Colorectal cancer (CRC) is the second most fatal and the third most diagnosed type of cancer worldwide. Despite having multifactorial causes, most CRC cases are mainly determined by dietary factors [
]. Nutrition has been estimated to cause more than one-third of cancer deaths, and that dietary factors are responsible for 70–90 % of all cases [
Plants have proved to be an important source of anti-cancer drugs [
]. Polyphenols are secondary metabolites widely present in plant kingdom that play promising role in cancer prevention and therapy [
]. Several studies using cancer cell lines and animal models of carcinogenesis have shown that a wide range of polyphenols possess anticancer properties including initiation of apoptosis through the regulation of cell death pathways, the suppression of cancer cell proliferation and metastasis through inhibition of anti-apoptotic molecules, and cell cycle arrest [
]. Although polyphenols are generally recognized as antioxidants, they also act as prooxidants inducing growth arrest and cell death through increasing ROS production [
The most important signaling pathways regulating cell proliferation and survival implicated in colorectal cancer involve Wnt/β-catenin, phosphatidyl-inositol-3-kinase (PI3 K), growth factor receptors/Ras/mitogen-activated protein kinases (MAPK), JAKs/STAT3 and especially nuclear factor κB (NF-κB) [
]. Induction of NF-κB transcription factor, caused by extracellular stimuli, passes through IκB kinase α (IKKα) and/or IKKβ activation [
]. The phosphorylation of IκB inhibitory proteins by IKK activated complex induces ubiquitination and degradation of the IκBs. The dissociated NF-κB complex relocates to the nucleus where it binds to DNA promoter region and activates genes involved in several cellular activities like cell growth, survival, angiogenesis, migration and metastasis [
]. Two major target genes of NF-κB, cell cycle cyclin D1 and Vascular Endothelial Growth Factor (VEGF), are known to play a vital role in tumor progression [
]. This perfectly correlates with the fact that inhibition of NFκB activity in colorectal cancer cells dramatically reduces cell growth in vitro and in vivo [
Considering this, several dietary natural phytochemical compounds have been found to be potent inhibitors of NF-κB pathway with anticarcinogenic properties [
Miller (quince) is recognized as a good and low-cost natural source of different classes of phenolic compounds responsible for its anti-oxidant, anti-ulcerative and anti-microbial activity [
]. We have previously showed that quince peel polyphenols have a potent anti-inflammatory effect in LPS-stimulated human macrophages and that such effect pass through inhibition of NF-κB activation [
]. Moreover, quince polyphenols were reported to present antiproliferative activity in human cancer cells [
]. Notwithstanding these various studies, the anti-tumor effect with mechanisms of action of
Miller has never been assessed. Here, we investigated the anti-colon cancer activity of polyphenolic extract from the Tunisian quince (
Miller). We found that both quince peel polyphenolic extract (Peph) and pulp polyphenolic extract (Puph) inhibits viability of human colon adenocarcinoma LS174 cells. However, Peph present the most potent antitumor effect through the blocking of cell growth and the induction of apoptosis and a cell cycle arrest accompanied with an increase of reactive oxygen species (ROS) production. Moreover, Peph extract significantly enhances the anti-cancer effect of 5-fluorouracil. This study suggests that Cy
Miller phenolic extract may have therapeutic applications for colon cancer treatment and that a quince rich regimen may prevent the development and the progress of colon cancer.
We have previously reported a potent anti-inflammatory effect of quince (
Miller) peel polyphenolic extract in LPS-stimulated human THP-1-derived macrophages [
]. Accumulating evidence shows that chronic inflammation is an important etiologic factor in the development of colorectal cancer [
] and several studies have shown that dietary polyphenols with anti-inflammatory properties are effective against different types of cancer [
]. Our aim was to investigate the anti-colon cancer effect of a non toxic natural extract from quince as an alternative to treat and prevent the growth and development of colorectal cancer. In this work we show, for the first time, that peel polyphenolic extract of
Miller goes further than an antiproliferative agent in colon cancer cells, displaying pro-apoptotic and anti-angiogenic activities along with the capacity to enhance chemotherapeutic effectiveness of 5-fluorouracil.
We first found that the Tunisian quince (
Miller) aqueous acetone extract presented a dose-dependent inhibition effect on the viability of colon LS174 adenocarcinoma cells, while no effect was observed in the non-tumorigenic cells. Our data indicate that the peel polyphenolic extract inhibited the proliferation of human LS174 colon cancer cells without any toxic effect, as assessed with LDH assay. This correlates with previous work done by Carvalho et al. [
] that reported an inhibition effect of the methanolic extract of
Miller on the proliferation of colon Caco-2 cancer cells. We have further found that quince peel polyphenols (Peph) presented higher antiproliferative effect than those extracted from the pulp (Puph). This might be explained by the difference in the phenolic composition of each extract. Indeed, according to the results we previously reported [
], total phenolic content of the pulp and peel parts of quince (
Miller) extracts ranged from 37 to 47 and 105 to 157 mg/100 g of fresh weight, respectively. Phenolic components level in the peel fraction is three times higher than the one of the pulp. Phytochemical investigations led to the identification of thirteen phenolic compounds in quince Peph extract [
]. Previous studies highlighted the anti-cancer properties of the isolated phenolic molecules in tumor cells. Chlorogenic acid, a phenolic molecule found in the quince peel polyphenolic extract, has been reported to induce apoptotic cell death in U937 leukemia cells [
]. Rutin, another major component of the peel quince polyphenolic extract (36 %), has been also reported to exert antitumoral effects in human colon cancer cells [
]. Moreover, quercetin and kaempferol, additional members of the quince peel polyphenols, effectively inhibits pancreatic tumour growth in vitro and in vivo via an increase in apoptosis [
]. All the remaining components of the peel quince polyphenolic extract like catechin and procyanidin have revealed promising chemopreventive and/or anticancer efficacy in several cell cultures and animal models [
]. The use of commercially purified compounds based on their concentration and percentage in 5 µg/ml of Peph extract have indicated that the antiproliferative effect of Peph involves total polyphenolic compounds (Table
). However, in the context of our study, we still do not know which combination of compounds from the peel phenolic extract is behind the antiproliferative effect (Table
; Additional file
: Figure S1). Our data suggest that a synergistic effect between different molecules may have contributed to the antiproliferative effect of the Peph extract, on LS174 adenocarcinoma cells. This supports the fact that there are usually more benefits to use a whole natural plant extract, with different pharmacologically active phytochemicals, than a single isolated compound. Indeed, total compounds within Peph extract could inhibit any toxic effects of one compound alone and have many different intracellular targets, which may act in a synergistic way to enhance specific activity. Additionally, the presence of multiple components may possibly decrease the chances of developing chemoresistance [
]. Moreover, natural extracts like quince can be administered orally to patients, as a safe mode of administration. Many works have focused on the bioavailability and polyphenols bioefficacy in humans [
]. Polyphenol concentrations in the colon can reach several hundred micromoles per liter, and together with a few carotenoids, polyphenols constitute the only dietary present in the colon, because vitamins C and E are absorbed in the upper segments of the intestine [
Based on the fact that the total peel polyphenolic extract of
Miller is endowed with the potent antiproliferative activity against human colon carcinoma LS174 cells along with the cited biological activities of its compounds, we tried to uncover the anticancer mechanisms of quince polyphenolic extract. Flow cytometry analysis showed an increase in the percentage of annexin-V positive cells when treated with quince Peph extract. Such apoptotic feature has been reported to be frequently associated with increased caspases activity [
]. However, treatment with a pan-caspase inhibitor (z-VAD-fmk) did not block the apoptotic cell death in Peph-treated LS174 cells. Furthermore, the Peph-induced apoptotic effect was not accompanied by processing of procaspase-3,-9, and PARP confirming that quince peel polyphenols induced LS174 cell apoptosis through a caspase-independent pathway. Endonuclease G (Endo G) and AIF are key factors, released by the mitochondria, that are able to induce a caspase-independent apoptosis [
]. Interestingly, the quince Peph extract-induced apoptosis of LS174 cells was associated with an increase of AIF level but no effect was observed concerning Endo G expression level (Data not shown). Our result is in agreement with several studies reporting that various chemotherapeutic agents induced AIF-mediated apoptosis in a large array of colon cancer cell lines [
]. Our data also correlate with a previous work done by Wang et al. [
] who reported that Berberine, an isoquinoline alkaloid derived from plants, induces caspase-independent cell death in colon tumor cells through activation of AIF. This latter is also a Janus protein that directly regulates ROS production in the mitochondria [
]. In accordance with this, we investigated the potential of quince peel polyphenolic extract to modulate the intracellular amount of ROS. Interestingly, we observed an increase of ROS level after 24 and 72 h of treatment with Peph extract. This result suggests that the pro-apoptotic effect of Peph extract may, at least in part, be caused by ROS production. Our data is in agreement with previous studies that reported a high level of ROS production in anti-cancer therapeutic strategies based on the activation of the AIF caspase-independent cell death [
Colon cancer has been also shown to be associated with an overexpression of growth promoting cell cycle regulators such as cyclin D1 [
]. An aberrant accumulation of this regulator occurs in about one-third of colorectal cancer [
]. Interestingly, we found that Peph induced the downregulation of cyclin D1. This data suggests that the antiproliferative effect of Peph phenolic extract pass through the inhibition of cyclin D1 expression, causing then the cell cycle arrest before the engagement of tumor cells into an apoptotic cell death. Multiple signaling pathways such as Wnt/β-catenin, MAPK, PI3 K, JAKs/STAT3 and NF-κB signaling pathways are implicated in colorectal cancer development [
]. We have then explored the possible modulation, by Peph extract, of NF-κB, PI3 K/AKT and MAPK cascades pathways including ERK, JNK, and p38MAPKs. Analysis of the indicated pathways by western blot demonstrated that MAPK activity was not involved in the antiproliferative and apoptotic effect of Peph extract. These data are consistent with those reported by Cho et al. [
] that found no involvement of the MAPK cascades in the caspase-independent apoptosis of A172 glioma cells induced by 15d-PGJ2. Our results also demonstrate that the PI3 K/AKT survival pathway was not affected by Peph extract. However, Peph extract treatment of the LS174 cells inhibited the NF-κB signaling pathway which is recognized as a key player in the initiation and propagation of CRC [
]. This data are also in accordance with a most recent review where authors reported that inhibition of the NF-κB signaling contributes to the anticancer effects of some therapeutic drugs to prevent colon cancer metastasis [
]. These data also correlate with our previous work using THP-1-derived macrophages. Indeed, we reported that one way by which quince Peph extract inhibits LPS-induced inflammation, is through the blockade of NF-κB activation [
]. NFκB orchestrates the expression of inflammatory cytokines, adhesion molecules and angiogenic factors [
]. Moreover, it has been shown that the inhibition of angiogenesis is mediated by the blocking of NFκB activation [
]. VEGF-A, the most important regulator of tumor angiogenesis, is the first-choice target of anti-angiogenic therapies [
]. Clinical studies on patients with CRC suggest that VEGF-A expression is significantly higher in metastatic tumors than in non metastatic tumors, and the increased VEGF-A expression is related to the worse prognosis [
]. In accordance with this, we have then investigated the potential of quince Peph extract to modulate the expression and secretion of VEGF-A. Interestingly, we found that quince polyphenols inhibited VEGF-A gene expression, as assessed by Real-Time quantitative PCR. We have also found that such effect was associated with a down-regulation of VEGF-A secretion. These data suggest that the inhibition effect, exerted by Peph extract on NFκB pathway caused the down-regulation of VEGF expression which may lead to the blocking of tumor-induced angiogenesis.
An understanding of the angiogenic pathways has progressed for the development of colorectal treatment [
]. Bevacizumab is a humanized monoclonal antibody which exerts its effect by inhibiting the effect of VEGF-A thus inhibiting it’s binding to the VEGF receptor and prevents angiogenesis [
]. However, the indicated drugs suffer from being expensive for a larger use, besides they present several harmful side effects. Our results suggest that quince peel polyphenolic extract could represent an alternative since it has chemopreventive and chemotherapeutic properties through its dual inhibitory effect of both colon cancer cell survival/proliferation and angiogenesis promoting molecules. Recent studies reported the promising possibilities of use of dietary compounds to sensitize tumors to chemotherapeutics agents [
]. Combinations of chemotherapeutical agents with secondary metabolites that have different modes of action can decrease the systemic toxicity because they allow lowering the concentrations of the most toxic therapeutic drug [
]. We have then assessed the effect of quince Peph extract in combination to 5-Fluorouracil (5-FU), a classical anticancer drug recommended for first-line treatment in CRC [
]. Our result demonstrate that the combined treatment of 5-FU with Peph extract caused a significant decrease in colony formation of colon cancer cells compared to 5-FU alone. This finding suggest that quince Peph extract may improve the therapeutic effects of 5-Fluorouracil.
In the light of our study, quince peel polyphenolic extract, a natural product appears to be a promising potential anti-tumoral agent. Actually, natural products are the source of many drugs in cancer therapy and approximately 75 % of the approved anticancer therapies have been derived from natural products [
Quince polyphenols extraction
Quince polyphenols preparations along with identification and quantification of their compounds using high-performance liquid chromatography with diode-array detection (HPLC–DAD) coupled on line to a mass spectrometer (MS) were performed as previously reported [
]. The thirteen identified pure compounds present in the quince peel polyphenolic extract (Peph) (Quercetin (Q), Rutin (R), (+)-Catechin (+C), (−)-Catechin (−C), Hyperin (H), Isoquercitrin (I), Chlorogenic acid (ChA), Cryptochlorogenic acid (CrA), Neochlorogenic acid (NeA),
-coumaric acid (PcA), Kaempferol (K), Kaempferol-3-
-glucoside(K3g) and Kaempferol-3-
-rutinoside (K3r)) were purchased from Sigma (Sigma, Aldrich).
Cell culture and treatment
Human colon adenocarcinoma LS174 cell line (CL-188), and non-tumourigenic cells human embryonic kidney HEK293 (CRL1573) and mouse embryonic fibroblasts NIH3T3 (CRL-1658) were obtained from American Type Culture Collection (
ATCC, Manassas, VA). The cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10 % fetal bovine serum (FBS; GIBCO) and 50 U/ml penicillin and 50 µg/ml streptomycin. Cells were seeded with an adequate cell density and treated with different concentrations of quince polyphenolic extract in triplicate and incubated for 24, 48 and 72 h.
Measurement of cell viability
Cell viability was determined with colorimetric MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay. The non-tumourigenic HEK293 and NIH3T3 cells and human colon adenocarcinoma LS174 cells were seeded in 96-well plates (1000 cells/well). After 24 h, the cells were treated with serial concentrations of quince polyphenolic extracts (1, 2.5, 5, 10, 15 and 20 µg/ml) or pure phenolic compounds at equivalent concentrations to that present in 5 µg/ml of Peph for various periods (24, 48 and 72 h). In this assay, a colorless tetrazolium salt (MTT) is cleaved and converted to blue formazan by the mitochondrial dehydrogenase in living cells. Thereafter, 50 µl of a MTT solution (1 mg/ml final) was added into each 96-well plate, and the cells were incubated for a further 3 h at 37 °C, 100 µl of DMSO was added to each well to dissolve the blue formazan. The optical density (OD) at 540 nm was measured with a microplate reader (MULTISKAN, Labsystems). The cell viability was expressed as percentage of the viable cell number in treated cells relative to mock-treated cells (control). Cell viability was calculated using the following formula: % cell viability = OD
control × 100.
LDH release assay
Cellular membrane integrity was monitored by the permeability assay based on the release of lactate dehydrogenase (LDH) into the media. LDH release from non-tumourigenic cells HEK293, NIH 3T3 and Human colon adenocarcinoma LS174 cells was determined by using the LDH Cytotoxicity Detection Kit-PLUS test (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s protocol. Briefly, cells were seeded in 12-well plates (10
5cells/well) and cultured for 72 h with different concentrations (5, 10 and 20 µg/ml) of quince polyphenolic extracts (Peph and Puph). The percentage release of LDH was calculated from the treated cells by comparing it with the maximum release obtained by 1 % Triton X-100 treatment (positive control) and the spontaneous LDH release (mock-treated cells considered as a negative control) as follows: Cytotoxicity (%) = (experimental value–negative control)/(positive control–negative control) × 100.
Cell cycle phase distribution analysis
LS 174 cells were seeded into six-well plates at a density of 2 × 10
5 cells/ml, grown for 24 h and then exposed for 24 and 72 h to 5 µg/ml of quince peel polyphenolic extract (Peph). Thereafter, cells were collected by centrifugation at 1000 rpm, washed twice with 1X ice-cold PBS containing 2 % bovine albumin serum (BSA) (Sigma) and resuspended in Hypertonic solution (20 mM HEPES, pH 7.2; 0.16 M NaCl; 1 mM EGTA; 0.05 % Triton X-100). After incubation at 4 °C, cells were washed, treated with propidium iodide (PI)/RNase staining solution (Cell Signaling Technology; Danvers, MA) and incubated for at least 30 min in the dark at 37 °C. Cell cycle distribution profiles were analyzed on a Becton–Dickinson FACScanto II flow cytometer and further analyzed with BD FACSDiva 6 software (Becton–Dickinson). The PI fluorescence signal at FL2-A peak versus counts was used to determine cell cycle distribution and the data were analyzed using the Modfit software. The percentage of cells in SubG0, G0/G
1, S and G
2/M was determined.
Detection and quantification of apoptosis was performed by the analysis of phosphatidylserine on the outer leaflet of apoptotic cell membranes using annexin V/PE apoptosis detection kit (BD-Pharmingen) according to the manufacturer’s protocol. Approximately 10
5 cells, treated with 5 µg/ml of Peph for 24 and 72 h or vehicle as a negative control were collected by centrifugation, and washed with PBS (1×). For caspase inhibitor activity assay, cells were pre-incubated with a pan-caspase inhibitor, Z-VAD-FMK (20 µM) (BD Pharmingen) for 2 h before Peph-treatment. LS174 cells were resuspended in 100 μl of binding buffer (1×) before addition of 4 μl of Annexin V conjugated to phycoerythrin (PE) and 4 µl of 7-AAD. The cell suspension was incubated in the dark rapidly for 15 min. Stained cells were analyzed on a Becton–Dickinson FACScanto II flow cytometer and further analyzed with BD FACSDiva 6 software (Becton–Dickinson). Cell death was quantitatively evaluated by measuring the proportion of annexin V-positive cells, regardless of their staining for 7-AAD in order to include both apoptotic and necrotic cell death. Values are given in percent of total cell number. Percentage of apoptotic cells (%) was calculated as follows: early apoptotic cells (%) + late apoptotic cells (%).
Measurement of reactive oxygen species (ROS)
The intracellular level of ROS was determined using a cell-permeable fluorogenic probe, CMH2DCF-DA (life technologies, Oregon, USA). This molecule passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases and its thiol-reactive chloromethyl group reacts with intracellular glutathione and other thiols. Subsequent oxidation yields a fluorescent adduct in the presence of ROS. LS174 cells were seeded in 96-well plates (2000 cells/well) and treated with 5 µg/ml of Peph for 24 and 72 h. Cells were washed with PBS (1×), resuspended in HBSS (GIBCO) and incubated with CMH2DCFDA (10 µM) at 37 °C for 30 min in dark. Fluorescence was detected with excitation and emission wavelength at 492 and 517 nm respectively.
Western blot analysis
At various times (24, 48 and 72 h) after 5 µg/ml of peel polyphenols treatment, the LS174 cells were collected and lysed at room temperature with 100 µl of Laemmli buffer (1×). Protein content of the cell lysates was quantified using the BCA method (Bicinchoninic Acid Protein Assay kit, Sigma). Equal amounts of protein (30 µg/sample) were separated electrophoretically by 10 % SDS-PAGE and blotted onto PVDF membranes (Immobilon-Millipore). The blots were probed with primary antibodies and incubated with a horseradish peroxidase-conjugated anti-IgG in a blocking buffer for 1 h. Primary antibodies anti-phospho-AKT, anti-AKT, anti-ERK
2, anti-phospho-JNK/SAPK, anti-JNK/SAPK, anti-phospho-p38, anti-phospho-IKKα/β, anti-IKKα, anti-PARP, anti-caspase 9, anti-caspase 3, anti-AIF, anti-Cyclin D1 and loading control anti-actin were from cell Signaling Technology (Danvers, MA, USA). Anti-phospho ERKs were from Sigma-Aldrich (L’Isle d’Abeau, Chesnes, France), and anti-horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from Promega (Madison, WI). After washing, the blots were developed with enhanced chemiluminescence (ECL) (Millipore) and exposed to X-ray film.
Real time quantitative RT-PCR
To evaluate the expression patterns of up-regulated or down-regulated genes after 5 µg/ml of peel polyphenols (Peph) treatment, the selected genes were chosen for further analysis using real-time quantitative reverse transcription-PCR (RTQ-RT-PCR) in a 96-well format. Real-time PCR measures of VEGF-A cDNA expression were obtained using a Light Cycler with a Fast Start DNA Master Mix SYBR Green. The 2
method was used to calculate the relative expression of gene, as previously described [
Determination of cytokine concentrations
Cell culture supernatants from LS174 cells treated with 5 µg/ml of peel polyphenolic extract (Peph) for 72 h were collected centrifuged at 10,000 rpm for 5 min and the cells were counted. Determination of the VEGF-A concentrations was carried out using an ELISA kit Quantikine human VEGF Immunoassay (Pierce, Rockford, USA) following the manufacturer’s guidelines and normalized to cell number.
Colony formation assay
For clonogenic assays, LS174 cells were plated in six-well plates (2 × 10
5cells/well) and treated the day after with 5 µg/ml of peel polyphenolic extract alone or in combination with 50 μM of 5-Fluorouracil for a period of 72 h. After removal of the medium containing antitumor drugs, cells were trypsinized and plated at low density (2000 cells per six-well plate). Cells were then cultivated for 10 days. Colonies were stained with crystal violet and clones for each condition were scored by Image J quantification software. Results are expressed as the number of colony forming cells per well and normalized to control (vehicle) in percentage (considered to represent 100 %).
Data from individual experiments are expressed as mean ± SE. Differences between means were evaluated using Student’s
t test. Differences with
p values of less than 0.05 were considered statistically significant.