Background An important role has emerged for calpain enzymes in regulating inflammation with one isoform, calpain-2, particularly important for macrophage activation. The goal of this study was to determine the therapeutic potential of a synthetic calpain-2 inhibitor, zLLY-CH2F, for colitis and inflammation associated colorectal cancer. Methods Mice were then subjected to the azoxymethane/dextran sulfate sodium (AOM/DSS) model of colitis and colitis associated cancer (CAC) incorporating intervention with daily injections of 0.75 mg/kg calpain-2 inhibitor beginning after the first signs of colitis. Results Calpain-2 inhibitor treatment alleviated weight loss and bloody diarrhea, and reduced inflammatory infiltration into colon tissues and inflammatory cytokine mRNA. Calpain-2 inhibitor intervention also reduced total CAC tumor volume by up to 70% in vehicle control mice and decreased cancer pathology scores of blinded histological colon tissue analyses. Mechanistic investigations showed that calpain-2 inhibition during macrophage activation reduced inhibitor of kappa beta (IκB) degradation and nuclear factor kappa beta (NFκB) nuclear localization as well as secretion of specific inflammatory cytokines. In addition, calpain-2 inhibitor treatment of CT26.WT mouse and HT-29 human colorectal cancer cells decreased proliferation, and reduced IκB degradation and NFκB translocation. Conclusions Overall, these findings suggest that intervention with a calpain-2 inhibitor may reduce colitis and CAC through a two-hit process of limiting macrophage activation as well as inhibiting growth of the colorectal cancer cells themselves.

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The response to hypoxia in tissues is regulated by the heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1). We have investigated the transcriptional effects of hypoxia-inducible factor 1 alpha (HIF-1α) in the heart by expressing an oxygen-stable form of HIF-1α in cardiac myocytes of transgenic mice. The result in most cases is regulation of an expected panoply of genes that restore homeostasis during hypoxia, with corresponding phenotypic changes including contractile dysfunction and increased capillary density. In mice that do not show this phenotype, the mRNA for protein kinase c binding protein 1 isoform 2 (PRKCBP1) was much more abundant, as was the protein, and chromatin immunoprecipitation shows a predominant binding of HIF to the promoter of this gene. Sequencing of the promoter region of the PRKCBP1 gene from the two phenotypes revealed an unexpected 480 bp insert in the HIF-resistant animals containing two canonical HIF binding sites. The protein co-immunoprecipitates with HIF and inhibits HIF transcriptional activity in cell culture. In FVB mice, that contain the promoter insert, PRKCBP is induced by ischemia and co-localizes with HIF in the infarct region. It may be responsible for the greater susceptibility of this strain to heart failure after infarction. We have confirmed with genetic, transcriptional, biochemical, and physiological data that Prkcbp1 inhibits HIF activity through direct interaction, in a mechanism mediated by transcriptional control.