Rat Prostate Cancer Research Articles

Induction of apoptosis in BCL-2-expressing rat prostate cancer cells using the Semliki Forest virus vector.

The Semliki Forest virus (SFV) vector is a transient RNA expression vector that has an inherent p53-independent apoptosis-inducing property. It is administered as recombinant SFV particles (rSFV) that undergo 1 round of replication only and express a gene cloned into the multicloning site. In our study we have investigated the ability of the SFV vector to induce apoptosis and inhibit tumour growth in rat prostate cancer (AT3-Neo) cells expressing the Bcl-2 oncogene (AT3-Bcl-2 cells), which normally inhibits apoptosis. rSFV expressing the enhanced green fluorescent protein (EGFP) gene (rSFV-EGFP), or recombinant RNA transfected into cells by electroporation, induced delayed apoptosis in AT3-Bcl-2 cells. SFV-mediated expression of a cloned pro-apoptotic Bax gene by the vector, however, enhanced apoptosis induction both in AT3-Bcl-2 cells and standard BHK-21 cells. Such Bax-expressing particles could be produced only at low titers compared to EGFP-expressing particles under standard conditions for particle production, but lowering the incubation temperature for particle production to 33 degrees C partially alleviated this effect. Bax-expressing particles were shown to inhibit the growth of AT3-Neo and AT3-Bcl-2 tumours in nude mice, as did high titre EGFP-expressing particles. It is concluded that SFV recombinant particles have potential as anti-tumour agents to treat apoptosis-resistant tumours.

Expression profiles of voltage-gated Na(+) channel alpha-subunit genes in rat and human prostate cancer cell lines.

Voltage-gated Na(+) channel (VGSC) activity has been implicated in prostate cancer (PC) metastasis. Although VGSCs can occur as multiple-subunit assemblies, the alpha-subunits (VGSCalphas) alone can encode functional channels. The VGSCalpha gene(s) responsible for the functional VGSCalpha expression in strongly metastatic PC cell lines is not known. Two reverse transcription-PCR (RT-PCR) methods, degenerate primer screening and a novel semi quantitative PCR (SQT-PCR) technique, were used. These methods enabled a detailed qualitative and quantitative investigation of VGSCalpha mRNA expression in rat (MAT-LyLu/AT-2) and human (PC-3/LNCaP) PC cells of markedly different metastatic potential. Expression of eight different VGSCalpha genes (SCN1A-4A, SCN7A-9A, and SCN11A) was determined in the PC cell lines. Most were expressed as multiple splice variants. SQT-PCR results were consistent with a basal level of VGSCalpha mRNA expression occurring in weakly metastatic (AT-2/LNCaP) cells, and this being greatly elevated in cells of stronger metastatic potential (MAT-LyLu/PC-3), primarily due to the elevated expression of the SCN9A gene (also termed PN1/hNe-Na). (1) Several VGSCalpha genes and their splice variants are expressed similarly in both rat and human PC cell lines. (2) Expression levels are much higher in the strongly metastatic (MAT-LyLu/PC-3) cells. (3) Levels of SCN9A mRNA specifically are predominant in MAT-LyLu and PC-3 cells; thus, SCN9A is highly likely to be the main source of the functional VGSC detected.

Identification of tumor metastasis suppressor region on the short arm of human chromosome 20.

Acquisition of metastatic ability by prostate cancer cells is the hallmark of their lethal trait and outcome. However, the genetic alterations underlying the clinical progression and pathogenesis of prostate cancer are not well understood. Several studies involving loss of heterozygosity (LOH) and comparative genomic hybridization analysis have identified distinctively altered regions on various human chromosomes, and genomic imbalance of chromosome 20 was implicated in progression and recurrence of prostate tumors. To examine the role of chromosome 20 in prostate neoplasms, we introduced this chromosome into highly metastatic rat prostate cancer cells using the microcell-mediated chromosome transfer technique. Introduction of the chromosome resulted in significant suppression of the metastatic ability of the hybrid cells, by as much as 98%, without any interference with the in vivo growth rate or tumorigenicity of primary tumor in SCID mice. Our STS-PCR analysis on 10 hybrid clones indicates that the suppressor activity of chromosome 20 is located in the p11.23-12 region. Further examination of the hybrid clones by experimental metastasis assay and histologic analysis as well as Matrigel invasion assay suggests the involvement of the suppressor region at an early stage of invasion and extravasation. We also investigated the status of the chromosome 20 suppressor region in pathology specimens from human prostate cancer patients and detected the frequent loss of this region in high-grade tumors. These results suggest the presence of a putative suppressor gene on human chromosome 20 that is functionally involved in development of prostate cancer metastases.

Directional movement of rat prostate cancer cells in direct-current electric field: involvement of voltagegated Na+ channel activity.

A two-part hypothesis has been tested, which proposes that (1) prostate cancer cells are galvanotactic (i.e. respond to an electric field by moving directionally) and (2) voltage-gated Na+ channel activity, which was shown previously to be expressed specifically by strongly metastatic cells, controls galvanotaxis. Two well-defined rat ('Dunning') cell lines, originally derived from the same prostate tumour but differing markedly in their metastatic ability, were used. Cells were exposed to exogenous direct-current electric fields of physiological strength (0.1-4.0 V cm(-1)), their reactions were recorded by light microscopy and analysed by a quantitative tracking method. Voltage-gated Na+ channel activity was modulated pharmacologically using a range of concentrations of a specific channel blocker (tetrodotoxin) or an opener (veratridine). The results showed that the highly metastatic MAT-LyLu cells responded to the application of the electric field strongly by migrating towards the cathode. By contrast, the weakly metastatic AT-2 cells gave no such response. Tetrodotoxin suppressed the galvanotactic response of the MAT-LyLu cells whereas veratridine enhanced it. Both compounds had little effect on the AT-2 cells. These results are consistent with functional voltage-gated Na+ channel expression occurring specifically in highly metastatic cells. This is also the first demonstration of control of galvanotaxis, in any cell type, by voltage-gated Na+ channel activity. The possible underlying mechanisms and the in vivo relevance of these findings are discussed.

Genetic prodrug activation therapy (GPAT) in two rat prostate models generates an immune bystander effect and can be monitored by magnetic resonance techniques.

Treatment of hormone refractory prostate cancer requires new treatment strategies. Genetic prodrug activation therapy (GPAT) may provide a new therapeutic avenue. In this study the antitumour efficacy of the gene encoding herpes simplex virus thymidine kinase (HSVtk) activating the prodrug ganciclovir (GCV) was compared in two models of ectopic (subcutaneous) rat prostate cancer. Both models, which differ in their characteristics, were previously shown to be weakly immunogenic but susceptible to immunotherapy. Tumour cell lines were stably transfected with HSVtk and were rendered highly sensitive to GCV. Little or no bystander killing effect was observed by tk-transfected cells on wild-type cells in vitro. However, a significant in vivo bystander effect was observed suggesting an immune-mediated response. Indeed, such an immune response was capable of slowing the growth of distant wild-type tumours and increased overall animal survival. A T helper 1 immune response was generated as a result of GCV activation and cell kill, demonstrated by the secretion of IFNgamma by cultured splenocytes in response to tumour cells. BrDU staining of tk-transfected cells treated with GCV in vitro suggested apoptotic cell death, but Annexin V staining was less marked for one of the cell lines. Serial in vivo monitoring by non-invasive magnetic resonance spectroscopy (MRS) of the tk-transfected MATLyLu tumours demonstrated a decreased ATP/Pi ratio (a measure of cell energy status) during growth and an increase in the ATP/Pi ratio during regression initiated by treatment with GCV. Further, significant differences were found in the phosphomonester (PME) to total phosphate (SigmaP) ratios in treated compared with untreated tumours, a result rarely seen in animal models, but commonly observed in patients. This study showed that a Th1-biased immune response generated by killing prostate tumour cells with tk/GCV can kill distant as well as local wild-type tumour cells. These findings suggest that GPAT may have a potential application in patients with both confined and metastatic prostate cancer and MRS may provide a method of monitoring response to treatment.

Molecular cloning and structural characterization of the rat thymosin β15 gene

Thymosin β15 is expressed in metastatic variants of Dunning rat prostatic carcinoma but not in a nonmetastatic variant. It is also upregulated in malignant human prostate cancers and was shown to be a predictive marker for patient outcome in prostate cancer. To explore the molecular mechanism of transcriptional regulation of thymosin β15, we isolated and characterized the rat thymosin β15 gene. The gene appears to exist as a single copy in the rat genome and to comprise three exons distributed over 2 kb. The transcription start site was defined by primer extension analysis at 30 bp upstream of the translation start site. Sequence analysis of the 5′-flanking region of the transcription start site revealed properties consistent with promoter activity. The promoter region is GC rich and contains numerous consensus transcription factor binding sites as well as a GC box, but no TATA box. The transcriptional activity of the 5′-flanking region was analyzed by transient transfection of rat prostate cancer cells with firefly luciferase-encoding gene expression vector constructs. The isolated 5′ region showed significant promoter activity. The identification of the thymosin β15 promoter could aid our understanding of the regulation of this gene and its enhanced expression in human cancer.

An intronic splicing silencer causes skipping of the IIIb exon of fibroblast growth factor receptor 2 through involvement of polypyrimidine tract binding protein.

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) transcripts involves the mutually exclusive usage of exons IIIb and IIIc to produce two different receptor isoforms. Appropriate splicing of exon IIIb in rat prostate cancer DT3 cells requires a previously described cis element (ISAR, for "intronic splicing activator and repressor") which represses the splicing of exon IIIc and activates the splicing of exon IIIb. This element is nonfunctional in rat prostate AT3 cells, which repress exon IIIb inclusion and splice to exon IIIc. We have now identified an intronic element upstream of exon IIIb that causes repression of exon IIIb splicing. Deletion of this element abrogates the requirement for ISAR in order for exon IIIb to be spliced in DT3 cells and causes inappropriate inclusion of exon IIIb in AT3 cells. This element consists of two intronic splicing silencer (ISS) sequences, ISS1 and ISS2. The ISS1 sequence is pyrimidine rich, and in vitro cross-linking studies demonstrate binding of polypyrimidine tract binding protein (PTB) to this element. Competition studies demonstrate that mutations within ISS1 that abolish PTB binding in vitro alleviate splicing repression in vivo. Cotransfection of a PTB-1 expression vector with a minigene containing exon IIIb and the intronic splicing silencer element demonstrate PTB-mediated repression of exon IIIb splicing. Furthermore, all described PTB isoforms were equally capable of mediating this effect. Our results support a model of splicing regulation in which exon IIIc splicing does not represent a default splicing pathway but rather one in which active repression of exon IIIb splicing occurs in both cells and in which DT3 cells are able to overcome this repression in order to splice exon IIIb.

Allogeneic whole-tumour cell vaccination in the rat model of prostate cancer.

To investigate cancer immunotherapy using whole allogeneic (differing tissue-type) tumour cells as vaccines in the rat prostate cancer model. Materials and methods Two rat models of prostate cancer were used; MAT-LyLu tumours which grow in Copenhagen rats and PAIII tumours which grow in Lobund-Wistar rats, with crossover of the cell lines to test allogeneic vaccination. The cell lines were immunologically characterized by flow cytometry. Irradiated tumour cells were administered as subcutaneous vaccines either before tumour challenge or after tumour establishment (both subcutaneous). A preparation of heat-killed Mycobacterium vaccae bacilli (SRL172) was used as an adjuvant to increase vaccine efficiency. Flow cytometry analysis of the cell lines showed that the PAIII cells had higher levels of major histocompatibility complex (MHC) class I and intercellular adhesion molecule (ICAM-1) expression than the MAT-LyLu cells. However, both tumour cell lines were rejected in their allogeneic hosts. Prophylactic vaccination with allogeneic MAT-LyLu cells protected against PAIII tumour challenge in Lobund-Wistar rats, with 80% of animals surviving for > 5 months, compared with 40% for animals receiving autologous cells. The immunity was prolonged, as rats were protected when rechallenged 5 months later. In Copenhagen rats allogeneic PAIII cells protected against the more aggressive MAT-LyLu tumour challenge only when the cells were combined with SRL172. Initial therapy experiments showed that vaccination with the cell lines mediated only limited tumour regression in the Lobund-Wistar rats. The allogeneic tumour cell vaccination model described is valuable for assessing the principle and efficacy of allogeneic prostate cancer cell vaccines for clinical use.

Down-regulation of TGF-beta1 production restores immunogenicity in prostate cancer cells.

The objective of this study is to determine if a non-immunogenic Dunning’s rat prostate cancer cell line, MATLyLu, can become immunogenic by reducing the endogenous production of TGF-β1. An expression construct containing a DNA sequence in an antisense orientation to TGF-β1 (TGF-β1 antisense) was stably transfected into MATLyLu cells. Following transfection, cellular content of TGF-β1 reduced from 70 to10 pg per 2 × 104cells and the rate of in vitro3H-thymidine incorporation increased 3–5-fold. After subcutaneous injection of tumour cells into syngeneic male hosts (Copenhagen rats), the tumour incidence was 100% (15/15) for the wild type MATLyLu cells and cells transfected with the control construct, but only 43% (9/21, P≤ 0.05) for cells transfected with TGF-β1 antisense. However, when cells were injected into immunodeficient hosts (athymic nude rats), the incidence of tumour development was 100% (10/10) for both the wild type MATLyLu cells and cells transfected with the control construct and 90% (9/10) for cells transfected with TGF-β1 antisense. These observations support the concept that MATLyLu cells are immunogenic, when the endogenous production of TGF-β1 is down-regulated. © 2000 Cancer Research Campaign

Laser-induced hyperthermia in rat prostate cancer: role of site of tumor implantation

Objectives. To investigate the importance of the site of tumor implantation on the treatment response to laser-induced hyperthermia (LIH) of rat prostate cancer (PCa), because interventional manipulations of PCa have been reported to increase metastatic dissemination. Methods. Seven to nine days after either subcutaneous or orthotopic implantation of MatLyLu cells, LIH (46.5°C) was induced using pulsed irradiations of a neodymium:yttrium-aluminum-garnet laser. Both local control and distant metastases were evaluated. Plasma metalloproteinase 9 (MMP-9) was tested as a possible marker of PCa progression and LIH response. Results. Twelve days after LIH treatment of subcutaneous tumors, the volumes were reduced by 64% after 8 minutes of irradiation, 73% after 10 minutes, 81% after 15 minutes, and 91.1% after 20 minutes. In the orthotopic model, the corresponding tumor reductions were 44% after 10 minutes, 61% after 20 minutes, and 65% after 30 minutes. Lung metastases were observed in only 1 animal with subcutaneous tumors. In contrast, 86% of the orthotopic tumor-bearing animals treated for 30 minutes had lung metastases compared with 23% of the untreated tumor-bearing rats. MMP-9 expression was detected in both orthotopic and subcutaneous tumor tissue and in the plasma of tumor-bearing rats. The prostate tissue of healthy rats and subcutaneous tumor-bearing rats was devoid of MMP-9. The plasma levels of MMP-9 showed a trend toward direct correlation with local tumor control but no correlation with the incidence of metastasis. Conclusions. These data emphasize the importance of the site of tumor implantation for evaluation of the efficacy of therapeutic interventions and may warrant further studies before widespread clinical application of LIH as monotherapy.

Antitumor efficacy of tumor-antigen-encoding recombinant poxvirus immunization in Dunning rat prostate cancer: implications for clinical genetic vaccine development.

One potential use for prostate-cancer-associated genes discovered through ongoing genetics studies entails the construction of virus- or plasmid-based recombinant vector vaccines encoding these new tumor-associated antigens (TAA) to induce TAA-specific immune responses for the prevention or therapy of prostate cancer. Clinical trials evaluating prototypes of such recombinant vaccines are under way. TAA-encoding recombinant vector vaccines, however, have not previously been evaluated in a prostate-cancer animal model. For assessment of the potential susceptibility of prostate cancer to genetic immunization strategies using TAA-encoding recombinant vectors, the antitumor efficacy of a model recombinant viral vector encoding a TAA was evaluated in rat Dunning prostate cancer. Recombinant vaccinia was chosen as a prototype virus vector encoding a TAA for these studies, and beta-galactosidase was chosen as a model target TAA. Dunning AT-2 cells were transduced with a retroviral vector to express beta-galactosidase, and the susceptibility of tumorigenic AT-2-lacZ cells to immunization with vaccinia-lacZ was measured using protection studies in Copenhagen and nu/nu rats. Stably transduced AT-2-lacZ cells expressing beta-galactosidase as measured by enzymatic substrate-based assays were found to retain their tumorigenicity in vivo despite abundant expression of rat major histocompatibility complex (MHC) class I. Immunization with model TAA-encoding recombinant vaccinia-lacZ conferred significant protection against subsequent growth of AT-2-lacZ cells in vivo (P = 0.01); however, the efficacy of such immunization was markedly dependent on the volume of tumor challenge. The antitumor efficacy of TAA-encoding recombinant vaccinia immunization was abrogated in nu/nu rats, suggesting a T-cell-dependent mechanism of activity. These studies suggest that prostate cancer may be a suitable target for immunization strategies using TAA-encoding recombinant vectors. Such immunization strategies may be more effective in settings of minimal cancer burden.

The Growth Regulatory Fibroblast IK Channel Is the Prominent Electrophysiological Feature of Rat Prostatic Cancer Cells

Physiological effectors for mitogenic cell growth control remain to be determined for mammalian tumor cells, particularly those derived from prostatic tissue. One such effector for mitogenic Ras/MAPK signaling in fibroblasts is an intermediate-conductance, calcium-activated potassium channel (FIK). In this study patch-clamp electrophysiology was used to show that both AT2.1 and MatLyLu rat prostate cancer cell lines express high levels of a current identified as FIK, based on the following criteria: activation by elevation of intracellular calcium, voltage independence, potassium selectivity, and block by charybdotoxin (ChTX) and the Stichodactyla helianthus potassium channel neurotoxin (StK). FIK current densities in AT2.1 and MatLyLu cells were comparable to the high levels seen in fibroblasts transfected with oncogenic Ras or Raf, suggesting hyperactivity of the Ras/MAPK pathway in prostatic cancer cells. Voltage-gated sodium current was present in most MatLyLu cells but absent from AT2.1 cells, and all AT2.1 cells had voltage-gated potassium currents. Thus, FIK is the main electrophysiological feature of rat prostatic cancer cells as it is for mitogenically active fibroblasts, suggesting it may play a similar growth regulatory role in both.

A study of membrane activity in rat prostate cancer cells: an evaluation of the FM1-43 dye technique.

A study was initiated to test whether the FM1-43 dye technique could be applied to the study of endocytic membrane activity in two rodent prostate cancer (MAT-LyLu and AT-2) cell lines of markedly different metastatic ability. The lipophilic dye FM1-43, which has frequently been used to monitor endo/exocytic activity in excitable cells was employed. We found, as in excitable tissues, that both strongly metastatic (MAT-LyLu) and weakly metastatic (AT-2) cells in culture take up FM1-43 to give vesicular staining of a variable pattern, which appeared to differ between the two cell lines. However, unlike excitable tissues, neither cell line subsequently released the dye. Indeed, both cell lines retained the dye through several rounds of cell division suggesting that dye incorporated by cells does not enter the endo/exocytotic cycle. Uptake of dye was independent of temperature, Na+/K+ gradients, pH or metabolism. We suggest that passive accumulation of FM1-43 can occur in cancer cells and should not, automatically, be interpreted as evidence of endocytosis.

Synthetic inhibitor of matrix metalloproteases decreases tumor growth and metastases in a syngeneic model of rat prostate cancer in vivo.

Members of the matrix metalloprotease (MMP) family are implicated in the progression of several malignancies including prostate cancer due to their ability to break down extracellular matrix (ECM) components. In this study, we have evaluated the ability of a synthetic MMP inhibitor (A-177430) to block tumor growth and metastases in a syngeneic model of rat prostate cancer. In an in vitro substrate assay, A-177430 exhibited nanomolar potency (IC(50) 2-6 nM) against the enzymatic activity of several MMPs. For in vivo studies, male Copenhagen rats were injected s.c. with Mat Ly Lu rat prostate cancer cells (1 x 10(6) cells ) into the right flank and animals were administered i.p.with different doses (10-100 mg/kg per day) of A-177430 for 16 days. Administration of A-177430 resulted in a dose-dependent decrease in tumor volume as compared to a control group of animals receiving vehicle alone. The maximum dose (100 mg/kg per day) of A-177430 exhibited complete arrest in tumor growth and prevented the development of macroscopic tumor metastases to lungs without exhibiting any noticeable side effects. Histologic examination of primary tumors from experimental animals showed extensive tumor necrosis and decreased tumor angiogenesis as determined by factor VIII staining of primary tumors following A-177430 treatment. These primary tumors from experimental animals also exhibited a significant increase in tumor cell DNA fragmentation as determined by TUNEL assay. Collectively, these results demonstrate the ability of MMP inhibitors to block tumor growth and metastases by blocking ECM degradation and by inhibiting tumor angiogenesis and promotion of prostate cancer cell apoptosis in vivo.

Vascular endothelial growth factor content in metastasizing and nonmetastasizing Dunning prostatic adenocarcinoma.

Tumor angiogenesis is important in progressive tumor growth and metastasis. In the normal rat prostate and in androgen-sensitive prostate tumors androgen ablation causes an involution of the vasculature and a decrease in the vascular endothelial growth factor (VEGF) levels before regression of the prostate gland. To examine whether angiogenesis and metastasis are regulated by VEGF in androgen-insensitive and metastasizing prostate tumors, five Dunning rat prostate cancer sublines were tested; the androgen-sensitive, nonmetastasizing R3327 PAP, and the androgen-insensitive, low metastasizing AT-1, and the three androgen-insensitive, metastasizing AT-2, AT-3, and MatLyLu Dunning prostatic adenocarcinomas. VEGF levels were quantified in the rat dorsolateral prostate and in the five Dunning sublines using competitive RT-PCR, Western blot, and Elisa. Vascular density was determined by factor VIII staining. VEGF mRNA was increased in all tumors compared with normal prostates. The two metastatic sublines AT-3 and MatLyLu and the nonmetastatic subline AT-1 showed the highest VEGF mRNA expression. VEGF protein levels in the prostate gland showed increased expression in the metastatic sublines, AT-2, AT-3, and MatLyLu, compared with the nonmetastatic AT-1 subline and the ventral prostate. VEGF proteins in serum were highest in the metastatic AT-3 subline. The vessel density was highest in the two highly metastatic sublines AT-3 and MatLyLu. Our results suggest that VEGF levels are associated with microvessel density and the previously established metastatic pattern of these rat prostate tumor systems.

Application of an improved cDNA competition technique to identify prostate cancer-associated gene.

A technique to improve cDNA library screening was developed by using mixed probes derived from two closely related cDNA populations of high-metastatic MAT-LyLu and low-metastatic AT-1 Dunning R3227 rat prostate cancer sublines. The technique required the generation of a cDNA library from each subline followed by polymerase chain reaction (PCR) amplification of the cDNA insert population. The PCR products derived from the first library were radiolabeled and mixed with an excess amount of PCR products from the second library. The mixture and an excess amount of both the lambda and pBluescript DNA were used as a probe to screen the first cDNA library. This mixed probe (designated the competition probe) differentially cross-hybridized with the plaque lift of the screened first cDNA library. Weak radioactive signals indicated the cross-hybridization of cDNA sequences common to the competition probe mixture and the first cDNA library, whereas strong signals implied unhybridized unique or abundant cDNA sequences in the first cDNA library. The reproducibility of this technique was confirmed by showing that the full-length cDNA clones were associated with the phenotype of the screened first cell line. The isolated clones were characterized as rat nucleolar protein, rat mitochondrial genes coding for 16S and 12S rRNAs, and rat tRNAs specific for valine and phenyl-alanine. This result is consistent with the fact that the first cell line, MAT-LyLu, is metabolically more active than are AT-1 cells because of higher gene dosage or amplification of nucleolar and mitochondrial RNA and its associated genes. Another clone which had a strong signal represented a novel gene associated with the MAT-LyLu cancer phenotype.

Effects of gadolinium ions upon rat prostatic cancer cell lines of markedly different metastatic potential

The effects of gadolinium chloride, a non-specific blocker of mechanosensitive ion channels (MSICs), upon the motility and proliferation of two Dunning rat prostatic tumour cell lines of markedly different metastatic potential were investigated. Gadolinium (2–10 μM) caused a dose-dependent increase in the distance moved in ‘wound’ assays over a 48-h testing period. The highly metastatic MAT-LyLu cell line was significantly more sensitive to Gd3+, the weakly metastatic AT-2 cells responding only at the highest concentration (10 μM) used. There was no effect on the cells’ proliferative rates. These data suggest that mechanosensitive channels could play a role in metastasis by modulating cell migration.

In vivo fluorescence imaging of the transport of charged chlorin e6 conjugates in a rat orthotopic prostate tumour.

Polymeric drug conjugates are used in cancer therapy and, varying their molecular size and charge, will affect their in vivo transport and extravasation in tumours. Partitioning between tumour vasculature and tumour tissue will be of particular significance in the case of photosensitizer conjugates used in photodynamic therapy, where this partitioning can lead to different therapeutic effects. Poly-l-lysine chlorine6 conjugates (derived from polymers of averageMr 5000 and 25 000) were prepared both in a cationic state and by poly-succinylation in an anionic state. A fluorescence scanning laser microscope was used to follow the pharmacokinetics of these conjugates in vivo in an orthotopic rat prostate cancer model obtained with MatLyLu cells. Fluorescence was excited with the 454–528 nm group of lines of an argon laser and a 570 nm long pass filter used to isolate the emission. Results showed that the conjugates initially bound to the walls of the vasculature, before extravasating into the tissue, and eventually increasing in fluorescence. The anionic conjugates produced tissue fluorescence faster than the cationic ones, and surprisingly, the largerMr conjugates produced tissue fluorescence faster than the smaller ones with the same charge. These results are consistent with differences in aggregation state between conjugates. © 1999 Cancer Research Campaign

Supraphysiological thermal injury in Dunning AT-1 prostate tumor cells.

To investigate the potential application of thermal therapy in the treatment of prostate cancer, the effects of supraphysiological temperatures (40-70 degrees C) for clinically relevant time periods (approximately 15 minutes) were experimentally studied on attached Dunning AT-1 rat prostate cancer cells using multiple assays. The membrane and reproductive machinery were the targets of injury selected for this study. In order to assess membrane injury, the leakage of calcein was measured dynamically, and the uptake of PI was measured postheating (1-3 hours). Clonogenicity was used as a measure of injury to the reproductive machinery 7 days post-injury after comparable thermal insults. Experimental results from all three assays show a broad trend of increasing injury with an increase in temperature and time of insult. Membrane injury, as measured by the fluorescent dye assays, does not correlate with clonogenic survival for many of the thermal histories investigated. In particular, the calcein assay at temperatures of < or = 40 degrees C led to measurable injury accumulation (dye leakage), which was considered sublethal, as shown by significant survival for comparable insult in the clonogenic assay. Additionally, the PI uptake assay used to measure injury post-thermal insult shows that membrane injury continues to accumulate after thermal insult at temperatures > or = 50 degrees C and may not always correlate with clonogenicity at hyperthermic temperatures such as 45 degrees C. Last, although the clonogenic assay yields the most accurate cell survival data, it is difficult to acquire these data at temperatures > or = 50 degrees C because the thermal transients in the experimental setup are significant as compared to the time scale of the experiment. To improve prediction and understanding of thermal injury in this prostate cancer cell line, a first-order rate process model of injury accumulation (the Arrhenius model) was fit to the experimental results. The activation energy (E) obtained using the Arrhenius model for an injury criterion of 30 percent for all three assays revealed that the mechanism of thermal injury measured is likely different for each of the three assays: clonogenics (526.39 kJ/mole), PI (244.8 kJ/mole), and calcein (81.33 kJ/mole). Moreover, the sensitivity of the rate of injury accumulation (d omega/dt) to temperature was highest for the clonogenic assay, lowest for calcein leakage, and intermediate for PI uptake, indicating the strong influence of E value on d omega/dt. Since the clonogenic assay is linked to the ultimate survival of the cell and accounts for all lethal mechanisms of cellular injury, the E and A values obtained from clonogenic study are the best values to apply to predict thermal injury in cells. For higher temperatures (> or = 50 degrees C) indicative of thermal therapies, the results of PI uptake can be used as a conservative estimate of cell death (underprediction). This is useful until better experimental protocols are available to account for thermal transients at high temperature to assess clonogenic ability. These results provide further insights into the mechanisms of thermal injury in single cell systems and may be useful for designing optimal protocols for clinical thermal therapy.

Bacillus Calmette‐Guerin Immunotherapy Significantly Prolongs Survival of Animals with Prostate Cancer

AbstractObjectives: Bacillus Calmette‐Guerin (BCG) is the treatment of choice for carcinoma in situ of the bladder, but its application to other cancers is very limited. The current study evaluates the effect of intralesional BCG in the PA3 rat prostate cancer model.Materials and Methods: One hundred five rats were randomized to four groups. Each group received 1 × 106 PA3 cells subcutaneously on experiment Day 0. On experimental Day 7, the groups received the first of six weekly treatments. Treatments consisted of saline solution, BCG 1 × 104 colony‐forming units (cfu), BCG 1 × 105 cfu, and BCG 1 × 106 cfu. All animals were monitored for tumor growth throughout the duration of the experiment.Results: Animals receiving treatments with BCG 1 × 106 cfu exhibited the greatest reduction in tumor volume ([mean ± SD] 4130.0 ± 1738.3 mm3; p = 0.0738), followed by those receiving BCG 1 × 104 cfu (4527.6 ± 1932.7 mm3; p = 0.1093) and BCG 1 × 105 cfu (4838.2 ± 1889.9 mm3; p = 0.5218), compared to those receiving the saline solution control (5445.0 ± 3119.5 mm3). Animal survival on Day 60 after transplantation was significantly increased in the group receiving BCG 1 × 106 cfu (19 [73.1%] of 26 animals; p = 0.0326) when compared to those receiving the saline solution control (12 [44%] of 27 animals). Treatment with BCG 1 × 105 cfu (11 [42.3%] of 26 animals survived; p = 0.5479) and BCG 1 × 104 cfu (13 [50%] of 26 animals survived; p = 0.4484) also reduced survival compared to the saline solution control, but not to the level of significance.Conclusions: The consistent reduction in tumor growth and animal mortality in animals receiving intralesional BCG demonstrates antitumor activity in this aggressive model of prostate cancer. The greatest efficacy was seen with the highest dose of BCG, suggesting that higher doses may be optimal in this model. BCG immunotherapy of prostate cancer warrants clinical evaluation.