Portfolio project Portfolio Project • The purpose of the portfolio project is to provide an assessment in which you are able to demonstrate your understanding and application of the CILOs. • The individual portfolio project will be measured against all four course intended learning outcomes; 1. Explain observations. [PILO 1.1] 2. Interpret assumptions. [PILO 1.1] 3. Evaluate sources. [PILO 2.3] 4. Propose hypothesis. [PILO 4.4] How to conduct your portfolio project • You must choose one of the research articles to answer for your portfolio project. • Once you have chosen a scientific article – post your choice on the BB discussion board to inform your instructor. • Portfolio projects should cover all CILO criterion and include references Students must choose one research article to base their portfolio project on 1. Caffeine and physical performance • https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059561 2. Substance addiction in KSA • https://medcraveonline.com/MOJAMT/MOJAMT-06-00145.pdf 3. GM tomato to treat cancer • https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175778 4. Smartphone-powered efficient water disinfection at the point of use • https://www.nature.com/articles/s41545-020-00089-9 Students Portfolio project should follow the structure below and cover all points • Identify the research question and rationale posed by the article • Provide a hypothesis and null hypothesis that applies to question (and papers associated) • Discuss the scientific rigor of the articles associated with your question (ie critically analyse and compare and contrast the articles) • Analyze the data provided in both papers to determine whether or not this accepts or rejects you null hypothesis (You must explain why and reference the data in this section) • Generate a conclusive scientific theory from the articles you are provided with and explain the assumptions of the theory identified. • Provide a conclusion, in which you must relate the findings of the the journal articles and provide areas of future research Feedback • Remember this is a summative assignment • Meaning you cannot receive formal feedback on your answers • Copying and pasting from each other or online sources is not acceptable • Plagiarism will result in a grade of Zero – all work should be your own • 1st of April 1pm is the deadline for submission RESEARCH ARTICLE Polyphenolic extract of InsP 5-ptase expressing tomato plants reduce the proliferation of MCF-7 breast cancer cells Mohammad Alimohammadi1☯, Mohamed Hassen Lahiani1☯, Diamond McGehee1☯, Mariya Khodakovskaya1,2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Biology, University of Arkansas at Little Rock, Little Rock, Arkansas, United States of America, 2 Institute of Biology and Soil Sciences, Far-Eastern Branch of Russian Academy of Sciences, Vladivostok, Russia ☯ These authors contributed equally to this work. * mvkhodakovsk@ualr.edu Abstract OPEN ACCESS Citation: Alimohammadi M, Lahiani MH, McGehee D, Khodakovskaya M (2017) Polyphenolic extract of InsP 5-ptase expressing tomato plants reduce the proliferation of MCF-7 breast cancer cells. PLoS ONE 12(4): e0175778. https://doi.org/ 10.1371/journal.pone.0175778 Editor: Rajeev Samant, University of Alabama at Birmingham, UNITED STATES Received: October 30, 2016 Accepted: March 9, 2017 Published: April 27, 2017 Copyright: © 2017 Alimohammadi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In recent years, by extensive achievements in understanding the mechanisms and the pathways affected by cancer, the focus of cancer research is shifting from developing new chemotherapy methods to using natural compounds with therapeutic properties to reduce the adverse effects of synthetic drugs on human health. We used fruit extracts from previously generated human type I InsP 5-ptase gene expressing transgenic tomato plants for assessment of the anti-cancer activity of established genetically modified tomato lines. Cellular assays (MTT, Fluorescent microscopy, Flow Cytometry analysis) were used to confirm that InsP 5-ptase fruit extract was more effective for reducing the proliferation of breast cancer cells compared to wild-type tomato fruit extract. Metabolome analysis of InsP 5-ptase expressing tomato fruits performed by LC-MS identified tomato metabolites that may play a key role in the increased anti-cancer activity observed for the transgenic fruits. Total transcriptome analysis of cancer cells (MCF-7 line) exposed to an extract of transgenic fruits revealed a number of differently regulated genes in the cells treated with transgenic extract compared to untreated cells or cells treated with wild-type tomato extract. Together, this data demonstrate the potential role of the plant derived metabolites in suppressing cell viability of cancer cells and further prove the potential application of plant genetic engineering in the cancer research and drug discovery. Data Availability Statement: All relevant data are within the manuscript and the Supporting Information files. Introduction Funding: Authors are grateful to Arkansas Space Grant Consortium for providing a stipend to Diamond McGehee. This project was supported by National Space Grant College Fellowship Program (NNXISAR7H) through Research Infrastructure Award provided by Arkansas Space Grant Consortium (award to MK). The funders had no role in study design, data collection and analysis, Cancer is one of the leading causes of death in humans. Scientific advances in recent years and the use of chemoprevention therapy has led to a significant reduction in death rates for different types of cancers [1–3]. Recently, natural compounds with cancer preventive properties have been more widely used in cancer therapy [4]. Natural compounds with antioxidant activity can be categorized into three major groups: compounds that can directly inhibit cell proliferation, compounds that affect tissues outside the cancer cells, and immune-stimulating compounds [5]. Epidemiological studies have shown a positive correlation between the long- PLOS ONE | https://doi.org/10.1371/journal.pone.0175778 April 27, 2017 1 / 21 Polyphenolic extract of InsP 5-ptas tomato plants reduce the proliferation of cancer cells decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. term consumption of fruits and vegetables containing naturally occurring antioxidants with a reduced risk of several types of cancer [6–10]. One of such naturally occurring antioxidants are polyphenols that can be found in various amounts in many types of fruits and vegetables [11–13]. They can be classified into two main groups according to their chemical structure: flavonoids and non-flavonoid compounds. These compounds are particularly valuable because of their high antioxidant activity [14–16]. Several clinical studies indicate that dietary intake of flavonoids and some other phenolic compounds such as caffeic acid and chlorogenic acid can significantly reduce the risk of multiple types of cancer including breast, lung, prostate, and pancreatic cancers [17–20]. Studies have also shown that the use of dietary phenolic compounds can have better preventive and therapeutic results compared to the common synthetic drugs used for cancer treatment since these natural compounds demonstrate less toxicity compared to synthetic chemo-preventive medicines [21]. Dietary flavonoids and other important phenylpropanoids naturally exist in plants. A good example of the commonly used crop plants with a high content of phenolic compounds is tomato (Solanum lycopersicum) [22]. Consumption of tomato has preventive and therapeutic effects on several types of diseases, including cancer [23]. The observed anti-cancer effects of tomato are mainly related to the properties of phenolic compounds that allow them to bind to or interact with a wide range of molecules, affect cell signaling processes, or even serve as a signaling molecule [24–27]. Several attempts have been made to improve the level of health promoting compounds in tomato through conventional breeding techniques as well as genetic engineering tools [28,29]. We recently generated transgenic tomato lines with increased biosynthesis of antioxidants such as lycopene, vitamin C and several flavonoids [30]. Particularly, the transgenic lines were generated by overexpression of InsP 5-ptase gene which affects the phosphoinositol stress signaling pathway through changes in the metabolism of InsP3, the key metabolite of the phosphoinositol pathway [31]. We also reported that the increase in metabolism of InsP3 in transgenic plants positively affects the biosynthesis of several flavonoids, such as chlorogenic acid and rutin, by changing the expression level of the main components of the light-signaling pathway that is linked to secondary metabolism in plants [32]. The observed increase in biosynthesis of phenolics and other secondary metabolites with antioxidant properties in InsP 5-ptase overexpressing transgenic plants suggest an increase in health beneficial properties of these transgenic tomato plants. Despite the obvious potential of the genetically enhanced crop plants with enhanced nutraceutical value, general concerns regarding consumption of food products containing genetically modified (GM) ingredients significantly limits the use of GM crops in medicine. In such circumstances, the extraction of desirable pharmaceuticals from GM crops can serve as an alternative approach to the direct consumption of GM crops [33– 35]. These compounds can then be purified and used in medicine as drugs or supplements. Here, we tested the anti-cancer activity of the total metabolite extract containing flavonoids and other phenolic compounds from InsP 5-ptase expressing tomato fruits in vitro. Anti-proliferative effects of extracts obtained from transgenic fruits on breast cancer cell line (MCF-7) were documented by a number of standard assays including cell viability assay, cell morphological analysis, and flow cytometry. Total transcriptome analysis of cancer cells treated with a mix of metabolites extracted from InsP 5-ptase fruits suggested possible pathways involved in anti-cancer effects of applied extracts. To identify metabolites that may play a role in the antiproliferative activity of InsP 5-ptase fruit extracts, we analyzed and compared extracts from wild-type tomato fruits (control) and extracts from transgenic fruits using LC-MS as a powerful and modern metabolomics tool. LC-MS data confirmed the up-regulation of a number of phenolic compounds with strong anti-proliferative potential in InsP 5-ptase fruit extracts. The design of our study is shown in Fig 1. PLOS ONE | https://doi.org/10.1371/journal.pone.0175778 April 27, 2017 2 / 21 Polyphenolic extract of InsP 5-ptas tomato plants reduce the proliferation of cancer cells Materials and methods Plant growth conditions InsP 5-ptase tomato lines were generated and described in details in our previous publication (30). Tomato (cv. Micro-Tom) seeds of control lines (wild-type, empty vector control, and transgenic lines (lines 6 and 7) were germinated in pots containing a combination of 75% Sun Gro Redi-earth ‘Plug and Seedling’ Mix (Sun Gro Horticulture, Bellevue, WA) and 25% sand. The seeds were germinated in a growth chamber under high-light conditions (800 μmol m-2 s-1) with intervals of 16 h light (25˚C) and 8 h dark (22˚C). The red fruits were collected between 6 to 8 weeks of growth under controlled environment and exposure to high-light. The red tomato fruits were immediately frozen in liquid nitrogen after harvest and stored at -80˚C or immediately used in the experiment. For phenolic extraction, fruit samples were immediately lyophilized and stored in the dark environment at room temperature before being used in the experiment. Total phenolic compounds extraction and quantification Total phenolics were extracted based on the method described by Ainsworth and Gillespie (2007) [36]. The colorimetric assay works based on the transfer of electrons in alkaline medium from phenolic compounds to phosphomolybdic/ phosphotungstic acid complexes. A three step sequential aqueous/methanol extractions method was used to extract Polyphenols, hydroxycinnamates, flavonoids, and their glycosides from lyophilized red tomato fruits. One milliliter of the methanol/water (2:1) solution was mixed with 100 mg of each fruit sample after which the samples were vortexed for 30 minutes. Next, the sample extracts were centrifuged at 10, 000 g for 5 minutes at 4˚C and the supernatant was collected. One milliliter of fresh extraction solution was added to the pellet and the above extraction process was repeated twice and at the end of each extraction, the supernatant was collected. It is important to protect the samples from light throughout the extraction process. Folin-Ciocalteau micro method as described by Slinkard et al. (1977) was used to measure the concentration of the phenolic compounds in the extraction solution [37]. Twenty microliters of the tomato extract were diluted in 1.58 ml of H2O and 100 μl of the Folin-Ciocalteau reagent was added to the solution. The mixture was vortexed for 5 minutes after which 300 microliters of sodium carbonate solution (250 μg/ml) were added to the reaction, mixed and incubated for 30 minutes at 4˚C. Following the incubation period, absorbance was read at 765 nm against the blank. A galic acid Fig 1. Overview of applied research strategy. Total metabolites of transgenic tomato fruits were analyzed for their potential anti-cancer properties by application of the total metabolite mixture to MCF-7 cancer cells followed by cell viability assays and transcriptome analysis of the cancer cells. The total tomato wild-type and transgenic fruit extracts were scanned for the potential metabolite with antioxidant and anti-cancer properties by using LC-MS. https://doi.org/10.1371/journal.pone.0175778.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0175778 April 27, 2017 3 / 21 Polyphenolic extract of InsP 5-ptas tomato plants reduce the proliferation of cancer cells calibration curve (0–1 g/L) was used as standard and the flavonoid concentrations were expressed as galic acid equivalents. Fruit tissue preparation for LC-MS analysis Whole, intact tomatoes were pulverized using a mortar and pestle with liquid nitrogen to keep tissue frozen. Approximately 20 mg of frozen tissue powder was then extracted using 80% methanol by using a bead beater for each of the three technical replicates for each line. Samples were then vortexed, sonicated, and centrifuged. The supernatant was filtered through 13 mm syringe filters into microcentrifuge tubes and dried overnight in a 45˚C vacufuge. Samples were then stored at -80˚C for future use. Extraction solution (80% methanol) was used to reconstitute samples to equal volumes. Samples were vortexed and sonicated to ensure all residues had dissolved before they were centrifuged. The supernatant was transferred to a labeled autosampler vial and analyzed immediately. Chromatography and mass spectrometry Samples were analyzed using a Grace C-18 (Grace Davison Discovery Sciences, USA) reverse phase column on an Agilent 1100 series LCMS (Agilent Technologies, Waldbronn, Germany) equipped with a G1379A degasser, G1312A binary pump, G1329A autosampler, G1316A column oven, G13158B diode array detector, and G2445C MS. The aqueous phase was acidified HPLC-grade water (0.05% formic acid), and the organic phase was HPLC-grade methanol. Ten microliter injections were pumped at 0.6 mL/min with the following elution gradient: 0–2 min, 5% B; 2–22 min, 75% B; 22–27 min, 75% B; 27–28 min, 5% B; 28–32 min. A fiveminute wash was included after each sample run. Negative-mode electrospray ionization was used to detect the metabolites by utilizing a trap mass spectrometer scanning of 100–1500 m/z. The target was set at 10,000 and maximum accumulation time at 100.00 ms with two averages. Chemstation software (http://www.agilent.com/en-us/products/software-informatics/ massspec-workstations/lc-ms-chemstation-software), provided with the Agilent machine used to analyze samples and collect data was used to convert files to netCDF format. Further conversion to mzXML format was completed with msConvert (http://proteowizard.sourceforge.net/ tools.shtml) [38]. Files were then loaded into MZmine software (http://mzmine.github.io/) and processed [39]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database, available at http://www.genome.jp/kegg/tool/map_pathway1.html, was used for tentative online compound identification and was completed through MZmine using the gap-filled peak list [40]. Further statistical analysis was carried out by uploading the identified peak list to Metaboanalyst (http:// www.metaboanalyst.ca/) for analysis and by comparison to publications [41–43]. More information regarding data structure can be found in S4 Fig. Cell culture conditions MCF-7 breast cancer cells (ATCC) were seeded in T-75 culture flasks (Thermo Scientific) and maintained in Dulbecco’s modified Eagle‘s medium (DMEM) media, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin. The culture plates were maintained at 37˚C with 5% carbon dioxide. The medium was changed every two days, and the cells were passaged at 80% confluency before the experiment. MTT assay Cells were divided into six groups: blank group (no cells), control group (no treatment) and four experimental groups (WT, EV, L6 and L7 lines extract treatments). Cells were seeded in PLOS ONE | https://doi.org/10.1371/journal.pone.0175778 April 27, 2017 4 / 21 Polyphenolic extract of InsP 5-ptas tomato plants reduce the proliferation of cancer cells 96-well plates 24 hours prior the experiment at the density of 104 cells/well. The next day, the medium was changed and metabolite extract (34 μg/μl was supplemented to the fresh medium. The cells were incubated 24 hours with the medium containing the metabolite extract. After the incubation period, 3-(4,5-dim ethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was then added to each well at the concentration of 5 mg/ml. The cells were incubated for 4 hours, after which the supernatant was replaced with 200 μL of dimethyl sulfoxide (DMSO). The absorption was measured at 570 nm using a micro-plate reader. The results were presented as OD 570–620 using the following formula: MTT OD 570–620 = (Mean A 570–560)—(Mean A of Blank) / (Mean A Negative Control)—(Mean A of Blank). The results are based on two independent experiment with each experiment consisting of 3 technical replicates. Double immuno-staining and microscopy MCF-7 were seeded into each well of Lab-Tek 2 chamber slide (Thermo Scientific Nunc. NY) and incubated for 4 hours at 37˚C (5x105 cells in each chamber) in a humidified, 5% carbon dioxide atmosphere to attach. Cells were divided into five groups: control group (no treatment) and four experimental groups (WT, EV, L6 and L7 lines extract treatments). Total metabolite extract (34 μg/μl) was then added to fresh DMEM medium and applied to the wells and incubated for 24 hours. After incubation, cells were washed once with phosphate buffer saline (PBS) and stained with 1% Acridine orange/Ethidium bromide solution in PBS for 1 minute. Chambers were then washed two times with PBS after which slides were detached from the chamber and air dried. Images were then taken by fluorescence microscopy. A Nikon Eclipse 90i microscope equipped with a 12V-100W halogen lamp, external transformer, flyeye lens built-in and NCB11, ND8, ND32 filters, was used to visualize the stained samples. The following Nikon filters were used: barrier filter BP365, reflector filter FT 395 and exciter filter LP395. Live/dead cells were counted using Image J software. Live cells fluoresce green (FITC/ green) and dead cells fluoresce red/orange (Texas Red/red). Flow cytometry analysis Cells of MCF-7 breast cancer cell line were seeded in 6-well plates at a density of 5 x105 cells per well and incubated for 24 hours at 37˚C in an incubator with 5% carbon dioxide to attach. After the initial seeding, the cells were incubated with fresh medium containing 34 micrograms per microliters of extract and were incubated for 24 hours at 37˚C. Next day, cells were trypsinized and collected in 2 ml tubes. Samples were centrifuged for 15 minutes at 1,300 rpm at 37˚C. The supernatant was discarded and the cells were resuspended by addition of cold PBS. Samples were briefly vortexed, transferred to Flow Cytometry tubes, and then placed on ice. Components of YO-PRO kit (Life Technologies) were used for labeling the samples. One sample was kept as a control (without label). One microliter of the YO-PRO -1 stock solution (Component A) was added directly to the mixture in each tube followed by one microliter of the propidium iodide (PI) stock solution (Component B). Labeled tubes were incubated on ice for 30 minutes. Cells were analyzed by flow cytometry using BD LSRFortessa Cell Analyzer (BD biosciences, CA) (to detect green (YO-PRO-1) and red (propidium iodide) signals. Gene expression analysis using microarray (Affymetrix platform) Breast cancer cell line MCF-7 cells were seeded in 6 well plates at a concentration of 106 cells/ well, 24 hours prior to the experiment. Afterwards, cells were washed two times with warm media and were incubated for 24 hours with fresh medium containing tomato extracts from different plant lines (WT, EV, L6, L7) at a concentration of 34 μg/μl. RNeasy Mini Kit (Qiagen Sciences, Maryland, USA) was used to isolate RNA samples with modification of standard PLOS ONE | https://doi.org/10.1371/journal.pone.0175778 April 27, 2017 5 / 21 Polyphenolic extract of InsP 5-ptas tomato plants reduce the proliferation of cancer cells Qiagen protocol. Cells were disrupted briefly by using Trizol reagent (Ambion, Grand Island, NY), and total RNA was extracted by using chloroform extraction method. After the RNA purification, on-column DNA digestion using the RNase-free DNase Kit (Qiagen Inc. Valencia, CA) was used to remove the residual DNA. The purity of RNA samples was confirmed by electrophoresis and the concentration was quantified by using Nanodrop spectrophotometer (Thermo Scientific. Wilmington, DE). Affymetrix Human Genome Arrays were used as the microarray platform. Biotinylated cRNA targets were synthesized by Affymetrix IVT Express target labeling assay as specified in the Affymetrix GeneChip Expression Analysis Technical Manual. Hybridization reactions to the Affymetrix Human GeneChips were carried out by Expression Analysis, Inc. (Durham, NC). Statistical analysis The cell viability data were analyzed by Tukey’s test and expressed as mean ±S.D. by which the significant differences (P value < 0.05) between groups were determined. To analyze the microarray raw data, column-wise normalization using a reference sample (control)was applied. The resulting data was then visualized using Multi Experiment viewer (Mev). The data was further analyzed by ANOVA and Tukey test with a p-value of 0.001 while assuming variance between variables are equal. The genes with significantly different expression were clustered by hierarchical clustering. In addition, all the known and unknown genes with changes in expression were functionally analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 and PANTHER for gene classification. Microarray data were deposited in GEO database (GEO number: GSE94548). Results Effect of InsP 5-ptase overexpression in tomatoes on total phenolic content in transgenic fruits InsP 5-ptase overexpressing (two independent transgenic lines) and control (two control lines) tomato fruits were tested for their total content of metabolites with phenolic nature. Results of the experiment demonstrated that the fruits of transgenic tomato lines contain more phenolic compounds (37% for L6, 50% for L7) compared to control lines (Fig 2). These results confirm Fig 2. Total phenolic content in mature (red) InsP 5-ptase expressing and wild-type tomato fruits. (A) Standard curve using galic acid (0–1000 mg) as a standard reagent. (B) Total phenolic content in the mature tomato fruits of InsP 5-ptase transgenic lines (L6 and L7) and control lines (WT and EV). The total phenolic content is expressed as mg/g galic acid equivalent. The different letters (a,b) means statistically different groups (p
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