Interleukin 8 expression regulates tumorigenicity and metastases in androgen-independent prostate cancer.

Interleukin 8 (IL-8) is mitogenic and chemotactic for endothelial cells. Within a neoplasm, IL-8 is secreted by inflammatory and neoplastic cells. The highly metastatic PC-3M-LN4 cell line overexpresses IL-8 relative to the poorly metastatic PC-3P cell line. We evaluated whether IL-8 expression by human prostate cancer growing within the prostate of athymic nude mice regulates tumor angiogenesis, growth, and metastasis. PC-3P cells were transfected with the full-length sense IL-8 cDNA, whereas PC-3M-LN4 cells were transfected with the full-sequence antisense IL-8 cDNA. Control cells were transfected with the neomycin resistance gene (Neo). In vitro, sense-transfected PC-3P cells overexpressed IL-8-specific mRNA and protein, which resulted in up-regulation of matrix metalloproteinase 9 (MMP-9) mRNA, and collagenase activity, resulting in increased invasion through Matrigel. After antisense transfection of the PC-3M-LN4 cells, IL-8 and MMP-9 expression, collagenase activity, and invasion were markedly reduced relative to controls. After orthotopic implantation, the sense-transfected PC-3P cells were highly tumorigenic and metastatic, with significantly increased neovascularity and IL-8 expression compared with either PC-3P cells or controls. Antisense transfection significantly reduced the expression of IL-8 and MMP-9 and tumor-induced neovascularity, resulting in inhibition of tumorigenicity and metastasis. These results demonstrate that IL-8 expression regulates angiogenesis in prostate cancer, in part by induction of MMP-9 expression, and subsequently regulates the growth and metastasis of human prostate cancer.     

Smad3 is overexpressed in advanced human prostate cancer and necessary for progressive growth of prostate cancer cells in nude mice.

PURPOSE: The purpose of this study was to investigate the potential role of Smad3, a key mediator of transforming growth factor-beta signaling, in progression of prostate cancer. EXPERIMENTAL DESIGN: Expression of Smad proteins was determined in human prostate cancer tissue array and cell lines. Growth and metastasis of cells overexpressing dominant-negative Smad3 (Smad3D) were studied to determine its role in tumor progression in mice. Cell growth, apoptosis, and expression of angiogenic molecules in tumor lesions were studied to determine potential pathways that Smad3 promotes tumor progression. RESULTS: Smad3 was overexpressed in human prostate cancer, which correlated with Gleason score and expression of proliferating cell nuclear antigen. Androgen-independent PC-3MM2 and DU145 cells expressed much higher levels of Smad3 than did androgen-dependent LNCaP, 22Rv1, and LAPC-4 cells. Overexpression of Smad3D in PC-3MM2 cells (PC-3MM2-Smad3D) had minimal direct effects on cell growth but attenuated effects of transforming growth factor-beta1 on gene expression and cell growth. Overexpression of Smad3D did not significantly alter tumor incidence but reduced tumor growth rate and metastasis incidence. Most cells in the control tumors, but not PC-3MM2-Smad3D tumors, were positively stained by an antibody to proliferating cell nuclear antigen. Microvessels and expression of angiogenic molecule interleukin-8 were significantly reduced in tumors from PC-3MM2-Smad3D cells. PC-3MM2-Smad3D tumors also expressed lower levels of vascular endothelial growth factor and platelet-derived growth factor. CONCLUSIONS: These data suggest that Smad3, through regulating angiogenic molecule expression in tumor cells, is critical for progression of human prostate cancer.     

Loss of responsiveness to transforming growth factor beta induces malignant transformation of nontumorigenic rat prostate epithelial cells.

Transforming growth factor (TGF)-betas are multifunctional growth factors, the properties of which include the potent inhibition of epithelial cell growth. Expression patterns of TGF-betas and TGF-beta receptors in the normal prostate indicate that these growth regulators play key roles in prostatic development and proliferative homeostasis. Importantly, TGF-beta receptor levels are frequently diminished in malignant human prostate tissue. To test the hypothesis that loss of TGF-beta responsiveness is causally involved in the tumorigenic process, we have used retroviral transduction to introduce a dominant-negative mutant type II TGF-beta receptor (DNR) into the premalignant rat prostatic epithelial cell line, NRP-152. High-level expression of the DNR abolished the ability of TGF-beta to inhibit cell growth, to promote cell differentiation, and to induce apoptosis, and it partially blocked the induction of extracellular matrix gene expression. When injected into nude mice, NRP-152-DNR cells formed carcinomas at 13 of 34 sites, compared with 0 of 30 sites for parental and control cells (P = 0.0001). We conclude that the type II TGF-beta receptor is an important tumor suppressor in the prostate, and furthermore, that loss of TGF-beta responsiveness can contribute early in the tumorigenic process by causing the malignant transformation of preneoplastic cells.     

Suppression of angiogenesis, tumorigenicity, and metastasis by human prostate cancer cells engineered to produce interferon-beta

We determined whether the IFN-beta gene can be used to suppress angiogenesis, tumor growth, and metastasis of human prostate cancer cells growing in the prostate of nude mice. Highly metastatic PC-3M human prostate cancer cells were engineered to constitutively produce murine IFN-beta subsequent to infection with a retroviral vector containing murine IFN-beta cDNA. Parental (PC-3M-P), control vector-transduced (PC-3M-Neo), and IFN-beta-transduced (PC-3M-IFN-beta) cells were injected into the prostate (orthotopic) or subcutis (ectopic) of nude mice. PC-3M-P and PC-3M-Neo cells produced rapidly growing tumors and regional lymph node metastases, whereas PC-3M-IFN-beta cells did not. PC-3M-IFN-beta cells also suppressed the tumorigenicity of bystander nontransduced prostate cancer cells. PC-3M-IFN-beta cells produced small tumors (3-5 mm in diameter) in nude mice treated with anti-asialo GM1 antibodies and in severe combined immunodeficient/Beige mice. Immunohistochemical staining revealed that PC-3M-IFN-beta tumors were homogeneously infiltrated by macrophages, whereas control tumors contained fewer macrophages at their periphery. Most tumor cells in the control tumors were stained positive by an antibody to proliferative cell nuclear antigen; very few were positively stained by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling. In sharp contrast, PC-3M-IFN-beta tumors contained fewer proliferative cell nuclear antigen-positive cells and many terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling-positive cells. Staining with antibody against CD31 showed that control tumors contained more blood vessels than PC-3M-IFN-beta tumors. PC-3M-IFN-beta cells were more sensitive to lysis mediated by natural killer cells in vitro or to cytostasis mediated by macrophages than control transduced cells. Conditioned medium from PC-3M-IFN-beta cells augmented splenic cell-mediated cytolysis to control tumor cells, which could be neutralized by antibody against IFN-beta. Collectively, the data suggest that the suppression of tumorigenicity and metastasis of PC-3M-IFN-beta cells is due to inhibition of angiogenesis and activation of host effector cells.     

Blockade of epidermal growth factor receptor signaling in tumor cells and tumor-associated endothelial cells for therapy of androgen-independent human prostate cancer growing in the bone of nude mice.

PURPOSE: We determined whether blockade of the epidermal growth factor receptor (EGF-R) signaling pathway by oral administration of the EGF-R tyrosine kinase inhibitor (PKI 166) alone or in combination with injectable Taxol inhibits the growth of PC-3MM2 human prostate cancer cells in the bone of nude mice. EXPERIMENTAL DESIGN: Male nude mice implanted with PC-3MM2 cells in the tibia were treated with oral administrations of PKI 166 or PKI 166 plus injectable Taxol beginning 3 days after implantation. The incidence and size of bone tumors and destruction of bone were determined by digitalized radiography. Expression of epidermal growth factor (EGF), EGF-R, and activated EGF-R in tumor cells and tumor-associated endothelial cells was determined by immunohistochemistry. RESULTS: Oral administration of PKI 166 or PKI 166 plus injectable Taxol reduced the incidence and size of bone tumors and destruction of bone. Immunohistochemical analysis revealed that PC-3MM2 cells growing adjacent to the bone expressed high levels of EGF and activated EGF-R, whereas tumor cells in the adjacent musculature did not. Moreover, endothelial cells within the bone tumor lesions, but not in uninvolved bone or tumors in the muscle, expressed high levels of activated EGF-R. Treatment with PKI 166 and more so with PKI 166 plus Taxol significantly inhibited phosphorylation of EGF-R on tumor and endothelial cells and induced significant apoptosis and endothelial cells within tumor lesions. CONCLUSIONS: These data indicate that endothelial cells exposed to EGF produced by tumor cells express activated EGF-R and that targeting EGF-R can produce significant therapeutic effects against prostate cancer bone metastasis.     

Tumor-infiltrating macrophages are involved in suppressing growth and metastasis of human prostate cancer cells by INF-beta gene therapy in nude mice.

PURPOSE: This study was to determine the role of tumor-infiltrating macrophages in IFN-beta-induced host defense against prostate cancer. EXPERIMENTAL DESIGN: Efficacy of adenovirus-mediated IFN-beta gene therapy against orthotopic xenografts of human prostate cancer was tested in macrophage-compromised nude mice. Immunohistochemistry and Northern blotting were used to elucidate mechanisms responsible for the IFN-beta gene therapy. RESULTS: PC-3MM2 human prostate cancer cells were inoculated into the prostates of nude mice. Intralesional injection of an adenoviral vector-encoding murine IFN-beta (AdmIFN-beta) but not control vector AdE/1 suppressed growth of PC-3MM2 tumors in a dose-dependent manner, with a maximal reduction of tumor weight by approximately 85% at 2 x 10(9) plaque-forming units. The therapy prevented metastasis, eradicated established metastases in some mice, and prolonged the survival of tumor-bearing mice. The efficacy of AdmIFN-beta therapy was reduced significantly in mice treated with macrophage-selective anti-Mac-1 and anti-Mac-2 antibodies. Moreover, the i.p. injection of the antibodies restored the tumorigenicity of PC-3MM2 cells stably engineered with murine IFN-beta gene. Tumor-infiltrating macrophages, significantly increased in AdmIFN-beta-injected lesions, were depleted by the antibodies. The therapy stimulated expression of the inducible nitric oxide synthase, down-regulated transforming growth factor-beta1 and interleukin-8, reduced microvessel density, and resulted in apoptosis of endothelial cells in the lesions. These effects of AdmIFN-beta were partially diminished in mice treated with the antibodies. CONCLUSIONS: These data suggest that macrophages play an important role in IFN-beta gene therapy and that intralesional delivery of the IFN-beta gene could be an effective therapy for clinically localized human prostate cancer.     

Responsiveness of human prostate carcinoma bone tumors to interleukin-2 therapy in a mouse xenograft tumor model.

We have tested an immunotherapy approach for the treatment of metastatic prostate carcinoma using a bone tumor model. Human PC-3 prostate carcinoma tumor cells were heterotransplanted into the femur cavity of athymic Balb/c nude mice. Tumor cells replaced marrow cells in the bone cavity, invaded adjacent bone and muscle tissues, and formed a palpable tumor at the hip joint. PC-3/IF cell lines, generated from bone tumors by serial in vivo passages, grew with faster kinetics in the femur and metastasized to inguinal lymph nodes. Established tumors were treated with systemic interleukin-2 (IL-2) injections. IL-2 significantly inhibited the formation of palpable tumors and prolonged mouse survival at nontoxic low doses. Histologically IL-2 caused vascular damage and infiltration of polymorphonuclear cells and lymphocytes in the tumor as well as necrotic areas with apoptotic cells. These findings suggest destruction of tumor cells by systemic IL-2 therapy and IL-2 responsiveness of prostate carcinoma bone tumors.     

Targeted Antivascular Therapy with the Apolipoprotein(a) Kringle V, rhLK8, Inhibits the Growth and Metastasis of Human Prostate Cancer in an Orthotopic Nude Mouse Model

Antivascular therapy has emerged as a rational strategy to improve the treatment of androgen-independent prostate cancer owing to the necessity of establishing a vascular network for the growth and progression of the primary and metastatic tumor. We determined whether recombinant human apolipoprotein(a) kringle V, rhLK8, produces therapeutic efficacy in an orthotopic human prostate cancer animal model. Fifty thousand androgen-independent human prostate cancer cells (PC-3MM2) were injected into the prostate of nude mice. After 3 days, these mice were randomized to receive the vehicle solution (intraperitoneally [i.p.], daily), paclitaxel (8 mg/kg i.p., weekly), rhLK8 (50 mg/kg i.p., daily), or a combination of paclitaxel and rhLK8 for 4 weeks. Treatment with paclitaxel or rhLK8 alone did not show significant therapeutic effects on tumor incidence or on tumor size compared with the control group. The combination of rhLK8 and paclitaxel significantly reduced tumor size and incidence of lymph node metastasis. Significant reduction in microvessel density and cellular proliferation and induction of apoptosis of tumor cells, and tumor-associated endothelial cells, were also achieved. Similarly, PC-3MM2 tumors growing in the tibia showed significant suppression of tumor growth and lymph node metastasis by the combination treatment with rhLK8 and paclitaxel. The integrity of the bone was significantly preserved, and apoptosis of tumor cells and tumor-associated endothelial cells was increased. In conclusion, these results suggest that targeting the tumor microenvironment with the antivascular effect of rhLK8 combined with conventional cytotoxic chemotherapy could be a new and effective approach in the treatment of androgen-independent prostate cancer and their metastases.     

腺病毒介导反义VEGF降低肿瘤诱导免疫抑制及增强IL-2的抗肿瘤效果

目的：本研究旨在探讨腺病毒介导的反义VEGF降低肿瘤诱导的免疫抑制及增强IL-2的抗肿瘤效果。方法：用反义VEGF修饰及反义VEGF与IL-2基因共同修饰MM45T，Li小鼠肝癌细胞，然后观察修饰后的肿瘤细胞的致瘤性；并在体内观察反义VEGF与IL-2基因诱导肝癌模型小鼠肿瘤细胞的凋亡，以及肿瘤组织中CD4^ ,CD8^ 细胞浸润情况。结果：反义VEGF降低了肝癌细胞MM45T.Li的致瘤性；增加了肿瘤组织周围CD4^ ,CD8^ 细胞浸润，改善了模型小鼠的免疫抑制状态，提高了IL-2抗肿瘤效果。结论：反义VEGF不仅可抑制新生血管的生成，而且可降低肿瘤诱导的免疫抑制，从而提高免疫系统对肿瘤细胞的识别，与IL-2免疫基因治疗相结合可明显提高对肿瘤的杀伤效果。     

腺病毒介导反义VEGF降低肿瘤诱导免疫抑制及增强IL-2的抗肿瘤效果

20 0 2年广东西医结合肿瘤学术年会暨全国肿瘤诊疗新技术研讨会由广东省中西医结合学会肿瘤专业委员会、广州抗癌协会生物治疗专业委员会和第一军医大学南方医院肿瘤中心联合举办。为国家和广东省学术活动Ｉ类会议 (将授予国家和广东省继续教育Ｉ类学分 )。这次会议将邀请国内 2 0多名著名专家报告肿瘤诊疗最新进展 ,交流各地在肿瘤防治研究中的新成果、新经验 ,提高我省和全国肿瘤防诊治水平 ,现将有关事项通知如下 :1.会议时间地点 :2 0 0 2年 8月 16～ 19日　广州市2 .专题报告主要内容 :(1)肿瘤综合治疗进展 ;(2 )造血干细胞“鸡尾酒疗…     

Enhanced radiation sensitivity in prostate cancer by inhibition of the cell survival protein clusterin.

PURPOSE: The purpose of this study is to evaluate the role of the cell survival gene clusterin in radiation-induced cell death in human LNCaP and PC-3 prostate cancer models. Experimental Design: Radiation sensitivities were compared in parental and clusterin-overexpressing LNCaP cells and in PC-3 cells and tumors treated with antisense or mismatch clusterin oligonucleotides. RESULTS: Clusterin-overexpressing LNCaP cells were less sensitive to irradiation with significantly lower cell death rates (23% after 8 Gy) compared with parental LNCaP cells (50% after 8 Gy) 3 days after irradiation. Clusterin expression in PC-3 cells after radiation was found to be up-regulated in a dose-dependent manner in vitro by 70% up to 12 Gy and in vivo by 84% up to 30 Gy. Inhibition of clusterin expression in PC-3 cells using antisense oligonucleotides (ASOs) occurred in a sequence- and dose-dependent manner and significantly enhanced radiation-induced apoptosis and decreased PC-3 cell growth rate and plating efficiency. Compared with mismatch control oligonucleotide treatment, clusterin ASO treatment enhanced radiation therapy and significantly reduced PC-3 tumor volume in vivo by 50% at 9 weeks. In addition, TUNEL staining revealed increased number of apoptotic cells in clusterin ASO-treated and irradiated PC-3 tumors, compared with treatment with mismatch control oligonucleotides plus radiation. CONCLUSIONS: These findings support the hypothesis that clusterin acts as a cell survival protein that mediates radioresistance through the inhibition of apoptosis. In vivo results further suggest that inactivation of clusterin using ASO technology might offer a novel strategy to improve results of radiation therapy for prostate cancer patients.     

Role of interleukin 10 and transforming growth factor beta1 in the angiogenesis and metastasis of human prostate primary tumor lines from orthotopic implants in severe combined immunodeficiency mice.

Transfection of primary human prostate tumor cells (i.e., HPCA-10a, 10b, 10c, and 10d lines) with the transforming growth factor (TGF)-beta1 gene stimulated anchorage-independent growth and promoted tumor growth, angiogenesis, and metastasis after orthotopic implantation in severe combined immunodeficiency mice. In contrast, interleukin (IL)-10 transfected cells or cells cotransfected with these two genes exhibited reduced growth rates and significantly reduced angiogenesis and metastasis after 8, 12, and 16 weeks. Enzyme-linked immunosandwich assays confirmed that the respective tumors expressed elevated levels of TGF-beta1 and IL-10 in vivo. ELISAs further showed that TGF-beta1 expression induced matrix metalloproteinases-2 (MMP-2) expression, whereas IL-10 down-regulated MMP-2 expression while up regulating TIMP-1 in the transfected cells. Also, tumor factor VIII levels correlated with TGF-beta1 and MMP-2 expression and inversely with IL-10 and TIMP-1 levels. More importantly, mouse survival was zero after 4-6 months in mice bearing TGF-beta1- and MMP-2-expressing tumors and increased significantly in mice implanted with IL-10- and TIMP-1-expressing tumors (i.e., to >80% survival). Analysis of the metastatic lesions showed that they expressed TGF-beta1 and MMP-2 but barely detectable levels of IL-10 or TIMP-1, suggesting that IL-10 and TIMP-1 might normally block tumor growth, angiogenesis, and metastasis.     

Responsiveness of experimental prostate carcinoma bone tumors to neutron or photon radiation combined with cytokine therapy

PURPOSE: To improve the outcome of radiotherapy for prostate carcinoma bone tumors, we investigated bone tumor irradiation with photons or neutrons followed by interleukin 2 (IL-2) therapy in a tumor model. METHODS AND MATERIALS: Implantation of PC-3 cells in nude mouse femur cavity induced a bone tumor that progressed to the formation of a palpable tumor, at the hip joint, by Day 20. Established bone tumors were irradiated with photons or neutrons, and a day later, mice were treated with IL-2 therapy for 3 weekly cycles. RESULTS: PC-3 bone tumors responded to radiation with photons or neutrons in a dose-dependent manner. Combination of photon or neutron radiation with IL-2 therapy increased tumor growth delay, compared to that with photons or neutrons alone. Radiation alone or combined with IL-2 significantly increased mouse survival compared to that with IL-2 or no treatment. After combined therapy, a complete inhibition of bone tumor growth was observed in 45% to 50% of the mice. Histologically, the combined therapy resulted in greater tumor destruction associated with fibrosis, new bone formation, and inflammatory infiltrates than that observed with each modality alone. CONCLUSIONS: The efficacy of tumor irradiation with neutrons or photons was enhanced by IL-2 therapy for the treatment of prostate carcinoma bone tumors.     

Inhibition of cyclooxygenase (COX)-2 expression by Tet-inducible COX-2 antisense cDNA in hormone-refractory prostate cancer significantly slows tumor growth and improves efficacy of chemotherapeutic drugs.

PURPOSE: Overexpression of the proinflammatory enzyme cyclooxygenase (COX)-2 is associated with the progression of various malignancies; the role of COX-2 in prostate cancer is less clear. The significance of COX-2 in prostate cancer growth and response to chemotherapy was investigated in an androgen-refractory prostate cancer cell line using a Tet-inducible antisense COX-2 expression system. EXPERIMENTAL DESIGN: An antisense COX-2 cDNA construct under the control of a doxycycline-inducible promoter was transfected into a prostate cancer cell line, PC-3ML. Modulations of cell growth, apoptosis, and chemosensitivity in the presence or absence of doxycycline were analyzed. Tumor incidence, growth rate, and response to two cytotoxic drugs, COL-3 [chemically modified tetracycline-3-(6-demethyl-6-deoxy-4-dedimethylamino-tetracycline)] and Taxotere (docetaxel), were investigated in tumor xenografts. Apoptotic incidences and tumor microvessel density in tumors were determined by immunohistochemistry. RESULTS: Conditional suppression of COX-2 in PC-3ML caused reduced cell proliferation, decreased levels of phosphorylated AKT, G(0)-G(1) arrest, and increased apoptosis and caspase-3 activity. Suppression of COX-2 increased Bax protein and decreased Bcl-x(L) protein in vitro. COX-2 antisense-expressing PC-3ML tumors showed a 57% growth delay compared with nontransfected or vector controls. Oral administration of COL-3 (40 mg/kg, oral gavage) or Taxotere (2.3 mg/kg, intraperitoneally; 3x per week) in tumor-bearing mice further slowed tumor growth (65% and approximately 94%, respectively). Compared with the control group, the occurrence of apoptosis in antisense COX-2 tumors was eight times higher, and the tumor microvessel density was three times lower. CONCLUSIONS: These results provide direct evidence that constitutive expression of COX-2 in prostate cancer has both angiogenic and cytoprotective functions. Suppression of tumor cell COX-2 is sufficient to enhance chemotherapy response in prostate cancer.     

Interleukin-8 is a molecular determinant of androgen independence and progression in prostate cancer

The proinflammatory chemokine interleukin-8 (IL-8) is undetectable in androgen-responsive prostate cancer cells (e.g., LNCaP and LAPC-4), but it is highly expressed in androgen-independent metastatic cells, such as PC-3. In this report, we show IL-8 functions in androgen independence, chemoresistance, tumor growth, and angiogenesis. We stably transfected LNCaP and LAPC-4 cells with IL-8 cDNA and selected IL-8-secreting (IL8-S) transfectants. The IL8-S transfectants that secreted IL-8 at levels similar to that secreted by PC-3 cells (100-170 ng/10(6) cells) were characterized. Continuous or transient exposure of LNCaP and LAPC-4 cells to IL-8 reduced their dependence on androgen for growth and decreased sensitivity (>3.5x) to an antiandrogen. IL-8-induced cell proliferation was mediated through CXCR1 and was independent of androgen receptor (AR). Quantitative PCR, immunoblotting, and transfection studies showed that IL8-S cells or IL-8-treated LAPC-4 cells exhibit a 2- to 3-fold reduction in PSA and AR levels, when compared with vector transfectants. IL8-S cells expressed 2- to 3-fold higher levels of phospho-EGFR, src, Akt, and nuclear factor kappaB (NF-kappaB) and showed increased survival when treated with docetaxel. This increase was blocked by NF-kappaB and src inhibitors, but not by an Akt inhibitor. IL8-S transfectants displayed a 3- to 5-fold increased motility, invasion, matrix metalloproteinase-9 and vascular endothelial growth factor production. LNCaP IL8-S cells grew rapidly as tumors, with increased microvessel density and abnormal tumor vasculature when compared with the tumors derived from their vector-transfected counterparts. Therefore, IL-8 is a molecular determinant of androgen-independent prostate cancer growth and progression.     

Adenovirus-mediated interferon-beta gene therapy suppresses growth and metastasis of human prostate cancer in nude mice

The purpose of this study was to determine the effects of interferon-beta (IFN-beta) gene transfer on the growth of PC3MM2 human prostate cancer cells in nude mice. Intralesional delivery of an adenoviral vector encoding murine IFN-beta (AdIFN-beta), but not a vector encoding bacterial beta-galactosidase (AdLacZ), suppressed PC3MM2 tumors in a dose-dependent manner. At the highest dose (2x10(9) plaque-forming units, PFU), a single injection of AdIFN-beta (but not AdLacZ) suppressed orthotopic PC3MM2 tumors and development of metastasis by 80%, and eradicated the tumors in 20% of mice. Immunohistochemical staining showed that AdIFN-beta-treated tumors contained fewer microvessels, fewer proliferating cells, and more apoptotic cells than did the control tumors. Compared with controls, tumors injected with AdIFN-beta expressed higher levels of IFN-beta and inducible nitric oxide synthase (iNOS) and lower levels of basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1). In vitro analysis indicated that expression of bFGF and TGF-beta1 in PC3MM2 cells could be suppressed by the nitric oxide donor sodium nitroprusside. These data suggest that intratumoral delivery of the IFN-beta gene with adenoviral vectors could be an effective therapy for prostate cancer and that tumor suppression by AdIFN-beta correlated with up-regulation of iNOS and down-regulation of angiogenesis.     

Knock-down of calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells

Calcitonin (CT) and its receptor (CTR) are expressed only in basal epithelium of benign prostate and in whole epithelium of malignant prostates. Also, CT and CTR mRNA levels in prostate cancers increase with an increase in tumor grade. We tested the role of the CT/CTR autocrine axis on the tumorigenicity of prostate cancer cells. We enforced the expression of CTR in CT-positive/CTR-deficient PC-3 cells. In contrast, we knocked down CTR expression in CT/CTR-positive PC-3M cells. The effect of CTR modulation on the oncogenicity was evaluated by the rate of cell proliferation, invasion, colony formation and in vivo growth in nude mice. Up-regulation of CTR in PC-3 cells and its down-regulation in PC-3M cells significantly altered their tumorigenicity. Intratumorally administered CTR RNAi in preexisting PC-3M xenografts markedly attenuated their further growth. This treatment also led to a remarkable decrease in endothelial cell populations in the tumors and increase in apoptotic, PCNA-negative cell populations. Tumors receiving CTR RNAi treatment displayed markedly lower levels of urokinase-type plasminogen activator, phospho-Akt and survivin, suggesting CTR activates uPA-uPAR axis and PI-3-kinase-Akt-survivin pathway. These results suggest an important role for CT-CTR autocrine axis in the progression of localized prostate tumor to a metastatic phenotype, and offer a potential therapeutic option for invasive cancers.     

Inhibition of angiogenesis by the antiepidermal growth factor receptor antibody ImClone C225 in androgen-independent prostate cancer growing orthotopically in nude mice.

In human androgen-independent prostate cancer (PCa), epidermal growth factor receptor (EGFR) regulates angiogenesis, tumor growth, and progression. In this study, we evaluated whether the blockade of EGFR by the anti-EGFR antibody ImClone C225 (IMC-C225) inhibited tumor growth and metastasis by inhibiting angiogenesis, and whether paclitaxel enhanced the results of therapy in androgen-independent PCa. PC-3M-LN4 PCa cells were implanted orthotopically in athymic nude mice and treated with i.p. IMC-C225 (1 mg twice a week) and/or paclitaxel (200 microg once a week). In vitro treatment of PC-3M-LN4 with IMC-C225 inhibited EGFR autophosphorylation without any significant antiproliferative effect. In contrast, in vivo therapy with IMC-C225 alone (P < 0.05) or in combination with paclitaxel (P < 0.005) significantly inhibited PCa growth and metastasis. Serum levels of interleukin (IL) 8 were lower after therapy, and IL-8 mRNA expression was down-regulated within the tumors after therapy. The down-regulation of IL-8 correlated with reduced microvessel density. IMC-C225 reduced tumor cell proliferation, enhanced p27(kip1) expression, and induced tumor and endothelial cell apoptosis. These studies indicate that IMC-C225 has significant antitumor effect in this murine model, mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. The simultaneous administration of paclitaxel enhanced this effect.