Enhanced tumorigenic and metastatic potential of an androgen-sensitive human prostate cancer cell line, LNCaP, by intratesticular inoculation in SCID mice

To establish a prostate cancer model expressing prostate-specific antigen (PSA) with metastatic potential, LNCaP or PC-3 cells were inoculated into the testis of SCID mice, resulting in a 100% rate of tumor formation. A significant increase in serum PSA was found in mice with LNCaP xenografts. Circulating tumor cells and micrometastases to organs such as lung, liver, spleen, and omentum were detected for both cell lines by PCR of the human beta-globin gene. Lymph node metastases occurred more frequently with PC-3 than LNCaP cells. This is the first report showing stable growth of LNCaP cells in mice with metastases to the regional lymph nodes. This model of prostate cancer should help to assess treatment strategies targeting PSA.     

Insulin-like growth factor binding protein-6 inhibits prostate cancer cell proliferation: implication for anticancer effect of diethylstilbestrol in hormone refractory prostate cancer

Diethylstilbestrol (DES) is a synthetic oestrogen, and its anticancer effects are exerted in androgen-dependent prostate cancer. The administration of DES decreases serum testosterone to castration levels. However, in androgen-independent prostate cancer patients, who are already orchiectomised, the administration of DES improves symptoms and decreases prostate-specific antigen (PSA). The mechanisms responsible for these direct inhibitory effects have been explained as biological actions not mediated by oestrogen receptors. We assessed the gene expression profiles of prostate cancer cells treated with DES, and investigated direct inhibitory effects of DES. DES inhibited the proliferation of LNCaP and PC-3 cells. cDNA microarray analysis showed that expression of many genes was downregulated by DES. However, insulin-like growth factor binding protein 6 (IGFBP-6) gene expression levels were upregulated in PC-3 cells. IGFBP-6 gene expression and protein levels significantly increased after DES treatment. Recombinant IGFBP-6 inhibited cell proliferation, and the inhibitory effect of DES was neutralised by anti-IGFBP-6 antibody. From the immunohistochemical analysis of IGFBP-6 using biopsy samples from androgen-independent prostate cancer, we found IGFBP-6 expression in androgen independent prostate cancer, and that DES treatment increased the IGFBP-6 staining intensity of the cancer cells in one sample. These findings suggested that DES induces IGFBP-6, which inhibits cell proliferation in an androgen-independent prostate cancer cell line, PC-3. IGFBP-6 therefore might be involved in the direct effects of DES in androgen-independent prostate cancer.     

Involvement of prostate specific antigen in the stimulation of LNCaP cell growth

Since its identification in 1979, prostatic specific antigen (PSA) has been used extensively as a serum marker for diagnosis and prognosis of prostate cancer. In addition, PSA is an immunohistochemical marker for the identification of prostatic tissues and cells in histological specimens. PSA is found in normal prostate, benign prostatic hypertrophy (BPH) tissue, in cancer of the prostate, and its metastases as well as in other hormone dependent cancers, such as breast and ovarian carcinoma. However, the importance of PSA as a regulator of cell growth generally has not been appreciated. The role of PSA in the development of prostate or other hormone-dependent cancers has remained unclear. We therefore examined the role of PSA in the control of cell growth using both the PSA positive cell line, LNCaP cells and the PSA negative cell line PC-3 and DU145. LNCaP cell growth was stimulated by the conditioned medium (CM) from LNCaP cells, but not by CM from PC-3 or DU145 cells. No such stimulation was observed when PC-3 or DU145 cells were exposed to CM from LNCaP cells nor from CM produced by their own lines. The stimulation of LNCaP cell growth by its own CM could not be attributed to the high level of insulin-like growth factor binding protein-2 (IGFBP-2) present in the CM since even higher level of IGFBP-2 was also found to be present in CM from both PC-3 and DU145 CMs. High level of PSA and 66 kDa epidermal growth factor (EGF) were present in LNCaP CM as measured by Western blotting. The stimulation of LNCaP cell growth by its own CM was eliminated partially by PSA or EGF antibody. Stimulation of DNA biosynthesis in LNCaP cells by LNCaP CM or pure PSA was also observed. These data indicate that PSA and EGF are involved in the growth regulation of PSA positive LNCaP cell line.     

Tissue prostate-specific antigen facilitates refractory prostate tumor progression via enhancing ARA70-regulated androgen receptor transactivation

Despite being well recognized as the best biomarker for prostate cancer, pathophysiologic roles of prostate-specific antigen (PSA) remain unclear. We report here that tissue PSA may be involved in the hormone-refractory prostate cancer progression. Histologic analyses show that the increased tissue PSA levels are correlated with lower cell apoptosis index and higher cell proliferation rate in hormone-refractory tumor specimens. By stably transfecting PSA cDNA into various prostate cancer cell lines, we found that PSA could promote the growth of androgen receptor (AR)-positive CWR22rv1 and high-passage LNCaP (hormone-refractory prostate cancer cells) but not that of AR-negative PC-3 and DU145 cells. Surprisingly, the protease activity of PSA is not crucial for PSA to stimulate growth and promote AR transactivation. We further showed that increased PSA could enhance ARA70-induced AR transactivation via modulating the p53 pathway that results in the decreased apoptosis and increased cell proliferation in prostate cancer cells. Knockdown of PSA in LNCaP and CWR22rv1 cells causes cell apoptosis and cell growth arrest at the G(1) phase. In vitro colony formation assay and in vivo xenografted tumor results showed the suppression of prostate cancer growth via targeting PSA expression. Collectively, our findings suggest that, in addition to being a biomarker, PSA may also become a new potential therapeutic target for prostate cancer. PSA small interfering RNA or smaller molecules that can degrade PSA protein may be developed as alternative approaches to treat the prostate cancer.     

Metastatic model for human prostate cancer using orthotopic implantation in nude mice.

BACKGROUND: Understanding the mechanism of prostate cancer metastasis is essential to the design of a more effective therapy. An effective therapy for this disease will depend on the development of a clinically relevant in vivo model. PURPOSE: We describe the development of such a model by using orthotopic implantation of human prostate cells in BALB/c nude mice. METHOD: We compared the tumorigenicity of and the incidence of metastasis of human prostate cancer PC-3M and LNCaP-FGC (LNCaP) cell lines subsequent to prostatic (orthotopic) or subcutaneous (ectopic) implantations in male nude mice. RESULTS: LNCaP cells produced tumors only in the prostate. Enhanced tumorigenicity at the orthotopic site was found for PC-3M cells. Lymph node metastases were observed in practically all mice given an injection of PC-3M cells in the prostate, but they were uncommon with subcutaneous injection of these cells. Bilateral orchiectomy did not alter the tumorigenicity of either PC-3M or LNCaP cells or the incidence of lymph node metastasis by PC-3M cells. LNCaP tumors in the mouse prostate (but not PC-3M tumors) elaborated detectable levels of human prostate-specific antigen (PSA) in the serum, even when tumors were small (1.5 mm in diameter). Immunohistochemistry analysis revealed the presence of the PSA marker in tissue sections of LNCaP but not of PC-3M tumors. CONCLUSIONS: The implantation of human prostate cancer cells in an ectopic environment does not permit expression of metastatic potential. In contrast, intraprostatic implantation does. IMPLICATIONS: These data suggest that the orthotopic injection of human prostate cancer cells into the nude mouse may provide a valuable model to study the biology and therapy of human prostate cancer.     

Increased manganese superoxide dismutase (SOD-2) is part of the mechanism for prostate tumor suppression by Mac25/insulin-like growth factor binding-protein-related protein-1

Increased expression of mac25/insulin-like growth factor binding-protein related protein-1 (IGFBP-rP1) in human breast and prostate epithelial cell lines results in the suppression of tumor growth. CDNA expression array analysis revealed increased manganese superoxide dismutase (SOD-2) expression in the mac25/IGFBP-rP1-transfected M12 human prostate cancer cell line compared to M12 control cells. SOD-2 has been postulated to be a tumor suppressor. SOD-2 was also increased in LNCaP cells stably transfected with mac25/IGFBP-rP1, but not in mac25/IGFBP-rP1-transfected PC-3 cells. Mac25 LNCaP cells had a marked decrease in tumor growth in nude mice compared to controls, but there was no difference in tumor growth in mac25 PC-3 cells compared to control. Phosphorylated Erk and Akt were increased in the M12 and LNCaP transfected mac25/IGFBP-rP1 cells but not PC-3 mac25. Inhibition of PI-3 kinase results in a marked decrease in viability of the M12-mac25 cells compared to M12 controls. Cells treated with H(2)O(2) result in an increase in phospho-ERK. Transfection of SOD-2 in M12 cells markedly decreased tumor growth, apoptosis, G1 delay in the cell cycle, and expression of senescence associated beta-galactosidase. These results suggest that one of the downstream mediators of the senescence-associated tumor suppression effect of mac25/IGFBP-rP1 is SOD-2.     

Thalidomide up-regulates prostate-specific antigen secretion from LNCaP cells

Thalidomide has been shown to have species- and metabolic-dependent antiangiogenic activity in vitro and in vivo, suggesting its potential in treating human angiogenesis-dependent pathologies such as solid tumors. Based on promising preclinical studies, thalidomide has entered phase II clinical trials for prostate, brain, breast cancer, and Kaposi's sarcoma. However, the antiangiogenic mechanism of action is largely unresolved, as are its effects on tumor-associated gene expression, cytokine secretion, etc. We have investigated the effects of thalidomide on: 1) the secretion of prostate-specific antigen (PSA) in a human androgen-dependent prostate cell line; 2) growth and viability of human prostate cells; and 3) differential gene expression profiles of thalidomide-treated vs untreated human prostate cells. A human androgen-dependent prostate carcinoma cell line (LNCaP) and a human androgen-independent prostate carcinoma cell line (PC-3) were incubated with thalidomide 0.6, 6, or 60 7g/mL for 5-6 days. Secreted PSA from LNCaP cells was measured using a commercial enzyme-linked immunosorbant assay. Cell viability studies were conducted in both LNCaP and PC-3 cells using the same thalidomide concentrations. Furthermore, the differential gene expression of thalidomide-treated LNCaP cells was compared to that of untreated control cells using a commercially available human cancer cDNA expression array system. Thalidomide-treated LNCaP cells demonstrated increased PSA/cell levels at all concentrations tested compared to untreated control cells. Thalidomide demonstrated a cytostatic effect in LNCaP cells but had no appreciable effect on PC-3 cell viability compared to untreated control cells. Comparison of cDNA expression arrays hybridized with thalidomide-treated LNCaP cDNA probes suggests that thalidomide may up- or downregulate expression of angiogenesis-related genes, i.e., vitronectin, but these differential effects require further verification. Thalidomide over a range of doses has demonstrated nontoxic, cytostatic activity in LNCaP cells and significant upregulation of LNCaP cell PSA secretion in vitro. Furthermore, preliminary data from cDNA nucleic acid arrays of thalidomide-treated LNCaP cells suggest that thalidomide upregulates a potential angiogenic modulatory protein, the vitronectin precursor, which may eventually link thalidomide's antiangiogenic activity with modulation of angiogenic vascular integrin pathways.     

HIV-1 protease inhibitor induces growth arrest and apoptosis of human prostate cancer LNCaP cells in vitro and in vivo in conjunction with blockade of androgen receptor STAT3 and AKT signaling

This study found that the HIV-1 protease inhibitor nelfinavir (NFV) induced growth arrest and apoptosis of human prostate cancer cells (LNCaP, DU145 and PC-3 cells), as measured by MTT and terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays, respectively, on the third day of culture. In addition, NFV blocked androgen receptor (AR) signaling in association with downregulation of nuclear levels of AR in LNCaP cells as measured by reporter assay and western blot analysis. As expected, NFV downregulated the level of the AR target molecule prostate specific antigen in these cells. Moreover, NFV disrupted STAT3 signaling; protease inhibitors blocked interleukin-6-induced phosphorylation of STAT3 and inhibited STAT3 DNA binding activity in LNCaP and DU145 cells, as measured by western blot analysis and enzyme-linked immunosorbent assay (ELISA), respectively. Furthermore, NFV blocked AKT signaling in prostate cancer cells as measured by kinase assay with glycogen synthase kinase-3alpha/beta as a substrate. Importantly, NFV inhibited the proliferation of LNCaP cells presented as tumor xenografts in BALB/c nude mice without side-effects. Taken together, NFV inhibited the proliferation of prostate cancer cells in conjunction with blockade of signaling by AR, STAT3, and AKT, suggesting that this family of compounds might be useful for the treatment of individuals with prostate cancer.     

Gene expression of angiogenic factors correlates with metastatic potential of prostate cancer cells

We hypothesize that expression of proangiogenic genes correlates with the metastatic potential of prostate cancer cells. LNCaP, DU-145, and PC-3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as we demonstrated by their capacity to invade an extracellular matrix, an established tumor invasion assay. The constitutive gene expression of the proangiogenic factors, vascular endothelial growth factor, intercellular adhesion molecule-1, interleukin-8, and transforming growth factor-beta2, was significantly greater in the more metastatic DU-145 and PC-3 cells as compared with LNCaP cells. Matrix metalloproteinase (MMP)-9 is thought to contribute to the invasive phenotype of tumor cells. PC-3 cells showed increased expression of MMP-9 and membrane type 4-MMP as compared with LNCaP and DU-145. Tissue inhibitors of metalloproteinase 1 and 4 gene expression were elevated in DU-145 and PC-3 cells, but paradoxically, LNCaP cells had undetectable levels of these genes. We transfected and overexpressed MMP-9 in poorly metastatic LNCaP cells and measured their invasive activity. Transient expression of human MMP-9 in LNCaP cells produced a 3-5-fold increase in MMP-9 activity with a comparable increase in invasiveness. Antisense ablation of the expression of MMP-9 in DU-145 and PC-3 cells produced concomitant inhibition of the gene expression of the proangiogenic factors, vascular endothelial growth factor, and intercellular adhesion molecule-1 (ICAM-1). Treatment of DU-145 and PC-3 cells with a selective chemical inhibitor of MMP-9 proteinase activity also inhibited their invasive activity. These results support our hypothesis that metastatic potential of prostate cancer cells correlates with expression of proangiogenic factors.     

CAPC negatively regulates NF-κB activation and suppresses tumor growth and metastasis

CAPC, also known as LRRC26, is expressed in normal prostate and salivary gland. We developed a mAb to CAPC and used it to characterize the protein and study its function. CAPC protein was detected in normal prostate and salivary gland, in several human breast cancer cell lines and in the prostate cancer cell line LNCaP. Knockdown of CAPC by siRNA in LNCaP cells enhanced anchorage-independent growth in soft agar. Conversely, overexpression of CAPC in MDA-231 breast cancer cells and A431 epidermoid cancer cells inhibited growth in soft agar and tumorigenesis in nude mice, and suppressed the metastasis of MDA-231 cells to the lung. Overexpression of CAPC downregulated NF-κB activity and its target genes, including GM-CSF (CSF2), CXCL1, IL8 and LTB1. It also suppressed genes encoding the serine protease mesotrypsin (PRSS3) and cystatin SN (CST1). CAPC expressing tumors showed a decrease in the number of proliferating cells and a large increase in ECM. The role of CAPC in the suppression of tumor growth and metastasis may be through its alteration of the tumor microenvironment.     

The Chinese medicinal herbal formula ZYD88 inhibits cell growth and promotes cell apoptosis in prostatic tumor cells

Prostate cancer is a leading cause of cancer death in American males. Currently, there is no curative therapy available once prostate cancer has metastasized. A major systemic therapy for metastatic prostate cancer is anti-androgen therapy. Unfortunately this therapy is only palliative and rarely curative, and eventually the tumor cells develop resistance to further hormone manipulation. It is therefore imperative to develop alternative effective therapies. In the present study, the effect of a Chinese herbal formula, ZYD88, on regulation of cell growth and cell apoptosis was examined in prostatic tumor cells. ZYD88 decreased cell viability of multiple prostatic tumor cell lines, DU-145, PC-3, MDA-PCa 2b and LNCaP in a time- and dose-dependent manner. It also produced a rapid and dose-dependent increase in caspase 3 activity in LNCaP and PC-3 cells, and induced DNA fragmentation in LNCaP cells, indicating cell apoptosis. In cotransfection assays, ZYD88 inhibited androgen-induced prostate specific antigen (PSA) gene promoter activity, and induced estrogen-target gene promoter activity. These data suggest that ZYD88 is a potential agent for prostate cancer therapy, and deserves further study.     

Prostaglandin E2 induces vascular endothelial growth factor secretion in prostate cancer cells through EP2 receptor-mediated cAMP pathway

Prostaglandin E2 (PGE2) has been shown to induce expression of vascular endothelial growth factor (VEGF) and other signaling molecules in several cancers. PGE2 elicits its functions though four G-protein coupled membrane receptors (EP1-4). In this study, we investigated the role of EP receptors in PGE2-induced molecular events in prostate cancer cells. qRT-PCR analysis revealed that PC-3 cells express a substantially higher level of EP2 and moderately higher EP4 than DU145 and LNCaP cells. LNCaP cells had virtually no detectable EP2 mRNA. EP1 and EP3 mRNAs were not detected in these cells. Treatment of prostate cancer cells with PGE2 (1 nM-10 microM) increased both VEGF secretion and cyclic adenosine monophosphate (cAMP) production. Levels of induction in PC-3 cells were greater than in DU145 and LNCaP cells. The selective EP2 agonist CAY10399 also significantly increased VEGF secretion and cAMP production in PC-3 cells, but not in DU145 and LNCaP cells. Moreover, PGE2 and CAY10399 increased mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) and Akt phosphorylation in PC-3 and DU145 cells, but not in LNCaP cells. However, neither the MAPK/Erk inhibitor U0126 nor the PI3K/Akt inhibitor LY294002 abolished PGE2-induced VEGF secretion in PC-3 cells. We further demonstrated that the adenylate cyclase activator forskolin and the cAMP anologue 8-bromo-cAMP mimicked the effects of PGE2 on VEGF secretion in PC-3 cells. Meanwhile, the adenylate cyclase inhibitor 2'5'-dideoxyadenosine, at concentrations that inhibited PGE2-induced cAMP, significantly blocked PGE2-induced VEGF secretion in PC-3 cells. We conclude that PGE2-induced VEGF secretion in prostate cancer cells is mediated through EP2-, and possibly EP4-, dependent cAMP signaling pathways.     

Evidence of determinant spreading in the antibody responses to prostate cell surface antigens in patients immunized with prostate-specific antigen.

PURPOSE: Prostate cancer consistently remains a difficult clinical problem. The development of novel therapy strategies for effective control and treatment of prostate cancer is essential. The prostate represents a unique site for immunotherapy, in part because prostate-specific immunity would most probably be without significant long-term sequellae. Antibodies and cell-mediated immunity, induced by either active or passive immunization, represent potential means to specifically target prostate tumor cells. EXPERIMENTAL DESIGN: The serum IgG response to cell surface antigens expressed on LNCAP [prostate-specific antigen (PSA)-positive] and PC-3 (PSA-negative) were analyzed in individuals with advanced disease receiving vaccinia- or fowlpox-expressed PSA (v-PSA or f-PSA, respectively) by flow cytometry. RESULTS: Sera from all seven patients in a Phase I study of v-PSA, collected prior to the third immunization, reacted with both prostate tumor cell lines. The majority of individuals (n = 12) in a Phase II trial of v-PSA and f-PSA developed sustainable antibody responses to cell surface antigens on the prostate tumor cell lines. The magnitude and kinetics of these responses were dependent on the immunization schedule. Of importance, the baseline serum of only one of nine patients tested had reactivity with nonprostate tumor cell lines. Sera from three normal males also lacked reactivity with prostate tumor cells. CONCLUSIONS: PSA vaccine constructs are immunogenic and induce antibody responses to a multitude of surface antigens on prostate tumor cell lines by epitope or determinant spreading after stimulation of the immune system by PSA immunization.     

血管内皮生长因子及其受体KDR在前列腺癌细胞中的表达研究

目的:探讨血管内皮生长因子(vascular endothelial growthfactor,VEGF)及其受体(KDR)在前列腺癌细胞中的表达及意义.方法:免疫细胞化学和Western blot检测体外培养的LNCaP,PC-3,PC-3M,DU-145和22RV1前列腺癌细胞中VEGF及其受体KDR的蛋白表达,RT-PCR检测mRNA,ELISA检测前列腺癌细胞培养上清VEGF蛋白含量.结果:免疫细胞化学染色显示VEGF和KDR在前列腺癌细胞LNCaP,PC-3,PC-3M,DU-145和22RV1中均有表达,但表达水平略有差别.LNCaP,PC-3和DU-145细胞中VEGF和KDR蛋白表达及其mRNA的含量显著高于PC-3M和22RV1(P＜0.01).细胞培养上清中VEGF蛋白含量结果与免疫细胞化学染色结果相同.结论:VEGF及其受体KDR在前列腺癌细胞中的表达量不近相同,但二者的表达对研究前列腺癌的发生发展及其机理有重要意义.     

Genistein affects androgen-responsive genes through both androgen- and estrogen-induced signaling pathways

This study examined the mechanisms by which the prostate cancer chemopreventive agent genistein modulates gene expression in LNCaP human prostate cancer cells. Expression of androgen- and estrogen-regulated genes was measured in LNCaP cells cultured in the presence or absence of hormonal stimulation and the presence or absence of genistein. Genistein strongly suppressed basal expression of androgen-responsive gene (ARG) mRNAs, including prostate-specific antigen (PSA) and Ste20-related proline-alanine-rich kinase (SPAK). However, genistein had little or no effect on basal expression of two other ARGs, beta2-microglobulin (B2M) or selenoprotein P (SEPP1). Culturing LNCaP cells in the presence of the synthetic androgen R1881-induced increases in PSA, SPAK, B2M, and SEPP1 mRNA levels. The R1881-induced expression of these genes was uniformly blocked by genistein. For PSA and SPAK, genistein also blocked or downregulated 17beta-estradiol-induced increases in mRNA expression. These results indicate that genistein selectively alters expression of ARG mRNAs in LNCaP cells through modulation of both androgen- and estrogen-induced signaling pathways.     

Folate-linked nanoparticle-mediated suicide gene therapy in human prostate cancer and nasopharyngeal cancer with herpes simplex virus thymidine kinase

For targeted gene delivery to human prostate cancer LNCaP and PC-3 cells and nasopharyngeal cancer KB cells, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated suicide gene therapy in the cells and xenografts with herpes simplex virus thymidine kinase (HSV-tk) and connexin 43 (Cx43). An NP-F-plasmid DNA complex (NP-F nanoplex) showed high DNA transfection efficiency in KB, LNCaP and PC-3 cells. Cell growth inhibition in the presence of ganciclovir (GCV) was enhanced with HSV-tk and Cx43 genes in LNCaP cells. In suicide gene therapy, the tumor growths of KB and LNCaP xenografts were significantly inhibited when an NP-F nanoplex of the HSV-tk gene, and HSV-tk and Cx43 genes, respectively, was injected intratumorally and GCV was administered intraperitoneally. These findings suggested that the NP-F is a potential target vector in prostate and nasopharyngeal cancer for suicide gene therapy.