Thorsten J. Maier

Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

Inhibition of prostaglandin E2 synthesis by SC-560 is independent of cyclooxygenase 1 inhibition

Prostaglandin E2 (PGE2) produced by cyclooxygenase‐2 (COX‐2) and microsomal prostaglandin E2 synthase‐1 (mPGES‐1) plays an important role in the pathophysiology of inflammation, pain, and fever. We investigated the actions of TNFα toward stimulation of PGE2 synthesis in primary spinal cord neurons. TNFα induced COX‐2 and mPGES‐1 expression in neurons, followed by formation of PGE2, which was blocked by a selective COX‐2 inhibitor. Surprisingly, the “selective COX‐1” inhibitor SC‐560 completely inhibited TNFα‐induced PGE2 synthesis in neurons at nanomolar concentrations. Moreover, SC‐560 inhibited PGE2 and thromboxane A2 synthesis in human mono‐cytes and platelets with IC50 of 1.8 nM and 2.5 nM, respectively. SC‐560 treatment neither altered TNFα‐induced COX‐2 or mPGES‐1 expression nor did the addition of the calcium ionophore A23187 or arachi‐donic acid reverse the inhibition by SC‐560. Moreover, no influence of SC‐560 on PGE2 synthase activities or PGE2 transport was seen. Most importantly, SC‐560 blocked TNFα‐induced PGE2 synthesis in COX‐1‐defi‐cient spinal cord neurons, demonstrating a COX‐1‐independent inhibition of PGE2 synthesis. Although SC‐560 inhibited LPS‐induced PGE2 synthesis in neurons and RAW264.7 macrophages in whole cell assays, no inhibition was observed in lysates of the same cells. Taken together our data demonstrate that SC‐560 acts at least in some cell types as an unselective COX inhibitor despite its selectivity toward COX‐1 under cell‐free conditions.—Brenneis, C., Maier, T. J., Schmidt, R., Hofacker, A., Zulauf, L., Jakobsson, P‐J., Scholich, K., Geisslinger, G. Inhibition of prostaglan‐din E2 synthesis by SC‐560 is independent of cyclo‐oxygenase 1 inhibition. FASEB J. 20, 1352–1360 (2006)

Inhibition of microsomal prostaglandin E2 synthase-1 as a molecular basis for the anti-inflammatory actions of boswellic acids from frankincense

BACKGROUND AND PURPOSE Frankincense, the gum resin derived from Boswellia species, showed anti‐inflammatory efficacy in animal models and in pilot clinical studies. Boswellic acids (BAs) are assumed to be responsible for these effects but their anti‐inflammatory efficacy in vivo and their molecular modes of action are incompletely understood.

Celecoxib inhibits 5-lipoxygenase

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor used in the therapy of inflammatory and painful conditions. Various COX-2-independent pharmacological effects, such as a chemo-preventive and tumor-regressive activity have been suggested, but the respective non-COX-2 targets of celecoxib are still a matter of research. We now demonstrate that celecoxib inhibits 5-lipoxygenase (5-LO), a key enzyme in leukotriene (LT) biosynthesis. Celecoxib suppressed 5-LO product formation in ionophore A23187-activated human polymorphonuclear leukocytes (IC(50) approximately 8 microM). Similarly, celecoxib inhibited LTB(4) formation in human whole blood (IC(50) approximately 27.3 microM). Direct interference of 5-LO with celecoxib was visualized by inhibition of enzyme catalysis both in cell homogenates and with purified 5-LO (IC(50) approximately 23.4 and 24.9 microM, respectively). Related lipoxygenases (12-LO and 15-LO) were not affected by celecoxib. Other COX-2 inhibitors (etoricoxib and rofecoxib) or unselective NSAIDs (non-steroidal anti-inflammatory drugs, diclofenac) failed to inhibit 5-LO. In rats which received celecoxib (i.p.), the blood LTB(4) levels were dose-dependently reduced with an ED(50) value approximately 35.2 mg/kg. Together, celecoxib is a direct inhibitor of 5-LO in vitro and in vivo. These findings provide a potential molecular basis for some of the described COX-2-independent pharmacological effects of celecoxib.

The anti-proliferative potency of celecoxib is not a class effect of coxibs.

Celecoxib, a COX-2 (cyclooxygenase-2)-selective inhibitor (coxib), is the only NSAID (non-steroidal anti-inflammatory drug) that has been approved for adjuvant treatment of patients with familial adenomatous polyposis. To investigate if the anti-proliferative effect of celecoxib extends to other coxibs, we compared the anti-proliferative potency of all coxibs currently available (celecoxib, rofecoxib, etoricoxib, valdecoxib, lumiracoxib). Additionally, we used methylcelecoxib (DMC), a close structural analogue of celecoxib lacking COX-2-inhibitory activity. Due to the fact that COX-2 inhibition is the main characteristic of these substances (with exception of methylcelecoxib), we conducted all experiments in COX-2-overexpressing (HCA-7) and COX-2-negative (HCT-116) human colon cancer cells, in order to elucidate whether the observed effects after coxib treatment depend on COX-2 inhibition. Cell survival was assessed using the WST proliferation assay. Apoptosis and cell cycle arrest were determined using flow cytometric and Western blot analysis. The in vitro results were confirmed in vivo using the nude mouse model. Among all coxibs tested, only celecoxib and methylcelecoxib decreased cell survival by induction of cell cycle arrest and apoptosis and reduced the growth of tumor xenografts in nude mice. None of the other coxibs (rofecoxib, etoricoxib, valdecoxib, lumiracoxib) produced anti-proliferative effects, indicating the lack of a class effect and of a role for COX-2. Our data emphasize again the outstanding anti-proliferative activity of celecoxib and its close structural analogue methylcelecoxib in colon carcinoma models in vitro and in vivo.

Pharmacological inhibition of platelet-tumor cell cross-talk prevents platelet-induced overexpression of cyclooxygenase-2 in HT29 human colon carcinoma cells.

Cyclooxygenase (COX)-2–derived prostanoids can influence several processes that are linked to carcinogenesis. We aimed to address the hypothesis that platelets contribute to aberrant COX-2 expression in HT29 colon carcinoma cells and to reveal the role of platelet-induced COX-2 on the expression of proteins involved in malignancy and marker genes of epithelial-mesenchymal transition (EMT). Human platelets cocultured with HT29 cells rapidly adhered to cancer cells and induced COX-2 mRNA expression, but not protein synthesis, which required the late release of platelet-derived growth factor and COX-2 mRNA stabilization. Platelet-induced COX-2-dependent prostaglandin E2 (PGE2) synthesis in HT29 cells was involved in the downregulation of p21WAF1/CIP1 and the upregulation of cyclinB1 since these effects were prevented by rofecoxib (a selective COX-2 inhibitor) and rescued by exogenous PGE2. Galectin-3, which is highly expressed in HT29 cells, is unique among galectins because it contains a collagen-like domain. Thus, we studied the role of galectin-3 and platelet collagen receptors in platelet-induced COX-2 overexpression. Inhibitors of galectin-3 function (β-lactose, a dominant-negative form of galectin-3, Gal-3C, and anti-galectin-3 antibody M3/38) or collagen receptor-mediated platelet adhesion (revacept, a dimeric platelet collagen receptor GPVI-Fc) prevented aberrant COX-2 expression. Inhibition of platelet-cancer cell interaction by revacept was more effective than rofecoxib in preventing platelet-induced mRNA changes of EMT markers, suggesting that direct cell-cell contact and aberrant COX-2 expression synergistically induced gene expression modifications associated with EMT. In conclusion, our findings provide the rationale for testing blockers of collagen binding sites, such as revacept, and galectin-3 inhibitors in the prevention of colon cancer metastasis in animal models, followed by studies in patients.

Activation of c-Jun-N-terminal-kinase is crucial for the induction of a cell cycle arrest in human colon carcinoma cells caused by flurbiprofen enantiomers

The unselective cyclooxygenase (COX) inhibitor S‐flurbiprofen and its—in terms of COX‐inhibition—“inactive” enantiomer R‐flurbiprofen have been previously found to inhibit tumor development and growth in various animal models. The underlying mechanisms are unknown. Here, we show that both R‐ and S‐flurbiprofen reduce survival of three colon cancer cell lines, which differ in the expression of COX‐2 (HCT‐15, no COX‐2; Caco‐2, inducible COX‐2; and HT‐29, constitutive COX‐2). The IC50 for S‐ and R‐flurbiprofen ranged from 250 to 450 µM. Both flurbiprofen enantiomers induced apoptosis in all three cell lines as indicated by DNA‐ and PARP‐cleavage. In addition, R‐ and S‐flurbiprofen caused a G1‐cell cycle block. The latter was associated with an activation of c‐Jun N‐terminal kinase (JNK), an increase of the DNA binding activity of the transcription factor AP‐1 and down‐regulation of cyclin D1 expression. Western blot analysis, as well as supershift experiments, revealed that the AP‐1 activation was associated with a change of AP‐1 composition toward an increase of JunB. The JNK inhibitor SP600125 antagonized R‐ and S‐flurbiprofen‐induced AP‐1 DNA binding, suppression of cyclin D1 expression, and the G1‐cell cycle block. However, JNK inhibition had no effect on flurbiprofen‐induced apoptosis. Hence, the cell cycle arrest is obviously mediated, at least in part, through JNK‐activation, whereas R‐ and S‐flurbiprofen‐induced apoptosis is largely independent of JNK. Although in vitro effects of R‐and S‐flurbiprofen were indistinguishable, only R‐flurbiprofen inhibited HCT‐15 tumor growth in nude mice, suggesting the involvement of additional in vivo targets, which are differently affected by R‐ and S‐flurbiprofen.

5-Lipoxygenase inhibitors induce potent anti-proliferative and cytotoxic effects in human tumour cells independently of suppression of 5-lipoxygenase activity

BACKGROUND AND PURPOSE Certain 5‐lipoxygenase (5‐LO) inhibitors exhibit anti‐carcinogenic activities against 5‐LO overexpressing tumour types and cultured tumour cells. It has been proposed therefore that 5‐LO products significantly contribute to tumour cell proliferation. To date, the relationship between the inhibitory mechanisms of 5‐LO inhibitors, which vary widely, and tumour cell viability has not been evaluated. This study addresses the anti‐proliferative and cytotoxic potency of a number of 5‐LO inhibitors with different inhibitory mechanisms in 5‐LO‐positive and 5‐LO‐negative tumour cells.

5-Lipoxygenase: Underappreciated Role of a Pro-Inflammatory Enzyme in Tumorigenesis

Leukotrienes constitute a group of bioactive lipids generated by the 5-lipoxygenase (5-LO) pathway. An increasing body of evidence supports an acute role for 5-LO products already during the earliest stages of pancreatic, prostate, and colorectal carcinogenesis. Several pieces of experimental data form the basis for this hypothesis and suggest a correlation between 5-LO expression and tumor cell viability. First, several independent studies documented an overexpression of 5-LO in primary tumor cells as well as in established cancer cell lines. Second, addition of 5-LO products to cultured tumor cells also led to increased cell proliferation and activation of anti-apoptotic signaling pathways. 5-LO antisense technology approaches demonstrated impaired tumor cell growth due to reduction of 5-LO expression. Lastly, pharmacological inhibition of 5-LO potently suppressed tumor cell growth by inducing cell cycle arrest and triggering cell death via the intrinsic apoptotic pathway. However, the documented strong cytotoxic off-target effects of 5-LO inhibitors, in combination with the relatively high concentrations of 5-LO products needed to achieve mitogenic effects in cell culture assays, raise concern over the assignment of the cause, and question the relationship between 5-LO products and tumorigenesis.

Effects of celecoxib on prostanoid biosynthesis and circulating angiogenesis proteins in familial adenomatous polyposis

Vascular cyclooxygenase (COX)-2-dependent prostacyclin (PGI2) may affect angiogenesis by preventing endothelial activation and platelet release of angiogenic factors present in platelet α-granules. Thus, a profound inhibition of COX-2-dependent PGI2 might be associated with changes in circulating markers of angiogenesis. We aimed to address this issue by performing a clinical study with celecoxib in familial adenomatous polyposis (FAP). In nine patients with FAP and healthy controls, pair-matched for gender and age, we compared systemic biosynthesis of PGI2, thromboxane (TX) A2, and prostaglandin (PG) E2, assessing their urinary enzymatic metabolites, 2,3-dinor-6-keto PGF1α (PGI-M), 11-dehydro-TXB2 (TX-M), and 11-α-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), respectively. The impact of celecoxib (400 mg b.i.d. for 7 days) on prostanoid biosynthesis and 14 circulating biomarkers of angiogenesis was evaluated in FAP. Intestinal tumorigenesis was associated with enhanced urinary TX-M levels, but unaffected by celecoxib, suggesting the involvement of a COX-1-dependent pathway, presumably from platelets. This was supported by the finding that in cocultures of a human colon adenocarcinoma cell line (HT-29) and platelets enhanced TXA2 generation was almost completely inhibited by pretreatment of platelets with aspirin, a preferential inhibitor of COX-1. In FAP, celecoxib profoundly suppressed PGE2 and PGI2 biosynthesis that was associated with a significant increase in circulating levels of most proangiogenesis proteins but also the antiangiogenic tissue inhibitor of metalloproteinase 2. Urinary PGI-M, but not PGE-M, was negatively correlated with circulating levels of fibroblast growth factor 2 and angiogenin. In conclusion, inhibition of tumor COX-2-dependent PGE2 by celecoxib may reduce tumor progression. However, the coincident depression of vascular PGI2, in a context of enhanced TXA2 biosynthesis, may modulate the attendant angiogenesis, contributing to variability in the chemopreventive efficacy of COX-2 inhibitors such as celecoxib.