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. 2014 Jan 1;74(1):243-52.
doi: 10.1158/0008-5472.CAN-13-2245. Epub 2013 Nov 12.

6-C-(E-phenylethenyl)-naringenin Suppresses Colorectal Cancer Growth by Inhibiting cyclooxygenase-1

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Free PMC article

6-C-(E-phenylethenyl)-naringenin Suppresses Colorectal Cancer Growth by Inhibiting cyclooxygenase-1

Haitao Li et al. Cancer Res. .
Free PMC article

Abstract

Recent clinical trials raised concerns regarding the cardiovascular toxicity of selective cyclooxygenase-2 (COX-2) inhibitors and cyclooxygenase-1 (COX-1) is now being reconsidered as a target for chemoprevention. Our aims were to determine whether selective COX-1 inhibition could delay or prevent cancer development and also clarify the underlying mechanisms. Data clearly showed that COX-1 was required for maintenance of malignant characteristics of colon cancer cells or tumor promoter-induced transformation of preneoplastic cells. We also successfully applied a ligand-docking computational method to identify a novel selective COX-1 inhibitor, 6-C-(E-phenylethenyl)-naringenin (designated herein as 6CEPN). 6CEPN could bind to COX-1 and specifically inhibited its activity both in vitro and ex vivo. In colorectal cancer cells, it potently suppressed anchorage-independent growth by inhibiting COX-1 activity. 6CEPN also effectively suppressed tumor growth in a 28-day colon cancer xenograft model without any obvious systemic toxicity. Taken together, COX-1 plays a critical role in human colorectal carcinogenesis, and this specific COX-1 inhibitor merits further investigation as a potential preventive agent against colorectal cancer.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest:

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
COX-1 is required for maintenance of malignant characteristics of colorectal cancer cells. A, Western bolt analysis of COX-1 and COX-2 expression in human colorectal cancer and normal colon epithelial cells (HCEC). B, COX-1 is required for anchorage-independent growth of colorectal cancer cells. C, COX-1 and COX-2 are equally important for tumorigenic properties in human colorectal cancer cells. Knockdown of COX-1 or COX-2 in colon cancer cells was analyzed by Western blot. Mock and knockdown cells were then subjected to anchorage-dependent growth, anchorage-independent growth and PGE2 production assays as described in “Materials and Methods”. Cell growth was evaluated by MTS assay. Data are presented as means ± S.E.M. (n = 4). Anchorage-independent cell growth was evaluated by colony formation in soft-agar. Data are presented as means ± S.E.M. from 3 independent experiments. Production of PGE2 in supernatant fractions was measured by ELISA. Data are presented as means ± S.E.M. (n = 4). The asterisks (***) indicate a significant (p < 0.001) difference compared to Mock group.
Figure 2
Figure 2
COX-1 is involved in neoplastic transformation. A, knockdown of COX-1 in JB6 CI41 cells was analyzed by Western blot. B, EGF- or TPA-induced cell transformation is dramatically reduced by COX-1 knockdown. JB6 cells were grown in soft agar in the absence or presence of EGF (10 ng/mL) or TPA (20 ng/mL) and colonies were counted as described in “Materials and Methods”. Data are presented as means ± S.E.M. from 3 independent experiments. The asterisks (***) indicate a significant (p < 0.001) difference compared to Mock group. C, knockdown of COX-1 partially blocks EGFR signal transduction in EGF-induced cell transformation. After starvation for 24 h, JB6 cells were stimulated with EGF (10 ng/mL) for 15 min. Cell lysates were subjected to Western blot analysis.
Figure 3
Figure 3
6CEPN is identified as a novel selective COX-1 inhibitor. A, chemical structure of 6CEPN. B, proposed molecular model of 6CEPN binding with COX-1. 6CEPN binds to the active site of COX-1 by forming 3 hydrogen bonds with Tyr355, Phe518 and Ser530. C, proposed molecular model of 6CEPN binding with COX-2. 6CEPN failed to occupy the active pocket of COX-2 (left panel) but might bind to COX-2 in another region (i.e., His207 and His388). D, 6CEPN binds with COX-1 and COX-2 in vitro. A pull-down assay was performed using recombinant COX-1 and COX-2 proteins. Proteins bound to the beads were analyzed by Western blotting. E, 6CEPN specifically inhibits COX-1 activity in vitro. The inhibitory activity of 6CEPN was evaluated using a COX Inhibitor Screening Kit (Cayman) according to the manufacturer’s instructions. Data are presented as means ± S.E.M. (n = 4). The asterisks (***) indicate a significant (p < 0.001) difference compared to each respective control group.
Figure 4
Figure 4
6CEPN targets COX-1 ex vivo. A, 6CEPN specifically inhibits COX-1 activity in HEK293T cells. The effector plasmids (COX-1 and COX-2) and control plasmid (pcDNA3.1) were transiently transfected into HEK293 cells using jetPEI reagent (Qbiogen) following the manufacturer’s instructions. After 24 h, transfection reagents were removed. Cells were then incubated with 6CEPN for 24 h, and supernatant fractions were collected for PGE2 measurement using an enzyme immunoassay kit (Cayman). Data are presented as means ± S.E.M. (n = 4). The asterisks indicate a significant (*, p < 0.05; ***, p < 0.001) difference compared to each respective control group. B, 6CEPN targets COX-1 in HT29 cells. COX-1 proteins in HT29 cell lysates were pulled down and analyzed by Western blotting (left panels). 6CEPN inhibits PGE2 production in HT29 cells (right panels). Production of PGE2 was measured by ELISA. Data are presented as means ± S.E.M. (n = 4). The asterisks (***) indicate a significant (p < 0.001) difference compared to control group. C, 6CEPN inhibits anchorage-independent growth of human colorectal cancer cells. HT29, HCT15 or DLD1 cells were grown in soft agar for 7 d and colonies counted as described in “Materials and Methods”. Data are presented as means ± S.E.M. from 3 independent experiments. The asterisks indicate a significant (**, p < 0.01; **, p < 0.01) difference compared to each respective control group.
Figure 5
Figure 5
Effect of 6CEPN on human umbilical vein endothelial cells. A, Western blot analysis of COX-1 and COX-2 protein expression in human umbilical vein endothelial cells. To simulate pro-inflammatory conditions observed in many vascular diseases, cells were incubated with 1 nM IL-1β for 8 h. B, effects of 6CEPN treatment on the ratio of TXB2 to 6-keto-PGF. HUVECs were seeded in a six-well-plate (6×105 cells per well). At 70–80% confluence, cells were pretreated with 1 mL fresh medium containing DMSO or compounds for 2 h and then IL-1β (1 nM) was added together with compounds for another 8 h incubation. Supernatant fractions were collected for prostaglandin measurement. Data are presented as means ± S.E.M. (n = 4). The asterisks indicate a significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001) difference compared to each respective control group.
Figure 6
Figure 6
Chemopreventive activity of 6CEPN in a HT29 colon cancer xenograft model. A, effect of 6CEPN on tumor growth. B, effect of 6CEPN on tumor mass. C, effect of continuous 6CEPN treatment on the ratio of TXB2 to 6-keto-PGF. D, effect of 6CEPN on body weight of mice. Chemopreventive activity of 6CEPN was evaluated in a 28-day colon cancer xenograft model. Before cell injection, mice were pretreated with 6CEPN for 14 days. 6CEPN dissolved in 5% (v/v) dimethyl sulfoxide (DMSO)/PEG400 was given to the mice by gavage every other day for a total of 42 days. Data are presented as means ± S.E.M. (n = 12 mice). The asterisks indicate a significant (*, p < 0.05; ***, p < 0.001) difference compared to vehicle control group.

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