Albert A. Geldof

Effect of age on functional P‐glycoprotein in the blood‐brain barrier measured by use of (R)‐[11C]verapamil and positron emission tomography

P‐glycoprotein (P‐gp) is an efflux transporter responsible for the transport of various drugs across the blood‐brain barrier (BBB). Loss of P‐gp function with age may be one factor in the development and progression of neurodegenerative diseases. The aim of this study was to assess the effect of aging on BBB P‐gp function. Furthermore, the relationship between BBB P‐gp activity and peripheral P‐gp activity in CD3‐positive leukocytes was investigated. Finally, plasma pharmacokinetics of carbon 11–labeled (R)‐verapamil was evaluated.

Cytotoxicity and neurocytotoxicity of new marine anticancer agents evaluated using in vitro assays

Purpose: New classes of anticancer drugs, isolated from marine organisms, have been shown to possess cytotoxic activity against multiple tumor types. Aplidine, didemnin B, and isohomohalichondrin B (IHB), among the more promising antitumor candidates, have been evaluated in the present study on a comparative basis in terms of their antiproliferative activity and neurotoxic effects in vitro. Methods: Using a panel of different human, prostatic cancer cell lines (DU 145, PC-3 and LNCaP-FGC) the effects of Aplidine, didemnin B, and IHB on tumor cell proliferation were tested in a colorimetric (XTT) assay and compared with the effects of vincristine, vinorelbine, and Taxol. Under analogous in vitro conditions these drugs were also monitored for neurocytotoxic effects using a PC 12 cell line based model. Results: Didemnin B and – especially – Aplidine were more effective in the inhibition of prostate cancer cell proliferation than vincristine, vinorelbine or Taxol at concentration levels between 5 and 50 pmol/ml. At these same concentrations, however, Didemnin B and Aplidine were also most potent in the in vitro neurotoxicity assays. IHB was found to exert even more potent antiproliferative activity (at concentration levels between 0.05 and 0.1 pmol/ml). However, neurotoxic effects were also found to be present at these levels. After drug withdrawal, the neurotoxic damage, inflicted by aplidine or IHB appeared to be more long lasting than after vincristine or vinorelbine exposure. Conclusions: These results point to high antiproliferative activity of aplidine and IHB in prostate cancer. At the same time, the data urge some caution in the clinical use of these agents because of potential neurotoxic side-effects. The use of a newly formulated Aplidine may involve a more favorable therapeutic profile.

Nerve growth factor stimulates in vitro invasive capacity of DU145 human prostatic cancer cells

The prevalence of nerve growth factor (NGF) production in different human prostatic tumor cell lines (DU145, PC-3, LNCaP-FGC) was investigated using a specific enzyme-linked immunosorbent assay (ELISA) and compared to that of different human and rat prostatic tissue samples. In addition, the biological effects of NGFβ addition to the human prostatic cancer cell cultures were investigated. The ELISA technique showed the DU145 cell line to secrete measurable levels of NGF in the culture medium. When neurite-outgrowth determination in a pheochromocytoma cell line was used as a bioassay, the NGF synthesized by DU145 cells was confirmed to exhibit functional biological activity. No effect of exogenously added NGF could be established on tumor cell proliferation, on the basis of either colorimetric tetrazolium-based staining assay or bromodeoxyuridine incorporation. Also the expression of prostate specific acid phosphatase was not influenced by NGF addition. However, the in vitro invasive capacity (Matrigel) of DU145 cells was significantly increased by inclusion of 50 ng or 100 ng NGFβ/ml culture medium. In view of the clinically well-known perinuural invasion of prostate cancer cells, the possible involvement of NGF as a (paracrine) factor in prostatic cancer metastatic behavior should be investigated further.

Progression Delay of Prostate Tumor Skeletal Metastasis Effects by Bisphosphonates

Prostate tumor cell metastasis to the axial skeleton was induced in male Copenhagen rats using the intravenous injection of syngeneic metastatic R3327-MATLyLu tumor cells and concomitant caval vein clamping. The proliferation of tumor cells in the lumbar region was monitored by the progression, within 19 days post tumor cell injection, of hindleg paralysis which appeared in these animals. Histology confirmed the presence of tumor cells within the lumbar spine in 100% of cases displaying hindleg paralysis. Treatment with either of the bisphosphonate drugs, Cl2MDP or APD, suppressed and delayed the development of hind leg paralysis. Bisphosphonate treatment may be expected to delay the onset of axial skeletal metastasis effects in prostate cancer patients.

Nerve-growth-factor-dependent neurite outgrowth assay; a research model for chemotherapy-induced neuropathy

The rat clonal pheochromocytoma cell line PC12 can be induced to display neurite outgrowth upon induction with nerve growth factor (NGF). This NGF-dependent neurite outgrowth assay looks a promising model for research on toxic neuropathies. Using this assay we demonstrated that cisplatin caused a dose-dependent reduction of NGF-dependent neurite formation. Increasing doses of NGF, however, proved to exert protective activity against this cisplatin effect at an intermediate and clinically relevant cisplatin concentration of 1 μg/ml. Even at a high cisplatin concentration (10 μg/ml), the protective action of NGF, although less adequate, was observed. The value and strength of this model for screening neuropathogenic effects of anticancer agents at the cellular level and the possibly therapeutic action of neurotrophins are discussed and demonstrated. Furthermore, in the light of the urgent need for adequate models for neuropathy research, the PC12 neurite outgrowth protection assay may contribute to our knowledge of the mechanisms underlying the development of neuropathy.

The metastatic potential of rat prostate tumor variant R3327-MatLyLu is correlated with an increased activity of N-acetylglucosaminyl transferase III and V

Enzyme activities of N‐acetylglucosaminyltransferase (GlcNAc‐Tase) I–V involved in N‐linked complex‐type carbohydrate synthesis were determined in a non‐metastatic hormone‐dependent rat prostate tumor (R3327‐H) and a related, hormone‐independent variant metastasizing to lymph nodes and lungs (R3327‐MatLyLu). In the metastasizing variant a significantly increased activity of both GlcNAc‐Tase III and GlcNAc‐Tase V was observed, whereas the activities of GlcNAc‐Tase I and II were essentially unchanged. The increase in activity of GlcNAc‐Tase III is particularly noteworthy since it indicates that elevated expression of this enzyme cannot be considered as an exclusive marker of hepatic malignancy.

Consideration of the use of 17β-N,N-diethylcarbamoyl-4-methyl-4-aza-5-α-androstan-3-one (4MA), a 5α-reductase inhibitor, in prostate cancer therapy

SummaryWe investigated the effect of 17β-N,N-diethylcarbamoyl-4-methyl-4aza-5α-androstan-3-one (4MA), a 5α-reductase inhibitor, on growth inhibition of androgen-sensitive rat prostatic tumour (R3327-H) and correlated it with changes in weight of normal androgen target tissues and with levels of androgens. Groups of male Copenhagen rats were treated for 28 days with a daily injection of various, increasing doses of 4MA (0.01–4.0 mg/day) and the results were compared with control (vehicle-treated) and with castrated animals. 4MA decreased tumour growth rate in a dose-dependent manner, which was reflected in a decreased incorporation of BrdUrd in DNA of glandular epithelial cells in the tumour. Normal prostate wet weight was also decreased after high-dose 4MA treatment while serum testosterone levels were not affected by 4MA treatment. Contrary to expectations, however, tissue levels of dihydrotestosterone in tumour and ventral prostate were still considerable in 4MA-treated animals. The tumour-inhibiting action of 4MA, therefore, has to be interpreted as not being purely due to 5α-reductase inhibition. On the other hand, it was not possible to demonstrate any direct tumoricidal effect of 4MA in vitro. The relevance of these findings in terms of the endocrine mechanism of action of 4MA on tumour growth is discussed.

A predictive role for noncancerous prostate cells: low connexin-26 expression in radical prostatectomy tissues predicts metastasis

Background:It is important to identify markers that predict whether prostate cancer will metastasise. The adjacent noncancerous cells (influenced by the tumour cells) may also express potential markers. The objective of this study was to determine the influence of cancer cells on noncancerous cells and to assess the value of the cell-communication protein connexin-26 (Cx26) as a marker to predict the development of metastasis.Methods:The effect of conditioned medium (CM) from PrCa cells on in vitro noncancerous cell proliferation, migration and invasion and Cx26 expression was determined. Connexin-26 expression was investigated in prostatectomy tissues from 51 PrCa patients by immunohistochemistry and compared with various clinicopathological parameters.Results:Proliferation, migration and invasion of noncancerous cells were influenced by CM from the PrCa cell lines. Importantly, a clear relation was found between low Cx26 expression in the noncancerous tissue in prostatectomy sections and the risk of development of metastasis (P<0.0002). Kaplan–Meier analysis showed a relation between low Cx26 expression in noncancerous tissues and time to biochemical recurrence (P=0.0002).Conclusion:Measuring Cx26 expression in the adjacent noncancerous tissues (rather than cancer tissues) of prostatectomy sections could help to identify high-risk patients who may benefit from adjuvant therapy to decrease the risk of metastasis.

Abstract 112: Synthesis and preclinical evaluation of radiolabeled alisertib as an investigational aurora kinase A PET tracer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: High expression of the aurora kinase A (AURKA) protein is associated with colorectal adenoma-to-carcinoma progression and with poor prognosis of patients with stage III colorectal cancer (CRC) and patients with CRC liver metastases. Several agents have been developed that specifically target AURKA kinase activity, among those the investigational agent alisertib. To identify CRC patients with high AURKA expression levels a dedicated imaging method would be preferred, since only then a whole body assessment of AURKA expression in primary tumors as well as in metastases can be achieved. Positron emission tomography (PET) using a dedicated radiotracer allows for this. To this end, [3H]alisertib has been synthesized for in vitro experiments and [11C]alisertib for in vivo imaging with PET in xenograft mouse models. Methods: [3H]alisertib was synthesized starting from [3H]methyl nosylate under similar conditions as [11C]alisertib, which was synthesized starting from [11C]CH3I in a two-step procedure, purified and formulated within 45 minutes. Four CRC cell lines with different levels of AURKA expression were selected, HCT116, SW480, SW1398 and Caco-2, and the in vitro dynamic uptake of [3H]alisertib in these cell lines was measured before and after siRNA-mediated AURKA downmodulation. Next, the uptake of [11C]alisertib was assessed with PET in xenografted mice using the same four CRC cell lines. Results: The synthesis of [3H]alisertib was optimized to an overall yield of 42%, while an overall yield of 23 ± 4%, starting from [11C]CH3I, was obtained in an optimized synthesis of [11C]alisertib. The CRC cell line with high expression of AURKA, Caco-2, showed a significantly higher uptake of [3H]alisertib compared to the lower expressing AURKA CRC cell lines, HCT116, SW480 and SW1398. In addition, the uptake of [3H]alisertib was reduced in all cell lines upon downmodulation of AURKA. The stability of [11C]alisertib in rodents was determined at 97.8% ± 1.3% intact tracer in blood at 45 minutes after iv injection (n=4). Preliminary data using a HCT116 xenograft mouse model indicated a tumor-to-background ratio of 1.56 ± 0.12, with an uptake of 1.00 %ID/g at 90 minutes after injection. PET studies with the SW480, SW1398 and Caco-2 xenografts are currently in progress. Conclusions: Both [3H]alisertib and [11C]alisertib were obtained with good purity and yield. In vitro studies using [3H]alisertib indicate good correlation of cellular uptake with AURKA expression. [11C]alisertib is stable in vivo and HCT116 xenografted mice show a fair tumor uptake of [11C]alisertib. Citation Format: Joost Verbeek, Jeroen ACM Goos, Albert A. Geldof, Annemieke C. Hiemstra, Otto S. Hoekstra, Gerrit A. Meijer, Steven Stroud, Daniel Bradley, Remond JA Fijneman, Albert D. Windhorst. Synthesis and preclinical evaluation of radiolabeled alisertib as an investigational aurora kinase A PET tracer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 112. doi:10.1158/1538-7445.AM2014-112

Abstract LB-14: Low connexin-26 predicts the development of metastasis after radical prostatectomy

Prostate cancer is the second most common cause of death in males. Not all tumors will become metastatic within a patients life time, but when metastasized no curative therapy is available. It is therefore important to identify molecules that predict whether the tumor will become metastatic. Connexin-26 (Cx26) is a gap-junction protein that is involved in cell-cell contacts and in cell adhesion. Cx26 is involved as a tumor suppressor in various tumor types. The aim of the present study was to determine Cx26 expression in localized prostate cancer and relate the expression to the development of metastasis. Cx26 was stained by immunohistochemical staining in radical prostatectomy tissues of 43 patients. The expression levels were scored by a pathologist. Cx26 expression was observed in 95% of the patients. No relation was found to Cx26 expression in the tumor and the development of metastasis (p=0.2). Interestingly, a clear relation was found between low Cx26 expression in benign epithelial cells within the same sections and the development of metastasis (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-14. doi:10.1158/1538-7445.AM2011-LB-14