W.M. van Weerden

Adrenal glands of mouse and rat do not synthesize androgens

Human adrenal glands produce considerable amounts of the C-19 steroids dehydroepiandrosterone (DHEA) and androstenedione. To investigate the capability of rodent adrenals to produce these steroids, cell suspensions of mouse and rat adrenal glands were incubated in the absence and presence of adrenocorticotropic hormone (ACTH). Corticosterone levels in the incubation medium increased dramatically in the presence of ACTH, but no significant amounts of 17-hydroxyprogesterone or androstenedione could be detected. This indicates that the adrenals of rat and mouse lack the enzyme 17 alpha-hydroxylase. Absence of plasma cortisol in the presence of high levels of corticosterone confirmed these data. Plasma levels of androstenedione were significantly decreased in castrated male rats as compared to levels observed in intact males, showing the contribution of the testes to the plasma content of androstenedione. Very low levels of androstenedione were observed in female, male and castrated male mice. Plasma concentrations of DHEA were not detectable in intact and castrated male mice and rats. It is concluded that rat and mouse lack the enzyme necessary to synthesize adrenal C-19 steroids and that the adrenals in these animals, therefore, do not contribute to plasma levels of androstenedione and DHEA.

Regulation of expression of Na+, K+-ATPase in androgen-dependent and androgen-independent prostate cancer

SummaryThe β1-subunit of Na+,K+-ATPase was isolated and identified as an androgen down-regulated gene. Expression was observed at high levels in androgen-independent as compared to androgen-dependent (responsive) human prostate cancer cell lines and xenografts when grown in the presence of androgens. Down-regulation of the β1-subunit was initiated at concentrations between 0.01 nM and 0.03 nM of the synthetic androgen R1881 after relatively long incubation times (> 24 h). Using polyclonal antibodies, the concentration of β1-subunit protein, but not of the α1-subunit protein, was markedly reduced in androgen-dependent human prostate cancer cells (LNCaP-FGC) cultured in the presence of androgens. In line with these observations it was found that the protein expression of total Na+,K+-ATPase in the membrane (measured by 3H-ouabain binding) was also markedly decreased. The main function of Na+,K+-ATPase is to maintain sodium and potassium homeostasis in animal cells. The resulting electrochemical gradient is facilitative for transport of several compounds over the cell membrane (for example cisplatin, a chemotherapeutic agent experimentally used in the treatment of hormone-refractory prostate cancer). Here we observed that a ouabain-induced decrease of Na+,K+-ATPase activity in LNCaP-FGC cells results in reduced sensitivity of these cells to cisplatin-treatment. Surprisingly, androgen-induced decrease of Na+,K+-ATPase expression, did not result in significant protection against the chemotherapeutic agent.

Disconnecting the Yin and Yang Relation of Epidermal Growth Factor Receptor (EGFR)-Mediated Delivery: A Fully Synthetic, EGFR-Targeted Gene Transfer System Avoiding Receptor Activation

The epidermal growth factor receptor (EGFR) is upregulated within a high percentage of solid tumors and hence is an attractive target for tumor-targeted therapies including gene therapy. The natural EGFR ligand epidermal growth factor (EGF) has been used for this purpose, despite the risk of mitogenic effects due to EGFR activation. We have developed a fully synthetic, EGFR-targeted gene delivery system based on PEGylated linear polyethylenimine (LPEI), allowing evaluation of different EGFR-binding peptides in terms of transfection efficiency and EGFR activation. Peptide sequences directly derived from the human EGF molecule enhanced transfection efficiency with concomitant EGFR activation. Only the EGFR-binding peptide GE11, which has been identified by phage display technique, showed specific enhancement of transfection on EGFR-overexpressing tumor cells including glioblastoma and hepatoma, but without EGFR activation. EGFR targeting led to high levels of cell association of fluorescently labeled polyplexes after only 30 min of incubation. EGF pretreatment of cells induced enhanced cellular internalization of all polyplex types tested, pointing at generally enhanced macropinocytosis. EGF polyplexes diminished cell surface expression of EGFR for up to 4 hr, whereas GE11 polyplexes did not. In a clinically relevant orthotopic prostate cancer model, intratumorally injected GE11 polyplexes were superior in inducing transgene expression when compared with untargeted polyplexes.

Human xenograft models as useful tools to assess the potential of novel therapeutics in prostate cancer

With docetaxel as effective chemotherapy for hormone refractory prostate cancer (HRPC), the number of new treatment combinations for HRPC is expanding demanding a fast-track screening system. This review elaborates on the use of xenograft models to select the most promising combination therapies for entering into phase II clinical trials.

Overexpression and gene amplification of BAG-1L in hormone-refractory prostate cancer.

BAG‐1L (Bcl‐2‐associated anthanogene 1) has been found to interact with androgen receptor (AR), and has been suggested to be involved in the development of prostate cancer. In order to determine the presence of genetic and/or expression alterations of BAG‐1L in prostate cancer, we analysed human prostate cancer cell lines and xenografts as well as patient samples of untreated, hormone‐naïve, and hormone‐refractory prostate carcinomas for sequence variations using denaturing high‐performance liquid chromatography (DHPLC), for gene copy number using fluorescence in situ hybridization (FISH), and for expression using both quantitative RT‐PCR and immunostaining. Only one sequence variation was found in all 37 cell lines and xenografts analysed. BAG‐1 gene amplification was detected in two xenografts. In addition, gene amplification was found in 6 of 81 (7.4%) hormone‐refractory clinical tumours, whereas no amplification was found in any of the 130 untreated tumours analysed. Additionally, gain of the BAG‐1 gene was observed in 27.2% of the hormone‐refractory tumours and in 18.5% of the untreated carcinomas. In a set of 263 patient samples, BAG‐1L protein expression was significantly higher in hormone‐refractory tumours than in primary tumours (p = 0.002). Altogether, these data suggest that amplification and overexpression of BAG‐1L may be involved in the progression of prostate cancer. Copyright

Variations in activin receptor, inhibin/activin subunit and follistatin mRNAs in human prostate tumour tissues.

The possible role of activin in the regulation of malignant prostatic growth was studied using RNAase protection assays of activin receptors, inhibin/activin subunits and follistatin mRNAs in the human prostatic carcinoma cell lines LNCaP-FGC, -R and -LNO, in human prostatic carcinoma xenografts and in human prostatic tissue. Activin receptor types IA (ActRIA), IB (ActRIB), IIA (ActRIIA) and IIB (ActRIIB) mRNAs were generally expressed in prostate pithelial cells, with significantly lower levels of ActRIB mRNA in prostate tumour aterial when compared to non-malignant tissue (P< 0.05; Mann–Whitney U -test). Inhibin/activin βA- and βB-subunit mRNA expression was also found in prostate tissue. Androgen-independent xenografts expressed significantly lower amounts of βB-subunit mRNA when compared to androgen-dependent xenografts (P< 0.05). While βB-subunit mRNA was expressed by LNCaP-FGC and -LNO cells, virtually no expression was found in the androgen-independent LNCaP-R line. Inhibin α-subunit mRNA levels were low or undetectable in all samples investigated. Follistatin mRNA was undetectable in LNCaP-sublines, while low levels were found in prostatic tissues. In androgen-independent LNCaP-R cells, activin inhibited cell growth in a dose-dependent manner. These results suggest that prostate tumour progression is accompanied by a decrease of the inhibitory effect of locally produced activin by either a decrease in the expression of activin βB-subunit mRNA or by a decrease of ActRIB mRNA levels.

Validation of transrectal ultrasonographic volumetry for orthotopic prostate tumours in mice

Orthotopic human prostate tumour models in athymic nude mice are regarded as being most suitable for fundamental and pre-clinical research on prostate cancer. The anatomic localization of the tumour in the pelvis, however, provides little possibility for monitoring tumour growth or regression. To assess time-related changes in orthotopic tumour volume, we applied transrectal ultrasonography (TRUS) to the murine prostate. This technique has the advantages of allowing accurate monitoring of tumours during therapeutic manipulations and a reduction of animal use due to a reduction of sacrificing endpoints. To validate the TRUS method, the mouse prostate reconstitution model, RM-9, and the prostate-specific antigen (PSA) producing human prostate cancer xenograft PC-346 were used. Volumetric calliper measurements were performed with a 30 MHz ultrasound probe designed for intra-arterial use in humans. Tumour weight, determined at various time-points, was found to be closely related to actual tumour weight (R = 0.99) and, in the PC-346 model, to the level of PSA in the plasma. Furthermore, the interobserver variation for TRUS was low for tumours above 50 mg. Thus, TRUS for murine prostate tumours proves to be an accurate, reproducible and sensitive method.

Ki‐67 expression and BrdUrd incorporation as markers of proliferative activity in human prostate tumour models

Abstract. The validity of the use of the monoclonal antibodies Ki‐67 and anti‐BrdUrd to evaluate proliferative activity of human prostate tumour models was studied. Growth of the transplantable PC‐82 and PC‐EW prostate tumours, as assessed by tumour volume measurements, was significantly correlated with the proliferative activity as reflected by BrdUrd incorporation into DNA (r= 0.64 and r= 0.78, respectively). The proliferative activity of PC‐82 tumours detected by Ki‐67 antigen expression paralleled the pattern observed with BrdUrd (r= 0.51) and a significant correlation (r= 0.60) between the results obtained with both markers was found. In growing PC‐82 and PC‐EW tumours only small variations in the Ki‐67 and BrdUrd indices were observed. In contrast, Ki‐67 expression in regressing PC‐82 tumours varied considerably (2.7 ± 2.2%). The BrdUrd index in regressing PC‐32 tumours showed less variation (1.3 ± 0.2%), but part of the BrdUrd‐positive cells were found in the stromal (murine) part of the regressing tissue. It is concluded that the Ki‐67 and BrdUrd proliferation markers are reliable parameters to monitor changes in growth of prostate tumour lines, but that in slow growing or regressing tumours Ki‐67 and BrdUrd data should be interpreted with caution.

A transductionally retargeted adenoviral vector for virotherapy of Her2/neu-expressing prostate cancer.

The efficacy of adenovirus (Ad)-based gene therapy of solid tumors, such as prostate cancer, is limited. One of the many problems is that the virus infects many different cell types in the body, resulting in high toxicity, whereas the target cancer cells are often less prone to wild-type Ad infection. Our aim was to develop genetically de- and retargeted Ad vectors to reduce off-target effects and increase target infection for prostate cancer. We have previously reported an Ad5 vector specific for the cancer-associated receptor Her2/neu, created by inserting Her2/neu-reactive Affibody(®) molecules (ZH) into the HI loop of a coxsackievirus and adenovirus receptor binding-ablated fiber (Ad[ZH/1]). In addition to virus retargeting to Her2/neu, this virus was further modified from wild-type Ad by changing the RGD motif in the penton base to EGD and by substitution of the KKTK motif in the third shaft repeat to RKSK, resulting in the vector Ad[ZH/3]. The ZH-containing vectors could be produced to high titers and were specific for their target, resulting in efficient infection and killing of Her2/neu-positive androgen-dependent PC346C prostate cancer cells in vitro. Here we show that the oncolytic Ad[ZH/3] vector significantly prolonged survival time and reduced serum prostate-specific antigen levels in an orthotopic prostate tumor model in nude mice to the same extent as wild-type Ad5. Our results show that Her2/neu targeting using Ad-based vectors for prostate cancer is feasible and may serve as a basis for the development of gene therapy of human prostate cancer as well as other Her2/neu-expressing cancers.

Assessment of the critical level of androgen for growth response of transplantable human prostatic carcinoma (PC-82) in nude mice

The androgen dependent prostatic carcinoma of human origin, PC-82, was used as a model system to investigate the effect of various levels of androgen on the growth of prostatic tumor tissue. Plasma testosterone levels in mice were correlated to tumor growth and intratumor concentrations of testosterone and 5 alpha-dihydrotestosterone. PC-82 tumor burden remained stable at plasma testosterone levels of 0.8 nmol/l., whereas tumor growth occurred at higher levels and tumor regression was observed at lower plasma levels. This critical level of testosterone corresponded with intratumor testosterone and 5 alpha-dihydrotestosterone concentrations of six to 10 and three to four pmol/gm. tissue, respectively, which are significantly above the levels found in castrated non-supplemented animals (3.1 and 1.4 pmol/gm. respectively). This indicates that remaining concentrations of dihydrotestosterone, which amount to two to three times the castrate level, are not stimulatory for tumor growth in the model of the androgen dependent PC-82 tumor.