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Quercetin Enhances Chemosensitivity to Gemcitabine in Lung Cancer Cells by Inhibiting Heat Shock Protein 70 Expression
Original Study
Quercetin Enhances Chemosensitivity to Gemcitabine in Lung Cancer Cells by Inhibiting Heat Shock Protein 70 Expression Seung Hyeun Lee,1 Eun Joo Lee,2 Kyung Hoon Min,2 Gyu Young Hur,2 Seung Heon Lee,2 Sung Yong Lee,2 Je Hyeong Kim,2 Chol Shin,2 Jae Jeong Shim,2 Kwang Ho In,2 Kyung Ho Kang,2 Sang Yeub Lee2 Abstract Quercetin is a bioflavonoid known for antioxidation and antiproliferation activities. We demonstrated that quercetin inhibited cancer cell growth and sensitized cancer cells to gemcitabine treatment by promoting apoptosis via inhibiting heat shock protein 70 expression. Our results suggest that quercetin might have potential to increase sensitivity to chemotherapy and that heat shock protein 70 could be a new target for lung cancer treatment. Background: Quercetin is a bioflavonoid with antiproliferative and proapoptotic activity in various cancer cells. However, little is known about the mechanism by which quercetin inhibits cancer growth or its potential role as a chemosensitizer in lung cancer cells. We investigated whether quercetin-induced inhibition of heat shock protein 70 (HSP70) is involved in its anticancer activity and whether it could modulate the responsiveness of lung cancer cells to chemotherapy. Materials and Methods: Various concentrations of quercetin and gemcitabine, either alone or in combination, were applied to lung cancer cells (A549 and H460 cells). We evaluated cell viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt assay, apoptotic activity by determining caspase-3 and caspase-9 activities, and HSP70 expression using Western blot analysis after treatment. Results: Quercetin reduced cell viability and suppressed HSP70 expression in both cell lines dose-dependently. Adding a fixed quercetin dose enhanced gemcitabine-induced cell death, which was related to increased caspase-3 and caspase-9 activities. Combination treatment with quercetin and gemcitabine downregulated HSP70 expression more prominently than treatment with quercetin or gemcitabine alone. Conclusion: Quercetin-induced HSP70 inhibition was involved in growth inhibition and sensitization to chemotreatment in lung cancer cells. Quercetin might have potential as a chemosensitizer in lung cancer treatment. Clinical Lung Cancer, Vol. -, No. -, --- ª 2015 Elsevier Inc. All rights reserved. Keywords: Apoptosis, Gemcitabine, Heat shock protein, Lung cancer, Quercetin
Introduction Lung cancer is the leading cause of cancer-related mortality, accounting for approximately 20% of all cancer deaths.1 Nonesmallcell lung cancer (NSCLC) accounts for approximately 80% of 1 Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, KEPCO Medical Center, Seoul, Republic of Korea 2 Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea
Submitted: Mar 20, 2015; Revised: May 10, 2015; Accepted: May 12, 2015 Address for correspondence: Sang Yeub Lee, MD, PhD, Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Korea University Anam Hospital, Korea University College of Medicine, 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, Korea Fax: þ82 2 929 2045; e-mail contact: [email protected]
1525-7304/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cllc.2015.05.006
all lung cancer cases. The prognosis of NSCLC is very poor, because 40% of cases are diagnosed in the advanced stage, and the 5-year survival rate is as low as 15%.2 Although recent advances in early diagnosis, chemotherapeutics, and surgical techniques have gradually improved survival, the rate of the NSCLC decline in mortality has plateaued in the past 2 decades.1 Among various factors, nonresponse and acquired resistance to chemotherapy are the most significant barriers to improvement of long-term outcomes. To overcome the limitations of the current treatment strategy, researchers have been actively searching for drugs that: (1) target particular pathways mainly involved in cancer progression; and (2) improve cancer cell sensitivity to chemotherapy and prevent the development of drug resistance.
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Quercetin and Chemosensitivity to Gemcitabine Heat shock proteins (HSPs) are a group of molecular chaperones that assist in protein-folding, modification, and transportation. HSP expression increases under stressful conditions, such as increased temperature, hypoxia, or radiation, to protect cells from injury.3 HSP70 is one of the main HSPs, and its overexpression has been reported in a wide range of malignant tumors. In addition, HSP70 overexpression has been related to drug resistance in colon cancer, breast cancer, and pancreatic cancer cells by inhibiting apoptosis.4-6 Studies have shown that inhibition of HSP70 expression potently induced apoptosis and inhibited the growth of many types of cancer cells.5,7,8 Increased HSP70 expression has also been described in serum and tissue samples from patients with NSCLC and is related to an adverse prognosis.9,10 However, studies on the anticancer effects of inhibition of HSP70 in lung cancer are very limited. Quercetin is a major bioflavonoid abundant in fruits and vegetables.11 Quercetin is characterized by its diverse biological activities, including antioxidation, proliferation, and proapoptosis.12 Numerous studies have reported a variety of beneficial effects of quercetin on cardiovascular disease, rheumatologic disease, and neurodegeneration.13-15 In addition, quercetin has cancer-preventive and anticancer effects on various types of cancer cells.15 Although quercetin is involved in multiple intracellular pathways, studies have shown that it induces apoptosis by downregulation of HSP70 expression in various cancer cells without affecting normal epithelial cells.6,16,17 Moreover, quercetin potentiates the growth inhibition of several chemotherapeutic agents, such as paclitaxel and cisplatin, through different intracellular mechanisms in different types of cancer cells.18,19 However, little is known about the anticancer effect of quercetin in lung cancer cells, and no studies have been conducted to investigate whether quercetin has a chemosensitizing effect on these cells. Considering a study that reported that quercetin and quercetin metabolites distribute at the highest concentrations in the lungs,20 lung cancer could be a good candidate for quercetin treatment. Thus, we performed this study to evaluate the following: (1) the growth inhibitory effect of quercetin; (2) the association between quercetin-induced growth inhibition and HSP70 expression; and (3) the possible role of quercetin as a chemotherapy sensitizer in lung cancer cells.
Korea). A Caspase-Glo assay kit was purchased from Promega (Madison, WI).
Cell Cultures and Treatment A549 and H460 cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37 C in a humidified atmosphere with 5% CO2. The cells were incubated in 96-well plates with 2 104 cells per well for viability measurements. The cells were plated in 6-well plates at a density of 2.5 105 cells per well to analyze protein and apoptosis, and were incubated overnight at 37 C. To determine cell viability and protein expression doseresponses, different concentrations of quercetin (10, 50, 100, and 200 mM) and gemcitabine (0.01, 0.1, 1, and 10 mg/mL) were dissolved in DMSO and added to the cells in serum-free medium, followed by a 24-hour incubation at 37 C.
Determining Cell Viability Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide salt (MTT) assay. The cells were incubated in 96-well plates at 1 104 cells per well overnight. After treatment with quercetin and gemcitabine, either alone or in combination at various concentrations for 24 hours, 10 mL of MTT (0.5 mg/mL) was added to each well, and the plate was incubated at 37 C to allow for the formation of blue formazan crystals. After 2 hours, residual MTT was carefully removed, and the crystals were dissolved by incubation with 150 mL of DMSO for 30 minutes. After shaking the plate for 1 hour, absorbance was measured at 570 nm. All experiments were repeated independently at least 3 times in quadruplicate.
Measuring Apoptotic Activity The Caspase-Glo luminescence-based assay was used to measure caspase-3 and caspase-9 activities according to the manufacturer’s instructions. Cells were seeded into 96-well white opaque plates and corresponding optically clear 96-well plates at 1 104 cells per well and allowed to adhere overnight. Then, the cells were treated with various concentrations of quercetin and gemcitabine, either alone or in combination for 24 hours. At the end of the incubation, 100 mL of Caspase-Glo reagent was added to each well. The plates were gently mixed and incubated at room temperature for 2 hours. Luminescence was read using a luminometer.
Measuring HSP70 Levels Using Western Blot Analysis
Materials and Methods Reagents and Chemicals
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Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone), dimethyl sulfoxide (DMSO), Ponceau S solution, and nitrocellulose membranes were purchased from Sigma-Aldrich (St Louis, MO). Gemcitabine was kindly donated by Eli Lilly Pharmaceuticals (Indianapolis, IN). Rabbit anti-b-actin and rabbit anti-b-tubulin were purchased from Abcam (Cambridge, UK). Goat antimouse immunoglobulin (Ig) G-horseradish peroxidase (HRP) and goat antirabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The human NSCLC cell lines A549 and H460 were obtained from the Korean Cell Line Bank (Seoul National University, Seoul, Korea). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT). Penicillin-streptomycin was acquired from WelGENE Inc (Daegu,
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Cell lysates were prepared by resuspending cells in lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), and 1% Triton X-100) for 20 minutes at 4 C and then cleared by centrifugation for 15 minutes at 13,000g. The supernatants were collected and stored at 80 C. Total protein concentration was measured with a Pierce bicinchoninic acid assay. Equal quantities of lysates were run on 10% sodium dodecyl sulfate-polyacrylamide gels and blotted to nitrocellulose membranes for the HSP70 Western blot analysis. Equal protein loading was confirmed by staining with Ponceau S. The membranes were blocked with 5% skim milk for 1 hour at room temperature and incubated with mouse monoclonal antiHSP70, rabbit anti-b-actin, and rabbit anti-b-tubulin to measure HSP70 levels. After 2 washes in Tris-buffered saline Tween-20, the blots were incubated with goat antimouse IgG-HRP and goat
Seung Hyeun Lee et al
Results
(Figure 1A). Viability was reduced by 22% at 0.01 mg/mL gemcitabine, and the reduction also tended to be dose-dependent (Figure 1B). Quercetin treatment reduced H460 cell viability by 20% at 10 mM, and a dose-dependent reduction in cell viability was observed with increasing concentrations. An approximate 67% reduction in cell viability was observed at 200 mM quercetin (Figure 1C). Gemcitabine treatment also reduced cell viability by 42% at 0.01 mg/mL, and cell growth was inhibited dosedependently with increasing concentrations (Figure 1D). Incubating either cell line with 0.1% DMSO had no effect on viability. H460 cells were more susceptible to quercetin treatment according to the 50% inhibitory concentration (IC50; 175.3 mM for A549 cells vs. 78.1 mM for H460 cells). Similarly, H460 cells were more susceptible to gemcitabine treatment (no IC50 for A549 cells vs. 0.1 mg/mL for H460 cells).
Cell Viability After Quercetin and Gemcitabine Treatment
Apoptotic Activity After Quercetin Treatment
Figure 1 shows the effects of a 24-hour treatment with quercetin and gemcitabine at varying concentrations on cell viability. A549 cell viability was reduced by 20% at a concentration of 10 mM quercetin and was further suppressed in a dose-dependent manner with increasing quercetin concentrations. An approximately 60% reduction in cell viability was observed at 200 mM quercetin
To evaluate the role of apoptosis on the inhibition of cell growth by quercetin, we measured and compared caspase-3 (effector caspase) and caspase-9 (initiator caspase) activities in quercetin treatments. In the A549 cell line, a 1.7-fold increase in caspase-3 activity was observed at 10 mM quercetin, and the activity increased in a dose-dependent manner, with maximum activity of 2.0-fold at
antirabbit IgG-HRP. After extensive washing, the immunoreactive bands were visualized using enhanced chemiluminescence (West-Q Femto Clean ECL Solution; GenDEPOT, Barker, TX). The data were normalized to the b-actin level and are presented as relative band density to b-actin using Scion Image software (version 4.0.2, Scion Corporation, Frederick, Maryland).
Statistical Analysis Data are expressed as mean standard error. Differences between the control and experimental test groups were analyzed with the unpaired Student t test. A P value < .05 was considered significant. The statistical analysis was performed using SPSS version 13.0 for Windows (SPSS Inc, Chicago, IL).
Figure 1 Effects of Quercetin and Gemcitabine on Cell Viability. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Salt Assay Demonstrated That Quercetin and Gemcitabine Treatment Reduced A549 Cell Viability in a Dose-Dependent Manner; This Reduction Was Significant at 10 mM and 0.01 mg/mL, Respectively (A and B). H460 Cell Viability Also Decreased With Both Treatments, and There Was a More Prominent Reduction in Viability Compared With That in A549 Cells (C and D). *P < .05 Compared With the Control. Bars Indicate Standard Errors
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Quercetin and Chemosensitivity to Gemcitabine Figure 2 Effects of Quercetin on Apoptosis. In A549 Cells, Caspase-3 and Caspase-9 Activities Increased Dose-Dependently From the Concentration of 10 mM (A and B). Caspase Activities in H460 Cells Also Increased in a Dose-Dependent Manner, and a More Prominent Increase Was Observed Compared With That in A549 Cells (C and D). *P < .05 Compared With the Control. Bars Indicate Standard Errors
200 mM (Figure 2A). Similar dose-dependent increase was observed in caspase-9 activity (Figure 2B). In the H460 cell line, a 1.6-fold increase in caspase-3 activity was observed at 10 mM quercetin, and a dose-dependent increase was observed, with a maximal increase of 2.9-fold at 200 mM (Figure 2C). Also, caspase-9 activity was increased dose-dependently, with a maximal increase of 3.1-fold at 200 mM (Figure 2D).
Heat Shock Protein 70 Expression After Quercetin Treatment Figure 3 shows the effect of quercetin on HSP70 expression. A Western blot analysis showed that quercetin inhibited of HSP70 expression in a dose-dependent manner in both cell lines. Relative HSP70 expression at 200 mM quercetin was reduced by 60% in A549 cells and by 82% in H460 cells. Quercetin-induced HSP70 inhibition was more prominent in H460 cells based on the IC50 value (165.5 mM vs. 95.3 mM).
Cell Viability After Combined Quercetin and Gemcitabine Treatment
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Figure 4 shows the effect of the combined treatment with quercetin and gemcitabine on cell viability. Both cell lines were
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incubated with various concentrations of gemcitabine in the presence of a fixed dose of quercetin (50 mM). A significant reduction in viability was observed at 0.01 mg/mL gemcitabine in the A549 (Figure 4A) and H460 (Figure 4B) cell lines compared with that of the gemcitabine-alone treatment (all P < .05). This decreased viability was more prominent with increasing gemcitabine concentrations, suggesting a dose-dependent relationship. In addition, the gemcitabine IC50 was not different between the cell lines after the combined treatment, suggesting that the difference in susceptibility between the 2 cell lines that occurred with the gemcitabine-alone treatment disappeared after the addition of quercetin.
Apoptotic Activity After Combined Quercetin and Gemcitabine Treatment We measured and compared caspase-3 and caspase-9 activities in gemcitabine alone and combined quercetin and gemcitabine treatments to evaluate the role of quercetin in apoptosis-mediated growth inhibition. In the A549 cell line, a 1.3-fold increase in caspase-3 activity was observed with 0.1 mg/mL gemcitabine, and activity increased in a dose-dependent manner, with maximum activity of 1.8-fold at 10 mg/mL. The combination with quercetin resulted in a significant increase in caspase-3 activity at 0.1 mg/mL
Seung Hyeun Lee et al Figure 3 Effects of Quercetin and Gemcitabine on Heat Shock Protein 70 (HSP70) Expression. A Western Blot Analysis Shows That Quercetin Treatment Reduced HSP70 Expression in a Dose-Dependent Manner in A549 Cells (A) and H460 Cells (B). *P < .05 Compared With the Control. Bars Indicate Standard Errors
compared with that of the gemcitabine-alone treatment, and a maximum 5.8-fold increase was observed at 10 mg/mL (Figure 5A). In the H460 cell line, a 2-fold increase in caspase-3 activity was observed for the 0.01 mg/mL gemcitabine-alone
treatment, and a dose-dependent increase was observed, with a maximal increase of 3.6-fold at 10 mg/mL. A significantly greater increase in caspase-3 activity was noted in the combined quercetin and gemcitabine treatment at 0.01 mg/mL compared with that of
Figure 4 Combined Effect of Quercetin and Gemcitabine on Cell Viability. The Combination of a Fixed Dose of Quercetin (50 mM) and Various Concentrations of Gemcitabine Led to a Significant Dose-Dependent Reduction in A549 Cell (A) and H460 Cell (B) Viability at All Concentrations. This Effect Was Significantly More Prominent Compared With That of Treatment With Gemcitabine Alone. *P < .05 Compared With Treatment With Gemcitabine Alone. Bars Indicate Standard Errors
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Quercetin and Chemosensitivity to Gemcitabine Figure 5 Combined Effect of Quercetin and Gemcitabine on Apoptosis. The Combined Treatment of Quercetin and Gemcitabine More Significantly Increased Caspase-3 Activity in the A549 (A) and H460 (B) Cell Lines With Doses as Low as 0.1 mg/mL Gemcitabine. In Addition, Caspase-9 Activity Increased Significantly at the Same Concentration of Gemcitabine in A549 (C) and H460 (D) Cells. *P < .05 Compared With the Control. Bars Indicate Standard Errors
the gemcitabine-alone treatment, and the increased activity was dose-dependent. The maximal increase was 9.2-fold at 1 mg/mL (Figure 5B). The gemcitabine-alone treatment did not affect caspase-9 activity in both cell lines. However, a dose-dependent increase of the activity was observed with a maximal increase of 2.2-fold at 10 mg/mL in A549 (Figure 5C) and H460 (Figure 5D) cells in the combined quercetin and gemcitabine treatment.
Heat Shock Protein 70 Expression After Combined Quercetin and Gemcitabine Treatment
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To validate the role of HSP70 expression on cell viability for the combined treatment, we compared HSP70 expression levels with 0.1 mg/mL of gemcitabine and 50 mM of quercetin, either alone or in combination. As shown in Figure 5, HSP70 expression decreased significantly after the combined treatment compared with the quercetin- or gemcitabine-alone treatment in the A549 (Figure 6A) and H460 (Figure 6B) cell lines. These data suggest that the augmented growth inhibition and increased apoptosis after the combined treatment is associated with downregulation of HSP70 expression.
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Discussion Our data demonstrate that quercetin alone inhibited the growth of lung cancer cells dose-dependently and enhanced the growth inhibition caused by gemcitabine in a combined treatment. The growth-inhibitory effect of quercetin was associated with downregulation of HSP70, and increased anticancer activity in the combined treatment was mediated by increased apoptotic activity via inhibition of HSP70 expression. To the best of our knowledge, this is the first study to elucidate the association between the anticancer activity of quercetin and HSP70 modulation and the chemosensitizing effect of quercetin, which was induced by HSP70-related apoptosis in the lung cancer cell lines. Quercetin is a major flavonoid that is abundant in a broad range of vegetables and fruits. It has attracted the attention of cancer researchers because it has a variety of biologic activities, including antioxidative, antiproliferative, proapoptotic, and antiangiogenic activities, which affect cancer development and progression.15 Quercetin exerts anticancer effects in different cancers through different mechanisms, including inhibition of oncogenic activation, modulation of signal transduction, and interactions with receptors;
Seung Hyeun Lee et al Figure 6 Heat Shock Protein 70 (HSP70) Expression in the Combined Quercetin (Q) and Gemcitabine (G) Treatment. A Combined Treatment With 50 mM Q and 0.1 mg/mL G (G D Q) Decreased HSP70 Expression Significantly in A549 (A) and H460 (B) Cells. *P < .05 Compared With That of the Treatment With G Alone. Bars Indicate Standard Errors
it downregulates mutant p53 expression in breast cancer,21 induces Gap 1 (G1)/Synthesis (S) arrest in colon and gastric cancer cells, and blocks the Gap 2 (G2)/Mitosis (M) transition in breast and lung cancer cells.21-24 In addition, quercetin-induced apoptosis is associated with inactivating the Akt-1 protein, altering expression of Bcl-2 family proteins, and activating the mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway in lung cancer cells.25 Moreover, quercetin inhibits cell invasion and induces apoptosis through a pathway involving HSPs.17 In particular, quercetin inhibits HSP70 expression by blocking heat shock transcription factor (HSF) 1 and HSF2 and by reducing HSP70 mRNA accumulation.26 Quercetin downregulates HSP70 expression in various cancer cell lines, including breast, pancreatic, and colon cancer cells.6,27,28 However, the association between HSP70 expression and quercetin-induced growth inhibition in lung cancer cells has never been evaluated. Our data show that quercetin inhibited growth of lung cancer cells by downregulating HSP70 expression. In addition, the decreased expression of HSP70 was involved in quercetin-induced chemosensitization by increasing apoptotic activity. Our results are supported by previous findings that demonstrated that the combination of quercetin with different chemotherapeutic agents improved the efficacy of many cancer therapies.29 Apoptosis occurs via 2 main pathways. The extrinsic pathway is characterized by activation of death receptors on the cell surface, whereas the intrinsic or mitochondrial pathway is independent of death receptor signaling. The latter pathway involves the release of cytochrome c from mitochondria and the formation of a complex of cytochrome c, cytoplasmic apoptotic protease activating factor (Apaf)-1, and procaspase-9.30,31 In our study, quercetin-induced HSP70 inhibition significantly increased caspase-3 activity in both
lung cancer cell lines, suggesting that the apoptotic pathway is responsible for quercetin-induced cell death. Our data are consistent with those of a previous study that showed that quercetin induced apoptosis and caspase-3 activity in H460 cells.32 Moreover, we determined that the combination of quercetin and gemcitabine significantly increased caspase-9 activity. These results indicate that quercetin-induced HSP70 inhibition exerts its anticancer effect mainly via involvement of the intrinsic branch of the apoptotic pathway and it acts on the antiapoptotic function upstream of caspase3 in lung cancer cells. In addition, our findings suggest that HSP70 might play a major role in caspase-dependent apoptosis, and that downregulating HSP70 might be one of the mechanisms of quercetininduced apoptosis during inhibition of lung cancer cell growth. Gemcitabine is a pyrimidine nucleoside analogue that inhibits DNA synthesis. It exerts anticancer activity by inducing apoptosis and has been widely used against a variety of tumors. The response rate to gemcitabine in the advanced stages of NSCLC is approximately 25%, and the response rate increases to 54% when it is combined with platinum.33,34 Although the mechanism related to gemcitabine resistance in NSCLC is not fully understood, evidence suggests that insufficient intracellular concentrations of the active moiety and alterations in the associated apoptotic mechanism might contribute to this resistance.35 Similar to previous studies that investigated the effect of quercetin on gemcitabinetreated fibrosarcoma cells and pancreatic cancer cells,36,37 the present data show that quercetin-induced HSP70 inhibition enhanced the anticancer activity of gemcitabine in lung cancer cells. Although further study is required, the current results suggest that quercetin might be a good candidate as a chemosensitizer that would allow dose reduction and minimize drug toxicity in lung cancer treatment.
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Quercetin and Chemosensitivity to Gemcitabine Another implication of the present data is that inhibition of HSP70 could be a novel target for NSCLC treatment. HSP70 overexpression has been described in serum and tissue samples from patients with NSCLC and is related to an adverse prognosis in those patients.9,10 Previous reports demonstrate that downregulating HSP70 using small molecules or compounds potently inhibit cell proliferation in different cancers.6,8,38 However, targeting HSP70 as a lung cancer treatment is less well studied compared with other types of cancer. Results of 2 recent studies suggest that HSP70 could be a novel target for lung cancer treatment. Wen et al demonstrated that the small molecule VER155008, which specifically inhibits HSP70 expression, inhibits NSCLC proliferation and cell cycle progression and sensitizes A549 cells to ionizing radiation.39 In that study, the antiproliferative effect of VER-155008 was synergized when combined with an HSP90 inhibitor. Furthermore, Endo et al demonstrated that ibuprofen enhances cisplatin-induced apoptotic cell death in A549 cells by suppressing HSP70 expression.40 In that study, enhanced anticancer activity was mediated by the mitochondrial apoptotic pathway, which is identical with our results. Taken together, modulating HSP70 expression could be a promising therapeutic strategy for lung cancer either alone or in combination with other treatment modalities. Several limitations of our study should be discussed. First, despite that our data showed the potential role of quercetin as a novel chemosensitizer, an in vivo study to validate the in vitro results was not performed. Second, the possible mechanism by which quercetin downregulates HSP70 expression was not evaluated simultaneously. Further study using a lung cancer xenograft model and the candidate molecules that might be involved in downregulating HSP70 expression is required for quercetin to be evaluated in a clinical trial, similar to another HSP70 inhibitor.41
Conclusion We identified a role for HSP70 in quercetin-induced growth inhibition and chemosensitization in lung cancer cells. Our data suggest a potential benefit of quercetin in the treatment of lung cancer and clinical implications of HSP70 as a novel target for future development of lung cancer therapeutics. Further investigation should be conducted to validate the present findings in vivo and to determine the effects of quercetin or other HSP70 inhibitors combined with different chemotherapeutic agents on lung cancer growth.
Clinical Practice Points Quercetin is a bioflavonoid known for antioxidation, anti-
inflammation, and antiproliferation activities. Previous studies have demonstrated that quercetin not only in-
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hibits proliferation of a broad range of cancer cells but also enhances sensitivity to chemotherapeutic agents. However, the effects of quercetin on lung cancer cells have rarely been studied, and the combined effects of quercetin with other chemotherapeutic agents on lung cancers cells have not been investigated. We demonstrated that quercetin inhibited cancer cell growth and sensitized cancer cells to gemcitabine treatment by promoting apoptosis via inhibiting HSP70 expression.
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Although further studies are needed to verify our results, our
results suggest potential roles of quercetin and HSP70 as a chemosensitizer and a new target for lung cancer treatment, respectively.
Disclosure The authors have stated that they have no conflicts of interest.
References 1. Jemal A, Siegel R, Xu J, et al. Cancer statistics, 2010. CA Cancer J Clin 2010; 60: 277-300. 2. Fu XL, Zhu XZ, Shi DR, et al. Study of prognostic predictors for nonesmall-cell lung cancer. Lung Cancer 1999; 23:143-52. 3. Ritossa F. Discovery of the heat shock response. Cell Stress Chaperones 1996; 1:97-8. 4. Vargas-Roig LM, Gago FE, Tello O, et al. Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy. Int J Cancer 1998; 79:468-75. 5. Nylandsted J, Rohde M, Brand K, et al. Selective depletion of heat shock protein 70 (Hsp70) activates a tumor-specific death program that is independent of caspases and bypasses Bcl-2. Proc Natl Acad Sci U S A 2000; 97:7871-6. 6. Aghdassi A, Phillips P, Dudeja V, et al. Heat shock protein 70 increases tumorigenicity and inhibits apoptosis in pancreatic adenocarcinoma. Cancer Res 2007; 67: 616-25. 7. Kaur J, Ralhan R. Induction of apoptosis by abrogation of HSP70 expression in human oral cancer cells. Int J Cancer 2000; 85:1-5. 8. Yang S, Chen J, Guo Z, et al. Triptolide inhibits the growth and metastasis of solid tumors. Mol Cancer Ther 2003; 2:65-72. 9. Huang Q, Zu Y, Fu X, et al. Expression of heat shock protein 70 and 27 in nonesmall-cell lung cancer and its clinical significance. J Huazhong Univ Sci Technolog Med Sci 2005; 25:693-5. 10. Suzuki K, Ito Y, Wakai K, et al. Serum heat shock protein 70 levels and lung cancer risk: a case-control study nested in a large cohort study. Cancer Epidemiol Biomarkers Prev 2006; 15:1733-7. 11. Hertog MG, Hollman PC, Katan MB, et al. Intake of potentially anticarcinogenic flavonoids and their determinants in adults in The Netherlands. Nutr Cancer 1993; 20:21-9. 12. Choi JA, Kim JY, Lee JY, et al. Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin. Int J Oncol 2001; 19:837-44. 13. Chirumbolo S. The role of quercetin, flavonols and flavones in modulating inflammatory cell function. Inflamm Allergy Drug Targets 2010; 9:263-85. 14. Ishizawa K, Yoshizumi M, Kawai Y, et al. Pharmacology in health food: metabolism of quercetin in vivo and its protective effect against arteriosclerosis. J Pharmacol Sci 2011; 115:466-70. 15. Russo M, Spagnuolo C, Tedesco I, et al. The flavonoid quercetin in disease prevention and therapy: facts and fancies. Biochem Pharmacol 2012; 83:6-15. 16. Larocca LM, Ranelletti FO, Maggiano N, et al. Differential sensitivity of leukemic and normal hematopoietic progenitors to the killing effect of hyperthermia and quercetin used in combination: role of heat-shock protein-70. Int J Cancer 1997; 73:75-83. 17. Wei YQ, Zhao X, Kariya Y, et al. Induction of apoptosis by quercetin: involvement of heat shock protein. Cancer Res 1994; 54:4952-7. 18. Choi JS, Jo BW, Kim YC. Enhanced paclitaxel bioavailability after oral administration of paclitaxel or prodrug to rats pretreated with quercetin. Eur J Pharm Biopharm 2004; 57:313-8. 19. Jakubowicz-Gil J, Paduch R, Piersiak T, et al. The effect of quercetin on pro-apoptotic activity of cisplatin in HeLa cells. Biochem Pharmacol 2005; 69: 1343-50. 20. de Boer VC, Dihal AA, van der Woude H, et al. Tissue distribution of quercetin in rats and pigs. J Nutr 2005; 135:1718-25. 21. Avila MA, Velasco JA, Cansado J, et al. Quercetin mediates the down-regulation of mutant p53 in the human breast cancer cell line MDA-MB468. Cancer Res 1994; 54:2424-8. 22. Ong CS, Tran E, Nguyen TT, et al. Quercetin-induced growth inhibition and cell death in nasopharyngeal carcinoma cells are associated with increase in Bad and hypophosphorylated retinoblastoma expressions. Oncol Rep 2004; 11:727-33. 23. Yang JH, Hsia TC, Kuo HM, et al. Inhibition of lung cancer cell growth by quercetin glucuronides via G2/M arrest and induction of apoptosis. Drug Metab Dispos 2006; 34:296-304. 24. Ranelletti FO, Ricci R, Larocca LM, et al. Growth-inhibitory effect of quercetin and presence of type-II estrogen-binding sites in human colon-cancer cell lines and primary colorectal tumors. Int J Cancer 1992; 50:486-92. 25. Nguyen TT, Tran E, Nguyen TH, et al. The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549 lung cancer cells. Carcinogenesis 2004; 25:647-59. 26. Hosokawa N, Hirayoshi K, Kudo H, et al. Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids. Mol Cell Biol 1992; 12:3490-8. 27. Kuo SM. Antiproliferative potency of structurally distinct dietary flavonoids on human colon cancer cells. Cancer Lett 1996; 110:41-8.
Seung Hyeun Lee et al 28. Jakubowicz-Gil J, Rzymowska J, Gawron A. Quercetin, apoptosis, heat shock. Biochem Pharmacol 2002; 64:1591-5. 29. Chen C, Zhou J, Ji C. Quercetin: a potential drug to reverse multidrug resistance. Life Sci 2010; 87:333-8. 30. Kroemer G, Reed JC. Mitochondrial control of cell death. Nat Med 2000; 6:513-9. 31. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91:479-89. 32. Youn H, Jeong JC, Jeong YS, et al. Quercetin potentiates apoptosis by inhibiting nuclear factor-kappaB signaling in H460 lung cancer cells. Biol Pharm Bull 2013; 36:944-51. 33. Braakhuis BJ, van Dongen GA, Vermorken JB, et al. Preclinical in vivo activity of 20 ,20 -difluorodeoxycytidine (gemcitabine) against human head and neck cancer. Cancer Res 1991; 51:211-4. 34. León L, Cueva-Banuelos JF, Huidobro G, et al. Gemcitabine, cisplatin and vinorelbine as induction chemotherapy followed by radical therapy in stage III nonesmall-cell lung cancer: a multicentre study of Galician-Lung-Cancer-Group. Lung Cancer 2003; 40:215-20.
35. Sève P, Dumontet C. Chemoresistance in nonesmall-cell lung cancer. Curr Med Chem Anticancer Agents 2005; 5:73-88. 36. Sliutz G, Karlseder J, Tempfer C, et al. Drug resistance against gemcitabine and topotecan mediated by constitutive hsp70 overexpression in vitro: implication of quercetin as sensitiser in chemotherapy. Br J Cancer 1996; 74:172-7. 37. Hyun JJ, Lee HS, Keum B, et al. Expression of heat shock protein 70 modulates the chemoresponsiveness of pancreatic cancer. Gut Liver 2013; 7:739-46. 38. Phillips PA, Dudeja V, McCarroll JA, et al. Triptolide induces pancreatic cancer cell death via inhibition of heat shock protein 70. Cancer Res 2007; 67: 9407-16. 39. Wen W, Liu W, Shao Y, et al. VER-155008, a small molecule inhibitor of HSP70 with potent anti-cancer activity on lung cancer cell lines. Exp Biol Med (Maywood) 2014; 239:638-45. 40. Endo H, Yano M, Okumura Y, et al. Ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein 70. Cell Death Dis 2014; 5:e1027. 41. Chugh R, Sangwan V, Patil SP, et al. A preclinical evaluation of Minnelide as a therapeutic agent against pancreatic cancer. Sci Transl Med 2012; 4:156ra39.
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Quercetin Enhances Chemosensitivity to Gemcitabine in Lung Cancer Cells by Inhibiting Heat Shock Protein 70 Expression Seung Hyeun Lee,1 Eun Joo Lee,2 Kyung Hoon Min,2 Gyu Young Hur,2 Seung Heon Lee,2 Sung Yong Lee,2 Je Hyeong Kim,2 Chol Shin,2 Jae Jeong Shim,2 Kwang Ho In,2 Kyung Ho Kang,2 Sang Yeub Lee2 Abstract Quercetin is a bioflavonoid known for antioxidation and antiproliferation activities. We demonstrated that quercetin inhibited cancer cell growth and sensitized cancer cells to gemcitabine treatment by promoting apoptosis via inhibiting heat shock protein 70 expression. Our results suggest that quercetin might have potential to increase sensitivity to chemotherapy and that heat shock protein 70 could be a new target for lung cancer treatment. Background: Quercetin is a bioflavonoid with antiproliferative and proapoptotic activity in various cancer cells. However, little is known about the mechanism by which quercetin inhibits cancer growth or its potential role as a chemosensitizer in lung cancer cells. We investigated whether quercetin-induced inhibition of heat shock protein 70 (HSP70) is involved in its anticancer activity and whether it could modulate the responsiveness of lung cancer cells to chemotherapy. Materials and Methods: Various concentrations of quercetin and gemcitabine, either alone or in combination, were applied to lung cancer cells (A549 and H460 cells). We evaluated cell viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt assay, apoptotic activity by determining caspase-3 and caspase-9 activities, and HSP70 expression using Western blot analysis after treatment. Results: Quercetin reduced cell viability and suppressed HSP70 expression in both cell lines dose-dependently. Adding a fixed quercetin dose enhanced gemcitabine-induced cell death, which was related to increased caspase-3 and caspase-9 activities. Combination treatment with quercetin and gemcitabine downregulated HSP70 expression more prominently than treatment with quercetin or gemcitabine alone. Conclusion: Quercetin-induced HSP70 inhibition was involved in growth inhibition and sensitization to chemotreatment in lung cancer cells. Quercetin might have potential as a chemosensitizer in lung cancer treatment. Clinical Lung Cancer, Vol. -, No. -, --- ª 2015 Elsevier Inc. All rights reserved. Keywords: Apoptosis, Gemcitabine, Heat shock protein, Lung cancer, Quercetin
Introduction Lung cancer is the leading cause of cancer-related mortality, accounting for approximately 20% of all cancer deaths.1 Nonesmallcell lung cancer (NSCLC) accounts for approximately 80% of 1 Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, KEPCO Medical Center, Seoul, Republic of Korea 2 Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea
Submitted: Mar 20, 2015; Revised: May 10, 2015; Accepted: May 12, 2015 Address for correspondence: Sang Yeub Lee, MD, PhD, Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Korea University Anam Hospital, Korea University College of Medicine, 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, Korea Fax: þ82 2 929 2045; e-mail contact: [email protected]
1525-7304/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cllc.2015.05.006
all lung cancer cases. The prognosis of NSCLC is very poor, because 40% of cases are diagnosed in the advanced stage, and the 5-year survival rate is as low as 15%.2 Although recent advances in early diagnosis, chemotherapeutics, and surgical techniques have gradually improved survival, the rate of the NSCLC decline in mortality has plateaued in the past 2 decades.1 Among various factors, nonresponse and acquired resistance to chemotherapy are the most significant barriers to improvement of long-term outcomes. To overcome the limitations of the current treatment strategy, researchers have been actively searching for drugs that: (1) target particular pathways mainly involved in cancer progression; and (2) improve cancer cell sensitivity to chemotherapy and prevent the development of drug resistance.
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Quercetin and Chemosensitivity to Gemcitabine Heat shock proteins (HSPs) are a group of molecular chaperones that assist in protein-folding, modification, and transportation. HSP expression increases under stressful conditions, such as increased temperature, hypoxia, or radiation, to protect cells from injury.3 HSP70 is one of the main HSPs, and its overexpression has been reported in a wide range of malignant tumors. In addition, HSP70 overexpression has been related to drug resistance in colon cancer, breast cancer, and pancreatic cancer cells by inhibiting apoptosis.4-6 Studies have shown that inhibition of HSP70 expression potently induced apoptosis and inhibited the growth of many types of cancer cells.5,7,8 Increased HSP70 expression has also been described in serum and tissue samples from patients with NSCLC and is related to an adverse prognosis.9,10 However, studies on the anticancer effects of inhibition of HSP70 in lung cancer are very limited. Quercetin is a major bioflavonoid abundant in fruits and vegetables.11 Quercetin is characterized by its diverse biological activities, including antioxidation, proliferation, and proapoptosis.12 Numerous studies have reported a variety of beneficial effects of quercetin on cardiovascular disease, rheumatologic disease, and neurodegeneration.13-15 In addition, quercetin has cancer-preventive and anticancer effects on various types of cancer cells.15 Although quercetin is involved in multiple intracellular pathways, studies have shown that it induces apoptosis by downregulation of HSP70 expression in various cancer cells without affecting normal epithelial cells.6,16,17 Moreover, quercetin potentiates the growth inhibition of several chemotherapeutic agents, such as paclitaxel and cisplatin, through different intracellular mechanisms in different types of cancer cells.18,19 However, little is known about the anticancer effect of quercetin in lung cancer cells, and no studies have been conducted to investigate whether quercetin has a chemosensitizing effect on these cells. Considering a study that reported that quercetin and quercetin metabolites distribute at the highest concentrations in the lungs,20 lung cancer could be a good candidate for quercetin treatment. Thus, we performed this study to evaluate the following: (1) the growth inhibitory effect of quercetin; (2) the association between quercetin-induced growth inhibition and HSP70 expression; and (3) the possible role of quercetin as a chemotherapy sensitizer in lung cancer cells.
Korea). A Caspase-Glo assay kit was purchased from Promega (Madison, WI).
Cell Cultures and Treatment A549 and H460 cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37 C in a humidified atmosphere with 5% CO2. The cells were incubated in 96-well plates with 2 104 cells per well for viability measurements. The cells were plated in 6-well plates at a density of 2.5 105 cells per well to analyze protein and apoptosis, and were incubated overnight at 37 C. To determine cell viability and protein expression doseresponses, different concentrations of quercetin (10, 50, 100, and 200 mM) and gemcitabine (0.01, 0.1, 1, and 10 mg/mL) were dissolved in DMSO and added to the cells in serum-free medium, followed by a 24-hour incubation at 37 C.
Determining Cell Viability Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide salt (MTT) assay. The cells were incubated in 96-well plates at 1 104 cells per well overnight. After treatment with quercetin and gemcitabine, either alone or in combination at various concentrations for 24 hours, 10 mL of MTT (0.5 mg/mL) was added to each well, and the plate was incubated at 37 C to allow for the formation of blue formazan crystals. After 2 hours, residual MTT was carefully removed, and the crystals were dissolved by incubation with 150 mL of DMSO for 30 minutes. After shaking the plate for 1 hour, absorbance was measured at 570 nm. All experiments were repeated independently at least 3 times in quadruplicate.
Measuring Apoptotic Activity The Caspase-Glo luminescence-based assay was used to measure caspase-3 and caspase-9 activities according to the manufacturer’s instructions. Cells were seeded into 96-well white opaque plates and corresponding optically clear 96-well plates at 1 104 cells per well and allowed to adhere overnight. Then, the cells were treated with various concentrations of quercetin and gemcitabine, either alone or in combination for 24 hours. At the end of the incubation, 100 mL of Caspase-Glo reagent was added to each well. The plates were gently mixed and incubated at room temperature for 2 hours. Luminescence was read using a luminometer.
Measuring HSP70 Levels Using Western Blot Analysis
Materials and Methods Reagents and Chemicals
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Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone), dimethyl sulfoxide (DMSO), Ponceau S solution, and nitrocellulose membranes were purchased from Sigma-Aldrich (St Louis, MO). Gemcitabine was kindly donated by Eli Lilly Pharmaceuticals (Indianapolis, IN). Rabbit anti-b-actin and rabbit anti-b-tubulin were purchased from Abcam (Cambridge, UK). Goat antimouse immunoglobulin (Ig) G-horseradish peroxidase (HRP) and goat antirabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The human NSCLC cell lines A549 and H460 were obtained from the Korean Cell Line Bank (Seoul National University, Seoul, Korea). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT). Penicillin-streptomycin was acquired from WelGENE Inc (Daegu,
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Cell lysates were prepared by resuspending cells in lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), and 1% Triton X-100) for 20 minutes at 4 C and then cleared by centrifugation for 15 minutes at 13,000g. The supernatants were collected and stored at 80 C. Total protein concentration was measured with a Pierce bicinchoninic acid assay. Equal quantities of lysates were run on 10% sodium dodecyl sulfate-polyacrylamide gels and blotted to nitrocellulose membranes for the HSP70 Western blot analysis. Equal protein loading was confirmed by staining with Ponceau S. The membranes were blocked with 5% skim milk for 1 hour at room temperature and incubated with mouse monoclonal antiHSP70, rabbit anti-b-actin, and rabbit anti-b-tubulin to measure HSP70 levels. After 2 washes in Tris-buffered saline Tween-20, the blots were incubated with goat antimouse IgG-HRP and goat
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Results
(Figure 1A). Viability was reduced by 22% at 0.01 mg/mL gemcitabine, and the reduction also tended to be dose-dependent (Figure 1B). Quercetin treatment reduced H460 cell viability by 20% at 10 mM, and a dose-dependent reduction in cell viability was observed with increasing concentrations. An approximate 67% reduction in cell viability was observed at 200 mM quercetin (Figure 1C). Gemcitabine treatment also reduced cell viability by 42% at 0.01 mg/mL, and cell growth was inhibited dosedependently with increasing concentrations (Figure 1D). Incubating either cell line with 0.1% DMSO had no effect on viability. H460 cells were more susceptible to quercetin treatment according to the 50% inhibitory concentration (IC50; 175.3 mM for A549 cells vs. 78.1 mM for H460 cells). Similarly, H460 cells were more susceptible to gemcitabine treatment (no IC50 for A549 cells vs. 0.1 mg/mL for H460 cells).
Cell Viability After Quercetin and Gemcitabine Treatment
Apoptotic Activity After Quercetin Treatment
Figure 1 shows the effects of a 24-hour treatment with quercetin and gemcitabine at varying concentrations on cell viability. A549 cell viability was reduced by 20% at a concentration of 10 mM quercetin and was further suppressed in a dose-dependent manner with increasing quercetin concentrations. An approximately 60% reduction in cell viability was observed at 200 mM quercetin
To evaluate the role of apoptosis on the inhibition of cell growth by quercetin, we measured and compared caspase-3 (effector caspase) and caspase-9 (initiator caspase) activities in quercetin treatments. In the A549 cell line, a 1.7-fold increase in caspase-3 activity was observed at 10 mM quercetin, and the activity increased in a dose-dependent manner, with maximum activity of 2.0-fold at
antirabbit IgG-HRP. After extensive washing, the immunoreactive bands were visualized using enhanced chemiluminescence (West-Q Femto Clean ECL Solution; GenDEPOT, Barker, TX). The data were normalized to the b-actin level and are presented as relative band density to b-actin using Scion Image software (version 4.0.2, Scion Corporation, Frederick, Maryland).
Statistical Analysis Data are expressed as mean standard error. Differences between the control and experimental test groups were analyzed with the unpaired Student t test. A P value < .05 was considered significant. The statistical analysis was performed using SPSS version 13.0 for Windows (SPSS Inc, Chicago, IL).
Figure 1 Effects of Quercetin and Gemcitabine on Cell Viability. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Salt Assay Demonstrated That Quercetin and Gemcitabine Treatment Reduced A549 Cell Viability in a Dose-Dependent Manner; This Reduction Was Significant at 10 mM and 0.01 mg/mL, Respectively (A and B). H460 Cell Viability Also Decreased With Both Treatments, and There Was a More Prominent Reduction in Viability Compared With That in A549 Cells (C and D). *P < .05 Compared With the Control. Bars Indicate Standard Errors
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Quercetin and Chemosensitivity to Gemcitabine Figure 2 Effects of Quercetin on Apoptosis. In A549 Cells, Caspase-3 and Caspase-9 Activities Increased Dose-Dependently From the Concentration of 10 mM (A and B). Caspase Activities in H460 Cells Also Increased in a Dose-Dependent Manner, and a More Prominent Increase Was Observed Compared With That in A549 Cells (C and D). *P < .05 Compared With the Control. Bars Indicate Standard Errors
200 mM (Figure 2A). Similar dose-dependent increase was observed in caspase-9 activity (Figure 2B). In the H460 cell line, a 1.6-fold increase in caspase-3 activity was observed at 10 mM quercetin, and a dose-dependent increase was observed, with a maximal increase of 2.9-fold at 200 mM (Figure 2C). Also, caspase-9 activity was increased dose-dependently, with a maximal increase of 3.1-fold at 200 mM (Figure 2D).
Heat Shock Protein 70 Expression After Quercetin Treatment Figure 3 shows the effect of quercetin on HSP70 expression. A Western blot analysis showed that quercetin inhibited of HSP70 expression in a dose-dependent manner in both cell lines. Relative HSP70 expression at 200 mM quercetin was reduced by 60% in A549 cells and by 82% in H460 cells. Quercetin-induced HSP70 inhibition was more prominent in H460 cells based on the IC50 value (165.5 mM vs. 95.3 mM).
Cell Viability After Combined Quercetin and Gemcitabine Treatment
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Figure 4 shows the effect of the combined treatment with quercetin and gemcitabine on cell viability. Both cell lines were
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incubated with various concentrations of gemcitabine in the presence of a fixed dose of quercetin (50 mM). A significant reduction in viability was observed at 0.01 mg/mL gemcitabine in the A549 (Figure 4A) and H460 (Figure 4B) cell lines compared with that of the gemcitabine-alone treatment (all P < .05). This decreased viability was more prominent with increasing gemcitabine concentrations, suggesting a dose-dependent relationship. In addition, the gemcitabine IC50 was not different between the cell lines after the combined treatment, suggesting that the difference in susceptibility between the 2 cell lines that occurred with the gemcitabine-alone treatment disappeared after the addition of quercetin.
Apoptotic Activity After Combined Quercetin and Gemcitabine Treatment We measured and compared caspase-3 and caspase-9 activities in gemcitabine alone and combined quercetin and gemcitabine treatments to evaluate the role of quercetin in apoptosis-mediated growth inhibition. In the A549 cell line, a 1.3-fold increase in caspase-3 activity was observed with 0.1 mg/mL gemcitabine, and activity increased in a dose-dependent manner, with maximum activity of 1.8-fold at 10 mg/mL. The combination with quercetin resulted in a significant increase in caspase-3 activity at 0.1 mg/mL
Seung Hyeun Lee et al Figure 3 Effects of Quercetin and Gemcitabine on Heat Shock Protein 70 (HSP70) Expression. A Western Blot Analysis Shows That Quercetin Treatment Reduced HSP70 Expression in a Dose-Dependent Manner in A549 Cells (A) and H460 Cells (B). *P < .05 Compared With the Control. Bars Indicate Standard Errors
compared with that of the gemcitabine-alone treatment, and a maximum 5.8-fold increase was observed at 10 mg/mL (Figure 5A). In the H460 cell line, a 2-fold increase in caspase-3 activity was observed for the 0.01 mg/mL gemcitabine-alone
treatment, and a dose-dependent increase was observed, with a maximal increase of 3.6-fold at 10 mg/mL. A significantly greater increase in caspase-3 activity was noted in the combined quercetin and gemcitabine treatment at 0.01 mg/mL compared with that of
Figure 4 Combined Effect of Quercetin and Gemcitabine on Cell Viability. The Combination of a Fixed Dose of Quercetin (50 mM) and Various Concentrations of Gemcitabine Led to a Significant Dose-Dependent Reduction in A549 Cell (A) and H460 Cell (B) Viability at All Concentrations. This Effect Was Significantly More Prominent Compared With That of Treatment With Gemcitabine Alone. *P < .05 Compared With Treatment With Gemcitabine Alone. Bars Indicate Standard Errors
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Quercetin and Chemosensitivity to Gemcitabine Figure 5 Combined Effect of Quercetin and Gemcitabine on Apoptosis. The Combined Treatment of Quercetin and Gemcitabine More Significantly Increased Caspase-3 Activity in the A549 (A) and H460 (B) Cell Lines With Doses as Low as 0.1 mg/mL Gemcitabine. In Addition, Caspase-9 Activity Increased Significantly at the Same Concentration of Gemcitabine in A549 (C) and H460 (D) Cells. *P < .05 Compared With the Control. Bars Indicate Standard Errors
the gemcitabine-alone treatment, and the increased activity was dose-dependent. The maximal increase was 9.2-fold at 1 mg/mL (Figure 5B). The gemcitabine-alone treatment did not affect caspase-9 activity in both cell lines. However, a dose-dependent increase of the activity was observed with a maximal increase of 2.2-fold at 10 mg/mL in A549 (Figure 5C) and H460 (Figure 5D) cells in the combined quercetin and gemcitabine treatment.
Heat Shock Protein 70 Expression After Combined Quercetin and Gemcitabine Treatment
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To validate the role of HSP70 expression on cell viability for the combined treatment, we compared HSP70 expression levels with 0.1 mg/mL of gemcitabine and 50 mM of quercetin, either alone or in combination. As shown in Figure 5, HSP70 expression decreased significantly after the combined treatment compared with the quercetin- or gemcitabine-alone treatment in the A549 (Figure 6A) and H460 (Figure 6B) cell lines. These data suggest that the augmented growth inhibition and increased apoptosis after the combined treatment is associated with downregulation of HSP70 expression.
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Discussion Our data demonstrate that quercetin alone inhibited the growth of lung cancer cells dose-dependently and enhanced the growth inhibition caused by gemcitabine in a combined treatment. The growth-inhibitory effect of quercetin was associated with downregulation of HSP70, and increased anticancer activity in the combined treatment was mediated by increased apoptotic activity via inhibition of HSP70 expression. To the best of our knowledge, this is the first study to elucidate the association between the anticancer activity of quercetin and HSP70 modulation and the chemosensitizing effect of quercetin, which was induced by HSP70-related apoptosis in the lung cancer cell lines. Quercetin is a major flavonoid that is abundant in a broad range of vegetables and fruits. It has attracted the attention of cancer researchers because it has a variety of biologic activities, including antioxidative, antiproliferative, proapoptotic, and antiangiogenic activities, which affect cancer development and progression.15 Quercetin exerts anticancer effects in different cancers through different mechanisms, including inhibition of oncogenic activation, modulation of signal transduction, and interactions with receptors;
Seung Hyeun Lee et al Figure 6 Heat Shock Protein 70 (HSP70) Expression in the Combined Quercetin (Q) and Gemcitabine (G) Treatment. A Combined Treatment With 50 mM Q and 0.1 mg/mL G (G D Q) Decreased HSP70 Expression Significantly in A549 (A) and H460 (B) Cells. *P < .05 Compared With That of the Treatment With G Alone. Bars Indicate Standard Errors
it downregulates mutant p53 expression in breast cancer,21 induces Gap 1 (G1)/Synthesis (S) arrest in colon and gastric cancer cells, and blocks the Gap 2 (G2)/Mitosis (M) transition in breast and lung cancer cells.21-24 In addition, quercetin-induced apoptosis is associated with inactivating the Akt-1 protein, altering expression of Bcl-2 family proteins, and activating the mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway in lung cancer cells.25 Moreover, quercetin inhibits cell invasion and induces apoptosis through a pathway involving HSPs.17 In particular, quercetin inhibits HSP70 expression by blocking heat shock transcription factor (HSF) 1 and HSF2 and by reducing HSP70 mRNA accumulation.26 Quercetin downregulates HSP70 expression in various cancer cell lines, including breast, pancreatic, and colon cancer cells.6,27,28 However, the association between HSP70 expression and quercetin-induced growth inhibition in lung cancer cells has never been evaluated. Our data show that quercetin inhibited growth of lung cancer cells by downregulating HSP70 expression. In addition, the decreased expression of HSP70 was involved in quercetin-induced chemosensitization by increasing apoptotic activity. Our results are supported by previous findings that demonstrated that the combination of quercetin with different chemotherapeutic agents improved the efficacy of many cancer therapies.29 Apoptosis occurs via 2 main pathways. The extrinsic pathway is characterized by activation of death receptors on the cell surface, whereas the intrinsic or mitochondrial pathway is independent of death receptor signaling. The latter pathway involves the release of cytochrome c from mitochondria and the formation of a complex of cytochrome c, cytoplasmic apoptotic protease activating factor (Apaf)-1, and procaspase-9.30,31 In our study, quercetin-induced HSP70 inhibition significantly increased caspase-3 activity in both
lung cancer cell lines, suggesting that the apoptotic pathway is responsible for quercetin-induced cell death. Our data are consistent with those of a previous study that showed that quercetin induced apoptosis and caspase-3 activity in H460 cells.32 Moreover, we determined that the combination of quercetin and gemcitabine significantly increased caspase-9 activity. These results indicate that quercetin-induced HSP70 inhibition exerts its anticancer effect mainly via involvement of the intrinsic branch of the apoptotic pathway and it acts on the antiapoptotic function upstream of caspase3 in lung cancer cells. In addition, our findings suggest that HSP70 might play a major role in caspase-dependent apoptosis, and that downregulating HSP70 might be one of the mechanisms of quercetininduced apoptosis during inhibition of lung cancer cell growth. Gemcitabine is a pyrimidine nucleoside analogue that inhibits DNA synthesis. It exerts anticancer activity by inducing apoptosis and has been widely used against a variety of tumors. The response rate to gemcitabine in the advanced stages of NSCLC is approximately 25%, and the response rate increases to 54% when it is combined with platinum.33,34 Although the mechanism related to gemcitabine resistance in NSCLC is not fully understood, evidence suggests that insufficient intracellular concentrations of the active moiety and alterations in the associated apoptotic mechanism might contribute to this resistance.35 Similar to previous studies that investigated the effect of quercetin on gemcitabinetreated fibrosarcoma cells and pancreatic cancer cells,36,37 the present data show that quercetin-induced HSP70 inhibition enhanced the anticancer activity of gemcitabine in lung cancer cells. Although further study is required, the current results suggest that quercetin might be a good candidate as a chemosensitizer that would allow dose reduction and minimize drug toxicity in lung cancer treatment.
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Quercetin and Chemosensitivity to Gemcitabine Another implication of the present data is that inhibition of HSP70 could be a novel target for NSCLC treatment. HSP70 overexpression has been described in serum and tissue samples from patients with NSCLC and is related to an adverse prognosis in those patients.9,10 Previous reports demonstrate that downregulating HSP70 using small molecules or compounds potently inhibit cell proliferation in different cancers.6,8,38 However, targeting HSP70 as a lung cancer treatment is less well studied compared with other types of cancer. Results of 2 recent studies suggest that HSP70 could be a novel target for lung cancer treatment. Wen et al demonstrated that the small molecule VER155008, which specifically inhibits HSP70 expression, inhibits NSCLC proliferation and cell cycle progression and sensitizes A549 cells to ionizing radiation.39 In that study, the antiproliferative effect of VER-155008 was synergized when combined with an HSP90 inhibitor. Furthermore, Endo et al demonstrated that ibuprofen enhances cisplatin-induced apoptotic cell death in A549 cells by suppressing HSP70 expression.40 In that study, enhanced anticancer activity was mediated by the mitochondrial apoptotic pathway, which is identical with our results. Taken together, modulating HSP70 expression could be a promising therapeutic strategy for lung cancer either alone or in combination with other treatment modalities. Several limitations of our study should be discussed. First, despite that our data showed the potential role of quercetin as a novel chemosensitizer, an in vivo study to validate the in vitro results was not performed. Second, the possible mechanism by which quercetin downregulates HSP70 expression was not evaluated simultaneously. Further study using a lung cancer xenograft model and the candidate molecules that might be involved in downregulating HSP70 expression is required for quercetin to be evaluated in a clinical trial, similar to another HSP70 inhibitor.41
Conclusion We identified a role for HSP70 in quercetin-induced growth inhibition and chemosensitization in lung cancer cells. Our data suggest a potential benefit of quercetin in the treatment of lung cancer and clinical implications of HSP70 as a novel target for future development of lung cancer therapeutics. Further investigation should be conducted to validate the present findings in vivo and to determine the effects of quercetin or other HSP70 inhibitors combined with different chemotherapeutic agents on lung cancer growth.
Clinical Practice Points Quercetin is a bioflavonoid known for antioxidation, anti-
inflammation, and antiproliferation activities. Previous studies have demonstrated that quercetin not only in-
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hibits proliferation of a broad range of cancer cells but also enhances sensitivity to chemotherapeutic agents. However, the effects of quercetin on lung cancer cells have rarely been studied, and the combined effects of quercetin with other chemotherapeutic agents on lung cancers cells have not been investigated. We demonstrated that quercetin inhibited cancer cell growth and sensitized cancer cells to gemcitabine treatment by promoting apoptosis via inhibiting HSP70 expression.
Clinical Lung Cancer Month 2015
Although further studies are needed to verify our results, our
results suggest potential roles of quercetin and HSP70 as a chemosensitizer and a new target for lung cancer treatment, respectively.
Disclosure The authors have stated that they have no conflicts of interest.
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