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. 2010 Mar;12(3):254-63.
doi: 10.1593/neo.91782.

Absence of glutathione peroxidase 4 affects tumor angiogenesis through increased 12/15-lipoxygenase activity

Manuela Schneider  1 , Markus Wortmann, Pankaj Kumar Mandal, Warangkana Arpornchayanon, Katharina Jannasch, Frauke Alves, Sebastian Strieth, Marcus Conrad, Heike Beck
Affiliations

Absence of glutathione peroxidase 4 affects tumor angiogenesis through increased 12/15-lipoxygenase activity

Manuela Schneider et al. Neoplasia. 2010 Mar.

Abstract

The selenoenzyme glutathione peroxidase 4 (GPx4) has been described to control specific cyclooxygenases (COXs) and lipoxygenases (LOXs) that exert substantiated functions in tumor growth and angiogenesis. Therefore, we hypothesized a putative regulatory role of GPx4 during tumor progression and created transformed murine embryonic fibroblasts with inducible disruption of GPx4 VSports手机版. GPx4 inactivation caused rapid cell death in vitro, which could be prevented either by lipophilic antioxidants or by 12/15-LOX-specific inhibitors, but not by inhibitors targeting other LOX isoforms or COX. Surprisingly, transformed GPx4(+/-) cells did not die when grown in Matrigel but gave rise to tumor spheroids. Subcutaneous implantation of tumor cells into mice resulted in knockout tumors that were indistinguishable in volume and mass in comparison to wild-type tumors. However, further analysis revealed a strong vascular phenotype. We observed an increase in microvessel density as well as a reduction in the number of large diameter vessels covered by smooth muscle cells. This phenotype could be linked to increased 12/15-LOX activity that was accompanied by an up-regulation of basic fibroblast growth factor and down-regulation of vascular endothelial growth factor A protein expression. Indeed, pharmacological inhibition of 12/15-LOX successfully reversed the tumor phenotype and led to "normalized" vessel morphology. Thus, we conclude that GPx4, through controlling 12/15-LOX activity, is an important regulator of tumor angiogenesis as well as vessel maturation. .

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Figures

Figure 1
Figure 1
Inducible deletion of GPx4 in transformed cells leads to rapid cell death, which can be prevented by Trolox or 12/15-LOX inhibitors. (A) MEFs isolated from conditional GPx4 knockout mice were transfected with a TAM-inducible GPx4 knockout system [7]. MERCreMER (MER = mutated estrogen receptor) is retained in the cytosol. On addition of TAM, MERCreMER translocates to the nucleus where Cremediated deletion of the floxed GPx4 alleles occurs. (B) Immunoblot analysis with a monoclonal antibody directed against murine GPx4 peptide shows strongly reduced GPx4 protein levels after TAM administration (upper panel). TAM-inducible GPx4 MEFs were transformed with the oncogenes c-Myc and Ha-rasV12 using lentivirus-mediated gene transfer. The expression of both oncogenes was confirmed by immunoblot analysis (lower panel). (C) TAM-induced cell death of GPx4 knockout MEFs can be prevented by Trolox or (D) by the general LOX inhibitor NDGA and the 12/15-LOX specific inhibitors Baicalein and PD146176 but not by the general COX inhibitor indomethacin. Cell viability was determined 72 hours after TAM-treatment by MTT assay. Trolox, NDGA, Baicalein, or PD146176 treatment caused a statistically significant increase in cell viability compared with cell viability in nontreated GPx4-deleted cells.
Figure 2
Figure 2
GPx4 knockout MEFs survive in BD Matrigel. TAM-induced GPx4 knockout MEFs and uninduced control cells were either embedded in BD Matrigel (A and B) or plated on normal culture dishes (C and D). After 14 days in BD Matrigel, GPx4 knockout cells formed tumor spheroids (B and E) that were comparable in appearance and number to tumor spheroids formed by control cells (A and E). Similar results were obtained when tumor cells were embedded in Growth Factor-reduced Matrigel (F). In contrast, GPx4 knockout cells plated on normal dishes died within 48 hours (D).
Figure 3
Figure 3
GPx4-deficient transformed MEFs are capable to form tumors in vivo (A–D) and display elevated tumor proliferation and apoptosis (H–M). (A–F) c-Myc/Ha-rasV12-transformed GPx4-deficient MEFs were implanted subcutaneously into C57BL/6 mice. Tumors were collected 16 days after implantation. Control and GPx4 knockout tumors did not show a significant difference in the macroscopic appearance (A–D), tumor volume (E), and tumor mass (F). (G) Deletion of GPx4 in the tumors was confirmed by immunoblot analysis. (H–J) Immunohistologic analysis revealed a higher proliferation rate in GPx4 knockout tumors (I) compared with control tumors (H) using PH3 antibody as marker for proliferation. (K–M) TUNEL staining demonstrated more apoptotic cells in tumors derived from GPx4-deficient tumor cells (L) compared with control tumors (M).
Figure 4
Figure 4
In vivo imaging by fpVCT showed no significant alterations for GPx4-null tumors. SCID mice with tumors derived from either GPx4-knockout cells or control cells were scanned over time using fpVCT in combination with the blood pool agent eXia 160. Representative pictures from fpVCT data sets show that 7 days after implantation of tumor cells, tumors with comparable volumes formed in both groups (A and C), whereas vessels could not be detected (B and D); red circles indicate tumor localization. Likewise, at the end of the experiment, 14 days after tumor cell implantation, there were no differences in tumor volume and tumor vascularization between the two groups. Note that the tumors of both groups showed similar vessel recruitment (arrows). Scale bars, 10 mm.
Figure 5
Figure 5
Altered tumor vascularization in GPx4-null tumors compared with control tumors. (A–D) Immunohistologic evaluation of CD31-stained tumor sections revealed higher vessel density, in particular more microvessels in GPx4 knockout tumors. (E–H) For analysis of functional vascular density and vessel diameter, GPx4-deficient cells were implanted into the dorsal skinfold chamber preparations in mice. The microvessel density in knockout tumors is significantly increased (E–G). However, newly formed vessels had thinner diameter and markedly reduced vessel lumina in contrast to control tumors (E, F, and H).
Figure 6
Figure 6
Vessels of GPx4-null tumors show diminished pericyte coverage. To further analyze vessel morphology, double staining for CD31 and αSMA was performed on tumor sections (A and B). We observed a reduced number of pericyte covered vessels in GPx4 knockout tumors when compared with control tumors (C).
Figure 7
Figure 7
Baicalein treatment reversed the vascular alterations in GPx4 knockout tumors. The increased microvessel density (A–C) as well as the reduced number of vessels covered by smooth muscle cells in GPx4 knockout tumors (D) could be reverted by Baicalein administration in vivo. Vascular alterations were evaluated by CD31 immunohistochemistry as well as by CD31/αSMA double staining. Note the strong reduction of the overall vascular area (C) as well as the increase of the number of smooth muscle cell covered vessels (D) after Baicalein treatment.
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References

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