Melatonin and prostate cancer cell proliferation: interplay with castration,epidermal growth factor,and androgen sensitivity

BACKGROUND: Potential modulatory effects of melatonin on the proliferation of androgen-sensitive LNCaP and androgen-insensitive PC-3 and DU 145 prostate cancer cells were reported recently. In this study, we investigated the effects of combined melatonin and castration on LNCaP tumor growth in vivo, the interactions between melatonin and epidermal growth factor (EGF) on LNCaP cell proliferation, and melatonin actions on the proliferation of PC-3 and DU 145 cells. METHODS: Tumor development and growth in castrated nude mice inoculated with LNCaP cells or in intact animals inoculated with DU 145 cells, with or without daily melatonin treatment, were monitored by observation and caliper measurement. MT(1) receptor expression in native or transfected prostate cancer cell lines was examined by immunocytochemistry or 2-[(125)I]iodomelatonin binding. Cyclin D1 expression in LNCaP cells was assessed by Western blotting, and cell proliferation was measured by thymidine incorporation and/or cell count. RESULTS: Melatonin treatment was associated with further decreases in LNCaP tumor incidence and growth rate in castrated nude mice. Melatonin and 2-iodomelatonin (a melatonin receptor agonist) attenuated EGF-stimulated increases in LNCaP cell proliferation and cyclin D1 levels. Melatonin had no effect on the proliferation or growth of MT(1) receptor-expressing DU 145 cells, and of PC-3 cells in which MT(1) receptor protein was undetectable. The proliferation of transfected PC-3 cells expressing MT(1) receptor was unaffected by 2-iodomelatonin. CONCLUSION: Together with previous data, the present results indicate synergistic action of melatonin and castration in inhibiting the growth of androgen-sensitive LNCaP tumor. Androgen-sensitive prostate cancer cell proliferation may be modulated by opposite changes in cyclin D1 levels induced by activated MT(1) and EGF receptors. In androgen-insensitive prostate cancer cells, MT(1) receptor-mediated signal transduction may become defective not only through changes in membrane receptor protein expression and/or functions, but also by means of alterations in downstream postreceptor signaling events.     

NK4基因转染对前列腺癌DU145细胞增殖、迁移、侵袭和凋亡的影响

研究HGF拮抗剂NK4在前列腺癌中的作用。将包含NK4cDNA的表达载体pBudCE4．1-EGFP-NK4转染到DU145细胞中。体外实验检测白分泌的NK4对肿瘤细胞增殖、迁移、转移及凋亡的影响。体内实验裸鼠分为三组,分别皮下种植DUl45、空质粒转染的Dul45和NK4转染的DUl45细胞,检测皮下肿瘤的大小、细胞凋亡及细胞增殖情况。体外实验结果显示转染NK4的DUl45细胞可以分泌NK4蛋白。自分泌的NK4抑制HGF诱导的肿瘤细胞增殖、迁移及转移,促进凋亡（P〈0．01）。NK4可以调节HGF受体c－Met及其下游ERKl与Akt1／2蛋白的活性。体内实验显示,NK4转染DUl45细胞组的肿瘤生长及细胞增殖受到抑制,同时肿瘤细胞凋亡增加。本实验显示包含NK4cDNA的表达载体转染前列腺癌细胞可以有效地调节肿瘤细胞的增殖、迁移、侵袭及凋亡。NK4作用于HGF／c－Met可以作为前列腺癌治疗的一个有效靶点。     

DU145 human prostate cancer cells express functional receptor activator of NFkappaB: new insights in the prostate cancer bone metastasis process

Prostate cancer metastases to bone are observed in around 80% of prostate cancer patients and represent the most critical complication of advanced prostate cancer, frequently resulting in significant morbidity and mortality. As the underlying mechanisms are not fully characterized, understanding the biological mechanisms that govern prostate cancer metastases to bone at the molecular level should lead to the determination of new potential therapeutic targets. Receptor activator of NFkappaB ligand (RANKL)/RANK/Osteoprotegerin (OPG) are the key regulators of bone metabolism both in normal and pathological condition, including prostate cancer bone metastases. In the present study, we demonstrated that human prostate cancer cell lines, DU145 and PC3 express biologically functional RANK. Indeed, soluble human RANKL (shRANKL, 100 ng/ml) treatment induced ERK 1/2, p38 and IkappaB phosphorylations in these cells. shRANKL administration also promoted DU145 and PC3 prostate cancer cell invasion in vitro. Whereas human OPG (hOPG) administration alone (100 ng/ml) had no marked effect, combined association of both agents abolished the RANKL-induced DU145 cell invasion. As RANKL had no direct effect on DU145 cell proliferation, the observed effects were indeed related to RANKL-induced cell migration. DU145 human prostate cancer cells promoted osteoclastogenesis of osteoclast precursors generated from mouse bone marrow. Moreover, DU145 cells produced soluble factor(s) that up-regulate the proliferation of MC3T3-E1 pre-osteoblasts through the activation of the ERK 1/2 and STAT3 signal transduction pathways. This stimulation of pre-osteoblast proliferation resulted in an increased local RANKL expression that can activate both osteoclasts/osteoclast precursors and prostate cancer cells, thus facilitating prostate cancer metastasis development in bone. We confirm that RANKL is a factor that facilitates metastasis to bone by acting as an activator of both osteoclasts and RANK-positive prostate cancer cells in our model. Furthermore, the present study provides the evidence that blocking RANKL-RANK interaction offer new therapeutic approach not only at the level of bone resorbing cells, but also by interfering with RANK-positive prostate cancer cells in the prostate cancer bone metastasis development.     

17alpha-estradiol inhibits LAPC-4 prostatic tumor cell proliferation in cell cultures and tumor growth in xenograft animals

BACKGROUND: Blockade of androgen activity is a major effective therapy for advanced prostate cancer. Estrogen analogs have been used for prostate cancer therapy for years presumably by inhibiting testosterone biosyntheses, but with considerable adverse events due to their classic estrogenic activity. With the discovery of the estrogen receptor (ER) beta and its presence in prostate tumor cells, evaluation of estrogen analogs with less classic estrogenic activity in prostate cancer therapy is emerging. METHODS: The effects of 17alpha-estradiol (alphaE2), a stereo-isomer of 17beta-estradiol (betaE2), on dihydrotestosterone (DHT)-induced cell growth and gene expressions were examined in androgen-dependent LAPC-4 prostatic tumor cells and in LAPC-4 xenograft animals, and compared to those of betaE2. RESULTS: Both alphaE2 and betaE2 attenuated DHT induction of PSA gene expression, cell proliferation, and cell growth in cultured LAPC-4 cells. The inhibition of cell proliferation was associated with a blockade of DHT-induced cyclin A and cyclin D1 expression by alphaE2 and betaE2. In LAPC-4 xenograft mice, alphaE2 significantly inhibited tumor growth without altering the plasma testosterone level, while betaE2 failed to inhibit tumor growth even though it significantly inhibited PSA gene expression. CONCLUSION: alphaE2 is an effective agent for inhibition of DHT-induced PSA, cyclin A, cyclin D1 gene expression, and cell proliferation in LAPC-4 cells, and tumor growth in LAPC-4 xenograft mice.     

Vitamin D3 analogue inhibits keratinocyte growth factor signaling and induces apoptosis in human prostate cancer cells.

BACKGROUND: Prostate cancer is a worldwide significant health care problem, due to its high incidence and mortality. In particular, androgen-independent tumors have the worst prognosis, because they are refractory to almost all kinds of available therapy. Hence, there is the need of new treatment opportunities targeting androgen-independent, growth factor-mediated, tumor signaling. One of these new promising opportunities is vitamin D3 and its related analogues. METHODS: We investigated the effect of a vitamin D3 analogue, analogue (V), on proliferation of several human prostate cancer cells in basal condition and after treatment with KGF, one of the intraprostatic growth factors that might participate in the progression of prostate cancer. In addition, in the androgen-independent cell line DU 145, we also studied the effect of analogue (V), KGF, and their mutual interaction on protein tyrosine phosphorylation, bcl-2 expression and apoptosis. RESULTS: Overall, we found that analogue (V) dose-dependently decreased basal and KGF-induced prostate cancer cell growth, although to a different extent. Maximal effect was obtained in DU 145 cells. In these cells, KGF stimulated tyrosine phosphorylation of a protein corresponding to its receptor, induced bcl-2 expression, and prolonged cell survival. Analogue (V) not only counteracted all these KGF-mediated events, but also decreased basal bcl-2 expression, therefore, allowing DU 145 cells to undergo an apoptotic program. CONCLUSIONS: Our results indicated that in prostate cancer cells analogue (V) decreased basal and KGF-induced cell proliferation. This effect, at least in DU 145 cells, is in part mediated by negative interactions with cell survival and KGF signaling.     

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.     

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.     

核小体结合蛋白调控Survivin基因对前列腺癌DU145凋亡的作用

目的探讨抑制人核小体结合蛋白1（NSBP1）基因后促进非激素依赖性前列腺癌细胞系DU145凋亡的作用机制。方法通过慢病毒lentivirus-NSBP1转染非激素依赖性前列腺癌细胞系DU145后,MTT法观察细胞活性变化,流式细胞仪检测细胞的凋亡情况,应用Western-blot技术检测NSBP1、Survivin蛋白的变化情况。结果抑制NSBP1表达水平后非激素依赖性前列腺癌细胞系DU145细胞活性降低,凋亡增加,同时Survivin蛋白的表达也随之降低。结论通过慢病毒转染抑制NSBP1的表达可以促进非激素依赖性前列腺癌细胞系DU145凋亡,NSBP1可能是通过调控Survivin基因的变化影响细胞的增值凋亡。     

Radiosensitivity of prostate cancer cells is enhanced by EGFR inhibitor C225

PurposeTo determine the direct effects of the epidermal growth factor receptor (EGFR) inhibitor C225 on the radiosensitivity of human prostate cancer cells.Experimental designHuman prostate cancer DU145 cells were irradiated with ⁶⁰Co (1.953 Gy/min) at various doses in the presence or absence of C225. The cellular proliferation and cell-survival rate were evaluated by MTT and colony-forming assays after irradiation. The cell-cycle distribution, cell apoptosis, and MAPK expression were investigated using FCM. The expression of Cyclin D1, CDK2, CDK4, and Survivin were determined by RT-PCR.ResultsThe RBE in the C225 group compared with that in the control group was 1.39. Cells treated with C225 and irradiated at 4 Gy predominantly exhibited G₀/G₁ phase arrest and significant decrease in the fraction of cells in the S phase in comparison with those in the control cells, respectively. An evidently higher apoptosis rate on irradiation at 4 Gy was observed in C225-treated cells compared with that in the control cells. Decreased cell proliferation and increased cell death were further supported by the down-regulation of cyclin D1, CDK2, CDK4, and survivin in C225-treated DU145 cells, as determined by RT-PCR. Furthermore, C225 significantly inhibited the phosphorylation of P38-MAPK in DU145 cells.ConclusionsThe EGFR inhibitor C225 increased the radiosensitivity of DU145 cells through antiproliferative effect, inhibition of clonal growth, G₀/G₁ phase arrest, apoptosis induction, and inhibition of EGFR-signaling pathways by the down-regulation of MAPK activation.     

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.     

熊果酸对前列腺癌细胞雄激素非依赖性的逆转作用

目的 探讨中药成分熊果酸对雄激素非依赖性前列腺癌(AIPC)的治疗作用及其机制.方法 应用熊果酸处理体外培养的人雄激素依赖性前列腺癌(ADPC)细胞株LNCaP和AIPC细胞株DU145,噻唑蓝(MTT)比色法检测细胞活性及对人工合成雄激素R1881的反应性,免疫细胞化学检测熊果酸对雄激素受体(AR)、糖皮质激素受体(GR)、前列腺特异性抗原(PSA)及成活因子HSP90和白细胞介素(IL)-6表达的影响,逆转录.聚合酶链反应(RT-PCR)检测熊果酸对DU145细胞AR mRNA表达的影响.结果 熊果酸对不同浓度雄激素下的LNCaP细胞均呈浓度和时间依赖性生长抑制,20 mg/L的熊果酸作用96 h对LNCaP细胞的抑制率近50%.0.1 nmoL/L的R1881为最适生长浓度,熊果酸作用后,LNCaP细胞生长的最适雄激素浓度上升了10倍;熊果酸对DU145细胞的生长有浓度和时间依赖性抑制效应,DU145细胞对AR阻断剂羟氟他胺缺乏反应,熊果酸作用同时再应用氟他胺比单纯熊果酸的作用更明显,对细胞抑制率明显上升.熊果酸作用后,LNCaP和DUl45细胞IL-6、HSF90表达均明显下降(P＜0.05),DU145细胞GR表达明显降低(P＜0.01),AR和PSA蛋白及AR mRNA出现再表达.结论 熊果酸能改善前列腺癌细胞对雄激素的反应性,使LNCaP细胞对雄激素的依赖性加强,并诱发了DU145细胞对雄激素的反应性,其部分机制是降低了GR、HSP90、IL-6的表达并促进AR再表达.     

Antitumor activity and downregulation of pro-angiogenic molecules in human prostate cancer cells by a novel thiazolidione compound

BACKGROUND: Current treatments for prostate cancer are effective in many patients with locally advanced disease, but many of these patients eventually have recurrence. It is therefore important to develop alternative therapeutic agents with improved efficacy and tolerability. We recently identified a synthetic thiazolidin compound, 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidione (DBPT), that induces apoptosis in human colon cancer cells, independent of p53 and P-glycoprotein status. Here, we investigated the antitumor properties and mechanisms of action of this compound in human prostate cancer cell lines. METHODS: The effect of DBPT on cell-cycle progression and apoptosis in LNCaP and DU145 cells was examined by flow cytometry and Western blotting. The effect of DBPT on pro-angiogenic molecules was analyzed by Western blotting and by an enzyme-linked immunosorbent assay. RESULTS: DBPT inhibited the growth of LNCaP and DU145 cells with 50% inhibitory concentrations ranging from 1.6 to 5.9 microM. Treating LNCaP and DU145 cells with DBPT led to a time-dependent cell-cycle arrest in the G(2)/M phase and increased levels of G(2)/M checkpoint proteins, such as cyclin B1, cdc25C, phosphorylated histone H(3), and MPM-2. DBPT induced the phosphorylation of Bcl-xL and Bim, and induced apoptosis, as evidenced by cleavage of caspase and poly (ADP-ribose) polymerase. DBPT also effectively induced apoptosis in Bcl-2-overexpressing DU145 cells. Furthermore, DBPT decreased hypoxia-inducible factor 1 alpha and vascular endothelial growth factor expression in LNCaP cells under both normoxia and hypoxia. CONCLUSIONS: DBPT can suppress proliferation, induce apoptosis, and down regulate pro-angiogenic molecules in prostate cancer cells, and might be useful in treating prostate cancer.     

ASF1B通过调控P53相关信号通路促进前列腺癌迁移和增殖的研究

目的研究组蛋白伴侣抗沉默功能1B（ASF1B）对前列腺癌（PCa）迁移和增殖能力的影响,探索ASF1B调控P53相关信号通路的具体分子机制。 方法根据TCGA数据库中的数据分析ASF1B在前列腺癌患者中的生存预后情况,使用细胞小干扰RNA（SiRNA）敲低ASF1B表达后,通过Transwell细胞迁移实验比较细胞的运动能力,通过CCK8实验和平板克隆实验比较细胞的增殖能力,使用细胞凋亡实验比较细胞的凋亡率,通过转录组二代测序比较敲低ASF1B后各个信号通路的改变情况,通过Western-Blot实验及Q-PCR实验比较P53相关信号通路分子的蛋白质和mRNA水平变化,以阐明ASF1B对前列腺癌细胞的迁移及增殖能力的影响。 结果SiRNA敲低ASF1B表达后,DU145和PC3细胞的运动能力降低,相同时间内穿过小室的细胞数减少。敲低ASF1B表达后,DU145和PC3细胞的细胞活力和集落形成个数及大小降低,细胞增殖能力下降。敲低ASF1B表达后,DU145和PC3细胞的凋亡率增加。敲低ASF1B表达后,PC3细胞的转录组二代测序结果提示P53信号通路受到调控（P<0.01）。敲低ASF1B表达后,PC3细胞内P53相关通路的P21、IGFBP3、BBC3表达量上升,Cyclin D、Snail、Slug表达量下降。 结论ASF1B以非P53依赖的形式通过P53相关信号通路调控前列腺癌细胞的迁移及增殖,ASF1B有望成为前列腺癌的诊断及治疗的新型靶点。     

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.     

Effects of adipocytes on the proliferation and differentiation of prostate cancer cells in a 3‐D culture model

Objective: To investigate how the mechanism of adipocyte–prostate cancer cell interaction affects the proliferation and differentiation of prostate cancer cells. Methods: An androgen‐dependent cell line (LNCaP), two androgen‐independent cell lines (PC‐3, DU145), and mature adipocytes harvested from male Wistar rats were used. Cancer cells were co‐cultured with the isolated mature adipocytes in 3‐D collagen gel matrix culture. The morphology and proliferative ability of the prostate cancer cells were examined. With regard to the activation of the phosphatidylinositol 3‐kinase (PI3K) pathway, the expression of phosphatase and tensin homologue deleted on chromosome ten (PTEN), Akt and Bad were determined by immunohistochemistry. Results: LNCaP cells co‐cultured with adipocytes formed larger clusters than those of the control. PC‐3 cells co‐cultured with adipocytes did not form larger clusters, but formed spherical and spindle‐shaped cells. The phosphorylation of Akt in PC‐3 cells was greater in the co‐cultured group compared with the controls, but there were no significant differences in the phosphorylation of Akt with regard to LNCaP and DU145 cells. Conclusions: Adipocytes could modulate the proliferation and differentiation of prostate cancer cell lines. Activation of the PI3K pathway might be involved in the prostate cancer cell–adipocyte interaction.     

Mitogenic signaling in androgen sensitive and insensitive prostate cancer cell lines

PURPOSE: To investigate the role of a specific mitogen activated protein kinase, extracellular signal-regulated kinase (ERK), in regulating cell proliferation induced by three potentially important prostate cancer mitogens that signal via different classes of receptors. MATERIALS AND METHODS: Androgen sensitive (LNCaP) and insensitive (PC-3) prostate cancer cell lines were used in these studies. Epidermal growth factor (EGF), lysophosphatidic acid (LPA), and dihydrotestosterone (DHT) were the mitogenic stimulants and AG1478, a receptor tyrosine kinase inhibitor, and PD98059, an inhibitor of MEK, were the chemical inhibitors used in this study. Cell proliferation was measured using the WST-1 assay and ERK expression and activation was determined by immunoblotting for phospho- and total ERK. RESULTS: In androgen-sensitive LNCaP cells, epidermal growth factor (EGF) and dihydrotestosterone (DHT) both enhanced cell proliferation. EGF-stimulation dramatically increased ERK phosphorylation while DHT did not. In the androgen-insensitive cell line, PC-3, EGF- and LPA-induced ERK phosphorylation and cell proliferation. Inhibition of EGF- and LPA- induced ERK activation with the EGF receptor inhibitor, AG1478, or the MEK inhibitor, PD98059, attenuated their proliferative effects. Neither inhibitor had an effect on DHT stimulated cell proliferation. CONCLUSIONS: These data demonstrate heterogeneity of mitogenic signaling in prostate cancer cells, and support the hypothesis that androgens and growth factors utilize divergent signaling pathways in prostate cancer to induce proliferation.