Homeodomain-interacting protein kinase-2 restrains cytosolic phospholipase A2-dependent prostaglandin E2 generation in human colorectal cancer cells.

PURPOSE: Homeodomain-interacting protein kinase-2 (HIPK2), a corepressor for homeodomain transcription factors, is a multifunctional kinase whose role in tumor cell survival is not completely clarified. We addressed whether HIPK2 restrains colon tumorigenesis by turning off cytosolic phospholipase A2 (cPLA2)-dependent prostaglandin E2 (PGE2) generation in the light of overwhelming evidence suggesting the contribution of this prostanoid in a variety of cancers. EXPERIMENTAL DESIGN: In the human colorectal cancer cell line, RKO, we studied the effect of RNA interference for HIPK2 (HIPK2i) on prostanoid biosynthesis, both in the absence and in the presence of the cPLA2 inhibitor arachidonyl trifluoromethyl ketone. We evaluated the role of HIPK2 in the cPLA2 gene regulation by reverse transcriptase-PCR, transcriptional activity, and chromatin immunoprecipitation analyses. The involvement of HIPK2 in tumorigenicity in vivo was studied by tumor growth of HIPK2i cells in nude mice. We compared the gene expression of HIPK2 and cPLA2 in human colorectal cancer specimens by reverse transcriptase-PCR. RESULTS: HIPK2 silencing was associated with rousing PGE2 biosynthesis that was profoundly suppressed by the cPLA2 inhibitor. HIPK2 overexpression, along with histone deacetylase-1, inhibited the cPLA2-luc promoter that is strongly acetylated in HIPK2i cells. The tumors derived from HIPK2i cells injected in nude mice showed noticeably increased growth compared with parental cells. HIPK2 mRNA levels were significantly higher in colorectal cancers of patients with familial adenomatous polyposis, which showed undetectable cPLA2 levels compared with sporadic colorectal cancer expressing cPLA2. CONCLUSIONS: Our findings reveal the novel mechanism of HIPK2 to restrain progression of human colon tumorigenesis, at least in part, by turning off cPLA2-dependent PGE2 generation.

New insights into COX-2 biology and inhibition.

It is now established that prostanoids play important roles in many cellular responses and pathophysiologic processes including modulation of the inflammatory reaction, erosion of cartilage and juxtaarticular bone, gastrointestinal cytoprotection and ulceration, angiogenesis and cancer, hemostasis and thrombosis, renal hemodynamics, and progression of kidney disease. The initial step in the formation of prostanoids, i.e., the conversion of free arachidonic acid (AA) to prostaglandin (PG)G(2) and then to PGH(2), is controlled by two PGH synthases (COX-1 and COX-2). Selective inhibitors of COX-2 (coxibs) have established efficacy in the treatment of pain and inflammation comparable to that of nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) but exhibit enhanced gastrointestinal safety. Several lines of evidence suggest a critical role of COX-2 expression in cancer and selective COX-2 inhibitors may represent novel chemopreventive tools. Moreover, it has been suggested that COX-2 inhibitors may contribute to maintain high levels of chemotherapeutics in tumor tissues by preventing the overexpression of the multidrug resistance protein MDR1/P-gp. The place of COX-2 inhibitors in neurological diseases continues to attract basic and clinical investigation. The possible involvement of COX-2 in neurodegeneration, substained by the results of epidemiological studies with nonselective NSAIDs, has not been confirmed by the results of initial clinical trials with coxibs in Alzheimer's disease. Recently, the involvement of COX-2 in endogenous cannabinoid system has been suggested. Interestingly, COX-2-mediated oxygenation of arachidonylethanolamide (anandamide, AEA) and 2-arachidonylglycerol (2-AG) provides diverse sets of novel lipids that are structurally related to prostaglandins.

Paracetamol effectively reduces prostaglandin E2 synthesis in brain macrophages by inhibiting enzymatic activity of cyclooxygenase but not phospholipase and prostaglandin E synthase.

Epidemiological studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) are neuroprotective, although the mechanisms underlying their beneficial effect remain largely unknown. Given their well-known adverse effects, which of the NSAIDs is the best for neurodegenerative disease management remains a matter of debate. Paracetamol is a widely used analgesic/antipyretic drug with low peripheral adverse effects, possibly related to its weak activity as inhibitor of peripheral cyclooxygenase (COX), the main target of NSAIDs. As microglia play an important role in CNS inflammation and pathogenesis of neurodegenerative diseases, we investigate the effect of paracetamol on rat microglial cultures. Although less potent than other NSAIDs, (indomethacin approximately NS-398 > flurbiprofen approximately piroxicam > paracetamol approximately acetylsalicylic acid), paracetamol completely inhibited the synthesis of prostaglandin E(2) (PGE(2)) in lipopolysaccharide-stimulated microglia, when used at concentrations comparable to therapeutic doses. The drug did not affect the expression of the enzymes involved in PGE(2) synthesis, i.e., COX-1, COX-2, and microsomal PGE synthase, or the release of the precursor arachidonic acid (AA). Paracetamol inhibited the conversion of exogenous AA, but not PGH(2), into PGE(2) indicating that the target of the drug is COX activity. Consistently, paracetamol inhibited with similar IC(50) the synthesis of PGF(2alpha) and thromboxane B(2), two other COX metabolites. Finally, none of the NSAIDs affected the productions of nitric oxide and tumor necrosis factor(alpha), two inflammatory mediators released by activated microglia. As paracetamol was reported to inhibit PG synthesis in peripheral macrophages with an IC(50) at least three orders of magnitude higher than in microglia, we suggest that this drug represents a good tool for treating brain inflammation without compromising peripheral PG synthesis.