Dunning R3327 Research Articles

Flow cytometric characterization of tumor subpopulations in three sublines of the dunning R3327 rat prostate tumor model

Subsets of tumor cells were characterized by mapping DNA ploidy patterns in correlation with established cell surface markers in three non-treated sublines of the Dunning R3327 prostate tumor system representing different progressional stages. Flow cytometry was used to analyze DNA-index, cell cycle distribution as well as multiparametric aquisition of single and combined cell surface markers in single cell suspensions of frozen tumor tissues. The three Dunning prostate tumor sublines clearly differ in their ploidy status. In addition each tumor subline displays a characteristic cell surface marker profile, which is correlated with the cell cycle phase and the amount of genomic alterations. In a feasibility study we have shown that cross-reacting antibodies to human cell surface markers stain discrete tumor subpopulations in three sublines of the Dunning tumor model. Although it remains presently uncertain, which cell surface markers are most suitable for cell sorting to display cancer initiating (CIC) properties following subcutaneous or orthotopic grafting, the model may be useful for mechanistic investigations of putative stem-like tumor subpopulations and their significance in response to radio- or chemotherapy.

T6.2 Phenotypic characterization of kidney podocytes from women with preeclampsia

A method for accurate prediction of prognosis in human prostatic cancer does not exist. The limitations of pathologic grading systems may result from the failure of standard pathological examination of fixed dead tissue to accurately assess the biological behavior of live tumor cells. Many of the sublines of the Dunning R-3327 rat adenocarcinoma are histologically similar yet differ widely in their metastatic potential. The nonmetastatic G, occasionally metastatic AT-1 and AT-2, and highly metastatic AT-3 and MAT-Lu Dunning sublines, and normal dorsal prostate were grown in culture and filmed by time-lapse videomicroscopy. Cell membrane ruffling, undulation and pseudopodal extension, vectoral translation, irregularity of pathway, and overall subjective motility (gestalt) were visually graded. Intra-assay, intra-observer, and inter-observer reproducibility were 75, 80 and 75% respectively. The combination of ruffling, pseudopodal extension and vectoral translation was most successful in identifying the six sublines. To validate this technique prospectively, five tumor sublines and two normal prostates were graded by 10 observers unfamiliar with the technique. Fifty-nine percent of unknowns were correctly identified when motility profiles were compared to previously developed standards by least sum of squares analysis. We devised a new technique for characterizing the motility of living prostate cells which was more accurate in identifying normal rat prostate and the Dunning sublines than standard pathological examination. Prostatic cancer cell motility may reflect biological behavior and metastatic potential and thus contribute to the assessment of an individual patient’s prognosis.

Ultrasound Molecular Imaging of VEGFR2 in a Rat Prostate Tumor Model Using BR55

To evaluate BR55, a new VEGFR2-specific ultrasound contrast agent, for imaging prostate tumors in an orthotopic model in the rat. Rat prostate adenocarcinoma were established by injection of G Dunning R-3327 tumor cells in one lobe of the prostate of Copenhagen rats. Imaging experiments were performed with BR55, SonoVue, and streptavidin-functionalized microbubbles coupled with an anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody using a clinical ultrasound scanner. Contrast enhancement in the tumor and healthy prostate was followed over time by intermittent imaging at low acoustic power. Signal quantification and statistical analysis were performed in the tumor and healthy tissue to compare the behavior of the 3 contrast agents. Immunohistochemistry was performed on the prostate and tumor specimen to determine the expression of VEGFR2. Comparable contrast enhancement was observed in tumors at peak intensity for BR55 and SonoVue. Then, once unbound microbubbles had cleared from the circulation, a strong enhancement of the tumor was obtained with BR55, whereas no significant microbubble accumulation was detected in the healthy prostate tissue. SonoVue microbubbles were rapidly eliminated, and no significant binding was observed in the tumor. The tumor to prostate ratio calculated after signal quantification was about 20 for the 3 doses of BR55 tested. The enhancement obtained with BR55 in the tumor was not significantly different from the one observed with antibody-coupled streptavidin microbubbles. Intense staining for VEGFR2 was detected in the tumor vessels by immunohistochemistry. This study showed that BR55 binding to prostate tumors resulted in a strong enhancement of the lesions as early as a few minutes after contrast injection, whereas minimal nonspecific accumulation occurred in the healthy part of the gland. BR55, like SonoVue, provide information on tissue perfusion during the early vascular phase, but BR55 binding to the tumoral endothelium allows to gain additional information by highlighting the sites of active angiogenesis. The late phase enhancement of the tumor should be particularly valuable for prostate cancer detection and for biopsy guidance.

Effect of Cyproterone Acetate in Comparison to Flutamide on the Ventral Prostate of Adult Male Castrated Copenhagen-Fisher Rats and on Dunning R-3327 H Tumors/Der Einfluß von Cyproteron-Acetat im Vergleich zu Flutamid auf die ventrale Prostata der erwachs

In this investigation the effect of CPA was tested in comparison to FL after the procedure of double blinding on the ventral prostate of 70 adult male castrated Copenhagen-Fisher rats and on the Dunning R-3327 H tumor. Total androgen blockade by castration plus CPA or by castration plus FL induced significant decrease in prostate weight compared to the androgen deprivation by castration alone. No significant difference between CPA and FL was observed. Furthermore it was impossible to exaggerate this effect with higher doses of CPA of FL. The Dunning R-3327 H tumor did not become palpable 60 days after inoculation of the tumor cells indicating that androgen deprivation by total androgen blockade by castration plus CPA or plus FL did not exhibit any proliferative activity on the hormone-sensitive Dunning R-3327 H tumor cells.

Correlation of radiation response with tumor oxygenation in the Dunning prostate R3327-AT1 tumor

To investigate the application of pretreatment oxygenation to the AT1 subline of the Dunning R3327 prostate tumor, which is more hypoxic and faster growing than the H1 subline previously studied. Dunning prostate R3327-AT1 tumors growing on Copenhagen rats were administered 30 Gy of X-ray radiation either with or without oxygen inhalation. Tumor oxygenation was sampled by (19)F nuclear magnetic resonance echo planar imaging relaxometry of the reporter molecule hexafluorobenzene, no more than 24 h before irradiation. Large tumors (>3.0 cm(3)) exhibited significantly greater hypoxic fractions and lower mean partial pressure of oxygen (pO(2)) than their smaller counterparts (<1.5 cm(3)). However, unlike the R3327-HI subline, large AT1 tumors generally did not respond to oxygen inhalation in terms of altered hypoxic fraction or response to irradiation. Although the tumors did not respond to oxygen inhalation, each tumor had a different pO(2), and there was a clear trend between level of oxygenation at time of irradiation and tumor growth delay, with considerably better outcome when mean pO(2) > 10 mm Hg. The comparatively small baseline hypoxic fraction in the group of small tumors was virtually eliminated by breathing oxygen, and the growth rate was significantly reduced for tumors on rats breathing oxygen during irradiation. These results further validate the usefulness of nuclear magnetic resonance oximetry as a predictor of response to radiation therapy.

Platinum(II) complexes interfering with testicular steroid biosynthesis: drugs for the therapy of advanced or recurrent prostate cancers? Preclinical studies

[Meso-1,2-bis(2,6-dihalo-3/4-hydroxyphenyl)ethylenediamine]platinum(II) complexes (meso-1-PtLL': 2,6-F(2),3-OH; meso-2-PtLL': 2,6-F(2),4-OH; meso-3-PtLL': 2,6-Cl(2),3-OH; meso-4-PtLL': 2,6-Cl(2),4-OH; L = OH(2), L' = OSO(3) or L,L' = Cl(2)) were designed with the aim to get drugs comprising both cytotoxic and testosterone level lowering potencies. It is assumed that such compounds are more efficient than the established endocrine therapeutic measures and can affect the development of hormone refractory prostate cancer (PC). With exception of meso-3-PtLL' all Pt-complexes and the comparison compound cisplatin significantly reduced the testosterone level in experiments on male rats. However, in the test on the Dunning R3327 PC of the rat only cisplatin and meso-4-PtLL' showed a significant anti-tumor activity at well-tolerated dose ranges. Meso-4-PtLL' also significantly extended the time to disease progression in comparison with orchiectomy in this tumor model. Interestingly, the relapsed tumor, too, responded to meso-4-PtLL' as demonstrated in a long-term study on orchiectomized rats bearing Dunning R3327 PC grafts. This effect cannot be ascribed to cytotoxic effects of meso-4-PtLL' because of its inactivity on the human LNCaP/FGC PC cell line. Therefore, the contribution of an additional mechanism to the anti-prostate cancer activity of meso-4-PtLL', presumably owing to its estrogenic potency, must be considered. This assumption was supported by test results with diethylstilbestrol (DES) (non-steroidal estrogen) on the Dunning R3327 PC of the rat relapsed after orchiectomy. This tumor model was strongly inhibited by DES. The possible mode of action of meso-4-PtLL' is thoroughly discussed.

Identification of ABR-215050 as lead second generation quinoline-3-carboxamide anti-angiogenic agent for the treatment of prostate cancer

Linomide, Figure 1, produces robust and consistent in vivo growth inhibition of prostate cancer models via its anti-angiogenic activity and inhibition of autoimmune encephalomyelitis models of multiple sclerosis (MS). MS clinical trials were discontinued because of unacceptable toxicity, due to dose-dependent induction of proinflammation. Therefore, linomide analogs were initially screened to determine their in vivo potency to inhibit growth of the Dunning R-3327 AT-1 rat prostate cancer model in rats and their potency to inhibit angiogenesis in a Matrigel assay in mice. Based upon its superior potency (i.e., 30- to 60-fold more potent than linomide) in these assays and its lack of a proinflammation in the Beagle-dog, ABR-215050 (tasquinimod), Figure 1, was characterized for dose-response ability to inhibit the growth of a series of four additional human and rodent prostate cancer models in mice. Pharmacokinetic analysis following oral dosing documented that blood and tumor tissue levels of ABR-215050 as low as 0.5-1 microM are therapeutically effective. This efficacy is correlated with inhibition of angiogenesis in a variety of assays (endothelial capillary tube formation, aortic ring assay, chorioallantoic membrane assay, real-time tumor blood flow and PO(2) measurements, tumor blood vessel density, and tumor hypoxic and apoptotic fractions). Based upon its robust and consistent anti-angiogenic activity and thus tumor growth, ABR-215050 has entered clinical trials for the treatment of prostate cancer.

Enhanced inhibitory effect of the matrix metalloproteinase inhibitor Ro 28-2653 in combination with estramustine and etoposide on the prostate carcinoma in the rat Dunning orthotopic tumor model

Therapeutic efficacy of the novel matrix metalloproteinase (MMP) inhibitor Ro 28-2653 has been shown in various models of different tumor entities. We hypothesized that the inhibitor effect of Ro 28-2653 on the tumor growth could be improved by combination with chemotherapeutic drugs and examined therefore the effect of Ro 28-2653 alone and in combination with etoposide or estramustine in the MatLyLu Dunning R-3327 rat tumor model characteristic for the androgen-independent prostate cancer (PCa). In vitro effects were estimated measuring the proliferation of MatLyLu cells incubated with the three agents alone or in combination using the XTT test. The in vivo effects of the agents alone or in combination were examined by measuring the tumor weight 18 days after tumor cell injection. The proliferation rate was only inhibited by etoposide while that effect was increased in combination with Ro 28-2653 and estramustine. Ro 28-2653 reduced the tumor weight by 86%. That effect was significantly increased in combination with etoposide to 92%. The inhibitory effect of the MMP inhibitor Ro 28-2653 on the tumor growth in the Dunning PCa model is enhanced by the standard chemotherapeutic drug etoposide. A combined application of both agents could be considered as potential tool to improve the therapy of patients with advanced PCa after failure of hormonal treatment.

Identification of androgen‐responsive genes that are alternatively regulated in androgen‐dependent and androgen‐independent rat prostate tumors

The vast majority of androgen-dependent prostate tumors progress toward incurable, androgen-independent tumors. The identification of androgen-responsive genes, which are still actively transcribed in the tumors of patients who have undergone androgen ablation, may shed light on the molecular mechanisms underlying this phenomenon. To address this question, we chose the Dunning R3327 rat model system, in which the progression from androgen-dependent to -independent tumors is represented by several transplantable prostate-derived tumors. Gene expression profiles were analyzed in normal rat prostates and in the prostates of rats 14 days after castration by use of microarrays containing approximately 5,000 oligonucleotides, together representing more than 4,800 known rat genes. These expression profiles were compared with similarly obtained expression profiles of androgen-dependent and androgen-independent rat prostate tumors. By doing so, a series of known and novel prostate cancer-associated androgen-responsive genes was identified. Within this series, we were able to identify several clusters of genes that are differentially regulated in the various prostate tumors. These genes may serve as (i) novel prognostic identifiers and (ii) novel therapeutic targets.

Magnetic fluid hyperthermia (MFH)reduces prostate cancer growth in the orthotopic Dunning R3327 rat model

Magnetic fluid hyperthermia (MFH) is a new technique for interstitial hyperthermia or thermoablation based on AC magnetic field-induced excitation of biocompatible superparamagnetic nanoparticles. Preliminary studies in the Dunning tumor model of prostate cancer have demonstrated the feasibility of MFH in vivo. To confirm these results and evaluate the potential of MFH as a minimally invasive treatment of prostate cancer we carried out a systematic analysis of the effects of MFH in the orthotopic Dunning R3327 tumor model of the rat. Orthotopic tumors were induced by implantation of MatLyLu-cells into the prostates of 48 male Copenhagen rats. Animals were randomly allocated to 4 groups of 12 rats each, including controls. Treatment animals received two MFH treatments following a single intratumoral injection of a magnetic fluid. Treatments were carried out on days 10 and 12 after tumor induction using an AC magnetic field applicator system operating at a frequency of 100 kHz and a variable field strength (0--18 kA/m). On day 20, animals were sacrificed and tumor weights in the treatment and control groups were compared. In addition, tumor growth curves were generated and histological examinations and iron measurements in selected organs were carried out. Maximum intratumoral temperatures of over 70 degrees C could be obtained with MFH at an AC magnetic field strength of 18 kA/m. At a constant field strength of 12.6 kA/m, mean maximal and minimal intratumoral temperatures recorded were 54.8 degrees C (centrally) and 41.2 degrees C (peripherally). MFH led to an inhibition of tumor growth of 44%-51% over controls. Mean iron content in the prostates of treated and untreated (injection of magnetic fluids but no AC magnetic field exposure) animals was 82.5%, whereas only 5.3% of the injected dose was found in the liver, 1.0% in the lung, and 0.5% in the spleen. MFH led to a significant growth inhibition in this orthotopic model of the aggressive MatLyLu tumor variant. Intratumoral deposition of magnetic fluids was found to be stable, allowing for serial MFH treatments without repeated injection. The optimal treatment schedules and temperatures for MFH need to be defined in further studies.

Susceptibility of Lobund-Wistar × Copenhagen hybrid rats to autochthonous prostate carcinogenesis

Transplantation of the Dunning R-3327 prostate tumor cell line is a common model of prostate cancer, though the rat strain (Copenhagen) from which the cell line was derived is resistant to spontaneous and chemically induced prostate cancer. To determine if susceptibility to chemically induced prostate carcinogenesis can be transferred from the susceptible Lobund-Wistar (LW) rat strain to the resistant Copenhagen (COP) strain, male COP rats and LW x COP hybrids were administered an intravenous dose (30 mg/kg) of methylnitrosourea (MNU) and implanted three times with silastic capsules containing testosterone propionate (25 mg each) at a 2-month interval. Serum was sampled at 3, 6, 9, and 12 months of age and assayed for testosterone. Serum testosterone was significantly but transiently elevated following implantation of capsules with testosterone propionate, but then decreased by 12 months of age to a level which was significantly less (P < or = 0.001) than in LW rats but not significantly different from COP rats. Prostate cancer did not develop in COP rats, but 36% of LW x COP hybrids developed prostate-seminal vesicle tumors which expanded into the dorsolateral lobes within 10 months of MNU administration; this compares to 90% of LW rats which develop such tumors following this same induction protocol. COP rats lack inherent susceptibility to development of prostate cancer; susceptibility may be transferred from LW rats, or resistance from COP rats, to LW x COP hybrids and is present in the haploid state, consistent with the two-mutation event hypothesis of Knudson which holds that two mutations are required at a genetic locus for development of some cancers.

Directionally specific paracrine communication mediated by epithelial FGF9 to stromal FGFR3 in two-compartment premalignant prostate tumors.

Tissue homeostasis in normal prostate and two-compartment nonmalignant prostate tumors depends on harmonious two-way communications between epithelial and stromal compartments. Within the fibroblast growth factor (FGF) family, signaling to an epithelial cell-specific FGF receptor (FGFR) 2IIIb-heparan sulfate complex from stromal-specific FGF7 and FGF10 delivers directionally specific instruction from stroma to epithelium without autocrine interference. Using a two-compartment transplantable prostate tumor model in which survival of stromal cells in vivo depends on epithelial cells, we show that signaling from epithelial FGF9 to stromal FGFR3 potentially mediates epithelial-to-stromal communication that also is directionally specific. FGF9 mRNA was expressed exclusively in the epithelial cells derived from well-differentiated, two-compartment Dunning R3327 rat prostate tumors. In contrast, FGFR3 was expressed at functionally significant levels only in the derived stromal cells. Competition binding and immunoprecipitation assays revealed that FGF9 only bound to an FGFR on the stromal cells. FGF9 also failed to covalently cross-link to clonal lines of stromal cells devoid of FGFR3 that expressed FGFR1 and FGFR2IIIc. Furthermore, FGF9 specifically stimulated DNA synthesis in stromal cells expressing FGFR3. These results demonstrate a directionally specific paracrine signaling from epithelial FGF9 and stromal FGFR3. Similar to the FGF7/FGF10 to FGFR2IIIb signaling from the stroma to the epithelium, the directional specificity from epithelium to stroma appears set by a combination of cell-specific expression of isoforms and cell-context specificity of FGFR isotypes for FGF.

Photodynamic Therapy of Prostate Cancer by Means of 5-Aminolevulinic Acid-Induced Protoporphyrin IX – In vivo Experiments on the Dunning Rat Tumor Model

Objective: In order to expand the use of photodynamic therapy (PDT) in the treatment of prostate carcinoma (PCA), the aim of this study was to evaluate PDT by means of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX) in an in vivo tumor model. Methods: The model used was the Dunning R3327 tumor. First of all, the pharmacokinetics and the localization of PPIX were obtained using fluorescence measurement techniques. Thereafter, PDT using 150 mg 5-ALA/kg b.w. i.v. was performed by homogenous irradiation of the photosensitized tumor (diode laser λ = 633 nm). The tumors were resected 2 days post-PDT and the extent of the necrosis was determined histopathologically. Results: The kinetics of PPIX fluorescence revealed a maximum intensity in the tumor tissue within 3 and 4.5 h post-application of 5-ALA. At this time, specific PPIX fluorescence could be localized selectively in the tumor cells. The PDT-induced necrosis (n = 18) was determined to be 94 ± 12% (range 60–100%), while the necrosis of the controls (n = 12) differs significantly (p < 0.01), being less than 10%. Conclusion: These first in vivo results demonstrate the effective potential of 5-ALA-mediated PDT on PCA in an animal model.

Comparison of different methods to assess the cytotoxic effects of cytosine deaminase and thymidine kinase gene therapy

Dunning R3327 AT-1 rat prostate tumor cells were transfected with a double-fusion suicide gene (CDglyTK) that coded for the cytosine deaminase from E. coli and the thymidine kinase (TK) from HSV-1. The resulting cell line AT-1/CDglyTK was incubated with 10 and 20 microg/ml 5-FC or 0.25 microg/ml GCV, or both 5-FC and GCV 96 hours before harvest. The MTS assay detected cell viabilities of 50+/-5 and 25+/-5% after 5-FC treatment, and 50+/-5% after GCV treatment. The dye exclusion and the colony-forming assay confirmed the data of the MTS assay with GCV (47+/-5 and 32+/-5%), but presented different results for the 5-FC incubation. We detected 100+/-1 and 85+/-5% viable cells after 10 microg/ml 5-FC, and 97+/-1 and 85+/-5% after 20 microg/ml 5-FC treatment, respectively. S-phase arrest in both suicide gene systems was noticeable and a significant increase in cell granularity was observed after incubation with GCV or GCV & 5-FC. This study demonstrates that 5-FC and the metabolized 5-FU act not only as genotoxic reagents, but also as RNA-directed agent, because of the recovery of the cells. On the other hand, a significant S-phase block could be observed after 24 hours incubation with GCV. This short time is enough to incorporate the genotoxic GCV metabolites in the nascent DNA to impair the cell cycle.

Cytosine deaminase versus thymidine kinase: a comparison of the antitumor activity.

The efficacy of single and combination suicide gene therapy was evaluated using a Herpes simplex virus thymidine kinase/ganciclovir system and Escherichia coli cytosine deaminase/5-fluorocytosine system on the rat prostate tumor cell line R3327 AT-1. The wild-type R3327 AT-1 cell line was transfected with a bifunctional fusion gene CDglyTK, which had the advantage that the resulting R3327 AT-1/CDglyTK cell line has the same amount of cytosine deaminase and thymidine kinase molecules. The percentage of viable R3327 AT-1/CDglyTK cells after 96 h incubation with 0.1 micro g/ml ganciclovir or 10 micro g/ml 5-fluorocytosine were 85% and 52% of controls, respectively. The cell viability when both suicide genes systems were activated was 43%. For in vivo analysis, Copenhagen rats were injected subcutaneously with R3327 AT-1 or R3327 AT-1/CDglyTK cells and treated with 30 mg/kg ganciclovir, 500 mg/kg 5-fluorocytosine, or both prodrugs together. A survival of 83% with the thymidine kinase/ganciclovir and 57% with the CD/5-FC could be observed. Only co-administration of thymidine kinase- and cytosine deaminase-specific prodrugs resulted in a 100% recurrence-free survival of the Copenhagen rats with a Dunning R3327 AT-1/CDglyTK prostate tumor and showed an additive cytotoxic effect. Calculation of the degree of activation and the potential of activation can be used to predict the success of a suicide gene therapy. In our case, the cytosine deaminase/5-fluorocytosine system had a low degree of activation (value 40), which is also found in the low response to 5- fluorocytosine in vivo (57% tumor free).

Solid stress facilitates spheroid formation: potential involvement of hyaluronan.

When neoplastic cells grow in confined spaces in vivo, they exert a finite force on the surrounding tissue resulting in the generation of solid stress. By growing multicellular spheroids in agarose gels of defined mechanical properties, we have recently shown that solid stress inhibits the growth of spheroids and that this growth-inhibiting stress ranges from 45 to 120 mmHg. Here we show that solid stress facilitates the formation of spheroids in the highly metastatic Dunning R3327 rat prostate carcinoma AT3.1 cells, which predominantly do not grow as spheroids in free suspension. The maximum size and the growth rate of the resulting spheroids decreased with increasing stress. Relieving solid stress by enzymatic digestion of gels resulted in gradual loss of spheroidal morphology in 8 days. In contrast, the low metastatic variant AT2.1 cells, which grow as spheroids in free suspension as well as in the gels, maintained their spheroidal morphology even after stress removal. Histological examination revealed that most cells in AT2.1 spheroids are in close apposition whereas a regular matrix separates the cells in the AT3.1 gel spheroids. Staining with the hyaluronan binding protein revealed that the matrix between AT3.1 cells in agarose contained hyaluronan, while AT3.1 cells had negligible or no hyaluronan when grown in free suspension. Hyaluronan was found to be present in both free suspensions and agarose gel spheroids of AT2.1. We suggest that cell–cell adhesion may be adequate for spheroid formation, whereas solid stress may be required to form spheroids when cell–matrix adhesion is predominant. These findings have significant implications for tumour growth, invasion and metastasis.British Journal of Cancer (2002) 86, 947–953. DOI: 10.1038/sj/bjc/6600158 www.bjcancer.com© 2002 Cancer Research UK

Efficacy of a synthetic lytic peptide in the treatment of prostate cancer

In the last several years, significant effort has been applied to identifying novel agents with effectiveness against prostate cancer. These studies were designed to determine the efficacy of one of these novel compounds, D2A21, in the treatment of an animal model of prostate cancer. Using the Mat-Ly-Lu(MLL) line of the Dunning R-3327 rat prostate adenocarcinoma model, the optimal dose, schedule and route of administration of D2A21 were established. A study involving the G line was used to further support these findings. In addition, hemotoxylin and eosin stained tissue samples were examined to investigate the extent of inhibition of lung metastases in animals injected with MLL cells. When D2A21 was injected intraperitoneally or subcutaneously, MLL and G cell tumor growth was inhibited 50-72% as demonstrated by both tumor volumes and weights. The optimal dosage of 0.179 mg/injection was established and it was determined to be most efficacious when administered five times per week. At this concentration, D2A21 appears to have no significant toxicity. Additionally, D2A21 increased the survival rate from only 25% to 70-75% in animals that were challenged with a large number of tumor cells. The peptide D2A21 is able to significantly inhibit tumor growth in rat models of prostate cancer. In addition, it can inhibit metastases and decrease deaths resulting from metastases in these animals.

Combined treatment of Dunning R3327 rat prostatic tumor with the 5alpha-reductase inhibitor PNU 157706 and the antiandrogen bicalutamide.

PNU 157706 [N-(1,1,1,3,3,3-hexafluorophenyl-propyl)-3-oxo-4-aza- 5alpha-androst-1-ene-17beta-carboxamide], a novel, potent and selective dual 5alpha-reductase inhibitor, was reported to be effective in inhibiting the growth of established tumors in the Dunning R3327 rat prostatic carcinoma model. We investigated the efficacy of treatment with PNU 157706 in combination with the antiandrogen bicalutamide in this prostatic tumor model. Rats with tumor diameters of about 1 cm were treated orally 6 days a week for 9 weeks with PNU 157706 (10 mg/kg per day) alone or in combination with bicalutamide (0.2 and 1 mg kg per day). Animals were killed 24 h after the last treatment, and ventral prostates were removed for testosterone (T) and dihydrotestosterone (DHT) determination. PNU 157706 reduced the growth of established tumors by 39%; bicalutamide proved ineffective at 0.2 mg/kg per day, but reduced tumor growth by 45% at a dose of 1 mg/kg per day. The combination of PNU 157706 with both doses of bicalutamide caused an additive tumor growth inhibition (50% and 64%). Castration resulted in marked tumor growth inhibition (72%). Ventral prostate weight was markedly reduced by PNU 157706 (78%) treatment and by bicalutamide (59% and 77%); combined treatment was as effective as castration. Prostatic DHT content was markedly reduced by PNU 157706 (88%), whereas prostatic T increased slightly (60%). Concomitant treatment with bicalutamide antagonized the T increase induced by PNU 157706 and did not modify the already remarkable suppression of DHT. These data show that the inhibitory effect of PNU 157706 and bicalutamide on Dunning prostatic tumor growth is additive, thus suggesting a possible role of PNU 157706 in the therapy of advanced prostate cancer, in combination with antiandrogens, to provide an effective peripheral androgen ablation therapy with minimal side effects.

Combined treatment with the 5 alpha-reductase inhibitor PNU 157706 and the antiandrogen flutamide on the Dunning R3327 prostatic carcinoma in rats.

The steroid 5 alpha-reductase enzyme catalyzes the conversion of testosterone to the potent androgen 5 alpha-dihydrotestosterone (DHT). PNU 157706, a novel, potent and selective dual 5 alpha-reductase inhibitor, was reported to be effective in inhibiting the growth of established tumors in the Dunning R3327 rat prostatic carcinoma model. We have studied the efficacy of combined treatment with PNU 157706 and the antiandrogen flutamide in this prostatic tumor in rats. Rats with tumor diameters of about 1 cm were treated orally 6 days a week for 9 weeks with PNU 157706 (10 mg/kg per day) alone or in combination with flutamide (1 and 5 mg/kg per day). Animals were killed 24 h after the last treatment and ventral prostates were removed for testosterone and DHT determination. PNU 157706 reduced the growth of established tumors by 36%; flutamide showed a slight effect at 1 mg/kg per day (24% inhibition), while at the dose of 5 mg/kg per day it reduced tumor growth by 48%. The combination of PNU 157706 with the lower dose of flutamide caused an additive tumor growth inhibition (60%) and the combination with the higher dose of flutamide resulted in a better inhibition of tumor growth (68%) than did either treatment alone. Castration resulted in marked tumor growth inhibition (76%). Ventral prostate weight was more markedly reduced by PNU 157706 treatment than by flutamide; combined treatment was as effective as castration. Prostatic DHT content was markedly reduced by PNU 157706 (93%), whereas prostatic testosterone increased (137%). Concomitant treatment with flutamide partially antagonized the testosterone increase induced by PNU 157706 and did not modify the already considerable suppression of DHT. These data show that the inhibitory effects of PNU 157706 and flutamide on Dunning prostatic tumor growth are additive, thus supporting the rationale of this combination therapy in advanced prostate cancer, in order to achieve adequate androgen blockade with minimal side-effects.

Methotrexate-albumin conjugate causes tumor growth delay in Dunning R3327 HI prostate cancer-bearing rats.

Based on the rationale of a preferred albumin uptake by tumors, conjugates comprising of rat serum albumin (RSA) as a drug carrier and of methotrexate (MTX) as chemotherapeutic drug were prepared. For a comparative study of MTX-RSA and MTX we chose a slow growing Dunning R3327 HI prostate cancer model. In a radiopharmacologic study blood kinetics and the tumor and organ distribution pattern of residualizingly labeled MTX-RSA were determined, and were found to be similar to that of residualizingly labeled RSA. The MTD was established for Copenhagen rats at a total four injections of 2 mg/kg MTX or MTX-RSA administered at days 0, 4, 8 and 12. Tumor volume measurements and tumor removal showed a small non-significant growth delay in the MTX treatment group, suggesting MTX resistance for the Dunning R3327 HI prostate carcinoma. In contrast, treatment with MTX-RSA resulted in a significant (50%) growth inhibition of the Dunning R3327 HI tumor. The cellular mechanisms responsible for MTX resistance in Dunning HI tumor cells is not known. The improved therapeutic effects seen during MTX-RSA treatment in this slow growing adenocarcinoma might be a result of prolonged tumor exposure time and an altered cellular uptake by a lysosomal route. MTX-albumin conjugates have shown antitumor activity exceeding that of MTX in several tumor xenografts in nude mice, including human prostate cancer. The recently initiated clinical development of MTX-human serum albumin will be continued and cancer of the prostate will be included as a potential target tumor during further clinical phase II testing.