Synthetic and mechanistic aspects of a new method for ruthenium-metalation of porphyrins and Schiff-bases.

A new method is presented for metalation of a wide range of free-base, neutral, cationic, and anionic porphyrins in refluxing dimethylformamide (DMF) using an easily prepared [Ru(DMF) 6](OTf) 3 complex, and comparisons are made with the more familiar metalation procedure using Ru 3(CO) 12. Both procedures generate Ru (II)(porp)(CO)L complexes (L = solvent); use of the Ru (III)-triflate precursor gives yields comparable to, or greater than, those obtained with the carbonyl, and generates no Ru-chlorin impurities. Mechanistic studies on the meso-tetraphenylporphyrin system reveal that the DMF furnishes the CO, which in the presence of essential water reduces the metal, and metalation likely occurs via a Ru (II)-CO species. Corresponding metalation of tetradentate Schiff-bases gives trans-[Ru (III)(Schiff- base)(DMF) 2]OTf complexes in yields of approximately 50%, a limitation being the accompanying hydrolysis of the Schiff-base through the presence of trace water.

Non-invasive evaluation of tumour hypoxia in the Shionogi tumour model for prostate cancer with 18F-EF5 and positron emission tomography.

OBJECTIVE: To evaluate hypoxia non-invasively in androgen-dependent (AD), regressing (6-days after castration, RG) and androgen-independent (AI) Shionogi tumours, using the radiolabelled tracer for hypoxia, 18F-EF5, and positron emission tomography (PET). MATERIALS AND METHODS: Groups of mice bearing AD, RG and AI Shionogi tumours were co-injected with 18F-EF5 and unlabelled EF5. The mice were imaged non-invasively with PET to examine the accumulation of 18F-EF5 in hypoxic regions of the tumour. The tumours were subsequently placed in a gamma-counter, or disaggregated for flow cytometry, to determine the levels of 18F-EF5 and the percentage of hypoxic cells present in the tumour, respectively. RESULTS: The mean (sd) levels of hypoxia in AD Shionogi tumours decreased significantly 6 days after androgen ablation as measured by flow cytometry, from 17.1 (4.77) to 1.74 (0.46)% (P=0.003). There were no significant differences in the levels of 18F-EF5 in the tissue between AD and RG tumours using region-of-interest analysis of PET images or gamma-counting, although the differences were significant when measured by flow cytometry. However, mean (sd) levels of hypoxia in AI Shionogi tumours were significantly higher than in AD tumours regardless of the analysis method; PET, 10.5 (4.93)x10(-5)) Bq/cm2 (P=0.017), flow cytometry, 42.98 (3.35)% (P<0.001), well count, 6.81 (1.17)x10(4) and 13.1 (1.99)x10(4) cpm/g, for AD and AI tumours, respectively (P<0.001). CONCLUSIONS: Differences in hypoxia between AD and AI, but not RG, Shionogi tumours can be detected non-invasively with 18F-EF5 and PET. As prostate tumours are hypoxic and the oxygen levels can change with androgen ablation, noninvasive imaging of hypoxia with PET and 18F-EF5 might ultimately have a prognostic and/or diagnostic role in the clinical management of the disease.

pH modulation using CsCl enhances therapeutic effects of vitamin D in LNCaP tumor bearing mice.

BACKGROUND: The downstream effects of pH modulation significantly impact the biological fate of chemotherapeutic agents and tumor responsiveness to therapy. We have studied the effects of cesium chloride (CsCl) on pH modulation and subsequent vitamin D treatment in vitamin D receptor (VDR) positive LNCaP tumors and VDR null MDA/LCC6 tumors in vivo. METHODS: Mice bearing LNCaP or MDA/LCC6 tumors were dosed orally with CsCl (150 mg/kg) or vitamin D (1 microg/kg) alone and in combination. Tumor volume and serum PSA (LNCaP only) were measured and intracellular pH was determined, using magnetic resonance spectroscopy (MRS), at tumor and leg muscle sites. Atomic absorption spectroscopy (AAS) was used to quantitate cesium in serum, organs, and tumor tissues. RESULTS: From day 10 onwards, statistically significant (P<0.01) differences were observed in all LNCaP treated groups as compared with control. CsCl co-administered with vitamin D, caused an apparent sensitization of efficacy in this tumor model. There were no correlating differences in serum PSA. Elevation of pH was statistically significant for all three treatment groups as compared with control. The pH measured in leg muscle was not influenced by CsCl treatment. Inhibition of tumor growth was not apparent in VDR null MDA/LCC6 tumors although intracellular tumor pH was shifted. Cesium was rapidly absorbed into serum and present in LNCaP tumors, prostates and other tissues after 1 hr, remaining for up to 24 hr. CONCLUSIONS: The data presented in this manuscript is the first report of chemosensitization by in vivo pH modulation using CsCl in mice bearing prostate or any other tumor xenograft.

Chemoprotection against N-nitrosomethylbenzylamine-induced mutation in the rat esophagus.

Prevention of esophageal cancer may be possible through dietary modification or supplementation. In this study we have investigated the mutation preventive properties of ellagic acid, green tea, and diallyl sulfide (DAS) against the mutagenicity of the nitrosamine N-nitrosomethylbenzylamine (NMBA) in the esophagus of the rat. In addition, the effect of the consumption of ethanol on the mutagenicity of NMBA was examined. NMBA is specific in inducing tumors in the rat esophagus and has been used in many studies investigating the mechanism and the prevention of this cancer. We found that the type of mutations induced by two 2-mg/kg subcutaneous injections of NMBA in the lacI gene of "Big Blue" rats is consistent with that found previously for nitrosamines in other systems and consists of G:C-->A:T transitions. We report that the addition of ellagic acid to the feed, replacing drinking water with green tea, and gavage with DAS significantly reduced the mutagenicity of NMBA. In contrast, the addition of 5% ethanol to the drinking water increased the mutagenicity of NMBA. This is consistent with findings that these compounds modulate NMBA-induced carcinogenesis in the rat.

Hypoxia in the androgen-dependent Shionogi model for prostate cancer at three stages.

The objective of this study was to investigate a possible relationship between androgen status and hypoxia in the Shionogi murine prostate tumor model, which is widely used to study the effects of androgen withdrawal on hormone resistance and radiation response. Binding of the nitroimidazole hypoxia marker EF5 was assessed using the Cy3-tagged monoclonal antibody ELK3-51. Three hours after injection of EF5 (30 mg/kg), tumors from the following three stages were excised: androgen-dependent, regressed tumors 7 days after castration, and androgen-independent. Half of each tumor was disaggregated for analysis by flow cytometry and the remainder was flash frozen. Statistically significant differences (P < 0.01) were found between androgen-dependent, regressed and androgen-dependent tumors: approximately 30, approximately 2 and approximately 50% hypoxic cells, respectively. Frozen sections from androgen-dependent tumors exhibited highly variable EF5 binding; regressed tumors showed very little or no binding; each section from androgen-dependent tumors showed high levels and uniformly distributed binding of EF5. There was no correlation between the degree of hypoxia and tumor weight (P > 0.1). The results from this preliminary study indicate that hypoxia may play an important role with respect to the timing of irradiation in prostate cancer treatments and possibly may be a useful prognostic tool. In addition, hypoxia may also be relevant to progression in this disease after androgen ablation.

HER-2/neu overexpression increases the viable hypoxic cell population within solid tumors without causing changes in tumor vascularization.

The effects of HER-2/neu overexpression on the tumor microenvironment in an aggressive breast cancer xenograft model were investigated. These studies focused on tumors derived following the subcutaneous injection of MDA-MB-435/LCC6 cells transfected with human c-erbB2 (LCC6(HER-2)) into SCID-Rag2M mice. LCC6(HER-2) tumors were more viable (H&E-stained tumor sections) than isogenic vector control tumors (LCC6(Vector)). Correspondingly, a 2.7-fold increase in trypan blue-excluding cells (P = 0.00056) and a 4.8-fold increase in clonogenic cells (P = 0.00146) were noted in cell suspensions derived from disaggregated LCC6(HER-2) versus LCC6(Vector) tumors. Tumor sections stained with the antibody detecting 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), a marker of hypoxia, showed a greater fraction of hypoxic tissue in LCC6(HER-2) tumors compared with control tumors. Flow cytometric analyses based on viable tumor cells (DNA content >/= 2N) in cell suspensions from disaggregated tumors confirmed that there were significantly more EF5-positive cells (i.e., hypoxic) in LCC6(HER-2) than in LCC6(Vector) tumors (16.41 +/- 8.1% and 5.96 +/- 4.1%, respectively; P = 0.0015). Protein levels of phosphorylated (Ser(536)) nuclear factor-kappaB p65 were significantly elevated in LCC6(HER-2) tumors (P = 0.00048), and a trend in increased hypoxia-inducible factor-1alpha protein levels was observed in LCC6(HER-2) compared with LCC6(Vector) tumors. Despite the substantial viable hypoxic cell fraction and a 1.7-fold increase of vascular endothelial growth factor protein (P = 0.05) in LCC6(HER-2) tumors, no significant differences were found (P > 0.05) between LCC6(HER-2) and LCC6(Vector) vasculature (CD31 staining and Hoechst 33342 perfusion). These results suggest that HER-2/neu overexpression may be linked with overall increased tumor viability and a significant increase in the population of viable hypoxic cells, which is not due to differences in tumor vascularization.

Enhanced radiation sensitivity in prostate cancer by inhibition of the cell survival protein clusterin.

PURPOSE: The purpose of this study is to evaluate the role of the cell survival gene clusterin in radiation-induced cell death in human LNCaP and PC-3 prostate cancer models. EXPERIMENTAL DESIGN: Radiation sensitivities were compared in parental and clusterin-overexpressing LNCaP cells and in PC-3 cells and tumors treated with antisense or mismatch clusterin oligonucleotides. RESULTS: Clusterin-overexpressing LNCaP cells were less sensitive to irradiation with significantly lower cell death rates (23% after 8 Gy) compared with parental LNCaP cells (50% after 8 Gy) 3 days after irradiation. Clusterin expression in PC-3 cells after radiation was found to be up-regulated in a dose-dependent manner in vitro by 70% up to 12 Gy and in vivo by 84% up to 30 Gy. Inhibition of clusterin expression in PC-3 cells using antisense oligonucleotides (ASOs) occurred in a sequence- and dose-dependent manner and significantly enhanced radiation-induced apoptosis and decreased PC-3 cell growth rate and plating efficiency. Compared with mismatch control oligonucleotide treatment, clusterin ASO treatment enhanced radiation therapy and significantly reduced PC-3 tumor volume in vivo by 50% at 9 weeks. In addition, TUNEL staining revealed increased number of apoptotic cells in clusterin ASO-treated and irradiated PC-3 tumors, compared with treatment with mismatch control oligonucleotides plus radiation. CONCLUSIONS: These findings support the hypothesis that clusterin acts as a cell survival protein that mediates radioresistance through the inhibition of apoptosis. In vivo results further suggest that inactivation of clusterin using ASO technology might offer a novel strategy to improve results of radiation therapy for prostate cancer patients.

F 11782, a novel catalytic inhibitor of topoisomerases I and II, induces atypical, yet cytotoxic DNA double-strand breaks in CHO-K1 cells.

F 11782, or 2'', 3''-bispentafluorophenoxyacetyl-4, 6'-ethylidene-beta-D glucoside of 4'-phosphate-4'-dimethylepipodopliyllotoxin 2N-methyl glucamine salt, is a novel fluorinated lipophylic epipodophylloid which has shown marked antitumour activity in vivo. In vitro studies have demonstrated a dual catalytic inhibitory activity of F 11782 against topoisomerases and I and II by an original mechanism involving interference with the DNA binding activity of these enzymes, without DNA intercalating properties. Nevertheless, the precise mechanism(s) of cytotoxicity of F 11782 remains unclear and recent studies have suggested that this cytotoxicity might result, at least in part, from an induction of DNA-strand breaks without stabilisation of cleavable complex. In this study, DNA damage induced by F 11782 and its repair by non-homologous recombination was investigated in CHO-K1 cells. The results suggest that the nature of such damage differs from that induced by etoposide, a structurally-related topoisomerase II poison and identify a high level of stability of the damage induced which may account, at least in part, for the superior preclinical anti-tumour activity of F 11782.