Resveratrol suppresses IGF-1 induced human colon cancer cell proliferation and elevates apoptosis via suppression of IGF-1R/Wnt and activation of p53 signaling pathways
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Obesity is a global phenomenon and is associated with various types of cancer, including colon cancer. There is a growing interest for safe and effective bioactive compounds that suppress the risk for obesity-promoted colon cancer. Resveratrol (trans-3, 4', 5,-trihydroxystilbene), a stilbenoid found in the skin of red grapes and peanuts suppresses many types of cancers by regulating cell proliferation and apoptosis through a variety of mechanisms, however, resveratrol effects on obesity-promoted colon cancer are not clearly established.
We investigated the anti-proliferative effects of resveratrol on HT-29 and SW480 human colon cancer cells in the presence and absence of insulin like growth factor-1 (IGF-1; elevated during obesity) and elucidated the mechanisms of action using IGF-1R siRNA in HT-29 cells which represents advanced colon carcinogenesis.
Resveratrol (100-150 μM) exhibited anti-proliferative properties in HT-29 cells even after IGF-1 exposure by arresting G0/G1-S phase cell cycle progression through p27 stimulation and cyclin D1 suppression. Treatment with resveratrol suppressed IGF-1R protein levels and concurrently attenuated the downstream Akt/Wnt signaling pathways that play a critical role in cell proliferation. Targeted suppression of IGF-1R using IGF-1R siRNA also affected these signaling pathways in a similar manner. Resveratrol treatment induced apoptosis by activating tumor suppressor p53 protein, whereas IGF-1R siRNA treatment did not affect apoptosis. Our data suggests that resveratrol not only suppresses cell proliferation by inhibiting IGF-1R and its downstream signaling pathways similar to that of IGF-1R siRNA but also enhances apoptosis via activation of the p53 pathway.
For the first time, we report that resveratrol suppresses colon cancer cell proliferation and elevates apoptosis even in the presence of IGF-1 via suppression of IGF-1R/Akt/Wnt signaling pathways and activation of p53, suggesting its potential role as a chemotherapeutic agent.
KeywordsResveratrol Colon Cancer Cell Human Colon Cancer Cell Suppress Cell Proliferation Resveratrol Treatment
Insulin like growth factor 1
T cell transcription factor 4
Murine double minute 2
Insulin like growth factor 1 receptor
Forkhead (Drosophila) homolog (rhabdomyosarcoma) like 1
Phosphoinositide 3 kinase
Protein kinase B
Glycogen synthase kinase 3 beta.
Nearly two-thirds of Americans are overweight or obese , and obesity elevates the risk for colon cancer [2, 3, 4, 5, 6, 7, 8, 9], the second leading cause of cancer deaths in the United States . Surgery, radiation and chemotherapy alone or in combination are the most common methods to treat different types of cancers, including colon cancer. These conventional therapies can prolong a patient's life span, but they may cause serious side-effects. Since colon cancer takes 10-20 years to progress beyond initiation stage , there exists an opportunity to prevent colon cancer progression through appropriate nutrition and exercise. A recent meta-analysis found that a 5 kg/m2 increase in body mass index (BMI) raises colon cancer risk by 24% in men . Thus, it is imperative to intensify our efforts to better understand the pathogenesis of colon cancer during obese conditions and try to develop novel, evidence-based and safe approaches for its prevention and treatment.
The adipokines and growth factors are involved in a wide range of physiological processes including general homeostasis, cell growth, metabolism, and are frequently deregulated during obese conditions . Over-activation of the IGF system is also commonly observed in obese conditions and plays a critical role in obesity-promoted colon cancer . IGF-1 has been linked to tumor growth in adults in a number of studies, including two large trials jointly conducted by Harvard Medical School and Brigham and Women's Hospital. A six-year study of 32,826 nurses, found that those with the highest levels of IGF-1 had a two-and-a-half times greater risk of colorectal cancer; another study of 14,916 male physicians also concluded that men run the same risk [15, 16]. This IGF system includes ligands, receptors, and ligand-binding proteins (IGFBPs). Over-nourishment and chronic hyperinsulinemia observed in obese conditions may deregulate colonocyte growth kinetics, as elevated insulin and suppressed IGFBP-1, and IGFBP-2 levels increase the pool of free or bioavailable IGF-1. A larger pool of bioavailable IGF-1 can activate the IGF-1 receptor (IGF-1R), thus may stimulate colonocyte proliferation [14, 17, 18, 19]. IGF-1R is over expressed during colon carcinogenesis, with the highest expression in the proliferating cells at the base of the colonic crypts . Upon IGF-1 binding, IGF-1R activates the PI3K/Akt cascade, which promotes G1 to S cell cycle progression  and elevates cell proliferation . One of the pathways activated by the PI3K/Akt cascade is the Wnt/β-catenin pathway . The Wnt/β-catenin pathway plays a central role in elevating colonocyte proliferation  and suppressing apoptosis in both humans and rodent models of experimentally induced colon cancer [25, 26]. Akt phosphorylates and inactivates GSK3β, allowing β-catenin, a downstream effector of Wnt pathway to translocate into the nucleus where it binds to TCF-4. The β-catenin/TCF-4 complex constitutes the master switch that regulates colonocyte proliferation .
Resveratrol, a stilbenoid derived mainly from the skin of grapes, is a potential dietary compound against various cancers - breast, colon, etc., in vitro and in vivo [28, 29]. Resveratrol works at a very low concentration in vivo (< 2 μM). However, in vitro studies require a much higher concentration (at least 25-50 μM) to elicit similar chemotherapeutic action . In a variety of cancer cells, resveratrol suppressed critical components of the PI3K/Akt pathway [31, 32, 33]. Recent reports suggest that resveratrol also promotes phosphorylation of p53 in a dose and time-dependent manner in human breast cancer cells . This impairs ability of p53 to bind to its negative regulator, MDM2 . Thus p53 is active and can cause either cell cycle arrest or apoptosis [34, 35]. P53 suppresses IGF-1R gene expression [36, 37] in Saos-2 (osteosarcoma-derived cells) and RD (rhabdomyosarcoma-derived cells) cells . Resveratrol also has been shown to suppress IGF-1 levels in mice consuming a high-fat diet . Low concentrations of resveratrol (< 40 μM) have also shown to inhibit Wnt signaling in colon cancer cells . However, the inhibitory efficacy of resveratrol against IGF-1 induced colonocyte proliferation and the molecular mechanisms of its action are not yet fully elucidated. We hypothesized that resveratrol suppresses colon cancer cell proliferation and elevates apoptosis after IGF-1 exposure through activation of p53 and suppression of IGF-1R/Akt/Wnt signaling pathways. In order to establish the efficacy of resveratrol against IGF-1 induced colon cancer growth, we utilized HT-29 and SW480 (IGF-1R constitutively active) human colon cancer cell lines and treated them with/without IGF-1 and/or resveratrol.
In this study, resveratrol at 100-150 μM suppressed IGF-1R/Akt and Wnt/β-catenin signaling pathway proteins in the HT-29 colon cancer cell lines. Similar results were observed with IGF-1R siRNA treatment. Resveratrol also activated p53 protein and suppressed levels of sp1, a protein that transcriptionally activates IGF-1R. Resveratrol elevated unphosphorylated (active) form of forkhead transcription factor (FKHRL1 protein), thereby contributing to cell cycle arrest. Overall our results demonstrate that resveratrol (i) suppresses IGF-1R levels, thus, downregulates Akt and Wnt/β-catenin signaling; (ii) elevates levels of active FKHRL1 and p27, and concomitantly suppresses cyclin D1 levels and (iii) activates p53 and suppresses sp1, thus suppressing cell cycle progression and elevating apoptosis in vitro even in the presence of free mitogenic IGF-1.
IGF-1 was obtained from the R&D Systems (Minneapolis, MN). Fetal bovine serum (FBS) was purchased from the Thermo Fisher Scientific (Pittsburgh, PA). All other cell culture supplies and resveratrol were purchased from the Sigma Chemical Company (St. Louis, MO).
HT-29 and SW480 colon cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained at 37°C with 5% CO2 and grown in Dulbecco's Modified Eagle's Medium F-12 (DMEM/F-12) supplemented with 2.2 g/L sodium bicarbonate, 0.2 g/L bovine serum albumin, 50 mL/L fetal bovine serum and 10 mL/L antibiotic and antimycotic solution.
Design of siRNA against IGF-1R
IGF-1R siRNA and inverted control duplex were designed as described  and were purchased from Ambion (Austin, TX). The sequences of IGF-1R siRNA duplex are: sense strand, 5'-CGACUAUCAGCAGCUGAAGTT-3'; antisense strand, 5'-CUUCAGCUGCUGAUAGUCGTT-3'. It is homologous to 168-186 nucleotides of human IGF-1R transcript. 5'-GAAGUCGACGACUAUCAGCTT-3' and 5'-GCUGAUAGUCGUCGACUUCTT-3' are sense and antisense sequences of inverted control duplex, respectively. The construct against IGF-1R mRNA was referred as IGF-1R siRNA, the inverted control or non specific siRNA was referred as siRNA control, and GAPDH siRNA was used as positive control.
HT-29 cells were seeded in DMEM/F-12 supplemented with 5% fetal bovine serum in six-well plates. Cells at 50% confluence were transfected with IGF-1R siRNA duplexes after 20-24 h, using Dharma FECT 4 reagent (Dharmacon Inc., CO) in antibiotic- and serum-free DMEM/F-12 medium. Three concentrations (50, 75 and 100 nM) of IGF-1R siRNA duplexes, 100 nM non specific siRNA and 100 nM GAPDH siRNA were used for initial optimization, and for subsequent assays 75 nM siRNA was used, as there was no difference in IGF-1R inhibition between 75 and 100 nM concentrations. Cells were harvested after 72 h of transfection using 1× lysis buffer, and then protein was extracted. The knockdown of IGF-1R was confirmed by Western blotting.
Cell proliferation assay
Cells were plated at a density of 5 × 104 cells per well in 12-well plates in Dulbecco's modified Eagle's medium F-12 (DMEM/F-12) containing 2.5% charcoal-stripped fetal bovine serum. After 24 h, cells were treated with DMSO (solvent control), 10 nM IGF-1 and/or resveratrol (150 μM), and anti-proliferative properties of resveratrol were evaluated after 24, 48 and 72 h. As colonocytes have greater exposure to the bioavailable IGF-1 in obese condition, in the combined treatments (for all the studies in the manuscript), the cells were pre-incubated with IGF-1 (10 nM) for 5 minutes followed by resveratrol treatment to check the effect of resveratrol when the cells are already primed to proliferate. Dose response studies with IGF-1 (5 - 20 nM) showed that 10 and 20 nM IGF-1 treatments did not differ (p < 0.05) in inducing cell proliferation (data not shown). Thus, we used 10 nM concentration of IGF-1 for subsequent experiments. In case of siRNA experiment, after 24 h of transfection, cells were treated with IGF-1 at 10 nM and/or resveratrol at 150 μM for 24 h. Cells were counted after 24 and 48 h using a Z1 Coulter Counter, Beckman Coulter (Fullerton, CA). Each experiment was carried out in triplicate, and results were expressed as mean ± SE.
The TUNEL (terminal transferase dUTP nick end labeling) assay was performed to determine the effect of resveratrol/IGF-1R siRNA on apoptosis. Cells (4 × 104) were seeded in four-chambered glass slides and after treatment for 12 h, the in situ cell death detection kit, POD (peroxidase), Roche Applied Science (Indianapolis, IN) was used according to the instruction manual protocol for fixed cells to identify apoptotic cells. Slides incubated without terminal deoxynucleotidyl transferase (TdT) served as a negative control, and slides treated with 1000 U DNAse I/ml for 10 min before TdT exposure served as a positive control. After the incubation of cells with POD and diaminobenzidene substrate, the percentage of apoptotic cells was calculated by counting the stained cells in 12 fields, each containing at least 50 cells .
Fluorescence-activated cell sorting analysis (FACS)
HT-29 cells were plated at a density of 15 × 105 cells per 100 mm plate and after 18 h treatment with either the control (DMSO), IGF-1 (10 nM), resveratrol (50 or 150 μM), or their combinations, cells were trypsinized and centrifuged. The pellet was resuspended with 1 ml of PI staining buffer containing 4 mM sodium citrate, 0.1% Triton X-100, 50 μg/ml propidium iodide and 200 μg/ml RNAse and incubated for 10 min at 37°C in the dark, and the final concentration of sodium chloride was adjusted to 0.15 M. Cells were analyzed using FACSCalibur flow cytometer and CellQuest Acquisition software, Becton Dickinson immunocytometry systems (San Jose, CA). Results were reported as per cent cells in each phase of the cell cycle.
Western blot analysis
HT-29 cells were seeded at a density of 1.5 × 105 cells/mL in Dulbecco's Modified Eagle's Medium F-12 with 2.5% charcoal-stripped fetal bovine serum for 24 h. Cells were treated with control, IGF-1, different concentrations of resveratrol (50, 100 and 150 μM) with and without IGF-1 (10 nM) for 24 h. Protein was extracted into a high-salt buffer containing 1% protease inhibitor cocktail from Sigma-Aldrich (St. Louis, MO), and protein concentrations were determined by a BCA Protein Assay kit from Pierce (Rockford, IL). Cell lysates (30 μg) were incubated at 98°C for 5 min and separated by Novex® 8-16% Tris-HCl gels from Invitrogen (Carlsbad, CA) at 120 V for 2 h in 1 × running buffer [25 mmol/L Tris, 192 mmol/L glycine, 0.1% SDS (pH 8.3)], and electrophoretically transferred to Immun-Blot PVDF membranes from Bio-Rad Laboratories (Hercules, CA) at 95 V for 35 min in 2× Tris-glycine transfer buffer (Novex, LC3675, Invitrogen) with 0.025% SDS. PVDF membranes were blocked with 2% bovine serum albumin from Fisher Scientific (Pittsburg, PA) for 1 h at room temperature. The membranes were incubated with rabbit polyclonal anti-sp1 antibody, Upstate Biotechnology (1:1,000; Lake Placid, NY), goat polyclonal anti-β-catenin antibody, rabbit polyclonal anti-GSK3β antibody, mouse monoclonal anti-cyclin D1 antibody, goat polyclonal anti-β-actin antibody, mouse monoclonal anti-MDM2 antibody, rabbit polyclonal anti-p27 antibody, rabbit polyclonal anti-IGF-1Rβ antibody and rabbit polyclonal anti-FKHRL1 antibody, all from Santa Cruz Biotechnology (1:500; Santa Cruz, CA) for 2 h at room temperature. Membranes were subsequently probed with bovine anti-goat, goat anti-rabbit or goat anti-mouse IgG-HRP conjugate secondary antibody, Santa Cruz Biotechnology (all 1:100,000). Target proteins were detected with Super Signal West Dura Extended Duration Substrate from Pierce (Rockford, IL). Membranes were scanned with ChemiDoc XRS (Bio-Rad) using Quantity One software (Bio-Rad) and the band intensities were calculated using Image J software, and normalized to β-actin, a loading control.
Analysis of variance and Fisher least square difference at 5% significance level determined the degree of significance among treatments. The results were expressed as mean ± SE for three replicates for each treatment.
Growth stimulatory/inhibitory effects of IGF-1 and resveratrol
Efficacy of IGF-1R siRNA in suppressing IGF-1 stimulated growth of colon cancer cells
Resveratrol but not IGF-1R siRNA induces apoptosis in HT-29 cells
Resveratrol down regulates IGF-1R signaling proteins
Sp1 is a critical mediator of IGF-1R transcription and upregulates IGF-1R promoter activity [38, 45]. Figure 7B illustrates the effect of IGF-1 and resveratrol on sp1 protein levels. Resveratrol downregulated sp1 protein and suppressed the levels of IGF-1R (Figure 7B, 6B). These results suggest that IGF-1R is an important molecular target for resveratrol in the suppression of colon cancer cell proliferation and also suggests the role of resveratrol in regulating IGF-1R transcription. To elucidate whether or not resveratrol affects the insulin receptor (IR), we determined IR protein levels. We did not observe down regulation of IR receptor, even with a high concentration of resveratrol (data not shown), which confirms selective targeting of IGF-1R by resveratrol.
Resveratrol activates tumor suppressor protein p53 and elevates activated forkhead transcription factor 1 levels
A critical tumor suppressor gene p53 regulates DNA repair, cell cycle progression and apoptosis . In cancer cells, p53 is generally degraded due to ubiquitination by MDM2, a negative regulator of the p53 . Our results show that resveratrol activates p53 and suppresses MDM2 levels in colon cancer cells (Figure 7C). The observed reduction in IGF-1R protein levels in resveratrol treatment may be in part due to p53 activation, because p53 is involved in transcriptional suppression of the IGF-1R gene, and regulation of the IGF-1R promoter activity through suppression of sp1 [38, 45].
FKHRL1, a member of the family of forkhead transcription factors, is a transcriptional activator that induces cell cycle arrest or upregulates apoptosis depending upon cellular context and type of stress . Unphosphorylated FKHRL1 was upregulated in cells treated with resveratrol (Figure 7D). P27, a downstream target of FKHRL1, is a cell cycle regulator protein that binds to and inactivates cyclin D1 . P27 was also upregulated by resveratrol treatment (Figure 7D). Combined, these results suggest that resveratrol may cause cell cycle arrest at the G0/G1-S phase transition via FKHRL1 pathway by upregulating p27 and inactivating cyclin D1.
Phytochemicals affect myriad intracellular targets; it is this quality that often makes them desirable as chemotherapeutic agents against cancer [17, 41, 48, 49, 50]. Studies in both in vitro and in vivo models suggest that resveratrol can suppress components of the IGF system [32, 33], which in turn may suppress cell proliferation and elevate apoptosis. Our results indicate that resveratrol suppresses cell proliferation and induces apoptosis even when the cells are primed to proliferate with IGF-1, a mitogen that is highly bioavailable during obesity. Resveratrol exerts its anti-proliferative and pro-apoptotic properties through suppression of IGF-1R/Akt/Wnt pathways and activation of p53 signaling.
Emerging evidence suggests that members of the insulin-like growth factors (IGFs) family, including IGF-I, IGF-II, IGF-IR, and the IGF-binding proteins (IGFBPs) play a central role in the development and progression of a variety of cancers during obesity, including colon cancer . IGF-1 binding to IGF-1R stimulates downstream proliferating pathways such as the PI3K/Akt  and Ras signaling  resulting in increased human colon cancer cell proliferation, thus suppressing IGF-1R might attenuate proliferation. Resveratrol suppressed IGF-1 stimulated HT-29 colon cancer cell proliferation. Pre-incubation with IGF-1 followed by resveratrol suppressed proliferation lower than that of control and was similar to that of treatment with resveratrol alone. This may be due to resveratrol's combined effect on IGF-1R signaling suppression, apoptosis and multiple other pathways. Similar results were also observed in SW480 cells with constitutively expressed IGF-1R. Indeed, FACS results show that treatment with resveratrol alone or in combination with IGF-1 arrests G0/G1-S phase progression. These results indicate that resveratrol may suppress cellular proliferation even when colon cancer cells are already exposed to high levels of IGF-1 during obese conditions.
Knockdown of IGF-1R using IGF-1R siRNA also suppressed cell proliferation comparable to resveratrol treatment even in the presence of IGF-1. Resveratrol induced apoptosis in HT-29 cells, but IGF-1R siRNA had no effect on apoptosis. However, resveratrol treatment had greater percent apoptosis in IGF-1R siRNA transfected cells compared to non specific siRNA. Whether provided alone or following incubation with IGF-1, resveratrol suppressed the IGF-1R/Akt/GSK3β signaling pathway and concomitantly suppressed nuclear translocation of β-catenin. Once inside the nucleus, β-catenin binds to TCF-4 and transcriptionally activates proliferative (e.g., cyclin D1) genes. Cyclin D1 is known to function as a regulatory subunit of CDK4 or CDK6 (cyclin dependant kinases), whose activity is required for G0/G1-S phase transition . Interestingly, IGF-1 pretreatment potentiated the resveratrol suppression of nuclear β-catenin. This may be because resveratrol targets actively proliferating cells and IGF-1 promotes number of proliferating cells via G1 to S cell cycle progression. This is in accordance with greater resveratrol suppression of proliferation in IGF-1 pretreated cells. IGF-1R siRNA transfection also affected the downstream Akt/GSK3β/β-catenin pathway similar to that of resveratrol. These results suggest that resveratrol exerts its anti-proliferative properties through suppression of IGF-1R, and is similar to IGF-1R siRNA in suppressing HT-29 cell proliferation but not in the induction of apoptosis.
In normal, unstressed cells, p53 levels are low due to continuous MDM2-mediated ubiquitination and degradation. IGF-1R activation further ensures low levels of p53 as the resulting increase in phosphorylated Akt increases the ubiquitination activity of MDM2 . However, p53 phosphorylation prevents p53 from binding to its negative regulator MDM2 . Reports suggest that p53 activation suppresses the IGF-1R mRNA levels . The observed reduction in IGF-1R protein levels in resveratrol treatment may be in part due to p53 activation, because p53 is involved in transcriptional suppression of the IGF-1R gene, and regulation of the IGF-1R promoter activity through suppression of sp1 [38, 45]. Resveratrol treatment exerted anti-proliferative effects without suppressing IR levels (data not shown), suggesting that resveratrol specifically targets IGF-1R in suppressing cell proliferation by activating p53 and suppressing sp1 levels.
Transcriptional activation of FKHRL1 proteins is regulated by the serine/threonine kinase Akt, which phosphorylates FKHRL1 and inactivates it [54, 55]. Induction of apoptosis or withdrawal of growth factors stimulates dephosphorylation and nuclear translocation of FKHRL1, leading to FKHRL1-induced gene-specific transcriptional activation [54, 56, 57, 58, 59, 60]. We studied the effect of resveratrol on FKHRL1. Resveratrol caused up-regulation of unphosphorylated active form of FKHRL1. This active form of FKHRL1 translocates to the nucleus and has been shown to induce apoptosis by upregulation of Fas-ligand expression and activation of the death receptor pathway . P27 is a direct downstream target of FKHRL1 and over expression of cytoplasmic FKHRL1 concomitantly elevates p27, an inhibitor of cyclin D1, thereby affecting cell cycle progression [47, 61]. Our data suggests that resveratrol up-regulates FKHRL1 in the cytoplasm and also increases p27 levels. Over expression of FKHRL1 resulted in an increased expression of p27 and thus potential inhibition of cyclin D1, and concurrent suppression of cell proliferation and cell cycle arrest as observed in this study.
However, resveratrol at lower concentrations (upto 50 μM) is not sufficient to elicit its chemotherapeutic properties in HT-29 cells. A lower dose of resveratrol is not strong enough to induce p53 activation (Figure 7C). As a result, the cancer cells upregulate the survival machinery through activation of survival pathways such as Akt/GSK3β/Wnt signaling at low doses of resveratrol (Figure 6B). However, at lower doses of resveratrol (50 μM), there is no significant effect on proliferation of HT-29 cells compared to control (Figure 1). At higher concentrations (100-150 μM), there is an elevation of activated p53 levels and suppression of IGF-1R/Akt/Wnt signaling pathways leading to suppressed cell proliferation, and enhanced apoptosis. Several studies indicate that for in vitro studies, carcinogenesis-modulating effects of resveratrol require the sustained presence of at least 5 to 100 μM [28, 30]. Even though several preclinical efficacy studies [28, 62, 63] on resveratrol have reported that very low plasma concentrations of resveratrol (20 nM - 2 μM) are sufficient to exert potent cancer chemotherapeutic efficacy and pharmacodynamic activity, such in vivo levels would be insufficient to induce anti-carcinogenic effects in in vitro studies. Detailed studies need to be conducted in order to determine the dose difference in resveratrol's anti-proliferative and pro-apoptotic effects observed in vivo vs. in vitro.
In conclusion, we have shown resveratrol to suppress IGF-1 (IGF-1 levels are elevated during obesity) induced cell proliferation and elevate apoptosis in human colon cancer cells, and elucidated the mechanisms of action using IGF-1R siRNA (Figure 8). Results of the current study will improve our understanding of the molecular pathways involved in the chemotherapeutic intervention of IGF-1 promoted colon tumorigenesis by the bioactive compound resveratrol. Understanding how IGF-1, Wnt/β-catenin and p53 pathways regulate colon cancer cell kinetics may help in the development of new diagnostic markers, prognostic markers, therapeutic targets and/or evidence based safe chemotherapeutic strategies against IGF-1 promoted cancers. We are working on detailed mechanistic studies and generating in vivo data to further decipher the underlying mechanism of the connection between obesity and colon cancer.
This project was supported by American Institute for Cancer Research (AICR 05B094) and National Research Initiative Grant (2009-55200-05197) from the USDA National Institute for Food and Agriculture. We sincerely thank Dr. Shaheen Khan for her critical comments.
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