Combined histone deacetylase and cyclooxygenase inhibition achieves enhanced antiangiogenic effects in lung cancer cells

Abstract

Prostaglandin E2 (PGE2) is an important pro-angiogenic and pro-proliferative cytokine and the key enzymes modulating its levels, cyclooxygenase (COX)-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) play important opposing roles in carcinogenesis. Previously we found loss of 15-PGDH expression in lung cancer and its reactivation leads to strong in vivo tumor-suppressive effect via an antiangiogenic mechanism. Here, we find that HDAC inhibitors (HDACI), such as trichostatin A (TSA) and vorinostat could reactivate 15-PGDH expression but overall induce PGE2 generation and this is the result of concomitant induction of COX-1 and -2 leading to functional promotion of endothelial cell proliferation and capillary formation. Direct TSA treatment inhibits endothelial cell proliferation and capillary formation in our study in line with prior reports as HDACIs have been shown to directly inhibit angiogenesis. The elevation of PGE2 levels induced by HDACI is potently neutralized by indomethacin (INN) or Celecoxib co-treatment and accordingly, angiogenesis is more effectively inhibited when using conditioned medium of co-treatment than either alone confirming that this effect is mediated via the PGE2 axis. Accordingly, blockage of EP2/4 receptors mitigates the stimulation of angiogenesis by excessive PGE2 generation mediated by TSA. In this study, we identify a potentially adverse effect of HDACIs through induction of both 15-PGDH and COX-2 leading to elevated PGE2 levels and thereby stimulation of angiogenesis. Co-treatment of TSA and INN shows more potent anti-angiogenic effects by inducing 15-PGDH and inhibiting COX-2. Overall, our results suggest that combined HDACI and COX inhibition should be explored clinically to achieve more meaningful benefits from HDACI therapy in lung cancer.

Figure 1

Expression of 15-PGDH is induced by TSA in lung cancer and normal bronchial epithelial cells. A. Quantitative RT-PCR of 15-PGDH expression in NHBE, H211, H23, H292, H322, Calu1 and H460 cells following 24 hours of TSA treatment. B. Immunoblotting of 15-PGDH in lung cancer cells following 24 hours of TSA treatment. (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 1

Expression of 15-PGDH is induced by TSA in lung cancer and normal bronchial epithelial cells. A. Quantitative RT-PCR of 15-PGDH expression in NHBE, H211, H23, H292, H322, Calu1 and H460 cells following 24 hours of TSA treatment. B. Immunoblotting of 15-PGDH in lung cancer cells following 24 hours of TSA treatment. (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 2

TSA induces PGE2 in lung cancer cell lines. A. The induction of PGE2 is time-dependent and increases as least until 24 hours of TSA treatment in H211 and H23 cells. B. PGE2 is also induced by TSA in H292 and H460 but not in Calu3 and H322 cells. (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 2

TSA induces PGE2 in lung cancer cell lines. A. The induction of PGE2 is time-dependent and increases as least until 24 hours of TSA treatment in H211 and H23 cells. B. PGE2 is also induced by TSA in H292 and H460 but not in Calu3 and H322 cells. (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 3

Expression of COX-1/2 is induced by TSA and vorinostat in lung cancer and bronchial epithelial cells. A. Quantitative RT-PCR of COX-1/2 expression in H211, H23 and NHBE cells after 24 hours of TSA treatment. B. Immunoblotting of COX-2 in H211 and H23 shows that COX2 induction by TSA is time dependent (I) and dose dependent (II). Vorinostat treatment leads to analogous Cox-2 induction (III). COX1/2 induction by TSA is also observed in H292, H322, Calu-1 and H460 cells (IV) C. COX-2 induction by TSA is also observed under both normoxic and hypoxic culture conditions (I). Quantitative real-time PCR confirm that HIF-1α is induced in H23 cells during hypoxic culture condition (II). (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 3

Expression of COX-1/2 is induced by TSA and vorinostat in lung cancer and bronchial epithelial cells. A. Quantitative RT-PCR of COX-1/2 expression in H211, H23 and NHBE cells after 24 hours of TSA treatment. B. Immunoblotting of COX-2 in H211 and H23 shows that COX2 induction by TSA is time dependent (I) and dose dependent (II). Vorinostat treatment leads to analogous Cox-2 induction (III). COX1/2 induction by TSA is also observed in H292, H322, Calu-1 and H460 cells (IV) C. COX-2 induction by TSA is also observed under both normoxic and hypoxic culture conditions (I). Quantitative real-time PCR confirm that HIF-1α is induced in H23 cells during hypoxic culture condition (II). (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 3

Expression of COX-1/2 is induced by TSA and vorinostat in lung cancer and bronchial epithelial cells. A. Quantitative RT-PCR of COX-1/2 expression in H211, H23 and NHBE cells after 24 hours of TSA treatment. B. Immunoblotting of COX-2 in H211 and H23 shows that COX2 induction by TSA is time dependent (I) and dose dependent (II). Vorinostat treatment leads to analogous Cox-2 induction (III). COX1/2 induction by TSA is also observed in H292, H322, Calu-1 and H460 cells (IV) C. COX-2 induction by TSA is also observed under both normoxic and hypoxic culture conditions (I). Quantitative real-time PCR confirm that HIF-1α is induced in H23 cells during hypoxic culture condition (II). (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 4

Combined treatment of HDACIs for 24h along with COX inhibitors neutralizes the induction of PGE2 by TSA in H211 and H23 cells. A. Analysis of PGE2 level by ELISA after combined treatment with TSA/vorinostat and INN for 24 hours in H211 and H23 cells B. Analysis of PGE2 by ELISA after combined treatment with TSA and celecoxib for 24 hours in H211 and H23 cells. (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 4

Combined treatment of HDACIs for 24h along with COX inhibitors neutralizes the induction of PGE2 by TSA in H211 and H23 cells. A. Analysis of PGE2 level by ELISA after combined treatment with TSA/vorinostat and INN for 24 hours in H211 and H23 cells B. Analysis of PGE2 by ELISA after combined treatment with TSA and celecoxib for 24 hours in H211 and H23 cells. (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 5

The stimulation of HUVEC cell growth by TSA is PGE2 mediated. A. MTS assay of HUVEC cell growth for 72 hours after pretreatment with EP2 (AH6809) or EP4 (L161,982) antagonists for 1 hour, followed by the addition of 10% conditioned medium- i.e. collected culture medium of H23 cells treated with TSA for 24 hours. B. Western blot shows that EP2 and EP4 receptors are strongly expressed in HUVEC cells. Both AH6809 and L161,982 treatment potently block ERK activation after PGE2 stimulation. C. MTS assay of HUVEC cell growth following treatment with different conditioned media. TSAc is the collected medium of H23 cells treated with TSA for 24 hours. INNc is the collected medium of H23 cells treated with INN for 24 hours. TSA+ is the direct treatment of TSA of HUVEC cells. (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 5

The stimulation of HUVEC cell growth by TSA is PGE2 mediated. A. MTS assay of HUVEC cell growth for 72 hours after pretreatment with EP2 (AH6809) or EP4 (L161,982) antagonists for 1 hour, followed by the addition of 10% conditioned medium- i.e. collected culture medium of H23 cells treated with TSA for 24 hours. B. Western blot shows that EP2 and EP4 receptors are strongly expressed in HUVEC cells. Both AH6809 and L161,982 treatment potently block ERK activation after PGE2 stimulation. C. MTS assay of HUVEC cell growth following treatment with different conditioned media. TSAc is the collected medium of H23 cells treated with TSA for 24 hours. INNc is the collected medium of H23 cells treated with INN for 24 hours. TSA+ is the direct treatment of TSA of HUVEC cells. (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 5

The stimulation of HUVEC cell growth by TSA is PGE2 mediated. A. MTS assay of HUVEC cell growth for 72 hours after pretreatment with EP2 (AH6809) or EP4 (L161,982) antagonists for 1 hour, followed by the addition of 10% conditioned medium- i.e. collected culture medium of H23 cells treated with TSA for 24 hours. B. Western blot shows that EP2 and EP4 receptors are strongly expressed in HUVEC cells. Both AH6809 and L161,982 treatment potently block ERK activation after PGE2 stimulation. C. MTS assay of HUVEC cell growth following treatment with different conditioned media. TSAc is the collected medium of H23 cells treated with TSA for 24 hours. INNc is the collected medium of H23 cells treated with INN for 24 hours. TSA+ is the direct treatment of TSA of HUVEC cells. (* p<0.05; ** p<0.01; *** p<0.001 ).

Figure 6

Effect of PGE2 induced by TSA on tumor angiogenesis. Matrigel tube formation assays show that direct TSA treatment by itself (TSA+) could reduce capillary tube formation of HUVEC cells while conversely the conditioned medium of TSA treated H23 cells (TSAc) could promote angiogenesis. The conditioned medium of H23 cells treated with the combination of INN and TSA (TSA+INNc) shows more significant reduction of capillary tube formation of HUVEC cells compared with other groups. Quantification is achieved by measurement of the length of vessel-like extensions from the explants. (* p<0.05; ** p<0.01; *** p<0.001 ).