatment of those cells by sorafenib for 48 hours resulted in a concentration-dependent inhibition of proliferation. Maximal inhibition was achieved with 25 μM of the compound. Cell number was reduced by 50% in both cell lines. We observed inhibition of expression of cyclin-dependent kinase 2 in LNCaP and PC3 cells. In addition, Mcl-1 protein, that is frequently overexpressed in prostate cancer, was down-regulated by sorafenib in both cellular models. Conclusions: Sorafenib caused inhibition of growth of prostate cancer cells regardless of their androgen sensitivity. Its inhibitory effects on cyclin- dependent kinase 2 and Mcl-1 imply that sorafenib regulates cell cycle progression and apoptosis in prostate cancer. On the basis of these results experiments with chemotherapy-resistant prostate cells are being performed. [Table: see text]

A Novel Androgen Receptor Splice Variant Is Up-regulated during Prostate Cancer Progression and Promotes Androgen Depletion–Resistant Growth

The androgen receptor (AR) plays a key role in progression to incurable androgen ablation-resistant prostate cancer (PCA). We have identified three novel AR splice variants lacking the ligand-binding domain (designated as AR3, AR4, and AR5) in hormone-insensitive PCA cells. AR3, one of the major splice variants expressed in human prostate tissues, is constitutively active, and its transcriptional activity is not regulated by androgens or antiandrogens. Immunohistochemistry analysis on tissue microarrays containing 429 human prostate tissue samples shows that AR3 is significantly up-regulated during PCA progression and AR3 expression level is correlated with the risk of tumor recurrence after radical prostatectomy. Overexpression of AR3 confers ablation-independent growth of PCA cells, whereas specific knockdown of AR3 expression (without altering AR level) in hormone-resistant PCA cells attenuates their growth under androgen-depleted conditions in both cell culture and xenograft models, suggesting an indispensable role of AR3 in ablation-independent growth of PCA cells. Furthermore, AR3 may play a distinct, yet essential, role in ablation-independent growth through the regulation of a unique set of genes, including AKT1, which are not regulated by the prototype AR. Our data suggest that aberrant expression of AR splice variants may be a novel mechanism underlying ablation independence during PCA progression, and AR3 may serve as a prognostic marker to predict patient outcome in response to hormonal therapy. Given that these novel AR splice variants are not inhibited by currently available antiandrogen drugs, development of new drugs targeting these AR isoforms may potentially be effective for treatment of ablation-resistant PCA.

CDNA microarray analysis of pigment epithelium‐derived factor‐regulated gene expression profile in prostate carcinoma cells

To clarify molecular mechanisms involved in the action of pigment epithelium-derived factor (PEDF) in hormone insensitive prostate cancer cells. Total ribonucleic acid from untreated and PEDF-treated cells was subjected to microarray analysis using BioStar 8464 microarray. Real-time polymerase chain reaction analysis was conducted to confirm the microarray data. Twenty-seven out of 8464 genes were found altered in both cell lines. Common gene responses altered by PEDF were identified and included genes known to alter cell signaling as well as genes involved in catalytic activity, cell proliferation, angiogenesis and apoptosis. Real-time reverse transcription polymerase chain reaction, in accordance with the microarray analysis, indicated that PEDF treatment caused an upregulation in the mRNA expression level of stanniocalcin 2, brain-specific angiogenesis inhibitor 2 and growth arrest, DNA-damage-inducible, alpha, and downregulation in the messenger ribonucleic acid level of fibroblast growth factor 3, teratocarcinoma-derived growth factor, neuropilin1, and endothelial Per/ARNT/Sim domain protein1, respectively. These findings demonstrate that PEDF administration causes significant changes in the gene expression of the prostate, providing insights into the potential role of PEDF in the treatment of prostate cancer.

KCNMA1 gene amplification promotes tumor cell proliferation in human prostate cancer

Molecular mechanisms of prostate cancer progression are poorly understood. Here, we studied gene amplification of the large conductance calcium-activated potassium channel alpha subunit (KCNMA1), which is located at the chromosomal region 10q22. Fluorescence in situ hybridization (FISH) revealed KCNMA1 amplification in 16% of 119 late-stage human prostate cancers and in the hormone-insensitive prostate cancer cell line PC-3. In contrast, KCNMA1 amplification was absent in 33 benign controls, 32 precursor lesions and in 105 clinically organ-confined prostate cancers. Amplification was associated with mRNA and protein overexpression as well as increased density of BK channel protein and beta-estradiol-insensitive BK currents in PC-3 cells as compared to non-amplified control cell lines. Specific blockade of BK channels by iberiotoxin or RNA(i) significantly inhibited K(+) currents and growth of PC-3 cells. The data demonstrate that 10q22 amplification drives KCNMA1 expression and cell proliferation. Thus, KCNMA1 qualifies as a promising diagnostic and therapeutic target in patients with prostate cancer.

Herbal therapy PC-SPES: in vitro effects and evaluation of its efficacy in 69 patients with prostate cancer.

We investigate the potential use of the phytotherapeutic PC-SPES to treat human prostate cancer, and evaluate its in vivo and in vitro activity, and clinical efficacy. PC-SPES was evaluated for its ability to induce apoptosis on prostate cancer cell lines LNCaP, PC3 and DU145. The effect of oral PC-SPES on growth of PC3 tumors present in male immunodeficient mice was studied. A total of 30 male nude mice were divided in 5 groups. In groups 1 control and 2 full dose therapy was started the same day of the tumor injection. In groups 3 control, 4 half dose and 5 full dose PC-SPES therapy was initiated 1 week after tumor injection. A total of 69 patients with prostate cancer were treated with 3 capsules of 320 mg. PC-SPES daily. Serum prostate specific antigen (PSA) responses and side effects were evaluated. All of the cultured prostate cancer cell lines had a significant dose dependent induction of apoptosis following exposure to an alcoholic PC-SPES extract. Immunodeficient mice xenografted with the PC3 cell line had reduced tumor volume compared with sham treated controls when they were treated with a PC-SPES extract from the time of tumor cell implantation (931 +/- 89 versus 1,424 +/- 685 mm.3, p not significant) but not when the treatment was begun 1 week after tumor cell implantation. The testis, prostate, bladder and seminal vesicles of the treated mice were significantly reduced in weight compared with the sham treated animals. Of the patients with prostate cancer 82% had decreased serum PSA 2 months, 78% 6 months and 88% 12 months after treatment with PC-SPES. Side effects in the treated patient population included nipple tenderness in 42% and phlebitis requiring heparinization in 2%. An extract of the phytotherapeutic agent PC-SPES proved to be active in inducing apoptosis of hormone sensitive and insensitive prostate cancer cells in vitro, and in suppressing the growth rate of a hormone insensitive prostate cancer cell line in vivo. The overwhelming majority of patients with prostate cancer treated with the agent experienced a decrease in serum PSA but also demonstrated a side effect profile comparable to estrogen treatment.

Influence of pertussis toxine on local progression and metastasis after orthotopic implantation of the human prostate cancer cell line PC3 in nude mice

Tumor cell migration is a fundamental process of metastasis. Pertussis toxine inhibits lysophosphatidic acid related cell migration by ADP-ribosylation of G proteins. We examined the influence of pertussis toxine (PTX) on progression and metastasis of the human hormone-insensitive prostate cancer cell line PC-3 after orthotopic implantation in nude mice. In 30 athymic male nude mice (NMRI) 5x10(5) PC-3 cells were injected into the dorsal prostate. After 7 d 15 mice received a total of six intraperitoneal injections of 5 micro g PTX/100 g body weight at an interval of 4 d. The other 15 mice received phosphate buffered saline and served as control. All mice were killed at 37 d followed by macroscopical and histological evaluation of local tumor growth and metastasis. In the control group tumorigenicity was 100% (15 out of 15). Mean weight of the tumor bearing unit of prostate and seminal vesicles was 541 mg (243-763 mg). The rate of positive lymphnodes was 100% with a mean transversal diameter of 3.9 mm (1.2-5.4 mm). In the PTX group local take rate was 100% with a mean weight of 251 mg (88-478 mg) (P two sided <0.0001). The rate of positive lymphnodes was 60% (9 out of 15) (P=0.017) with a mean transversal diameter of 2.3 mm (1.0-4.5 mm). PTX following orthotopic implantation of the human hormone-insensitive PC-3 cell line significantly reduces local tumor growth as well as metastasis to locoregional lymphnodes.

Induction of Apoptosis by Diethylstilbestrol in Hormone-Insensitive Prostate Cancer Cells

Diethylstillbestrol (DES) and diethylstilbestrol diphosphate (DESdP) are effective agents for the treatment of advanced prostate cancers. Tumor-inhibiting effects of DES and DESdP are presumed secondary to suppression of androgen production in vivo. Little is known, however, about the direct cellular mechanisms of the tumor inhibition. Estrogens have been reported not only to stimulate growth but also to disrupt microtubule formation in prostate cancer cells. The study was designed to examine and compare mechanisms of in vitro growth inhibition of DES and DESdP in human androgen-insensitive prostate cancer cells (DU145, 1-LN, and PC-3) and human androgen-sensitive prostate cancer cells (LNCaP) and to examine estrogen receptor modulation of such effects. The cytotoxic effects of DES and DESdP were examined in vitro by use of a standard microculture tetrazolium assay to quantitate numbers of viable cells. Immunofluorescence microscopy, DNA fragmentation analysis, and fluorescence flow cytometry were used to investigate microtubules, the induction of apoptosis, and changes in cell cycle distribution. The degree of estrogen receptor positivity of untreated and treated cells was determined by immunohistochemistry and quantitative image analysis. LD50 levels (the dose at which 50% of cells are no longer viable) in the concentration range of 19-25 microM were observed for both DES and DESdP in all cell lines examined. DESdP-induced growth inhibition was found to be dependent on heat-labile phosphatases present in fetal calf serum. DES-induced cytotoxicity was not affected by the presence of 17 beta-estradiol, and it was not dependent on the presence of estrogen receptor. Estrogen receptor-positive cells and estrogen receptor-negative cells were equally responsive to DES. PC-3 cells stained with fluorescent anti-tubulin, phalloidin (actin stain), and 4',6-diamidino-2-phenylindole (DNA stain) showed no inhibition of microtubules or actin filaments but revealed the presence of apoptotic bodies in the nuclei. Fluorescence flow cytometry of nuclear DNA content of propidium iodide-stained nuclei from androgen-insensitive prostate cancer cells treated with 15 or 30 microM DES or DESdP revealed an increase in relative numbers of hypodiploid (apoptotic) nuclei, a depletion of G1- and S-phase cells, and an accumulation of cells in G2/M phase. Conversely, androgen-sensitive cells contained a lower percentage of hypodiploid nuclei but no accumulation of cells in G2/M phase. Direct cytotoxic effects of DES in prostate cancer cells are estrogen receptor independent and do not involve disruption of microtubule architecture but do involve the promotion of cell cycle arrest and apoptosis. These are the first data confirming direct cytotoxic effects of DES and DESdP in prostate cancer cells via an apoptotic mechanism. IMPLICATIONS. These results suggest that DES and DESdP have potential value as agents against androgen-insensitive prostate neoplasms through induction of an apoptotic cascade.