Contragestazol （DL111-IT） inhibits proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo

Aim： To evaluate the antiproliferative activity of contragestazol （DL111-IT） on the human prostate cancer cell line PC3 in vitro and in vivo and to elucidate its potential molecular mechanisms. Methods： The cell killing ability of DL111-IT was measured by the 3-（4,5-dimethylthia-zol,2-yl）-2,5-diphenyltetrazolium bromide （MTT） reagent assay method and the tumor xenograft model. The cell cycle was analyzed by flow cytometry and protein expression, including retinoblastoma （pRb）, cyclin-dependent kinase 4 （CDK4） and cyclin D 1, was detected by Western blotting. Results： DL111-IT exhibited high efficiency on cell growth inhibition of the human androgen-independent prostate cancer cell line PC3. The drug concentration that yielded 50 % cell inhibition （IC50 value） was 9.9 mg/mL. In the PC3 tumor xenograft study, DL111-IT （1.25 mg/kg-20.0 mg/kg） given once a day for 10 days significantly inhibited tumor growth, with the inhibition rate ranging from 21% to 50 %. Flow cytometric analysis indicated that DL111-IT could cause GI arrest in the PC3 cell line, but not apoptosis. DL111-IT enhanced pRb expression and down-regulated CDK4 and cyclin D 1 expression, suggesting that cell cycle regulation might contribute to the anticancer property of DL 111- IT. Conclusion： DL111-1T inhibits the proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo by a cell cycle regulation pathway.     

Suppression of LNCaP prostate cancer xenograft tumors by a prostate-specific protein tyrosine phosphatase,prostatic acid phosphatase

BACKGROUND: Although the molecular mechanism of androgen-independent prostate cancer growth and progression has been gradually elucidated, there is limited effective treatment for this prevalent disease. Human prostatic acid phosphatase (PAcP), a major protein tyrosine phosphatase in prostate epithelium, plays a critical role in regulating the growth of prostate cancer cells. In prostate carcinomas, the expression of cellular PAcP decreases. To explore directly the possible therapeutic potential of cellular PAcP, we investigated the suppression effect of PAcP by utilizing cDNA direct intratumoral administration in androgen-independent LNCaP xenograft tumors. METHODS: An androgen-independent LNCaP cell model (C-33 and C-81 cells) and stable subclones of PAcP cDNA-transfected C-81 cells (LNCaP-23 and LNCaP-34 cells) were used for the experiments. We examined the growth property and expression of PAcP and c-ErbB-2 of these different LNCaP cells in vitro and in vivo. We subsequently investigated the growth suppression effect of PAcP cDNA intratumoral injection in pre-established C-81 xenograft tumors, and analyzed the expression of PAcP, prostate-specific antigen (PSA), proliferating cell nuclear antigen (PCNA), and c-ErbB-2 in the tumors by immunohistochemistry and Western blotting. RESULTS: The different LNCaP cells exhibited different growth property and tumorigenicity, both in cell culture and xenograft. Biochemical characterizations revealed that the level of cellular PAcP correlated negatively with the growth property of different LNCaP cells, while the level of tyrophosphorylated c-ErbB-2 had an inverse correlation with cellular PAcP. The single intratumoral administration of the wild type PAcP cDNA showed a significant suppression effect on C-81 xenograft tumor growth, compared to vector alone-injected control (P<0.05). In the tumors injected with this PAcP cDNA, the PAcP expression was detected 1 week (wk) after injection, but was undetectable at 6 wk, which inversely correlated with the level of tyrophosphorylated c-ErbB-2 and the degree of cell proliferation indicated by PCNA staining. CONCLUSIONS: Our results clearly demonstrated that cellular PAcP has a suppression effect on the growth of androgen-independent LNCaP xenograft tumors. This effect occurs at least partly through the dephosphorylation of c-ErbB-2 by PAcP, the prostate-specific protein tyrosine phosphatase. The data indicates that human PAcP could be utilized in the corrective gene therapy for a subgroup of androgen-independent human prostate cancer cells that lack cellular PAcP expression.     

米非司酮诱导雄激素非依赖性前列腺癌细胞凋亡的实验研究

目的探讨米非司酮在体外诱导雄激素非依赖性前列腺癌DU-145、PC-3细胞凋亡的作用。方法采用四甲基偶氮唑蓝法检测1,10,50和100μmol／L米非司酮作用于前列腺癌DU-145、PC-3细胞24～120h的吸光度（A）值,用流式细胞仪检测10μmol／L米非司酮作用24和48h后DU．145、PC-3细胞凋亡率的变化;采用免疫组化法检测米非司酮作用DU-145、PC．3细胞后bax、bcl-2、血管内皮生长因子（VEGF）蛋白表达的变化。结果1μmol／L米非司酮组的A值与对照组相比,差异无统计学意义（P〉0．05）;10,50和100μmol／L米非司酮组的A值与对照组比较,差异有统计学意义（P〈0．01）;米非司酮对前列腺癌DU-145、PC-3细胞的抑制作用呈时间-剂量依赖性。10μmol／L米非司酮作用前列腺癌DU-145细胞24和48h的凋亡率分别为15．3％和30．4％,PC-3细胞的凋亡率分别为22．2％和32．0％。经10μmol／L米非司酮作用后,DU-145、PC-3细胞中VEGF和bcl-2蛋白的表达明显减少,而bax的表达显著增加（P〈0．05）。结论米非司酮以时间．剂量依赖性方式抑制激素非依赖性前列腺癌DU-145和Pc-3细胞的增殖,其作用可能是通过降低VEGF蛋白的表达。从而下调bcl-2、激活bax蛋白的表达来实现。     

MK2206 potentiates cisplatin-induced cytotoxicity and apoptosis through an interaction of inactivated Akt signaling pathway

ObjectivesTo improve conventional chemotherapeutic efficacy, it is important to detect new molecular markers for chemosensitivity and possible accelerating cell-killing mechanisms. In this study, we investigated how MK2206, an allosteric Akt inhibitor, enhances the cisplatin (CDDP)-induced cytotoxicity and apoptosis in urothelial cancer cells.Materials and methodsWe examined bladder cancer cell lines for the expression of phospho(p)-Akt and its downstream targets by Western blot. The potential antitumor effects were analyzed by MTT assay in vitro and by subcutaneous xenograft models in vivo. The cell invasion was examined by transwell invasion assay, and the activities of the Akt signaling pathway and expression of apoptosis-related proteins were measured by Western blot.ResultsThe expression of p-Akt and its downstream targets was increased in invasive bladder cancer cell lines vs. in noninvasive bladder cancer cell lines. MK2206 (500 nM) inhibited cell invasion in UMUC3 cell line and significantly increased the susceptibility of bladder cancer cell lines to CDDP. When used in combination with CDDP, MK2206 (500 nM) enhanced CDDP-induced cytotoxicity and apoptosis, with suppressed expression of p-Akt and its downstream targets. In vivo MK2206 combined with CDDP effectively suppressed tumor growth in subcutaneous xenograft models.ConclusionsThese results suggest that concomitant use of MK2206 could promote the CDDP-induced cytotoxicity and apoptosis in urothelial cancer cell lines through the inhibited expression of the Akt pathway. This combined treatment may provide a new therapeutic option to enhance chemosensitivity in bladder cancer.     

Increased fatty acid synthase as a therapeutic target in androgen-independent prostate cancer progression

BACKGROUND: Fatty acid synthase (FAS) performs the anabolic conversion of dietary carbohydrate or protein to fat. FAS expression is low in most normal tissues, but is elevated in many human cancers, including androgen-sensitive and androgen-independent prostate cancer. METHODS: Immunohistochemical evaluation of FAS expression was performed in human prostate cancer specimens under various states of androgen ablation. In vitro and in vivo prostate cancer models were evaluated for FAS expression and activity under androgenic and androgen-depleted conditions, and were tested for sensitivity to antimetabolite drugs that target fatty acid synthesis. RESULTS: While FAS expression in the prostate was androgen responsive, it persisted or was reactivated in human prostate carcinoma after androgen ablation, and was high in 82% of lethal tumors examined at autopsy. Similar patterns of FAS expression and fatty acid synthesis were seen in cell culture and xenograft models of human prostate cancer. Pharmacologic inhibition of FAS resulted in a dose-dependent reduction of tumor growth in these models, including fourfold inhibition of an androgen-independent human prostate cancer xenograft with little associated toxicity. CONCLUSIONS: The data suggest that FAS expression/FA synthesis provides an important functional aspect of the malignant phenotype in prostate cancer, perhaps supporting cell growth or survival. FAS expression may be upregulated by alternate signaling pathways important for prostate cancer growth under androgen withdrawal. The re-emergence of FAS expression and activity during the development of androgen independence demonstrate that FAS may serve as a novel target for antimetabolite therapy in prostate cancer.     

MALAT1对膀胱癌UMUC3细胞功能的影响及下游基因的筛选

【摘要】目的 研究MALAT1对膀胱癌UMUC3细胞系增殖、迁移、侵袭、克隆形成能力的影响,探讨MALAT1在膀胱癌中的作用机制。方法 化学合成针对MALAT1的siRNA,脂质体法转染UMUC3细胞。反转录实时定量聚合酶链式反应(RT-qPCR)测定转染效率,MTS法检测增殖变化,Transwell法检测迁移、侵袭改变,克隆形成实验观察克隆形成能力变化,5-乙炔基-2′-脱氧尿苷(EDU)法检测增殖及周期改变,基因芯片筛选潜在作用通路。结果 干扰MALAT1表达后,膀胱癌UMUC3细胞株增殖、侵袭、迁移、克隆形成能力下降,基因芯片结果发现肿瘤通路相关基因MMP9等明显改变。 结论 MALAT1促进UMUC3膀胱癌细胞的增殖、侵袭、迁移、克隆形成能力,提示可能与其引起肿瘤通路相关基因的激活和失活相关。     

三氧化二砷对前列腺癌细胞株PC-3生长影响的研究

目的 :探讨三氧化二砷 (As2 O3 )对雄激素非依赖性人前列腺癌细胞株PC 3生长的影响及其机制。　方法 :通过光学显微镜观察As2 O3 处理前后培养的PC 3细胞生长和形态的变化 ,采用MTT法了解不同浓度As2 O3 作用后PC 3细胞的生长抑制曲线 ,应用流式细胞仪Annexin V FITC/PI双染法分析不同浓度As2 O3 处理的PC 3细胞的凋亡情况。　结果 :As2 O3 作用后 ,PC 3细胞形状变圆 ,体积变小 ,胞质透亮度下降 ,部分细胞脱落悬浮于培养基中。在7.81 2 5、1 5 .6 2 5、31 .2 5 0、6 2 .5 0 0、1 2 5、2 5 0、5 0 0 μmol/LAs2 O3 作用 4 8h和 72h后 ,MTT法检测细胞生长抑制率分别为(0 .0 6± 0 .99)、(1 5 .0 1± 1 .1 2 )、(2 9.2 1± 1 .31 )、(34.32± 1 .1 4 )、(4 0 .5 1± 1 .81 )、(6 9.39± 1 .74 )、(73.1 9± 2 .4 1 ) %和(0 .0 4± 1 .5 1 )、(1 6 .1 9± 1 .0 4 )、(4 3.6 1± 1 .1 2 )、(5 6 .6 6± 1 .2 3)、(73.1 3± 2 .6 1 )、(85 .2 2± 1 .74 )、(91 .4 1± 2 .81 ) %。用流式细胞仪检测经过 0 (对照组 )和 0 .1、1 .0、3.0、5 .0、2 0 .0、5 0 .0 μmol/LAs2 O3 作用 4 8、72h后的PC 3细胞 ,凋亡率分别为 0 .87%、5 .33%、8.94 %、9.6 6 %、1 2 .5 6 %、4 5 .5 9%、6 9.0 9%和 0 .1 3%、1 3.4 9%、     

Anti‐PSMA immunotoxin as novel treatment for prostate cancer? High and specific antitumor activity on human prostate xenograft tumors in SCID mice

BACKGROUND: Expression of the prostate specific membrane antigen (PSMA) is highly restricted to prostate epithelial cells. Therefore, toxin-based immunotherapy against this antigen may represent an alternative therapeutic option for prostate cancer. For these purposes, the effects of the recombinant anti-PSMA immunotoxin A5-PE40 on prostate tumor growth were investigated in vitro and in vivo. METHODS: The in vitro binding and cytotoxicity of A5-PE40 were tested on the PSMA-expressing prostate cancer cell line C4-2 and on the PSMA-negative cell line DU145 by flow cytometry and WST assays. The binding of the immunotoxin to SCID mouse xenografts and to various mouse organs was examined by Western blot analysis. In vivo, the antitumor activity of the immunotoxin was tested by injecting A5-PE40 in mice bearing C4-2 or DU145 xenografts. RESULTS: In vitro, a specific binding of A5-PE40 to C4-2 cells could be shown with a concentration-dependent cytotoxicity (IC(50) value=220 pM). In the next step, a specific binding of the immunotoxin to C4-2 xenografts could be demonstrated. In contrast, no binding on mouse organs expressing high homologous mouse PSMA was found. The treatment of mice with C4-2 tumors caused a significant inhibition of tumor growth in vivo, whereas DU145 xenografts remained totally unaffected. CONCLUSIONS: A5-PE40 represents a recombinant anti-PSMA immunotoxin with potent antitumor activity in mice bearing human prostate cancer xenograft tumors. Therefore, A5-PE40 could be a promising candidate for therapeutic applications in patients with prostate cancer.     

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.     

Therapy for pancreatic cancer with a recombinant humanized anti-HER2 antibody (herceptin)

The HER2/neu oncogene is overexpressed in human pancreatic cancer, but the clinical significance of that overexpression is uncertain. In the present study we investigated the antitumor efficacy of Herceptin, a new recombinant humanized anti-HER2/neu antibody, which exhibits cytostatic activity on breast and prostate cancer cells that overexpress the HER2 oncogene. That antibody may retard tumor growth in certain patients with those diseases. We quantified HER2 expression in various human pancreatic cancer cell lines and studied the bioactivity of this antibody both in vitro and in vivo. Growth inhibition by Herceptin was observed in vitro in cell lines with high levels of HER2/neu expression. Cell lines with low levels of this protein did not respond significantly to the antibody. In vivo we studied two different pancreatic cancer cell lines in an orthotopic mouse model of the disease. Herceptin treatment suppressed tumor growth in the MIA PaCa-2 tumor cell line, which expressed high levels of HER2/neu. These data suggest that Herceptin treatment of patients with pancreatic cancer who express high levels of the HER2/neu oncogene may be reasonable. Supported by the Ronald S. Hirshberg Pancreatic Cancer Research Foundation, Los Angeles, Calif. Presented at the Forty-First Annual Meeting of The Society for Surgery of the Alimentary Tract, San Diego, Calif., May 21–24, 2000.     

A neutralizing anti-fibroblast growth factor (FGF) 8 monoclonal antibody shows anti-tumor activity against FGF8b-expressing LNCaP xenografts in androgen-dependent and -independent conditions

BACKGROUNDS: Fibroblast growth factor 8-isoform b (FGF8b) has been detected in human clinical sex-organ related cancers including hormone-refractory prostate cancer. There are, however, few relevant experimental models. A murine monoclonal anti-FGF8 antibody, KM1334, has been shown to neutralize FGF8b and inhibit the growth of androgen-dependent mouse mammary SC-3 cells in vitro and in vivo. In the present study, we evaluated the anti-tumor activity of KM1334 against androgen-dependent and -independent progression of FGF8b-expressing human prostate cancer xenografts. METHODS: FGF8b cDNA was transfected into androgen-dependent human prostate cancer cell line LNCaP, and its xenograft tumors were established subcutaneously in SCID mice with or without castration. KM1334 at the dose of 400 microg/head was injected twice weekly. RESULTS: FGF8b-expressing LNCaP cells secreted FGF8b, showed enhanced level of Erk1/2 phosphorylation, and showed more potent growth properties than mock-expressing cells in vitro and in vivo. KM1334 reduced these properties in vitro, inhibited tumorigenecity in vivo (T/C=0.33), and showed anti-tumor activity against established tumors (T/C=0.47) of FGF8b-expressing cells. FGF8b-expressing LNCaP tumors were androgen-dependent. However, they recurred as androgen-independent FGF8b positive tumors after castration. KM1334 also inhibited the growth of established FGF8b-expressing tumors in the androgen-independent states (T/C=0.47). CONCLUSIONS: These results indicate that humanized monoclonal antibodies, conserving the paratope of KM1334, are a promising candidate for therapy of FGF8b-expressing clinical prostate cancers. Follow-up studies using xenograft models with clinical FGF8b-expressing tumors are required to validate these early findings.     

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.     

Small interference RNA targeting heat-shock protein 27 inhibits the growth of prostatic cell lines and induces apoptosis via caspase-3 activation in vitro

OBJECTIVES: To evaluate synthetic small interference RNA (siRNA) compounds targeting heat-shock protein 27 (Hsp27) as an alternative approach to Hsp27 'knockdown' in prostate cancer cells, as Hsp27 expression is highly up-regulated in prostate cancer cells after androgen withdrawal or chemotherapy, to become uniformly highly expressed in androgen-independent (AI) prostate cancer. MATERIALS AND METHODS: We recently showed that targeting Hsp27 by a 2'-methoxyethyl modified phosphorothioate antisense oligonucleotide, OGX-427, inhibits Hsp27 expression and enhances hormone- and chemotherapy in prostate cancer xenograft models. In the present study, a 'gene walk' screening different siRNAs was initially used in PC-3 and LNCaP cells to determine the most potent sequence to down-regulate Hsp27 mRNA and protein levels. The effects of Hsp27 silencing on in vitro growth rates were studied by tetrazolium-blue and crystal violet assays. Apoptosis was determined by single-stranded DNA nuclear and cleaved caspase-3 immunostaining, as well as flow cytometry. Spotted microarrays with 14,000 human oligonucleotides were used to examine changes in gene expression. RESULTS: Low concentrations of 1 nm siRNA decreased Hsp27 mRNA levels by 19-fold and suppressed protein expression to undetectable levels. Silencing of Hsp27 in prostate cancer cells by siRNA # 2 increased apoptotic rates 2.4-4 fold and caused 40-76% inhibition of cell growth in LNCaP and PC-3 cells. Characteristic cleavage of caspase-3 occurred after treatment with Hsp27 siRNA (1 nm). cDNA microarray analysis from LNCaP and PC-3 cell lines revealed differential gene expression profiles after Hsp27 down-regulation that could be used to identify various survival pathways involved in androgen-dependent and AI growth. CONCLUSIONS: These findings illustrate the potential utility of Hsp27-silencing therapy and highlight Hsp27 siRNA strategies as a novel and highly effective tool, with the potential for future targeted therapy in enhancing the efficacy of chemotherapy in advanced prostate cancer.     

Induction of apoptosis in human bladder cancer cells in vitro and in vivo caused by FTY720 treatment

PURPOSE: FTY720 is a unique immunosuppressant that induces apoptosis in activated lymphocytes but not in other hematopoietic cells. We examined whether FTY720 has anticancer effects on human bladder cancer cells by inducing apoptosis and we investigated its molecular pathway. MATERIALS AND METHODS: We used the 3 human bladder cancer cell lines T24, UMUC3 and HT1197, and the human fibroblast derived cell line CRL-2096 (American Type Tissue Collection, Rockville, Maryland) in this study. The difference in drug susceptibility to FTY720 in cancer cells and fibroblasts was examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cell growth assays. FTY720 induced apoptosis was determined by morphological analysis under light and electron microscopy, and DNA electrophoresis, and its molecular pathway was evaluated by Western blot analysis focusing on the p42/p44 mitogen activated protein kinase pathway. We then tested the in vivo effect of this agent using 2 mouse models of human bladder cancer xenograft. RESULTS: FTY720 treatment in vitro induced selective apoptosis in cancer cells at a concentration of less than 10 microM. Morphological analysis revealed features characteristic of apoptosis, including small cytoplasm with fragmented nuclei and condensed chromatin. DNA electrophoresis confirmed apoptosis, as evidenced by a distinct oligosomal ladder. Western blot analysis revealed that the agent significantly inhibited hepatocyte growth factor induced p42/p44 mitogen activated protein kinase activity. The in vivo anticancer effect was clearly confirmed by significantly decreased tumor growth without notable side effects in the 2 xenograft models. CONCLUSIONS: FTY720 treatment may induce selective apoptosis in vitro as well as in vivo in cancer cells. We suggest that FTY720 is a potent and clinically applicable anticancer agent for bladder cancer.     

Vascular endothelial growth factor A,secreted in response to transforming growth factor-β1 under hypoxic conditions,induces autocrine effects on migration of prostate cancer cells

Hypoxia and transforming growth factor-β1 (TGF-β1) increase vascular endothelial growth factor A (VEGFA) expression in a number of malignancies. This effect of hypoxia and TGF-β1 might be responsible for tumor progression and metastasis of advanced prostate cancer. In the present study, TGF-β1 was shown to induce VEGFA₁₆₅ secretion from both normal cell lines (HPV7 and RWPE1) and prostate cancer cell lines (DU145 and PC3). Conversely, hypoxia-stimulated VEGFA₁₆₅ secretion was observed only in prostate cancer cell lines. Hypoxia induced TGF-β1 expression in PC3 prostate cancer cells, and the TGF-β type I receptor (ALK5) kinase inhibitor partially blocked hypoxia-mediated VEGFA₁₆₅ secretion. This effect of hypoxia provides a novel mechanism to increase VEGFA expression in prostate cancer cells. Although autocrine signaling of VEGFA has been implicated in prostate cancer progression and metastasis, the associated mechanism is poorly characterized. VEGFA activity is mediated via VEGF receptor (VEGFR) 1 (Flt-1) and 2 (Flk-1/KDR). Whereas VEGFR-1 mRNA was detected in normal prostate epithelial cells, VEGFR-2 mRNA and VEGFR protein were expressed only in PC3 cells. VEGFA₁₆₅ treatment induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in PC3 cells but not in HPV7 cells, suggesting that the autocrine function of VEGFA may be uniquely associated with prostate cancer. Activation of VEGFR-2 by VEGFA₁₆₅ was shown to enhance migration of PC3 cells. A similar effect was also observed with endogenous VEGFA induced by TGF-β1 and hypoxia. These findings illustrate that an autocrine loop of VEGFA via VEGFR-2 is critical for the tumorigenic effects of TGF-β1 and hypoxia on metastatic prostate cancers.