The present study focused on the elucidation of the putative anticancer potential of quercetin. The anticancer activity of quercetin at 10, 20, 40, 80 and 120 µM was assessed
The development or identification of compounds capable of killing transformed or cancer cells, without being toxic to their normal counterparts, is of utmost importance, and has gained the increasing interest of scientists worldwide. Since antiquity, plants have been considered rich sources of chemicals, with immense therapeutic potential. During recent years, some of these plant-derived compounds or phytochemicals have been shown to be highly competent anticancer agents, in addition to being effective against many other diseases (
Cancer, following cardiovascular diseases, is the main cause of mortality and morbidity in Europe. The key characteristics of this aggressive disease are uncontrolled growth and the spread of transformed cells (
Throughout history, plant extracts and their purified active components have been the backbone of cancer chemotherapeutics (
Natural polyphenols are a large and abundant group of phytochemicals found in herbal beverages and food (
Among anticancer and cancer preventing drugs, flavonoids are the most studied ones. These compounds can interfere with specific stages of the carcinogenic process, inhibit cell proliferation and induce apoptosis in several types of cancer cells (
Quercetin (3,3′,4′,5,7-pentahydroxyflavone) belongs to polyphenolic flavonoids which are abundantly found in apples, red grapes, onions, raspberries, honey, cherries, citrus fruits and green leafy vegetables, and exerts various biological effects, including antioxidant, anticancer, antiviral, apoptosis-inducing, protein kinase C-inhibitory, cell cycle modulatory and angiogenesis inhibitory effects. Indeed, quercetin is a unique compound due to its potential to combat cancer-related diseases in a multi-targeted manner (
Furthermore, it has been suggested that the chronic administration and daily intake of quercetin may be useful for prevention of some cancer types (
The aim of this study was to investigate the anticancer effects of quercetin
We used the following cancer cell lines: CT-26 which is a mouse colon carcinoma cell line that is widely used for drug development (
Dilution series (10, 20, 40, 80 and 120 µM) of quercetin were prepared and used for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. All 9 cancer cell lines were seeded at a density of 1.5×104 cells/well and treated with a range of concentrations in triplicate in 96-well cell culture plates, whereupon cell proliferation was assessed using a standard MTT assay. Specifically, the growth inhibitory activity of quercetin was determined using MTT, which correlates the cell number with the mitochondrial reduction of MTT to a blue formazan precipitate. In brief, the cells were plated in 96-well plates and allowed to attach overnight. The medium was then replaced with serum-free medium containing the test compounds and cells were incubated at 37°C for 72 h. The medium was then replaced with fresh medium containing 1 mg/ml MTT. Following incubation at 37°C for 2–4 h, the wells were aspirated, the dye was solubilized in DMSO and the absorbance was measured at 570 nm using a BioTek Instruments EL800 Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). The viability of cells was compared with that of the control cells.
For the evaluation of apoptosis using the Annexin V/PI method, the LNCaP, CT-26, MOLT-4 and Raji cells were seeded in 12-well plates (2×105 cells/well). The cells were cultured and incubated (with 5% CO2 and 95% air) at 37°C. Various concentrations of quercetin (10, 20, 40, 80 and 120 µM were dissolved in DMSO and incubated with the cells for 48 h. DMSO in culture medium never exceeded 0.1% (v/v), the concentration known not to affect cell proliferation. The Annexin V-FITC/PI apoptosis kit (Abcam, Cambridge, MA, USA) was used. For this purpose, the cells were incubated with 5 µl Annexin V-FITC and 5 µl PI for 5 min in the dark. The treated cells were analyzed using a Partec PAS flow cytometer (Sysmex Partec GmbH, Gorlitz, Germany).
Female BALB/c mice, aged 6–8 weeks (weighing, 20–25 g) were obtained from Zabol University of Medical Sciences (Zabol, Iran). The animals were maintained in a temperature and humidity-controlled room. On day 1, the animals were shaved on the back flank. In the shaved right flank of each mouse, 3×105 CT-26 or MCF-7 cells in 50 µl PBS were injected subcutaneously, as previously described (
In addition, the animal survival rate was evaluated up to 40 days. Furthermore, for apoptosis evaluation in the animals, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed. The study protocol was approved by the Ethics Committee of Zabol University of Medical Sciences. All experimental procedures conformed to the declaration of Helsinki and were conducted in accordance with recent legislation of National Institutes of Health guide for the care and use of laboratory animals.
Implanted tumor tissues were collected and apoptosis was detected using TUNEL assay. Based on the instructions of the manufacturer (Roche Diagnostics, Basel, Switzerland), paraformaldehyde-fixed blocks were embedded in paraffin, cut into 4-µm thick slices, and incubated with TUNEL reaction mixture containing TdT and fluorescein-dUTP. Prior to incubation of the slices with TUNEL mixture, their permeability was enhanced by proteinase solution. The TUNEL signal was then detected by an anti-fluorescein antibody conjugated with alkaline phosphatase (
Statistical analysis was performed using the Student's t-test. Data are presented as the means ± standard deviation of 3 independent treatments. A value of P<0.05 was considered to indicate a statistically significant difference.
MTT assay was used to evaluate the viability of all 9 cancer cell lines following 24, 48 and 72 h of treatment with quercetin at 10, 20, 40, 80 and 120 µM (
In continuation, we examined the apoptotic rate in a panel of 4 cell lines of high and low sensitivity to quercetin (i.e., CT-26 and LNCaP, as well as MOLT-4 and Raji, respectively). Our results revealed that quercetin initiated the apoptotic process in these cells in a dose-dependent manner (
In continuation, we evaluated the
At the end of the experiment (36 days following treatment), all surviving animals were sacrificed, and tumors from all animals were dissected and the poly-D-lysine-coated coverslips for TUNEL assay were positioned. An increase in the percentage of apoptotic cells in the treated as compared to the control groups was observed; however, it did not reach statistical significance (P>0.05;
In spite of many advances in cancer therapy, cancer is still one of the major causes of mortality worldwide. Natural products, particularly flavones found in the human diet, have been found to exert anti-proliferative and apoptosis-promoting effects against cancer cells (
Previous studies have shown that grape stem extracts have an ability to inhibit the growth of colon (HT29), breast (MCF-7), renal (Caki-1) and thyroid (K1) cancer cell lines (
Furthermore, quercetin induces pro-apoptotic effects via different mechanisms involving antioxidant effects and the suppression of p53 gene and BCL-2 protein (
The role of quercetin in apoptosis mediated by p53 has been studied in many cancer cell lines. When p53 is inhibited, cells become more susceptible toward cytotoxicity induced by quercetin (
Quercetin has also been found to modulate the PI3K/Akt/mTOR pathway (
Quercetin exerts anticancer effects through the cell death domain mechanism at the cell surface (
The expression of heat shock proteins (HSPs) in almost all forms of cancer is elevated (
The anticancer effects of quercetin have been confirmed in many studies (
An
Importantly, quercetin can exert tumor suppressive effects by interfering with the cell cycle. The molecular targets of this flavonoid include p27, topoisomerase II, p21 and cyclin B (
Furthermore, quercetin has been shown to inhibit NF-κB-evoked pathways of cell survival and reduce pro-inflammatory cytokine expression that finally leads to cancer formation (
Quercetin seems to play an inhibitory role in angiogenesis in human prostate tumor growth. A recent study using an animal model, indicated that low doses of quercetin inhibited the following angiogenic stages: proliferation and migration, as well as the invasion and tube formation of endothelial cells. Protein expression analysis of prostate cancer cells treated with quercetin revealed the inhibition of the VEGF-induced phosphorylation of VEGFR-2 and its downstream targets, such as mTOR, Akt, and ribosomal S6 kinase (
The oral administration of quercetin is recommended for cancer prevention. It has been shown that a diet supplemented with 2% quercetin significantly reduced the onset of colorectal cancer (
In conclusion, our results demonstrate that quercetin inhibits the growth of a panel of 9 cancer cell lines with various IC50 values. Cell growth inhibition was attributed to the induction of apoptosis, evident in the CT-26, PC-12, LNCaP and PC-3 cancer cell lines. Furthermore, our results demonstrated that quercetin reduced CT-26 and MCF-7 tumor volume in a mouse model and increased animal survival; however, we did not verify increased
Special thanks to Zabol University of Medical Sciences for their financial support.
Apoptotic rate determined by Annexin V/PI staining in (A) CT-26, (B) LNCaP, (C) MOLT-4 and (D) Raji cell lines following 48 h of treatment with quercetin at 120 µM. Early apoptotic cells are Annexin V-positive and PI-negative (lower right quadrant). PI, propidium iodide.
Induction of apoptosis in LNCaP, CT-26, MOLT-4 and Raji cell lines treated with quercetin as assessed by flow cytometer Annexin V/PI. The results revealed that quercetin significantly induced apoptosis in comparison to the control group (***P<0.001). PI, propidium iodide.
(A)
Percentage apoptosis in tumor slices from mice bearing CT-26 and MCF-7 tumors treatment with various concentration of quercetin (50, 100 and 200 mg/kg; intraperitoneally) as assessed by TUNEL assay.
Data obtained from MTT assay on the cell viability of different cell lines treated with quercetin (10, 20, 40, 80 and 120 µM) for 24, 48 and 72 h.
A, Cells treated for 24 h | |||||
---|---|---|---|---|---|
Cell line | 10µM | 20 µM | 40 µM | 80 µM | 120 µM |
CT-26 | 94.2±4.4 | 83.5±3.7 | 75.1±4.2 | 65.8±5.5 | 49.7±5.9 |
LNCaP | 96.8±5.4 | 90.3±4.6 | 71.7±2.2 | 61.5±3.4 | 45.1±5.0 |
PC3 | 96.4±5.0 | 87.6±4.9 | 80.1±4.6 | 75±4.4 | 73.2±4.1 |
PC12 | 91.1±6.5 | 84.5±6.0 | 68.5±6.8 | 57.1±6.3 | 40.3±4.4 |
MCF-7 | 94.8±6.1 | 90.2±5.9 | 77.5±5.1 | 66.4±4.7 | 47.1±4.2 |
MOLT-4 | 88.6±3.6 | 70.2±4.1 | 57.5±4.0 | 46±2.8 | 42.8±3 |
U266B1 | 95.1±4.9 | 73.1±5 | 53.8±4.6 | 47.5±3.3 | 33.8±4.7 |
Raji | 85.6±4.1 | 80.1±3.2 | 68.1±2.8 | 42.9±3.2 | 29.4±4.6 |
CHO | 97.7±5.2 | 91.3±5.5 | 74.4±4.1 | 75.4±4.1 | 70.5±5.2 |
B, Cells treated for 48 h | |||||
Cell line | 10 µM | 20 µM | 40 µM | 80 µM | 120 µM |
CT-26 | 87.4±5.4 | 77.7±5.9 | 70.3±4.1 | 61.5±3.2 | 42.1±3 |
LNCaP | 91.5±6.3 | 84.2±5.1 | 66.6±5.7 | 46.7±4.9 | 38.5±3.8 |
PC3 | 89.9±3.6 | 77.6±3.2 | 70.7±2.8 | 52.2±3.3 | 28.5±3.4 |
PC12 | 94.4±5 | 80.8±3.4 | 62.5±4.6 | 44.5±3.2 | 30.7±3.8 |
MCF-7 | 81.3±4.1 | 70.2±3.1 | 55.5±3.4 | 39.6±3.7 | 25.2±2.1 |
MOLT-4 | 70.6±2.8 | 52.5±2.6 | 43.1±1.9 | 33.3±2.5 | 21.6±1.4 |
U266B1 | 68.5±2.3 | 53.4 ±1.8 | 37.2±2 | 33.4±1.6 | 20.4±2.1 |
Raji | 60.6±3.6 | 49.5±2.3 | 30.3±2.4 | 26.4±2.3 | 14.6±3.3 |
CHO | 97.4±4.4 | 81.3±3.4 | 64.6±2.8 | 45.8±2.6 | 21.9±3.5 |
C, Cells treated for 72 h | |||||
Cell line | 10 µM | 20 µM | 40 µM | 80 µM | 120 µM |
CT-26 | 65.5±1.5 | 55.8±1.9 | 35.9±0.83 | 29.7±1.1 | 25±2.3 |
LNCaP | 58.4±2.9 | 51.4±2.6 | 39±1.9 | 36.1±2.2 | 30.7±2 |
PC3 | 61.7±2.1 | 57.3±1.8 | 46.9±1.4 | 36.2±0.9 | 31.5±3.7 |
PC12 | 50.4±3.6 | 47.2±2.6 | 40.8±1.8 | 31.9±2.3 | 22.1±1.1 |
MCF-7 | 51.6±3.2 | 48.5±2.9 | 35.7±2.5 | 30.8±3 | 19.1±1.4 |
MOLT-4 | 11.5±0.5 | 10.2±0.45 | 10±0.37 | 5.2±0.48 | 2.1±0.9 |
U266B1 | 15.9±0.8 | 13.3±0.65 | 4.8±0.72 | 6.8±1.1 | 5.5±0.38 |
Raji | 5.5±0.4 | 2.7±0.8 | 1.3±0.25 | 0.25±0.12 | 0.18±0.09 |
CHO | 57.8±3.9 | 52.4±3.2 | 39.2±2.7 | 28.5±2.1 | 20.7±3.7 |
IC50 values (in µM) for the studied cell lines following treatment with various concentrations of quercetin (10, 20, 40, 80, 120 µM) for 24, 48 and 72 h.
Cell line | 24 h | 48 h | 72 h |
---|---|---|---|
CT-26 | 118.1±5.55 | 97.5±4.31 | 27.2±1.52 |
LNCaP | 110.7±4.30 | 72.6±5.15 | 21.7±2.31 |
PC3 | >120 | 81.9±3.27 | 36±1.98 |
PC12 | 99.3±6.11 | 65.2±4 | 11.8±2.27 |
MCF-7 | 105.4±5.2 | 52.5±3.28 | 13.7±2.61 |
MOLT-4 | 64.9±3.5 | 28.6±2.23 | 2.91±0.54 |
U266B1 | 54.3±4.5 | 25±1.96 | 6.13±0.73 |
Raji | 66.5±3.57 | 19.2±2.83 | 3.52±0.46 |
CHO | >120 | 70.7±3.44 | 23.4±3.11 |
IC50 values were calculated as previously described by Entezari Heravi