Rat Prostate Cancer Research Articles

Androgen regulation of prostate cancer cell FGF-1, FGF-2, and FGF-8: Preferential down-regulation of FGF-2 transcripts

Using quantitative RT-PCR, we found that T1 rat prostate cancer cell relative FGF-1 transcript content was about 180-fold greater than that of FGF-2. This difference in transcript content was not representative of T1 cell relative FGF-1 and FGF-2 protein content which showed, at most, only a 4- to 5-fold greater FGF-I content. Testosterone caused time-dependent down-regulation of prostate cancer cell FGF-2 transcript content without influencing either FGF-1 or FGF-8 transcript content or TI cell proliferation. Moreover, testosterone-mediated down-regulation of prostate cancer cell FGF-2 transcripts did not result in a statistically significant change in 21.5 or 17.0 kD FGF-2 isoform content. By contrast, an approximately 20% statistically significant decrement in 19.5 kD FGF-2 isoform content was demonstrable following 24 h testosterone treatment. However, following 72 h testosterone treatment, T1 cell 19.5 kD FGF-2 isoform content was not statistically significantly different from that of control. It is probable that the modest and variable decrement in 19.5 kD isoform content is not physiologically significant and is attributable to artifact resulting from difficulty quantifying this minor component of the FGF-2 isoforms. Transient transfection analysis showed that androgen caused concentration-dependent increases in MMTV-LTR regulated expression of chloramphenicol acetyl transferase activity. Consequently, the failure of androgen to affect either T1 cell FGF-1 and FGF-8 transcript content or Ti cell proliferation could not be attributed to defective androgen receptor function. Moreover, the absence of a close relationship between T1 cell FGF-2 transcript and FGF-2 protein content implies that FGF-2 transcript content is not the dominant determinant of prostate cancer cell FGF-2 protein content. Testosterone-mediated down-regulation of prostate-cancer-cell gene expression may have significance for clinical management of human disease that is treated by androgen ablation. The possibility that such ablation may enhance aggressiveness of “androgen-independent” cells by selective up-regulation of gene expression merits further consideration.

Localization of prostate cancer metastasis‐suppressor activity on human chromosome 17

BACKGROUND Prostate cancer is the most commonly diagnosed malignancy in American men. Currently, it is difficult to accurately predict the clinical course of histologically localized prostatic cancer in the individual patient. Identification of markers for metastatic potential of prostate cancer may improve the diagnosis and treatment of this disease. We have previously demonstrated that human chromosome 17 (17pter–q23) suppresses the metastatic ability of AT6.1 rat prostatic cancer cells. In this study we report on the further localization of the metastasis suppressor activity encoded by human chromosome 17. METHODS A series of AT6.1-17 microcell hybrids was constructed using microcell-mediated chromosomal transfer of human chromosome 17 into highly metastatic AT6.1 cells. Hybrids which had spontaneously deleted regions of chromosome 17 were analyzed by PCR for the presence of 32 sequence-tagged sites (STS) markers as well as the prostate cancer tumor-suppressor loci reported on 17q. In addition, we examined a number of candidate genes and markers that previously have been mapped to chromosome 17. The in vivo metastatic potential of these AT6.1-17 deletion hybrids was determined. RESULTS We have localized metastasis-suppressor activity to a ∼70-centiMorgan (cM) portion of chromosome 17, consisting of three distinct regions of 30 cM (D17S952→D17S805), 6 cM (D17S930→D17S797), and 34 cM (D17S944→D17S784). Three of the four markers on 17p13, including HIC1 and TP53, and 12 of the 13 markers in 17q21–23, including BRCA1 (D17S855) and NM23 (NME1), were not retained in the conserved ∼70-cM metastasis-suppressor region. CONCLUSIONS These results support a role for a novel metastasis-suppressor gene(s) or a novel metastasis-suppressor function on chromosome 17. Complementary candidate gene and positional cloning approaches are being used to identify the gene(s) within the ∼70-cM conserved region responsible for metastasis suppression. Prostate 33:271–280, 1997. © 1997 Wiley-Liss, Inc.

Mechanism and role of growth arrest in programmed (apoptotic) death of prostatic cancer cells induced by thapsigargin.

More than 95% of metastatic androgen independent prostatic cancer cells per day are in a proliferatively quiescent G0 state [Berges et al.: Clin Cancer Res 1:473-480, 1995] limiting their responsiveness to anti-proliferative chemotherapeutic agents. Novel therapeutics capable of activating the programmed (apoptotic) death pathway in these cells without requiring entrance into the proliferative cell cycle are urgently needed. Thapsigargin (TG) treatment of rapidly growing androgen independent prostatic cancer cells arrests such cells in G0 and induces their programmed death. This raises not only the issue of the mechanism for such growth arrest, but also whether this programmed death is simply a response of rapidly growing cells to growth arrest making cytotoxicity still dependent upon the initial rate of cell proliferation. To resolve the mechanism of TG induced growth arrest, rat AT3.1 prostatic cancer cells were analyzed for RNA and protein expression of the growth arrest gene, gadd153, intracellular free Ca2+ levels (Cai), and cell cycle distribution on exposure to TG alone and in combination with Ca2+ chelation induced by BAPTA-AM or BAPTA-AM/EGTA. To resolve whether growth arrest is required for TG cytotoxicity, primary cultures of proliferatively quiescent, human prostatic cancer cells were exposed to TG. Co-treatment of androgen independent AT-3 rat prostatic cancer cells with the Cai chelator BAPTA plus TG prevented growth arrest, as monitored by DNA flow cytometry, and failure to induce mRNA and protein for gadd153, demonstrating that growth arrest is due to Cai elevation, not depletion of intracellular Ca2+ pools. In addition, proliferatively quiescent G0 primary cultures of human prostatic cancer cells were resistant to anti-proliferative agents, but could be induced to undergo programmed death by TG as documented by morphological criteria and 14C-labeled DNA fragmentation assays. These results demonstrate that TG with its ability to elevate Cai induces proliferating prostate cancer cells to growth arrest. Such Cai dependent growth arrest is not required, however, since TG can induce the programmed death of proliferatively quiescent G0 prostatic cancer cells without requiring either growth arrest or progression through the proliferative cell cycle.

Plasminogen activator and matrix metalloproteinase production and extracellular matrix degradation by rat prostate cancer cells in vitro: correlation with metastatic behavior in vivo.

The plasminogen activation (PA) and metalloproteinase (MMP) system are involved in tumor cell migration and invasion. The proteolytic activity of cell lines originating from the rat Dunning R-3327 prostate tumor was analyzed by measuring in vitro extracellular matrix degradation, enzyme activity, and mRNA levels of enzymes, inhibitors, and receptors, and compared with their known metastatic behavior in vivo. Only the highly metastatic sublines AT-3, MATLu, and MATLyLu showed a high extracellular matrix degradation mediated by urokinase-type plasminogen activator (u-PA). Relatively high levels of u-PA were present in the aggressive cell lines. u-PA receptor mRNA was produced in all cells, and all but AT-1 produced LDL-receptor-related-protein (LRP) mRNA. t-PA mRNA was only found in HIF and MATLu. In gelatin, zymography lysis was observed at 72 kD and 74-76 kD in MATLu and MATLyLu cells, respectively. MMP-2 mRNA was present in all cell lines except AT-1 and AT-2, and MMP-3 mRNA was present in AT-2, AT-3, and MATLu. These in vitro experiments show that in different rat prostate cancer sublines, proteolytic activity and u-PA-mediated extracellular matrix degradation correlate with their known metastatic behavior in vivo.

Location of KAI1 on the short arm of human chromosome 11 and frequency of allelic loss in advanced human prostate cancer.

We recently isolated the KAI1 gene, a metastasis suppressor gene for prostate cancer, from human chromosome region 11p13-cen-containing rat prostate cancer cells. The present study was performed to further locate the region of the KAI1 gene on the short arm of chromosome 11, and to examine whether loss of this region is significant during progression of human prostate cancer. The small portion of human chromosome 11 (i.e., 11p13-cen) was reintroduced into highly metastatic rat prostate cancer cells by using microcell-mediated chromosome transfer. Loss of heterozygosity (LOH) at polymorphic microsatellite loci on the human chromosome 11 was examined in human prostate cancer tissues. The minimum region of human chromosome 11 that contained the KAI1 gene was located on the proximal region of 11p11.2 divided by the D11S554 locus. The percentage of LOH or allelic imbalance at the D11S1344 locus, which is located on the same region as the KAI1 locus, in metastasis tissues from autopsy cases who died from metastatic prostate cancer was 70% (7 of 10 informative cases), whereas the percentages in primary tumors from the same cases and from cases with clinically localized prostate cancer were 33% (3 of 9 informative cases) and 8% (1 of 12 informative cases), respectively. These findings demonstrate a high frequency of LOH or allelic imbalance at the centromeric region of 11p, which contains the KAI1 gene in advanced prostate cancer.

Induction in human osteoblastic cells (SaOS2) of the early response genes fos, jun, and myc by the amino terminal fragment (ATF) of urokinase.

Previous studies have demonstrated that overexpression of urinary plasminogen activator (uPA) in rat prostate cancer cells results in increased skeletal metastases, which are primarily of the osteoblastic variety. The osseous activation induced by the metastases appears to be mediated through the amino terminal fragment (ATF) of uPA, which lacks the catalytic domain and can act as a growth factor for osteoblasts. To explore further the mechanism of action of uPA in bone cells, we evaluated the effects of ATF on modulating the expression of various proto-oncogenes. Human-osteoblast-derived osteosarcoma cells, SaOS2, were treated with graded doses of ATF for 10-120 min, and effects on early response proto-oncogenes were monitored. ATF increased c-myc, c-jun, and c-fos gene expression in a time-dependent manner for up to 60 min, after which mRNA levels fell. The maximum induction was seen in c-fos gene expression, which was found to be dose dependent. This effect of ATF was localized to its growth-factorlike domain. Examination of the half life of these transcripts in the presence of the transcriptional inhibitor actinomycin D demonstrated that ATF does not alter the stability of c-fos mRNA in these bone cells. Nuclear run-off assays indicated that ATF effects were due to stimulation of c-fos gene transcription. An increase in c-fos protein levels was correlated with the augmentation of its mRNA in ATF-treated SaOS2 cells. Pretreatment of SaOS2 cells with the protein tyrosine kinase inhibitor herbimycin and recombinant soluble uPA receptor (uPAR) caused a significant reduction in the ability of ATF to induce c-fos expression. These results demonstrate a novel role for uPA in activating early response proto-oncogenes, in particular c-fos, which plays an important role in bone cell growth and differentiation and may be a key factor in the signal transduction pathway of ATF.

Expression and function of the leucine zipper protein Par-4 in apoptosis.

The prostate apoptosis response-4 (par-4) gene was identified by differential screening for genes that are upregulated when prostate cancer cells are induced to undergo apoptosis. The par-4 gene is induced by apoptotic signals but not by growth-arresting, necrotic, or growth-stimulatory signals. The deduced amino acid sequence of par-4 predicts a protein with a leucine zipper domain at its carboxy terminus. We have recently shown that the Par-4 protein binds, via its leucine zipper domain, to the zinc finger domain of Wilms' tumor protein WT1 (R. W. Johnstone et al., Mol. Cell. Biol. 16:6945-6956, 1996). In experiments aimed at determining the functional role of par-4 in apoptosis, an antisense par-4 oligomer abrogated par-4 expression and activator-driven apoptosis in rat prostate cancer cell line AT-3, suggesting that par-4 is required for apoptosis in these cells. Consistent with a functional role for par-4 in apoptosis, ectopic overexpression of par-4 in prostate cancer cell line PC-3 and melanoma cell line A375-C6 conferred supersensitivity to apoptotic stimuli. Transfection studies with deletion mutants of Par-4 revealed that full-length Par-4, but not mutants that lacked the leucine zipper domain of Par-4, conferred enhanced sensitivity to apoptotic stimuli. Most importantly, ectopic coexpression of the leucine zipper domain of Par-4 inhibited the ability of Par-4 to enhance apoptosis. Finally, ectopic expression of WT1 attenuated apoptosis, and coexpression of Par-4 but not a leucine zipperless mutant of Par-4 rescued the cells from the antiapoptotic effect of WT1. These findings suggest that the leucine zipper domain is required for the Par-4 protein to function in apoptosis.

Intracellular volume and apparent diffusion constants of perfused cancer cell cultures, as measured by NMR.

Diffusion NMR spectroscopy was used to study intracellular volume and apparent water diffusion constants in different cell lines (DU145, human prostate cancer; AT3, rat prostate cancer; MCF-7, human breast cancer; RIF-1, mouse fibrosacroma). The cells were grown on various matrices (collagen sponge, collagen beads, polystyrene beads) which enabled continuous growth in perfused high density cell culture suitable for NMR studies. In perfused cell systems, the attenuation of the water signal versus the squared gradient strength was fitted by the sum of two decaying exponentials. For the slowly decaying component the apparent water diffusion constant at 37 degrees C was 0.22 (+/-0.02) x 10(-9) s/m2 for all cell lines at diffusion times > 100 ms. It continuously increased up to 0.47 (+/-0.05) x 10(-9) s/m2 when the diffusion time was decreased to 8 ms, indicating restricted diffusion. No significant effect of the matrices was observed. The fractional volume of the slow component as determined from the biexponential diffusion curve correlated with the relative intracellular volume, as obtained from the cell density in the sample and the cell size as measured by light microscopy. Therefore, this simple NMR approach can be used to determine intracellular volume in perfused cell cultures suitable for NMR studies. Using this information in combination with spectroscopic data, changes in intracellular metabolite concentration can be detected even when the cellular volume is changing during the experiment. The apparent diffusion constant for the fast diffusing component varied with growth matrix, cell density and cell type and also showed the typical characteristics of restricted diffusion (increase of apparent diffusion constant with time).

Elevated expression of calcium-binding protein p9Ka is associated with increasing malignant characteristics of rat prostate carcinoma cells.

Northern and Western blotting techniques were used to study expression of the mRNA and corresponding protein product of the S100-related calcium-binding molecule p9Ka in 6 different metastatic cell lines of the Dunning R3327 rat prostate cancer model. In cells with the lowest metastatic capability (G cells), p9Ka mRNA was barely detectable. In 2 weakly metastatic cell lines (AT-1 and AT-2), p9Ka transcript amounts were, respectively, 6.29 +/- 0.74 and 5.55 +/- 1.11 times that detected in the G cells. In 3 highly metastatic cell lines (AT-3, MAT-LyLu and MAT-Lu), the amounts of p9Ka mRNA were, respectively, 12.85 +/- 2.82, 13.06 +/- 1.69 and 11.62 +/- 1.81 times that expressed in the G cells. Western blot analyses detected no p9Ka protein in the G cells. The amounts of p9Ka protein expressed by tumour cells of intermediate metastatic capability (AT-1 and AT-2) were 3.4 +/- 1.3 microg and 3.3 +/- 1.4 microg, respectively, per 1 x 10(6) cells. The amounts of p9Ka protein expressed by the tumour cells of highest metastatic capability (AT-3, MAT-LyLu and MAT-Lu) were 8.3 +/- 1.1 microg, 8.7 +/- 1.6 microg and 9.6 +/- 1.7 microg, respectively, per 1 x 10(6) cells. Our data reveal a direct association between the elevated expression of mRNA and the p9Ka protein amounts and the increased metastatic capability of individual prostatic cancer cell lines. We suggest that calcium-binding protein p9Ka may play an important role in the metastatic behaviour of rat prostate cancer.

THE EFFECT OF TREADMILL EXERCISE ON THE DEVELOPMENT OF PROSTATE CANCER IN RATS 1195

It is now evident that both acute and chronic exercise have an effect on the immune system. Moderate exercise training has been shown to significantly increase serum immunoglobulins, monocytes, as well as NK cell number and activity. The influence of physical activity on prostate cancer is a topic which has not been well investigated. Therefore, the purpose of this study was to examine the effect of moderate treadmill exercise on the development of prostate cancer in rats. Forty male Lobund-Wistar rats were randomly assigned to aerobic exercise (AE: n=20) or control (C: n=20) groups. Aerobic exercise consisted of running on a motor-driven treadmill at 20 m/min (73.2 ± 12.6% VO2 max), 15 min/day, three times weekly for six weeks. Following exercise, all animals were inoculated with 1 × 106 transplantable rat prostate cancer (PA-III) subcutaneously on the back, above the sacral region (day = 0). The AE group exercised an additional 2 weeks post inoculation. Max VO2 did not change in either group during the initial six-week training period (AE: 65.52 ± 6.16 vs 65.72 ± 4.29; C: 65.79 ± 4.34 vs. 64.36 ± 4.86, ml/kg/min). At day 30, the number of rats displaying tumor (AE: 20 vs C: 18) and the total tumor volume (AE: 7305.2 ± 3313.4 vs C: 9134.9 ± 6509.6, mm3) did not differ between groups. Lung tissue collected from 10 AE and 10 C at day 30 showed no differences in metastatic lesions. In conclusion, the treadmill exercise protocol was not sufficient to elicit a training response and had no effect on the incidence or growth of prostate cancer in rats at day 30. Future studies need to consider a greater dose of exercise in order to determine its efficacy.

Suppression of invasive ability of highly metastatic rat prostate cancer by introduction of human chromosome 8.

Introduction of human chromosome 8 to a highly metastatic subline (AT6.2) from the Dunning R-3327 rat prostate cancer resulted in suppression of metastatic ability of the resultant microcell hybrids (AT6.2-8 clones) [12]. The present study has been performed to clarify which step of metastasis was suppressed in the microcell hybrids. Northern blot analysis of E-cadherin and alpha-catenin, in vitro invasion assay, and intra-venous metastasis assay by injection of tumor cells into the lateral tail vein of nude mice were performed. No detectable expressions of either E-cadherin or alpha-catenin were found in either AT6.2 parental or AT6.2-8 microcell hybrid clones. In the invasion assay, invasiveness of AT6.2-8 hybrid clones was less than that of the AT6.2 parental clone. In the intravenous metastasis assay, no significant differences in the number of lung metastases were observed among these cell lines. Introduction of human chromosome 8 to AT6.2 cells shows suppression of invasiveness and no suppression of cell dissociation or process after entry into blood circulation. This suggests that human chromosome 8 contains suppressor gene(s) for the invasive ability of prostate cancer.

Rat and human maspins: structures, metastatic suppressor activity and mutation in prostate cancer cells.

The rat homologue of human maspin cDNA was cloned. The deduced amino acid sequence of rat maspin was homologous to human maspin with 88% of the amino acids conserved. Rat maspin mRNA was detected in rat mammary gland, vagina, urinary bladder, thymus, small intestine, skin, ventral prostate, seminal vesicles, and thyroid but not in many other organs, such as heart, lung, liver, brain and kidney. Rat maspin cDNA retrovirally introduced into highly metastatic Dunning AT3.1 rat prostate cancer cells did not suppress metastasis of these tumor cells in Copenhagen rats. Maspin mRNA was detected in 5/10 human prostatic carcinoma tissue samples. Two human prostate cancer cell lines, PC-3 and LNCaP, and two human prostatic carcinoma and two benign prostatic hyperplasia tissue samples contained maspin mRNA having an isoleucine to valine mutation at amino acid 319.

Influence of High-Intensity Focused Ultrasound on the Development of Metastases

The hypothesis that exposure of a solid tumor to high-intensity focused ultrasound (HIFU) could lead to an increase of metastases was investigated in an animal model. The highly metastatic AT-6 Dunning R3327 rat prostate cancer subline was implanted into the hind limb of Fisher-Copenhagen rats and was exposed to 1 pulse/mm2 tumor (acoustical power 1,000 W/cm2) delivered by an experimental machine (Ablatherm, Technomed, France), or the animals were sham treated, as soon as the tumor had reached a volume of 175-225 mm3. The tumor-bearing leg was amputated 24 h later and the number of metastases examined 12 weeks thereafter at autopsy. Metastases were seen in 3 (23%) of the 13 animals exposed to HIFU and in 4 (25%) of the 16 sham-treated animals; this is not significantly different. There was also no significant difference in weight of the lungs that contained metastases between sham-treated and HIFU-treated animals. Metastatic spread of animal tumors with a high metastatic potential is not enhanced by HIFU exposure.

Cytotoxic Effects of High Energy Shock Waves on Cancer Cells Linked to Metallic Beads Vehicled by Monoclonal Antibodies

Previous studies have reported that high energy shock waves (HESW), generated by an electrohydraulic lithotriptor, may have some utility as a cancer treatment modality. Furthermore, it has been described that shock waves propagating in a fluid, show demolitive effects at the level of the interface of a solid fragment immersed in the fluid. In this study, we demonstrate that it is possible to enhance the antineoplastic effects of HESW if treated cells or tissues are linked to monoclonal antibodies (MoAbs) conjugated with metallic beads (MB) (about 1 mu of diameter) and specific for a cancer cell surface determinant. A leukemic cell line was used to study the effects of HESW on cells linked to MB. A fresh human breast cancer specimen was used to perform the assay on tumor tissue. MB linked treated cell viability, growth curve, cloning efficiency and Bromodeoxyuridine incorporation were reduced in comparison to cells treated with HESW alone. Our data suggest that the presence of solid fragments vehicled by MoAbs on a cancer cell surface is able to synergize with the limited antineoplastic effects of HESW.

Glucocorticoid receptor function possibly modulates cell-cell interactions in osteoblastic metastases on rat skeleton.

We analysed the glucocorticoid receptor (GR) function and its ability to modulate cell-cell interactions between the PA-III rat prostate cancer and UMR 106 osteoblast-like rat osteosarcoma cells as an in vitro model for studying GR function in PA-III cell-induced tumor and blastic reaction in rat bone. Intact GR was detected by ligand binding assays, DNA band-shift, and GR trans-activation analysis of PA-III and UMR 106 cells transiently transfected with the mouse mammary tumor virus thymidine kinase-chloramphenicol acetyltransferase reporter gene. Dexamethasone and transforming growth factor beta 1 (TGFbeta1) inhibited the growth of PA-III and UMR 106 cells. Dexamethasone's inhibition of PA-III and UMR 106 cells was reversed by anti-TGFbeta1 antibody and exogenous insulin-like growth factor I (IGF-I). Exogenous IGF-I, urokinase-type plasminogen activator (uPA), UMR 106 conditioned media (CM) and PA-III CM stimulated the proliferation of PA-III and UMR 106 cells. The mitogenic activity exerted by uPA and PA-III CM in UMR 106 cells was completely neutralized by anti-IGF-I specific antibody. In addition, dexamethasone up-regulated TGFbeta1 mRNA and down-regulated uPA mRNA expression in PA-III cells without affecting TGFbeta1 and uPA mRNA expression in UMR 106 cells. These data suggested that TGFbeta1, uPA, and IGF-I mediate at least in part cell-cell interactions and GR function in PA-III prostate cancer and UMR 106 osteosarcoma cells.

Mapping of metastasis suppressor gene(s) for rat prostate cancer on the short arm of human chromosome 8 by irradiated microcell-mediated chromosome transfer.

Our previous studies demonstrated that human chromosome 8 contains metastasis suppressor gene(s) for rat prostate cancer. However, it is still unknown which portion of human chromosome 8 is associated with suppression of metastatic ability, because all of the clones in which metastatic ability is suppressed contain at least one copy of intact human chromosome 8. In the present study, we used the irradiated microcell-mediated chromosome transfer technique to enrich for specific chromosomal arm deletions of selected chromosomes. The resultant series of human chromosomes 8 with a variety of chromosomal deletions was introduced into highly metastatic Dunning rat prostate cancer cells. All of the resultant microcell hybrids showed reduced metastatic ability. To obtain a smaller size of human chromosome 8 and to locate further the region of metastasis suppressor gene(s), the most reduced size of human chromosome 8 that was generated with the initial irradiated chromosome transfer was retransferred into the Dunning cancer cells without irradiation. The resultant microcell hybrids were analyzed to determine which portion of human chromosome 8 suppressed the metastatic ability of the recipient cells. This analysis demonstrates that the portion of human chromosome 8 containing metastasis suppressor gene(s) for rat prostate cancer cells lies on human chromosome segment 8p21-p12, where frequent allelic losses have been detected in allelotype analyses of human prostate cancer. This suggests that one of the metastasis suppressor genes for rat prostate cancer on human chromosome 8 may also play an important role in the progression of human prostate cancer.

Inhibition of prostate cancer growth by 9-aminocamptothecin and estramustine

Hormone-refractory prostate cancer continues to be associated with a very poor prognosis. Agents that interact with the nuclear matrix have been demonstrated to have activity against hormone-refractory prostate cancer. It was the aim of this study to assess the activity of estramustine, an estradiol-nitrogen mustard conjugate, and 9-aminocamptothecin (9-AC), a topoisomerase I inhibitor, in a preclinical model of hormone-refractory prostate cancer. We used the Dunning rat prostatic adenocarcinoma model to demonstrate that the combination of estramustine and 9-AC interacts at the level of the nuclear matrix to inhibit the growth of prostate cancer cells. We demonstrate that the combination of these two agents at pharmacologically achievable doses are cytotoxic to rat and human prostate cancer cells in vitro and in vivo in the rat. The combination of the two drugs was significantly more cytotoxic than either drug alone. We have instituted a Phase II clinical trial in patients with hormone-refractory prostate cancer using 9-AC based on these preclinical findings.

Antisense RNA to the type I insulin-like growth factor receptor suppresses tumor growth and prevents invasion by rat prostate cancer cells in vivo.

Prostate carcinoma is the second leading cause of death from malignancy in men in the United States. Prostate cancer cells express type I insulin-like growth factor receptor (IGF-IR) and prostate cancer selectively metastazises to bone, which is an environment rich in insulin-like growth factors (IGFs), thereby supporting a paracrine action for cancer cell proliferation. We asked whether the IGF-IR is coupled to tumorigenicity and invasion of prostate cancer. When rat prostate adenocarcinoma cells (PA-III) were stably transfected with an antisense IGF-IR expression construct containing the ZnSO4-inducible metallothionein-1 transcriptional promoter, the transfectants expressed high levels of IGF-IR antisense RNA after induction with ZnSO4, which resulted in dramatically reduced levels of endogenous IGF-IR mRNA. A significant reduction in expression both of tissue-type plasminogen activator and of urokinase-type plasminogen activator occurred in PA-III cells accompanying inhibition of IGF-IR. Subcutaneous injection of either nontransfected PA-III or PA-III cells transfected with vector minus the IGF-IR insert into nude mice resulted in large tumors after 4 weeks. However, mice injected with IGF-IR antisense-transfected PA-III cells either developed tumors 90% smaller than controls or remained tumor-free after 60 days of observation. When control-transfected PA-III cells were inoculated over the abraded calvaria of nude mice, large tumors formed with invasion of tumor cells into the brain parenchyma. In contrast, IGF-IR antisense transfectants formed significantly smaller tumors with no infiltration into brain. These results indicate an important role for the IGF/IGF-IR pathway in metastasis and provide a basis for targeting IGF-IR as a potential treatment for prostate cancer.

Increased Phospholipid Fatty Acid Remodeling in Human and Rat Prostatic Adenocarcinoma Tissues

Purpose To study the mechanism of diminished arachidonic acid levels in malignant prostatic tissues. Materials and Methods Benign and malignant prostate tissues were obtained from human radical prostatectomy specimens and from rats using Pollard's Lobund/Wistar rat prostate cancer model. Fatty acid composition and a variety of enzyme activities involved in maintaining phospholipid fatty acid composition were compared in malignant and benign prostatic tissues. Results Decreased arachidonic acid levels, previously reported in human prostate cancer, were present in malignant rat as well as in human tissues. There were 21 percent and 26 percent decreases of arachidonic acid levels in the rat and human malignant tissues compared with benign tissues. Fatty acid desaturase activity was undetectable. Fatty acyl-CoA hydrolase and synthetase activities were not altered in the malignant tissues. However, there was a 2-fold increase in phospholipase A 2 activity and a 4- to 12-fold increase in fatty acyl-CoA lysophosphatidylcholine acyltransferase activity in malignant rat and human prostatic tissues. Conclusions These data indicate that, in malignant prostate tissues, the fatty acid remodeling mechanism is activated through the deacylation-reacylation cycle. This process may be a result of increased use of arachidonic acid for the formation of prostaglandins that may be crucial for the further development and growth of the malignant tissues.

There are multiple forms of glyceraldehyde-3-phosphate dehydrogenase in prostate cancer cells and normal prostate tissue.

We analyzed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in normal and malignant human prostate tissues, normal rat prostate, and Dunning R-3327 rat prostate cancer cell lines. We detected multiple forms of GAPDH in Dunning cell lines by two-dimensional protein electrophoresis and Western analysis. Five forms of GAPDH that differed by isoelectric point were detected for each of the two metastatic Dunning cell lines, while four or fewer forms were detected for Dunning cell lines with low metastatic ability. We also detected multiple forms of GAPDH in normal and malignant human prostate specimens by two dimensional protein electrophoresis and immunohistochemical analysis. GAPDH was undetectable in normal human prostate secretory epithelium by immunohistochemistry, but was abundant in nuclei of normal basal cells and stromal cells. In human prostate cancer specimens, there was a rough correlation between cytoplasmic staining for GAPDH and tumor grade, but GAPDH staining was extremely heterogeneous. GAPDH was abundant in nuclei of some high-grade human prostate tumors. Both of the human prostate cancer bone metastases analyzed with immunohistochemistry had markedly elevated cytoplasmic GAPDH expression. We conclude that multiple forms of GAPDH may play diverse roles in normal prostate tissue and in prostate cancer.