20-HETE-producing enzymes are up-regulated in human cancers

Abstract

Background: 20-Hydroxyeicosatetraenoic acid (20-HETE), a metabolite of arachidonic acid (AA) produced by the CYP4A and CYP4F enzyme families has been reported to induce mitogenic and angiogenic responses both in vitro and in vivo, and inhibitors of this pathway reduced growth of brain and kidney tumors.

Materials and methods: Real-Time PCR, western blot and immunohistochemistry were used to compare the expression of CYP4A/F mRNA and protein levels in human cancer tissue samples versus normal controls. Liquid chromatography/mass spectrometry analysis (LC-MS/MS) was performed to measure 20-HETE formation in tumor homogenates. Activation of Ras in human proximal tubule epithelial cells (HRPTEC) treated with stable agonist of 20-HETE was measured using a Ras pull-down detection kit.

Results: The expression of CYP4A/4F genes was markedly elevated in thyroid, breast, colon, and ovarian cancer samples in comparison to matched normal tissues. Furthermore, the levels of the CYP4F2 protein and of 20-HETE were higher in ovarian cancer samples compared to normal control tissues. A stable 20-HETE agonist induced activation of the small-GTPase Ras in HRPTEC cells.

Conclusion: The present finding of elevated expression of CYP4A/F enzymes in human cancer tissue suggests that 20-HETE inhibitors and antagonists may be useful in the treatment of cancer.

Figure 1

Expression of CYP4A and CYP4F genes at mRNA level is elevated in human cancer tissue samples. TissueScan cancer survey panels containing pre-normalized cDNA from multiple cancer tissues. (A) Thyroid; (B) breast; (C) colon; (D) ovary, were used to run real-time PCR reaction to analyze the expression of CYP4A and CYP4F genes at the mRNA level. The tissue samples used for each type of TissueScan panel, obtained from independent patients, cover various disease stages, and matched normal control tissues are included for comparison between normal and disease samples. The amount of PCR product is correlated with the amount of original RNA transcript and data the expressed in arbitrary units. *indicates tumor samples demonstrating striking up-regulation in CYP4A/F enzymes.

Figure 2

Expression of CYP4F2 gene is elevated in ovarian cancer tissue samples. TissueScan cancer survey panel containing pre-normalized cDNA from ovarian cancer tissues, was used to run real-time PCR reaction to analyze the expression of CYP4F2 gene at the mRNA level. The tissue samples used for ovary TissueScan panel were obtained from independent patients, covering various disease stages, and matched normal control tissues are included for comparison between normal and disease samples. The amount of PCR product is correlated with the amount of original RNA transcript and data are expressed in arbitrary units. A representative of two independent experiments is shown. *indicates tumor samples demonstrating striking up-regulation in CYPF2 enzyme.

Figure 3

Expression of CYP4F2 enzyme at protein the level is up-regulated in ovarian cancer tissue samples. (A) Western blot analyses of OncoPair INSTA-Blot PVDF membrane containing; 14 lanes of alternating ovary tumor (T) and matched normal adjacent (NA) tissue protein lysates from 7 patient donors using isoform specific CYP4F2 antibody. (B) Immunohistochemical staining for CYP4F2 (brown precipitate) in ovarian cancer (left panel) and matched normal tissue (right panel). Cell nuclei and cytoplasm were counterstained with hematoxylin and eosin.

Figure 4

Increased metabolism of AA by CYP4A/F enzymes in ovarian cancer tissue samples. 1 mg/ml of (A) normal ovary and (B) human ovary cancer tissue homogenates was incubated with exogenous AA (40 μM), 1 mM NADPH and 2 μM indomethacin for 1 h at 37°C in the presence of 100% O₂. AA metabolites were further extracted and subjected to LC–MS/MS, as described in Materials and Methods to detect levels of 20-HETE.

Figure 5

20-HETE signaling in HRPTEC cells through activation of the small-GTPase Ras. HRPTEC cells were serum-starved overnight before the addition of 20 μM WIT003 for the times indicated (min). GTP-bound active Ras was isolated from the stimulated lysates by utilizing an Active GTPase pull-Down and a Detection Kit as described in Materials and Methods, followed by western blot analysis with the p-21Ras antibody (lower panel). The GST fusion protein (~42 kDa, upper panel), was detected by staining the membrane with Ponceau S to demonstrate equal loading. Untreated cells and EGF-treated cells were used as negative and positive controls respectively, and a representative of three separate experiments is shown.