Despite its limited success, 5-fluorouracil (5-FU) remains the primary chemotherapy agent for the treatment of esophageal cancer. Quercetin has been demonstrated to inhibit the growth of transformed cells. The present study was conducted to examine whether quercetin combined with conventional chemotherapeutic agents would improve the therapeutic strategy for esophageal cancer. In this study, an MTT assay was used to determine the effects of quercetin on the proliferation of EC9706 and Eca109 cells. Annexin V-FITC/propidium iodide (PI)-stained fluorescence-activated cell sorter (FACS) analysis was used to detect the apoptotic fraction of treated cells, and western blot analysis was used to examine the protein levels. The results of our study demonstrated that quercetin in combination with 5-FU significantly inhibited growth (P<0.05) and stimulated apoptosis (P<0.005) in EC9706 and Eca109 esophageal cancer cells compared with quercetin or 5-FU alone. These changes were associated with the decreased expression of a phosphorylated inhibitory molecule of NF-κB (pIκBα), which was activated by exposure to 5-FU alone. We suggest that inclusion of quercetin to the conventional chemotherapeutic agent 5-FU may be an effective therapeutic strategy for esophageal cancer.
Esophageal cancer is a highly aggressive malignant disease, which is generally diagnosed at an advanced stage. Esophageal cancer has an extremely poor prognosis with a 5-year survival rate of less than 10% (
5-Fluorouracil (5-FU) remains the most effective chemotherapeutic option available for the treatment of advanced esophageal cancer. In numerous patients, esophageal tumors are either inherently resistant to chemotherapy, or the tumors develop resistance during treatment when 5-FU is administered alone or in conjunction with other agents (
Quercetin, the major constituent of the flavonol subclass of flavonoids (
Human esophageal cancer cells (EC9706 and Eca109) were cultured in RPMI-1640 medium supplemented with heat inactivated 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. Cell lines were maintained in a humidified incubator containing 5% CO2 at 37°C. The cells were passaged twice a week at an initial density of 1×106 cells/ml.
An MTT assay was conducted according to the method described by Kawada
The cells were harvested by trypsinization and washed twice with cold PBS. Once the cells had been centrifuged at 1,000 x g for 5 min, the supernatant was discarded and the pellet was resuspended in binding buffer at a density of 1.0×105–1.0xl06 cells/ml. Following this, 100 μl of the sample solution was transferred to a 5-ml culture tube and incubated with 5 μl of FITC-conjugated Annexin V and 5 μl of PI for 15 min at room temperature in the dark. Subsequently, 400 μl of binding buffer was added to each sample and the samples were analyzed by FACS using CellQuest Research Software (Largo, FL, USA).
EC9706 and Eca109 cells were collected following centrifugation at 500 x g for 5 min, and the pellets were resuspended in lysis buffer containing 1% NP40, 1 mM phenylmethylsulfonyl fluoride, 40 mM Tris-HCl (pH 8.0), 150 mM NaCl and 1 mM NaOH at 4°C for 15 min. Cell lysates were resolved on 12.5% SDS-polyacrylamide gels and transferred to nitrocellulose membranes according to the manufacturer’s instructions. Antibody binding was detected using an enhanced chemiluminescence kit (ECL) with a hyper-ECL film. The antibodies against pIκBα and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
The values shown are the mean ± standard error of mean (SEM). Statistical analyses were conducted using the Student’s t-test and one-way ANOVA. P<0.05 was considered to indicate a statistically significant difference and statistical calculations were conducted using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA).
A dose-response study was conducted for EC9706 and Eca109 cancer cells in response to quercetin. Quercetin inhibited the growth of both cell lines in a dose-dependent manner, revealing a maximum inhibition of 60% at 100–200 μM concentrations (
Cells were stained with Annexin V-FITC and PI. A FACS analysis was conducted to distinguish and quantify the percentage of viable and apoptotic cells following treatment with 0.2 mM 5-FU, 100 μM quercetin or a combination of both drugs, for 48 h. Quercetin demonstrated a dose-dependent increase in apoptosis in EC9706 and Eca109 cells (
To investigate whether the effect of quercetin on EC9706 and Eca109 cells was mediated through the NF-κB signaling pathway, we examined the effects of 50 μM quercetin and 0.2 mM 5-FU on the expression levels of a phosphorylated inhibitory molecule of NF-κB (pIκBα) using western blot analysis. As shown in
It has been well established that the reduced capacity of tumor cells to undergo apoptotic cell death plays a key role in the pathogenesis of cancer and in therapeutic failure (
NF-κB is primarily retained in the cytoplasm as an inactive complex through direct binding of IκB. Recent data indicate that activation of NF-κB represents a principal pathway in inducible chemoresistance. Cell exposure to various stimuli results in the phosphorylation and degradation of IκBα, the translocation of active NF-κB to the nucleus (
In our study, 5-FU and quercetin demonstrated a dose-dependent induction of apoptosis in addition to the inhibition of EC9706 and Eca109 cell growth. Although the anticancer effect of quercetin was mild, the addition of quercetin to 5-FU significantly enhanced the cytotoxic and apoptotic cellular responses, even at low concentrations of 5-FU. This demonstrates that these cells may be more sensitive to 5-FU when combined with quercetin.
Based on studies suggesting that NF-κB is constitutively active in a number of cancer cell lines (
The last series of experiments was conducted to determine the potential mechanisms by which quercetin inhibits NF-κB activation. We revealed that the addition of quercetin significantly enhanced the cytotoxic and apoptotic responses to 5-FU. To confirm that quercetin plays a converse role in the 5-FU-mediated activation of NF-κB, we sought to determine the extent to which NF-κB was affected by quercetin and 5-FU, alone or in combination. Consistent with other studies, 5-FU increased the expression of pIκBα in EC9706 and Eca109 cells, but expression of pIκBα was inhibited when quercetin was combined with 5-FU, although quercetin had little effect on its own.
Our data demonstrated that quercetin acts synergistically with 5-FU to induce apoptosis in esophageal cancer cells. This may be attributed to the attenuation of NF-κB activation, as demonstrated by the decrease in pIκBα expression. These data suggest that the addition of quercetin to 5-FU may potentially be a superior therapeutic strategy for the treatment of esophageal cancer.
Dose-dependant inhibition of growth (as determined by an MTT assay) of esophageal cancer cells EC9706 and Eca109 in response to quercetin. The cells were incubated for 48 h in the absence (control) or presence of quercetin. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Dose-dependant apoptosis at 48 h in EC9706 and Eca109 cells with quercetin. The values represented are the mean ± SEM of 3–4 replicates. *A statistically significant value compared with the control group (P<0.05). SEM, standard error of mean.
Apoptosis induction at 48 h in EC9706 and Eca109 cells with 100 μM quercetin, 0.2 mM 5-FU or 0.1 mM 5-FU + 50 μM quercetin. The values represent the mean ± SEM of 3-4 replicates. *A statistically significant value compared with the control group (P<0.05); #A statistically significant value compared with the 5-FU or quercetin only groups (P<0.05). 5-FU, 5-fluorouracil; SEM, standard error of mean.
Western blot analysis of EC9706 and Eca109 cells. Lane 1, control; lane 2, 5-FU; lane 3, 5-FU + quercetin; lane 4, quercetin. Phosphorylated IκBα is increased in the presence of 5-FU and decreased in the presence of quercetin. β-actin expression was used as a loading control. 5-FU, 5-fluorouracil; IκBα, an inhibitory molecule of NF-κB.