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Carcinogenesis. 2016 April; 37(4): 385–396.
Published online 2016 January 19. doi:  10.1093/carcin/bgw009
PMCID: PMC4806710

An ornamental plant targets epigenetic signaling to block cancer stem cell-driven colon carcinogenesis


Phytochemicals modulate key cellular signaling pathways and have proven anticancer effects. Alcea rosea (AR; Hollyhock) is an ornamental plant with known anti-inflammatory properties. This study explored its role as an anticancer agent. The AR seed extract (AR extract) inhibited proliferation and colony formation in a dose- and time-dependent manner and promoted apoptosis as was evidenced by cleavage of PARP and increased expression of Bax accompanying reduced levels of BCL-xl protein in HCT116 and SW480 cells, respectively. In addition, AR extract-arrested cells at Go/G1 phase of cell cycle and exhibited decreases in Cyclin D1. AR extract-treated cells exhibited reduced number and size of colonospheres in a dose-dependent manner concomitant with decreases in cancer stem cell (CSC) markers ALDH1A1 and Dclk1. Relative levels of β-catenin, Notch-ICD, Hes1 and EZH2 were also attenuated by AR extract. TOP-flash reporter activity, a measure of Wnt signaling, decreased significantly in response to treatment while overexpression of wild type but not mutant EZH2, reversed the inhibitory effects. Moreover, WIF1 (a Wnt antagonist) promoter activity increased dramatically following treatment with AR extract which phenocopied increases in WIF1 reporter activity following EZH2 knockdown. In vivo, AR extract attenuated tumor growth due probably to reduced levels of EZH2, β-catenin, CyclinD1 and Ki-67 along with reduced levels of CSC markers. Since partial purification via HPLC yielded a prominent peak, efforts are underway to identify the active ingredient(s). Taken together, the results clearly suggest that AR extract/active component(s) can be an effective preventative/therapeutic agent to target colon cancer.


Colorectal cancer (CRC) is the third most common cause of cancer mortality worldwide in both males and females (1). Despite recent advances in therapeutic interventions, about 40% of CRC patients are still expected to die due mainly to metastasis (1) resulting in a 5-year survival rate of <13% (2). Although early stage CRC can be surgically resected, advanced stage CRC frequently recurs and becomes fatal, even in patients receiving combination chemotherapy (3). A cascade of genetic events eventually leads to the development of this deadly disease (4). While some forms of CRC are heritable (5), most sporadic CRC cases are associated with diet and lifestyle (6).

It is being increasingly understood that tumors originate from a small subset of cancer cells, termed cancer stem cells (CSCs), and these are capable of initiating and sustaining tumor growth, as well as promoting cell invasion and drug resistance (7). Several reports support the idea that cancers can be considered as a stem cell disease (8) although recent developments also provide evidence that non-stem cells have the potential to dedifferentiate and to reacquire stem cell properties (9) suggesting that the ‘bottom-up’ or ‘top-down’ models of CRC may not be mutually exclusive. CSCs were first identified in hematologic malignancies and most recently in several solid tumors, including CRC (8). Specific markers have been correlated to the colon CSC phenotype including CD133, CD44, CD166, CD24, aldehyde dehydrogenase 1 (ALDH1A1) and double cortin-like kinase-1 (Dclk1) (10). Both Wnt/β-catenin and Notch signaling pathways play crucial roles in transforming colon epithelial cells into cancer cells and self-renewing CSCs (11,12). Since CSCs have the ability to self-renew and maintain the tumor (13), failure to eliminate CSCs may be critical for metastasis and cancer relapse following therapeutic treatment (14). Therefore, targeting CSCs may represent a key therapeutic strategy to target diseases that are maintained by these CSC populations.

In the past, a large number of substances derived from plants have been studied in antitumor research fields and many have proven to exhibit chemopreventative properties which could be used as adjuvant chemotherapy. Alcea rosea (AR) is an ornamental plant belonging to the Malvaceae family. It is popularly known as Holyhock and is widely grown in gardens and parks in the Southern Europe and Asia. Several pharmacological studies have reported that this plant possesses anti-inflammatory, antibacterial and analgesic effects (15). In Iranian traditional medicine, the roots of AR are used as medicine for a wide range of ailments, including bronchitis, diarrhea, constipation, inflammation, severe coughs and angina (15). In this study, we tested the hypothesis that AR will inhibit proliferation of colon cancer cells and suppress the growth of tumor xenografts by targeting CSCs. Indeed, AR seed extracts blocked colon cancer cell proliferation, promoted cell death via apoptosis and inhibited both Notch and Wnt/β-catenin signaling to inactivate CSCs. In vivo as well, AR seed extracts targeted CSCs to dramatically suppress the growth of tumor xenografts. These results clearly suggest that the systematic use of AR seed extracts and/or compound(s) purified from the extracts can be an effective preventative/therapeutic strategy to target CRC.

Materials and methods

Preparation of extract

The fresh seeds of Alcea rosea were dried at 30°C. The dried material was then powdered by mortar and pestle and passed through a sieve of 0.3mm mesh size. The powder was extracted with ethyl acetate for 48h using Soxhlet apparatus at 64.7°C. The extract was then concentrated with the help of a rotary evaporator under reduced pressure and the solid extract so obtained was stored in refrigerator. At the time of use, stock solution was prepared at a concentration of 50mg/ml in dimethyl sulfoxide.

Cells and cell culture

Human CRC cell lines, HCT116 and SW480 were purchased from American Type Culture Collection (ATCC, Manassas, VA). All the cell lines used in this study were within 20 passages after receipt or resuscitation (3 months of non-continuous culturing). The cell lines were not authenticated as they came from national repositories. Cells were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum (Sigma-Aldrich) and 1% antibiotic–antimycotic solution (Mediatech Inc.) at 37°C in a humidified atmosphere containing 95%O2/5% CO2.

Proliferation assay

Cell proliferation was measured by Hexoseaminidase assay (16). Cells were plated at a density of 5000 per well and incubated overnight in 96-well plates with 10% fetal bovine serum supplemented Dulbecco's modified Eagle's medium culture medium before treatment. Cells were treated with either dimethyl sulfoxide or increasing doses of AR (0–100 μg/ml). The optical density (OD) at 405nm was measured by using a Biotek plate reader (Biotek Instruments Inc). The percentage of cell viability was calculated as ODdrug/ODcontrol × 100%.

Cell cycle analysis

Cells were synchronized by serum starvation for 24h and then cultured in serum-containing medium for another 24h before harvesting for flow cytometric analysis. Cultured cells were trypsinized and fixed with 70% ethanol at 4°C overnight. Cells were permeabilized with phosphate-buffered saline (PBS) containing 1mg/ml propidium iodide (Sigma-Aldrich), 0.1% Triton X-100 (Sigma-Aldrich) and 2mg DNase-free RNase (Sigma-Aldrich) at room temperature. Flow cytometry was done with a FACSVerse cytometer (BD Biosciences, San Jose, CA) capturing 10 000 events for each sample. Histograms were analyzed for cell-cycle compartments, and the percentage of cells at each phase of the cell cycle was calculated using CellQuest (BD Biosciences, San Jose, CA) analysis software.

Apoptosis detection by Annexin V-FITC/propidium iodide staining

Annexin V staining for apoptosis detection was performed according to Van et al. (17). Briefly, 105 cells/ml were cultured overnight and then treated with AR extract for 12, 24 and 48h. At the end of incubation, cells were trypsinized and washed twice with PBS and centrifuged at 200g. Cell pellet was resuspended in binding buffer (10mM Hepes, pH 7.4, 140mM NaCl and 2.5mM CaCl2) containing Annexin V-FITC/PI (Invitrogen, Carlsbad, CA), and was incubated in the dark for 15min at room temperature. The binding buffer was added to the stained cells and cells were analyzed immediately by FACSVerse (BD Biosciences, San Jose, CA) analysis. At least 10 000 counts were recorded in each analysis.

Clonogenicity assay

For clonogenic assays, single-cell suspensions were generated for each cell line and 500 viable cells were seeded into six-well tissue culture plates. Cells were allowed to adhere for 16h and then treated with AR extract for 48h. Cells were then washed with PBS and incubated in fresh medium for 5–8 days after seeding, depending on each individual cell line. The colonies obtained were washed with PBS and fixed in 10% formalin for 10min at room temperature and then washed with PBS followed by staining with 1% crystal violet. For the HCT116 cells, colonies were stained after 5 days while for SW480 cells, colonies were stained after 8 days. The colonies were compared to untreated cells and counted using ImageJ software (National Institute of Health). Colonies of greater than 50 cells were counted to determine the surviving fraction. Surviving fractions were normalized to the plating efficiency of each cell line (18).

Colonosphere assay

To obtain colonospheres, cells from monolayer were disaggregated with Trypsin-EDTA (Cellgro) to a single cell suspension, passed through 40 μm cell strainer and plated at a density of 2500 cells/cm3 and grown in non-adharent culture conditions. Cells were treated with 25 μg/ml of AR extract and allowed to grow for 7 days in Dulbecco's modified Eagle's medium medium (Sigma) supplemented with 20ng/ml bFGF (Invitrogen), 1ml/50ml of 50× B27 supplement (Invitrogen), 20ng/ml of EGF (Invitrogen) in low attachment 24 well plates (CytoOne) in humidified incubator at 37°C, 5% CO2. Colonospheres were ezymatically disaggregated as mentioned above and re-plated at clonogenic densities to obtain secondary colonospheres without further addition of AR extract. Same procedure was applied to obtain tertiary colonospheres. Colonospheres were counted with 10× objective on microscope (Jenco).

Western blot

HCT116 and SW480 cells were washed with ice-cold PBS and lysed in RIPA buffer supplemented with mixture of protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL). The protein concentration was determined by the BCA protein assay (Biorad). About 30–50 μg of cell lysates were subjected to SDS-PAGE and electrotransferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were blocked with 5% bovine serum albumin or 5% non-fat dry milk in Tris-buffered saline (20mM Tris–HCl and 137mM NaCl, pH 7.5) for 1h at room temperature (21°C). Immunoantigenicity was detected by incubation of the membranes overnight with the appropriate primary antibodies (0.5–1.0 μg/ml in 5% BSA or 5% non-fat dry milk). After they were washed, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and developed using enhanced chemiluminescence (ECL Detection System, GE) according to the manufacturer’s instructions.

In vivo tumor growth

For in vivo studies, male athymic BALB/c nude mice (5 weeks old, 20 g) were purchased from NCI mouse repository, Frederic, MD and housed in pathogen-free condition throughout experimental duration. All surgical and care procedures administered to the animals were in accordance with the recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experimental protocols were approved by the University of Kansas Medical Center Animal Care and Use Committee. For xenograft studies, 5×106 HCT 116 cells suspended in 100 μl of PBS were injected subcutaneously into both flanks of each mice. Within a week of tumor cell inoculation, palpable tumor was observed and mice were injected either PBS or 200mg/kg of AR extract daily intraperitoneally (N = 5 each group) for 21 days. Tumor size at injection site was measured after every 7 days by caliper measurement of two perpendicular diameters of the implants and tumor volume calculated using following formula: 1/2 (length × width2). The animal body weights were also recorded simultaneously. On day 28, the animals were killed, and the tumors were removed, weighed and either stored in fixing solutions or snap-frozen in liquid nitrogen for further analyses.

Immunofluorescence and immunohistochemistry

The cells were grown on coverslips and treated with AR extract for 48h. After formalin fixing, the cells were incubated with primary antibody followed by fluorescein isothiocyanate-conjugated secondary antibody. Cell images were captured under a fluorescent microscope.

Tumor xenografts were fixed in 10% neutral buffered formalin before paraffin embedding. Embedded paraffin blocks were cut into a section of 4 μm, deparaffinized and blocked with Avidin/Biotin for 20min. The slides were then incubated with primary antibodies, followed by corresponding secondary antibodies and then developed with 3,3′diaminobenzidine (DAB; Sigma-Aldrich). Finally, the slides were counterstained with hematoxylin. Cell images were observed under a bright field microscope.

The colonospheres were harvested and fixed in Thinprep preservcyt solution (Hologic Inc., MA) followed by embedding, histological processing and staining as mentioned above.

High performance liquid chromatography analysis

AR extract was analyzed using high performance liquid chromatography (HPLC) on an isocratic Shimadzu 10A HPLC System equipped with diode array detector and fraction collector. Chromatographic elution was performed on a Phenominex Kinetex 2.6µ C18 100A Column (100×4.6mm) at a flow rate of 0.5ml/min for 20min using various solvents including Solvent A (0.1% Formic acid in water), Solvent B (100%, methanol), Solvent C (95% Solvent A and 5% Solvent B), Solvent D (80% Solvent A and 20% Solvent B).


All values are expressed as mean ± SEM. Differences among groups were analyzed using an unpaired two-tailed t test. A P < 0.05 was considered statistically significant.


Inhibition of colon cancer cell proliferation by AR extract

The human colon cancer cells, HCT 116 and SW480, were used to evaluate the growth inhibitory effect of AR extract. Cells were treated with various concentrations of AR extract for 1–3 days and cell viability was determined by hexoseaminidase assay. As shown in Figure 1A and andB,B, AR extract inhibited growth of both cell lines in a dose and time dependent manner. Microscopic images of HCT116 and SW480 cells at various concentrations of AR extract are shown in Figure 1C and andD.D. These data demonstrate that AR extract suppresses growth of colon cancer cells in culture.

Figure 1.
AR extract inhibits colon cancer cell proliferation. (A, B) Cell proliferation assay. HCT116 (A) and SW480 (B) cells were incubated with increasing doses of AR extract (0–100 µg/ml) for up to 72h and analyzed for cell proliferation (n ...

AR extract promotes a time-dependent cell cycle arrest at Go/G1 phase in human colon cancer cells

Given the growth inhibitory role of AR extract on proliferation of colon cancer cells, we next examined time-dependent effect of AR extract on cell cycle profiles by flow cytometry. At 24 and 48h following AR extract treatment, there was a G0–G1 arrest in both HCT116 and SW480 cells (Figure 2A). Western blot analysis of cell cycle proteins in HCT116 and SW480 revealed reduced expression of Cyclin B and Cyclin D1 in both the cell lines at 24h and particularly at 48h post-treatment (Figure 2B). Expression of Cyclin A2 that promotes both cell cycle G1/S and G2/M transitions, while reduced in HCT116, appeared elevated in SW480 cells at 48h (Figure 2B).

Figure 2.
AR extract induces cell death through apoptosis. (A) Cell cycle analysis. Time-dependent effect of AR extract on cell cycle profiles by flow cytometry. (B) Western blotting. Lysates from HCT116 and SW480 incubated with 50 µg/ml AR extract were ...

AR extract triggers cell apoptosis in human colon cancer cells

To explore the mechanism by which AR extract induced cell death, we performed flow cytometry to measure apoptosis. HCT116 cells were incubated with AR extract for 12, 24 and 48h, and the apoptosis was measured with Annexin-V/PI double staining. The Annexin/PI double staining showed the induction of apoptosis by AR extract as evidenced by the augmented percentages of cells in apoptotic phase (Figure 2C). To further investigate the mechanisms involved in AR-mediated apoptosis, western blotting for apoptosis-related proteins was done. After treatment with indicated concentrations of AR extract for 48h, PARP cleavage, the hallmark of apoptosis, in the AR-treated cells was prominent. Expression of antiapoptotic Bcl-xl protein was markedly decreased while that of proapoptotic Bax displayed an increased trend (Figure 2Di). Finally, the level of full length Caspase 3 declined in AR-treated cells while cleaved Caspase 3 bands at 19 and 17kDa, respectively, exhibited increases in relative abundance in response to AR extract (Figure 2Dii). The data verified that AR extract-induced cell death was due to induction of apoptosis.

AR extract affects self-renewal characteristics of colon CSCs

Self-renewal is one of the characteristics of CSCs. Clonogenic assays are used to analyse formation of colonies from a single cell, a process that requires reproductive integrity and represents the self-renewal potential of CSCs (19). We treated both cell lines with AR extract for 48h before they were allowed to grow in regular medium for a week. The results showed that AR extract suppressed the formation of colonies in dose dependent manner in both HCT116 and SW480 cells, respectively (Figure 3A1 and Aiii). Figure 3Aii and 3Aiv represent surviving fractions following AR extract treatment compared to controls.

Figure 3.
Effect of AR extract on self-renewal characteristics of colon CSCs. (AiAiv) Cells were incubated with specified concentration of AR extract for 48h and subsequently allowed to grow into colonies for 5 and 8 days, respectively. Number of colonies ...

Spheroid formation assay is yet another assay to analyze the self-renewal ability of CSCs. In this assay, cells are allowed to grow in non-adherent plates and serum free CSC medium to examine their ability to develop into spheroids/colonospheres (20). AR extract treatment of HCT116 cells significantly inhibited colonosphere size as well as number in a dose-dependent manner. (Figure 3Bi and Bii). In addition, to assess the impact of AR extract on the replicative potential of colon stem cells, we serially plated cells from primary colonospheres (Generation 1) for two more generations (Generations 2 and 3) without further addition of AR extract. Colonosphere formation in AR treatment group was progressively inhibited at each passage, due probably to exhaustion of adult stem cells (Figure 3Ci and Cii). Indeed, immunoflorescence showed decreased Dclk1 (a marker of tumor-initiating cells) staining in AR-treated colonospheres than in controls (Supplementary Figure 1A, available at Carcinogenesis Online).Western blot analyses demonstrated that Dclk1, CD44 and ALDH1A1 expression was markedly reduced in cells in 2D culture treated with AR extract (Figure 3Ciii). Taken together, these results indicate that AR extract has the ability to suppress CSC renewal.

AR extract inhibits Wnt/β-catenin and Notch signaling pathways

We and others have shown existence of a functional cross talk between Wnt/β-catenin and the Notch signaling pathways that share several common downstream targets (21–23). Moreover, these morphogenic pathways are also critical regulators of intestinal stem cell fate (24). Hence, we were interested in investigating the effect of AR extract on Wnt/β-catenin and Notch signaling in these cells. As observed by western blotting, relative levels of β-catenin were significantly reduced following treatment with AR extract in both HCT116 and SW480 cells (Figure 4A). TOP-flash reporter activity, a measure of Wnt signaling, decreased significantly in response to AR extract while increases in reporter activity following GSK-3β inhibition was used as a positive control (Figure 4B). We have recently shown that WIF1 (a Wnt antagonist) is epigenetically regulated by a histone methyl transferase, EZH2 (25). During measurement of WIF1 promoter activity in the presence of AR extract, a significant increase was observed and that this increase was attenuated by ectopic overexpression of wild-type EZH2 (Figure 4C). Neither low nor high doses of AR extract were able to reverse this inhibitory effect. Interestingly, ectopic overexpression of kinase-mutant of EZH2 led to elevated WIF1 promoter activity which was further enhanced by treatment with AR extract (Figure 4C). Since AR extract also reduced the relative levels of EZH2 in treated cells (data not shown), we hypothesized that epigenetic mechanisms may be integral to the chemopreventative effects of AR extracts. To demonstrate that, we overexpressed EZH2 in HEK293T and HCT116 cells, treated them with AR extract followed by spheroid assay. We observed significant reduction in the number of colonospheres with AR extract, thus corroborating our hypothesis (Figure 4D upper panel). Lower panels represent quantitation of number of spheroids in the two cell lines as well as Western blot to show EZH2 overexpression.

Figure 4.
AR extract inhibits Wnt/β-catenin and Notch signaling pathways. (A) Western blotting. Western blot analysis of lysates obtained from HCT116 and SW480 cells treated with 50 μg/ml of AR extract for 24 and 48h (n = 3). (B) Topflash Reporter ...

We next explored whether AR extract targets Notch signaling in HCT116 and SW480 cells. As depicted in Figure 4D, relative levels of Notch intracellular domain (NICD) and its downstream target Hes1 decreased significantly in both cell lines following treatment with AR extract (Figure 4E). Next, we investigated the NICD transcriptional activity for Hes1 promoter driving the luciferase reporter gene (Hes1-Luc). The reporter assays demonstrated that the transcriptional activities of NICD in both cell lines in the presence of AR extract were markedly lower than vehicle-treated controls (Supplementary Figure 1B, available at Carcinogenesis Online). During immunofluorescence staining, significantly lower levels of NICD and nuclear Hes1 were observed in the AR treated cells compared to controls (Figure 4Fi–4Gii). Finally, Hes1 staining of colonospheres also showed significant decrease in intensity following AR treatment compared to control (Supplementary Figure 1C, available at Carcinogenesis Online). These results suggest that AR extract also targets Notch signaling in colon cancer cells.

Effect of AR extract on tumor development

The preceding data prompted us to assess the antitumor activity of AR extract in vivo. HCT116 colon cancer cell xenograft tumors were allowed to develop and grow for 1 week, following which AR extract was administered intraperitoneally daily for 3 weeks. Treatment with AR extract significantly inhibited the growth of tumor xenografts (Figure 5A). In addition, while there was no apparent change in animal body weight, both the tumor weight and tumor volume in AR extract treated mice were significantly lower than vehicle-treated mice (Figure 5B and andC).C). To check how AR extract affected the growth of xenograft tumor cells, we stained tumor xenograft sections with a marker of cell proliferation, Ki-67. As depicted in Figure 5D, while control group exhibited intense nuclear staining for Ki-67, AR extract significantly attenuated Ki-67 staining. These decreases in cell proliferation in AR extract treated xenografts were probably due to significant downregulation of CyclinD1 and c-Myc levels in immunostained samples (Figure 5E). This is consistent with AR extract’s antiproliferative role in vitro (see Figure 1). Immunohistochemistry for caspase 3 demonstrated that AR treatment promoted induction of apoptosis in xenograft tumor cells (Figure 5F). It is well established that the metastatic dissemination of primary tumors is directly linked to patient’s survival and accounts for about 90% of all colon cancer deaths (26). Whether the AR extract has antimetastatic properties is not clear. Given that metastasis is almost always preceded by epithelial–mesenchymal transition, we have investigated the effect of AR extract on epithelial/mesenchymal markers such as E-cadherin and Vimentin and the results clearly suggest that AR extract induces expression of E-cadherin to promote epithelialization of cancer cells (Supplementary Figure 1D, available at Carcinogenesis Online).

Figure 5.
AR extract inhibits tumor growth in HCT116 tumor xenografts. (A) Representative pictures of tumor xenografts in nude mice treated with AR extract or PBS (n = 3). Arrows indicate tumor area. (B) Average weight of mice (upper panel) and average weight of ...

AR extract blocks Wnt/β-catenin and Notch pathways thereby affecting expression of stem cell markers in tumor xenografts

During analysis of tumor xenografts for components of both Wnt/β-catenin and Notch pathways, AR treatment significantly attenuated staining intensities of β-catenin, NICD and Hes1 (Figure 6A and B). Western blot analyses further confirmed decreases in relative levels of NICD and Hes1 in AR extract-treated tumor samples (Figure 6C). AR extract also blocked increases in EZH2 compared to vehicle-treated samples (Figure 6D) consistent with similar effects in vitro (see Figure 4C; data not shown). Finally, Western blots also revealed significant reduction in the relative levels of both stem cell markers ALDH1A1, CD44, and Dclk1 (Figure 6E; upper panel) as well as pluripotency marker cMyc (Figure 6E; lower panel) in AR extract treated tumors compared to vehicle control. Reduced levels of Dclk1 and cMyc were further confirmed via immunohistochemical analyses (Figure 6F and G). These data clearly suggest that AR extract blocks pathways critical for CSCs maintenance in vivo thereby affecting the growth of colon cancer cell xenograft.

Figure 6.
(A, B) Effect of AR extract treatment on components of Wnt and Notch pathway. Representative images of tumor xenograft sections stained for β-catenin (A) and NICD and Hes1 (B) through immunohistochemistry (n = 3). (C) Western blotting. Western ...

Partial purification of AR extract via HPLC

Partial purification of AR extract via HPLC revealed a major peak along with several minor peaks and efforts are underway to identify these peaks and whether they have better anticancer properties than the original extract (Supplementary Figure 1E, available at Carcinogenesis Online).


Phytochemicals are important targets in anticancer research due to drug resistance and toxic effects of current chemotherapy. There are various reports which suggest that phytochemicals have potent in vitro and in vivo chemopreventative properties {Reviewed in [27]}. In the current study, we demonstrate that the ethyl acetate extract of AR seeds possesses anticancer activity in vitro and in vivo in murine model of xenograft tumors and may have potential as chemopreventative/therapeutic agent against colon cancer.

The data presented in the current study show that AR extract selectively inhibited the proliferation of colon cancer cells and suppressed formation of colon cancer cell colonies. AR extract-triggered Go/G1 phase arrest may be another reason for the inhibition of cell growth. Cyclin D1 is over-expressed in many human cancers. We observed significant decreases in relative levels of Cyclin D1 in both cell lines as well as in tumor xenografts which probably prevented G0–G1/S phase transition by promoting G0/G1 arrest. Resistance to apoptotic programming, particularly in response to commonly used gemcitabine or platinum-based regimens, is the hallmark of many cancers that results in high rates of treatment failures. Indeed, deregulated apoptotic pathways play a central role in developing CRC (28,29). AR extract reduced the cell viability in part due to the induction of apoptosis, as was shown by annexin V-FITC/PI-staining method and western blots for pro- and antiapoptotic proteins. Moreover, protein levels of Bax were significantly upregulated, while levels of Bcl-2, a major antiapoptotic protein of Bcl-2 family, were greatly reduced by AR extract, further implicating intrinsic pathways in the regulation of AR extract-induced apoptosis.

The hypothesis that stem cells drive tumorigenesis in colon cancer raises questions as to whether current treatments are able to efficiently target the CSCs. In this scenario, the elimination of CSCs represents an effective strategy to suppress tumor growth. In the present study, in vitro clonogenic and sphere formation assays were performed and the inhibitory effects of AR extract on the self-renewal capacity of colon CSCs were analyzed (14). AR extract suppressed the colony and sphere formation of colon cancer cell lines and downregulated CSC markers CD44, ALDH1A1 and Dclk1 in vitro and in vivo further suggesting that targeting CSCs via AR extract may be an important strategy to reduce CRC onset and/or progression.

CSCs use many of the same signaling pathways that are found in normal stem cells, such as Wnt, Notch, and Hedgehog (Hh). The Wnt/β-catenin signaling pathway is critical for promoting the self-renewal capacity of CSCs (30), and aberrant Wnt/β-catenin signaling is an early event in most human cancers including colon (31–35). Wnt activity can be regulated by microenvironment and can define CSCs (36). Thus, β-catenin has been reported to be localized to the nucleus in cells expressing CD133 and CD44 (37). Interestingly, we observed that AR extract downregulated relative levels of β-catenin both in vitro and in tumor xenografts thereby corroborating with earlier studies wherein, natural compounds have been shown to suppress Wnt/β-catenin signaling in human cancers to antagonize the self-renewal capacity of colon cancer cells (38–40). Likewise, Notch signaling is involved in maintaining cancer-initiating cells in the colon and the inhibition of Notch may prove beneficial in the treatment of CRC (12,41). Hence, compounds inhibiting Notch pathways can be implicated in the elimination of CSCs, which would likely improve treatment outcome (42). We have previously shown that Honokiol, a biphenolic compound, targets Notch signaling to inhibit colon CSCs (43). Results of the present study clearly demonstrate that AR extract significantly blocks Notch signaling by downregulating NICD generation leading to reduced levels of Hes1. These results corroborate well with known inhibitors of Notch pathway including several phytomolecules from dietary sources like resveratrol, curcumin and genistein (44–47).

It is generally understood that lifestyle and environmental or dietary exposure may contribute to cancer risk through epigenetic mechanisms. Indeed, epigenetic alterations are increasingly recognized as valuable targets for the development of cancer therapies (48). The polycomb repressive complex 2 (PRC2) is a key epigenetic regulator that catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3) via the histone methyltransferase, EZH2, which confers stemness and regulates differentiation during embryonic development. Given these roles of EZH2 and H3K27me3, plastic and dynamic features of cancer cells, especially CSCs, may be closely associated with this epigenetic mechanism. We and others have previously shown that β-catenin signaling is epigenetically regulated by EZH2 (25,49) and that this happens through Wnt antagonist WIF1 (25). In the current study, EZH2 levels were significantly reduced following treatment with AR extract both in cell lines (data not shown) as well as in tumor xenografts while AR extract significantly upregulated EZH2-regulated WIF1 reporter activities thereby antagonizing Wnt/β-catenin signaling. Following spheroid assay in cell lines overexpressing EZH2, AR extract antagonized spheroid growth. Finally, AR extract also promoted epithelialization of cancer cells by inducing expression of E-cadherin. Thus, AR extract provides an interesting prospect of this epigenetic therapy to target CSCs with significant potential to reduce cancer development and/or progression.

In conclusion, our results indicate that AR seed extracts can efficiently inhibit the growth of colon cancer cells in vitro and antagonize the growth of tumor xenografts in vivo. The underlying mechanism of AR extract may be related to targeting CSC stemness that is potentiated via inhibitory effects on Wnt/β-catenin and Notch signaling, respectively. Wnt/β-catenin may itself be regulated epigenetically by EZH2. As HPLC analysis resulted in a single major peak, further purification and characterization of AR extract may help us identify compound(s) that possess potent chemopreventative/therapeutic capabilities to target CRC.

Supplementary material

Supplementary Figure 1 can be found at


National Cancer Institute (1R01CA185322-01A1 to S.U.); University of Kansas Cancer Center (Pilot project to S.U.); University of Kansas Medical Center (Start-up funds to SU).

Conflict of Interest Statement: None declared

Supplementary Material

Supplementary Data:



Alcea rosea
cancer stem cell
colorectal cancer
phosphate-buffered saline


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