Cranberry Polyphenols in Esophageal Cancer Inhibition: New Insights

Abstract

Esophageal adenocarcinoma (EAC) is a cancer characterized by rapidly rising incidence and poor survival, resulting in the need for new prevention and treatment options. We utilized two cranberry polyphenol extracts, one proanthocyanidin enriched (C-PAC) and a combination of anthocyanins, flavonoids, and glycosides (AFG) to assess inhibitory mechanisms utilizing premalignant Barrett's esophagus (BE) and EAC derived cell lines. We employed reverse phase protein arrays (RPPA) and Western blots to examine cancer-associated pathways and specific signaling cascades modulated by C-PAC or AFG. Viability results show that C-PAC is more potent than AFG at inducing cell death in BE and EAC cell lines. Based on the RPPA results, C-PAC significantly modulated 37 and 69 proteins in JH-EsoAd1 (JHAD1) and OE19 EAC cells, respectively. AFG treatment significantly altered 49 proteins in both JHAD1 and OE19 cells. Bioinformatic analysis of RPPA results revealed many previously unidentified pathways as modulated by cranberry polyphenols including NOTCH signaling, immune response, and epithelial to mesenchymal transition. Collectively, these results provide new insight regarding mechanisms by which cranberry polyphenols exert cancer inhibitory effects targeting EAC, with implications for potential use of cranberry constituents as cancer preventive agents.

Keywords: Barrett’s esophagus; anthocyanins; cranberry polyphenols; esophageal adenocarcinoma; flavonoids; glycosides; proanthocyanidins; reverse phase protein array.

Figure 1

AFG induces cell death in EAC cell lines. Viability of (A) JHAD1 and (B) OE19 cells was assessed using the live stain Calcein-AM following treatment with AFG (200 and 400 µg/mL). The data are reported as the mean ± standard error of the mean (SEM) of at least four wells per treatment. Each panel contains representative fluorescent images of viable JHAD1 and OE19 cells at each time point assessed. Viability was analyzed by One-way ANOVA with Tukey’s post-hoc test (p < 0.05) where multiple conditions were assessed. Treatments were significantly different from a = vehicle, b = AFG (200 µg/mL) and c = AFG (400 µg/mL). AFG, fraction comprised of anthocyanins, flavonoids and glycosides; VEH, vehicle; EAC, esophageal adenocarcinoma; JHAD1, JH-EsoAd1; OE19, OE19 esophageal adenocarcinoma cell line; h, hours.

Figure 2

Cranberry polyphenols induce cell death in progressor BE cell line CP-B. (A)Viability of CP-B cells was assessed using the following treatment with C-PAC (75, 100 and 150 µg/mL) or AFG (300, 400, 500 and 600 µg/mL). The data are reported as the mean ± SEM of at least three wells per treatment. (B) Representative fluorescent images of viable CP-B cells at each time point assessed. Viability was analyzed by One-way ANOVA with Tukey’s post-hoc test (p < 0.05) where multiple conditions were assessed. Treatments were significantly different from a = vehicle, b = C-PAC (75 µg/mL), c = C-PAC (100 µg/mL), d = C-PAC (150 µg/mL), e = AFG (300 µg/mL), f = AFG (400 µg/mL), g = AFG (500 µg/mL) and h = AFG (600 µg/mL). C-PAC, proanthocyanidin rich fraction; BE, Barrett’s esophagus; CP-B, isolated from a “progressor” or patient who progressed to esophageal adenocarcinoma.

Figure 3

Cranberry polyphenols induce cell death in non-progressor BE cell line CP-C. (A) Viability of CP-C cells was assessed following 24 h treatment with C-PAC (75, 100 and 150 µg/mL) or AFG (300, 400, 500 and 600 µg/mL). The data are reported as the mean ± SEM of at least three wells per treatment. (B) Representative fluorescent images of viable CP-C cells at each time point assessed. Viability was analyzed by One-way ANOVA with Tukey’s post-hoc test (p < 0.05) where multiple conditions were assessed. Treatments were significantly different from a = vehicle, b = C-PAC (75 µg/mL), c = C-PAC (100 µg/mL), d = C-PAC (150 µg/mL), e = AFG (300 µg/mL), f = AFG (400 µg/mL), g = AFG (500 µg/mL) and h = AFG (600 µg/mL).

Figure 4

Cranberry proanthocyanidins modulate multiple process networks and pathway maps in JHAD1 cells. All significantly upregulated (red; n = 21) and downregulated (blue; n = 16) proteins from RPPA in JHAD1 cells were used to generate heatmaps following C-PAC (75 µg/mL) treatment for 24 h (p < 0.05). Upregulated and downregulated proteins are represented in Cluster 1 and 2, respectively. Below the heatmap is a table containing the top 10 modulated process networks and pathway maps for all proteins significantly changed with C-PAC treatment. Representative proteins in each process network or pathway map are listed. RPPA, reverse phase protein array.

Figure 5

AFG modulates multiple process networks and pathway maps in JHAD1 cells. All significantly upregulated (red; n = 22) and downregulated (blue; n = 27) proteins from RPPA in JHAD1 cells were used to generate heatmaps following AFG (400 µg/mL) treatment for 24 h (p < 0.05). Upregulated and downregulated proteins are represented in Cluster 1 and 2, respectively. Below the heatmap is a table containing the top 10 modulated process networks and pathway maps for all proteins significantly changed with AFG treatment. Representative proteins in each process network or pathway map are listed.

Figure 6

Cranberry proanthocyanidins modulate multiple process networks and pathway maps in OE19 cells. All significantly upregulated (red; n = 30) and downregulated (blue; n = 39) proteins from RPPA in OE19 cells were used to generate heatmaps following C-PAC (75 µg/mL) treatment for 24 h (p < 0.05). Upregulated and downregulated proteins are represented in Cluster 1 and 2, respectively. Below the heatmap is a table containing the top 10 modulated process networks and pathway maps for all proteins significantly changed with C-PAC treatment. Representative proteins in each process network or pathway map are listed.

Figure 7

AFG modulates multiple process networks and pathway maps in OE19 cells. All significantly upregulated (red; n = 25) and downregulated (blue; n = 24) proteins from RPPA in OE19 cells were used to generate heatmaps following AFG (400 µg/mL) treatment for 24 h (p < 0.05). Upregulated and downregulated proteins are represented in Cluster 1 and 2, respectively. Below the heatmap is a table containing the top 10 modulated process networks and pathway maps for all proteins significantly changed with AFG treatment. Representative proteins in each process network or pathway map are listed.

Figure 8

Polyphenols modulate several proteins related to P53, inflammation and metastasis in premalignant and EAC cell lines. CP-B, CP-C, JHAD1 and OE19 cells were treated with C-PAC (75 µg/mL, AFG (400 µg/mL) or vehicle and lysates isolated at 24 h following treatment. Western blotting was performed using commercially available antibodies to proteins of interest. Expression values were normalized to the appropriate loading control, GAPDH or HSP60, and fold change from vehicle was calculated using Imagelab. Bands with no detectable expression are denoted as ND. The asterisk (*) next to the P53 results in OE19 cells denote a faster migrating protein due to an insertion and duplication in the TP53 coding region as described in the results and discussion. FL and CL denote full-length and cleaved proteins, respectively. The plus (+) sign denotes treatment group.

Figure 9

Polyphenols modulate several proteins related to DNA damage, cell cycle and growth arrest in premalignant and EAC cell lines. CP-B, CP-C, JHAD1 and OE19 cells were treated with C-PAC (75 µg/mL), AFG (400 µg/mL) or vehicle and lysates isolated at 24 h following treatment. Western blotting was performed using commercially available antibodies to proteins of interest. Expression values were normalized to the appropriate loading control, GAPDH or HSP60, and fold change from vehicle was calculated using Imagelab. Bands with no detectable expression are denoted as ND. FL and CL denote full-length and cleaved proteins, respectively. The plus (+) sign denotes treatment group.

Figure 10

Polyphenols modulate several proteins involved in inflammation, DNA damage and cell cycle in premalignant and EAC cell lines. CP-B, CP-C, JHAD1 and OE19 cells were treated with C-PAC (75 µg/mL), AFG (400 µg/mL) or vehicle and lysates isolated at 24 h following treatment. Western blotting was performed using commercially available antibodies to proteins of interest. Expression values were normalized to GAPDH and fold change from vehicle was calculated using Imagelab. Bands with no detectable expression are denoted as ND. CL denotes a cleaved protein. The plus (+) sign denotes treatment group.