Graphene Enhances Cellular Proliferation through Activating the

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Graphene enhances cellular proliferation through activating EGFR Wei Liu, Cheng Sun, Chunyang Liao, Lin Cui, Haishan Li, Guangbo Qu, Wenlian Yu, Naining Song, Yuan Cui, Zheng Wang, Wenping Xie, Huiming Chen, and Qunfang Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05923 • Publication Date (Web): 20 Jun 2016 Downloaded from http://pubs.acs.org on June 25, 2016

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Journal of Agricultural and Food Chemistry

Graphene enhances cellular proliferation through activating EGFR

Wei Liu†, Cheng Sun‡, Chunyang Liao‡, Lin Cui‡, Haishan Li†, Guangbo Qu‡, Wenlian Yu†, Naining Song†, Yuan Cui†, Zheng Wang†, Wenping Xie†, Huiming Chen†*, Qunfang Zhou‡*

†

Institute of Chemical Safety, Chinese Academy of Inspection and Quarantine,

Beijing, China, 100124 ‡

Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,

Beijing, China, 100085

*Correspondence to [email protected] and [email protected]

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ACS Paragon Plus Environment


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Abstract

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Graphene has promising applications in food packaging, water purification, and

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detective sensors for contamination monitoring. However, the biological effects of

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graphene are not fully understood. It is necessary to clarify the potential risks of

5

graphene exposure to human beings through diverse routes like foods. In the present

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study, graphene, as the model nanomaterial, was used to test its potential effects on

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the cell proliferation based on multiple representative cell lines including HepG2,

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A549, MCF-7 and Hela cells. Graphene was characterized by Raman spectroscopy,

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particle size analyzer, atomic force microscope (AFM) and transmission electron

10

microscope (TEM). The cellular responses to graphene exposure were evaluated

11

using flow cytometry, MTT and Alamar-blue assays. Rat cerebral astrocyte cultures,

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as the non-cancer cells, were used to assess the potential cytotoxcity of graphene as

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well. The results showed that graphene stimulation enhanced cell proliferation in all

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tested cell cultures and the highest elevation in cell growth was up to 60%. Western

15

blot assay showed that the expression of epidermal growth factor (EGF) was

16

up-regulated upon graphene treatment. The phosphorylation of EGF receptor (EGFR)

17

and the downstream proteins, ShC and extracellular regulating kinase (ERK) were

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remarkably induced, indicating the activation of MAPK/ERK signaling pathway was

19

triggered. The activation of PI3 kinase p85 and AKT showed that PI3K/AKT

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signaling pathway was also involved in graphene induced cell proliferation, causing

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the increase of cell ratios in G2/M phase. No influences on cell apoptosis were

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observed in graphene treated cells when compared to the negative controls, proving 2


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the low cytotoxicity of this emerging nanomaterial. The findings in this study

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revealed the potential cellular biological effect of graphene, which may give useful

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hints on its biosafety evaluation and the further exploration of the bioapplication.

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Keywords: graphene, cell proliferation, EGFR, cytotoxicity, biosafety

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Introduction

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Graphene is a kind of two dimensional nanomaterial and it is composed of

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sp2-hybridized carbon atoms hexagonally arranged in single atom thickness. It has a

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large surface area on both sides of the planar axis and possesses unique electrical,

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thermal, and mechanical characters1. Since the production of graphene layers was

34

proposed by mechanically peeling sheets off graphite crystals in 2004, the

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application of this emerging nanomaterial has been widely explored in several fields

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such as electronics and biomedicine2, 3. The usage of graphene in food packaging is

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becoming more and more popular as graphene nanoplates are heat resistant and can

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efficiently prevent the migration of oxygen, CO2, and water vapor4. Graphene based

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material was also reported to kill pathogenic microbes in food and drinking water5-8.

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Graphene based sensors are effective for the contamination monitoring in food and

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water9, 10. All these usages evidently cause the exposure of graphene to human beings.

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It is thus necessary to understand the potential biological effects of this emerging

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nanomaterial.

44

In view of the cytotoxicity of graphene-based materials, there is no consensus in

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recent studies. Some people believe that the derivatives of graphene are highly

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biocompatible without any significant toxicity, while others pointed out that obvious

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cytotoxicity was induced by this kind of nanomaterial in several cell lines11-14. For

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instance, Ryoo et al showed that graphene substrates improved gene transfection

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efficacy in NIH-3T3 fibroblasts without inducing notable deleterious effects14.

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Chang et al found that graphene oxide (GO) did not enter A549 cells and showed no 4


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obvious toxicity to A549 cells15. On the contrary, Wang et al demonstrated that

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water-soluble GO had obvious toxicity to human fibroblast cells such as decreasing

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cell adhesion, inducing cell apoptosis, entering into lysosomes, mitochondrion,

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endoplasm, and cell nucleus when the dose was higher than 50 µg/mL16. Biris et al

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demonstrated that graphene sheets induced cytotoxic effects on phaeochromocytoma

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(PC-12) cells while its toxicity was much lower than CNTs17. Different cytotoxicities

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were observed for graphene in diverse cell types. Qu et al demonstrated that GO

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inhibited the proliferations of J774A.1 and RAW 264.7 cells at the doses ranging

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from 2.5 to 80 µg/mL, but they did not observe any impairments were induced by

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GO at similar doses on the cell growth or survival of the other tested cell lines

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including mouse Hepa 1-6 hepatocytes, human Hepa G2 hepatocytes, or human

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MB-MDA-231 breast cancer cells18. Altogether, most of the toxicological studies

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focused on the exploration of the potential biological effects of GO materials and

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people believed that GO materials were more toxic than graphene as they could form

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stable colloidal suspensions in cell culture systems19. However, it was pitifully

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inadequate to make the arbitrary conclusion about the exposure risks of graphene

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based on the data from GO materials regarding its current wide applications and the

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distinct characters of these two kinds of nanomaterials.

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The aim of this study was to investigate the cellular biological effects of a well

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prepared graphene. The cell proliferations of multiple cell cultures were firstly

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screened by graphene stimulations. The most interesting finding was that the

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graphene treatment caused significant elevation in cell proliferation in all tested cell 5



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cultures. The investigation on the underlying molecular mechanisms showed that

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both MAPK/ERK and PI3K/AKT signaling pathways were involved in graphene

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induced cell growth. To our knowledge, this was the first report that fully elucidated

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graphene enhanced cellular proliferation on the molecular mechanisms.

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Materials and methods

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Graphene preparation and characterization

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Graphene is purchased from XFNANO Materials Tech Co., Ltd (Nanjing, China).

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Stocking graphene suspension is prepared in DMSO or cell culture medium with 0.5%

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FBS (for cell apoptosis and cell cycle assays) at concentration of 1×103µg/mL. The

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suspension is dispersed by ultrasonic for 5 min to obtain homogeneous concentration

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before treatment.

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The graphene was characterized by raman spectrum (Horiba Jobin Yvon LabRAM

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HR800, Japan). Zeta potential was measured at 10 µg/mL in water, DMSO,

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RPMI1640 medium with 10% FBS respectively by a laser particle size analyzer

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(Malvern Nano ZS, Nalvern, U.K.). The morphologies of graphene were observed

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by transmission electron microscopy (TEM, Hitachi H-7500, Japan) and atomic

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force microscope (AFM, Veeco Dimension 3100, Plainview, NY).

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Cell Culture

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The five types of commercial available cell lines used in this study were human lung

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adenocarcinoma ephithelial cells (A549), human hepatocellular carcinoma cells

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(HepG2), human breast adenocarcinoma cells (MCF-7), human cervical carcinoma 6


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Cells (Hela) and Chinese-hamster ovary cells (CHO-K1). A549, HepG2 and Hela

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cells were maintained in RPMI1640 medium (Hyclone, Logan, UT, USA). MCF-7

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cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Hyclone),

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CHO-K1 cells were cultured in Dulbecco’s Modified Eagle’s Medium supplied with

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F12 (DMEM/F12; Hyclone). The primary cultured astrocyte cells were kindly

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provided by Dr. Qunfang zhou from RCEES, CAS, Beijing and maintained in

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DMEM F12 medium (Hyclone)20. All medium was supplemented with 10% fetal

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bovine serum (FBS; Gibco, Grand Island, NY, USA) and 100 U/mL

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penicilli/streptomycin (Gibco). All cell lines were grown at 37 °C in a 5% CO2

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humidified environment. The cells at a logarithmic phase of growth were used for

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the exposure tests.

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Cell viability

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The cell viability was examined by three different methods. For MTT assay, the

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protocol was adapted from report of Mosmann et al21. Cells were exposed to

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graphene of 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL for 24 hrs. At the end of

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exposure, 150 µL methyl- thiazoletetrazolium (MTT; Amresco, Solon, OH, USA)

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solutions (0.5 mg/mL in cell culture medium) were added to each well and the cells

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were incubated at 37 °C for 4 h. After incubation, MTT solution was aspirated and

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the cells were treated with 150 µL dimethyl sulfoxide (DMSO; Amresco). The plates

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were slightly shaken at room temperature until the crystals were dissolved.

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Absorbance

116

spectrophotometer (Thermo Electron, Varioskan Flash, USA).

was

measured

at

490

nm

using

a

7


multimode

microplate


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For Alamar blue assay, cells were exposed to graphene of 1.25, 2.5, 5, 10, 20, 40,

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80 and 160 µg/mL for 24 hrs. At the end of exposure, 1/10th volume of

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AlamarBlue® reagent (Life Technologies) was added directly to cells in culture

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medium. Then cells were incubated for 2 hours at 37 °C in dark. Fluorescence

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emission at 590nm (excited at 540 nm) was measured by multimode microplate

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spectrophotometer (Thermo Electron, Varioskan Flash, USA).

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For cell counting, cells were seeded in 6-well plates with 105 cells/well. After 12

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hrs’ culturing, cells were treated with graphene at doses of 10, 20, 40, and 80 µg/mL.

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After 24 hrs, live cell counting was performed with a hemocytometer after trypan

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blue staining.

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Cell apoptosis and cell cycle

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Cell apoptosis and necrosis were analyzed by double staining with Annexin V-FITC

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and PI, 100 U/ml TNF-a (Calbiochem, San Diego, CA, USA) was used as the

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positive control. After 24 h of exposure, the cells were collected and stained using

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Annexin V-FITC Apoptosis Detection Kit (Bender, Austria). Then, the samples were

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detected by the flow cytometer (FACSCalibur, Becton Dickenson, CA, USA).

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Cell cycle progression was also analyzed using the flow cytometer. Cells were

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seeded in 6-well plates with 105 cells/well and starved for 48 hrs in medium with 0.5%

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FBS for synchronization, then changed to normal medium and treated with graphene.

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After 24 h of exposure, the collected cells were washed by PBS and fixed using 70%

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ethanol overnight. Ethanol was removed by centrifugation (1500 rpm, 5 min) and the

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cells were washed twice with PBS. Cells were then resuspended in PBS containing 8


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RNase (1 mg/ml; Sigma, St Louis, MO, USA) and propidium iodide (PI, 50 mg/ml;

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Sigma) and kept at 37 °C in the dark for 30 min, then analyzed by measuring the

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amount of PI-labeled DNA on the flow cytometer.

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Western bloting

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Cells were seeded in 6-well plates with 105 cells/well and treated with 0, 10, 20, 40

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µg/mL graphene, DMSO was used as solvent control with a same dose as in 40

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µg/mL graphene (0.25 %). After treated for 24 hrs, cells was washed with PBS for 3

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times and lysed with RIPA lysis buffer. The same amount of protein extracts were

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loaded for the separation on 10% SDS-PAGE, then transferred to nitrocellulose

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membranes. The membranes were blocked with 5% non-fat milk in TBST at room

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temperature for 1 hr. The primary antibody was used at 1:1000 dilution and

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incubated overnight at 4 °C with gentle shake. The secondary antibody was used at

151

1:1000 dilution and incubated for 2 hr at room temperature.

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using enhanced chemiluminescence. The tested proteins were Phospho-EGFR,

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Phospho-ERK (P-ERK), ERK, Phospho-PI3 Kinases p85, Phospho-AKT, AKT,

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Phospho-ShC and β-actin. All of the antibodies were purchased from Cell Signaling

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Technology.

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miRNA silencing and RT-PCR analysis

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HepG2 cells were seeded in 6-well plates with 105 cells/well and transfected with (1)

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miRNA-7 (synthesized by Sangon Biotech, Shanghai, PRC) in Lipofectamine2000

159

(Lipo2000, Invitrogen) (2) negative control with Cy3 tag (Cy3-NC, synthesized by

160

Sangon Biotech, Shanghai, PRC) in Lipo2000. Cells were continue incubated for 9


Blots were developed


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24-hrs after transfection. The transfection efficiency was investigated by confocal

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laser scanning microscopy (CLSM, Zeiss 510, Germany).

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Transfected cells were treated with 40 µg/mL graphene. Relative quantifications of

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mRNA expression in the genes EGFR, PI3K and Akt were calculated using RT-PCR

165

assay.

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Total RNAs were extracted from cells using Trizol (Invitrogen). RT-PCR analysis

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was performed as described in reference22. GAPDH was used as an internal control.

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Primers are as follows: GAPDH forward: 5’- CAT CAG CAA TGC CTC CTG

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CAC-3’, reverse: 5’-TGA GTC CTT CCA CGA TAC CAA AGTT-3’. EGFR forward:

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5’-CTT GAT TCC AGT GGT TCT GCT TC-3’, reverse: 5’-CAT CCC CTC CGT

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TTC TTC TTT-3’. PI3K forward: 5’-CTG TGT GGG ACT TAT TGA GGT GGT-3’,

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reverse: 5’-ACT GAT GTA GTG TGT GGC TGT TGA-3’. Akt forward: 5’-TGT

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GAA GGA GGG TTG GCT GC-3’, reverse: 5’-ACT GCG CCA CAG AGA AGT

174

TGTT-3’.

175

Statistical Analysis

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The difference of experimental data between two groups was assessed using the

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two-tailed Student's t-test. Data were shown in mean±SD, n≥3. p< 0.05 was

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considered statistically significant.

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Results and Discussion

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Characterization of graphene

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The commercially available graphene was specified with no functionalization or

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oxidation. Careful characterization was performed before it was submitted to the cell 10


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stimulations. The results based on atomic force microscopy (AFM) observation

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showed that the thickness of this graphene was about 1.2 nm (Fig.1A and B). The

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morphology provided by transmission electron microscopy (TEM) demonstrated that

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the graphene was consisted of ultra-thin layers (Fig.1C). Raman spectrum analysis

187

showed the featured peaks of graphene at 1580 cm-1 (G peak) and 2680 cm-1(2D

188

peak) (Fig.1D), indicating the character of multi layers for this

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result was consistent with the thickness of individual graphene sheet (1.2 nm)

190

obtained by AFM measurement. The characterization of zeta potential showed that

191

this graphene was negatively charged in water and dimethyl sulfoxide (DMSO),

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while it turned nearly neutral in cell culture medium (Table 1). The stability of

193

graphene suspensions was evaluated and the results showed that graphene was well

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dispersed in DMSO and cell culture medium after ultrasonic for 5 min.

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Graphene induced cell proliferation in diverse cell cultures

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As reported, liver and lung are the important target tissues which graphene family

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nanomaterials may bioaccumulate in and induce damnification25. A long-term in

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vivo study revealed that the graphene quantum dots mainly accumulated in liver,

199

spleen, lung, kidney, and tumor sites26. Graphene was accumulated initially in the

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RES, liver, and spleen after intravenous injection27. Graphene oxides at a dose of 0.4

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mg induced granulomas in the lungs, liver, spleen, and kidney in mice16. Moreover,

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graphene oxides showed good biocompatibility with red blood cells28. In vivo

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ﬂuorescence imaging revealed that graphene was high uptaken in tumor in several

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xenograft tumor mouse models29. Based on the liver/lung-targeting and tumor-uptake 11


graphene23, 24. This


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of graphene, we chose cell lines (HepG2 and A549) derived from liver and lung as

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the main models to study the in vitro toxicity of graphene. Several other cells were

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also selected to discuss the cell type selection for the graphene cytoxicity test.

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To assess the potential cellular biological effects of the graphene, we performed

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24-hr stimulation on four cell lines including HepG2, A549, Hela and MCF-7 with

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the graphene at a series of concentration gradient (1.25-160 µg/mL). The cell

211

viability based on MTT assay showed that obvious cell proliferation was induced for

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all four cell lines in the tested dose range (Fig. 2A). Comparatively, three cell lines

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including HepG2, A549 and Hela cells showed more sensitive to graphene treatment

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when compared with MCF-7. For example, significant increase in cell proliferation

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was observed in HepG2 cells exposed to 10 µg/mL graphene, while the elevation in

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cell growth didn’t become significant for MCF-7 until it was stimulated by 160

217

µg/mL graphene. Approximate 20-60% of cell proliferation increase was observed in

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HepG2 in the tested range of 10-160 µg/mL graphene and it climaxed at the

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stimulation of 80 µg/mL graphene with the increase ratio of 60%. The typical

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dose-dependent manner was clearly exhibited in this cellular response induced by

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graphene. Likewise, A549 and Hela cells responded to graphene exposure in the

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similar way to HepG2.

223

Nevertheless, the argument was proposed by Liao et al that MTT assay would be

224

influenced by GS/GO materials as they could reduce the [MTT]+ cations by donating

225

the electrons30. The potential disturbance of graphene on MTT assay was evaluated

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by the direct incubation of this nanomaterial with MTT dye and the result showed no 12


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effect was observed during the tested range of 0-160 µg/mL graphene (data not

228

shown). This confirmed that the cell proliferation induced by graphene observed

229

above was real instead of the false positive results from the failure of MTT assay in

230

evaluation of some nanomaterials’ effects.

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The cellular phenotype of graphene induced cell proliferation was further tested

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by some alternative methods including Alamar-blue assay and hemocytometer

233

counting. The results in Fig. 2B and C showed the similar trends in cell proliferation

234

were exhibited in all tested cell cultures upon graphene treatments, the sensitivities

235

in cell responses were varied though. These findings further confirmed the

236

phenotype of the enhanced cellular proliferation upon graphene stimulation.

237

As we know, those four cell lines used above are all derived from diverse tumor

238

tissues, and may have some specific distinct characters when compared to the normal

239

tissue cells. The subcultures from the primary rat cerebral astrocytes were used to

240

evaluate the potential effects of graphene on normal tissue cells. The results from

241

Alamar-blue assay showed graphene stimulation caused the significant increase in

242

the proliferation of astrocytes as well (Fig. 2D) and this cellular response was also

243

elevated in the dose-dependent manner. This was consistent with the findings from

244

the other tested cell lines, proving that graphene induced cell proliferation occurred

245

in both tumor derived cell lines and normal cells.

246

The effects of graphene on cell cycle progression

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We investigated the apoptosis and necrosis using FITC-Annexin V and PI staining

248

and detected it by flow cytometry. No change was observed between cells treated 13



249

with and without graphene (data not shown). Cell cycle was detected by flow

250

cytometry and PI staining. We found no obvious changes in phase percentage in

251

graphene treated A549 cells in comparison with the untreated cells (Fig. 3D). While,

252

in HepG2 cells, there is increase of G2/M percentages and concomitantly decrease of

253

S fractions (Fig. 3 E). This result revealed the enhancement of transition from S

254

phase to G2/M phase upon graphene treatment, which would also contribute to the

255

cellular proliferation.

256

The attachment of graphene on cell membrane

257

Previous studies31 have indicated that graphene exhibited excellent compatibility

258

with red blood cells and platelets. It didn’t disturb the plasma coagulation pathways.

259

Minimal alterations were induced in the cytokine expression of human peripheral

260

blood mononuclear cells upon graphene stimulation. Others reported the obvious

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cytotoxicity of graphene and GO was often due to the good dispersion of graphene

262

materials in cell culture medium and the substantial cellular uptake of the

263

nanomaterials18. As for the graphene used in this study, it had poor dispersion in the

264

cell culture medium and obvious aggregation was formed on the outer membrane of

265

the cells after 24-hr exposure (Fig. 4). Very few graphene was found in cells based

266

on TEM searching (Fig 5). The cellular uptake of graphene could be hindered by

267

both aggregation and the lamellar shape of graphene. Therefore, the poor solubility

268

of graphene may block its potential toxic effects from exogenous invader for cells.

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Similar results in Sayan’s study showed that A549 cells had no uptake of graphene

270

nanoribbons32. Jaworski et al also pointed out that graphene platelets did not enter 14


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into the cells33. Nevertheless, as we can see from Fig.4 and Fig 5 (B, C, D), plenty of

272

graphene sheets deposited closely to or attached on the cell membrane (indicated by

273

the yellow arrow), which could pose direct stimulations to the cells through the

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physical contact.

275

Graphene activated EGFR/MAPK signaling pathways

276

As we all know that the activation of members of epidermal growth factor receptor

277

family (EGFR) is a kind of transmembrane tyrosine kinases. They play critical roles

278

in regulating cell proliferation, differentiation, and migration34, 35. Considering the

279

phenotype of graphene enhanced cellular proliferation and the membrane contact

280

with graphene aggregates, it could be speculated that EGFR could be influence and

281

involved in this biological process. Using HepG2 and A549 as the cell models, we

282

evaluated the secretion of EGF in the cell culture medium after graphene stimulation

283

using commercially available ELISA kit. Time-course study showed that

284

extracellular EGF levels were significantly higher than the negative controls after

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24-hr exposure of 40 µg/mL graphene (p

Graphene Enhances Cellular Proliferation through Activating the

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