The Investigation of Lipoxygenases as Therapeutic Targets in Malignant Pleural Mesothelioma

Abstract

Advanced malignant pleural mesothelioma (MPM) has an extremely poor prognosis with limited chemotherapy options, therefore the identification of new therapeutic targets would aid in disease management. Arachidonic acid is metabolised by cyclooxygenase and lipoxygenase enzymes. The lipoxygenase isoenzymes 5-LOX and 12-LOX have been implicated in carcinogenesis. We aimed to examine 5-LOX and 12-LOX protein expression in a large retrospective series of mesothelioma samples. Further to this, the in vitro cytotoxic effects of lipoxygenase pathway inhibitors were investigated in mesothelioma cells. Archival samples from 83 patients with MPM were examined by immunohistochemistry for expression of the 5-LOX and 12-LOX proteins. The MTS assay was used to assess cell viability following 72 h treatment with the lipoxygenase pathway inhibitors baicalein, licofelone, MK-886 and zileuton in the MPM cell lines NCI-H2052, NCI-H2452 and MSTO-211H. Positive 12-LOX protein expression was recorded in 69/83 (83%) and positive 5-LOX expression was observed in 56/77 (73%) of MPM tissue samples. Co-expression of 5-LOX with 12-LOX was seen in 46/78 (58%) of MPM samples. Positive expression of 5-LOX, 12-LOX and COX-2 proteins was identified in the NCI-H2052, NCI-H2452 and MSTO-211H MPM cell lines. Baicalein (12-LOX and 15-LOX inhibitor) was effective in 3/3 MPM cell lines at low concentrations with an IC50 range of 9.6 μM to 20.7 μM. We have demonstrated that the 5-LOX and 12-LOX proteins are expressed in a significant proportion of MPM samples (73% and 83% respectively) and may represent novel therapeutic targets in this disease. We have demonstrated that the inhibition of the LOX pathway using baicalein may be effective as a novel treatment for MPM, however further human pharmacokinetic studies are required in order to establish whether the concentration used in vitro is clinically achievable.

Keywords: Arachidonic acid; Cyclooxygenase; Immunohistochemistry; Lipoxygenase; Mesothelioma.

Fig. 1

Expression of 12-LOX protein demonstrated by IHC using primary antibody ab23678 (Abcam). a Negative control (antibody omitted) (× 100). b Positive control (colorectal cancer with adjacent normal mucosa, × 100). c, d 12-LOX expression in non-reactive benign pleural tissue (× 100 and × 400 respectively). e Epithelial subtype of MPM demonstrating strong positive expression (× 400). f Biphasic subtype of MPM demonstrating negative expression (× 400)

Fig. 2

Expression of 5-LOX protein demonstrated by IHC using primary antibody ab169755 (Abcam). a Negative control (antibody omitted) (× 100). b Positive control (colorectal cancer with adjacent normal mucosa, × 100). c Benign pleural tissue with non-reactive mesothelial cells, which can be seen as an organised strip of mesothelial cells on the surface (red arrows), exhibiting no expression of 5-LOX protein. Inflammatory cells (black arrow) can be seen in the connective tissue (× 400). d Benign pleural tissue with reactive mesothelial cells and inflammatory cells demonstrating positive expression for 5-LOX (× 400). e Epithelial subtype of MPM demonstrating positive expression (× 400). f Epithelial subtype of MPM demonstrating negative expression (× 400)

Fig. 3

Effect of COX-2, LOX and FLAP inhibitors on the viability of MPM cells. a Immunoblotting analysis of the NSCLC cell line A549 and the MPM cell lines NCI-H2452, NCI-H2052 and MSTO-211H. b-f Cell proliferation (MTS) assays to investigate the single-agent effect in MPM and A549 cells of a COX-2 inhibitor (celecoxib), a 12-LOX/15-LOX inhibitor (baicalein), a FLAP inhibitor (MK-886), a 5-LOX inhibitor (zileuton) and a dual COX/LOX-5 inhibitor (licofelone) respectively. Following treatment for 72 h, cell viability was determined using the MTS reagent and expressed as a ratio of cell viability in comparison to the relevant control (cells treated with <0.1% dimethyl sulfoxide). Each data point is the mean of 18 replicates and error bars represent the standard error of the mean. The IC50 values generated are shown in Table 2

Fig. 4

Effect of combining celecoxib with baicalein on the viability of MPM cells. Cell proliferation (MTS) assays were performed to investigate the combined effects of 3 μM celecoxib (COX-2 inhibitor) with 10 μM baicalein (12-LOX and 15-LOX inhibitor) in the NSCLC cell line A549 and the MPM cell lines NCI-H2452, NCI-H2052 and MSTO-211H. Following treatment for 72 h, cell viability was determined using the MTS reagent. The data represents the mean and standard deviation of six replicates from at least three independent experiments and the statistical significance of the combination versus celecoxib-alone is shown (ns – not significant; * p = 0.01 to 0.05; *** p = 0.0001 to 0.001). The Chou-Talalay combination index (3.38 in A549; 1.72 in NCI-H2452; 1.36 in NCI-H2052; 0.73 in MSTO-211H) indicates a synergistic effect of celecoxib and baicalein in the MSTO-211H cells at the selected doses