Chemoprevention of Colorectal Cancer by Artocarpin, a Dietary

Artocarpus heterophyllus is an evergreen tree distributed in tropical regions, and its fruit (jackfruit) is well-known as the world's largest tree-bor...
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Chemoprevention of Colorectal Cancer by Artocarpin, a Dietary Phytochemical from Artocarpus heterophyllus Guochuan Sun,† Zongping Zheng,† Mee-Hyun Lee,‡ Yijuan Xu,† Soouk Kang,‡ Zigang Dong,‡ Mingfu Wang,§ Zhennan Gu,† Haitao Li,*,† and Wei Chen*,†,⊥ †

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China The Hormel Institute, University of Minnesota, Austin 55912, United States § School of Biological Sciences, The University of Hong Kong, Hong Kong, China ⊥ Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048, China ‡

S Supporting Information *

ABSTRACT: Artocarpus heterophyllus is an evergreen tree distributed in tropical regions, and its fruit (jackfruit) is well-known as the world’s largest tree-borne fruit. Although A. heterophyllus has been widely used in folk medicines against inflammation, its potential in cancer chemoprevention remains unclear. Herein we identified artocarpin from A. heterophyllus as a promising colorectal cancer chemopreventive agent by targeting Akt kinase. Phenotypically, artocarpin exhibited selective cytotoxicity against human colon cancer cells. Artocarpin impaired the anchorage-independent growth capability, suppressed colon cancer cell growth, and induced a G1 phase cell cycle arrest which was followed by apoptotic as well as autophagic cell death. Mechanistic studies revealed that artocarpin directly targeted Akt 1 and 2 kinase activity evidenced by in vitro kinase assay, ex vivo binding assay as well as Akt downstream cellular signal transduction. Importantly, oral administration of artocarpin attenuated colitis-associated colorectal tumorigenesis in mice. Taken together, artocarpin, a bioactive component of A. heterophyllus, might merit investigation as a potential colorectal cancer chemopreventive agent. KEYWORDS: Artocarpus heterophyllus, artocarpin, colorectal cancer, Akt

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Akt could directly phosphorylate Bad to promote cell survival,10 and PI3K/Akt signaling is required for anchorage-independent growth ability which is one of malignant characteristics of cancer cells.11 Therefore, great interest exists in the development of novel PI3K/Akt inhibitors for both CRC chemoprevention and therapy.12 During the course of chemopreventive agents identification, dietary phytochemicals draw extensive attention for their relative safety.13 Artocarpus heterophyllus is an evergreen tree distributed in tropical regions and famous for its fruit (jackfruit) as the world’s largest tree-borne fruit.14 A. heterophyllushas been widely used as traditional folk medicine against inflammation, diarrhea, ulcers, and tapeworm infection, but its potential in chemoprevention remains largely unknown.15 In our previous study, the extracts of A. heterophyllus were prepared and evaluated for in vitro cytotoxicity against a panel of human cancer cells. On the basis of bioassay-guided fractionation, artocarpin was identified as the active principle.16 The objective of this study was to examine the potential of artocarpin in colon cancer chemoprevention. Herein, we reported that artocarpin attenuated colitis-associated colorectal tumorigenesis in mice without any obvious systemic toxicity. On the basis of the evidence from three different subsequent methodological approaches

INTRODUCTION Colorectal cancer (CRC) is the third most common cancer in the United States in 2016.1 Despite major improvements in early detection and chemotherapeutic regimens, little change in CRC mortality has occurred over the past 50 years. Fortunately, colorectal carcinogenesis is a multistep process that offers opportunities for preventive interventions.2 The critical role of cyclooxygenase-2 (COX-2) has been well-established in CRC, and selective COX-2 inhibitors have long been regarded as an elegant example of from bench to bedside for colorectal cancer chemoprevention.3 However, recent clinical trials indicated that long-term COX-2 inhibition might increase the risk of cardiovascular events,3,4 and animal studies further established that COX-2 plays a cardioprotective role in the late phase of ischemic preconditioning. Therefore, identifying novel preventive agents is thought to be an essential step forward, which is largely dependent upon the discovery of precise molecular targets for colorectal cancer chemoprevention.5 The phosphoinositide 3-kinase (PI3K)/Akt pathway have been recently implicated in the etiology of either sporadic or colitis-associated CRC.6−8 For example, PIK3CA mutations were high frequently in CRC patients (32%). Cell proliferation self-sufficiency and avoidance of apoptosis are common events during colon carcinogenesis,9 and thus mutant PIK3CA is likely to function as an oncogene through activation of its downstream signal transduction cascade including Akt, mTOR, Bad, and β-catenin. PI3K/Akt signaling might cooperate with Wnt to activate β-catenin at the early stage of colorectal tumorigenesis,7 © 2017 American Chemical Society

Received: Revised: Accepted: Published: 3474

January 26, 2017 April 7, 2017 April 9, 2017 April 9, 2017 DOI: 10.1021/acs.jafc.7b00278 J. Agric. Food Chem. 2017, 65, 3474−3480

Journal of Agricultural and Food Chemistry

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(in vitro, ex vivo, and in vivo), artocarpin might exert its chempreventive activity against CRC by directly targeting Akt.

Article

MATERIALS AND METHODS

Materials, Chemicals, and Reagents. Artocarpin was isolated from A. heterophyllus as described previously.16 All primary antibodies were from Cell Signaling Technology (Danvers, MA). Recombinant active Akt 1 and Akt 2 were purchased from Millipore (Billerica, MA). All chemicals and reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Cell Culture. All human colon cell lines (CCD-18Co, DLD1, HCT116, HCT15, HT29, and SW480) were obtained from American Type Culture Collection (Manassas, VA) and maintained following their instructions. Necrotic cell death was determined by trypan blue exclusion test. Cell Viability. Cell viability was measured by MTS assay. CCD-18Co, DLD1, HCT116, HCT15, HT29, and SW480 cells were seeded (5 × 103 cells) in 96-well plates and cultured overnight. After treatment for 48 h, 20 μL of CellTiter96Aqueous One Solution (Promega Corporation, Madison, WI) were added, and the optical density was determined at 492 nm. Cell Growth. Cell growth was measured by MTS assay. HT29 and HCT15 cells were seeded (1 × 103 cells) in 96-well plates with or without drugs. After incubation for various times, 20 μL of CellTiter96Aqueous One Solution were added, and the optical density was determined at 492 nm. Anchorage-Independent Cell Growth. Anchorage-independent growth ability was measured by soft agar formation assay.17 DLD1, HCT115, and HT29 cells (8 × 103) were suspended in BME medium (0.33% agar) and plated over a layer of solidified BME (0.5% agar) in a 6-well plate. After incubation for 14d, and colonies in soft agar were

Figure 1. Cytotoxicity of artocarpin on human colon cancer cells. Human colon adenocarcinoma cells (DLD1, HCT15, HCT116, HT29, and SW480) and normal colon fibroblast cells (CCD-18Co) were treated with artocarpin for 48h, and cell viability was measured by MTS assay. Data are presented as mean ± SEM of three independent experiments.

Figure 2. Effects of artocarpin on the anchorage-independent cell growth. Artocarpin suppressed anchorage-independent growth of human colorectal cancer cells. Colon cancer cells were grown in soft agar for 14 days and colonies counted as described in the Materials and Methods. Data are presented as mean ± SEM of three independent experiments. The asterisks indicate a significant difference compared with each respective control group (***, p < 0.001). 3475

DOI: 10.1021/acs.jafc.7b00278 J. Agric. Food Chem. 2017, 65, 3474−3480

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Journal of Agricultural and Food Chemistry counted by using a microscope with the Image-Pro Plus software program (Media Cybernetics, Rockville, MD). Flow Cytometry Analysis. HT29 cells (2.5 × 105) were seeded on 60 mm plates and cultured overnight for attachment. After drug treatment, cells were collected and fixed in 70% ethanol and stored at-20 °C overnight. Propidium iodine staining of DNA was conducted to determine cell-cycle distribution using a BD FACSCalibur Flow Cytometer (San Jose, CA). In Vitro Kinase Assay. The assay was conducted at 30 °C for 30 min with either active Akt 1 or 2 protein (30 ng) and different doses of artocarpin. Crosstide was used as substrate peptide (30 μmol/L). After reaction, 20 μL aliquots were transferred onto p81 paper, and radioactive incorporation was determined by using LS6500 scintillation counter from Beckman Coulter (Fullerton, CA). In Vitro Pull-Down Assay. Artocarpin-Sepharose 4B beads were prepared following GE Healthcare Biosciences’s protocol. HT29 cell lysates (500 μg) were incubated with Artocarpin-Sepharose 4B beads (100 μL, 50% slurry) or Sepharose 4B beads only at 4 °C with gentle rocking overnight, and proteins bound to the beads were analyzed by Western blotting. In Vitro COX Enzyme Assay. The inhibition of artocarpin on COX activity was evaluated by using a COX Inhibitor Screening Kit from Cayman Chemical Company (Ann Arbor, MI) according to the manufacturer’s instructions. Celecoxib was used as positive control. Western Blot. Proteins (30 μg) were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After blocking, the membranes were hybridized with primary antibodies overnight at 4 °C. After further hybridization with a secondary antibody, the protein bands were visualized using a chemiluminescence reagent from GE Healthcare Biosciences (Pittsburgh, PA) Mouse Colitis-Associated Colorectal Tumorigenesis Model. An mouse colitis-associated colon carcinogenesis model was adopted for colorectal cancer chemoprevention study as described previously.18 Male Balb/c mice (5-week-old) were injected i.p. with 10 mg/kg azoxymethane (AOM). After 1 week, dextran sodium sulfate (DSS) at 3% was administered in the drinking water for 7 days, and then mice allowed to recovery for 14 days. Such cycle above was repeated twice. Artocarpin (100 mg/kg) or vehicle (DMSO) was administered by gavage every day. Mice were euthanized at week 16. The colons were harvested, flushed with PBS and opened longitudinally along the main axis. Polyps were counted using a stereomicroscope. After gross examination, the colons were cut into pieces at about 1 cm intervals and fixed in formalin (pH 7.4). Fixed tissues were then embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin according to standard protocols. Immunohistochemistry staining for phosphorylated (p)-Akt (Ser 473), p-GSK3β (Ser 9), or p-Bad (Ser136) was performed using an ABC complex kit (PK-6100, Vector Laboratories, Burlingame, CA). Sections were counterstained with Harris’s hematoxylin. For antibody-negative controls, the primary antibodies were substituted with normal rabbit serum. Statistical Analysis. All values are expressed as mean values ±SEM unless indicated otherwise. Statistical analysis was performed by using the Prism 5.0 statistical package. Turkey’s multiple comparison posthoc test was used to compare data between 2 groups, one-way ANOVA and the Bonferroni correction were used to compare data between 3 or more groups, and p < 0.05 was considered significant.

selectivity, artocarpin might be a potential colorectal cancer chemopreventive agent. Artocarpin Impaired the Anchorage-Independent Growth Capability. Anchorage-independent growth ability is a characteristic feature of cancer cells.9 Compared with control group, artocarpin treatment resulted in a significant dosedependent reduction in colony formation in soft agar (Figure 2). It should be pointed out that artocarpin suppressed anchorageindependent growth might not due to general cytotoxicity as the effective concentration range for inhibiting colony formation only weakly affected cell viability. For example, artocarpin at 5 μmol/L inhibited anchorage-independent growth more than 70% in tested cell lines. All of those findings indicated that artocarpin dramatically impaired the malignant potential of human colon cancer cells. Artocarpin Induced Apoptosis and Autophagy. To address the mechanism underlying the antitumor activity of artocarpin, we examined the effects of artocarpin on cell deaths. We noticed that after drug treatment, colon cancer cells still excluded trypan blue, a finding that excluded the possibility of

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RESULTS Cytotoxicity of Artocarpin on Human Colon Cancer Cells. Initially, we examined the cytotoxicity of artocarpin in a panel of human colorectal cell lines (Figure 1). Artocarpin exhibited potent cytotoxicity against human colon cancer cells with IC50 values at around 15 μmol/L. In contrast, nonmalignant human colon fibroblast cells (CCD-18Co) were much less sensitive to the cytotoxic effects of artocarpin at a similar concentration. In addition, artocarpin also suppressed colon cancer cell growth in a dose-dependent manner (Figure S1 of the Supporting Information, SI). In terms of potency, spectrum, and

Figure 3. Effects of artocarpin on apoptosis and autophagy. A, Human colon adenocarcinoma cells were treated with artocarpin as described. After treatment, colon cancer cells were lysed for Western blot. B, Neither caspase inhibitor nor autophagy inhibitor reversed cell viability reduction induced by artocarpin. HT29 cells were treated with artocarpin at 20 μM for 48 h in the presence of pan-caspase inhibitor (Z-VAD-FMK) or autophagy inhibitor (3-MA) at 10 μM, a concentration at which they completely abolished the occurrence of apoptosis and autophagy. Cell viability was evaluated by MTS assay. Data are presented as mean ± SEM (n = 6) of are presentative experiment performed in triplicate. 3476

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Figure 4. Artocarpin induces G1 phase cell cycle arrest. A, Artocarpin induced G1 phase cell cycle arrest. B, Quantitation of cells in the sub-G1 phase. HT29 cells were treated with DMSO (0.1%, v/v) or artocarpin as described. After treatment, cells were collected and stained by propidium iodide (PI) and subjected to flow cytometry for cell cycle analysis.

general necrosis.19 Further study suggested that artocarpin potently induced apoptosis and autophagy, evidenced by the cleavage of PARP and the up-regulation of LC3B expression (Figure 3A). In terms of sensitivity and cellular response, HT-29 was selected for subsequent mechanistic study, and treated with artocarpin at 20 μmol/L, at which inhibits 50−75% cell viability. On the basis of the findings above, we questioned whether apoptosis or autophagy afforded for artocarpin associated cell viability reduction. Accordingly, we pretreated HT29 cells with pan-caspase inhibitor (Z-VAD-FMK) or autophagy inhibitor (3-methyladenine, 3-MA) at 10 μM, a concentration at which they completely abolished the occurrence of apoptosis and

autophagy (Figure 3B). To our surprise, Both of them failed to reverse viability reduction by artocarpin. Artocarpin Induced G1 Phase Cell Cycle Arrest. Next, we checked the effect of artocarpin treatment on cell cycle distribution. As in Figure 4, artocarpin induced a significant G1 phase cell cycle arrest which was followed by apoptosis, evidence by the sub-G1 phase. Most likely, the persistent G1 phase cell arrest mediated antiproliferative effect of artocarpin, and the following apoptotic and autophagic events should be secondary events of such mitotic block.20 Artocarpin Targeted Akt 1 and Akt 2 Kinase Activity. To identify potential target of artocarpin, in silico screening was conducted by using a reverse-docking approach. Among candidates 3477

DOI: 10.1021/acs.jafc.7b00278 J. Agric. Food Chem. 2017, 65, 3474−3480

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Figure 5. Artocarpin directly targets Akt. A, Artocarpin inhibits Akt 1 and Akt 2 kinase activity in vitro. Data are presented as means ± SEM (n = 4).The asterisks indicate a significant difference compared with control (***, p < 0.001). B, Artocarpin binds to Akt in HT29 cells. C, Artocarpin inhibits Akt activation and the downstream effect or pathway. HT29 cells were treated with artocarpin at 20 μM for 48 h. After treatment, colon cancer cells were lysed for Western blot. D, Effect of artocarpin on EGFR signaling transduction. After starvation for 24 h, HT29 were stimulated with EGF (10 ng/mL) for 15 min. Cell lysates were subjected to Western blot analysis.

established that PI3K/Akt activation might be a common event in colorectal tumorigenesis, whereas it was attenuated by artocarpin intake evidenced by a substantial inhibition of p-Akt (Ser473) or its downstream signaling molecules such as p-GSK3β (Ser 9) and p-Bad (Ser136).

predicted, Akt attracted our attention for its implication both in G1 phase cell cycle arrest and the etiology of CRC.20 We then performed in vitro kinase assay and established that artocarpin potent suppressed Akt 1 and 2 kinase activities (Figure 5A). Artocarpin could bind to endogenous Akt, evidenced by an observation of Akt binding with artocarpin-Sepharose 4B beads, but not with Sepharose 4B beads alone (Figure 5B). Consistent with findings above, artocarpin also affected p-Akt (Ser473) and p27Kip1 protein expression (Figure 5C), which normally functions as an integral brake of the G1/S transition.20 Artocarpin Suppressed Colitis Associated Colorectal Tumorigenesis in Mice. We eventually examined the potential of artocarpin in colon cancer chemoprevention in vivo by using a colitis associated colon tumorigenesis animal model (Figure 6). In this connection, we observed that oral administration of artocarpin for 16 weeks significantly increased the survival rate, and reduced the multiplicity of colonic neoplasms by 56% (P < 0.001). Importantly, our immunohistochemistry data

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DISCUSSION PI3K/Akt pathway has been recently implicated in CRC, and thereby has attracted considerable attention as an oncology drug discovery target. In the present study, we identified artocarpin from A. heterophyllus as a natural Akt inhibitor. Although it might partly explain why artocarpin attenuated colitis-associated colorectal tumorigenesis in animal study, whether this is a primary association might remain unclear. For example, in addition to Akt and its downstream signaling molecules, artocarpin was also affected the phosphorylation of AMPK, MEK, and ERK as well as EGFR downstream signaling cascades (Figure 5D) a finding that pointed to alternate mechanisms at play. Considering the fact that 3478

DOI: 10.1021/acs.jafc.7b00278 J. Agric. Food Chem. 2017, 65, 3474−3480

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Figure 6. Artocarpin attenuated colitis-associated colon carcinogenesis. Chemopreventive activity of artocarpin on colorectal carcinogenesis was evaluated in Balb/c mice as described in the Materials and Methods. A, Experimental protocol for colitis-associated colon carcinogenesis model. B, Effect of artocarpin on body weight of mice. C, Effect of artocarpin on survival ratio of mice. D, Representative hematoxylin and eosin staining. E, Effect of artocarpin on the ratio of colon length to weight. F, Effect of artocarpin on the burden of colonic neoplasms. Data are presented as mean values ± SEM (n = 15). The asterisks indicate a significant difference compared with control (***, p < 0.001). G, Effect of artocarpin on the expression patterns of p-Akt (Ser437), p-GSK3β (Ser 9) and p-Bad (Ser136) in colon mucosa. Original magnification: 200×. Representative photographs for each antibody and each group are shown.

nonmalignant human colon fibroblast cells (CCD-18Co) at concentrations greater than 25 μmol/L. Actually, those data should be interpreted carefully as CRC is believed to arise from colonic epithelial mucosa.2 To this end, the relative toxicity of artocarpin should be evaluated in normal colon epithelial cells rather than fibroblasts. Moreover, the herbal parent of artocarpin, A. heterophyllus, has been widely used in folkmedicines for many years without untoward toxicity. During the entire period of this study, artocarpin was also well tolerated in mice. Therefore, while speculative, artocarpin might be relatively nontoxic as well. Another caveat of this study is that although it works well in vitro, the doses of artocarpin required to prevent colorectal tumorigenesis may be too high. Chemically, artocarpin belongs to flavonoids, and thus phenomenon above might partly due to its poor water-solubility. In further rational drug design, the introduction of the phosphate ester moiety or the boronic acid group is strongly suggested. In summary, the present study demonstrated that artocarpin derived from A. heterophyllus effectively prevented colorectal tumorigenesis and might merit further investigation as a colorectal cancer chemopreventive agent. Our data suggested that the observed chemopreventive effect of artocarpin on CRC might, at least in part, be explained by targeting the pro-survival PI3K/Akt/p27Kip1 signaling axis.

A. heterophyllus has been widely used as traditional folk medicine against inflammatory diseases, the chemopreventive activity of artocarpin might partly due to its anti-inflammatory properties. In this connection, artocarpin was found to be a COX inhibitor with a mild to moderate COX-1 selectivity index (Figure S2). Taken together, most likely, artocarpin might serve as a multitarget drug in colorectal cancer chemoprevention. Although our current studies are intriguing, several issues still need to be further addressed. For example, our data were mainly based on cell and animal model studies, and thus the first and most important question is whether those findings accurately predict the chemopreventive efficacy of artocarpin in human trials. To verify whether artocarpin-rich food intake would better serve the interest of high-risk colorectal cancer patients, clinical studies need to be performed. Even cell lines are commonly adapted to explore mechanistic relevant of diseases, translatability of such results is still a major concern given that in vivo system would be much more complex. To further elucidate mechanism of action of artocarpin, more rigorous experiments should be conducted to clarify its pharmacokinetics properties in vivo as well as its potential effect on inflammation. Another issue is the relative efficacy and toxicity of artocarpin in chemoprevention. In this study, we observed that artocarpin possessed substantial toxicity to 3479

DOI: 10.1021/acs.jafc.7b00278 J. Agric. Food Chem. 2017, 65, 3474−3480

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00278. Figure S1. Artocarpin lowered human colon cancer cell growth. HT29 and HCT15 cell growth was evaluated by MTS assay as described in the Materials and Methods. Data are presented as mean values ± SEM (n = 4). The asterisks indicate a significant difference compared with Control group (***, p < 0.001). Figure S2. Artocarpin inhibits COX activity in vitro. The inhibitory activity of artocarpinon COX-1 and -2 was evaluated using a COX Inhibitor Screening Kit (Cayman) according to the manufacturer’s instructions. Data are presented as mean values ± SEM (n = 4). The asterisks indicate a significant difference compared with Control group (*, p < 0.05; ***, p < 0.001) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-510-85197302, E-mail: [email protected] (H.L.). *Tel: +86-510-85912087, E-mail: [email protected] (W.C.). ORCID

Wei Chen: 0000-0003-3348-4710 Author Contributions

W.C., H.L., Z.G., M.W., and Z.D. designed and supervised the experiments. G.S. and H.L. prepared the manuscript. G.S., H.L., Z.Z., Y.X., M.-H.L., and S.K. performed experiments. Funding

This work was supported by the National Natural Science Foundation of China (81402366, 31530056, National Youth 1000 Talents Plan), Jiangsu Specially Appointed Professor, and the Fundamental Research Funds for the Central Universities (JUSRP11549 and JUSRP51501). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AOM, azoxymethane; COX-2, cyclooxygenase-2; CRC, colorectal cancer; DSS, dextran sodium sulfate; EGFR, the epidermal growth factor receptor; PARP, poly(ADP-ribose) polymerase

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REFERENCES

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DOI: 10.1021/acs.jafc.7b00278 J. Agric. Food Chem. 2017, 65, 3474−3480