Introduction

Colorectal cancer (CRC) is the third most common cancer and the second highest cause of cancer-related death worldwide with rapidly increasing rates due to many risk factors including smoking, physical inactivity, obesity, red processed meat and excessive alcohol consumption [1]. Progress in diagnosis and treatment has had a positive effect on improving overall survival, with more patients being diagnosed in the early stage of the disease, but the outcomes of patients diagnosed with advanced stage disease remains quite poor [2]. Understanding the molecular genesis of CRC is essential for identification of novel molecular targets that might be useful to define the prognosis of CRC patients [3]. Different methods have been introduced to substantially reduce the incidence and mortality of colon cancer cell lines. The search for new compounds having anti-cancer effects in foods and plant medicines gives rise to realistic and promising approach for treatments [4]. A variety of compounds have undergone clinical trials against colon cancer based on this strategy [5, 6]. Flavonoids are naturally occuring molecules that are abundant in fruits, vegetables, nuts, seeds, and beverages such as tea and wine. Studies have shown various biological activities of flavonoids as inhibiting related cancer enzymes, antioxidant, and immune response activities [7–11]. These properties could explain the beneficial effects of these natural molecules that have already been used in hypertension, inflammation, and cancer treatments [12]. Quercetin in the glycoside form is the most abundant bioflavonoid compound [13]. Quercitrin is a flavonoid with antinflammatory activity in experimental colitis, associated with an antioxidative action [14]. Recent studies demonstrated that quercitrin exhibits scavenger and anti-oxidant properties that increase the potential for quercitrin being an anti-carcinogenic [15]. Although different studies indicated that quercitrin is a potent antioxidant, the action of this compound is not well understood [16–18]. Hence, we proposed to investigate whether quercitrin has apoptotic and antiproliferative effects in DLD-1 colon carcinoma cell lines.

Material & Methods

Cell Culture

The Dukes’ type C colorectal adenocarcinoma carcinoma cell line DLD-1 was kindly provided by Dr.Yusuf Baran (Department of Molecular Biology and Genetics, IYTE). DLD-1 cell line was cultured in DMEM medium containing 1 % penicillin/streptomycin and 10 % fetal bovine serum at 37 °C in 5 % CO2.

Measurement of Cell Growth and Cytotoxicity

To detect the effect of quercitrin on cell viability after treatment, a WST-1 cell proliferation assay was performed. The WST-1 conversion assay (Roche, Germany) is based on the mitochondrial function of intact cells that enables them to metabolise the stable tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) to a soluble violet formazan product. Quanities of 1x104 cells/well were seeded into 96-well plates containing 100 μl of the growth medium in the absence or presence of increasing concentrations of quercitrin and then incubated at 37 °C in 5 % CO2 for 24, 48 and 72 h. After the incubation period, cells were treated with 10 μl of WST-1 for 4 h. Dye accumulation was measured at 450 nm using a Multiscan ELISA reader (Thermo Fisher Scientific, Germany). Viability was calculated by subtraction of the mean values without WST-1 from those with WST-1 substrate and was expressed as a percentage of control. Data was confirmed by three other independent experiments.

Cytotoxic effects of quercitrin in the dose and time dependent manner were colorimetrically determined with a “CytoTox 96R Non-Radioactive Cytotoxicity Assay” kit from Promega (Madison, WI). Cell treatment to prepare for cytotoxicity test was done as described for the WST-1 assay. Culture medium (10 μl) was then transferred to a 96-well microtiter plate. The levels of lactate dehydrogenase (LDH) were determined by adding 50 μl fresh substrate mix, incubating in a dark at room temperature for 30 min, then adding 50 μl stop solution, and measuring optical density (OD) at 490 nm with a microplate reader (Bio-Rad, Hercules, CA). The natural color of chemicals at 490 nm was corrected by subtracting the OD values of the corresponding chemical × concentration medium that were treated and measured in triplicates in the same manner as with the cells. Data was confirmed by three other independent experiments.

Detection of Apoptotic Nucleosomes and Necrotic DNA Release

The Cell Death Detection Elisa plus (CDDE) kit is a photometric enzyme-immunoassay to determine the quality and quantity of DNA in terms of necrosis and cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) related to apoptosis in the supernatant of treated cells (Roche, Germany). Application according to manufacturer’s instruction provides concurrent detection of apoptosis and necrosis in the same well. Colour development of samples was determined by enrichment factor of the amount of DNA fragments in cytoplasm or cell supernatant indicating apoptosis or necrosis, respectively, and expressed relative to untreated cells. In order to investigate possible interference with the assay, particles were added to the cells as two fold higher than the final concentration used in above experiment (160 μg/cm2). Subsequently, the samples were centrifuged (10 min, 200 g) conformance with the CDDE kit protocol. The supernatant was then mixed 1:1 with lysate of 50 μm quercitrin treated cells for 48 and 72 h using the lysis buffer included in the kit, and analysed according to manufacturer’s instructions.

Analysis of Phosphatidylserine Exposure and Cell Permeability

During apoptotic cell death, phosphatidylserine (PS), which is a phospholipid component of the inner-leaflet of cell membrane, becomes available at the cell surface. This early marker of apoptotic cell death can be detected by staining with the green fluorescent dye Annexin V-FITC (BD Pharmingen, Germany), a fluorochrome-conjugated Ca2 + −dependent PS-binding protein. In combination with the 7-AAD-DNA-staining, dye exclusion of vital cells permits a discrimination between apoptotic and necrotic cells. Cells treated in 24-well dishes were centrifuged (200 g, 5 min, 4 °C), washed with HBSS−/− (GIBCO, Germany) and stained with 150 μl of buffer (10 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 5 mM KCl, 2 mM CaCl) containing 5 μl Annexin V-FITC (1 mg/ml) and 1.5 μM 7-AAD at 37 °C. After 10 min, an additional 500 μl of ice-cold buffer was added by the time the cells were in the ice. After centrifugation (200 g, 5 min, 4 °C) cells were suspended in 250 μl buffer and analysed immediately by flow cytometry using the green-collecting fluorescent channels FL-1 for Annexin V-FITC and FL-3 for 7-AAD, as described above. Quadrant separation in the fluorescent channels FL-1 (Annexin V-FITC) versus FL-3 (7-AAD) represents events of necrotic and apoptotic cells. 7-AAD positive (Q1 + Q2); apoptotic cells (Q3, Annexin V-FITC positive and 7-AAD negative) and viable cells (Q4, fluorescent negative) were expressed as percentage of total cells.

Analysis of Caspase-3 Enzyme Activity

Changes in caspase-3 enzyme activity of the cells, which are important signs of apoptosis, were examined using the caspase-3 colorimetric assay kit (BioVision Research Products,USA). This assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate DEVD-pNA, which can be recognized by caspase-3. In short, the cells (5 × 105 cells/2 mL/well) that were induced to undergo apoptosis were collected by centrifugation at 1,000 rpm for 10 min. The cells were lysed by adding 50 μl of chilled cell lysis buffer and incubated on ice for 10 min before centrifugation at 10,000 g for 1 min. Supernatants were transferred to new Eppendorf tubes, and 150 μl of cell lysis buffer was added to dilute the samples. The reaction mixture was prepared in 96-well plates by adding 50 μl of reaction buffer (containing 10 mM DTT), 50 μl of sample, and 5 μl of DEVD-pNA substrate and incubated for 2 h at 37 °C in CO2 incubator. At the end of this period, the plate was read under 405 nm wavelengths using an Elisa reader (Thermo Electron Corporation Multiskan Spectrum, Finland). The absorbance values are normalized to protein concentrations determined using a Bradford assay as described previously.

Determination of Changes in Mitochondrial Membrane Potential

We also examined the loss of MMP, another important sign of apoptosis, in response to quercitrin treatment for 48 h in DLD-1 and MRC5 cells using the JC-1 Mitochondrial Membrane Potential Detection Kit (Cayman Chemicals, USA). This kit uses JC-1, a unique cationic dye, to signal the loss of the MMP. JC-1 accumulates in the mitochondria which stain red in nonapoptotic cells, while in apoptotic cells the MMP collapses, and thus the JC-1 remains in the cytoplasm as a monomer that stains green under fluorescent light. The cells (5 × 105 cells/2 mL) were induced to undergo apoptosis and collected by centrifugation at 1,000 rpm for 10 min. Supernatants were removed, pellets were homogenized by 200 μl of medium, and 20 μl of JC-1 dye was added onto the cells; then, the cells were incubated at 37 °C in 5 % CO2 for 30 min. Then, they were centrifuged at 400 g for 5 min, supernatants were removed, and 200 μl of assay buffer was added onto the pellets and vortexed. Then, this step was repeated once more. Afterwards, all pellets were re-suspended with 320 μl assay buffer and 100 μl from each of them was added into the 96-well plate as triplicates. In healthy cells, the aggregate in red form has absorption/emission maxima of 560/595 nm, whereas in apoptotic cells the monomeric green form has absorption/emission maxima of 485/535 nm. The plate was read at these wavelengths using a fluorescence Elisa reader (Thermo Varioskan Spectrum, Finland). The green/red (485/560) values were calculated to determine the changes in MMP.

Statistical Analysis

Results are expressed as the mean standard error of the mean (SEM). The data were analyzed using one-way ANOVA.

Results

Quercitrin Inhibited Proliferation of DLD-1 Colorectal Carcinoma Cell Line in a Time- and Dose-Dependent Manner

To determine the antiproliferative effects of quercitrin on DLD-1 colorectal carcinoma, the cells were incubated with increasing concentrations of quercitrin for 24, 48, 72 h using a WST-1 cell proliferation assay. The results of these assays showed that there were time- and dose- dependent decreases in cell proliferation when compared with controls only in the colorectal carcinoma cell line (Fig. 1).

Fig 1
figure 1

Effects of quercitrin on cell proliferation of DLD-1 cells. The WST-1 assays were performed using triplicate samples in three independent experiments. Statistical significance was determined using two-way analysis of variance, p < 0.05 (*) and p < 0.01 (**) were considered significant

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Quercitrin has Cytotoxic Effects on DLD-1 Colorectal Carcinoma Cell Line in a Time- and Dose-Dependent Manner

To determine the cytotoxic effects of quercitrin on DLD-1 colorectal carcinoma, the cells were incubated with increasing concentrations of quercitrin for 24, 48, 72 h using LDH assay. The results of these assays showed that there were time- and dose- dependent increases in cytotoxicity. (Fig. 2).

Fig 2
figure 2

Effects of quercitrin on cell cytotoxicity of DLD-cells. The LDH cytotoxicity assays were performed using triplicate samples in three independent experiments. Statistical significance was determined using two-way analysis of variance, and p < 0.05 (*) and p < 0.01 (**) were considered significant

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Quercitrin Increases Nucleosomal Enrichment Factor in Time and Dose Dependent Manner

To determine the apoptotic effects of quercitrin on DLD-1 cells, these cells were incubated with increasing concentrations of quercitrin for 48 and 72 h and changes in nucleosomal enrichment factor (EF) were analyzed. There were subsequently 3.95 and 5.85 fold increases in EF in response to 48 and 72 h incubation with 50 μM quercitrin on DLD-1 cells, as compared with untreated cells (Fig. 3).

Fig 3
figure 3

Effects of quercitrin on apoptosis related nucleeosomal enrichment factor of DLD-1 cells. The Cell Death detection plus ELISA were performed using triplicate samples in at least two independent experiments. Statistical significance was determined using two-way analysis of variance, and p < 0.05 was considered significant

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Quercitrin Increases Caspase-3 Enzyme Activity in Time and Dose Dependent Manner

To determine the apoptotic effects of quercitrin on DLD-1 cells, these cells were incubated with increasing concentrations of quercitrin for 48 and 72 h. Changes in caspase-3 enzyme activities were analyzed. There was 1.5 fold increases in caspase3 activity in response to 48 h incubation with 50 μM quercitrin on DLD-1 cells, as compared with untreated cells (Fig. 4). Quercitrin induced apoptosis in a dose-dependent manner could be related with caspase-3 activity.

Fig 4
figure 4

Effects of quercitrin on caspase3 activity in DLD-1 cells. Changes in caspase-3 enzyme activity in response to increasing concentrations of quercitrin in DLD-1 cells. The results are the means of two independent experiments. p < 0.05 was considered significant

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Quercitrin Induces the Loss of Mitochondrial Membrane Potential in a Time- and Dose- Dependent Manner

To assess the loss of MMP, DLD-1 cells, a JC-1 MMP assay was performed after the cells were exposed to 50 μM quercitrin for 48,72 h. The results of this assay revealed the 1.8 and 1.36 fold increases in loss of MMP in response to 50 μM quercitrin for 48 and 72 h DLD-1 cells respectively, as compared with untreated cells (Fig. 5).

Fig 5
figure 5

Effects of quercitrin on membrane potential in DLD-1 cells. Loss of mitochondrial membrane potential in response to increasing concentrations of quercitrin in DLD-1 cells. The results are the means of two independent experiments. p < 0.05 was considered significant

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Quercitrin Causes the Modulation of the Cell Membrane Resulting in the Translocation of PS from the Inner to Outer Leaflet in a Time and Dose Dependent Manner

In order to confirm the results of caspase-3 activity and loss of MMP, FITC AnnexinV/PI double staining was performed in DLD-1 cells and The cells were exposed to 50 μM quercitrin for 48 h was done. The results demonstrated that 50 μM quercitrin increased the apoptotic cell death as compared with the untreated control group (Fig. 6–7).

Fig 6
figure 6

Effects of quercitrin (0 to 50 microM) on apoptosis in DLD-1 cells. FITC AnnexinV/PI double staining of DLD-1 cells treated with increasing concentrations of quercitrin (a). Flow cytometric analysis of apoptosis in DLD-1 cells (b). Early apoptotic cells labelled with Annexin-V but not PI (shown in lower right quadrant) and apoptotic cells labelled with Annexin-V and PI were found in the upper right quadrant in flow cytometric graphics. The results are the means of two independent experiments. p < 0.05 was considered significant

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Fig 7
figure 7

Effects of quercitrin (0 to 50 microM) on apoptosis in DLD-1 cells. FITC AnnexinV/PI double staining of DLD-1 cells treated with increasing concentrations of quercitrin (a). Flow cytometric analysis of apoptosis in DLD-1 cells (b). Early apoptotic cells labelled with Annexin-V but not PI (shown in lower right quadrant) and apoptotic cells labelled with Annexin-V and PI were found in the upper right quadrant in flow cytometric graphics. The results are the means of two independent experiments. p < 0.05 was considered significant

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Discussion

Quercetin is the most ubiquitous flavonoid in nature, including foods, which is usually present in a glycosylated form such as quercitrin. It possesses various potentials as an antioxidant, anti-inflammatory; however, the underlying mechanisms leading to its anticancer effects is still unclear. Comalada et al. analyzed the potency and efficacy of both quercetin and quercitrin as anti-inflammatory compounds. They examined the in vitro effects of quercitrin in the initiation and progression of the inflammation process in the oxidative stage of colon carcinoma. Upregulated nitric oxide synthase was inhibited in these rat colitis, in addition NF-ĸB inactivation was detected [19].

The research on macrophage activity of quercetin and quercitrin indicated that quercetin, instead of quercitrin, is able to repress the proliferation and activation of macrophages by inhibiting cytokine and nitric oxide formation, thus down regulating the NFĸB pathway [20]. This study in rat collitis confirmed the protective and therapeutic activity of quercitrin [21]. Ding et al. examined the effects of quercitrin on neoplastic JB6 cells, the results demonstrated that the TPA-induced neoplastic transformation is blocked in tumor cells. They also showed that quercitrin stimulated the activation of NF-E2-related factor (Nrf2), which is essential in the regulation of cellular protective genes, and blocked damage in DNA due to UVB [13].

Despite much inflammatory and protective cell researches, there are limited studies about the anticancer effects of quercitrin in the literature. Epidemiologic studies have indicated that dietary flavonoids could have a role in oral cancer. Brownning et al. examined whether quercitrin directly affected cell proliferation in a human oral squamous carcinoma cell line. They found that quercitrin had no response to antiproliferative activity as a result of minimal cellular uptake and no hydrolysis of glycosides [22]. Xu and et al. studied the molecular mechanisms of apoptosis on a prostate cancer cell line PC3 induced by quercitrin and found that quercitrin possessed little effect on inducing apoptosis [23].

In this study, we determined that quercitrin has antiproliferative, cytotoxic and apoptotic affects as would be desirable for a strategy for in cancer chemotherapy on colon carcinoma cell lines in a dose and time dependent manner. Also, we found that quercitrin exerts an apoptotic effect through the loss of mitochondrial membrane potential. Nonfunctional mitochondria played a role in the induction of apoptosis and could be essential in the apoptotic pathway [24]. We hypothesized that quercitrin affects colon cancer cell lines by immune modulation.

In conclusion, taken together, all these data showed the antiproliferative and apoptotic effects of quercitrin on colon cancer cells. Based on the above results, quercitrin appears to activate specific intracellular death-related pathways in DLD-1 cells, leading to a loss of mitochondrial cell potential and inducing apoptosis. Our findings suggest the possible use of quercitrin against human colon cancer.