LNCaP Tumor-bearing Mice Research Articles

1-11C-Acetate as a PET Radiopharmaceutical for Imaging Fatty Acid Synthase Expression in Prostate Cancer

Although it is accepted that the metabolic fate of 1-(11)C-acetate is different in tumors than in myocardial tissue because of different clearance patterns, the exact pathway has not been fully elucidated. For decades, fatty acid synthesis has been quantified in vitro by the incubation of cells with (14)C-acetate. Fatty acid synthase (FAS) has been found to be overexpressed in prostate carcinomas, as well as other cancers, and it is possible that imaging with 1-(11)C-acetate could be a marker for its expression. In vitro and in vivo uptake experiments in prostate tumor models with 1-(11)C-acetate were performed both with and without blocking of fatty acid synthesis with either C75, an inhibitor of FAS, or 5-(tetradecyloxy)-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase (ACC). FAS levels were measured by Western blot and immunohistochemical techniques for comparison. In vitro studies in 3 different prostate tumor models (PC-3, LNCaP, and 22Rv1) demonstrated blocking of 1-(11)C-acetate accumulation after treatment with both C75 and TOFA. This was further shown in vivo in PC-3 and LNCaP tumor-bearing mice after a single treatment with C75. A positive correlation between 1-(11)C-acetate uptake into the solid tumors and FAS expression levels was found. Extensive involvement of the fatty acid synthesis pathway in 1-(11)C-acetate uptake in prostate tumors was confirmed, leading to a possible marker for FAS expression in vivo by noninvasive PET.

Establishment of a highly tumorigenic LNCaP cell line having inflammatory cytokine resistance

Human androgen-dependent prostate cancer LNCaP cells are low tumorigenic even in immunodeficient mice and were killed by the synergistic effect of inflammatory cytokines, IL-β and IL-6. To establish a highly tumorigenic LNCaP cell line, we isolated the cytokine-resistant LNCaP-CR cell line and examined the phenotypes. The parental LNCaP cells were induced to commit apoptosis by the addition of IL-1β and IL-6, but LNCaP-CR cells showed strong resistance against the cytokine action. However, LNCaP-CR cells did not exhibit any resistance to various antitumor drugs investigated. While LNCaP cells formed only palpable tumors in SCID mice, LNCaP-CR cells readily made tumors and their growth was significantly higher than that of LNCaP cells. Moreover, LNCaP tumor-bearing mice gained the weight gradually, but LNCaP-CR tumor-bearing mice significantly lost their body weight. LNCaP-CR cells still responded to androgen action and expressed AR, erbB2, IL-1R, IL-6R, gp130, STAT3, p21, Bcl-2 and caspase-3 as well as LNCaP cells. These results indicate that LNCaP-CR cell line is a new type of tumorigenic LNCaP cell lines and should be useful for identifying responsible genes of tumorigenicity, cytokine resistance, and also cachexia.

Increased Hsp27 after Androgen Ablation Facilitates Androgen-Independent Progression in Prostate Cancer via Signal Transducers and Activators of Transcription 3–Mediated Suppression of Apoptosis

One strategy to improve therapies in prostate cancer involves targeting cytoprotective genes activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. The objectives of this study were to define changes in Hsp27 levels after androgen ablation and to evaluate the functional relevance of these changes in AI progression. Using a tissue microarray of 232 specimens of hormone-naïve and post-hormone ablation-treated prostate cancer, we found that Hsp27 levels increase after androgen ablation to become highly expressed (>4-fold, P < or = 0.01) in AI tumors. Hsp27 overexpression rendered LNCaP cells highly resistant to androgen withdrawal both in vitro and in vivo. Tumor volume and serum prostate-specific antigen levels increased 4.3- and 10-fold faster after castration when Hsp27 was overexpressed. Treatment of LNCaP tumor cells in vitro with Hsp27 antisense oligonucleotides (ASO) or short-interfering RNA suppressed Hsp27 levels in a dose-dependent and sequence-specific manner increased the apoptotic sub-G0-G1 fraction and caspase-3 cleavage >2-fold, as well as decreased signal transducers and activators of transcription 3 (Stat3) levels and its downstream genes, c-fos and sPLA-2. The cytoprotection afforded by Hsp27 overexpression was attenuated by Stat3 knockdown using specific Stat3 ASO. Coimmunoprecipitation and immunofluorescence confirmed that Hsp27 interacts with Stat3 and that Stat3 levels correlated directly with Hsp27 levels. Hsp27 ASO treatment in athymic mice bearing LNCaP tumors significantly delayed LNCaP tumor growth after castration, decreasing mean tumor volume and serum prostate-specific antigen levels by 57% and 69%, respectively. These findings identify Hsp27 as a modulator of Stat3-regulated apoptosis after androgen ablation and as a potential therapeutic target in advanced prostate cancer.

Efficacy of an intratumoral controlled release formulation of clusterin antisense oligonucleotide complexed with chitosan containing paclitaxel or docetaxel in prostate cancer xenograft models

To develop and evaluate an injectable, controlled release delivery system for a phosphorothioate antisense oligonucleotide (ASO) based on complexed ASO:chitosan dispersed in a biodegradable polymeric paste for intratumoral treatment of solid tumors. Clusterin ASO was complexed with chitosan particles and incorporated into a paste based on a 60:40 blend of methoxy-poly(ethylene glycol) (MePEG) and triblock copolymer of poly(D: ,L: -lactic acid-co-caprolactone)-PEG-(D: ,L: -lactic acid-co-caprolactone). In vitro release profiles of clusterin ASO into phosphate-buffered saline at 37 degrees C were obtained under sink conditions and assayed by anionic exchange high-performance liquid chromatography. In vivo efficacy studies were carried out in human prostate PC-3 and LNCaP tumors grown subcutaneously in mice. Paste formulations of clusterin ASO with or without paclitaxel or docetaxel were injected intratumorally and tumor volumes and serum prostate specific antigen (PSA) levels were measured. Controlled release of clusterin ASO was obtained over several weeks. The rate and extent of ASO release was proportional to the ratio of ASO to chitosan in the paste. Treatment of mice bearing PC-3 tumors with clusterin ASO plus paclitaxel or docetaxel paste had reduced mean tumor volume by greater than 50% at 4 weeks. Treatment of mice bearing LNCaP tumors with clusterin ASO plus paclitaxel reduced mean tumor volume and serum PSA level by more than 50% and 70%, respectively. Complexation of clusterin ASO with chitosan and incorporation into polymeric paste with paclitaxel or docetaxel produced in vitro controlled release of the ASO and in vivo efficacy over 4 weeks following a single intratumoral injection in solid human prostate tumors in mice.

Nitroxide tempo, a small molecule, induces apoptosis in prostate carcinoma cells and suppresses tumor growth in athymic mice

In previous studies, nitroxide tempo (2, 2, 6, 6-tetramethyl-piperidine-1-oxyl), a small molecule, induced cell death in cancer cells. The current study examined the antineoplastic properties of tempo in the human hormone-dependent/hormone-independent prostate carcinoma models (LNCaP, DU-145, and PC-3). The apoptotic effects of tempo were examined by the flow cytometric analysis of cells labeled with fluorescein isothiocyanate-conjugated annexin-V, and by electron microscopy. Enzymatic assays were performed to measure the activities of 2 cysteine proteases, i.e., caspase-9 and caspase-3, in tempo-treated cells. The effects of tempo on cell proliferation and on cell cycle distribution profiles were measured by the flow cytometric assay using immunofluorescent staining of incorporated 5'-bromo-2'-deoxyuridine (BrdU) coupled with 7-amino-actinomycin D (7-AAD) staining of total DNA. The number of proliferating cells was also determined independently by enzyme-linked immunosorbent assay using chemiluminescent detection of incorporated BrdU. Subcutaneous growth of human prostate carcinoma in athymic mice was monitored after intratumoral administration of tempo into tumor-bearing mice. In addition, cell viability assays were performed to compare the cytotoxic effect of a combination of doxorubicin or mitoxantrone and tempo with single agents. Tempo treatment of prostate carcinoma cells caused a significant increase in the number of apoptotic cells compared with control groups (tempo, 2.5 mM, 24 hours: DU-145, approximately 3.4-fold; PC-3, approximately 6-7-fold; tempo 1 mM, 24 hours: LNCaP, approximately 12-fold). Tempo-induced loss of cell viability was blocked partially or completely after pretreatment of cells with actinomycin-D or cycloheximide, suggesting a de novo macromolecule synthesis-dependent mechanism of cell death. Electron microscopy revealed aggregation and marginalization of chromatin in the nuclei of a large number of tempo-treated LNCaP cells. Tempo treatment of LNCaP cells resulted in enhanced activities of caspase-9 (tempo, 5 mM, 15 hours: approximately 2-fold) and caspase-3 (tempo, 2.5 mM, 24 hours: approximately 12-fold). Tempo treatment also led to an enhanced number of cells in G2/M phase of the cell cycle (tempo, 5.0 mM, 24 hours: DU-145, approximately 1.6-fold; PC-3, approximately 1.5-fold; LNCaP, approximately 5.3-fold), and decreased BrdU incorporation indicative of a decline in the number of proliferating cells (tempo, 2.5 mM, 24 or 48 hours; DU-145, approximately 2-3-fold; PC-3, approximately 1.2-fold; LNCaP, approximately 5-10-fold). Administration of tempo into LNCaP tumor-bearing mice resulted in a significant inhibition of tumor growth (percent initial tumor volume [Day 30, n = 4]: vehicle, 845.35 +/- 272.83; tempo, 9.72 +/- 9.72; tempo vs. vehicle, P < 0.02). In hormone-refractory prostate carcinoma cells, a combination of relatively low doses of tempo and doxorubicin or mitoxantrone caused enhanced cytotoxicity as compared with single agents. These data demonstrated that nitroxide tempo induced apoptosis and activated a caspase-mediated signaling pathway in prostate carcinoma cells. Tempo treatment also caused cell cycle arrest in G2/M phase and decreased the number of proliferating cells (S phase). Tempo treatment of tumor-bearing mice led to inhibition of tumor growth, suggesting that tempo is a novel member of the small-molecule family of antineoplastic agents.

Potent CYP17 inhibitors: improved syntheses, pharmacokinetics and anti-tumor activity in the LNCaP human prostate cancer model

A facile preparation of azolyl steroids, VN/85-1 and VN/87-1 (potent inhibitors of CYP17) has been developed. This process without tedious chromatographic separations improved the overall yields from 55 and 45% to 70 and 65% for VN/85-1 and VN/87-1, respectively. Pharmacokinetic studies of VN/85-1 were conducted in male SCID mice. Following subcutaneous (s.c.) administration of 100 mg/kg of VN/85-1, peak plasma level of 16.73 μg/ml occurred after 45 min, and the compound was cleared rapidly with a t 1/2 of 52.34 min. The bioavailability of VN/85-1 after s.c. administration was 83.0%. VN/85-1 was also rapidly metabolized to the corresponding 3-oxo-4-ene analog, 17-(1 H-imidazol-1-yl)androsta-4,16-diene-3-one (VN/108-1). In our attempt to optimize the anti-tumor efficacy of these two CYP17 inhibitors, we studied their anti-tumor efficacies in male SCID mice bearing LNCaP tumor xenografts, utilizing various drug doses and drug scheduling. Three times a day dose regimen (3× dose regimen) of VN/85-1 was more effective than a once daily dose. In contrast, 3× dose regimen doses of VN/87-1 were less effective than the once daily dose. However, at their effective dosage regimes, VN/85-1 and VN/87-1 were each as effective as castration and more effective than finasteride or casodex, an anti-androgen used for prostate cancer (PC) therapy. For all of the treatments, there was a strong correlation between the tumor volumes and other associated parameters, such as, tumor weights, and serum testosterone (T) and PSA levels. These results indicate that VN/85-1 or VN/87-1 may be useful in the treatment of hormone-dependent prostate cancer.

NE-10 neuroendocrine cancer promotes the LNCaP xenograft growth in castrated mice.

Increases in neuroendocrine (NE) cells and their secretory products are closely correlated with tumor progression and androgen-independent prostate cancer. However, the mechanisms by which NE cells influence prostate cancer growth and progression, especially after androgen ablation therapy, are poorly understood. To investigate the role of NE cells on prostate cancer growth, LNCaP xenograft tumors were implanted into nude mice. After the LNCaP tumors were established, the NE mouse prostate allograft (NE-10) was implanted on the opposite flank of these nude mice to test whether NE tumor-derived systemic factors can influence LNCaP growth. Mice bearing LNCaP tumors with or without NE allografts were castrated 2 weeks after NE tumor inoculation, and changes in LNCaP tumor growth rate and gene expression were investigated. After castration, LNCaP tumor growth decreased in mice bearing LNCaP tumors alone, and this was accompanied by a loss of nuclear androgen receptor (AR) localization. In contrast, in castrated mice bearing both LNCaP and NE-10 tumors, LNCaP tumors continued to grow, had increased levels of nuclear AR, and secreted prostate-specific antigen. Therefore, in the absence of testicular androgens, NE secretions were sufficient to maintain LNCaP cell growth and androgen-regulated gene expression in vivo. Furthermore, in vitro experiments showed that NE secretions combined with low levels of androgens activated the AR, an effect that was blocked by the antiandrogen bicalutamide. Because an increase in AR level has been reported to be sufficient to account for hormone refractory prostate cancers, the NE cell population ability to increase AR level/activity can be another mechanism that allows prostate cancer to escape androgen ablation therapy.

Pharmacokinetic profile of 3β-hydroxy-17-(1 H-1,2,3-triazol-1-yl)androsta-5,16-diene (VN/87-1), a potent androgen synthesis inhibitor, in mice

The objective of this study was to assess the pharmacokinetics and bioavailability of 3β-hydroxy-17-(1 H-1,2,3-triazol-1-yl)androsta-5,16-diene (VN/87-1) in normal male mice and in SCID mice bearing human LNCaP tumor xenografts. VN/87-1 is a novel potent steroidal inhibitor of human testicular 17-α-hydroxylase/C 17,20-lyase. The steroid also shows anti-androgenic activity and inhibits the growth of human prostate cancer cell lines (LNCaP) in vitro and in vivo. Male Balb/c mice were given a single oral, subcutaneous (s.c.) or intravenous (i.v.) bolus dose of VN/87-1 (25, 50 or 100 mg/kg). Male SCID mice bearing LNCaP tumor xenografts were injected with a single s.c. dose of VN/87-1 (50 mg/kg). The animals were sacrificed at various times up to 24 h after drug administration and blood was collected. The plasma samples were prepared and analyzed by a reversed phase HPLC system equipped with a diode array detector. A non-compartmental pharmacokinetic approach was used to evaluate the plasma level versus time data. Following i.v. administration of VN/87-1, the plasma levels declined exponentially with an elimination half-life of 1.2±0.03 h. The absolute bioavailability of the 50 mg/kg dose after oral or s.c. administration was 12.08±2 or 57.2±4.5%, respectively. VN/87-1 is a high clearance (5.0±1.3 l/h per kg) compound in mice and its volume of distribution was relatively large (6.5±1.2 l/kg). The pharmacokinetic parameters of VN/87-1 were not significantly altered in SCID mice bearing human LNCaP tumor xenografts. VN/87-1 is well absorbed from the subcutaneous site compared with absorption from the gastrointestinal tract and shows linear kinetics at doses up to 100 mg/kg.

VASCULAR ENDOTHELIAL GROWTH FACTOR EXPRESSION AND TUMOR ANGIOGENESIS ARE REGULATED BY ANDROGENS IN HORMONE RESPONSIVE HUMAN PROSTATE CARCINOMA: : EVIDENCE FOR ANDROGEN DEPENDENT DESTABILIZATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR TRANSCRIPTS

The hormonally regulated growth of some human carcinomas represents an important therapeutic target. We report that androgens modulate the angiogenic activity of hormone responsive human prostate cancer. To define further the critical mechanisms underlying hormone responsiveness we examined the angiogenic mediator, vascular endothelial growth factor messenger (m) RNA and protein in response to androgens in vitro as well as the angiogenic response of xenografts of human prostate cancer after androgen withdrawal in vivo. In vitro androgen deprivation of LnCaP prostate cancer cells led to decreased vascular endothelial growth factor mRNA and protein expression as well as a 5-fold destabilization in vascular endothelial growth factor mRNA transcripts. In addition, androgen withdrawal inhibited the hypoxic induction of vascular endothelial growth factor mRNA. In mice bearing LnCaP tumors castration resulted in a rapid decrease in mRNA expression and markedly reduced tumor neovascularization. These findings implicate sex steroids as an important stimulus for vascular endothelial growth factor regulation in hormone sensitive tumors and demonstrate the reversal of neovascularization after hormone withdrawal as an early event in the tumor response to therapy.

Tumor-specific and photodependent cytotoxicity of hypericin in the human LNCaP prostate tumor model.

Hypericin (HYP) has been reported to have photodependent cytotoxic activity in a variety of cancer cell lines. However, this activity has yet to be rigorously tested in vivo in tumor models. In this study LNCaP, PC-3 and DU-145 cells were used to test the cytotoxic effects of HYP in vitro, precursory to an in vivo study designed to investigate the effects of HYP in an established murine model for prostate cancer. Specifically, the model used employs immunocompromised nude mice bearing the LNCaP solid tumor xenograft. In vitro cytotoxicity experiments indicated that the dose causing 50% lethality for HYP in LNCaP, PC-3 and DU-145 cells were 2.07, 2.15 and 2.23 microM, respectively, following irradiation with red light (590 nm) for 30 min at a fluence rate of 0.1 J/cm2/s. Cells treated with HYP in the absence of photoirradiation showed no signs of cytotoxicity. A tissue distribution study was also carried out using the LNCaP solid tumor model to determine whether or not HYP is distributed to the target tissue. HYP was broadly distributed in tissues studied, including LNCaP tumor xenograft tissue. Furthermore, tumor tissue eliminated HYP at a slower rate than any of the other tissues examined. Interestingly, HYP levels were maintained in serum 24 h after oral administration (5 mg/kg dose). A pilot study designed to examine the efficacy of HYP treatment in nude mice bearing LNCaP tumors conducted over 28 days suggested that HYP, in combination with photoirradiation, inhibits both tumor growth and the elevation of prostate-specific antigen levels. Although the results reported for the current studies are preliminary they do provide evidence for an application of HYP PDT to prostate cancer which warrants further investigation.

Intermittent androgen deprivation.

Intermittent androgen deprivation is a controversial approach to management of prostate cancer. Preclinical models have demonstrated delay in time to prostate-specific antigen (PSA) progression in athymic mice bearing LNCaP tumors and a delay in time to androgen independence in androgen-dependent Shionogi carcinoma tumors in castrated animals exposed to intermittent androgen. Phase II clinical trials have demonstrated improved sexual function and quality of life in men discontinuing androgen deprivation. The average percentage of time spent off androgen deprivation ranges from 37% to 58%. Most men respond to retreatment with hormonal therapy. Current ongoing phase III clinical trials of intermittent versus continuous androgen deprivation in men with metastatic disease or recurrent disease after localized therapy will assess the comparative impact on quality of life and survival. Final analyses of these critical trials will define the ultimate role of this approach in prostate cancer. In the interim, intermittent androgen deprivation should be considered an experimental approach.

Experimental studies for immunoscintigraphy of adenocarcinoma of the prostate with 125I-labeled monoclonal antibody to gamma-seminoprotein

We established LNCaP tumor in male nude mice after inoculation of LNCaP cells. The tumor produces the organ-specific glycoproteins in the human prostate, gamma-seminoprotein (gamma-Sm), which has been verified by immunohistochemistry and enzyme immuno assay. The LNCaP tumor is composed of 2 distinct portions, namely GEL/FLUID portion and SOLID portion. The GEL/FLUID portion contains much more gamma-Sm than the SOLID portion does. The monoclonal antibody against gamma-Sm (murine IgG1, K) was radio-labeled with iodine-125 by lactoperoxidase method followed by passage over column chromatography. Finally the conjugates with specific activity of 49.8-56.6 KBq/micrograms were obtained. Biodistribution of 125I-labeled monoclonal antibody to gamma-Sm (125I-gamma MAb) was evaluated by whole-body autoradiography (ARG) and by determination of label uptake (DLU) by the LNCaP tumor using the well type gamma counter. The ARG were obtained 1-9 days after intravenous administrations of 32-38 micrograms of 125I-gamma MAb to 12 nude mice bearing LNCaP tumor. The DLU were evaluated 3, 4 and 7 days after intravenous administrations of 18-25 micrograms of 125I-gamma MAb to 9 nude mice bearing LNCaP tumor. The DLU by organs (GEL/FLUID portion, SOLID portion, liver, kidney, spleen, lung, etc.) were expressed as F-values (%) which were calculated by the following formula: F = radioactivity per gram of tissue (dpm/g) divided by injected dose per gram of animal (dpm/g) x 100 and tissue-to-blood ratios (T/B). The specific uptake by the LNCaP tumors, especially in the GEL/FLUID portion, was observed on ARG, with optimal images of the tumor evaluated 4-7 days after administrations. The F-values and T/B of the LNCaP tumors were always higher than those of other organs. Especially T/B of GEL/FLUID portion were of 6.7 and 6.3, evaluated 4 and 7 days after administration of 125I-gamma MAb respectively. These results suggest that the radio-labeled gamma MAb is applicable for immunoscintigraphy of adenocarcinoma of the prostate.