European Journal of Pharmacology

Manuscript Draft

Manuscript Number: EJP-51850

Title: Curcumin Modulates the AKT and PTEN Expression to Suppress Glioma

Cell Proliferation

Article Type: Research Paper

Section/Category: Neuropharmacology and analgesia

Keywords: Glioblastoma; Curcumin; PTEN; AKT; Mechanism

Abstract: Glioma is the most common neoplasm in the brain. Curcumin，as a

known polyphenolic compound extracted from turmeric，is clinically used

in the chemoprevention and treatment of some cancers. However, the degree

to which curcumin is involved in mediating the treatment of glioma and

the possible mechanisms of such an effect remains unknown. we detected

cell viability of glioma cells under curcumin by MTT assay and examined

the invasion and migration ability. The proliferation and apoptotic

proteins were detected. We made glioma-xenograft model and detected the

proliferation under curcumin treatment.We found that curcumin inhibited

the proliferation of U251 and U87 cells of glioma. Curcumin up-regulated

the invasion and migration of U251 and U87 cells. We detected that

curcumin decreased p-AKT and p-mTOR protein expression of U251 and U87

cells. Curcumin promotes apoptosis of U251 and U87 cells. We used some

databases to analyze the effect of PTEN gene expression on the survival

rate of glioma patients and to analyze the interaction between proteins.

Further, we found that curcumin promoted the PTEN and p53 expression, as

the tumor suppressor genes. In addition, we administered curcumin to U87-

xenograft and found that curcumin decreased the tumor volume, caused

necrosis of tumor tissue, and significantly enhanced the PTEN and P53

expression in vivo. These results indicate that curcumin may inhibit

proliferation by increasing the p-AKT/p-mTOR pathway, and promotes

apoptosis by enhancing the PTEN and p53 expression. Our study can provide

new insights into the molecular mechanisms by which curcumin can inhibit

glioma and its targeted interventions.

Curcumin Modulates the AKT and PTEN Expression to Suppress Glioma

Cell Proliferation

Zexia Wang1,2

, Fei Liu1,2

, Liangzhu Yu1, Hongli Xia

3, Mincai Li

1,2#, Zhenwu Hu

1# 1 School of Basic Medical Sciences, Hubei University of Science and Technology, Xianning, 437100, P.R.China,

2 School of Pharmacy, Hubei University of Science and Technology, Xianning, 437100, P.R. China,

3 The Central Hospital of Xianning, Hubei University of Science and Technology, Xianning, 437100, P.R.China,

#Correspondence to School of Basic Sciences, Hubei University of Science and Technology, Xianning, 437100,

P.R.China

The email address for all author

Zexia Wang 454544454@qq.com

Fei Liu 2440032910@qq.com

Liangzhu Yu yuliangyu73@163.com

Hongli Xia mobei_hu@126.com

Mincai Li mincaili@163.com

Zhenwu Hu huzhenwu@hotmail.com

ManuscriptClick here to view linked References

Abstract

Background Glioma is the most common neoplasm in the brain. Curcumin，as a known

polyphenolic compound extracted from turmeric，is clinically used in the chemoprevention

and treatment of some cancers. However, the degree to which curcumin is involved in

mediating the treatment of glioma and the possible mechanisms of such an effect remains

unknown.

Methods we detected cell viability of glioma cells under curcumin by MTT assay and

examined the invasion and migration ability. The proliferation and apoptotic proteins were

detected. We made glioma-xenograft model and detected the proliferation under curcumin

treatment.

Results We found that curcumin inhibited the proliferation of U251 and U87 cells of glioma.

Curcumin up-regulated the invasion and migration of U251 and U87 cells. We detected that

curcumin decreased p-AKT and p-mTOR protein expression of U251 and U87 cells.

Curcumin promotes apoptosis of U251 and U87 cells. We used some databases to analyze

the effect of PTEN gene expression on the survival rate of glioma patients and to analyze

the interaction between proteins. Further, we found that curcumin promoted the PTEN and

p53 expression, as the tumor suppressor genes. In addition, we administered curcumin to

U87-xenograft and found that curcumin decreased the tumor volume, caused necrosis of

tumor tissue, and significantly enhanced the PTEN and P53 expression in vivo.

Conclusions These results indicate that curcumin may inhibit proliferation by increasing the

p-AKT/p-mTOR pathway, and promotes apoptosis by enhancing the PTEN and p53

expression. Our study can provide new insights into the molecular mechanisms by which

curcumin can inhibit glioma and its targeted interventions.

Key Words: Glioblastoma; Curcumin; PTEN; AKT; Mechanism

Introduction

Glioma is a common primary neoplasm, including astrocytoma, oligoastrocytomas,

oligodendroglioma and glioblastomas[1]. Glioblastoma (GB) is one of the most aggressive

solid tumors patients with these tumors have a very poor prognosis. The poorly differentiated

polymorphic glioma cells have diverse morphologies, nuclear atypia, and undergo active

nuclear division. Their histological features show the different morphology such as necrosis,

vascular proliferation and thrombosis[2] The current treatment methods include the tumor

resection, chemotherapy, radiotherapy, anti-vascular targeted drugs. Because current

therapeutic strategies are ineffective it is important to find new treatments or better drugs.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), isolated

from the spice turmeric of Curcuma，a natural polyphenolic compound [3,4]. This hydrophobic

compound has been used to treat a variety of human diseases, such as inflammatory,

neoplastic and some neurodegenerative diseases[5,6]. Previous studies showed that

curcumin inhibited cytotoxicity by decreasing oxidative damage caused by reactive oxygen

species[7,8,9]. Some studies have shown that curcumin has therapeutic properties and

preventive effects on many types of neoplasms. These reports demonstrated that curcumin

regulates several signaling pathways, including STAT3 and NF-κB transcription factor in the

proliferation, angiogenesis and metastasis[7,10]. However, the role of curcumin in gliomas is

unclear and further clinical studies are required.

Our study was designed to discover the potential ability of curcumin to suppress glioma

in vitro and in vivo. Our results suggest that curcumin inhibits the proliferation of glioma, and

that this effect is associated with the inhibition of AKT/mTOR signaling pathway and PTEN

activation.

Materials and Methods

Materials

The curcumin was purchased from Cayman Chemical Company (Ann Arbor, MI, USA).

Anti-AKT, anti-mTOR, anti- caspase-3,anti-beclin,anti-PTEN,anti-p53 and anti-AT1R

antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All

secondary antibodies were from Proteintech Group (USA). Trizol reagent and the enhanced

chemiluminescence kit were from Beijing Applygen Technologies (Beijing, China). All

procedures using animals were conducted as described previously[11] and were in

accordance with the National Institutes of Health Guide for the Care and Use of Laboratory

Animals (NIH publication 85-23, revised 1996) and approved by the institutional animal care

and use committee at the Hubei University of Science and Technology, China.

Glioma cell culture.

The glioma cell lines, U251and U87, were cultured in Dulbecco's modified Eagle's medium

(DMEM) both supplemented with 10% fetal bovine serum (FBS) as described previously[12].

The cells were incubated under humidified conditions at 37˚C and 5% CO2. The medium

was changed every 3 days. The cells were passaged at 95% confluency for the following

experiments.

Cell proliferation assay.

The cell proliferation assay was examined as described previously[13,14]. U251and U87cells

were seeded into 96-well tissue culture plates and were incubated with 0, 10, 20 and 40 µM

curcumin. After incubation for 24-, 48-, and 72- h at 37˚C, the viability was detected with a 3

‑(4,5‑dimethyl- ylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) assay. The glioma

cells are cultured in media containing 0.5 mg/ml MTT for 2h. Dimethyl sulfoxide was added

to dissolve the formazan products and the absorbance was measured

spectrophotometrically at a 550 nm wavelength. Three replicates were performed.

Wound healing assays

The wound healing assays were performed by the cell migration as described prevously[15].

Cells were seeded into 6-well plates and cultured until the confluence reached 90%. A sterile

10-μl pipette tip generated a scratch through each well. The wound closure was observed

after 0- and 24-h and photographed using a (insert make and model) microscope. The

number of U251 and U87 cells that migrated was calculated in 5 high-magnification fields

(200×) that were randomly selected. Three replicates were performed, and the data were

averaged for statistical analysis.

Transwell migration and invade assays

The migration assay was performed using a transwell system (Corning Costar, USA) as

described previously[12]. A total of 3×104 cells in 100 μL of serum-free media were added to

the top chamber 8-μm Costar polycarbonate transwells, and 500 μL of DMEM,

supplemented with 10% FBS and curcumin, were added to the bottom chamber. Culture

supernatant with DMSO served as the control group. After being incubated for 4 h, the cells

on the upper membrane surface were removed and the cells that migrated were fixed with 5%

(wt/vol) glutaraldehyde and stained with 0.1% (wt/vol) crystal-violet solution. Filter inserts

were inverted and the migratory cell number was determined by visualizing the

crystal-violet-stained cells directly on undersides of the inserts using a (insert make and

model here) microscope. Three replicates were performed, and the data were averaged for

statistical analysis.

Adhesion assay

Adhesion assay was performed as previously described[12] The 96-well plates were

preincubated with 20 μg/mL fibronectin (FN) fragment for 4 h, and 200 μL of U251 and U87

cells, with or without curcumin, were incubated for 60 min. The U251 and U87cells were

added at a density of 5 × 105 cells/well for 45 min at 37°C. The non-adhesive U251 and U87

cells were washed 3 times. The number of U251 and U87 cells that adhered to fibronectin

was calculated.

Western blots

The Western blot assay was performed as described previously[13,14]. Following dosing of

0-,10- and 20-µM curcumin treatment for 24 h, the protein was extracted with lysis buffer and

obtained after centrifugation. Equal amounts of protein extracts were separated by 10%

SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were

incubated with primary antibodies at 4˚C overnight. Bands were visualized using horseradish

peroxidase-conjugated anti-rabbit IgG and an enhanced chemiluminescence reagent,

according to the manufacturer’s instructions. GAPDH was used as an internal control.

Proteins were quantified by densitometric analysis.

Cell apoptosis assay.

The apoptosis assay was performed as described previously[14]. Following dosing of 0-,10-,

and 20-µM curcumin for 24 h, the cells were washed three times. An Annexin V-fluorescein

isothiocyanate (FITC)-propidium iodide (PI) apoptosis kit was used to detect apoptosis. Cells

were washed three times and suspended at a density of 3x106 cells/ml in 1X Annexin

V-binding buffer. Annexin V-FITC and PI buffer were added to the cells which were incubated

for 15 min at room temperature. Cells treated with DMSO were used as control.

Database analysis

The relationship between PTEN expression and survival in glioma was analyzed by

PrognoScan database (http://www.prognoscan.org/)[16]. The threshold was set as cox

p-value<0.05. The correlation of PTEN expression and survival of glioma was analyzed by

Kaplan-Meier plotter database (http://linkedomics.org/ analysis/)[17]. The hazard ratio with 95%

confidence intervals and log-rank p-value were analyzed.

Antitumor activity assay in vivo

Antitumor activity was performed as previously described[9]. Female nude mice were

housed with pathogen-free conditions. All experimental procedures conformed to the animal

experiment guidelines of the Animal Care and Welfare Committee of Hubei University of

Science and Technology. U87 xenografts were performed by subcutaneous injections of

2.0×106 U87 cells in nude mice[9]. When the tumor volume reached approximate 100 mm3,

the mice were randomly divided into two groups (n = 5) and intraperitoneal. injections of

curcumin (60 mg/kg) or saline was administered daily for 14 days. The tumor size and

animal body weights were measured every 2 days. Tumor volume was calculated as

described[9]. The mice were sacrificed and the tumors were removed and weighed at the end

of the experiment.

Statistical analysis

All results were presented as mean ±SEM of at least three independent experiments.

Groups were analyzed using one-way ANOVA with Bonferroni’s post-hoc tests. Statistical

significance was set as P < 0.05.

Results

1 Curcumin inhibits glioblastoma proliferation in a time- and dose-dependent manner.

Using an MTT assay, we measured the proliferation of U251 and U87 cells that were

treated with 0-, 10, 20-, and 40- µM curcumin for 24-,48-, and 72-h. As shown in Fig.1A, the

glioblastoma cells become small and appeared unhealthy due to the increasing

concentrations of curcumin. Treatments with 10- and 20-µM curcumin for 24 h significantly

inhibited cell proliferation, compared to the control group in both the U251 (Fig. 1B) and U87

(Fig. 1C) cells. Our results indicated that curcumin downregulates glioblastoma proliferation

in both U251 and U87 cells.

2 Curcumin inhibits the migration and invasion of glioblastoma in vitro

To detect the effects of curcumin on metastasis, the wound healing assay was used. The

morphological changes in migrating cells are presented in Fig. 2A. Following treatment with

0-, 10-, and 20-µM of curcumin for 24 h, the migration of U251 and U87 cells was inhibited

(Fig. 2B).

Next, we examined the invasion ability using the transwell assay. The invaded cells were

stained as shown in Fig. 2C. As displayed in Fig. 2D, treatments with10- and 20-µM of

curcumin significantly inhibited the invasion capacities of both U251 and U87 cells. These

data indicate that curcumin inhibits the migration of GB cells in a concentration-dependent

manner.

Next, we measured the adhesion ability of U251 and U87 cells. The adherent cells are

shown in Fig.3E. As displayed in the Fig.3F, treatments with10- and 20-µM of curcumin

inhibited the adhesion ability of both U251 and U87 cells.

3 Curcumin inhibits PI3K/mTOR protein activation

Since curcumin can inhibit the proliferation of glioblastoma, we determined whether

curcumin regulates the expression of AKT/mTOR in the cellular survival pathways of GB. To

this end we examined the protein expression of AKT/mTOR. As shown in Fig. 3A, no

differences between the non-phosphorylated (total) protein of AKT and mTOR in U251 cells

in response to curcumin were observed. As phosphorylation is the activator, we also

measured the phosphorylation levels of P13K/mTOR signaling. As shown in Fig.3B, the

curcumin treatment group significantly decreased the phosphorylation of AKT(p-AKT) and

mTOR(p-mTOR) in U251 cells. In addition, the PI3K/AKT inhibitor LY294002 markedly

decreased the p-AKT and p- mTOR protein expression.

4 Curcumin enhances the apoptosis of glioblastoma cells.

We also determined whether curcumin-induced apoptosis in glioblastoma. The apoptosis of

U251 and U87cells was measured using the annexin V and PI staining (Fig. 4A) and was

quantified by the fluorescence intensity (Fig. 4B). The results suggested that treatment

with10- and 20-µM curcumin significantly enhanced the apoptosis in both U251 and U87

cells.

Next, we determined the protein expression of the apoptotic proteins, caspase-3 and

beclin-1. Western bolt analysis showed that the caspase 3 and beclin-1 proteins were

markedly enhanced in U251 and U87 cells (Fig. 4C). Our results suggested that curcumin

enhanced apoptosis in glioblastoma cells.

5 Curcumin restores PTEN expression in glioblastoma cells.

PTEN is widely distributed in glioma patients and this fact prompted us to analyze whether

the PTEN affected the survival of glioma patients. We used the PrognoScan database to

predict the overall survival (OS) of patients with brain tumors. The result of the analysis

showed that the glioma database (GSE4412) was statistically significant for OS

(p=0.000706) (Fig. 5A). The analysis result was confirmed in the linkedomics database

(sample 396). There was a significant difference in OS between the tumor patients and the

normal group (p= 0.001613) for the survival of glioma patients (Fig. 5B). These results

indicated that the low PTEN expression in the glioma patients confers a poor prognosis.

Next, we determined the effect of curcumin on the PTEN protein expression. As shown in

Fig. 5C, treatments with 10- and 20-µM of curcumin enhanced the PTEN expression in both

U251 and U87 cells.

PTEN is a tumor suppressor and also interacts with other important proteins. Since curcumin

can enhance apoptosis in glioblastoma cells, we tested whether curcumin could attribute to

the restoration of the P53 expression in glioblastoma. As shown in the Fig.5C, the P53

protein expression increased significantly with curcumin treatment in U87 cells.

6 Curcumin inhibited tumor growth and increased the PTEN expression in

xenografts

To detect the inhibitory effect of curcumin on tumorigenesis in vivo, nude mice were used

to establish a xenograft tumor model. As shown in Fig. 6A, curcumin treatment significantly

inhibited tumor growth. In addition, tumor weight was significantly lower in the

curcumin-treated group, compared the control group (Fig. 6B). Hematoxylin and eosin (HE)

staining was used to show the morphological changes of tumor tissue in the xenograft tumor

model. We found that the tumor tissue in the curcumin-treated group contained focal

necrosis and had a reduction in angiogenesis (Fig. 6C). Further, the PTEN and P53

expression were significantly higher in the curcumin-treated group, compared to the control

group (Fig. 6D). Therefore, these data indicate that curcumin has a significant inhibitory

effect on glioma growth in vivo, and the mechanism appears to be the promotion of PTEN

and P53 expression.

Discussion

We investigated the biological effects of curcumin in glioma cells and evaluated its

potential efficacy in vitro (italicize) and in vivo (italicize), which are caused by inhibitory

effects on tumors. We found that curcumin has a dose-dependent effect on proliferation and

migration of glioma cells and inhibits significantly the proliferation, migration, and

invasiveness of glioma cells. The inhibition was associated with the proliferating activity of

the AKT/mTOR signaling pathway under curcumin treatment. We also found that

curcumin-induced apoptosis and significantly regulated the expression of PTEN and P53 in

glioma cells. We also found that curcumin significantly inhibited tumor volume and caused

tumor necrosis, while restoring PTEN expression.

Our study indicates that curcumin inhibits the proliferation of glioblastoma cells in a

dose-dependent manner. We also found that curcumin decreased the migration and

invasion of glioma cells in vitro (italicize). The effect of antitumor of curcumin is consistent

with previous reports[18,19].

We also analyzed the possibility that the inhibition of tumor proliferation may be the

result of action of curcumin on the AKT/mTOR pathway. It is known that AKT/mTOR

activation can promote cell proliferation and has been confirmed to contribute to the

biological behaviors for many tumors[20,21]. The growth of tumors is associated with the

activation of the AKT/mTOR pathway[19,22]. We found that AKT/mTOR expression is

up-regulated to promote the proliferation of glioma cell. Given this observation, we

measured the proliferation, migration and invasion of U251 and U87 glioma cells, and found

that curcumin significantly inhibited these biological process in glioma cells.. In aggregate,

these data indicate that curcumin inhibits the proliferation, migration and invasion of glioma

cells in vitro (italicize) and was associated with the inhibition of AKT/mTOR activation.

Many chemotherapy drugs have the ability to induce cell apoptosis[23]. Apoptosis might

be mediated by intrinsic or extrinsic signaling pathways[24] The apoptosis of glioblastoma

was enhanced by curcumin treatment in the present study. Furthermore, caspase 3 and

beclin-1 are key regulators for intrinsic apoptotic signaling pathways[25,26] and induce the

mitochondrial release of caspase-activated cytochrome C[27]. We found that

curcumin-induced apoptosis and was associated with decreased caspase 3 and beclin-1.

These findings suggest that curcumin can induce intrinsic apoptotic signaling involved in the

activation of apoptosis[28].

The PTEN gene is known as a negative regulator of AKT/mTOR signaling for

tumorigenesis[29,30]. PTEN inhibition is associated with invasive tumor growth, enhanced

metastasis, and poor prognosis for GB patients[31]. Our study showed that curcumin inhibited

the effects of phosphorylation of AKT and enhanced PTEN. Curcumin promoted the

expression of PTEN, in vitro. These findings are consistent with the inhibition of tumor

growth following r curcumin treatment in vitro (italicize) and in vivo[9,32].

The tumor suppressor protein, P53, may inhibit the growth of cancer cells by regulating

various molecular mechanisms, including apoptosis and DNA repair[33]. P53 decreased

proliferation, migration and invasion in many tumor cells[20]. Our study demonstrates that

curcumin enhances P53 expression in vitro and in vivo.

In summary, there are many cellular mechanisms by which curcumin can exert its

beneficial effects against GB. First, curcumin reduces the AKT/mTOR pathway and inhibits

GB cell proliferation and migration. Secondly, curcumin induces apoptosis. Curcumin

increases PTEN expression and reverses P53 expression. Finally, curcumin can

significantly inhibit tumor growth, induce tumor apoptosis, and up-regulates PTEN

expression in vivo.

Conclusion

Our findings show that curcumin treatment partially inhibits the proliferative activity of

glioma cells and suppresses the invasive behavior by down-regulating AKT/mTOR activity,

thereby inducing apoptosis of glioma cells and increasing PTEN and P53 expression.

Furthermore, this positive response to curcumin is dose-dependent and was confirmed in

U87-xenografts in vivo. These results suggest that further explorations in evaluating the

potential use of curcumin for the treatment of GB and other malignant tumors are warranted.

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Acknowledgments Not applicable.

Authors’ contributions LM and WZ designed the study. LM, WZ and LF performed the

experiment. XH analyzed the data. YL HZ contributed to the writing the manuscript. All

authors have read and approved the final manuscript.

Competing interests The authors declare that they have no competing interests.

Availability of data and materials All data generated or analyzed during this study are

included in this published article.

Funding This work was supported by the grants from the Fund of the Science and

Technology Department of Hubei Province (2018CFB737) and the Found of Hubei

University of Science and Technology (2018xzy02,2019xz01). No specific funding was

received for this study.

Declaration

Ethics approval and consent to participate All animals’ procedures were conducted in

accordance with the National Institutes of Health Guide for the Care and Use of Laboratory

Animals (NIH publication 85-23, revised 1996) and approved by the institutional animal care

and use committee at the Hubei University of Science and Technology, China. No ethical

approval for the use of the human glioma cell lines was required.

Figure Legends

Figure 1 The effects of curcumin on the proliferation and viability of U251 and U87 cell lines.

The anti-proliferative effects of curcumin (0-, 10-, 20- and 40-µM) on U251 and U87 cell lines

were assessed by MTT assay after 48 h of treatment( U251 in A). The values are given as

mean ± S.E. of three independent experiments for U251(B) and U87(C) cell. *P<0.05 and

**P<0.01, compared to control group.

Figure 2 Curcumin inhibits the migration, invasion, and adherence of U251 and U87 cells in

vitro. After U251 and U87 cells were treated with different concentrations of curcumin, the

migration ability was determined by the wound healing assay (A). The invasion of the glioma

was measured by the transwell assay (C) and cell adherence was evaluated by the ECM

assay (E). The migration (B), invasion (D) and adherence (F) values are given as mean ±

S.E. for three independent experiments for U251 and U87 cells. *P<0.05 and **P<0.01,

compared to control group.

Figure 3. Curcumin suppresses AKT/mTOR activation in U251 cells. After U251 cells were

treated with 20 µM of curcumin or 25 µM of LY294002, the levels of AKT, p-AKT, mTOR,

p-mTOR and GAPDH were determined by the Western blot analysis A). The quantitative

analysis of AKT, p-AKT, mTOR and p-mTOR was conducted (B). Values are given as mean

± S.E. for three independent experiments. *P<0.05 and **P<0.01, compared to control

group.

Figure 4. Curcumin enhances apoptosis in glioma cells. After U251 cells were treated with

10- and 20-µM of curcumin, apoptotic cells were visualized by the Annexin V and PI staining

(A).The quantitative analysis of apoptotic cells was performed (B). The apoptosis proteins,

caspase-3 and beclin-1, were measured by the Western blot analysis (C). Values are

given as mean ± S.E. for three independent experiments. *P<0.05 and **P<0.01, compared

to control group.

Figure 5. The functional analysis for the PTEN gene and the PTEN protein expression after

curcumin treatment. The overall survival of the PTEN gene in the glioma patients was

plotted from PrognoScan database(p-value=0.000706)(A) and was plotted from

Kaplan-Meier Plotter database(p-value=0.001613) (B). The PTEN and P53 protein were

detected by the Western blotting and were analyzed (C). Values are given as mean ± S.E.

for three independent experiments. *P<0.05, compared to control group.

Figure 6 Curcumin effectively inhibited the tumor growth and enhanced the PTEN and P53

expression in the xenograft tumor model of glioma. The tumor volume was detected io

different days(A). The tumor size and wet tumor weight (B) were measured at the time of

dissection. The histological features of the tumors were examined by HE (C). The PTEN and

P53 protein were detected by the Western blots and were quantified (D). Values are given

as mean ± S.E. for three independent experiments. *P<0.05, compared to control group.

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