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Title:Molecular mechanisms of bioactive compounds in fruits and vegetables that inhibit pancreatic cancer and their relationship with inflammation
Author(s):Johnson, Jodee
Director of Research:Gonzalez de Mejia, Elvira
Doctoral Committee Chair(s):Jeffery, Elizabeth H.
Doctoral Committee Member(s):Gonzalez de Mejia, Elvira; Schuler, Mary A.; Nakamura, Manabu T.
Department / Program:Nutritional Sciences
Discipline:Nutritional Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Pancreatic cancer
Abstract:For all stages of pancreatic cancer, the 5-year survival rate from the time of diagnosis is 6%, with the median survival rate being only 6 months. This poor prognosis for pancreatic cancer is due to its aggressive nature and, too frequently, to its late diagnosis due to the absence of clinical symptoms at early stages in this disease. A potential therapeutic target in pancreatic cancer is the serine/threonine kinase glycogen synthase kinase-3β (GSK-3β) that is known to translocate into the nuclei of pancreatic cancer cells and act as a key regulator of NF-κB transcriptional activity. The constitutively active NF-κB resulting from misregulation of this signaling cascade is an essential component in the progression and tumorigenic properties of this disease. Fruit and vegetable bioactive compounds have the potential to be used as therapeutic agents for pancreatic cancer due to their anti-oxidant, -carcinogenic, -inflammatory, -proliferation and -progression properties. The objective of this research was to examine the effect of bioactive compounds present in fruits and vegetables on pancreatic cancer proliferation, apoptosis, and the role of the GSK-3β/NF-κB signaling pathway using in vitro and in vivo models. In aim 1, we evaluated the ability of fruit and vegetable bioactive compounds (flavonoids, limonoids, phenolic acids and ascorbic acid) to bind within GSK-3β's catalytic site and inhibit its activity using an in vitro enzymatic assay and computational modeling. Of the 22 bioactive compounds tested, the flavonoids luteolin, apigenin, and quercetin had the highest inhibitory effects on GSK-3β activity, with 50% inhibitory values of 1.5, 1.9, and 2.0 µM, respectively. Molecular dockings were then performed to determine the potential interactions of each flavonoid with GSK-3β. Luteolin, apigenin and quercetin were predicted to fit within the binding pocket of GSK-3β with low interaction energies (-76.4, -76.1, and -84.6 kcal•mol-1, respectively). Our results indicated that several flavonoids inhibit GSK-3β activity and suggest that these have potential to suppress the growth of pancreatic tumors. In aim 2, we tested the inhibitory potential of fruit and vegetable bioactive compounds on the proliferation of BxPC-3 and PANC-1 pancreatic cancer cells, in vitro, and gene and protein expressions of molecular markers in the GSK-3β/NF-κB signaling pathway. Of the 22 bioactive compounds tested, apigenin and luteolin were the most effective at inhibiting pancreatic cancer cell proliferation (IC50 = 23 and 31 µM for BXPC-3 cells, respectively, and IC50 = 71 and 90 µM for PANC-1 cells, respectively, for 24 h). Further investigation with apigenin into the molecular mechanism by which these flavonoids induced pancreatic cell death demonstrated that it was through the GSK-3β/NF-κB signaling pathway. Apigenin arrested cell cycle at G2/M phase (36% and 32% at 50 µM for BxPC-3 and PANC-1, respectively) with concomitant decrease in the expression of cyclin B1. Apigenin activated the mitochondrial pathway of apoptosis (44% and 14% at 50 µM for BxPC-3 and PANC-1, respectively) and modified the expression of apoptotic proteins. Apigenin highly upregulated the expression of cytokine genes IL17F (114.2-fold), LTA (33.1-fold), IL17C (23.2-fold), IL17A (11.3-fold), and IFNB1 (8.9-fold) in BxPC-3 cells, which potentially contributed to the anti-cancer properties. These results suggest that flavonoids have a protective role in pancreatic tumorigenesis. In aim 3, we assessed the potential interactions of either apigenin or luteolin with the anti-cancer activity of chemotherapeutic drugs in BxPC-3 cells in vitro, and evaluated their mechanisms of action related to inhibition of the GSK-3β/NF-κB signaling pathway. The effect of simultaneous treatment of flavonoids and chemotherapeutic drugs as well as a range of flavonoid pretreatment times (0, 6, 24 and 42 h) and concentrations (0-50 µM) on pancreatic cancer cell proliferation were assessed using the MTS cell proliferation assay. Simultaneous treatment with either flavonoid (13, 25 or 50 µM) and the chemotherapeutic drug 5-fluorouracil (5-FU, 50 µM) or gemcitabine (Gem, 10 µM) for 60 h resulted in mostly less-than-additive effects on inhibition of cell proliferation. Pretreatment for 24 h with 13 µM of either apigenin or luteolin, followed by the addition of either 5-FU or Gem for 36 h was determined to optimally inhibit cell proliferation. These combinations led to 67 or 60% inhibition when apigenin or luteolin was used as a pretreatment for 5-FU and 66 or 69% inhibition when they were each used as a pretreatment for Gem. Pretreatment of cells with 11-19 µM of either flavonoid for 24 h resulted in 43-72% growth inhibition when followed by 5-FU (50 µM, 36 h) and 59-73% growth inhibition when followed by Gem (10 µM, 36 h). Pretreatment of cells with 11-15 µM of either flavonoid for 24 h resulted in 15-35% inhibition when followed by cisplatin (Cis, 10 µM, 36 h) and 36-63% growth inhibition when followed by oxaliplatin (Oxa, 0.1 µM, 36 h). Isobolographic analyses using the IC20s or IC30s for the flavonoids and chemotherapeutic drugs demonstrated an additive effect for either flavonoid in combination with 5-FU or Gem and an antagonistic effect when combined with Cis. When apigenin was used in combination with Oxa this caused an additive effect, while luteolin in combination with Oxa caused an antagonistic effect. Luteolin (15 µM, 24 h) pretreatment followed by Gem (10 µM, 36 h), significantly decreased the protein expressions of nuclear GSK-3β and NF-κB p65 and increased pro-apoptotic cytosolic cytochrome c. The results suggest that pretreatment of pancreatic cancer cells for 24 h with low concentrations of flavonoids effectively aid in the anti-cancer activity of chemotherapeutic drugs through inhibition of the GSK-3β/NF-κB signaling pathway leading to increased apoptosis. In aim 4, we evaluated the potential of luteolin to enhance the anti-tumor effects of Gem on pancreatic cancer using an orthotopic mouse model. Male athymic nude mice (6 wk old) were injected with BxPC-3 human pancreatic cancer cells directly into the pancreas and randomized into four treatment groups: (1) control (n = 14); (2) luteolin [84 mg/kg body weight (bw)] 7 times/wk for wk 1 and 5 times/wk for wks 2-6 (n = 12) by intraperitoneal (i.p.) injection; (3) Gem (125 mg/kg bw) twice/wk by i.p. injection for wks 2-6 (n = 14); and (4) luteolin (84 mg/kg bw) 7 times/wk for wk 1, followed by luteolin 5 times/wk and Gem (125 mg/kg bw) 2 times/wk for wks 2-6 by i.p. injection (n = 12). The combination treatment of luteolin and Gem significantly lowered (p = 0.048) the pancreatic tumor mass compared to the control group. Luteolin, Gem and their combination significantly reduced proliferating cell nuclear antigen expression by 25%, 37% and 37%, respectively. Immunohistochemical and western blot analyses showed that combination treatment led to a significant reduction in the expressions of K-Ras (46%, p=0.0006), GSK-3β (34%, p=0.014), p(Tyr216)GSK-3β (16%, p=0.033), p(Ser311)NF-κB p65 (27%, p=0.036) and the Bcl-2/Bax ratio (68%, p=0.006) while significantly increasing the expressions of cytochrome c (44%, p=0.035) and caspase 3 (417%, p=0.003). The combination treatment of luteolin and Gem promoted apoptotic cell death in pancreatic tumor cells through inhibition of the GSK-3β/NF-κB signaling pathway leading to a reduction in the Bcl-2/Bax ratio, release of cytochrome c and activation of caspase 3. These results suggest the potential for luteolin to enhance the anti-tumor activity of Gem in a pancreatic cancer in vivo model. In summary, we first demonstrated that fruit and vegetable flavonoids, specifically luteolin, apigenin and quercetin, were able to optimally bind within the catalytic site of GSK-3β and inhibit its activity. Next, the inhibitory effects of these bioactive compounds against the pancreatic cancer cell lines BxPC-3 and PANC-1 were determined, showing that apigenin and luteolin were the most potent. The inhibitory effects of the flavonoids on GSK-3β enzymatic activity and on BxPC-3 cell proliferation significantly correlated (r = 0.87 for 24 h exposure and r = 0.86 for 48 h exposure) suggesting that the flavonoids may be decreasing pancreatic cancer in vitro through inhibition of GSK-3β. However, the correlations were not statistically significant with the inhibitory results of the PANC-1 cells, potentially due to the higher tumor grade of this cell line. Investigation into the specific molecular mechanism of action demonstrated that apigenin is inhibiting proliferation of pancreatic cancer cells due to suppression of the GSK-3β/NF-κB signaling cascade and the induction of apoptosis. From there, we showed the ability of apigenin and luteolin to enhance the anti-cancer activity of chemotherapeutic drugs 5-FU, Gem and Oxa in BxPC-3 cells, and showed that this enhancement was due to inhibition of the GSK-3β/NF-κB signaling pathway. Lastly, these in vitro findings were translated to an in vivo model of pancreatic cancer demonstrating the ability of luteolin to enhance the anti-tumor effects of Gem through inhibition of the GSK-3β/NF-κB signaling pathway leading to increased apoptosis. In conclusion, the results from this research demonstrate the potential of fruit and vegetable flavonoids, specifically apigenin and luteolin, to protect against pancreatic carcinogenesis, and provide the foundation for future studies in this area.
Issue Date:2013-08-22
Rights Information:Copyright 2013 Jodee Johnson
Date Available in IDEALS:2013-08-22
Date Deposited:2013-08

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