Novel inhibitory effect of Omega-3 fatty acids regulating pancreatic cancer progression

Abstract

Pancreatic cancer is a devastating malignancy in great need of new and more effective treatment approaches. In recent years, studies have indicated that nutritional interventions, particularly nutraceuticals, may provide novel avenues to modulate cancer progression. Here, our study characterizes the impact of ω-3 polyunsaturated fatty acids, eicosapentaenoic acid, and docosahexaenoic acid, as a nutraceutical intervention in pancreatic cancer using a genetically engineered mouse model driven by KrasG12D and Trp53R172H. This model closely resembles human pancreatic carcinogenesis, offering a disease relevant platform for translational research. Our findings showed that ω-3 polyunsaturated fatty acids intervention (using a diet supplemented with 6% cod liver oil) significantly reduced tumor volume as well as lung and liver metastasis and a trend toward improved survival rate compared with control treated mice. This antitumoral effect was accompanied by distinct changes in tumor membrane fatty acid profile and eicosanoids release. Furthermore, the eicosapentaenoic acid and docosahexaenoic acid intervention also reduced malignant histological parameters and induced apoptosis without affecting cell proliferation. Of note is the significant reduction in tumor fibrosis that was associated with decreased levels of Sonic Hedgehog, a major ligand controlling this cellular compartment in pancreatic cancer. All together our results demonstrate the impact of eicosapentaenoic acid and docosahexaenoic acid as antitumor regulators in pancreatic cancer, suggesting potential for ω-3 polyunsaturated fatty acids as a possible antitumoral dietary intervention. This research opens new avenues for integrating nutraceutical strategies in pancreatic cancer management.

Keywords: Sonic Hedgehog; nutraceutical intervention; pancreatic cancer; ω-3 polyunsaturated fatty acids.

Graphical Abstract

Figure 1

Dietary ω-3 fatty acids suppress pancreatic tumor growth and metastasis in the KPC mouse model. (A) Experimental design of in vivo model: since weaning (30 days after birth), KPC animals received two different diets sufficient in essential fatty acids: control group and the Hω-3 fatty acids content. The animals were sacrificed after 180 days of feeding with the experimental diets and the parameters of tumor pancreatic progression in mice were evaluated: tumor volume and metastases, biochemical and molecular tumor analysis, and histopathology. (B) Macroscopic representative view of pancreas from KPC mice fed with control and Hω-3 diets. (C) Bars represent pancreatic volume recorded after necropsy. The values were obtained from independent samples and represent the mean ± SEM (standard error of the mean); asterisks indicate significant differences compared with the control group (P < .05; n = 8–10). (D, E) Representative micrographs of metastases in lung and liver tissue observed in KPC mice fed with control and Hω-3 diets (H&E, 100× and 400×). (F, G) Bars represent the number of microscopic metastases in the lung and liver of KPC mice (mean ± SEM); asterisks indicate significant differences compared with the control group (P < .05; n = 6–8). (H) Kaplan–Meier curve survival (n = 8–12).

Figure 2

Dietary ω-3 fatty acids modulate TC membrane lipid profile, eicosanoid metabolism, and PPARγ expression. (A) Bars represent the percentage of fatty acids in pancreatic TC membranes: the ω-3 EPA (C20:5) and DHA (C22:6) acids and the ω-6 AA (C20:4). Values represent mean ± SEM, asterisks indicate significant differences compared with the control group (P < .05; n = 3). (B, C) Bars represent the content (in ng) of eicosanoids in tumor membranes: 5(S)-HEPE, derived from the eicosapentaenoic acid (C20:5, ω-3 fatty acid) and 12(S)-HETE, derived from the AA (C20:4, ω-6 fatty acid). Values represent mean ± SEM (n = 3). (D) Representative micrographs at 100x and 400x magnifications showing PPARγ immunostaining in pancreatic tumor samples from KPC mice fed with Control and Hω-3 diets. (E) Bars represent the number of PPARγ-positive cells by immunohistochemistry in micrographs of pancreas sections from each dietary group. The values were obtained from independent samples divided into 10 sections each and represent the mean ± SEM; asterisks indicate significant differences compared with the control group (P < .05; n = 5)

Figure 3

Histopathological assessment of pancreatic tumor differentiation in KPC mice fed an Hω-3 diet. (A) Representative H&E micrographs of pancreatic tissues from KPC mice after control and Hω-3 dietary treatment (100× and 400×). We analyzed architectural variables of pancreatic tumor tissues: (1) necrosis, (2) vascularization, (3) dysplastic glandular pattern and solid pattern, (4) anisokaryosis and anisocytosis, and (5) hyperchromatic and prominent nucleoli. Malignant typical lesions of high undifferentiated adenocarcinoma were observed in KPC mice of the control diet; moderately differentiated pancreatic adenocarcinoma and glands were observed in KPC animals from Hω-3 experimental group (H&E stain, 100× and 400×). (B) Bars represent cancer differentiation grade of pancreatic tumor observed in KPC mice: well-differentiated, moderately differentiated, and poorly differentiated pancreatic adenocarcinoma. Asterisks indicate significant differences compared with the control group (P < .05; n = 6–8)

Figure 4

The effect of ω-3 fatty acid-rich diet on apoptosis and proliferation. (A) Representative micrographs of apoptotic cells detected by TUNEL in pancreatic sections (indicated by positive TUNEL staining) from KPC mice fed with control and Hω-3 diets (100x and 400x). (B) Bars represent the percentage of apoptotic cells in pancreatic tumor sections from each experimental group; the values were obtained from 10 independent sections per animal and represent the mean ± SEM; asterisks indicate significant differences compared to the Control group (P < 0.05; n=5). (C) Representative micrographs of Ki67 cells in pancreatic tumor sections from KPC mice fed with control and Hω-3 diets (100x and 400x). (D) Bars represent the percentage of proliferative cells in micrographs of pancreas sections (Ki67-positive cells) for each dietary group of mice; the values were obtained from independent samples divided into 10 sections each and represent the mean ± SEM (ns; n=5)

Figure 5

The impact of an ω-3 fatty acid-rich diet on fibrosis and SHH expression. (A) Representative micrographs of levels of fibrosis (indicated by Masson-positive signal) in pancreatic samples from KPC mice fed with control and Hω-3 diets (Masson stain, 100x and 400x). (B) Bars represent the percentage of fibrotic-positive areas (Masson-positive staining) in pancreatic tissues of KPC mice fed with control and Hω-3 diets; the values were obtained from independent samples divided into 10 sections each and represent the mean ± SEM; asterisks indicate significant differences compared to the control group (P < 0.05; n=10). (C) Representative micrographs of Shh expression detected by immunohistochemistry (indicated by positive immunostaining) in pancreatic samples from both experimental groups (100x and 400x). (D) Bars represent the number of Shh-positive cells by immunohistochemistry in micrographs of pancreas sections from each dietary group. The values were obtained from 10 independent tumor sections each and represent the mean ± SEM; asterisks indicate significant differences compared to the control group (P < 0.05; n=5). (E) Bars represent expression of Shh mRNA in pancreatic tumor tissue of KPC mice fed with control and Hω-3 diets determined using qPCR and is shown as Fold Change (mean ± SEM); asterisks indicate significant differences compared to control group (P < 0.05; n=3)