Phytocompounds Curcumin, Quercetin, Indole-3-carbinol, and Resveratrol, modulate

Lactate-Pyruvate level along with cytotoxic activity in HeLa Cervical Cancer cells

Sarita Pani, Amrita Sahoo, Abhilipsa Patra and Priya Ranjan Debata*
P. G. Department of Zoology, North Orissa University, Sriram Chandra Vihar, Takatpur,
Baripada, Mayurbhanj, Odisha, -757003

*
Corresponding author’s E.mail. prdebata@gmail.com

Authors Sarita Pani and Amrita Sahoo contributed equally.

Acknowledgment
The work was partially supported by the Ramalingaswami Fellowship Program, DBT Govt.
of India, and a research grant received from DST Govt. of Odisha. The authors declare there
is no conflict of interest in this work.

Abstract:
Aims: Cancer cells meet their energy need by predominantly increased uptake of glucose,
high rate of glycolysis, and increased production of lactate even in the presence of adequate
oxygen. This process was proposed by Otto Warburg and named after him as the Warburg
effect. The Development of drugs that target glucose intake and aerobic glycolysis or lactic
acid secretion of cancer cells is a newer approach for drug discovery. We have tested five
purified plants derived compounds such as Curcumin (C), Quercetin (Q), Ellagic acid (E),
Resveratrol (R), and Indole-3-carbinol (I3C) in HeLa cells for cytotoxicity, inhibition of
metastasis, and modulation of lactate-pyruvate metabolism.
Main method: Standard biochemical methods were used for glucose, lactic acid, and pyruvic
acid measurement. The cell viability was determined by MTT assay. Cell migration was
checked by wound healing assay.

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through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/bab.2061.

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Key findings: A dose-dependent cytotoxic effect and inhibition of cell migration were
observed in all the tested compounds. A decrease in the lactate and increase in pyruvate level
was observed in all the tested compounds except Ellagic acid.
Significance: Our finding suggests that tested phytocompounds are associated with the
metabolic reprogramming of cancer cells and execute the cytotoxic effect. These compounds
could be used for cancer prevention and therapy.
Keywords: HeLa cells, Cervical cancer, Warburg effect, Lactate-pyruvate, Curcumin

1. Introduction
Cervical cancer is the fourth most common malignancy and cancer-related death among
women worldwide [1]. The most common etiological factor is the persistent infection of
human papillomavirus (HPV) which is commonly transmitted via sexual contact [2]. There
are approximately 15 carcinogenic human papillomaviruses (HPV) types and the infection
causes cancer in the genital regions like vulvar, vaginal, cervix, anal, and penile cancers as
well as some head and neck cancers [3]. HPV oncoproteins E6 and E7 help in the degradation
of two host cell tumor suppressor proteins p53 and pRb, respectively. It is presumed that the
loss of function of these two important tumor suppressor proteins is involved in the process
of carcinogenesis.
Cancer cells meet their energy need by predominantly increased uptake of glucose and a very
high rate of glycolysis even in the presence of adequate oxygen [4]. This process is called as
“Warburg effect”. The development of drugs that target glucose uptake and aerobic
glycolysis or lactic acid secretion by cancer cells is a newer approach for drug discovery.
Warburg effect is identified by high glucose uptake and lactate release and is currently
observed as a hallmark of all cancer cells [5]. This is the basis of the diagnosis of cancer by
using position emission tomography (FDG-PET). According to Warburg’s principle, cancer
cells depend on aerobic glycolysis and is the major approach of glucose metabolism instead
of oxidative phosphorylation. The rate of glucose metabolism through aerobic glycolysis is
much more so that the generation of lactate from glucose occurs 10-100 times rapidly than
the complete oxidation of glucose in the mitochondria [6]. This metabolic alteration is the
advantage for cancer cells to remain alive through hypoxic conditions, frequently found in
tumors, and to hold their anabolic requirements [7]. The pyruvate generated during glycolysis
is converted into lactate by the enzyme lactate dehydrogenase (LDH). The LDH level is

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higher in various types of cancer and an elevated level of LDH is believed to be a negatively
prognostic biomarker [8].

The high level of lactate produced in tissues or cells can be shuttled to adjacent sides
including heart and skeletal muscle for utilization through gluconeogenesis or become a
substrate for other reactions. This was proposed by George Brooks in 1984 and called
“lactate shuttle”. Lactate is also used as fuel in many tissues including muscle, liver, heart,
and brain [9]. Lactate induces angiogenesis through HIF- 1α and VEGF pathway. The
transport of lactate into mitochondria is carried out by four isoforms of monocarboxylate
transport protein (MCT) and lactate is oxidized to pyruvate by lactate dehydrogenase (LDH)
[10].
Plants are the source of drugs for the treatment of various diseases including cancer.
Important plant derived compounds used in the treatment of cancer include vinblastine,
vincristine, paclitaxel, topotecan, etc. Many of these compounds are also under investigation
for a future treatment option. It has been reported that phytocompounds such as Curcumin,
Ellagic acid, Quercetin, Indole-3-carbinol and Resveratrol have various biological activities
including anticancer properties. Curcumin is a known Phyto polyphenolic compound
extracted from the rhizome of the Curcuma longa plant. Many studies have proved that it has
anti-cancer properties tested in various cell lines and animal models. It is an edible compound
and has various therapeutics properties like antioxidant, anti-inflammatory, and analgesic
activity [11][12]. Quercetin (3, 3´, 4´, 5, 7-pentahydroxy flavones) is widely distributed in
onions, apples, berries, and tea [13]. Quercetin has been showing a wide spectrum of
antioxidant, anti-inflammatory, antiproliferative properties [14]. Indole-3-carbinol is a
glucosinolate breakdown product found in cruciferous vegetables such as cauliflower,
broccoli, and cabbage. This compound has been documented as anticarcinogenic, anti-
inflammatory, antifungal, and antibacterial effects [15][16]. Ellagic acid is found in a wide
spectrum of fruits, nuts, and various berries like raspberries, strawberries, etc. It has been
reported to possess various effects, including anti-inflammatory [17], anti-oxidative [18], and
antiviral [19] activities. Resveratrol (3, 5, 4′-trihydroxytrans-stilbene) is a well-known
polyphenolic phytoalexin that is naturally present in grapes, mulberries, and peanuts [20]
with demonstrated anticancer properties [21].

In this work, we have tested several purified plants derived compounds for anticancer
properties such as cytotoxicity, inhibition of metastasis, and modulation of lactate/pyruvate

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level in HeLa cervical cancer cells. Our results reveal except Ellagic acid the other four
compounds modulate lactate pyruvate level in HeLa cervical cancer cells.

2.0. Materials and methods

Cell line and culture

HeLa cells were purchased from the National Centre for Cell Sciences (NCCS), Pune. The
cells were cultured in high-glucose DMEM (Gibco, USA) containing 10% fetal bovine serum
(Gibco, USA), 1% penicillin-streptomycin (Gibco, USA) at 37°C, under a 5% CO2 humified
incubator.
Drugs
The Curcumin, Quercetin, Ellagic acid, Indole-3-carbinol, and Resveratrol were purchased
from MP Biomedical, dissolved in DMSO as a stock concentration of 40mmol/L, and stored
in the dark at−20°C. Ellagic Acid was solubilized in water containing 1% triethanolamine
with an initial stock solution of 40 mM [22]. The final concentrations used for the different
experiments were prepared by diluting the stock solution with high-glucose DMEM
supplemented with 1X insulin-transferrin-selenium (Gibco, USA) and 1% Penicillin-
Streptomycin.
3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay
Cell viability was determined by MTT assay. HeLa cells (2000) per well seeded in 96-well
plates (Tarson, India). Cells were incubated overnight in a humified CO2 incubator. Second-
day cells were treated in various concentrations (12.5, 25, 50, 75, and 100 μmole/L) of
Curcumin, Quercetin, Indole-3-carbinol, Resveratrol, Ellagic acid for 96 h. Thereafter, each
well was treated with 50μL of MTT solution (Sigma-Aldrich) dissolved in PBS (5 mg/mL),
and the wells were incubated for 4 h at 37°C. The formazan crystals were solubilized in 80μL
of DMSO and were kept in a slow shaking for 1 h. Optical density was measured at 570nm
on a microplate reader (Bio-Rad). All experiments were done in triplicate and four
independent times. The IC50 calculation was carried through Non-linear regression analysis
using GraphPad Prism software. Different concentration of drugs used in the study was
transformed into log of the concentrations. The absorbance obtained from MTT assay was
converted to percent of the carrier. Thereby the viability of the carrier treated cells was
calculated as 100%. Similarly, the viability of the drug-treated cells was decreased in
percentage that to the carrier. Graphs were obtained taking Log of the concentration in the X-
axis and percent viability on the Y-axis.
Wound healing assay

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A wound-healing assay was carried out to study the migration/movement of cells in a cell
population. HeLa cells were seeded in a 12 well plate at a density of 1×105 cells/well and
were cultured overnight in high glucose DMEM containing 10% FBS, 1% PS, maintained in
a humidified CO2 incubator. A confluent monolayer of cells was washed with PBS and a
scratch was generated using a 200μL tip. Then the cells were treated with 5μM of each drug
along with a carrier. Cells were viewed under the inverted microscope with a 20x objective
lens attached with a camera (Nikon D5600). Photomicrographs were taken at 0 h, 24 h, and
48 h. Open areas not containing cells after the scratch was analyzed using the software
TScratch [23]. The migration rate is expressed as the percentage of area reduction of the
wound, which increases as cells migrate over time [24].
Glucose assay using the DNS method
HeLa cells (4000 cells/well) were plated in 96 well plates. The next day the cells were treated
with 20 µM concentration of each of the compounds along with control. After 24 h, cells
were washed with PBS, lysed with RIPA buffer, and centrifuged at 10000 rpm. The
supernatant was deproteinized using 8% perchloric acid (PCA) and centrifuged. The
deproteinized supernatant was used for estimation of reducing sugar using DNS (2, 4 dinitro
salicylic acid), a method [25]. Briefly, 100µL supernatant was mixed with 1% DNS, the
volume was adjusted to 1mL with distilled water and boiled for 15 minutes. The color
changed to the dark brown indicating presence of reducing sugar. This was allowed to cool
and 40% Potassium Sodium tartrate (PST) was added to stabilize the color. Absorbance was
measured at 595nm using a spectrophotometer. A standard curve was prepared using
anhydrous glucose and linearity was verified in a range of 0-1mg/mL (r2= 0.98) was found to
be statistically significant.
Determination of Intracellular and Extracellular Lactate and Pyruvate

Intracellular and extracellular lactate and pyruvate concentrations were measured using a
modified method [26]. The remaining lysates from glucose assay were used for the
determination of lactate and pyruvate. The extracellular Lactate and Pyruvate was measured
from the used culture media. For lactate estimation 40µl lysate was mixed in a glycine and
hydrazine buffer (90mM final concentration, pH 9.2), excess of NAD+ (2.4mM final) was
added with the final volume adjusted to 500 µL in distilled water. The reaction was catalyzed
by the addition of 01U/µl (0.1mg) of the Lactate dehydrogenase enzyme (LDH). A blank was
prepared to contain everything except the LDH enzyme. The absorbance of the blank was
measured at 340nm using a UV-Visible Spectrophotometer. Similarly, the absorbances of the

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test samples were also measured at 340nm in every one-minute interval for a total period of
ten minutes. The increase in absorbance at 340 nm is due to the reduction of NAD+ to
NADH by LDH. The maximum absorbance (maximum sample absorbance – blank
absorbance=ΔA340) was taken for calculation using the formula Lactate (mM)=

( )
. Where 6.22 is the millimolar extinction

coefficient of NADH at 340 nM. For the determination of pyruvate, 40µl lysate was mixed in
a tris buffer (0.2M, pH 7.3), excess of NADH (10mM) was added with a final volume
adjusted to 500 µL with distilled water. The reaction was catalyzed by the addition of 0.1U/µl
of Lactate dehydrogenase enzyme (LDH). Absorbance was measured using a UV-Visible
Spectrophotometer at 340 nm. The reduction in absorbance at 340nm due to the oxidation of
NADH to NAD+ was determined using the formula Pyruvate (mM)=
( )
( )
.

Calculation of NAD/NADH Ratio

The NAD+/NADH ratio was determined by a method described by [27] using the formula
Keq [pyruvate]eq[NADH]eq[H+]/([Lactate]eq[NAD+] eq) =1.11610211, where pH is 7.0, If
LDH catalysed reaction is allowed to proceed to equilibrium, the final products and reactants
could be expressed by the equation: Apparent Keq = Keq [H+], where Keq
=[pyruvate]eq[NADH]eq/([Lactate] eq[NAD+]eq) = 1.1161027 or [NAD+]/[NADH] =
[pyruvate] eq/(Keq[Lactate]eq) .

Statistical Analysis
Statistical Analysis for checking the significant difference between control and tested
compounds was analyzed by student’s t-test comparing (Carr. Vs drugs) with a P value less
than 0.05.
RESULTS:
Cytotoxic effects of Curcumin (C), Ellagic acid (E), Indole-3-carbinol (I3C), Quercetin
(Q), Resveratrol (R) and in HeLa cells:
Although similar types of results are published by us and others, these experiments are
conducted new for this manuscript [28]. The cytotoxic effects of C, R, E, I3C, and Q were
examined in HeLa cervical cancer cells. Cells (2000) were plated in 96 well plates, treated
with increasing concentration of C, R, E, I3C, Q (12.5, 25, 50, 75, 100μmole/L) for 96 h.
Cells were observed under an inverted microscope with morphological changes which

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include cell death, shrinkage of cells, and cell rounding in a dose-dependent manner (Figure
1). Cell viability was measured using MTT based assay. The data obtained from at least four
trials with triplicates were converted to percent control and plotted in GraphPad prism. The
result showed all the five compounds induced cytotoxicity in HeLa cells. The IC50 value of
Curcumin was calculated as 23.86 µM, Ellagic acid was 15.84 µM, Indole-3-carbinol was
52.09 µM, Quercetin was 26.41 µM and Resveratrol was 43.36 µM.
Curcumin (C), Ellagic acid (E), Indole-3-carbinol (I3C), Quercetin (Q) inhibit cell
migration in HeLa cells
The cell migration was evaluated using a wound-healing assay as described in the materials
and methods. The wound width was measured using the Tscratch program and the data is
represented as the percentage of wound closure. Results show a higher percentage of wound
closure in the carrier treated wells both in 24 h and 48 h due to cell migration. But in C, E,
I3C, and Q treated wells and a significant decrease in the wound closure thus inhibiting cell
migration. However, Resveratrol alone at 20 µM concentration shows poor inhibitory effects
on HeLa cell migration (Figure 2). Asterisks indicate a significant difference between Carrier
Vs drugs.
Curcumin (C), Indole-3-carbinol (I3C), Quercetin (Q) and Resveratrol (R) modulate
Glucose uptake in HeLa cells.
Intracellular glucose concentration was measured in HeLa cells treated with C, E, I3C, Q, and
R for 24 h. A sublethal dose of 20 µM concentrations of C, E, I3C, Q, and R was used for the
treatment. Intracellular glucose concentration was measured in the cell lysate. The results
showed there was a decrease in the intracellular glucose concentration in the C, I3C, Q and R
treated cells but in Ellagic acid treated cells there was no change in intracellular glucose
concentration (Figure 3). The data is represented in the Mean ± Standard Error. Asterisks
indicate a significant difference between Carrier Vs drugs.
Curcumin (C), Indole-3-carbinol (I3C), Quercetin (Q), and Resveratrol (R) reduces the
intracellular-extracellular lactate level and increases pyruvate level in HeLa cervical
cancer cells.
Effect of Curcumin, Ellagic acid, Indole-3-carbinol, Quercetin, and Resveratrol on
intracellular and extracellular lactate - pyruvate concentration were measured in HeLa cells.
A subtoxic dose of 20 µM was treated to HeLa cells for 24 h. The data showed a significant
reduction of lactate level and an increase in the pyruvate level except Ellagic acid treated
cells (Figure 4). The data is represented in the Mean ± Standard Error. Asterisks indicate a
significant difference between Carrier Vs drugs.

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Curcumin, Quercetin, Resveratrol, Indole-3-carbinol, alter NADH/NAD+ ratio in
cervical cancer cells
NADH/NAD+ ratio of HeLa cells treated with Curcumin (C), Ellagic acid (E), Indole-3-
carbinol (I3C), Quercetin (Q), and Resveratrol (R) were calculated as mentioned in the
materials and methods. The results show Curcumin, Quercetin, Resveratrol, Indole-3-carbinol
decrease the intracellular NADH-NAD+ ratio however, no change in the NADH-NAD+ ratio
of Ellagic acid treated cells. There is a significant reduction of extracellular NADH-NAD+
ratio in all five compounds treated cells (Figure 5). Asterisks indicate a significant difference
between Carrier Vs drugs.
Discussion
The rapid proliferation of cancer cells is associated with a high demand for precursor
molecules for various anabolic pathways including ribose sugar for nucleotides, glycerol, and
citrate for lipids, various amino acids, and NADPH. Cancer cells undergo metabolic
reprogramming to mitigate the high energy demand as well as the need for precursor
molecules for rapid cell division. One such metabolic alteration is the carbohydrate
metabolism, where cancer cells uptake a high quantity of glucose than the normal cells. The
increased uptake of glucose is also associated with a high rate of glycolysis even in the
presence of adequate oxygen. In cancer cells, 50% of energy is generated in the cytoplasm
via glycolysis, while others from the mitochondrial respiratory chain [29]. Cancer cells
depend on the inefficient glycolytic mode of ATP synthesis. (2ATP/glucose) instead of
respiration that produces more ATP/ glucose (36ATP/glucose) [30]. Glucose metabolism
increases reactive Oxygen Species that damages the mitochondrial membrane and depresses
respiration [31]. Cancer cells produce a 70-fold increase in lactate accumulation when cells
are cultured in 13mM glucose [32]. The end products of aerobic glycolysis are converted into
lactate. Thus, cancer cells have a high concentration of lactate with them. This metabolic
alteration in cancer cells has been recognized as a potential therapeutic target. Lactate is the
main element responsible for acidosis of the extracellular microenvironment due to the
constant lactate and proton shuttling from cancer cells to the extracellular space [33].
Curcumin, Quercetin, Ellagic acid, Resveratrol, and Indole 3-carbinol are well known for
their potential anticancer activities [34] [35] [36]. Various drugs targeting cancer metabolism
are under clinical trials [37]. Curcumin has been reported to cause metabolic alteration
leading to a decrease in the rate of glycolysis and induce cell death in cancer [38]. Treatment
of high dose metformin inhibits tumor growth in a xenograft model of cancer [39]. Dietary
polyphenols such as Resveratrol, Quercetin, Epicatechin gallate are associated with alteration

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in the metabolic processes especially fatty acid metabolism [40]. Our result indicates dietary
polyphenols such as Curcumin, Quercetin, Resveratrol, Ellagic acid, and I3C cause metabolic
alterations in HeLa cells. These compounds show cytotoxic activity and also inhibit cell
migration. Besides this, the compounds except for ellagic acid decrease the uptake level of
glucose and also decreases lactate production and increases the pyruvate level in the cells,
showing a reversal of the Warburg effect. Curcumin, Resveratrol, Quercetin, Indole-3-
carbinol influenced the inhibition of metabolism was evident at 20µM concentration,
revealed the sensitivity of cancer cells to Curcumin, and these phytocompounds and its
reliance on aerobic glycolysis. The decrease in glucose consumption and lactate production
obstructs the growth of cancer cells because glycolytic flux provides metabolic synthesis in
cancer cells to produce macromolecules for daughter cancer cells.
The results are preliminary and show a new direction of the anti-cancer mechanism of
Curcumin, Quercetin, Resveratrol, Ellagic acid, and Indole-3-carbinol. Although these
phytocompounds have been proved for having anti-cancer properties through various types of
processes, their association in metabolic reprogramming could be targeted for cancer therapy
without harming normal cells. Metabolic renovation in cancer cells is one such process. In
brief, this study resolves a new anticancer role of these phytocompounds and attracts further
research in utilizing these phytocompounds and its cognates for successful clinical inhibition
of metabolic alteration in cancer cells.
Conclusion
Natural dietary phytocompounds have been widely studied for cancer prevention and
treatment. Cancer cells alter their energy metabolism by an increase in the uptake of glucose
and switch to aerobic glycolysis. The end product of glycolysis is pyruvate but in cancer cells
it is converted to lactate. In our study, we have shown that natural phytocompounds such as
Curcumin, Indole-3-carbinol, Quercetin and Resveratrol cause modulation of the Warburg
effect in which there is increase in the level of pyruvate and decrease in the level of lactate in
HeLa cells. These tested phytocompounds Curcumin, Resveratrol, Quercetin, and Indole-3-
carbinol will continue to be a promising and active research area in the future.
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Figure Legend

Fig. 1: Dose-dependent response of Curcumin, Ellagic acid, Quercetin, Indole-3-carbinol,

and Resveratrol on the viability of HeLa cells (A) Brightfield analysis of HeLa cells treated
with the carrier (Control) or 20 µM concentrations of each of the compounds. (B) Graphs
showing the viability of HeLa cell as determined by tetrazolium salt (MTT) assay after 96 h
of exposure of the compounds with indicated concentrations. The concentrations were
converted to Log scale and plotted in X-axis. The Y-axis contains the absorbance normalized
to control. IC50 values were generated using GraphPad prism program and the value is
indicated in the graph.

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This article is protected by copyright. All rights reserved.
Fig. 2: Migration of HeLa cells Curcumin, Ellagic acid, Quercetin, Indole-3-carbinol, and
Resveratrol. (A) Photomicrograph of wound healing assay at the time point of 0 h, 24 h, and
48 h. (B) Quantification of wound closure in the carrier and treated cells.

Fig. 3: Intracellular glucose concentrations in HeLa cells treated with the Carrier, Curcumin,
Ellagic acid, Indole 3-carbinol, Quercetin, and Resveratrol for 24 h. Intracellular glucose
concentration significantly lower in Curcumin, Quercetin, Resveratrol, and Indole 3-carbinol
treated cells but no change of glucose concentration was observed in ellagic acid treated cells.

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Fig. 4: A. Intracellular and extracellular Lactate level in HeLa cervical cancer cells treated
with 20 μM Curcumin, Ellagic acid, Quercetin, Indole-3-carbinol, and Resveratrol. B.
Intracellular and extracellular Pyruvate level in HeLa cervical cancer cells treated with 20μM
Curcumin, Ellagic acid, Quercetin, Indole-3-carbinol, and Resveratrol.

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Fig. 5: Intracellular and extracellular NADH/NAD Ratio in HeLa cervical cancer cells
treated with 20 μM Curcumin, Ellagic acid, Quercetin, Indole-3-carbinol, and Resveratrol.

Conflict of Interest
There is no conflict of interest in this work.

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Graphical abstract

Research Highlight
 Curcumin, Ellagic acid, Quercetin, Indole-3-carbinol and Resveratrol promotes cell
death
 Curcumin, Ellagic acid Quercetin, Indole-3-carbinol and Resveratrol inhibits cell
migration
 Curcumin, Quercetin, Indole-3-carbinol and Resveratrol decreases glucose uptake
 Curcumin, Quercetin, Indole-3-carbinol and Resveratrol decreases lactate production

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