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. 2015 May;8(5):444-54.
doi: 10.1158/1940-6207.CAPR-14-0359. Epub 2015 Feb 23.

Nrf2-dependent suppression of azoxymethane/dextran sulfate sodium-induced colon carcinogenesis by the cinnamon-derived dietary factor cinnamaldehyde

Min Long  1 , Shasha Tao  2 , Montserrat Rojo de la Vega  2 , Tao Jiang  2 , Qing Wen  3 , Sophia L Park  2 , Donna D Zhang  4 , Georg T Wondrak  4
Affiliations

Nrf2-dependent suppression of azoxymethane/dextran sulfate sodium-induced colon carcinogenesis by the cinnamon-derived dietary factor cinnamaldehyde

Min Long et al. Cancer Prev Res (Phila). 2015 May.

Abstract

The progressive nature of colorectal cancer and poor prognosis associated with the metastatic phase of the disease create an urgent need for the development of more efficacious strategies targeting colorectal carcinogenesis. Cumulative evidence suggests that the redox-sensitive transcription factor Nrf2 (nuclear factor-E2-related factor 2), a master regulator of the cellular antioxidant defence, represents a promising molecular target for colorectal cancer chemoprevention. Recently, we have identified cinnamon, the ground bark of Cinnamomum aromaticum (cassia cinnamon) and Cinnamomum verum (Ceylon cinnamon), as a rich dietary source of the Nrf2 inducer cinnamaldehyde (CA) eliciting the Nrf2-regulated antioxidant response in human epithelial colon cells, conferring cytoprotection against electrophilic and genotoxic insult. Here, we have explored the molecular mechanism underlying CA-induced Nrf2 activation in colorectal epithelial cells and have examined the chemopreventive potential of CA in a murine colorectal cancer model comparing Nrf2(+/+) with Nrf2(-/-) mice. In HCT116 cells, CA caused a Keap1-C151-dependent increase in Nrf2 protein half-life via blockage of ubiquitination with upregulation of cytoprotective Nrf2 target genes and elevation of cellular glutathione. After optimizing colorectal Nrf2 activation and target gene expression by dietary CA-supplementation regimens, we demonstrated that CA suppresses AOM/DSS-induced inflammatory colon carcinogenesis with modulation of molecular markers of colorectal carcinogenesis. Dietary suppression of colorectal cancer using CA supplementation was achieved in Nrf2(+/+) but not in Nrf2(-/-) mice confirming the Nrf2 dependence of CA-induced chemopreventive effects. Taken together, our data suggest feasibility of colorectal cancer suppression by dietary CA, an FDA-approved food additive derived from the third most consumed spice in the world.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: The authors have no conflict of interest to disclose.

Figures

Figure 1
Figure 1
Molecular mechanism underlying CA-modulation of Nrf2 activity in HCT116 cells. A, For Oxidative Stress RT2 Profiler™ PCR expression array analysis, cells were exposed to CA (20 μM, 24h) followed by gene expression analysis. upper panel: scatter blot depiction of CA-induced gene expression (versus untreated); cut-off lines: threefold up- or down-regulation; the insert shows the chemical structure of cinnamaldehyde; bottom panel: numerical expression changes [n=3, mean ± SD; (p<0.05)]. B, Upregulation of Nrf2 target gene-encoded proteins in response to CA exposure was examined in HCT116 cells treated with CA (1–20 μM; 24 h), and cell lysates were used for immunoblot analysis. Equal loading was assessed by immunodetection of β-actin. In addition, Nrf2 protein levels were examined (CA, 1–20 μM; 4h). C, Viability was monitored in HCT116 cells exposed to CA (0–20 μM; 24 h) using flow cytometric analysis as detailed in Materials and Methods. D, Intracellular total glutathione levels relative to untreated control were determined in HCT116 cells exposed to CA (1–20 μM; 24 h) using the luminescent GSH-Glo™ glutathione assay as detailed in Materials and Methods. Data are expressed as means ± SD. Means without a common letter differ (ANOVA with Tukey’s post hoc test; *p < 0.05; n = 3). E, CA-modulation of ARE-luciferase reporter gene expression was assessed employing Keap1-C151S and Keap1-WT cotransfections. In addition, a Keap1-siRNA against the 3′-untranslated region was cotransfected to suppress endogenous Keap1. The transfected cells were then treated with CA (10 μM), As(III) (10 μM), SF (5 μM), or tBHQ (50 μM) for 16 h, before the measurement of firefly and Renilla luciferase activities. Data are expressed as means ± SD (*p < 0.05 control vs. compound treatment; #p < 0.05 Keap1-WT vs. Keap1-C151S). An aliquot of cell lysates was used for immunoblot analysis. F, CA-modulation of Nrf2-ubiquitination was assessed by cotransfecting cells with plasmids encoding the indicated proteins (Nrf2, HA-Ub). Cells were then treated with SF, CA, or tBHQ (5 μM; 4 h) along with MG132 (10 μM; 4 h) before cell lysates were collected for the ubiquitination assay. For detection of ubiquitin-conjugated Nrf2, anti-Nrf2 immunoprecipitates were analyzed by immunoblotting using an anti-HA antibody. G, CA-modulation of Nrf2 protein half-life. After cells were left untreated or treated with CA (10 μM; 4 h), cycloheximide (50 μM) was added and cells were harvested at the indicated time points (0–60 min). Cell lysates were subjected to immunoblot analysis using anti-Nrf2 and anti-tubulin antibodies. Band intensity was quantified using Quantity One software and plotted against the time after cycloheximide treatment.
Figure 2
Figure 2
Dietary CA-supplementation upregulates murine colon epithelial Nrf2 levels. A, Eight-week-old mice (Nrf2+/+) were fed with standard diet or received supplemented diet (CA 0.1%; CA 0.5%; three mice, each). After five days of dietary supplementation, animals were sacrificed on day 6, colon tissue was harvested, and Nrf2 and Nrf2 target gene expression at the protein level was examined by immunoblot analysis. The intensity of the bands was quantified using Quantity One software (mean ± SD; * p< 0.05). B, Colon tissue was subjected to H&E and IHC (Nrf2, NQO1) analysis confirming supplementation-induced Nrf2 and NQO1 upregulation. Per treatment group, one representative specimen is depicted (400 x magnification). As a negative control, staining was performed with omission of primary antibody.
Figure 3
Figure 3
Dietary CA supplementation decreases AOM/DSS-induced colitis in Nrf2+/+ but not Nrf2−/− mice. A, Eight-week-old Nrf2+/+ and Nrf2−/− mice were fed with standard diet or received diet supplemented with 0.5% CA. AOM injection was performed at the beginning of week 9, followed by three cycles of DSS (2.5 %) administration in drinking water. CA-supplementation was initiated one week before AOM injection and then maintained after AOM injection for one (CA2) or ten (CA11) weeks. Eleven weeks after the initiation of the experiment, mice were sacrificed for analysis. B, CA supplementation significantly decreased loss of crypts and maintained mucosal crypt organization in Nrf2+/+ mice only, as demonstrated by H&E staining (100 x magnification). C, Histopathological assessment of colon mucosa in Nrf2+/+ and Nrf2−/− mice was performed following the chronic colitis scoring system described in Materials and Methods. Horizontal lines indicate median values (*p<0.05; Mann-Whitney test); N.S.: not significant. D, AOM/DSS-induced weight loss was significantly attenuated by CA11 supplementation in Nrf2+/+ mice. The graph indicates average body weight (mean + SD) per treatment group at the beginning and at the end of the experimental regimen (eleven weeks total duration; *p < 0.05 versus control; #p < 0.05 versus AOM/DSS;). E–F, AOM/DSS-induced body weight loss in Nrf2+/+ versus Nrf2−/− mice was analyzed as a function of CA supplementation. At the end of the treatment regimen, the relative weight loss was compared between treatment groups; the difference between AOM/DSS versus untreated control was numerically defined as 1 (*p < 0.05).
Figure 4
Figure 4
Dietary CA-supplementation suppresses AOM/DSS-induced colon carcinogenesis in Nrf2+/+ but not Nrf2−/− mice. At the end of the treatment regimen, total tumor number (distal, middle, proximal combined) per mouse was determined as a function of treatment regimen. Horizontal lines indicate median values for every treatment group. CA-supplementation regimens (CA2 and CA11) caused a significant suppression of tumor multiplicity in Nrf2+/+ but not in Nrf2−/− mice (*p < 0.05; Mann-Whitney test; N.S.: not significant).
Figure 5
Figure 5
Dietary CA-supplementation modulates tissue markers of AOM/DSS-induced colon tumorigenesis. A, At the end of the experiment, colon tissue was harvested. Tumor tissue was processed for H&E staining and immunohistochemical analysis. Per treatment group, one representative specimen out of at least ten tumors is depicted (200 x magnification). B–E, IHC analysis of tumor specimens from Nrf2+/+ and Nrf2−/− mice was performed following the scoring system described in Materials and Methods. Horizontal lines indicate median values of markers of proliferation (Ki67), oxidative DNA damage (8-oxodG), inflammatory dysregulation (COX-2), and polyamine synthesis (ODC); (*p < 0.05; Mann-Whitney test; N.S.: not significant)
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