Neuroblastoma is a challenging childhood malignancy, with a very high percentage of patients relapsing following acquisition of drug resistance, thereby necessitating the identification of mechanisms of drug resistance as well as new biological targets contributing to the aggressive pathogenicity of the disease. In order to investigate the molecular pathways that are involved with drug resistance in neuroblastoma, we have developed and characterised cisplatin resistant sublines SK-N-ASCis24, KellyCis83 and CHP-212Cis100, integrating data of cell behaviour, cytotoxicity, genomic alterations and modulation of protein expression. All three cisplatin resistant cell lines demonstrated cross resistance to temozolomide, etoposide and irinotecan, all of which are drugs in re-initiation therapy. Array CGH analysis indicated that resistant lines have acquired additional genomic imbalances. Differentially expressed proteins were identified by mass spectrometry and classified by bioinformatics tools according to their molecular and cellular functions and their involvement into biological pathways. Significant changes in the expression of proteins involved with pathways such as actin cytoskeletal signalling (p = 9.28E-10), integrin linked kinase (ILK) signalling (p = 4.01E-8), epithelial adherens junctions signalling (p = 5.49E-8) and remodelling of epithelial adherens junctions (p = 5.87E-8) pointed towards a mesenchymal phenotype developed by cisplatin resistant SK-N-ASCis24. Western blotting and confocal microscopy of MYH9, ACTN4 and ROCK1 coupled with invasion assays provide evidence that elevated levels of MYH9 and ACTN4 and reduced levels of ROCK1 contribute to the increased ROCK1-independent migratory potential of SK-N-ASCis24. Therefore, our results suggest that epithelial-to-mesenchymal transition is a feature during the development of drug resistance in neuroblastoma.

A series of 2-cycloalkyl- and 2-alkyl-3-(hydroxymethyl)-1-methylindoloquinones and corresponding carbamates have been synthesized and substituted in the 5-position with a variety of substituted and unsubstituted aziridines. Cytotoxicity against hypoxic cells in vitro was dependent upon the presence of a 5-aziridinyl or a substituted aziridinyl substituent for 3-hydroxymethyl analogues. The activity of 5-methoxy derivatives was dependent upon the presence of a 3-(carbamoyloxy)methyl substituent. Increasing the steric bulk at the 2-position reduced the compounds' effectiveness against hypoxic cells. A 2-cyclopropyl substituent was up to 2 orders of magnitude more effective than a 2-isopropyl substituent, suggesting possible radical ring-opening reactions contributing to toxicity. Nonfused 2-cyclopropylmitosenes were more effective than related fused cyclopropamitosenes reported previously. The reduction potentials of the quinone/semiquinone one-electron couples were in the range -286 to -380 mV. The semiquinone radicals reacted with oxygen with rate constants 2-8 x 10(8) dm3 mol-1 s-1. The involvement of the two-electron reduced hydroquinone in the mediation of cytotoxicity is implicated. The most effective compounds in vitro were the 2-cyclopropyl and 5-(2-methylaziridinyl) derivatives, and of these, 5-(aziridin-1-yl)-2-cyclopropyl-3-(hydroxymethyl)-1-methylindole-4 ,7-dione (21) and 3-(hydroxymethyl)-5-(2-methylaziridin-1-yl)-1,2-dimethylindole+ ++-4,7-dione (54) were evaluated in vivo. Both compounds showed antitumor activity both as single agents and in combination with radiation, with some substantial improvements over EO9 (3) at maximum tolerated doses and as single agents against the RIF-1 tumor model and comparable efficacy in the KHT tumor model.

A series of indolequinones bearing a variety of leaving groups at the (indol-3-yl)methyl position was synthesized by functionalization of the corresponding 3-(hydroxymethyl)indolequinone, and the resulting compounds were evaluated in vitro as bioreductively activated cytotoxins. The elimination of a range of functional groups-carboxylate, phenol, and thiol-was demonstrated upon reductive activation under both chemical and quantitative radiolytic conditions. Only those compounds which eliminated such groups under both sets of conditions exhibited significant hypoxia selectivity, with anoxic:oxic toxicity ratios in the range 10-200. With the exception of the 3-hydroxymethyl derivative, radiolytic generation of semiquinone radicals and HPLC analysis indicated that efficient elimination of the leaving group occurred following one-electron reduction of the parent compound. The active species in leaving group elimination was predominantly the hydroquinone rather than the semiquinone radical. The resulting iminium derivative acted as an alkylating agent and was efficiently trapped by added thiol following chemical reduction and by either water or 2-propanol following radiolytic reduction. A chain reaction in the radical-initiated reduction of these indolequinones (not seen in a simpler benzoquinone) in the presence of a hydrogen donor (2-propanol) was observed. Compounds that were unsubstituted at C-2 were found to be up to 300 times more potent as cytotoxins than their 2-alkyl-substituted analogues in V79-379A cells, but with lower hypoxic cytotoxicity ratios.

Traditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in our understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results. Here, we demonstrated the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs.

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Summary The vaso-active drug hydralazine causes a considerable increase in the cytotoxic effect of melphalan towards the KHT tumour in mice. The enhancement in response, measured as the concentration of melphalan required to achieve a given tumour response, is 3.0 and 2.35 when determined using the regrowth delay assay and the technique for determining surviving fraction in vitro following treatment in vivo respectively. In contrast, measurement of systemic toxicity shows that the addition of hydralazine only causes a small increase (ER= 1.15) in melphalan damage. This suggests that the drug combination may have some therapeutic benefit. The tumour specificity for the action of hydralazine is supported by the finding that binding of 3H-misonidazole is increased in tumours but not in other tissues when mice are treated with hydralazine. Increased binding of labelled misonidazole is associated with an increase in the level and duration of hypoxia, which will occur as a consequence of changes in tumour blood flow brought about by hydralazine. However, hypoxia per se is not responsible for the enhanced effect of melphalan, since the agent BW12C, which also induces substantial tumour hypoxia as a result of changing the 02 affinity of haemoglobin, has no effect on melphalan tumour cytotoxicity.There have been various reports showing that vasoactive drugs can significantly affect the nature of blood flow in both experimental rodent and human tumours (Algire & Lagallais, 1951;Cater et al., 1962;Kruuv et al., 1967; Vorhees & Babbs, 1982;Knapp et al., 1985). Reduced blood flow in tumours can cause lowering of the oxygen status of the tumour, thereby causing radiation resistance (Kruuv et al., 1967). This so-called 'stealing' effect has recently been exploited by Chaplin and Acker (1987) in order to increase the anti-tumour effect of the bio-reductive agent, RSU 1069 (Adams et al., 1984) a compound which is activated under hypoxic conditions to give a species 100 x more toxic than the parent compound (Stratford et al., 1986).Reduction of blood flow in tumours may also be potentially useful for enhancing the effects of some anti-cancer drugs. The rationale for this is that, administration of the vaso-active drug at the time at which the chemotherapeutic agent has reached its maximum tumour concentration, will inhibit loss of active drug from the tumour. This could increase the overall exposure of the tumour cells to the cytotoxic drug. This paper describes the results of a study of the effect of the vasoactive agent hydralazine on the cytotoxic action of melphalan (L-phenylalanine mustard, L-PAM) towards the KHT sarcoma in mice. Materials and methodsMice and tumours Eight to 12 week old male Category IV C3H/He mice, obtained from NIMR, Mill Hill, London in 1984 and subsequently bred 'in-house', were used in the present experiments. The KHT sarcoma (Kallman et al., 1967), provided by Dr P. Twentyman, MRC, Cambridge in 1983, was maintained by inoculation of a tumour brei into the gastrocnemius muscle of female mice. Generall...

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Sunmnary Mice were injected with tritiated misonidazole (750mgkg-1), killed after 24h and the excised tissues prepared for autoradiography (ARG) to identify sites of accumulation. The previously reported high grain count associated with bound misonidazole metabolite(s) was observed in the liver. The ratio of grain count in the emulsion above the centrilobular hepatocytes to the count over connective tissue (stroma) was 12. A higher count ratio for 'target' cells to stroma was observed in the following cells/tissues: meibomian gland (ducts 110, acini 65), oesophagus (keratinised layer 60), incisor (enamel organ 17), nasal septum (subepithelial glands 13). For some of these tissues the explanation might appear to lie with localised hypoxia, but for others which were probably normoxic there is as yet no obvious reason for these findings.Misonidazole (MISO) sensitises hypoxic cells to the effects of ionising radiation. Further, its reduced metabolite(s) can bind firmly to these cells, which are commonly found in tumours (Garrecht & Chapman, 1983; Brown, 1975). The metabolic steps which lead to this binding are incompletely understood (Franko et al., 1982;Mason, 1982), but they lead, subsequent to an at least two-electron nitroreduction, to covalent binding to adjacent cellular macromolecules (Varghese & Whitmore, 1980;Chapman et al., 1983;Rauth, 1984).Although MISO was initially shown in mice to be an efficient radiosensitiser of tumour cells subsequent clinical trials revealed neurotoxicity at a dose level below that required for tumour radiosensitisation. MISO will also chemosensitise tumours, and the preferential binding of radionuclide-labelled MISO in the hypoxic regions of tumours points to the potential use of suitable derivatives in tumour imaging (Urtasun et al., 1986). The presence of hypoxic areas in many tumours has led to the suggestion that anticancer drugs could be developed which would be activated by the hypoxic environment, i.e. bioreductive drugs (Hall & Roizin-Towle, 1975).In the present work we have sought to identify those normal tissues which might, like tumours, have cells which accumulate MISO metabolites. Such accumulation could arise from hypoxia, a high reductase activity or some other mechanism. The skin, liver, intestine and cartilage are normal tissues which on occasion have been reported to have a low oxygen tension (Hendry, 1979;Bohlen, 1980;Langler et al., 1982). The accumulation of metabolites of radiosensitisers or bioreductive drugs in any quantity in normal healthy tissues might well cause problems when these compounds are used in diagnosis or therapy.MISO, labelled with tritium in the side chain, was injected into mice and the microscopic distribution in tissues 24 h later was examined by ARG. The advantage of 3H over the available alternative of 14C (ring label) in ARG is that the shorter pathlength of the tritium fl-particle allows more precise localisation of the cells containing the adduct(s). In the present study it is likely that the observed activity (grains) was due to ...