Increased Levels of NF-kB-Dependent Markers in Cancer-Associated Deep Venous Thrombosis

Abstract

Several studies highlight the role of inflammatory markers in thrombosis as well as in cancer. However, their combined role in cancer-associated deep vein thrombosis (DVT) and the molecular mechanisms, involved in its pathophysiology, needs further investigations. In the present study, C-reactive protein, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1β), matrix metalloproteases-9 (MMP-9), vascular endothelial growth factor (VEGF), tissue factor (TF), fibrinogen and soluble P-selectin, were analyzed in plasma and in monocyte samples from 385 cancer patients, of whom 64 were concomitantly affected by DVT (+). All these markers were higher in cancer patients DVT+ than in those DVT-. Accordingly, significantly higher NF-kB activity was observed in cancer patients DVT+ than DVT-. Significant correlation between data obtained in plasma and monocyte samples was observed. NF-kB inhibition was associated with decreased levels of all molecules in both cancer DVT+ and DVT-. To further demonstrate the involvement of NF-kB activation by the above mentioned molecules, we treated monocyte derived from healthy donors with a pool of sera from cancer patients with and without DVT. These set of experiments further suggest the significant role played by some molecules, regulated by NF-kB, and detected in cancer patients with DVT. Our data support the notion that NF-kB may be considered as a therapeutic target for cancer patients, especially those complicated by DVT. Treatment with NF-kB inhibitors may represent a possible strategy to prevent or reduce the risk of DVT in cancer patients.

Fig 1. Cytokines secretion in monocytes from cancer patients with and without DVT.

Levels of IL-6, TNF-α, IL-1β, and VEGF were measured in supernatants of purified monocytes from cancer patients with and without DVT by a sensitive enzyme-linked immunosorbent assay (ELISA). The results are shown as the means ± SD.

Fig 2. Nuclear factor (NF)–kB p65 subunit activity in monocytes from cancer patients with and without DVT and effect of DHMEQ.

The activated NF-kB p65 subunit was significantly higher in cancer patients DVT+ and DVT- than in healthy controls (P< 0.0001). (ANOVA test). NF-kB p65 subunit was significantly higher in cancer patients DVT+ than in those DVT- (P< 0.001) (t-test). To examine the effects of DHMEQ, monocytes were treated with 10 μg/mL DHMEQ. As control experiments, DMSO was used instead of DHMEQ. Monocytes from cancer patients DVT+ were more responsive to DHMEQ than those from DVT-. Intriguingly, no effect of DHMEQ there was in healthy monocytes. The results are shown as the means ± SD. OD, optical density; DVT, Deep Vein thrombosis; DMSO, dimethyl sulfoxide; DHEMQ, dehydroxymethylepoxyquinomicin.

Fig 3. Effect of dehydroxymethylepoxyquinomicin (DHMEQ) on lipopolysaccharide (LPS)-induced nuclear factor (NF)–kB p65 activation in monocytes from cancer patients with and without DVT.

Nuclear extracts were prepared from monocytes, pretreated and not with DHMEQ (10 μg/mL) for 2 h and after which stimulated with 100 ng/ml LPS for 24 hour, using a Nuclear Extract Kit. The stimulation of monocytes with LPS at 100 ng/mL induce un significant increase of the nuclear NF-kB p65 protein level in all groups or cancer patients DVT+, DVT- and in healthy controls, compared to unstimulated cells, (P<0.0001). NF-kB p65 subunit was significantly higher in cancer patients DVT+ than in those DVT- (P< 0.0001) (t-test). DVT, Deep Vein thrombosis.

Fig 4. Effects of dehydroxymethylepoxyquinomicin (DHMEQ) on cytokines release by monocytes from cancer patients with and without DVT.

Monocytes were treated or not with 10 μg/mL DHMEQ, after which the amounts of interleukins (IL)-6, tumor necrosis factor alpha (TNF-α), IL-1β and vascular endothelial growth factor (VEGF) secreted were measured by enzyme-linked immunosorbent assay (ELISA). Monocytes were incubated for 24 hr. The treatment with DHMEQ induces the decrease of all molecules in both groups of cancer patients with and without DVT compared to untreated cells (P<0.0001), (t-test). The results are shown as the means ± SD. DVT, Deep Vein thrombosis;

Fig 5. Serum-dependent activation of nuclear factor (NF)–NF-kB p65 subunit in healthy monocytes.

Monocytes from 25 healthy controls were evaluated for NF-kB activation after they were cultured for 20 hours in medium supplemented with either 40% serum from three groups. Monocytes (5x10⁵) from 25 healthy donors were cultured for 20 hours in medium (RPMI 1640) supplemented with either 40% serum derived from 64 cancer patients DVT+ and 257 DVT- with the highest cytokines plasma levels (> 75th percentile) or 40% serum derived from 100 healthy donors with the lowest cytokines values (< 25th percentile). The incubation of healthy monocytes with pooled sera derived from cancer patients DVT+ (sCADVT+) or DVT- (sCADVT-) induced a significant increase of NF-kB activity compared with that derived from healthy controls (HM) after treatment with sera from healthy controls (SH) (P<0.0001). An higher NF-kB p65 subunit activation was observed in monocytes stimulated by sera from cancer patients DVT+ compared to that stimulated by sera derived from DVT- (P<0.001) (t-test). No NF-kB p65 subunit activation was observed in monocytes stimulated with sera derived from healthy controls.