SummaryThe anti-tumour effects and mechanism of action of combretastatin A-4 and its prodrug, combretastatin A-4 disodium phosphate, were examined in subcutaneous and orthotopically transplanted experimental colon tumour models. Additionally, the ability of these compounds to directly interfere with endothelial cell behaviour was also examined in HUVEC cultures. Combretastatin A-4 (150 mg kg -1 , intraperitoneally (i.p.)) and its water-soluble prodrug (100 mg kg -1 , i.p.) caused almost complete vascular shutdown (at 4 h), extensive haemorrhagic necrosis which started at 1 h after treatment and significant tumour growth delay in MAC 15A subcutaneous (s.c.) colon tumours. Similar vascular effects were obtained in MAC 15 orthotopic tumours and SW620 human colon tumour xenografts treated with the prodrug. More importantly, in the orthotopic models, necrosis was seen in vascularized metastatic deposits but not in avascular secondary deposits. The possible mechanism giving rise to these effects was examined in HUVEC cells. Here cellular networks formed in type I calf-skin collagen layers and these networks were completely disrupted when incubated with a non-cytotoxic concentration of combretastatin A-4 or its prodrug. This effect started at 4 h and was complete by 24 h. The same non-cytotoxic concentrations resulted in disorganization of F-actin and β-tubulin at 1 h after treatment. In conclusion, combretastatin A-4 and its prodrug caused extensive necrosis in MAC 15A s.c. and orthotopic colon cancer and metastases, resulting in anti-tumour effects. Necrosis was not seen in avascular tumour nodules, suggesting a vascular mechanism of action.

Germ line mutations in the IntroductionThe Adenomatous polyposis coli (APC) tumor suppressor gene is mutated in the hereditary form of colorectal cancer, familial adenomatous polyposis (FAP), and in the majority of sporadic colorectal cancers in humans. 1-3 FAP individuals inherit one mutant APC allele and tumorigenesis proceeds following loss of the second APC allele. In sporadic colorectal cancers, tumorigenesis involves somatic inactivation of both APC alleles.Much of our insight into the functions of APC has come from analysis of murine models. The mouse Apc protein exhibits 90% amino acid (aa) homology to human APC. 4 Several different mouse models of intestinal polyposis exist, each harboring different truncating mutations of the Apc gene. The Apc Min/ϩ mouse is one of the most commonly used models to investigate the molecular mechanisms of intestinal tumorigenesis. Apc Min/ϩ mice carry a heterozygous germ line mutation at codon 850 of the Apc gene, 4 and loss of the normal Apc allele in intestinal epithelium precedes adenoma formation. 5 These animals develop adenomas along the length of the small intestine and colon. Apc Min/Min homozygotes arrest development at approximately 5 days after coitus and die in utero. 6 Apc is a large 310-kDa protein characterized by the presence of several different functional domains that mediate interactions with numerous protein partners. 7 Apc is postulated to have many functions including roles in microtubule dynamics, cell cycle control, cell adhesion, and chromosomal stability. 8 However, the tumor suppressor function of Apc appears to lie in its regulation of cellular levels of the protooncogene ␤-catenin. 9-11 ␤-Catenin has dual roles in both cadherin-mediated cell adhesion and in cell signaling as a transcriptional effector of the canonical Wnt signaling pathway. In this pathway, Apc forms part of a highmolecular weight phosphorylation/destruction complex with axin, glycogen synthase kinase 3␤ (GSK-3␤), protein phosphatase 2a, and GBP/Frat1 (GSK-3-binding protein/frequently rearranged in advanced T-cell lymphomas 1) that targets ␤-catenin for degradation via the ubiquitin/proteasome system. [12][13][14][15] In response to a Wnt signal, ␤-catenin escapes phosphorylation by the Apc complex and translocates to the nucleus where it activates expression of target genes through the Tcf/Lef-1 (T-cell factor 1/lymphoid enhancer factor 1) transcription factors. 16,17 Apc can also shuttle between nucleus and cytoplasm further regulating the subcellular localization and turnover of ␤-catenin. 18,19 In the intestinal epithelium, loss of function of Apc promotes tumorigenesis via constitutive activation of ␤-catenin/Tcf-4-mediated transcription of downstream targets including the growth promoting genes c-Myc 20 and cyclin D1. 21 The Wnt signaling pathway is an evolutionarily conserved mechanism that governs cell fate decisions and patterning during embryogenesis and in adult tissues. Wnt ligands are secreted glycoproteins that act on members of the frizzled and low-density lipo...

Summary Anti-vascular effects of the novel Vinca alkaloid, vinflunine have been investigated in the MAC 15A transplantable murine colon adenocarcinoma model and compared with those induced by the most recently identified clinically useful third generation Vinca. Administration of the maximum tolerated dose of either vinflunine (50 mg kg -1 ) or vinorelbine (8 mg kg -1 ) resulted in significant tumour growth delay with subsequent histological analysis revealing substantial haemorrhagic necrosis. This suggested possible anti-vascular effects and these were confirmed by Hoechst 33342 perfusion studies. Vinflunine, currently undergoing Phase I trials in Europe, was found to be at least as effective as the clinically active vincristine and vinorelbine in this model and, remarkably, produced anti-vascular effects at doses much lower than the maximum tolerated dose. Although vinflunine caused apoptosis in HUVEC monolayer cultures this event did not occur within the first 8 hours of exposure whereas vascular shutdown in vivo was observed within the first 4 hours.

Cyclooxygenase-2 (Cox-2) is expressed predominantly by stromal cells in intestinal adenomas from the Apc(Min/+) mouse model of familial adenomatous polyposis. We investigated the mechanistic basis of stromal cell Cox-2 expression in Apc(Min/+) mouse adenomas, as well as Cox-2 expression and activity in histologically normal (HN) Apc(Min/+) mouse intestine, in order to gain further insights into regulation of Cox-2 as a potential chemoprevention target. Upregulation of Cox-2 in intestinal tumours is not an intrinsic feature of Apc(Min/+) macrophages as bone marrow-derived Apc(Min/+) macrophages did not exhibit an abnormality in Cox-2 expression or activity. Intestinal permeability to lactulose or mannitol was similar in Apc(Min/+) mice and wild-type littermates, implying that macrophage activation by luminal antigen is unlikely to explain stromal cell Cox-2 induction. Moreover, stromal cells exhibited differential expression of Cox-2 and inducible nitric oxide synthase, suggesting 'alternative' (M2) rather than 'classical' (M1) macrophage activation. Flow cytometric sorting of isolated stromal mononuclear cells (SMNCs), on the basis of M-lysozyme and specific macrophage marker expression, demonstrated that macrophages, neutrophils and non-myelomonocytic cells all contributed to lamina propria prostaglandin (PG) E(2) synthesis. However, the majority of PGE(2) synthesis by macrophages was via a Cox-2-dependent pathway compared with predominant Cox-1-derived PGE(2) production by non-myelomonocytic cells. SMNCs from HN Apc(Min/+) intestinal mucosa exhibited similar levels of Cox-2 mRNA and protein, but produced more Cox-2-derived PGE(2) than wild-type cells at 70 days of age. There was an age-dependent decline in PGE(2) synthesis by Apc(Min/+) SMNCs, despite tumour progression. These data suggest that other Cox-2-independent factors also control PGE(2) levels during Apc(Min/+) mouse intestinal tumorigenesis. Regulation of macrophage Cox-2 expression and other steps in PGE(2) synthesis (e.g. PGE synthase) are valid targets for novel chemoprevention strategies that could minimize or avoid systemic COX-2 inhibition.