澳洲茄边碱对前列腺癌细胞增殖的抑制作用及机制研究

目的：研究澳洲茄边碱(solamargine,SM)对人前列腺癌激素非依赖细胞增殖的影响及可能的作用机制.方法用不同浓度 SM(0、1、2、4、6、8、10μmol/L)处理 DU145和 PC3细胞, MTT法检测 SM 对细胞生长的影响,Western blot 检测 SM 对相关信号通路蛋白 p38 MAPK、ERK1/2 MAPK、MUC1表达的影响.结果6μmol/L SM作用24 h后DU145和PC3细胞活力分别为(52.53±9.05)％、(56.28±2.36)％,并具有时间和剂量依赖;10μmol/L SM作用24 h后细胞活力分别为(27.36±2.72)％、(32.07±2.53)％.流式细胞术分析显示不同浓度 SM(0、4、6、8μmol/L)处理 PC3细胞能引起 PC3细胞阻滞在 G1期,G1期细胞比例分别为(52.61±0.50)％、(52.96±1.49)％、(66.16±2.84)％和(69.03±2.38)％.且能激活 MAPK信号通路减少下游蛋白 MUC1的表达.结论 SM能明显抑制DU145和PC3细胞生长,该作用可能与 MAPK信号通路的激活以及下游 MUC1蛋白表达下调有关.     

鱼藤素诱导人前列腺癌细胞凋亡及其机制研究

目的 观察鱼藤素对于激素抵抗型前列腺癌(HRPC)细胞PC3和DU145增殖、细胞周期和凋亡的影响并探讨其机制.方法 设阴性对照组(有细胞但不加药),空白对照组(无细胞仅有培养液),阳性对照组(渥曼青霉素100 nmoL/L),及鱼藤素分别10、100、1 ìmol/L共6组.CCK-8法进行细胞毒性实验,检测细胞生长抑制率.流式细胞术检测细胞周期和凋亡,Westem-blot检测Akt、MAPK及其磷酸化蛋白表达,探讨药物作用机制.结果 10 nmol/L～1 ìmol/L鱼藤素对PC3细胞均有生长抑制作用,呈现明显的时间、浓度依赖性,对DU145细胞则无此作用.鱼藤素使PC3细胞出现G2/M期阻滞现象并引起浓度依赖性的凋亡,而未改变DU145细胞的周期分布也不能诱导其凋亡.鱼藤素能够阻断P13K/AKT通路而对MAPK通路无影响.结论 鱼藤素通过阻断PI3K/AKT通路实现抑制PC3细胞增殖、诱导凋亡的作用.两株细胞间实验结果 的差异是因为其PI3K/AKT通路活化状态的差异造成的.     

Inactivation of the NF2 tumor suppressor protein merlin in DU145 prostate cancer cells

BACKGROUND: The neurofibromatosis 2 (NF2) tumor suppressor gene product merlin is an important regulator of contact-dependent cell proliferation. Phosphorylation of merlin at serine 518 (Ser518) by the Rac effector p21-activated kinase (PAK) inactivates merlin's growth suppressing function, and is regulated by cell-culture conditions, including cell density, cell/substrate attachment, and growth factor availability. We examined the regulation of merlin expression and merlin phosphorylation in prostate cancer cells. METHODS: Phosphorylation of merlin in five prostate cancer cell lines (LNCaP, DU145, PC3, 22RV1, and LAPC-4) was examined by Western blotting using anti-phospho-merlin (Ser518) antibody. The activity of PAK, an upstream regulator of merlin phosphorylation, was measured by Western blotting using phospho-PAK (Ser141) antibody. The effects of various cell-culture conditions on the phosphorylation levels of merlin and PAK were analyzed. RESULTS: Both merlin expression and phosphorylation were low in LNCaP, PC3, 22RV1, and LAPC-4 prostate cancer cells. In DU145 cells, total and phosphorylated merlin were abundant, but phosphorylation was not inhibited by high cell density, serum withdrawal, the addition of hyaluronic acid or inhibition of CD44 expression, all of which are reported to inhibit merlin phosphorylation in non-neoplastic cells. PAK activation was elevated in DU145 cells and the addition of a PAK-specific inhibitor peptide but not the Rac1-specific inhibitor NSC23766 inhibited both PAK and merlin phosphorylation. CONCLUSIONS: Merlin is inactivated in DU145 prostate cancer cells by PAK-mediated constitutive phosphorylation, identifying a novel mechanism of merlin inactivation in neoplastic cells.     

The survival effect of prolactin on PC3 prostate cancer cells

OBJECTIVES: Recent studies suggest a paracrine/autocrine loop involving prolactin (PRL) within the human prostate. The aims of this study were to determine the effects of PRL on the growth and survival of prostate cancer cells and the intracellular signalling mechanisms underlying such effects. METHODS: The effect of PRL on proliferation of LNCaP, PC3 and DU145 was assessed by Coulter counting. The effect of PRL on TRAIL-, staurosporine- and flavopiridol-induced apoptosis was assessed by Timelapse microscopy and Annexin V binding. The status of the PRL receptor (PRL-R) and Akt/PKB (protein kinase B) activity were assessed by Western blotting. RESULTS: All three cell lines expressed both the short and long forms of the PRL receptor. Although, no significant effect of PRL on the proliferation of these cells was found, PRL partially inhibited TRAIL-induced apoptosis in PC3 cells. PRL also enhanced the phosphorylation of Akt/PKB in these cells. CONCLUSIONS: PRL had no significant effect on the proliferation of PC3, DU145 and LNCaP, but inhibited TRAIL-induced apoptosis in PC3 cells, possibly via enhanced Akt/PKB phosphorylation in PC3 cells. Further investigations are underway to determine the survival effect of PRL on the other two prostate cancer cell line.     

Prostate cancer cell proliferation is influenced by leptin

BACKGROUND: Obesity is considered a risk for many cancers. Serum leptin levels are often elevated in obese people. Leptin acts as a mitogenic agent in many tissues; therefore, it may act to promote cancer cell growth. We previously demonstrated that leptin acts as a growth factor for prostate cancer cells in vitro. The purpose of this study was to characterize leptin receptor isoform mRNA expression in leptin-treated DU145 and PC-3 prostate cancer cell lines. Expression levels of SOCS-3, a known leptin-inducible suppressor of leptin signaling, and known mitogenic signaling pathways of PI3K and ERK were also analyzed METHODS: DU145 and PC-3 cells were treated with 0, 4, 40, or 80 ng/ml leptin for 0, 0.5, 1, 2, 4, 24, or 48 h. Multiplex RT-PCR was performed to determine mRNA levels of the short (huOB-Ra) or the long (huOB-Rb) OB-R isoforms or SOCS-3. p-Akt and p-ERK were determined by Western blot. Cell viability and apoptosis were determined by MTT and nucleosomal fragmentation assay RESULTS: DU145 and PC-3 expressed huOB-Ra, huOB-Rb, and SOCS-3 mRNA. huOB-Ra mRNA levels increased in PC-3 at 48 h (P < 0.01); however, no significant changes were observed in DU145. huOB-Rb mRNA levels decreased at 48 h in DU145; however, a twofold increase at 48 h (P < 0.01) was observed with PC-3 and was dose-dependent (P < 0.05). Leptin increased SOCS-3 mRNA in DU145 at 24 and 48 h (P < 0.05) and in PC-3 at 1 h (2-fold) and 48 h (fivefold; P < 0.01). Leptin up-regulated p-Akt in a time- and dose-dependent manner in the DU145 prostate cancer cells via a suppression of apoptosis. Leptin up-regulated p-ERK in a time-dependent manner in PC-3 cells CONCLUSIONS: In prostate cancer cells, the mitogenic effects of leptin are not a consequence of altered receptor isoform mRNA expression. No defect in SOCS-3 signaling was observed, and proliferation appears to be working through the PI3K and MAPK leptin receptor-activated pathways, depending on cell type. Leptin stimulation may be selective for either pathway to suppress apoptosis, thereby enhancing prostate cancer growth.     

表皮生长因子信号通路对前列腺癌细胞DU145表面整合素α5、β1的调控

目的 探讨表皮生长因子（EGF）信号通路对雄激素非依赖型前列腺癌细胞系DU145表面整合素α5、β1的调控.方法 应用流式细胞术、逆转录聚合酶链反应（RT-PCR）和Western蛋白印迹（Western blot）法测定EGF、丝裂原活化蛋白激酶（MAPK）信号通路抑制剂PD98059对DU145细胞整合素α5和β1表达的影响;采用细胞粘附能力测定和Transwell小室法检测EGF及PD98059对DU145细胞粘附能力和迁移能力的影响.结果 EGF上调DU145细胞表面整合素β1亚基的蛋白和mRNA的表达,分别为对照组的231%和248%（方差值为9.0和24.6,P均＜0.01）,并可促进DU145细胞对纤维连接蛋白（Fn）的粘附和迁移能力.而PD98059则具有相反的作用,下调DU145细胞表面整合素β1亚基的蛋白和mRNA的表达,分别为对照组的60%和63%（方差值为6.3和15.3,P均＜0.01）,并可抑制DU145细胞对Fn的粘附和迁移能力.EGF和PD98059对整合素α1的表达无明显影响.结论 EGF可能通过MAPK信号通路上调前列腺癌DU145细胞表面整合素β1亚基的表达,从而促进DU145细胞对Fn的粘附能力和迁移能力;此调节作用主要发生在mRNA转录水平.     

表皮生长因子受体阻断对前列腺癌细胞增殖的影响

目的：探讨阻断表皮生长因子受体（EGFR）对前列腺癌（PCa）细胞增殖的影响。方法：人PCa细胞株DU－145，分别加或不加抗EGFR单抗C225（20nmol/L)阻断EGFR体外细胞培养7d，收集细胞、计数以观察对PCa雄激素非依赖增殖的影响，并采用免疫沉淀和Western blot方法，探讨阻断EGFR后不同时相点磷酸化有丝分裂原激活下蛋白激酶MAPK，及p27^kipl的表达变化。结果：阻断EGFR与对照相比较可使DU－145细胞增殖被抑制达35％，8h后磷酸化MAPK的表达水平开始降低，p27^kip1表达开始升高，至24h最明显。结论：阻断EGFR可抑制PCa细胞增殖，可能的机制是MAPK的活性降低，使p27^kip1的表达升高而细胞周期被阻。     

Epidermal growth factor receptor mRNA levels in human prostatic tumors and cell lines.

The mitogenic activity of epidermal growth factor (EGF) is mediated by a cell surface receptor (EGF-R) which has been identified in human prostate tissues. Because of conflicting reports on the relative levels of EGF-R in prostate tumors as measured by binding of radiolabelled EGF, we have examined EGF-R expression at the level of the specific messenger RNA using a sensitive RNase protection assay. Expression of the mRNA for EGF-R was higher in carcinoma (CaP, N = 38) than in benign prostatic hyperplasia (BPH, N = 35) samples (p less than 0.01). The highest levels of EGF-R mRNA were found in the human prostatic carcinoma cell lines, PC-3 and DU145. Among the CaP samples, there was an association of higher EGF-R mRNA levels with higher tumor extent and dedifferentiation. Since EGF has also been found in prostatic tissues, the enhanced expression of the EGF-R gene may play a role in the growth of prostate tumors, possibly by an autocrine pathway.     

Expression of cyclin DI in human prostate cancer cell lines

Deregulation of cyclin expression has been found in many tumors. In this report, we studied expression of cyclin DI in three human prostate cancer cell lines: the androgen-dependent LNCaP and the androgen-independent PC3 and DU 145 cell lines. Northern blot analysis showed that DU145 and PC3 cells expressed more abundant cyclin DI than LNCaP cells. Southern blot analysis showed no evident gene amplification or rearrangement of cyclin DI in any of these cell lines. Serum starvation and replenishment were used in the cell culture to study the regulation of expression of cyclin DI. Cyclin DI mRNA expression was detected by Northern blot analysis when LNCaP cells grew in medium with serum but was not detected after serum withdrawal; however, cyclin DI mRNA was induced after serum was added. Cyclin DI mRNA expression by PC3 and DU 145 cells was detected both when they grew in medium with serum and after serum withdrawal, although expression decreased greatly after 24 hours in the PC3 cell line. Immunoprecipitation and immunohistochemical staining also showed that cyclin D I protein was always expressed in PC3 and DU 145 cells under different growth factor environment, whereas it decreased significantly in LNCaP cells deprived of serum and the level resumed again when serum was re-added. This suggests that expression of cyclin DI is regulated by exogenous growth factors in LNCaP cell line and becomes constitutive in PC3 and DU 145 cell lines.     

Differential involvement of the Fas receptor/ligand system in p53-dependent apoptosis in human prostate cancer cells

BACKGROUND: The objective of this study was to characterize the involvement of the Fas receptor/ligand system in p53-dependent apoptosis in human prostate cancer cells. METHODS: The effects of adenovirus-mediated p53 gene transfer (Ad5CMV-p53) into human prostate cancer LNCaP, DU145, and PC3 cells on their growth, apoptosis and Fas receptor/ligand expression were examined by the MTT assay, DNA fragmentation assay, and Northern blot analysis, respectively. The sensitivity of these cells to an agonistic anti-Fas receptor antibody (CH11) and the effects of an antagonistic anti-Fas ligand antibody (4H9) on Ad5CMV-p53-induced apoptosis were analyzed by the MTT assay and DNA fragmentation assay. RESULTS: Ad5CMV-p53 treatment resulted in substantial growth inhibition, induction of apoptosis and up-regulation of Fas receptor as well as Fas ligand mRNA expression in LNCaP, DU145 and PC3 cells. Despite the abundant expression of Fas receptor in all of these cells, CH11 induced apoptosis only in PC3 cells. Furthermore, 4H9 partially blocked the apoptosis induced by Ad5CMV-p53 in PC3 cells, but not in LNCaP and DU145 cells. CONCLUSIONS: The Fas receptor/ligand system is differentially involved in p53-dependent apoptosis in prostate cancer cells; therefore, reintroduction of wild-type p53 into prostate cancer cells may induce apoptosis through Fas receptor/ligand interaction as well as through an alternative pathway.     

Effect of an EGF-R selective tyrosine kinase inhibitor and an anti-androgen on LNCaP cells: identification of divergent growth regulatory pathways

BACKGROUND: The effect of an EGF-R selective tyrosine kinase (EGF-RTK) quinazoline inhibitor ZM252868 was determined on the androgen-sensitive human prostatic tumour cell line LNCaP, which can also respond via the EGF-R-regulated growth pathway for cell proliferation. Potential interaction or 'cross-talk' between steroid and the growth factor mitogen-activated protein kinase (MAPK) signalling pathway was also investigated. METHODS: The responses of LNCaP cells to various growth factors in the absence and presence of the EGF-RTK inhibitor and/or steroid and anti-androgen Casodex, was determined using cell population analysis. The effect of the inhibitor on the expression of androgen receptor, EGF-R and activated MAPK was assessed immunocytochemically and changes in the MAPK signalling cascade were also determined using Western blotting techniques. RESULTS: The ZM252868 inhibitor had no effect on LNCaP basal growth. At 100 nM and 1 microM concentrations, the inhibitor reduced the marked EGF- and TGF-alpha-stimulated LNCaP cell growth by 60% and to basal levels, respectively. Both bFGF- and 5alpha-DHT-stimulated growth were unaffected in this concentration range. The inhibitor (1 microM) decreased the expression of immunoreactive EGF-R but had no effect on androgen receptor levels. Activation of MAPK by EGF was noted, being down-regulated by the inhibitor at a concentration of 1 microM. MAPK was not activated by 5alpha-DHT. The anti-androgen Casodex reduced 5alpha-DHT-stimulated cell growth but had no effect on EGF-R mediated LNCaP growth or EGF-stimulated activated MAPK activity. Treatment with EGF and 5alpha-DHT in combination produced an additive effect on cell proliferation, with the anti-androgen and the EGF-RTK inhibitor only reducing the 5alpha-DHT- or EGF-stimulated portion of growth, respectively. CONCLUSIONS: The study demonstrated the efficacy and selectivity of the ZM252868 inhibitor in inhibiting EGF-R mediated LNCaP cell growth. Additionally, no interaction between androgen and EGF-R mediated growth pathways was determined.     

PI3K/Akt signaling regulates p27(kip1) expression via Skp2 in PC3 and DU145 prostate cancer cells, but is not a major factor in p27(kip1) regulation in LNCaP and PC346 cells

BACKGROUND: We compared the involvement of PI3K/PTEN/Akt signaling in the regulation of the cell-cycle regulator p27(kip1) and investigated the mechanism of PI3K/PTEN/Akt modulation of p27(kip1) in the prostate cancer cell lines LNCaP, PC346, PC3, and DU145. METHODS: PI3K/PTEN/Akt signaling was manipulated by wortmannin or specific siRNA. The effects on PI3K/Akt downstream effectors and p27(kip1) expression were monitored on RNA and protein levels. RESULTS: PI3K/Akt inhibition in LNCaP and PC346 cells hardly affected p27(kip1) expression. As shown in LNCaP cells, p27(kip1) expression inversely correlated with Skp2 expression, but Skp2 was not regulated by Akt. Blocking PI3K/Akt signaling in PC3 cells resulted in decreased Skp2 protein expression and increased p27(kip1). Downregulation of PTEN in DU145 cells also showed PTEN/Akt-dependent regulation of Skp2 and p27(kip1). CONCLUSIONS: In PC3 and DU145 cells, Skp2 is the main determinant in the PI3K/Akt-dependent regulation of p27(kip1). In LNCaP and PC346 cells, PI3K/Akt signaling is not a major factor in p27(kip1) regulation.     

Downregulation of androgen receptor expression by luteolin causes inhibition of cell proliferation and induction of apoptosis in human prostate cancer cells and xenografts

BACKGROUND: Androgen receptor (ARs) play a crucial role in the development and progression of prostate cancer. Recent studies have suggested that prostate cancer cell proliferation is inhibited by AR downregulation. Our aim was to investigate how luteolin, a natural flavonoid, affects cell growth and AR expression in prostate cancer cells and xenografts. METHODS: We assessed prostate cancer cell (LNCaP, DU145, and PC-3) proliferation and apoptosis by MTT assay, flow cytometric analysis, and Western analysis. AR function was measured by evaluating the AR target molecule, prostate-specific antigen (PSA), by RT-PCR, Western blotting, and enzyme-linked immunosorbent assay. We determined the mechanism of AR downregulation with cycloheximide chase assays, proteasome inhibitor, and coimmunoprecipitation experiments. The effects of luteolin on growth inhibition in vivo were examined by LNCaP xenografts in SCID mice. RESULTS: Luteolin significantly repressed prostate cancer cell proliferation and induced apoptosis in LNCaP cells. PC-3 and DU145 cells were less susceptible to luteolin-mediated growth inhibition. Luteolin simultaneously suppressed intracellular and secreted PSA levels and repressed AR mRNA and protein expression in a dose- and time-dependent manner. Luteolin reduced the association between AR and heat-shock protein 90, causing AR degradation through a proteasome-mediated pathway in a ligand-independent manner. Luteolin also suppressed LNCaP xenograft tumor growth in SCID mice. CONCLUSION: Luteolin-mediated AR downregulation contributes to the inhibition of cell proliferation and the induction of apoptosis in LNCaP human prostate cancer cells, suggesting that AR is a molecular target for luteolin-mediated anticancer activity. Luteolin may act as a chemopreventive or chemotherapeutic agent for prostate cancer.     

Inhibition of Wnt signaling downregulates Akt activity and induces chemosensitivity in PTEN-mutated prostate cancer cells

BACKGROUND: The cross-talk between Wnt signaling and the Akt pathway in prostate cancer (Pca) is still unclear. In the present study, we found that WIF-1 downregulates the Akt pathway and also enhances chemosensitivity in PTEN-null Pca cells. METHODS: Wnt inhibitory factor-1 (WIF-1), an inhibitor of Wnt proteins, was transfected into PC-3 and DU145 Pca cells. RESULTS: Akt was phosphorylated in PTEN-null PC-3 cells but underphosphorylated in PTEN-expressed DU145 cells. The levels of phosphorylated Akt in WIF-1 overexpressing PC-3 cells were lower than those in native or control vector-transfected PC-3 cells. However, WIF-1 showed no additional inhibition of already reduced Akt activity in DU145 cells. Overexpression of WIF-1 resulted in sensitizing PC-3 cells for paclitaxel to induce apoptosis. DU145 cells were more sensitive to paclitaxel but were not affected by WIF-1 transfection. The PI3K inhibitor LY294002 seemed to restore the chemosensitivity of native PC-3 cells like WIF-1 did. CONCLUSIONS: Our results show that Wnt signaling is involved in Akt activation in Pca cells. Our data also indicate the possibility that Wnt and its signaling pathway can be therapeutic targets for PTEN-mutated advanced Pca.     

Constitutive activation of the 41- and 43-kDa mitogen-activated protein (MAP) kinases in the progression of prostate cancer to an androgen-independent state

AIM: The 41- and 43-kDa mitogen-activated protein kinases (MAPK; ERK2 and ERK1, respectively) play pivotal roles in the mitogenic signal transduction pathway. We previously demonstrated that constitutive activation of the MAPK cascade was related to the carcinogenesis of human tumors. In this study, we examined whether constitutive activation of MAPK was related to the progression to androgen independence of prostate cancer. METHODS: MAPK activation was examined by the appearance of phosphorylated forms and an in vitro kinase assay in four human (androgen-dependent and independent) prostate cancer cell lines and rat prostate cancer cell line Dunning (androgen-sensitive G line, and androgen-independent AT-3, AT-6 sublines). In addition, when androgen-dependent mouse Shionogi Carcinoma 115 (SC115) cells were serially cultured without androgen to obtain androgen-independent cells, the time and degree of MAPK activation were examined. RESULTS: One of three human androgen-independent cell lines (DU145) showed constitutive activation of MAPK, while an androgen-dependent cell line (LNCaP) did not show MAPK activation. While MAPK were not activated in an androgen-sensitive Dunning G cell line, MAPK were activated in androgen-independent sublines (AT-3 and AT-6) derived from a G cell line. In addition, when SC115 cells were serially cultured without androgen, the cells 16-24 weeks after androgen removal showed MAPK activation. Furthermore, in subcloned cells, MAPK activation was observed even in the cells maintained for 9 weeks in medium without testosterone. CONCLUSIONS: The present fi ndings suggest that constitutive activation of MAPK may be associated with the acquisition of hormone independence in prostate cancer and that clonal selection after androgen removal and hormone-independent growth through the MAPK signal transduction pathway could begin at a relatively early period in the individual cells.     

Inhibitory effects of digitalis on the proliferation of androgen dependent and independent prostate cancer cells

PURPOSE: Digitalis or cardiac glycosides have been noted to induce tumor static or oncolytic effects in various types of cancer. We evaluated the effects and underlying mechanisms of cardiac glycosides, including digoxin, digitoxin and ouabain, on the proliferation of hormone dependent and independent prostate cancer cell lines. MATERIALS AND METHODS: Cell proliferation of the 3 human prostate cancer cell lines LNCaP, DU145 and PC3 was measured by 3-(4,5-dimethylthiazol-2-yle)2,5-diphenyltetralozium bromide (Sigma Chemical Co., St. Louis, Missouri) colorimetric assay. The cytotoxic effects of digitalis on prostate cancer cells were determined by lactate dehydrogenase measurements of the culture medium. Intracellular Ca2+ was measured by a dual wavelength spectrometer system. The percent of apoptotic cells after digitalis treatment was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and flow cytometry. RESULTS: Digoxin, digitoxin and ouabain significantly inhibited the proliferation of LNCaP, DU145 and PC3 cells at a dose of 1 or 10 microM. after 1 to 4 days of culture. Cytotoxicity of digitalis on the DU145 and LNCaP cells was dose dependent but cytotoxicity was not obvious in PC3. Digitalis (1 microM.) significantly increased intracellular Ca2+ in LNCaP and DU145 after 12 hours of culture but PC3 cells needed a 24-hour treatment to show any effect. In the apoptosis measurement digitalis at a dose of 1 and 10 microM. also significantly increased the percent of apoptotic cells in the LNCaP, DU145 and PC3 cell lines. Normal control human glomerular epithelial cells showed no response to digitalis treatment at all tested doses. CONCLUSIONS: Digitalis may inhibit the proliferation of prostate cancer cell lines, although the 3 cell lines showed varied sensitivity to digitalis. These effects are possibly the result of a mechanism involving sustained elevation of the concentration of intracellular Ca2+ and of apoptosis.     

Anti-proliferative and apoptotic effects of anandamide in human prostatic cancer cell lines: implication of epidermal growth factor receptor down-regulation and ceramide production

BACKGROUND: Anandamide (ANA) is an endogenous lipid which acts as a cannabinoid receptor ligand and with potent anticarcinogenic activity in several cancer cell types. METHODS: The inhibitory effect of ANA on the epidermal growth factor receptor (EGFR) levels expressed on the EGF-stimulated prostatic cancer cells LNCaP, DU145, and PC3 was estimated by ELISA tests. The anti-proliferative and cytotoxic effects of ANA were also evaluated on these human prostatic cancer cell lines by growth tests, flow cytometric analyses, trypan blue dye exclusion assays combined with the Papanicolaou cytological staining method. RESULTS: ANA induced a decrease of EGFR levels on LNCaP, DU145, and PC3 prostatic cancer cells by acting through cannabinoid CB(1) receptor subtype and this leaded to an inhibition of the EGF-stimulated growth of these cells. Moreover, the G(1) arrest of metastatic DU145 and PC3 growth was accompanied by a massive cell death by apoptosis and/or necrosis while LNCaP cells were less sensitive to cytotoxic effects of ANA. The apoptotic/necrotic responses induced by ANA on these prostatic cancer cells were also potentiated by the acidic ceramidase inhibitor, N-oleoylethanolamine and partially inhibited by the specific ceramide synthetase inhibitor, fumonisin B1 indicating that these cytotoxic actions of ANA might be induced via the cellular ceramide production. CONCLUSIONS: The potent anti-proliferative and cytotoxic effects of ANA on metastatic prostatic cancer cells might provide basis for the design of new therapeutic agents for effective treatment of recurrent and invasive prostatic cancers.