The present study demonstrates the mechanism of 2 flavonol glycosides, hyperoside and rutin, in the induction of apoptosis in HT-29 human colon cancer cells through the bioactivity-guided fractionation and isolation method. The chemical structure of hyperoside and rutin, isolated from the roots of
Colorectal cancer is the second most prevalent malignancy and the leading cause of cancer-associated mortality worldwide (
Although the toxicity of chemotherapy is a major obstacle for the successful treatment of cancer, chemo and radiation therapies are the major tools for cancer treatment, at present. For these reasons, the identification of safe components with a high selectivity for killing cancer cells from natural sources is required (
In a previous report, the anti-proliferative effect of a 70% ethanol extract of the root of
Flavonoids are polyphenol compounds that are commonly identified in plants and have gained considerable interest and attention in recent years, due to the bioactive functions they possess. Among these flavanoids, hyperoside and rutin are composed of flavan-3-ol glycosidic linkages, which are molecules of joined together glucose and glucose and rhamnose, respectively (
Numerous previous studies aimed to identify anticancer components from natural sources (
Propidium iodide (PI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Merck Millipore (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Primary antibodies included anti-mouse β-actin (sc-47778), and rabbit monoclonal anti-Bcl-2 (sc-492) and anti-Bax (sc-493) (dilution, 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit monoclonal antibodies against cysteinyl aspartate-specific cleaved caspase-3 (9661), −8 (8592) and −9 (7237) and poly-(ADP-ribose) polymerase (PARP; 5625) antibodies were purchased from Cell Signaling Technologies, Inc. (Danvers, MA, USA). Secondary antibodies included goat anti-rabbit IgG-horseradish peroxidase (HRP; sc-2004) and goat anti-mouse IgG-HRP (sc-2005) (dilution, 1:2,000), and were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All other chemicals and reagents were of the highest analytical grade.
The roots of
The uncorrected melting points (MPs) were determined using Mitamura-Riken Kogyo MEL-TEMP apparatus (Mitamura Riken Kogyo Inc., Tokyo, Japan). Ultraviolet-visible (UV-Vis) spectra were obtained using a JP/U3010 spectrometer (Hitachi, Ltd.,Tokyo, Japan) and infrared (IR) spectra were obtained on a FT/IR-5300 spectrometer (Jasco International Co., Ltd., Tokyo, Japan). The electron ionization (EI) and fast-atom bombardment-mass spectrometry (FAB-MS) spectra were obtained on a JMS-700 spectrometer (JEOL, Ltd., Tokyo, Japan). The nuclear magnetic resonance (NMR) spectra were measured on an Avance-500 spectrometer (500 MHz; Bruker Corporation, Billerica, MA, USA), and the chemical shifts were referenced against tetramethylsilane (TMS) and deuterium dimethylsulfoxide (DMSO-d6) as NMR solvents. Column chromatography (CC) was run on a silica gel 60 (70–230 or 230–400 mesh; Merck Millipore) and Sephadex LH-20 (25–100 mm; GE Healthcare Life Sciences, Uppsala, Sweden). Thin layer chromatography (TLC) was performed on silica gel 60F254 and RP-18254S plates. TLC plates were visualized using UV light, stained with FeCl3, aniline hydrogen phthalate and 20% H2SO4, and then heated at 70–80°C for 5 sec.
The dried powdered roots of
The human colon cancer HT-29 cell line was obtained from the Korean Cell Line Bank (Seoul, Korea). The normal colon epithelium FHC cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). HT-29 cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium, supplemented with 10% Invitrogen fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 units/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). FHC cells were maintained in Dulbecco's modified Eagle medium nutrient mixture F-12 (DMEM)/F-12, which contained 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 100 ng/ml hydrocortisone, supplemented with 10 % FBS, 100 units/ml penicillin and 100 µg/ml streptomycin. All of the cell lines were cultured in a humidified chamber with 5% CO2 at 37°C. Cell counts were performed using a hemocytometer from Hausser Scientific (Horsham, PA, USA).
The cytotoxic effects of hyperoside and rutin on the HT-29 and FHC cell lines were estimated colorimetrically using the MTT method, which is based on the reduction of tetrazolium salt by mitochondrial dehydrogenase in viable cells (
The nuclear morphology of the cells was observed using Hoechst 33342 DNA-specific blue fluorescent dye. The viable cells were stained homogeneously, whereas the apoptotic cells that had undergone chromatin condensation or nuclear fragmentation were not stained (
An Annexin V/PI double staining assay was carried out in order to distinguish between the early and late apoptosis stages. The stages were determined using an ApoScan™ Annexin V-FITC apoptosis detection kit (BioBud, Seoul, Korea) in hyperoside and rutin-treated HT-29 cells. The cells were trypsinized, harvested and washed with PBS. The cells were resuspended in 1X binding buffer (500 µl) and incubated with 1.25 µl of Annexin V-fluorescein isothiocyanate (concentration, 200 µg/ml) at room temperature for 15 min. The supernatant was then removed following centrifugation at 400 × g for 10 min. The cells were resuspended in 500 µl of 1X binding buffer and the cell suspensions were then stained with 10 µl PI (concentration, 30 µg/ml) at 4°C in the dark. Fluorescence was quantified using FACSCalibur flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The levels of early and late apoptosis were determined as the percentage of Annexin V+/PI− or Annexin V+/PI+ cells, respectively.
Western blot analyses were performed, as previously described (
Cells were treated with dihydroxyflavone at 10 µg/ml for various lengths of time, and were treated with hyperoside and rutin at final concentrations of 0, 100 and 200 µm. Total RNA was isolated from the cells using Gibco TRIzol® reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, and quantitated using spectrophotometry. Total RNA (5 µg) was reverse transcribed into cDNA by incubating with Invitrogen SuperScript®™ RNase H reverse transcriptase (Thermo Fisher Scientific, Inc.). PCR was conducted in a reaction composed of 40 µg of 1.25X reacting mix, 1 µl enzyme mix, 1 µl forward primer (200 nM), 1 µl reverse primer (200 nM) and RNA. cDNA was performed with following conditions: cDNA synthesis at 45°C for 30 min, followed by denaturation at 94°C for 2 min. A total of 40 cycles of PCR amplification were then performed with following conditions: 94°C for 15 sec, 60°C for 30 sec, and 68°C for 1 min. The last cycle was followed by final extension step at 72°C for 5 min.
The primer pairs (Bionics, Seoul, Korea), forward and reverse, respectively, were as follows: β-actin, 5-CCTCTATGCCAACACAGTGC-3 and 5′-ATACTCCTGCTTGCTGATCC-3; Bcl-2, 5-AGCTGCACCTGACGCCCTTCA-3′ and 5-AGCCAGGAGAAATCACAGAGG-3; Bax,5-ATGGACGGGTCCGGGGAGCAG-3 and 5-CAGTTGAAGTTGCCGTCAGA-3. PCR was performed for 40 cycles. Temperature cycling was initiated with each cycle, using the Takara PCR Thermal Cycler Dice (Takara Bio, Inc., Otsu, Japan), as follows: β-actin, 98°C for 10 sec (denaturation), 55°C for 30 sec (annealing), 72°C for 1 min (extension); Bcl-2, 98°C for 10 sec, 60°C for 30 sec, 72°C for 1 min; Bax, 98°C for 10 sec, 60.5°C for 30 sec and 72°C for 1 min. The amplified products were resolved on 1% agarose gels, stained with ethidium bromide (Sigma-Aldrich) and photographed under ultraviolet light using a Mini BIS image analysis system (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel).
SPSS software version 22.0 (IBM SPSS, Armonk, NY, USA) was used to analyse the data. The results were subjected to analysis of variance, followed by the Tukey range test in order to analyze the differences between conditions. In each case, the P<0.05 was considered to indicate a statistically significant difference. All measurements were made in triplicate, and all values are given as the mean ± standard deviation.
The dried and ground compounds of the root of
The spectral data were as follows: Pale yellowish powder (MeOH); FeCl3 and aniline hydrogen phthalate color reaction, positive; MP, 197–202°C; UV (MeOH) λmax, 280 (4.54), 364 (4.36) nm; IR vmax (KBr), cm−1: 3,380 (OH), 1,660 (α,β-unsaturated C=O), 1,612, 1,490 (aromatic C=C), 1,240 (aromatic C-O), 1,062 and 1,012 (glycosidic C-O); FAB-MS, 465 [M+H]+ m/z; 1H-NMR (500 MHz, DMSO-d6): δ 8.27 (1H, s, 5-OH), 7.66 (1H, dd,
The spectral data were as follows: Pale yellowish powder (MeOH); FeCl3 and aniline hydrogen phthalate color reaction, positive; MP, 202–204°C; UV λmax (MeOH), 268 (4.50), 375 (4.26) nm; IR vmax (KBr), cm−1: 3,360 (OH), 1,670 (α,β-unsaturated C=O), 1,600, 1,512 (aromatic C=C), 1,240 (aromatic C-O), 1,060 and 1,015 (glycosidic C-O); FAB-MS, 611 [M+H]+m/z; 1H-NMR (500 MHz, DMSO-d6): δ 7.68 (1H, d,
In the preliminary investigation, the EtOAc-soluble fractions of the roots of
The apoptogenic properties of the compounds were investigated through morphological changes in HT-29 cells. Nuclear Hoechst 33342 staining was performed in order to determine whether the anti-proliferative effect of hyperoside and rutin was due to apoptosis. As shown in
In order to quantify the percentage of apoptotic cells, flow cytometry analysis was performed using double staining with Annexin V and PI. The Annexin V−/PI− population was considered to account for unaffected cells, the Annexin V+/PI− population for early apoptosis, Annexin V+/PI+ for late apoptosis and Annexin V−/PI+ for necrosis. The results showed that the treatment of the cells with hyperoside and rutin significantly increased the percentage of apoptotic cells compared with untreated control cells (
In order to study the effects of hyperoside and rutin on apoptosis in HT-29 cells, the expression levels of apoptosis regulatory proteins, including Bcl-2 and Bax were examined. The mitochondrial pathway is an important apoptosis pathway as it regulates the apoptotic cascade via a convergence of signaling at the mitochondria. Bcl-2 interacts with the mitochondrial plasma membrane and prevents mitochondrial membrane pores from opening during apoptosis, blocking the signals of apoptotic factors (
The disruption of the mitochondrial plasma membrane by hyperoside and rutin was followed by the activation of the cleaved caspases-3, 8 and 9 and target protein PARP, respectively (
RT-PCR analysis was used to examine any alterations in Bax and Bcl-2 expression. During apoptosis, Bcl-2, a negative regulator of apoptosis, prevents mitochondrial membrane pores from opening; however, the positive regulator, Bax, produces the opposite effect. As a result, hyperoside and rutin increased Bax expression, but decreased the expression of Bcl-2 in a concentration-dependent manner (
Uncontrolled proliferation is a significant biological feature of cancer cells, and the inhibition of cell proliferation may achieve the arrest of tumor growth (
Apoptosis is an extremely important phenomenon due to the maintenance of cellular homeostasis by the regulation of cell division and cell death (
Caspase-3 is one of the key executioners of apoptosis, and is either partially or totally responsible for the proteolytic cleavage of numerous proteins, including PARP (
Chemical structure of (A) hyperoside and (B) rutin.
Cytotoxic effect of (A) hyperoside and (B) rutin on normal human colon cells. Cell viability at the indicated concentrations of hyperoside and rutin in human normal colon cells from the FHC cell line was assessed at 24 h, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays.
Cytotoxic effect of (A) hyperoside and (B) rutin on HT-29 human colon cancer cells. Cell viability at the indicated concentrations of hyperoside and rutin on human colon adenocarcinoma cells from the HT-29 cell line was assessed at 24 h, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. *P<0.05 vs. control cells.
Induction of apoptosis by (A) hyperoside and (B) rutin on HT-29 human colon cancer cells. The formation of apoptotic bodies in Hoechst 33342-stained cells were observed using fluorescent microscopy (magnification, ×400).
Induction of apoptosis by (A) hyperoside and (B) rutin on HT-29 human colon cancer cells. Flow cytometric analysis was performed on HT-29 human colon cancer cells that were incubated with hyperoside and rutin for 24 h. The right bottom quadrant represents Annexin V-stained cells (early-phase apoptotic cells). The top right quadrant represents PI - and Annexin V-stained cells (late-phase apoptotic cells). Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.
Effect of (A) hyperoside and (B) rutin on the expression of apoptosis-associated proteins on HT-29 human colon cancer cells. Cell lysates were electrophoresed and Bax and Bcl-2 were detected using western blot analysis. (C) Statistical analysis of apoptosis. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.*P<0.05 vs. control cells.
Expressions of caspase-related proteins by (A) hyperoside and (B) rutin on HT-29 human colon cancer cells. Cell lysates were electrophoresed, and cleaved caspases-3, - 8 and - 9 and PARP were detected using immune-blotting analysis. PARP, poly adenosine diphosphate ribose polymerase.
Expression of mRNA levels by (A) hyperoside and (B) rutin in HT-29 human colon cancer cells. Total RNA was electrophoresed, and Bax and Bcl-2 were detected by reverse transcripton-polymerase chain reaction. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.