Trichostatin A causes p53 to switch oxidative-damaged colorectal cancer cells from cell cycle arrest into apoptosis.

Many studies aim at improving therapeutic efficacy by combining strategies with oxidative stress-inducing drugs and histone deacetylase (HDAC) inhibitors in colorectal cancer. As p53 and p21(WAF1) are essential in oxidative stress-induced DNA damage, we investigated epigenetic regulation of p21(WAF1) promoter. Firstly, HCT116 p53(+)/(+) and p53(-)/(-) colorectal cancer cells were treated with H(2)O(2) for 6 hrs and 24 hrs (early/late response). Chromatin immunoprecipitation revealed transcriptional transactivation of p21(WAF1) in HCT116 p53(+)/(+) cells as shown by increased binding of p53 and acetylated H4 around two p21(WAF1) promoter sites, the responsible element (RE) and the Sp1 site, while both proteins bound preferentially on the RE. Interestingly, H3 was not involved, suggesting H4-specific transactivation of the p21(WAF1) promoter. H(2)O(2) addition resulted in G(2)/M arrest of both HCT116 cell lines without significant cell death. To investigate whether a HDAC inhibitor strengthens G(2)/M arrest, we pretreated cells with Trichostatin A (TSA). In HCT116 p53(+)/(+) cells, we found (i) remarkably increased acetylated H4 around both p21(WAF1) promoter regions, especially at the Sp1 site; (ii) increased acetylation of p53 at lysines 320 and 382;(iii) displacement of HDAC1 from the Sp1 site, thus inhibiting its repression effect and increasing p53 binding.p53 seems to trigger H4-acetylation around the p21(WAF1) promoter because there was nearly no H4 acetylation in HCT116 p53(-)/(-) cells. For the first time we show that there is a time-dependent TSA mode of action with increased p53-dependent histone H4 acetylation at the p21(WAF1) promoter in early response, and decreased acetylation in late response. Reduced p53-triggered transactivation of p21(WAF1) in late response allows cells to re-enter cell cycle, and TSA causes p53 to simultaneously induce apoptosis.

Modulation by polyamines of apoptotic pathways triggered by procyanidins in human metastatic SW620 cells.

We showed previously that inhibition of polyamine catabolism with the polyamine oxidase inhibitor MDL 72527 (MDL) potentiates the apoptotic effects of apple procyanidins (Pcy) in SW620 cells. Here we report that Pcy caused an activation of the intrinsic apoptotic pathway through enhanced polyamine catabolism and mitochondrial membrane depolarization. MDL in the presence of Pcy caused a profound intracellular depletion of polyamines and exerted a protective effect on mitochondrial functions. MDL potentiation of Pcy-triggered apoptosis was reversed by addition of exogenous polyamines. In addition, MDL in combination with Pcy activated the extrinsic apoptotic pathway through enhanced TRAIL-death receptor (DR4/DR5) expression. Potentiation of Pcy-triggered apoptosis by MDL was inhibited when cells were exposed to specific inhibitors of DR4/DR5. These data indicate that the depletion of intracellular polyamines by MDL in the presence of Pcy caused a switch from intrinsic to extrinsic apoptotic pathways in human colon cancer-derived metastatic cells.

Close localization of DAP-kinase positive tumour-associated macrophages and apoptotic colorectal cancer cells.

The death-associated protein kinase (DAP-kinase) is a cytoskeleton-associated protein crucially involved in the induction of early apoptotic pathways. Aberrant hypermethylation of the DAP-kinase promoter plays a major role in tumorigenesis. We aimed to investigate the inactivation of DAP-kinase and its association with apoptotic cell death in 94 colorectal carcinomas. DAP-kinase promoter hypermethylation and mRNA expression were investigated using methylation-specific PCR and real-time RT-PCR, respectively. The expression of DAP-kinase, Fas, and Fas-ligand (FasL) proteins was studied by immunohistochemistry and immunofluorescence. Apoptosis of tumour cells was investigated using the TUNEL assay. DAP-kinase was expressed in tumour cells and tumour-invading macrophages and was closely associated with high numbers of apoptotic tumour cells. DAP-kinase expression co-localized with FasL overexpression in tumour-associated macrophages, and aberrant promoter hypermethylation was verified in more than 50% of carcinomas. There was a tendency for proximal tumours to show DAP-kinase promoter methylation more frequently (p = 0.07). Promoter methylation resulted in a decrease or loss of DAP-kinase protein expression in tumour cells and tumour-associated macrophages. Simultaneously, a decreased apoptotic count and loss of Fas/FasL expression was observed in tumour cells. Our study is the first to demonstrate DAP-kinase expression in invading tumour-associated macrophages in colorectal cancer. The presence of similar expression levels of DAP-kinase in tumour cells and associated macrophages, and their dependence on the promoter methylation status of the tumour cells, suggests cross talk between these cell types during apoptotic cell death.

Effects of fasting and refeeding on jejunal morphology and cellular activity in rats in relation to depletion of body stores.

BACKGROUND: Intestinal mucosa atrophy following a period of starvation characterized by the mobilization of fat stores for energy expenditure (phase II) worsen after a long fast marked by an increase in protein catabolism (phase III). However, the morphology of the jejunum is completely restored after 3 days of refeeding. The aim of this study was to determine the mechanisms involved in the rapid jejunal restoration following the critical phase III. METHODS: Jejunal structure was observed through conventional and environmental scanning electron microscopy, whilst cellular dynamics were studied using classical optic microscopy tools and immunohistochemistry. RESULTS: Mucosal structural atrophy during fasting proved to worsen over the two phases. During phase II, apoptosis is still present at the tip of the villi, the number of mitosis in crypts showed a 30% decrease and a transient drop in cell migration is observed. During phase III, however, an 85% rise in mitosis was noticed along with an increase in cell migration and the disappearance of apoptotic cells at the villus tips. This increased cell renewal continues after food ingestion. CONCLUSIONS: Starved rats appeared to be in a phase of energy sparing in phase II, with depressed cellular events in the intestinal mucosa. In phase III, however, the preservation of functional cells and the early increase in crypt cell proliferation should prepare the mucosa to refeeding and could explain why jejunal repairs are complete after 3 days of refeeding following either phase II or phase III.