YC-1 Prevents Tumor-Associated Tissue Factor Expression and Procoagulant Activity in Hypoxic Conditions by Inhibiting p38/NF-κB Signaling Pathway

Abstract

Tissue factor (TF) expressed in cancer cells has been linked to tumor-associated thrombosis, a major cause of mortality in malignancy. Hypoxia is a common feature of solid tumors and can upregulate TF. In this study, the effect of YC-1, a putative inhibitor of hypoxia-inducible factor-1α (HIF-1α), on hypoxia-induced TF expression was investigated in human lung cancer A549 cells. YC-1 selectively prevented hypoxia-induced TF expression and procoagulant activity without affecting the basal TF levels. Surprisingly, knockdown or pharmacological inhibition of HIF-1α failed to mimic YC-1's effect on TF expression, suggesting other mechanisms are involved. NF-κB, a transcription factor for TF, and its upstream regulator p38, were activated by hypoxia exposure. Treatment of hypoxic A549 cells with YC-1 prevented the activation of both NF-κB and p38. Inhibition of p38 suppressed hypoxia-activated NF-κB, and inhibited TF expression and activity to similar levels as treatment with an NF-κB inhibitor. Furthermore, stimulation of p38 by anisomycin reversed the effects of YC-1. Taken together, our results suggest that YC-1 prevents hypoxia-induced TF in cancer cells by inhibiting the p38/NF-κB pathway, this is distinct from the conventional anticoagulants that systemically inhibit blood coagulation and may shed new light on approaches to treat tumor-associated thrombosis.

Keywords: MAP kinases; cancer; hypoxia; procoagulant activity; tissue factor.

Figure 1

YC-1 inhibits hypoxia-induced TF expression in human cancer cell lines. Human lung cancer A549, breast cancer MDA-MB-231, and oral cancer Ca9-22 cells were pretreated with DMSO (vehicle control) or YC-1 (10–100 μM) for 1 h, and then incubated under hypoxic (A) or normoxic (B) conditions for 24 h. The protein expression of TF was evaluated by Western blotting. (C) A549 cells were pretreated with DMSO or YC-1 and exposed to hypoxia or normoxia for 4 h. The mRNA levels of TF were determined by real-time PCR. All results are presented as mean ± SEM (n = 3). * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 2

YC-1 reduces hypoxia-induced TF procoagulant activity in A549 cells. A549 cells were pretreated with DMSO or YC-1 for 1 h, and then incubated under hypoxic or normoxic conditions for 24 h. (A) The cell surface TF activity was measured by a coupled amidolytic assay of TF-dependent factor Xa generation; (B) Cancer cell-induced plasma clotting was determined by tilt tube assay. Anti-TF antibody (20 μg/mL) was used as positive control. (C) A549 cells treated with DMSO or YC-1 (100 μM) in normoxia or hypoxia were harvested (1 × 10⁵ cells/mL) and mixed with platelet suspension (3 × 10⁸ platelets/mL). Platelet aggregation was induced by adding human plasma (0.25%). All results are presented as mean ± SEM (n = 3). * = p < 0.05, ** = p <0.01, *** = p < 0.001.

Figure 3

YC-1 inhibits hypoxia-induced TF via a HIF-1α-independent manner in A549 cells. (A,B) YC-1 inhibits HIF-1α accumulations and decreases HIF-1α nuclear translocation in response to hypoxia. A549 cancer cells were treated with DMSO or YC-1 for 1 h and incubated under normoxia or hypoxia for 24 h. Whole-cell lysates (A) and the nuclear fractions (B) were subjected to Western blotting for HIF-1α; In (B), ORC2 was used as a loading control in the nuclear fractions; (C) YC-1 inhibits hypoxia-induced upregulation of VEGF. A549 cancer cells pretreated with DMSO or YC-1 were exposed to hypoxia for 4 h. The mRNA levels were determined by real-time PCR. All results were presented as mean ± SEM (n = 3). ** = p < 0.01. Knockdown (D) or pharmacological inhibition (E) of HIF-1α fails to prevent hypoxia-induced TF expression. A549 cancer cells were transiently transfected with si-HIF-1α (50 nM) or negative control siRNA (D), or treated with the HIF-1α inhibitor CAY10585 (10 μM) for 1 h (E), followed by exposure to hypoxia for 24 h. The protein levels of TF in the cell lysates were determined by Western blotting.

Figure 4

YC-1 activates cyclic nucleotide-dependent protein kinases in A549 cells. (A) A549 cells were pretreated with YC-1 for 1 h and incubated under normoxia or hypoxia for another 1 h. Cell lysates were subjected to Western blotting for phospho-VASP. (B,C) A549 cells were pretreated with the PDE inhibitor IBMX (100 μM), the sGC inhibitor ODQ (10 μM), or the PKA inhibitor H89 (5 μM) for 30 min, then treated with YC-1 or BAY 41-2272 (BAY) for 1 h and incubated under hypoxia for another 24 h. TF expression was determined by Western blotting.

Figure 5

YC-1 inhibits hypoxia-induced NF-κB activation in A549 cells. A549 cancer cells were pretreated with YC-1 for 1 h and exposed to normoxia or hypoxia. Protein levels of NF-κB p65 in the cytosolic and nuclear fractions (A) and the phosphorylation of NF-κB p65 and IκBα in the whole-cell lysates (B) were determined by Western blotting. (C) A549 cells were pretreated with the NF-κB inhibitor RO 106-9920 for 1 h and exposed to hypoxia for 24 h. TF expression was determined by Western blotting.

Figure 6

YC-1 prevents hypoxia-induced TF through inhibition of the p38/NF-κB pathway. (A) Effects of specific inhibitors of MAPKs and Akt on hypoxia-induced TF expression. A549 cells were treated with U0126 (10 μM), SP600125 (10 μM), SB202190 (10 μM), and wortmannin (0.1 μM) for 1 h, and exposed to hypoxia for 24 h. Cell lysates were subjected to Western blotting for TF. (B,C) YC-1 inhibits hypoxia-stimulated p38 activation. Cells pretreated with YC-1 were exposed to hypoxia for 15 min (B) or 2 h (C). Cell lysates were subjected to Western blotting for p38 and MAPKAPK2. (D) The effect of YC-1 on ERK activation in hypoxic conditions. A549 cells were treated as in (B), and the cell lysates were subjected to Western blotting for ERK. (E) The p38 inhibitor prevents hypoxia-induced NF-κB activation. A549 cells treated with SB202190 (10 μM) or YC-1 (50 μM) were exposed to hypoxia for 2 h. Cell lysates were subjected to Western blotting for NF-κB. (F) The p38 and NF-κB inhibitors prevent hypoxia-induced TF activity. A549 cells treated with SB202190 (10 μM) or Ro 106-9920 (10 μM) were exposed to hypoxia for 24 h, then the TF-dependent factor Xa generation was determined. Data are presented as mean ± SEM (n = 3). * = p < 0.05, ** = p < 0.01. (G,H) The p38 activator rescues YC-1′s effect on hypoxia-induced TF expression and procoagulant activity. A549 cells were pretreated with YC-1 (50 μM) in the absence or presence of anisomycin (0.1 μM) and exposed to hypoxia for 24 h. The protein levels (G) and activity (H) of TF were determined by Western blotting and TF-dependent factor Xa generation, respectively. Data are presented as mean ± SEM (n = 3). ** = p < 0.01, *** = p < 0.001.