Abstract

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Quercetin is one of the most abundant dietary flavonoids widely present in many fruits and vegetables. Previous in vitro studies has shown that quercetin acts as an antioxidant and anti-inflammatory agent and it has potent anticarcinogenic properties as apoptosis inducer.
In this study we examined apoptotic effects of quercetin on the K562 erythroleukemia cell line. K562 cells were induced to apoptosis by hydrogen peroxide and quercetin. Cell viability and apoptosis level were assessed by annexin V and PI staining method using flow cytometry. Viability of K562 cells was increased by low dose of quercetin (5-100 μM) during 3 hours. High dose of quercetin at toxic doses (100-500 μM) during 24 hours resulted with decrease of K562 cell viability as expected (P2O2 (150, 300, 600 µM). K562 cells were protected from H2O2induced apoptosis by the Quercetin (P
As indicated in previously studies, reduction of superoxides by the free radical scavengers such as quercetin could be beneficial for prevention of cancer but consumption of flavonoid free radical scavengers during cancer treatment may weaken effect of the chemotherapeutics and radiotherapy.

Introduction

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Reactive oxygen species (ROS) are highly reactive molecules generated predominantly during cellular respiration and normal metabolism. These side products can damagemany biological molecules; they can change protein functions, damage DNA material and cause lipid peroxidation of cell membranes (1). In general, the reducing environment inside cells helps to prevent free radical mediated damage. This reducing environment is maintained by the action of antioxidant enzymes and substances, such as superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione, vitamins C and E (2, 3).
Oxidative stress as a dysbalance between the production of free radicals and antioxidant system of the cells is involved in the pathogenesis of the deadly diseases including cancer and cardiovascular diseases (4). Excessive production of ROS may lead to programmed cell death and may accelerate the process of aging (5).
For a long time, reactive oxygen species (ROS) have been considered harmful mediators of inflammation owing to their highly reactive nature. However, there are an increasing number of findings suggesting that ROS can play role in anti-inflammatory and prevent autoimmune responses (6).
Neutralization of free radical is important to prevent the damage of cells, tissues and organs. Control of free radical neutralizations are under the control of internal antioxidant enzymes such as SOD, NADH, NADPH (4). Destruction of balance between the oxidant and antioxidants depends on age, environmental conditions and some mutations. Uses of herbal sourced ingredients to improve balance of antioxidant system are common way, between the public.
Flavonoids are polyphenolic compounds that occur ubiquitously in foods of plant origin (7). It has been reported that some flavonoids such as rutin (quercetin-3-rutinoside) and quercetin shows antioxidant activity (8).
Quercetin is one of the most abundant dietary flavonoids. It can be found in apples, black, green and buckwheat tea, onions, raspberries, red grapes, cherries, citrus fruits as well as in some well known medical plants (ginkgo biloba, cranberries and St John’s wort) (9, 10).
In previous in vitro studies has shown that quercetin acts as an antioxidant and anti-inflammatory agent and that it has potent anticarcinogenic properties as apoptosis inductor.
In the normal conditions, herbal sourced antioxidants intake could be beneficial for cancer prevention (11, 12), coronary artery disease prevention (13) and moderation of aging process. But what will happen if cancer patient takes artificial herbal sourced antioxidants such as quercetin which is abundantly present in many herbs, fruit and grain includes. Will it facilitate anticancer therapy or not?
To examine this, K562 erythroleukemia cell line was induced to apoptosis by hydrogen peroxide and effect of quercetin over the apoptosis and viability of K562 cells was observed.

Methods

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Cell Culture
K562 cells (Erythroleukemia cell line derived from a chronic myeloid leukemia patient in blast crisis) were incubated at 37°C in RPMI 1640 medium (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) containing 10% FCS (Gibco-Invitrogen Ltd., Paisley, Scotland) in a humidified 5% CO2 atmosphere. Cell count was made with trypan blue. Viability of K562 cells were 85-90%.
Apoptosis induction by quercetin
Effect of quercetin (Sigma-Aldrich, St. Louis, MO, USA) on the cell viability of K562 was evaluated with different Quercetin concentration (5-500 µM) through 3 hours with Flow cytometer (Becton-Dickinson, San Jose, CA, USA). Toxic dose of Quercetin concentrations (100-500 µM) was evaluated through 24 hours.
Apoptosis induction by hydrogen peroxide
K562 cells were induced to apoptosis by with hydrogen peroxide (H2O2 Riedel de Haen, D3016, Seelzel, Germany) in different concentration (150, 300, 600 µM) and protective effect of Quercetin (100 µM) was analyzed through 24 hours. For this aim, protective dose of Quercetin (100 µM) was added on to K562 cell (106 cells in 10 mL RPMI 1640, %10 FCS, penicilin/streptavidin), then hydrogen peroxide were added.
Annexin V staining assay
After induction with H2O2 and Quercetin, K562 cells were washed twice with PBS and suspended in 1x ?binding buffer (10 mM HEPES, 140 mM NaCl, and 5 mM CaCl2 at pH 7.4) at a concentration of approx. 1 x 105 cells/ml. 5 ml of Annexin V-FITC and 10 ml of propidium iodide was added to both control and induced cell suspension. After incubation at room temperature for 10 minutes at dark, the fluorescence of the cells was determined immediately with a flow cytometer (FACS Calibur, Becton-Dickinson, San Jose, CA, USA).
Statistical Analysis:
Measurements were repeated more than 4 times and student t-test was used for statistical analysis.

Results

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For the time-course and dose-response experiments, K562 erythroleukemia cells were treated with 5, 10, 20, 40, 80, 100, 200, 300, 400 and 500 μM of quercetin for 3. Cell viability was assessed by Annexin V/PI flow cytometry assay. Figure 1 shows that quercetin (In the 5-200 µM doses for 3 hours) caused slight increase of K562 cells viability (Figure 1) but high doses of quercetin (over than 200 µM) caused to increase of apoptotic cell death (P
For second experiment K562 cells were treated with 100, 200, 300, 400 and 500 µM of quercetin for 24 h. Toxic doses of quercetin (doses higher than 100 µM, 24 hours) resulted with decrease of K562 cell viability as expected (P
In third probe, hydrogen peroxide (H2O2) was applied in dose 150, 300 and 600 µM for 24h. Kind of ROS, hydrogen peroxide (H2O2) induced apoptosis of K562 cells had been shown previously and similar results was obtained in our study (P
To determine preventive role of quercetin (free radical scavenger flavonoid) against to H2O2 (ROS) induced apoptosis, 100 µM of quercetin was added into cell culture medium (106 cell in the 10 ml RPMI 1640 include %10 FCS, penicillin-streptomicin), 20 minute before the 150, 300, 600 µM H2O2treatment. K562 cells viability were maintained by the Quercetin from the H2O2 induced apoptosis (Figure 3), (P

Discussion

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In previous studies quercetin was implied as an apoptotic activator and antioxidant as well as protective agent against various types of cancer (14). It was shown that quercetin can inhibit proliferation of tumor cells and reduce the number of aberrant crypt foci in colon tumors (15) that it can facilitate programmed cell death in lung carcinoma (16) and colonorectal tumor cells (17). Other researchers had showed that quercetin acts as a free radical scavenger and protects cells from oxidizier molecules (18, 19). As known, oxidative stress and free radicals are important activators for apoptosis (20).
On the other hand, studies conducted in last ten years showed that quercetin can act as an antiapoptotic agent as well (21). In a few studies it has been shown that quercetin can partially prevent H2O2 - inducedapoptosis (22) and it was suggested that protective effects of quercetin against oxidative injuries of some cells may be achieved via modulation of mitochondrial dysfunction and inhibition of caspase activity (23).
In this study, our results showed that higher dose of quercetin than 200 μM reduce K562 cell viability (which considered as toxic doses by Cao et al, 2007 (24)) but low doses of quercetin (2O2.
Free radical generation and inactivation’s are under the strict control of oxidoreductive reactions. Excessive superoxide production can accelerate cell death, DNA damage and can lead to cancer, also excessive inactivation of superoxides can disturb apoptosis signals. Inactivation of radiation and environmental sourced superoxides by the herb sourced flavonoid free radical scavengers seems beneficial for cancer prevention (11) but if cancer is already present, as known as free radicals are important for apoptosis signals. For this reason, uncontrolled consumption of flavonoid free radical scavengers such as quercetin during chemothrapy may weaken the effect of chemotherapeutics and radiotherapy rather then help it.
It has been theorized that cancer risk reduction may be achieved by greater consumption of phytochemical-rich fruits and vegetables (25, 20). However, results of our research suggest that antioxidants with free radical scavenger properties can interfere and attenuate success of anticancer therapy so antioxidant intakes should be strictly controlled in the cancer diagnosed patients.

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Source(s) of Funding

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This project was supported by Marmara University scientific research project unit (BAPKO) and The Scientific and Technological Research Council of Turkey (TUBITAK)

Competing Interests

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none

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