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. 2019 Jan 25;43(2):117–124. doi: 10.1002/cbin.11055

Quercetin preferentially induces apoptosis in KRAS‐mutant colorectal cancer cells via JNK signaling pathways

Yiwen Yang 1, Tao Wang 2, Degao Chen 3, Qizhao Ma 1, Yanjiang Zheng 1, Shiping Liao 1, Yufang Wang 1,✉, Ji Zhang 1,✉
PMCID: PMC13397398  PMID: 30203888

Abstract

Colorectal cancer (CRC) is the third most common type of cancer, and its incidence and mortality are markedly increasing worldwide. Oncogenic mutations of KRAS occur in up to 40% of CRC cases and pose a great challenge in the treatment of the disease. Quercetin is a dietary flavonoid that exerts anti‐oxidant, anti‐inflammatory, and anti‐cancer properties. The current study investigated the anti‐proliferative effect of quercetin on CRC cells harboring mutant or wild‐type KRAS. The effect of quercetin on cell viability was investigated by MTT and colony formation assays, and apoptosis was detected using flow cytometry by labeling cells with Annexin V‐FITC. The expression of the relevant proteins was examined by Western blotting. The data revealed that KRAS‐mutant cells were more sensitive to quercetin‐induced apoptosis than wild‐type cells. Caspase activation was involved in quercetin‐induced apoptosis. In addition, quercetin selectively activated the c‐Jun N‐terminal kinase (JNK) pathway in KRAS‐mutant cells, while inhibition of phospho‐JNK by SP600125 blocked quercetin‐induced apoptosis. The results of the present study suggest that treatment with quercetin, a common flavonoid in plants, is potentially a useful strategy for the treatment of CRCs carrying KRAS mutations.

Keywords: apoptosis, colorectal cancer, JNK, KRAS mutation, quercetin

Abbreviations

AKT

serine threonine protein kinase

ERK

extracellular signal‐regulated kinase

JNK

c‐Jun N‐terminal kinase

MAPK

mitogen‐activation protein kinase

Introduction

Colorectal cancer (CRC) is one of the most common malignancies worldwide (Brenner et al., 2014; Ferlay et al., 2015). Despite recent advances in early diagnosis and the development of molecularly targeted therapies, the overall survival times of patients with metastatic colon cancers remain disappointing (Dienstmann et al., 2015). This may be associated with oncogenic mutations of KRAS that drive the activation of multiple downstream signaling pathways important for cell survival and proliferation (Dienstmann et al., 2015). Activating mutations in the KRAS gene are observed in nearly 40% of cases and are usually associated with therapeutic resistance to conventional and targeted chemotherapies, as well as poor prognosis (Lievre et al., 2008; Tejpar et al., 2012).

The dietary flavonoid quercetin (3,3′,4′,5,7‐pentahy‐droxyflavone) is a bioactive compound that is widely distributed in a number of fruits, vegetables and plants (Li et al., 2015a). It possesses low intrinsic toxicity (Okamoto, 2005) and has a relatively high bioavailability when administered orally (Gugler et al., 1975; Cao et al., 2015). As a major representative of the flavonol subclass, quercetin acts as a powerful anti‐oxidant by scavenging free radicals and chelating transition metal ions (Mendoza and Burd, 2011). As a result, quercetin may aid in the prevention of various diseases, including cancer, chronic inflammation and atherosclerosis (Murota and Terao, 2003). Free radicals, such as reactive oxygen species, influence key cellular processes, including proliferation, apoptosis and senescence, which are implicated in the development of cancer. It has been previously reported that quercetin suppresses proliferation and induces apoptosis in colon cancer (Zhang et al., 2015b), breast cancer (Deng et al., 2013; Steiner et al., 2014; Sun et al., 2014), glioma (Pan et al., 2015), melanoma (Cao et al., 2015), gastric cancer (Du et al., 2015; Zhang et al., 2015a), pancreatic cancer (Zhou et al., 2010; Lee et al., 2015), and ovarian cancer (Yang et al., 2015). These results suggest that quercetin may be a candidate drug for early cancer therapy. However, the effects of quercetin on colorectal tumors harboring KRAS mutations have rarely been investigated. In the current study, we hypothesized that quercetin may selectively inhibit proliferation and induce apoptosis in KRAS‐mutant CRC cells. The results demonstrated that CRC cells containing a KRAS mutation were more sensitive to quercetin treatment than cells possessing the wild‐type KRAS gene.

Materials and methods

Materials

Quercetin was purchased from Chengdu Must Bio‐Technology Co., Ltd (Chengdu, China). The general caspase inhibitors Z‐VAD‐fmk (FMK001), Z‐LEHD‐fmk (FMK008) and Z‐DEVD‐fmk (FMK004), were purchased from R&D Systems China Co. Ltd. Primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Other chemicals were from Sigma–Aldrich (Merck Millipore, Shanghai, China).

Cell lines and culture condition

Human colon cancer cell lines, DLD‐1KRASG13D and DLD‐1KRASWT were kindly provided by Dr. Kevin Haigis. The DLD‐1KRASG13D cell line contains the KRASG13D mutation, one of the most commonly mutated sites in KRAS. DLD‐1KRASWT is the isogenic cell line carrying the wild‐type KRAS gene. The other cell lines were kindly provided by Kunming Cell Bank, Chinese Academy of Sciences. All the cells were cultured in Dulbecco's modified Eagle's medium (SH30243.01; Hyclone; GE Healthcare Life Sciences, Logan, UT) containing 5% fetal calf serum (10270‐106; E.U. Approved, South American; Thermo Fisher Scientific, Inc.), penicillin (10,000 U/mL) and streptomycin (10,000 µg/mL) (SV30010; Hyclone; GE Healthcare Life Sciences). Individual cell line authentication was regularly confirmed using the AmpFISTR Identifiler PCR Amplification Kit from Applied Biosystems (Thermo Fisher Scientific, Inc., Waltham, MA) and GeneMarker v1.91 software (SoftGenetics LLC, State College, PA). Resulting cancer cell line STR profiles were cross compared and, where available, matched with the American Type Culture Collection's online databases.

Cell viability assay

Cell viability was assessed by 3‐(4,5‐dimethylthylthiazol‐2‐yl)‐2,5‐diphenyltetra‐zolium bromide (MTT) colorimetric assay. Cells were seeded in 96‐well plates and treated with quercetin at the indicated concentrations for 72 h. At the end of the treatment, 100 µL of 2 mg/mL MTT was added to each well and cells were incubated at 37°C for 4 h. Cell viability was determined by measuring the absorbance at 570 nm using a microplate reader (Bio‐Rad Laboratories, Inc., Hercules, CA), with the following formula: Cell viability (%) = absorbance of experimental group/absorbance of control group × 100%.

Colony formation

A colony formation assay was used to determine the effect of the cytotoxic agents. Cells in the exponential growth phase were harvested, diluted, and seeded at a density of 1,000 cells per well into a 6‐well plate. After 12 h, cells were treated with quercetin at the indicated concentrations, and continuously incubated in fresh medium at 37°C in 5% humidified CO2. After incubation for 10–14 days, cells were washed, fixed with methanol for 15 min, and stained with 0.5% crystal violet for 15 min at room temperature. A colony was defined as consisting of ≥50 cells and all visible colonies were counted. Following air‐drying, colonies in each well were countered by ImageQuant™ TL software v7.0. Subsequently, stained colonies were dissolved by 33% acetic acid. The absorbance of the crystal violet solution from each well was determined using a spectrophotometer (Bio‐Rad Laboratories, Inc.).

Western blot analysis

Cells were lysed with RIPA buffer (50 mM Tris HCl, 150 mM NaCl, 2 mM EDTA, 1% NP‐40, 0.1% SDS, pH 7.4) and denatured by boiling with Lameili sample buffer. Thirty microgram protein of each sample was separated on SDS–PAGE gel and transferred to nitrocellulose membranes (Millipore, USA). Then membranes were blocked with PBST containing 5% BSA and incubated with the primary antibodies of PARP (#9532, dilution: 1:8,000), cleaved caspase‐3 (#9664S, dilution: 1:10,000), ERK1/2 (#9102S, dilution: 1:10,000), phospho‐ERK1/2 (#4377, dilution: 1:2,500), AKT (#9272S, dilution: 1:8,000), phospho‐AKT (#4051S, dilution: 1:4,000), JNK (#9252S, dilution: 1:10,000), phospho‐JNK (#9255S, dilution: 1:3,000) from Cell Signaling Technology. Additional antibodies included beta‐actin (A5316, dilution: 1:10,000) from Sigma–Aldrich. Subsequently, membranes were incubated with appropriate secondary antibodies of horseradish peroxidase (HRP) conjugated goat anti‐rabbit IgG (#31466, dilution: 1:3000) from Thermo Scientific or HRP conjugated goat anti‐mouse IgG (ab19195, dilution: 1:3000) from abcam and visualized by enhanced chemiluminescence (ECL) reagents (Millipore, USA) using ImageQuant™ LAS (GE Healthcare Life Sciences).

Apoptosis assay

Apoptosis was determined using flow cytometry by labeling cells with Annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide (PI). Briefly, cells were treated with quercetin or vehicle control at the indicated concentrations for 24 h. Following treatment, cells were harvested, stained for 20 min with 1 mL of 20 µg/mL PI containing 1 mg/mL RNase in PBS, and conjugated with Annexin V‐FITC using a Roche Annexin V‐FLUOS Staining kit (cat no. 11858777001; Roche Diagnostics, Basel, Switzerland), according to the manufacturer's instructions. Annexin V‐positive/PI‐positive and Annexin V‐positive/PI‐negative cell populations were defined as apoptotic cells.

Statistical analysis

Data are expressed as the mean ± standard error. Statistical analysis was performed using SPSS software (SPSS, Inc., Chicago, IL, USA). The Student's t‐test was used to assess differences among different groups. P < 0.05 was considered to indicate a statistically significant difference.

Results

Quercetin reduces the cell viability of KRAS‐mutant cells

DLD‐1KRASG13D and DLD‐1KRASWT cells were treated with quercetin at various concentrations for 48 h. As presented in Figure 1A, quercetin (100 μM) reduced the viability of DLD‐1KRASG13D and DLD‐1KRASWT cells to 34.6% and 71.8%, as measured using MTT assays; DLD‐1KRASG13D cells appeared to be more sensitive to quercetin‐induced reduction in viability than DLD‐1KRASWT cells. The different responses of the cells to quercetin were also reflected by the effect on long‐term cell survival, as demonstrated in the clonogenic assay (Figures 1B and 1C). The increased susceptibility of KRAS‐mutant colon cancer cells to quercetin was confirmed in additional two KRAS‐mutant cell lines (SW480KRASG12V and HCT116 KRASG13D) and three KRAS‐wild‐type cell lines (Colo205KRASWT, WIDRKRASWT, and HT29KRASWT; Figure 1D). Taken together, these results indicated that KRAS‐mutant colon cancer cells were more susceptible to quercetin treatment compared with the wild‐type cancer cells.

Figure 1.

Figure 1

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Quercetin inhibits cell proliferation in colorectal cancer cells in vitro. (A) Effects of quercetin on proliferation in DLD‐1KRASG13D and DLD‐1KRASWT cells. Following quercetin treatment at the indicated concentrations (25–100 μM), or 5% ethanol treatment as the vehicle control, for 72 h, DLD‐1KRASG13D and DLD‐1KRASWT proliferation was assessed in a 96‐well plates by MTT assay. The data are presented as the mean ± SEM of three individual experiments. (B and C) Quercetin inhibits CRC cells growth in colony formation assays. DLD‐1KRASG13D and DLD‐1KRASWT were treated with quercetin and colony formation was assessed after 14 days. (B) Wells were stained with crystal violet dye and the absorbance was measured at 540 nm. Data represent the means ± SEM. (C) Representative images of the colony formation assay. (D) Inhibition of proliferation by quercetin in a panel of colorectal cancer cell lines. Cells were treated with quercetin or 5% ethanol for 72 h and then subjected to measurement by MTT assay. The data are presented as the mean ± SEM of three individual experiments. SEM, standard error of the mean.

Quercetin induces apoptosis in KRAS‐mutant cells

Whether the induction of apoptosis was involved in the quercetin‐mediated inhibition of cell growth in DLD‐1KRASG13D and DLD‐1KRASWT cells was also investigated. Flow cytometric analysis suggested that apoptosis of KRAS‐mutant cells was induced by quercetin treatment (Figure 2A); the percentage of apoptotic cells was increased from 3.7% (0 μM quercetin) to 24.3% (100 μM quercetin). Cleaved‐PARP, a inactivated form of PARP protein, is usually considered to be a marker of apoptosis. In the current study, the amount of cleaved‐PARP was increased as the concentration of quercetin increased 50 µM in the DLD‐1KRASG13D cells, but no obvious change in cleaved‐PARP protein was observed in the DLD‐1KRASWT cells following treatment with quercetin (Figure 2B). Consistent with the results of cell viability analysis, quercetin‐induced apoptosis was increased in KRAS‐mutant cells compared with wild‐type cells (Figure 2C).

Figure 2.

Figure 2

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Apoptosis assay of KRAS‐mutant and KRAS‐wild‐type cell lines treated with quercetin. (A) Apoptosis analysis of DLD‐1KRASG13D and DLD‐1KRASWT treated with quercetin and stained with Annexin V. Flow cytometry results are the representative of three independent experiments. (B) Western blot analysis to detect cleaved PARP proteins in DLD‐1KRASG13D and DLD‐1KRASWT cells treated with quercetin. (C) Induction of apoptosis by quercetin in a panel of colorectal cancer cell lines. Cells treated with quercetin (25, 50, and 100 μM) or 5% ethanol for 24 h were subjected to Annexin V staining using flow cytometry. Values are presented as the mean + standard error of three individual experiments.

Quercetin induces caspase‐dependent apoptosis

Caspase‐3 activation is one of the essential steps during apoptosis. To understand the mechanisms of quercetin‐induced apoptosis, caspase‐3 protein was detected by Western blot assay. As demonstrated in Figure 3A, the level of cleaved‐caspase‐3 was markedly increased in DLD‐1KRASG13D cells as the concentration of quercetin increased; however, the effect of quercetin on caspase‐3 was less pronounced in DLD‐1KRASWT cells. The caspase inhibitors Z‐DEVD (caspase‐3 inhibitor), Z‐IETD (caspase‐8 inhibitor), and Z‐LEHD (caspase‐9 inhibitor) were used to confirm the functional role of caspases in quercetin‐induced cell death. The percentage of apoptotic cells in quercetin‐treated groups were decreased by all three caspase inhibitors (Figures 3B and 3C), demonstrating that quercetin‐induced apoptosis is caspase‐dependent and suggesting that the intrinsic (mitochondria‐mediated) and extrinsic (death receptor‐mediated) apoptotic pathways are both involved in quercetin‐induced apoptosis.

Figure 3.

Figure 3

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Quercetin induces caspase‐dependent apoptosis in colorectal cancer cells. (A) Quercetin induces caspase activation. Whole cell lysates from DLD‐1KRASG13D and DLD‐1KRASWT cells treated with quercetin (50 and 100 μM) were subjected to western blot analysis. The data presented are representative of three individual experiments. (B and C) DLD‐1KRASG13D and DLD‐1KRASWT cells were pretreated with the caspase‐3 inhibitor, Z‐DEVD‐fmk (8 µmol/l), the caspase‐9 inhibitor, Z‐LEHD‐fmk (20 µmol/l), or the caspase‐8 inhibitor, Z‐IETD (10 µmol/l), for 1 h before adding quercetin (100 μM) for another 24 h. Apoptosis was measured by Annexin V and propidium iodide staining using flow cytometry. Data are presented as the mean ± standard error of three individual experiments.

KRAS downstream signaling pathways regulate quercetin‐induced apoptosis in KRAS‐mutant CRC cells

Various signaling pathways affect apoptosis and are associated with RAS, including AKT, extracellular signal‐regulated kinase (ERK) and JNK. To clarify the status of these pathway during quercetin‐induced apoptosis, ERK, mitogen‐activation protein kinase (MAPK) 7 (MEK), JNK, AKT and the phosphorylated forms of these proteins were detected in KRAS‐mutant and KRAS‐wild‐type cell lines. The cells were treated with 50 µM and 100 µM quercetin for 24 h and proteins of the KRAS‐associated signaling pathways were detected by Western blot analysis. β‐actin was used as an internal‐control.

As demonstrated in Figure 4A, phosphorylated‐ERK and total ERK, and phosphorylated‐MEK and total MEK were not markedly altered following quercetin treatment in either of the cell lines. The DLD‐1KRASG13D cell line had a higher basal level of phosphorylated‐JNK than wild‐type cells, which was further increased upon quercetin treatment, whereas no such changes were observed in DLD‐1KRASWT cells line upon quercetin treatment. Additionally, DLD‐1KRASG13D cells had a higher basal level of phosphorylated‐AKT, which was decreased upon treatment with the highest quercetin concentration. In the isogenic wild‐type cells, there was no change in phosphorylated‐AKT levels following quercetin treatment.

Figure 4.

Figure 4

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AKT and JNK signaling pathways regulate quercetin‐induced apoptosis in KRAS‐mutant colorectal cancer cells. (A) Expression changes of proteins involved in KRAS‐associated signaling pathways following quercetin treatment. DLD‐1KRASG13D and DLD‐1KRASWT were treated with 50 and 100 µM quercetin for 24 h. ERK, MEK, JNK, AKT, and their phosphorylated forms were detected by Western blot analysis. Western blot images are the representative of three independent experiments. (B) JNK inhibitor, SP600125, inhibits the phosphorylation of JNK, but has no effect on the expression of total JNK. The data shown are representative of three individual experiments. (C) Inhibition of JNK rescues DLD‐1KRASG13D cells from quercetin‐induced apoptosis. DLD‐1KRASG13D cells were pretreated with SP600125 (20 μM) 1 h before adding quercetin (100 μM) for another 24 h, respectively. Apoptosis was measured by Annexin V and PI staining using flow cytometry. Data are presented as the mean + standard error of three individual experiments. p‐, phosphorylated; ERK, extracellular signal‐regulated kinase; MEK, mitogen‐activated protein kinase 7; JNK, c‐Jun N‐terminal kinase; AKT, AKT serine/threonine kinase.

To further delineate the pathways involved in quercetin‐induced apoptosis, the effects of a JNK inhibitor, SP600125 (20 µM), were evaluated. SP600125 suppressed the phosphorylation of JNK (Figure 4B) and prevented apoptosis induced by quercetin (Figure 4C). The results indicate that the JNK signal transduction pathway is involved in quercetin‐induced apoptosis.

Discussion

KRAS‐mutant tumors are particularly resistant to chemotherapy and various targeted therapeutic agents. In fact, KRAS mutation is an exclusion criterion for treatment with an epidermal growth factor receptor inhibitor (Siddiqui and Piperdi, 2010; Wheeler et al., 2010; Dienstmann et al., 2015). Directly targeting mutant KRAS is a challenge for drug design. Research on RAS protein trafficking and modification suggested a number of druggable targets, such as farnesyltransferase (Geryk‐Hall et al., 2010). However, farnesyltransferase inhibitors yielded disappointing results in clinical trials (Geryk‐Hall et al., 2010). Another approach is to target downstream pro‐survival pathways, including the MAPK and AKT pathways, among others (Yuen et al., 2012). However, in clinical practice, inhibition of both pathways leads to dose‐limiting toxicity (Pratilas and Solit, 2010; Little et al., 2013). Despite these failures, considering the prevalence of KRAS mutation in CRC, there are continuous efforts to target aberrant KRAS signaling (Ledford, 2015).

Quercetin is a flavonoid that is frequently found in fruits, vegetables, leaves, and grains. Research into the anti‐carcinogenic potential of flavonoids with animal and cellular model systems indicates that quercetin has a protective role against the progression of various types of cancer (Rajesh et al., 2015; Srinivas, 2015). An impressive body of information exists on the anti‐tumor action of quercetin. Since flavonoids have a higher concentration in the gut (especially in colorectal mucosa) than in other tissues (Mendoza and Burd, 2011), the current study investigated whether quercetin is an effective agent against CRC. The current study revealed that quercetin selectively induces apoptosis in a KRAS‐mutant CRC cell line, but not in its isogenic wild‐type line. This is particularly notable and is consistent with the recent finding that Asian populations has the lowest KRAS mutation rate while consume more vegetables, fruits and tea (enriched with quercetin and other flavonoids) than other populations, such as in the United States (Giovannucci, 2002; Center et al., 2009; Morrison et al., 2011; Morrison et al., 2013).

Upon further biochemical investigation, it was identified that activation of JNK signaling and inhibition of the AKT pathway may be an underlying mechanism of the selective effects of quercetin. AKT is a major pro‐survival and anti‐apoptotic pathway downstream of KRAS. It can activate the mechanistic target of rapamycin pathway, which is a master regulator of cell growth, protein translation and other macromolecule synthesis (Temraz et al., 2015), and can directly inhibit apoptosis (Li et al., 2015b). KRAS‐mutant tumors experience increased oncogenic stress and, thus, have higher levels of phosphorylated‐AKT and are more susceptible to AKT perturbation. JNK signaling can induce pro‐apoptotic or anti‐apoptotic effects, depending on the cellular stimuli, parallel signaling events and the overall apoptotic signaling strength (Dhanasekaran and Reddy, 2008). In the case of quercetin treatment, it induced sustained activation of JNK, together with inhibition of the AKT pathway. Phosphorylated‐JNK translocates to the nucleus and then phosphorylates and transactivates c‐Jun, consequently leads to the formation of the activator protein‐1 dimers. The transcription of a wide variety of proteins, many of which are known pro‐apoptotic proteins, are subsequently activated (Dhanasekaran and Johnson, 2007).

In summary, the current study demonstrated that quercetin suppresses the proliferation of CRC cells and induces apoptosis, particularly in cells harboring activated KRAS mutation. This effect is, at least partially, due to the inhibition of AKT and the activation of JNK signaling. These findings provide novel insights into the anti‐CRC molecular mechanism of quercetin, and further support the potential use of quercetin in KRAS‐mutant CRC treatment.

Acknowledgments and funding

We thank Professor Xudong Zhang and Lei He of Newcastle University for giving kindly advice. This work was supported by research grants from the Natural Science Foundation of China (81471551 and 81571861).

Yiwen Yang and Tao Wang contributed equally to this work.

Contributor Information

Yufang Wang, Email: wangyufang@scu.edu.cn.

Ji Zhang, Email: zhangj@scu.edu.cn.

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