PROTECTING AGAINST COLORECTAL CANCER BY ANTHOCYANIN-ENRICHED TRANSGENIC TOMATOES IN THREE-DIMENSIONAL CELL CULTURE MODEL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016397
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506

Performing Department
Human Nutrition & Foods

Non Technical Summary
The goal of this study is to delineate evidence and mechanisms by which the purple transgenic tomatoes may protect against cancer through anthocyanins in a novel three-dimensional cell culture model. Successful completion of this study will provide scientific evidence for the health benefits of purple tomatoes and develop novel cell culture model for anticancer screening. In addition, this study will encompass regular publications in reputable journals and train postdoctoral fellow and graduate/undergraduate students. Another important impact of this work is to promote agricultural farming and marketing opportunities for novel tomato varieties as functional food products for cancer prevention.

Animal Health Component

20%

Research Effort Categories

Basic

60%

Applied

20%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	1460	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

anthocyanins

transgenic tomatoes

3d cell culture

colorectal cancer prevention

Goals / Objectives
To define anti-cancer activity and underlying mechanisms of anthocyanin-enriched purple tomatoes as new value-added healthy products in novel three-dimensional cell culture model

Project Methods
Anthocyanins, the natural pigments in plant foods, have been associated with cancer prevention because of their anti-inflammatory and anti-oxidant properties. However, food content of anthocyanins is typically low and the mechanism by which they exert anti-cancer is still unclear.This project is collaborated by faculty in Nutrition, Horticulture, and Grain Science for the nutraceutical benefits of the anthocyanin-enriched transgenic tomatoes. We have developed a new anthocyanin-enriched transgenic tomato (AETT) in Dr. Park's lab. The chemical structures and profiles of anthocyanins in AETT has been analyzed in the Wang lab (Su et al, 2016). Furthermore, a novel 3-dimensional (3D) cell culture model has been established by Dr. Sun (Huang et al. 2012 & 2013, Liang et al. 2017). Our previous studies demonstrated that treatment with anthocyanins in human normal colonic epithelial FHC cells resulted in a dose-dependent decrease in cytokine TNFa-induced cellular mitogenic signaling, while treatment of AETT extracts in colonic adenocarcinoma SW480 cells resulted in a decrease of cell growth due to cytostatic mechanism. These observations, coupled with our pilot study that dietary administration of 10-30% AETT for 40 days significantly inhibited azoxymethane-induced TNFa production and aberrant crypt foci (ACF) formation in colonic mucosal tissues of CF-1 mice, constitutes our overall hypothesis that AETT can prevent colorectal cancer, primarily by suppression of inflammatory cytokine-induced signaling.Two specific objectives will be tested in this study: 1) Study the anti-inflammatory effect of AETT extracts in FHC cells versus chemo-protective effect in SW480 cells; 2) Investigate the underlying mechanisms of inflammatory and cellular signaling pathways in a novel 3D cell culture model.Aim 1. Study the anti-inflammatory effect of AETT extracts in normal colonic epithelial FHC cells in comparison with a chemoprotective effect in adenocarcinoma SW480 cells:AETT anthocyanin extraction: Wild type, Del/Ros1, and CHI×Del/Ros1 tomato samples will be grown in K-State Department of Horticulture greenhouses. For each line, anthocyanins will be extracted according to pur previous publication (Su et al 2016), which will be used for cell culture studies.HPLC-MS/ESI analysis: As described in our previous publications (Ayella et al. 2007; Su et al. 2017; Shen et al. 2017),Cell culture and treatments: As described by our previous publications (Higuchi and Wang 1995, Wang et al. 2000, Qu et al. 2005, Ayella et al. 2011).Selective knockdown of PI3K and Ras gene expression by siRNA: Both p110a and p85b isomers of PI3K- and both Kras and Hras isomers of Ras-specific siRNA as well as the control siRNA sequences will be synthesized according to the sequences reported by Kim et al. (2005). Briefly, cultured FHC or SW480 cells at 5x105 will be transfected with 60 mM PI3K- or Ras-specific siRNA or the control siRNA using X-tremeGENE siRNA transfection reagent (Roche, Mannheim, Germany) according to the manufacturer's recommendation. After 24 hrs, the cells will be treated with AETT extracts for 24-72 hrs and then subjected to Western blotting and all the other routine molecular biology and bioinformatics analyses.Selective inhibition of PI3K and Ras expression by pharmacologic inhibitors: The PI3K inhibitor LY294002 and the Ras inhibitor farnesylthiosalicylic acid will be obtained from Sigma Chemical Co. (St. Louis, MO). Inhibitors will be dissolved in DMSO and diluted at the time of use in culture medium. The final concentration of DMSO in all cultures will be kept at 0.2%. Cultured FHC or SW480 cells at 5x105 will be treated with 10 mM of inhibitors (LY294002 or farnesylthiosalicylic acid, respectively). After 24 hrs, the cells will be treated with AETT extracts for 24-72 hrs and then subjected to Western blotting and all the other routine molecular biology and bioinformatics technique analyses.Cell viability will be measured by trypan blue staining as described in our previous publication (Qu et al. 2005, Ayella et al. 2010).Routine Molecular Biology analyses: We will determine the multiple molecular markers of the impacted signaling pathways in the treated cell lysates. All the methods and techniques proposed in this grant have been established and/or routinely used in the PD's laboratory. A detailed procedure of each method has been described in our previous publications, including a pull down assay for activated Ras (Xie et al. 2007), immune complex assay for ERK activity (Wang et al. 2000; Xie et al 2007), Western blot for Hras, Kras, and PI3K (Wang et al. 2000; Xie et al. 2007, Ping et al. 2010), immunohistochemistry for PCNA and caspase-3 (Xie et al. 2007, Ping et al. 2010), gel shift assay for AP-1:DNA and NFkB:DNA binding (Przybyszewski et al. 2001 and 2010).State-of-the-Art Bioinformatics tools: Affymetrix genome microarray assay confirmed by RT-PCR or real-time PCR has been described in details in our previous publications (Lu et al. 2007, Xie et al. 2007; Standard et al 2014).Aim 2. Investigate ex vivo anticancer efficacy and mechanisms of inflammatory and cellular signaling pathways that are potentially conferring protection by dietary AETT in 3D cell culture model:Hydrogel preparation. H9e hydrogel peptide solution and PG works solution will be provided by Dr. Sun's lab (Huang et al. 2012; Huang et al. 2013).Fluorescence,Atomic force microscopy, Rheological tests, Cell culture and anticancer efficacy tests,Cell viability will be conducted according to previously published methods (Huang et al. 2012; Huang et al. 2013).All the other methods, including the routine molecular analyses and -omics tool for the signaling biomarkers have been described above in Aim 1.??

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The unique purple tomato varieties developed for farmers, the new value-added healthy products for consumers, and the novel 3-dimensional cell culture model for scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The studies planned in the next 3rd year will address the rest of the specific Aim 1 and start specific Aim 2. Briefly, we will continually address the underlying mechanisms of anthocyanin-enriched transgenic tomatoes in human colonocytes cultured by using the established ex vivo 3D and in vitro 2D systems. More routing molecular biology analyses for understanding the underlying molecular mechanisms will be continually applied. All the planned studies will be conducted as proposed.

Impacts
What was accomplished under these goals? By means of this 5-year USDA hatch support, we developed novel transgenic tomato lines and determined the anthocyanin profiles and contents in these transgenic tomato lines in the 1st year. In this past 2nd year, LC-MS/MS analysis has been applied for the potential metabolites of anthocyanins in the cultured cells. In addition, we continually tested the anti-inflammatory effect of AETT extracts in normal colonic epithelial FHC cells and adenocarcinoma SW480 cells. We have also established 3D cell culture system with both FHC and SW480 cells. The potential mechanisms of cancer prevention by AETTs via using selective knockdown PI3K and Ras genes have been initially assessed. All the conducted studies have not been modified from the original proposal.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhao J, Xu Y, Wang W, Griffin J, Roozeboom K, Wang D. Bioconversion of industrial hemp biomass for bioethanol production: A review. Fuel. 2020; 281:118725
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Xu Y, Li J, Zhao J, Wang W, Griffin J, Li Y, Bean S, Tilley M, Wang D. Hempseed as A Nutritious and Healthy Food Source: A Review. International Journal of Food Science and Technology. 2020; 16859558 (DOI: 10.1111/ijfs.14755).
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhao J, Xu Y, Wang W, Griffin J, Wang D. High ethanol concentration (77 g/L) of industrial hemp biomass achieved through optimizing the relationship between ethanol yield/concentration and solid loading. ACS Omega. 2020; (DOI: 10.1021/acsomega.0c03135).
• Type: Book Chapters Status: Accepted Year Published: 2021 Citation: 9. Xu J, Wang W. Cereal grains and the prevention of cancer. In: Improving the Nutritional and Nutraceutical Properties of Wheat and Other Cereals (Editor Trust Beta) Burleigh Dodds Science Publishing, 2021; in press
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Lei Wu, Xin Guo, Siau Yen Wong, Peiran Lu, Steven D. Hartson, Denis M. Medeiros, Weiqun Wang, Stephen L. Clarke, Edralin A. Lucas, Brenda J. Smith, Winyoo Chowanadisai, Dingbi Lin. Deficiency of ?-carotene oxygenase 2 induces mitochondrial fragmentation and activates the STING-IRF3 pathway in the mouse hypothalamus. Journal of Nutritional Biochemistry. 2020; 88:108542
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kelly Gude, Channa B. Rajashekar, Brianna Cunningham, Qing Kang, Weiqun Wang, Myungjin Lee, Cary Rivard, Eleni Pliakoni. Effect of high tunnel coverings on antioxidants of breaker and light red tomatoes at harvest and during ripening. Agronomy. 2020; 10:1639.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhao J, Xu Y, Wang W, Griffin J, Wang D. Bioethanol conversion of liquid hot water, acid and alkali pretreated industrial hemp biomass: Physicochemical and morphological properties, delignification, sugar recoveries, enzymatic hydrolysis, and bioethanol fermentation. Bioresource Technology. 2020; 309:123383.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Carter T, Qi G, Wang W, Nguyen A, Cheng N, Yong Min JY, Lee SJ, Yoo J, Atala A, Sun X. Self-Assembling Peptide Solution Accelerates Hemostasis. Advances in Wound Care. 2020; (DOI: 10.1089/wound.2019.1109).
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Xu J, Zhang Y, Wang W, Li Y. Advanced Properties of Gluten-Free Cookies, Cakes, and Crackers: A Review. Trends in Food Science & Technology. 2020; 103:200-213.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The unique purple tomato varieties developed for farmers, the new value-added healthy products for consumers, and the novel 3-dimensional cell culture model for scientific communityThe unique purple tomato varieties developed for farmers, the new value-added healthy products for consumers, and the novel 3-dimensional cell culture model for scientific community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?this study encompasses regular publications in reputable journals and trains postdoctoral fellow and graduate/undergraduate students How have the results been disseminated to communities of interest?These accomplishments have been orally presented in IFT2019 and also have been published in multiple top-notch journals including Toxicology in vitro. What do you plan to do during the next reporting period to accomplish the goals?The studies planned in the next year will address the rest of the specific Aim 1. Briefly, we will continually address the anti-inflammatory effect of anthocyanin-enriched transgenic tomatoes in human colonocytes. Both 3D and 2D cell culture systems will be used to determine the cellular uptake and metabolism of anthocyanins. All the planned studies will be conducted as proposed.

Impacts
What was accomplished under these goals? By means of this USDA hatch support, this project has been completed in part by addressing the specific aim 1. Briefly, we have developed novel transgenic tomato lines. The prominent anthocyanin profiles and contents in these transgenic tomato lines have been assessed by our established HPLC-MS/MS. Seven anthocyanins including two new anthocyanins have been identified. Furthermore, we have established 3D cell culture system with human colorectal SW480 cells. One of phytochemical phenolic acids has been tested in this novel 3D system when compared with classic 2D cell culture model. The anthocyanins have also been extracted from anthocyanin-enriched transgenic tomato lines, which are on-going tested in this 3D cell culture system. The potential mechanisms of cancer prevention by anthocyanin-rich transgenic tomatoes will be further assessed. These accomplishments have been orally presented in IFT2019 and also have been published in multiple top-notch journals including Toxicology in vitro. All the conducted studies have not been modified from the original proposal.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Xu J, Qi G, Sui C, Wang W*, Sun X. 3D h9e peptide hydrogel: an advanced three-dimensional cell culture system for anticancer prescreening of chemopreventive phenolic agents. Toxicology in Vitro. 2019; 61(7):104599
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Su X, Griffin J, Xu J, Ouyang P, Zhao Z, Wang W*. Identification and quantification of anthocyanins in purple-fleshed sweet potato leaves. Heliyon. 2019; 5(6):e01964
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Xu J, Smith S, Smith G, Wang W, Li Y. Glyphosate contamination in grains and foods: an overview. Food Control. 2019; 106:106710
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Xu S, Shen Y, Xu J, Qi G, Chen G, Wang W, Sun X, Li Y. Antioxidant and anticancer effects in human hepatocarcinoma (HepG2) cells of papain-hydrolyzed sorghum kafirin hydrolysates. Journal of Functional Foods. 2019; 58:374-382
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Cox S, Noronha L, Herald T, Bean S, Lee SH, Perumal R, Wang W, Smolensky D. Evaluation of ethanol-based extraction conditions of sorghum bran bioactive compounds with downstream anti-proliferative properties in human cancer cells. Heliyon. 2019; 5(5):e01589
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Xu J, Su X, Li Y, Sun X, Wang D, Wang W*. Response of bioactive phytochemicals in vegetables and fruits to environmental factors. European Journal of Nutrition & Food Safety. 2019; 9(3):233-247
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Woolley A, Sumpter S, Lee M, Xu J, Barry S, Wang W, Rajashekar CB. Nutritional quality: mineral nutrients and phytochemicals in lettuce and tomato grown in high tunnel. American Journal of Plant Sciences. 2019; 10:125-38
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Xu J, Wang W, Li Y. Dough properties, bread quality, and associated interactions with added phenolic compounds: A review. Journal of Functional Foods. 2019; 52:629-39
• Type: Book Chapters Status: Published Year Published: 2019 Citation: Xu J, Li Y, Wang W. Corn. In: Bioactive Factors and Processing Technology for Cereal Foods (Editors Wang J, Sun B, and Cao R) Springer Nature Singapore Pte Ltd., 2019; p 33-52
• Type: Book Chapters Status: Published Year Published: 2019 Citation: Davis H, Su X, Shen Y, Xu J, Wang D, Smith JS, Aramouni F, Wang W*. Phenotypic diversity of colored phytochemicals in sorghum accessions with various pericarp pigments. In: Polyphenols in Plants (Editor Ronald Ross Watson) 2nd Edition. Academic Press, 2019; p 123-31