Selective inhibitors of CYP2J2 related to terfenadine exhibit strong activity against human cancers in vitro and in vivo

Abstract

The cytochrome P450 epoxygenase, CYP2J2, converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs). We found recently that this enzyme is dramatically up-regulated in a variety of established human carcinoma cell lines and in human cancerous tissue and promotes the neoplastic phenotype. In the present study, we tested the hypothesis that specific inhibitors of CYP2J2 related to the drug terfenadine are effective antitumor agents. Four of these inhibitors (compounds 4, 5, 11, and 26) were tested for effectiveness in vitro and in vivo. In Tca-8113 cells, the CYP2J2 inhibitors decreased EET production by approximately 60%, whereas they had no effect on CYP2J2 mRNA or protein expression. Compound 26 inhibited the proliferation of human tumor cells, reduced their ability to adhere, invade, and migrate, and attenuated activation of epithelial growth factor receptor signal and kinases and phosphatidylinositol 3 kinase/Akt pathways. Inhibition of CYP2J2 also significantly potentiated human tumor cell apoptosis and caused a corresponding increase in caspase-3 activity and change in expression of apoptosis-related proteins Bax and Bcl-2. In murine xenograft models using MDA-MB-435 cells, treatment with compound 26 significantly repressed tumor growth, decreased lung metastasis, and was associated with increased expression of the anticancer genes CD82 and nm23, without causing toxicity. These data suggest that CYP2J2 inhibitors hold significant promise for use in treatment of neoplastic diseases.

Scheme 1.

Synthesis of terfenadone derivatives. The schematic shows synthesis and chemical structures of compounds 4, 5, 11, and 26.

Fig. 1.

Effect of terfenadone derivatives on CYP2J2 expression and activity. A, CYP2J2 mRNA levels. Total RNAs were isolated from Tca-8113 cells treated with compounds 4, 5, 11, and 26 (10 μM each) for 24 h. Semiquantitative analysis of the expression of CYP2J2 mRNA was done using a multiplex RT-PCR technique as described under Materials and Methods. B, CYP2J2 protein levels. Tca-8113 cells were treated with 5 or 10 μM C26 for 24 h. Proteins from cell lysates were then subject to Western blot analysis as described under Materials and Methods. C, 14,15-DHET and 20-HETE levels in Tca-8113 cells. Levels of 14,15-DHET and 20-HETE were determined as described under Materials and Methods over a 24-h period after treatment of cells with C26 (10 μM). D, 14,15-DHET levels in BAECs, H9C2 and Tca-8113 cells. 14,15-DHET levels in cells treated with C26 (5 μM) for 24 h were determined. Results shown are mean ± S.E. (n = 5); *, p < 0.05 versus control, representative of three independent experiments.

Fig. 2.

Effect of CYP2J2 inhibitors on cell proliferation. A, effect of C26 (10 μM) on number of Tca-8113, HeLa, A549 and MDA-MB-435 tumor cells. B, effect of C26 on number of HepG2 cells. C, effect of C26 (10 μM) on number of HEK293 cells, BAECs, and TC-1 cells. D, effect of CYP2J2 inhibitors on number of Ki67 immunopositive Tca-8113 cells. Cells were treated with vehicle and one of the CYP2J2 inhibitors (10 μM each), and the numbers of Ki67-positive cells per HPF were determined as described under Materials and Methods. E, effect of 11,12-EET (200 nM) on the antiproliferative effect of C26 (10 μM) in Tca-8113 cells. Growth inhibition studies for A, B, C, and E were performed using the MTT assay, as described under Materials and Methods. Data are expressed as percentage of untreated controls, which is set at 100% ± S.E. (n = 5); *, p < 0.05 versus control; #, p < 0.05 versus C26.

Fig. 3.

C26 increases human tumor cell apoptosis. A, caspase-3 activity in Tca-8113 cells. Cells were treated with 5 and 10 μM C26 for 18 h, lysed, and analyzed spectrophotometrically for caspase activity. Data are reported as mean absorption relative to control ± S.E. (n = 3). B, identification of apoptotic cells by TUNEL staining. Tca-8113 cells were treated with compounds 4, 5, 11, and 26 for 24 h and then fixed with paraformaldehyde. DNA fragments and double-strand breaks in apoptotic cells were labeled with biotinylated nucleotides and detected using the In Situ Apoptosis Detection Kit. Apoptotic cells with their characteristic fragmented chromatin exhibit a dark-blue nuclear staining. Graph represents quantification of the percentage of cells that were TUNEL positive. Data are reported as mean ± S.E. (n = 5). C, analysis by flow cytometry of Tca-8113 cells treated with C26 (10 μM) using annexin V-FITC and propidium iodide. The lower left quadrant represents nonapoptotic cells, the lower right quadrant is representative of early apoptotic cells (annexin positive, propidium iodide negative), and the upper right quadrant represents annexin V and propidium iodide-positive late apoptotic or necrotic cells. Graph represents the mean number of annexin-positive Tca-8113 cells expressed as percentage of control untreated cells ± S.E. (n = 3); *, p < 0.05 versus control.

Fig. 4.

CYP2J2 inhibitors decrease the adhesion, migration, and invasion of Tca-8113 to fibronectin. A, number of cells adhering to fibronectin after treatment with 5 and 10 μM C26 for 30 min to 2 h. B, number of cells adhering to fibronectin 2 h after treatment with 10 μM compounds 4, 5, 11, or 26. Fibronectin adhesion assay was performed as described under Materials and Methods. C, migration of Tca-8113 cells treated with 5 and 10 μM C26 for 4 h. D, migration of MDA-MB-435 cells treated with 10 μM C26 over a 4-h time period. E, effect of 11,12-EET (200 nM) on the antimigratory effect of C26 (10 μM) in Tca-8113 cells. The Boyden chamber migration assay was performed as described under Materials and Methods. F, cells were treated with 10 μM compounds 4, 5, 11, or 26 for 4 h. Thereafter, invasiveness was evaluated by quantifying the number of cells invading the Matrigel in a Boyden chamber as shown. Data are expressed as percentage of untreated controls, which is set at 100% ± S.E. (n = 5); *, p < 0.05 versus control; #, p < 0.05 versus C26.

Fig. 5.

Effect of C26 on tumor growth and metastasis. Athymic mice were inoculated with MDA-MB-435 cells, and the tumors were allowed to grow to approximately 40 mm³. The mice were then randomized to control versus C26 treatment. Mice were dosed orally with vehicle or C26 (0.25 mg/kg/day) for 30 days. A, 14,15-DHET levels in the urine of nude mice during treatment with C26 or vehicle control (n = 6). B, 20-HETE levels in the urine of nude mice during treatment with C26 or vehicle control. 14,15-DHET and 20-HETE were detected by ELISA according to the manufacturer's instructions (n = 6). C, tumor volume as measured weekly in control and C26-treated mice. Tumor volume was monitored by digital caliper on a weekly basis and calculated as length × width² × π/6 (n = 6). D, tumor and body weight of mice 30 days after randomization into control and C26 treatment groups (n = 6). E, cumulative survival curve of control and C26-treated mice (n = 10). F, average volume of lung metastases for each group (n = 6). Results shown are mean ± S.E. (n = 6); *, p < 0.05 versus control.

Fig. 6.

Effect of C26 treatment on cell signaling pathways. MDA-MB-435 cells were treated with 200 nM EET and/or 10 μM C26 for 24 h and then analyzed for protein expression and phosphorylation. A, decreased phosphorylation of the EGFR in MDA-MB-435 cells after C26 treatment is rescued by EETs. B, decreased PI3 kinase protein expression in MDA-MB-435 cells after C26 treatment is rescued by EETs. C, decreased phosphorylation of the Akt in MDA-MB-435 cells after C26 treatment is rescued by EETs. D, effects of EET and C26 treatments on apoptosis-related proteins in MDA-MB-435 cells. E, effects of EET and C26 treatments on metastasis-related proteins in MDA-MB-435 cells.