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. 2012 Sep;33(9):1639-46.
doi: 10.1093/carcin/bgs195. Epub 2012 Jun 7.

Epithelial-mesenchymal transition increases tumor sensitivity to COX-2 inhibition by apricoxib

Amanda Kirane  1 , Jason E Toombs, Jill E Larsen, Katherine T Ostapoff, Kathryn R Meshaw, Sara Zaknoen, Rolf A Brekken, Francis J Burrows
Affiliations

Epithelial-mesenchymal transition increases tumor sensitivity to COX-2 inhibition by apricoxib

Amanda Kirane et al. Carcinogenesis. 2012 Sep.

Abstract

Although cyclooxygenase-2 (COX-2) inhibitors, such as the late stage development drug apricoxib, exhibit antitumor activity, their mechanisms of action have not been fully defined. In this study, we characterized the mechanisms of action of apricoxib in HT29 colorectal carcinoma. Apricoxib was weakly cytotoxic toward naive HT29 cells in vitro but inhibited tumor growth markedly in vivo. Pharmacokinetic analyses revealed that in vivo drug levels peaked at 2-4 µM and remained sufficient to completely inhibit prostaglandin E(2) production, but failed to reach concentrations cytotoxic for HT29 cells in monolayer culture. Despite this, apricoxib significantly inhibited tumor cell proliferation and induced apoptosis without affecting blood vessel density, although it did promote vascular normalization. Strikingly, apricoxib treatment induced a dose-dependent reversal of epithelial-mesenchymal transition (EMT), as shown by robust upregulation of E-cadherin and the virtual disappearance of vimentin and ZEB1 protein expression. In vitro, either anchorage-independent growth conditions or forced EMT sensitized HT29 and non-small cell lung cancer cells to apricoxib by 50-fold, suggesting that the occurrence of EMT may actually increase the dependence of colon and lung carcinoma cells on COX-2. Taken together, these data suggest that acquisition of mesenchymal characteristics sensitizes carcinoma cells to apricoxib resulting in significant single-agent antitumor activity.

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Figures

Fig. 1.
Fig. 1.
Pharmacokinetics, target inhibition and antitumor activity of apricoxib. (A) Concentrations of apricoxib in the plasma (solid squares, dashed line) or tumor tissue (open symbols, solid lines) of HT29 tumor-bearing nude mice after a single oral dose of 10 (squares) or 30 (triangles) mg/kg were determined using high-performance liquid chromatography mass spectrometry analysis. Also shown for comparison is the concentration of apricoxib in the plasma of human patients after a single 400mg oral dose of apricoxib (solid circles, dotted line). Data are the mean of readings from three animals; errors were 6–32% of the mean and are not shown. (B) Pharmacodynamic response in tumor tissue. HT29 tumor-bearing mice were treated with vehicle or apricoxib (oral, daily 21×) and tumors were stained with an anti-COX-2 antibody. Data are displayed as mean ± SEM and represents 5 images per tumor with 5 tumors per group evaluated. *P ≤ 0.02 versus control by one-way ANOVA. Inset: Time course of inhibition of PGE2 production by HT29 cells in vitro after exposure to apricoxib (0.5 µM). HT29 tumor-bearing nude mice were randomized into 4 groups of 10 animals each. Mice were treated for up to 70 days with vehicle (1% CMC), apricoxib (daily oral) or cetuximab (biweekly I.P.) as indicated. Effects on tumor growth rate (C) and animal survival (D) are shown.
Fig. 2.
Fig. 2.
Apricoxib inhibits tumor cell proliferation in vitro and in vivo. Inhibition of proliferation in monolayer culture (black bars in A, left panel in C) or under anchorage-independent conditions (gray bars in A, right panel in C) of HT29 cells (A) or NSCLC lines (C) was determined using the MTS assay or clonogenic assay, respectively. Cells were seeded in log-growth phase, treated with serial dilutions of apricoxib in quadruplicate and cell or colony number determined 4 (MTS) or 14 (clonogenic assay) days later. (B) Apricoxib-induced changes in PCNA expression were determined by western blot in cultures of HT29 cells grown under standard conditions (top panel) or following growth on a collagen matrix in the presence of 20ng/ml TGF-β (bottom panel). (D) HT29 tumor-bearing mice were treated with vehicle or apricoxib (oral, daily 21×) and tumors were stained with an antiphospho-histone H3 antibody. Data are displayed as mean ± SEM, total magnification ×200 (B); *P ≤ 0.0001 versus control by t-test. Representative images are also shown (E).
Fig. 5.
Fig. 5.
Apricoxib reverses EMT. The effects of apricoxib on expression of epithelial (E-cadherin) and mesenchymal (ZEB1) markers in vitro was assessed by western blot (A) or immunocytochemistry (B) in cultures of HT29 cells grown under standard conditions or following growth on a collagen matrix in the presence of 20ng/ml TGF-β. (C–F) HT29 tumor-bearing mice were treated with vehicle or apricoxib (oral, daily 21×) and tumors were stained with an antibodies against E-cadherin (C,F), vimentin (D,F) or ZEB-1 (E). Data are displayed as mean ± SEM and were analyzed as described in the legend to Fig. 1 (B); *P ≤ 0.0003, **P ≤ 0.02, ***P ≤ 0.05 versus control by one-way ANOVA. Representative double-stained images are also shown (F).
Fig. 3.
Fig. 3.
Apricoxib induces tumor cell apoptosis in vitro and in vivo. Apricoxib-induced apoptosis in vitro was assessed by western blot for cleaved PARP (A) or immunocytochemistry with antibodies against cleaved caspase-3 (B) in cultures of HT29 cells grown under standard conditions or following growth on a collagen matrix in the presence of 20ng/ml TGF-β. (C–E) HT29 tumor-bearing mice were treated with vehicle or apricoxib (oral, daily 21×) and tumors were stained by TUNEL (C) or with an antibody against cleaved caspase-3 (D). Data are displayed as mean ± SEM, total magnification ×200 (B); *P ≤ 0.0005, **P ≤ 0.0041, ***P ≤ 0.009, versus control by t-test. Representative images are shown (E).
Fig. 4.
Fig. 4.
Apricoxib modulates VEGF levels in vitro and in vivo. (A) The effect of 1 µM apricoxib on human VEGF production in vitro was assessed by ELISA. (B) HT29 tumor-bearing mice received a single oral dose of 10mg/kg apricoxib and plasma was collected at various times thereafter and assayed for murine VEGF by ELISA (R&D Systems). (C–E) HT29 tumor-bearing mice were treated with vehicle or apricoxib (oral, daily 21×) and tumors were excised, lysed and assayed for human VEGF by ELISA (C) or were fixed, embedded and stained with antibodies to CD31 (green) and NG2 (red), quantification of microvessel density is displayed as mean ± SEM (D) and were analyzed as described in the legend to Fig. 1(B). Representative images of CD31 and NG2 in tumors from mice treated with control or apricoxib (10 and 30mg/kg) are shown (E), total magnification ×400.
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References

    1. Hanahan D, et al. (2011). Hallmarks of cancer: the next generation Cell 144 646–674 - PubMed
    1. Sobolewski C, et al. (2010). The role of cyclooxygenase-2 in cell proliferation and cell death in human malignancies Int. J. Cell Biol .,Article ID 215158 - PMC - PubMed
    1. Wang D, et al. (2010). Eicosanoids and cancer Nat. Rev. Cancer 3 181–193 - PMC - PubMed
    1. De Groot D.J, et al. (2007). Non-steroidal anti-inflammatory drugs to potentiate chemotherapy effects: from lab to clinic Crit. Rev. Oncol. Hematol., 61 52–69 - PubMed
    1. Park S.W, et al. (2011). The effects of the stromal cell-derived cyclooxygenase-2 metabolite prostaglandin E2 on the proliferation of colon cancer cells J. Pharm. Exper. Therap ., 336 516–523 - PubMed

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