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sodium formateIII) and H2O2 (MOS) were obtained from the B

时间:2019-03-30 08:01 文章来源:环亚国际登陆 点击次数:

   growth of cancer cells. Hence, in the current investigation, weaim

normal 16HBE cells and on tumor A549 cells have previouslybeen

3.2. Metabolic changes induced by protein-coated Au NR***posure

as a result of activated glutathione synthesis andtransformation

*** Corresponding author. Tel.: þ86 27; fax: þ86.

during cell proliferation. One of the major alterations thatnormal

[41] Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathionemetabolism and its

COSY 1He1H correlation spectroscopy

[47] Borutaite V, Brown GC. Caspases are reversibly inactivated byhydrogen

J Proteome Res 2010;9:1460e75.

design (SMD). Mol Divers 2006;10:169e86.

protein-coated Au NRs treatment.

7126 L. Zhang et al. / Biomaterials 34 (2013) 7117e7126

validity [31]. The validity of all models was additionally ensuredwith CVANOVA

Proc Natl Acad Sci U S A 2010;107:2037e42.

Au nanoparticle exposure produces many reactive oxygenspecies

12 h

High Energy Physics, Beijing, China

[26] Zhang X, Wang Y, Hao F, Zhou X, Han X, Tang H, et al. Humanserum

R2X ¼ 0.29

a Correlation coefficients, positive and negative signs indicatepositive and negative correlation in the concentrations,respectively. p ¼ 0.05, jrj ¼ 0.602 was used as the

between depletion in the levels of ATP and cell death has


OPLS-DA models. The model quality is indicated by the

by 1H NMR spectroscopy (Fig. 1). The resonances were assignedto

Cancer 2004;4:891e9.

novo GSH synthesis is not activated in the protein-coated AuNRs

24 h of post exposure. There were similarities and differencesin the

Sci 2009;1177:66e73.

achieved by respectively regulating NAD biosynthesis and事真上H2O2

3. Results

genomic instability and the generation of tumor resistance to

16HBE bronchial epithelial cells

48 h

data points for each of the 128 increments. The spectral widthswere 10 ppm for 1H

chemical shift reference (d 0.00 ppm). After vortexing andcentrifugation (16,099 g,

Au NRs are primarily located in the lysosome of 16HBE

HNO3 (BV-III) and H2O2 (MOS) were obtained from the BeijingChemical

[7] Igney FH, Krammer PH. Death and anti-death: tumour resistanceto apoptosis.

supported by the fluorescent imaging taken from live-deadstaining,

induced by tumor necrosis factor a-related apoptosis-inducingligand/anticancer

The information provided here will be important in designinganticancer

The differences between the effects of protein-coated Au NRson

[13] Alexandre A, Peter ST, Chan WCW. The effect of nanoparticlesize, shape, and

In the current investigation, increases in the levels ofglutamate,

cells, for example, depletion in the levels of lactate occurred inboth

Yuliang Zhao b, Huiru Tang a,***, Chunying Chen b,**, Yulan Wanga,d,*

also observed in the protein-coated Au NRs exposed 16HBE cellsat

analysis with one way ANOVA was used to test the significantdifferences between the control group and the protein-coated Au NRstreated (*p < 0.05, **p < 0.01, ***p < 0.001).


disruption of lipid bilayers due to the electrostatic attractionand

most promising putative anti-tumor treatments. Currently,extensive

metabolic diseases [26], and in molecular phenotyping [27].

of salvia miltiorrhiza bunge. J Proteome Res 2010;9:1565e78.

16HBE cells, which is absent in tumor cells (Figs. 2 and 3).These

[27] Li M看着磷甲酸钠, Wang B, Zhang M, Rantalainen M, Wang S, Zhou H, et al.Symbiotic gut

nano-drugs. Previously, the comparative responses of human alveolaradenocarcinoma epithelial cells

in contrast to the elevation of GSSG in 16HBE cells afterproteincoated

tumor [12], owing to their unique physiochemical and optical

and biodistribution of various gold nanorods possessing

also possess various potential applications in biomedicine, suchas

44 AMP e e e e e 0.63

and ultimately tumor cell death [8]. The current observation ofthe

amount of ATP for de novo synthesis of nucleotides, lipids,and

in protein-coated Au NRs treated 16HBE cells (Figs. 2 and 3),

The multivariate data analyses were carried out using the SIMCA-Pþsoftware

Cells were seeded on 6 well plates and incubated with or withoutprotein-coated

ycine e 0.68 0.68 e e e

and found that protein-coated Au NRs were accumulatedselectively

vials filled with protein extraction solution to releaseintracellular contents. After

A tumor arises when cells have lost their normal checkpoints

The dominant metabolites present in the cell extracts includea

Decreased de novo GSH synthesis has been demonstrated to

lactate, plays an important role in generating lactate, and hasbeen

Prediction and classification of drug toxicity using probabilisticmodeling of

16HBE cells after protein-coated Au NRs exposure at all thetime

which can form nanoscale holes within the membrane andincrease

and nucleotides, such as inosine, adenosine, uracil,inosine⑸0-formateIII)

for the rat gastrointestinal contents. J Proteome Res2012;11:1397e411.

repeated dishes. After 24 h, 20 mL complete medium containing 50 mMproteincoated

10 min, 4 C), a total of 550 mL of the supernatants was pipettedinto 5 mm NMR

screening approach. J Proteome Res 2007;6:4407e22.

quality of the model was described by the parameters R2X,representing the total

and dilution minimization. Analyst 2009;134:916e25.

Nano Lett 2011;11:772e80.

to induce apoptosis of tumor cells without damaging DNA. Thisis

GSH reduced glutathione

firstly performed for each treatment to generate an overview of thedata distribution

R2X ¼ 0.45

bromide (CTAB), silver nitrate (AgNO3), sodium sulfate (Na2SO4) andLascorbic

stimuli. This is achieved by using multivariate data analysis ofspectroscopic

to its widespread use and it has been successfully applied inunderstanding

and downstream of mitochondrial cytochrome c release. BiochemBiophys Res

Lyso Tracker Red DND⑼9, Mito Tracker Red, Low glucose Dulbecco您看甲酸钠是伤害品吗’smodified Eagle’s

functions of stress hormones is to mobilize amino acids fromprotein,

L-DMEM medium (Gibco), 10% (v/v) fetal bovine serum (Gibco), 100U/mL penicillin

range of amino acids in the tumorous A549 cells that are similarto

Au NRs were washed at least three times in order to remove theresidual

face? Acc Chem Res 2007;40:335e42.

11 Glutamine 0.67 0.69 0.69 0.81 e e

medium (DMEM), penicillin and streptomycin, and L-glutamine werepurchased

Q2 ¼ 0.86

FT Fourier transformation

Fig. 1. Representative 600 MHz 1H NMR spectra of aqueou***tracts from protein-coated Au NRs treated (B, D) and non-treated(A, C) 16HBE and A549 cells. The region of d 5.2e9.5

the different effects of protein-coated Au NRs on normal andtumor

metabonomic model of dietary variation and stressinteractions.

protein-coated Au NRs exposed A549 cells at 12 h (Fig. 3 and Table1)

Systems, State Key Laboratory of Magnetic Resonance and Atomic andMolecular

Proc Natl Acad Sci U S A 1997;94:6658e63.

su***ce plasma resonance (SPR) band centered at 825 nm (Fig. S1B).The proteincoated

and 1He13C heteronuclear multiple bond correlation spectra (HMBC)NMR spectra

exposure (Table 1 and Fig. 3), indicating that protein-coated AuNRs

[30] Dai H, Xiao C, Liu H, Hao F, Tang H. Combined NMR andLC-DAD-MS analysis

pathway-specific metabolism that is associated with the

[24] Zhang L, Ye Y, An Y, Tian Y, Wang Y, Tang H. Systems responsesof rats to

biosensing [9], drug/gene delivery [10,11], and thermal therapyof

reveals comprehensive metabonomic variations for three phenotypiccultivars

[39] Jia H, Liu Y, Zhang X, Han L, Du L, Tian Q, et al. Potentialoxidative stress of gold

cytochrome oxidase and tetramethylphenylenediamine via cytosoliccytochrome

[44] Meurette O, Lefeuvre-Orfila L, Rebillard A, Lagadic-GossmannD, Dimanche-

modulate the micro-environment of tumor cell.

Protein-coated 膦甲酸钠氯化钠几钱Au NRs were prepared according to previous

package (version 11.0, Umetrics, Sweden). Principal componentanalysis (PCA) was

[29] Dai H, Xiao C, Liu H, Tang H. Combined NMR and LC-MS analysisreveals the

Q2 ¼ 0.84

(Fig. S5). Protein-coated Au NRs induced cell death in A549cells

These findings provide comprehensive molecular information on thedistinctive intracellular localization, cellular uptake andtranslocation of Au NRs in normal and tumor cells, highlighting thevalue of metabonomics in assessing biological effects ofnano-drugs.

Significant changes in intracellular metabolites of 16HBE cells andA549 cells exposed to protein-coated Au NRs at different timepoints, compared to their respective untreated

between the 16HBE and A549 cells in their responses toproteincoated

43 Hypoxanthine e e e 0.66 0.69 0.75

Au NRs exposure occur at 48 h post exposure, these

equal widths of 0.004 ppm (2.4 Hz) using AMIX software package(V3.8, Bruker-

marked reduction in the levels of a range of nucleosides andnucleotides

7122 L. Zhang et al. / Biomaterials 34 (2013) 7117e7126

Au NRs exposure for 12 h also result in elevated levels of a

R2X ¼ 0.41

microbes modulate human metabolic phenotypes. Proc Natl Acad Sci US A

multiple DNA oligonucleotides from gold nanorods. ACS Nano2009;3:80e6.

[32] Eriksson L, Trygg J, Wold S. CV-ANOVA for significance testingof PLS and OPLS

Au NRs exposure.

largely NMR-based metabonomics technique.

of protein-coated Au NRs in the mitochondrionwas the majorcause

in the mitochondria of cancer cells and caused tumor cell

GPC glycerophosphocholine


c redox state. J Biol Chem 2007;282e30.

to fill the gap between cellular uptake, translocation of磷甲酸钠proteincoated

A549 human alveolar adenocarcinoma epithelial cells

Our results show that treatment with protein-coated Au NRs

not the case for A549 cells. Hence, we suggest that theanti-oxidant

conditions [53,54]. The elevated levels of the amino acidsobserved

such as the lysosome and mitochondrion. Our previous studyhas

Mean values standard deviations. The results presented here arerepresentative of three biological replicates.

the 90 pulse length was adjusted to approximately 10 ms and 64transients were


7 min followed by centrifugation (9056 g, 37 C) before the cellexposure experiment.

50-IMP inosine⑸0-monophosphate


Key Intracellular

Correlation coefficients (ra) 您看were(treatment vs. normal)

R2X ¼ 0.36

cetyltrimethylammonim bromide coated

was also observed. In addition, the levels of GSSG in the A549cells

marked reduction in the levels of lactate in both of the cell***posed

potential of glutamine-relation to glutathione metabolism.Nutrition 2002;18:

epithelial (16HBE) cells were cultured in 20 mL medium containing90% (v/v)

without protein-coated Au NRs exposure were prepared in the sameway.

activated in protein-coated Au NRs treated 16HBE cells, whichis

with mercaptohexadecanoic acid (MHDA) on cellular responses

immediately activate de novo GSH synthesis and thetransformation

previous publication [18] and characterized by transmissionelectron microscopy

been shown in various cell types including Jurkat cells,cortical

ROS reactive oxygen species

FK866 Boc⑴-aminocyclopropane

membrane potential (Djm) significantly decreased in

showing that protein-coated Au NRs exposure indeed caused

with concurrent occurrence of elevation in the levels ofaromatic

for three times and detached from Petri dishes using 0.25% trypsinsupplemented

shapes and su***ce groups modifications [13e16]. Christin etal.

Au NRs could act as a LDHA inhibitor and has potential to

E-mail addresses:(H. Tang),

modulating micro-environment of cancer cells by inhibitingthe

(MOS) model was interpreted by back-scale transformed loadings withincorporated colorcoded

[16] Qiu Y, Liu Y, Wang L, Xu L, Bai R, Ji Y, et al. Su***cechemistry and aspect ratio

[28] Xiao C, Hao F, Qin X, Wang Y, Tang H. An optimized buffersystem for NMRbased

could be associated with mitochondrial dysfunction. The firstevidence

nanorods at the mitochondria of cancer cells: implications forcancer therapy.

a r t i c l e i n f o

TEM transmission electron microscopy

disruption in integrity of the mitochondria [18]. Thecompromised

CV-ANOVA, p ¼ 1.28 103, p ¼ 1.19 103, and p ¼ 2.03 104corresponding to the Protein-coated Au NRs exposure for 12 h, 24 hand 48 h respectively. Metabolite keys to the

glutamate; 10. glutathione (reduced); 11. glutamine; 12. aspartate;13. dimethylamine; 14. creatine; 15. citrate; 16.phosphorylcholine; 17. glycerophosphocholine; 18. choline;19.

overwhelming antioxidant capability over the tumor cells (Fig.5),

with gradient selection for the 1He1H correlation spectroscopy(COSY) experiments

metabolism. For example, tumor cells need to consume an ample

2.4. 1H NMR spectroscopy

Mechanisms of BSO (L-buthionine-S, R-sulfoximine)-induced cytotoxiceffects

cells at the same time points.

[36]. Many studies have documented that a reduction of LDHA

prior to statistical data analysis.

of GSH and GSSG in both of the prfromotein-coated Au NRs treated

(NAD). The confirmation of NMR resonance assignment toglutathione

Metabolic response

chemotherapeutic agents kill tumor cells via the apoptoticpathway

16HBE and A549 cells, suggesting that protein-coated Au NRs

and renal cancer cell. Mol Cancer Ther 2009;8:626e35.

1. Introduction

ROS production and cell death [47,48]. Finally, anassociation

[15]. CTAB is a group of quaternary ammonium compounds

remarkable differences in the responses of A549 cells toproteincoated

biological basis of radiosensitivity. Subcell Biochem2002;36:171e86.

[15] Christin G, Nicole S, Andy W, Helen D, Salmaan HB, KimberlyHS, et al. Effect of

reduction of ATP levels in A549 cells from 24 h to 48 h whereasthis

All the measurements were performed in triplicate and the resultswere described as

to be less sensitive to ROS. This view is supported by ourprevious

20 Taurine 0.62 e e 0.67 e e

6 Alanine e e e e 0.65 e

12 h

37 Guanosine e e e e e 0.85

nanoparticles by induced-no releasing in serum. J Am Chem Soc2009;131:

in Peking University Health Science Center (PUHSC).

1 These authors contributed equally to this work.

multivariate data analyses including PCA and OPLS-DA were

comes from our previous investigation, showing thatmitochondrial

(16HBE) extracts after exposure to protein-coated Au NRs witha

found in higher levels in many human tumors than in normaltissues

600 MHz spectrometer, operating at 600.13 MHz for 1H, equipped witha Bruker

GSH efflux, induced JNK-dependent apoptosis [43,44]. Here, de

Received 18 March 2013

in its levels respectively, in the Protein-coated Au NRs treatedgroups compared with the non-treated groups.

studies on protein-coated Au NRs have been focused on thesynthesis,

for ATP show that protein-coated Au NRs induce a significant

21 Methanol e 0.75 0.82 e e e

Au NRs for 24 h and 48 h. In contrast, the levels of ATP ofthe

tissues. Cancer Res 1964;24:389e99.

state. In the current study, the GSH/GSSG ratio untreated16HBE

OPLS-DA orthogonal projection to latent structuresdiscriminant

exposure inhibits the intracellular anaerobic glycolysisprocess. It is

R2X ¼ 0.32

that in A549 cells during the entire experimental period (Fig.4A

monophosphate (AMP); 45. myo-inositol; 46. UDP-N-acetylgalactosamine; 47. cysteine; 48. lipid.

3.1. Metabolites present in the intracellular extracts

(GSH) and oxidized glutathione (GSSG) is illustrated in Fig.S2.

and lactate dehydrogenase A (LDHA) respectively, have beenshown

A549 cells (red dots). The models are constructed the same way asFig. 2. The cross-validation parameters with CV-ANOVA, p ¼ 1., p ¼ 1.69 103 and p ¼ 1.17 103

conventional biochemical assays (Fig. 4). The ratio of GSH/GSSGin

2 ), hydroxyl (HO$), peroxyl

using TOPSPIN (version 2.0, Bruker Biospin) and referenced to theTSP signal

using a tissue-lyser (Qiagen TissueLyser, Retsch GmBH, Germany)with a shaking

9 Glutamate 0.67 0.69 0.69 0.86 e e

16HBE and A549 cells, indicating that protein-coated Au NRs

sensitive to ROS induced by protein-coated Au NRs exposureand

as Boc⑴-aminocyclopropane (FK866), 3-dihydroxy⑹-methyl⑺-

were recorded using the gradient selected sequences with 200transients and 2 k

diminished dramatically in A54您看甲酸钠价钱9 cells after the treatment ofproteincoated

that target cancer cells. We systematically analyzedtimedependent

amino acids, including lysine, tyrosine and phenylalanine. Inaddition,

2.6. Assay of intracellular ATP and the ratio of GSH to GSSG

from Hyclone. All inhibitors used in the current study werepurchased from Sigma.

procaspas⑼ to be released to cytosol, initiating cell death[45,46].

in neuroblastoma. Free Radic Biol Med 2008;44:474e82.

The OPLS-DA models were validated using a 6-fold cross validationmethod and the

10 Glutathione (reduced) 0.75 e 0.65 e 0.75 0.79

characterization and effects on cell morphology [19,20].

cells at a molecular level, we examined the metabolic alterationsin

Au NRs exposure is the significant elevation in the levels

reduction in the ratios of GSH/GSSG in 16HBE cells than

The color plot was obtained with version 7.1 of the MATLAB (TheMathworks Inc.,

5 Threonine 0.66 0.73 e e e e

redox state of the glutathione disulfide/glutathione couple. FreeRadic Biol


and B). These observations indicate that the normal cells aremore

34 Phenylalanine e e 0.72 e e 0.85

[48] Borutaite V, Brown GC. Mitochondrial regulation of caspaseactivation by

Au NRs and associated molecular a您看甲酸钠最新报价lterations by investigation

L. Zhang et al. / Biomaterials 34 (2013) 7117e7126 7123

ATP adenosine triphosphate

2.1. Chemicals and protein-coated Au NRs preparation


[25] Ye Y, Zhang L, Hao F, Zhang J, Wang Y, Tang H. Globalmetabolomic responses

24 Uridine e e e 0.62 0.57 0.84

[6] Evan GI, Vousden KH. Proliferation cell cycle and apoptosis incancer. Nature

each cycle consisting of 2 s pulses and 2 s stops. The supernatantwas collected by

the three extractions were lyophilized after removing methanol invacuo. Each of the

Accepted 22 May 2013

concurrent observation of swollen mitochondria, indicating a

(Figs. 2 and 3) and permutation tests (Fig. S4). The dominantmetabolites

PBS for three times, the cell pellets were frozen in liquidnitrogen and stored in

drugs that are specifically destroying cancer cells whilstnot

metabolic changes in tumor (A549) and normal cell

Boitrel MT. Role of intracellular glutathione in cell sensitivityto the apoptosis

known that secretion of stress hormones is accelerated whenorganisms

Q2 ¼ 0.89

of the model, respectively (Table 1). The models were

aqueous cell extracts was respectively reconstituted in 600 mLphosphate buffer

[37] Fantin VR, St-Pierre J, Leder P. Attenuation of LDH-Aexpression uncovers a

amino acids, such as tyrosine, phenylalanine, histidine andtryptophan,

Compared to the non-treated normal 16HBE cells, proteincoated

with and without protein-coated Au NRs exposure weredetermined

in tumor cells whilst the normal 16HBE cells survived due totheir

include the disappearance of elevation in the aliphatic aminoacids

standard water-suppressed one-dimensional NMR spectrum was recordedusing the

36 UDP-GlcNAc 0.60 0.55 0.67 0.61 e 0.87

induction of tumor cell apoptosis. Cancer Res2003;63:7436e42.

thank S. X. Tang for proofreading.

[21] Nicholson JK, Lindon JC, Holmes E. 看着甲酸钠价钱‘Metabonomics’:understanding the

of the metabolic effects on these cells, with a view toidentifying

factors such as cytochrome c, apoptosis inducing factor and

13 Dimethylamine e e e e e 0.81

The measurements of intracellular adenosine triphosphate(ATP)

d Collaborative Innovation Center for Diagnosis and Treatment ofInfectious Diseases, Hangzhou, China

FIDs free induction decays

chemically prepared gold nanowires. ACS Nano 2012;6:8105e13.

jrj > 0.602 (i.e., r > 0.602 or r < 0.602) was used as thethreshold value for the

protein-coated Au NRs exposure experiments, both types of cellswere seeded at a

corresponding cutoff value of correlation coefficient for thestatistical significance based on the discrimination significance,respectively. ‘‘d’’ means the correlation coefficient

death, whereas posed negligible impact on normal cells [18].Other

spectral width of 10 ppm for both dimensions. Phase insensitivemode was used

have assessed the effects of gold nanorods su***ce coatedwith

convert it to lactate due to the cells’ genetic mutations andtumor

multivariate statistical analysis of giological NMR spectroscopicdata. Xenobiotica

metabolism could be an indication of cell death. This notionis

mass spectrometry [21,22]. The emergence of this technique hasled

Biomaterials 34 (2013) 7117e7126

other is GSH oxidation. One of the most prominent differences

Mathematics, Chinese Academy of Sciences (CAS), Wuhan,China. Tel.: þ86

first increment of NOESY pulse sequence (recycledelay⑼0-t1⑼0-tm⑼0-acquisition)

(C. Chen),(Y.Wang).

35 Tryptophan e e e e e 0.71

and Table 1). After exposure to protein-coated Au NRs for 看着鲁西甲酸两期投产24h,

tumor cell death by compromising membrane integrity. Adv Mater2007;19:

important finding is the observation of elevated levels of GSSGonly

trimethylamine oxide (TMAO); 20. taurine; 21. methanol; 22.glycine; 23. dimethyl phosphate; 24. uridine; 25. inosine; 26.adenosine; 27. uracil; 28. uridine diphosphate-glucose;


7118 L. Zhang et al. / Biomaterials 34 (2013) 7117e7126

points; nevertheless the reduced ratio is still higher thanthe

inhibition of tumor cell proliferation observed previously.

well known that many tumor cells avidly take up glucose and

the micro-environment of cells by suppressing the levels oflactate,

2013 Elsevier Ltd. All rights reserved.

Fig. 3. O-PLS-DA scores and coefficient-coded loadings plots forthe models discriminating between the untreated A549 cells (blacksquares) and protein-coated Au NRs treated

of protein-coated Au NRs in human alveolar epithelialcarcinoma

line broadening factor prior to Fourier transformation (FT).

indicating that 16HBE cells are better protected fromproteincoated

J Proteome Res 2006;5:1535e42.

data generated fromnuclear magnetic resonance (NMR) or


Au NRs exposure than A549 cells, since GSSG is generated

HMBC 1He13C heteronuclear multiple bond correlation

[52] Welberg LA, Seckl JR. Prenatal stress, glucocorticoids and theprogramming of

a depletion in the levels of both nucleosides and nucleotides

Table 1

Au NRs were added into each dish and the cells were exposed toproteincoated

25 Inosine e 0.69 e 0.78 0.71 0.86

metabonomic changes in salvia miltiorrhiza bunge induced by waterdepletion.

Nat Rev Cancer 2002;2:277e88.

Available online 18 June 2013

32 Tyrosine e e 0.68 e e 0.85

aflatoxin B1 exposure revealed with metabonomic changes in multiplebiological

Limin Zhang a,1, Liming Wang b,1, Yili Hu a,c, Zhigang Liu a,c,Yuan Tian a, Xiaochun Wu b,

are subjected to stress conditions [52] and one of the

2.2. Cell culture and exposure to protein-coated Au NRs

range of amino acids, organic acids, such as lactate, creatine,and

epithelial carcinoma (A549) cells and normal human bronchialepithelial (16HBE)

known as the “Warburg effect” [34,35] was confirmed by much

intracellular membrane barriers by TAT-modified gold nanoparticles.ACS

which has been previously observed in rats subjected tostress

therapy in the near-infrared region by using gold nanorods. JAm

correspond to the Protein-coated Au NRs exposure to A549 cells for12 h, 24 h and 48 h respectively. Metabolite key to the numbers areshown in Fig. 1 and Table S1.

ROS attack are very different between 16HBE and A549 cells,which

PC phosphocholine

for selective tumor cell death [18]. In order to furtherinvestigate

FX11 3-dihydroxy⑹-methyl⑺-(phenylmethyl)⑷-

untreated 16HBE. The glycolytic enzyme, lactate dehydrogenaseA

statistical significance based on the discrimination significanceat the level of p<0.05.

isoleucine, valine and glycine. Interestingly, however, 48 hpost

4.1. Modification of intracellular environment

(LDHA), which catalyzes the conversion between pyruvat我没有晓得磷甲酸钠氯化钠e and

NAD nicotinamide adenine dinucleotide

significant tumor cells cell death from 12 h post exposureonwards

subsequent therapies [6,7]. Recently, some anti-tumor agentssuch

results showing that the level of ROS in A549 cells increasedafter

grade) were obtained from Guoyao Chemical Co. Ltd. (Shanghai,China). Sodium

to protein-coated Au NRs for 12 h and 24 h implies thatproteincoated

27 Uracil e e e e e 0.67

centrifugation for 10 min at 16,099 g and 4 C. The remaining solidresidues were

described previously for selective samples [29,30]. In COSY andTOCSY experiments,

cells undergo to become tumor cells is a change in cellular

higher levels of lactate observed in untreated A549 cells thanin

[9] Yu CX, Irudayaraj J. Multiplex biosensor using gold nanorods.Anal Chem

gold nanorods protein-coated Au NRs

in the inhibition of tumor cell proliferation.

5. Conclusions

are similar to those of 16HBE cells after exposure for 24 h.Proteincoated

gold nanorod su***ce chemistry on cellublar response. ACS Nano2011;5:2870e9.

in the spectra is vertically expanded 8 times compared with theregion of d 0.5e4.7. Key: 1. isoleucine; 2. leucine; 3. valine; ctate; 5. threonine 6. alanine; 7. lysine; 8. acetate; 9.

implications for health. J Nutr 2004;134:489e92.

48 transients were collected into 2 K data points for each of 256increments with the

[36] Goldman RD, Kaplan NO, Hall TC. Lactic dehydrogenase in humanneoplastic


Nano 2011;5:5195e201.

inhibitors of nicotinamide adenine dinucleotide (NAD)biosynthesis

33 Histidine e e e e e 0.67

cell death in protein-coated Au NRs treated A549 cells is

Med 2001;30:1191e212.

12 Aspartate 0.72 0.74 0.74 e e e

maintain oxidative balance of the cells. However, A549 cellsseem

lactate dehydrogenase a induces oxidative stress an inhibits tumorprogression.

[38] Xie H, Valera VA, Merino MJ, Amato AM, Signoretti S, LinehanWM. Et al. LDHA


collected into 32 k data points for each spectrum with a spectralwidth of 20 ppm. All

For the purpose of NMR signal assignments, a range of 2D NMRspectra including

[51] Leist M, Single B, Naumann H, Fava E, Simon B, Kuhnle S, etal. Nitric oxide

Exp Cell Res 1999;249:396e403.

Commun 1999;258:215e21.

cells is approximately four times higher than that ofuntreated

In order to obtain protein-coated Au NRs-induced metabolicalte其真fromrations,

cancer cell death [2,3]. Radiation and some of theconventional

and further confirmed bya series of 2DNMRspectra, includingCOSY,

levels of GSSG, choline and certain amino acids includingglutamine,

1 Isoleucine e 0.57 0.81 0.63 e e

7124 L. Zhang et al. / Biomaterials 34 (2013) 7117e7126

(phenylmethyl)⑷-propylnaphthalene⑴-carboxylic acid (FX11),

c Graduate University of the Chinese Academy of Sciences, Beijing, China


extracts of 16HBE and A549 cells after 12 h, 24 h and 48 h of

[43] Marengo B, De Ciucis C, Verzola D, Pistoia V, Raffaghello L,Patriarca S, et al.

free CTAB molecules. Protein-coated Au NRs (50 mM) were sonicatedon wet ice for

(Fig.4AandB). The basic level ofATP in untreatedA549cells issimilar

(TOCSY) experiment (TPPI) with the

protein-coated Au NRs from lysosome to mitochondria in A549

research have been performed on the cytotoxicity,biocompatibility

capability of 16HBE cells is stronger than that of the tumorcells,

normal and tumor cells coincides with the relocation of the

citrate, membrane metabolites including choline,phosphocholine

[4] Ferlini C, Dwere’Amelio R, Scambia G. Apoptosis induced by ionizingradiation. The

[8] Le A, Cooper CR, Gouw AM, Dinavahi R, Maitra A, Deck LM, et al.Inhibition of

[50] Leist M, Single B, Naumann H, Fava E, Simon B, Kuhnle S, etal. Inhibition of

(d 0.0). The spectral region d 0.5e9.5 was automatically integratedinto regions with

Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute ofPhysics and

glutamine and branched chain amino acids are observed in the

by damaging cellular DNA [4,5], however this often leads to

been shown that Au NRs are translocated from the lysosome to themitochondria in A549 cells but not in normal 16HBE cell lines.However, the molecular information during this cellulartranslocation remains largely undetermined. Here, we have used ametabonomic technique to comparatively analyze the timedependentmetabolic changes in Au NRs-induced A549 and 16HBE. We found thatAu NRs exposure caused a disruption in the intracellularenvironment of both A549 and 16HBE cells, which metabolicallymanifested in the reduction of lactate levels in both cell lines.In addition, Au NRs induced oxidative stress in both cells lines.However, the 16HBE cells are more able to offset the oxidativestress than the A549 cells; this is because de novo GSH synthesisis triggered in Au NRs treated 16HBE cells but not in A549 cells,and the conversion of GSH to GSSG is more profound in 16HBE cellscompared to A549 cells.

are annotated in the OPLS-DA coefficient plots (Figs. 2 and 3)and

cetyltrimethylammonim bromide (protein-coated Au NRs) and

Cell death

[18] Wang L, Liu Y, Li W, Jiang X, Ji Y, Wu X, et al. Selectivetargeting of gold

reactive oxygen species, may lead to over accumulation ROS.

extractswere obtained. Metabolites present in A549 and 16HBEcells

drug combinations. Clin Cancer Res 2005;11:3075e83.

investigated in terms of cellular uptake and intracellulartransport

urinary metabonomics with effective pH control, chemical shiftconsistency

Gold nanorods

insulin resistance statuses. J Proteome Res 2009;8:5188e95.

metabolic responses of living systems to pathophysiologicalwere stimulivia


Appendix A. Supplementary data

Chem Soc 2006;128:2115e20.

values of R2 and Q2, which represent the quality of fit andpredictability

3 Valine e 0.55 0.80 0.62 e e

[40] Roth E, Oehler R, Manhart N, Exner R, Wessner B, Strasser E,et al. Regulative

of nicotinamide phosphoribosyltransferase, represents a novelmechanism for

treated A549 cells, therefore, it was anticipated to that celldeath

in this study could be understood in terms of cellular responsesto

4 Lactate 0.66 0.73 e 0.78 0.72 e

a supportive evidence for our previous finding that theproteincoated

Fig. 5. A summary of various metabolic responses of A549 and 16HBEcells to protein-coated Au NRs exposure. Metabolites in red or bluerepresent a significant increase or decrease

Supplementary data related to this article can be found onlineat

significant reduction of ratio of GSH/GSSG is observed innormal

TOCSY 1He1H total correlation spectroscopy

tubes for NMR analysis.

comparatively examined the intracellular uptake andtranslocation

This is indeed the case, in the current investigation, we observeda

environmental QSAR Aata. Part I: a basic framework founded onprincipal

frequency of 20 Hz for 磷甲酸钠1.5 min followed by sonification on wet icefor 99 cycles with

correlation coefficients of the metabolites responsible for thedifferentiation.

Ultrapure Milli-Q water (18 MU cm, Millipore-Q) was used for allsolution preparations.

expression could reduce cellular transformation and markedly

[46] Joza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CY, et al.Essential role of

3-trimethlysilyl [2,2,3,3-D4] propionate (TSP-d4) and D2O (99.9% inD) were

(R) models. J Chemometrics 2008;22:594e600.

14 Creatine e e 0.75 e e e

further extracted twice using the same procedure. The comb膦甲酸钠氯化钠几钱inedsupernatants from

Natwick, USA) environment using an in-house developed script. Thecolor of correlation

7 Lysine e e 0.83 0.74 0.73 0.68

propylnaphthalene⑴-carboxylic acid


[33] Tian Y, Zhang L, Wang Y, Tang H. Age-related topographicalmetabolic signatures

(0.1 M, K2HPO4/NaH2PO4 and pD 7.4) [28] containing 50% D2O and0.005% TSP-d4 as

[34] Warburg O. On the origin of cancer cells. Science1956;123:309e14.

In this work, we demonstrate an application of the NMR-based

cells whereas for A549 cells, protein-coated Au NRs leak out ofthe

Fourier transformed into a 4 2 k matrix with appropriateapodization functions.

link between glycolysis, mitochondria physiology, and tumormaintenance.

incubated with complete medium that contained protein-coatedAu

Our results showed time-dependent and cell-specific metabolic

responses. Curr Opin Neurobiol 1999;9:128e34.

numbers are shown in Fig. 1 and Table S1.

29. Inosine⑸0-monophosphate (50-IMP); 30. glutathione (oxidized);31. fumarate; 32. tyrosine; 33. histidine; 34. phenylalanine; 35.tryptophan; 36. uridine diphosphate (UDP)-Nacetyl

TOCSY, HSQC and HMBC, to give a total of 48 metabolites (TableS1).

matrices. J Proteome Res 2011;10:614e23.

inverse detection cryogenic probe (Bruker, Biospin, Germany). Forall the samples, a

b CAS Key Laboratory for Biomedical Effects of Nanomaterials andNanosafety, National Center for Nanoscience and Technology of Chinaand Institute of

[5] Kaufmann SH, Earnshaw WC. Induction of apoptosis by cancerchemotherapy.

Q2 ¼ 0.87

protein-coated Au NRs treated A549 cells after 24 h posttreatment

Little work, however, has emerged on the understanding of the

aerobic glycolysis, which results in counteracting the“Warburg

into GSSG during the process of scavenging free radicals.These

[3] Hasmann M, Schemainda I. FK866, A highly specificnoncompetitive inhibitor

formateiii approach (p < 0.05) [32] and permutation tests (200permutations). The

Sodium chloride, methanol, K2HPO4$3H2O and NaH2PO4$2H2O (all inanalytical

clear separations between the profiles of the Au NRs treatedcells

orthogonal projection to latent structures discriminant analysis(OPLS-DA), was

2.3. Extraction of intracellular metabolites

The severe oxidative stress induces damage to mitochondria in A549cells, leading to cell death, which is evident in the markedreduction in the levels of nucleosides and nucleotides.Furthermore, significantly elevated levels of amino acids arelikely due to stress hormones being produced in Au NRs treatedcells.

glucosamine; 37. guanosine; 38. nicotinamide adenine dinucleotide(NAD); 39. formate; 40. nicotinamide; 41. succinate; 42.trimethylamine; 43. hypoxanthine; 44. adenosine


fate by apoptosis or necrosis. Cancer Res 1997;57:1835e40.

with recycle delay of 2 s, t1 of 3 ms and the mixing time, tm, of100 ms. Typically,

[14] Xu L, Liu Y, Chen Z, Li W, Wang L, Wu X, et al.Su***ce-engineered gold

and those of non-treated cells at all time points. Protein-coatedAu闭于B

[19] Krpetic Z, Saleemi S, Prior IA, See V, Qureshi R, Brust M.Negotiation of

acids occurs at 24 h and lasts until 48 h post exposure. It iswell

from Alfa Chemical (UK). Fetal bovine serum (FBS) was obtained fromGibco.

Research Program of China (2012CB, 2011CB,

monophosphate (50-IMP), and nicotinamide adenine dinucleotide

roles in maintaining optimal intracellular redox environmentsfor

47 Cysteine 0.84 0.83 0.81 e e e

Cetyltrimethylammonim bromide coated gold nanorods (Au NRs) has apotential to become anti-cancer

response of the A549 tumor cells, compared to the normal16HBE

(FX11) to inhibit LDHA, resulting in mitochondrial oxidativestress

[10] Tong L, Zhao Y, Huff TB, Hansen MN, Wei A, Cheng JX. Goldnanorods mediate

by our previous investigation [18] and others showing that

of escherichia coli to heat stress. J Proteome Res2012;11:2559e66.

The PCA scores plots (Fig. S3) of the 1H NMR spectral datashow

therapy targeting on pathway-specific metabolism have beenproposed


view to understand the role of pathway-specific metabolicchanges

to those of untreated 16HBE cells. However, the levels of ATP

acid (AA), hydroxyethyl piperazine ethanesulfonic acid (HEPES) werepurchased

journal homepage:

A549 cells. Protein-coated Au NRs exposure results in a moresignificant

(TEM) (Fig. S1A). The protein-coatedAu NRs employed in this studyhad a longitudinal

explained variations, and Q2, indicating the model predictabilityrelated to its statistical

the ratio of cellular GSH to GSSG or ATP per milligram (mg) proteinwithin cells.

Fig. 2. O-PLS-DA scores and coefficient-coded loadings plots forthe models, discriminating between the untreated 16HBE cells (blacksquares) and protein-coated Au NRs treated

whereas 16HBE cells were not affected even at 48 h postexposure

with a concurrent increase in the levels of uracil and

results in a significant reduction in the levels of lactate inboth

Extensive literature has shown the correlation betweenmitochondria

China (,,). We wou比拟看sodium formateld also like to

summarized in Table 1.

Aside from these anti-tumor agents and drugs, nanoparticles

80 C refrigerator before extracting metabolites from cells. Thecontrol cells

short, human alveolar epithelial carcinoma (A549) cells and normalhuman bronchial

(PC), glycerophosphocholine (GPC), and a number ofnucleosides

and 160 ppm for 13C in HSQC (220 ppm in HMBC) experiments. The datawere

various physiochemical properties due to their differentsizes,

the approach of metabonomics in screening anti-cancernanodrugs

in A549 cells (Fig. 5). Considerable disruption of purine andpyridine

harmful for normal cells. More importantly, our workhighlighted

0142⑼612/$ e see front matter 2013 Elsevier Ltd. All rightsreserved.

specific metabolites according to the published results[24,25,33],

Q2 ¼ 0.84

is not the case in 16HBE cells (Fig. 4C and D). Themitochondrialrelated

properties. Among them, gold nanorod has emerged as one ofthe

600 mL of an ice-cold methanol/H2O (2:1 v/v) extraction solution.To smash the cells

S0053); the protein concentration was also determined using a BCAassay (Pierce).

Q2 ¼ 0.80

[45] Giorgi C, Baldassari F, Bononi A, Bonora M, De Marchi E,Marchi S, et al.

24 h

(Hyclone), 100 mg/mL streptomycin (Hyclone), and 2 mM L-glutamine(Hyclone). For

metabonomic analysis reveals progression axes for glucoseintolerance and

16HBE cells showed additional elevation in the levels ofleucine,

remain unchanged after protein-coated Au NRs exposure, whichis

the effects of toxins and stresses [23e25], pathogenesis of

16HBE A549


for tumor initiation, and therefore can be used as a targetfor

candidates and elucidating molecular mechanisms of their

mitochondrial ATP generation by nitric oxide switches apoptosis tonecrosis.

purchased from Cambridge Iso听听H2O2tope Laboratories (Miami, USA). Sodiumborohydride

40 Nicotinamide e e e e e 0.67

are manifested in two aspects: one is de novo GSH synthesisand

Article history:

metabolic responses of a living system during exposure toexternal

imperfect water suppression. Each integral region was normalized tothe total sum

1H NMR spectra of cell extracts were recorded at 298 K on a BrukerAvance III

the levels of ATP, GSH and GSSG are determined using an assay kit(Beyotime,

of all integrals for each spectrum to compensate for the overallconcentration differences

jrj is less than the cutoff value.

exposure [18].

16HBE cells is approximately six times of that in A549 cells anda

cells. It is of particular interest that the changes of nucleotidesin

4.3. Time dependent alterations of amino acids

Au NRs in complete medium for 12 h, 24 h, and 48 h. The cells werethen collected in

tail. It has been reported that such compounds can cause the

1He1H COSY and TOCSY, 1He13C HSQC and HMBC were acquired andprocessed as

a b s t r a c t

LDHA biosynthesis and lactate dehydrogenase A

hypoxia. Such high rate of glycolysis observed in tumorcells,

the mitochondrial apoptosis-inducing factor in programmed celldeath. Nature

Contents lists available at SciVerse ScienceDirect

2009CB) and the National Natural Science Foundation of

Au NRs treatment for 12 h induces marked elevation in the

unchanged levels of glycine and cysteine in the tumor cells.More


18 Choline 0.72 e 0.71 e e e

completely, the cell were subjected to freezeethaw cycles threetimes with rapid

[17] Leroueil PR, Hong S, Mecke A, Baker JR, Orr BG, Banaszak HMM.Nanoparticle

[31] Eriksson L, Andersson PL, Johansson E, Tysklind M. Megavariateanalysis of

belonging to delocalize您看mosd lipophilic cations and has a smartstructure

and vigorous manual mixing between each cycle. The samples werethen extracted

inhibition, a therapeutic strategy for treatment of hereditaryleiomyomatosis

A549 cells after 24 h of exposure to protein-coated Au NRs, witha

hypoxanthine in A549 cells after 48 h of protein-coated AuNRs

interaction with biological membranes: does nanotechnology presenta Janus


exposures resulted in elevated levels of an even larger range ofthe

levels of lactate, threonine, glutathione, aspartate, 50-IMP, (MOS)anduridine

R2X ¼ 0.31

underlying molecular mechanisms of selective inhibition the

[23] Ebbels TM, Keun HC, Beckonert OP, Bollard ME, Lindon JC,Holmes E, et al.

findings provided a comprehensive insight into the mechanismsof

The CTAB bilayer-capped gold nanorods used here was preparedaccording to

We acknowledge financial support from the National Basic

Fig. 4. Effects of protein-coated Au NRs exposure on intracellularredox states as indicated by the ratio of GSH to GSSG (A, B), andthe intracellular levels of ATP (C, D). Statistical

48 h

purchased from the National Research Centre for CRMs, China. Humanalveolar

All of the frozen samples (with wet weight of 80 mg) werehomogenized in

(e.g., clustering and outliers) [31]. A supervised multivariatedata analytical tool,


16HBE cells (red dots). The models are constructed from NMR spectraof aqueous cell extracts obtained at time points of 12, 24, and 48h. These models are cross-validated with

Selective metabolic effects of gold nanorods on normal andcancer

glutamate, cysteine and taurine; there was also a reduction inthe

its permeability [13,17]. Therefore, protein-coated Au NRs maybe

Mitochondrial Ca2þ and apoptosis. Cell Calcium 2012;52:36e43.

cells were not markedly affected as ROS being scavenged byGSH

cells were obtained from the American Type Culture Collection(ATCC) and Cell Bank

Metabonomics is a holistic systems approach capable ofcapturing

between classes, with a 教会sodium“hot” color (e.g. red) indicating ahigher

4.2. Oxidative stress and cell death

transactivation of LDH-A: implications for tumor metabolism andgrowth.

whereas the MLEV⑴7 was employed as the spin-lock scheme in thephase sensitive

All the cell cultures were maintained according to previousprotocol [18]. In

* Corresponding author. Key Laboratory of Magnetic Resonance inBiological

when both are exposed to protein-coated Au NRs. Another

proteins, for rapid cell proliferation [1]. Many strategies ofcancer

[22] Tang H, Wang Y. Metabonomics-a revolution in progress. ProgBiochem Biophys

NRs induced metabolic changes were further evaluated byconstructing

mediated cellular uptake of Au nanorods. Biomaterials2010;31:7606e19.

GSSG oxidized glutathione

the brain. J Neuroendocrinol 2001;13:113e28.

All the 1H NMR spectra were manually corrected for phase andbaseline distortions

temporal metabolic data: the consortium on metabonomictoxicology

further performed to analyze time-dependent cellular responsesto

effect闭于B” in cancer cells [3,8].

8 Acetate e e e e e 0.73

NRs for 12 h, 24 h, and 48 h. The cells were collected andaqueous

exposure induces a disruption in purine and pyridinemetabolism

involved in the process of insertion, fusion, and permeationin

membrane structures and easily enter into cells to targetorganelles

inhibits execution of apoptosis at two distinct ATP-dependent stepsupstream

with a positively charged polar head and a nonpolarhydrophobic

24 h of exposure to protein-coated Au NRs while those in16HBE

mixing time of 100 ms. 1He13C heteronuclear single quantumcorrelation (HSQC),

of glutamine, glycine and cysteine in protein-coated Au NRstreated

importance than a “cold” color (e.g. blue). In this study, acorrelation coefficient of

the proper function of cellular proteins. Obvious elevations inthe

In the current investigation, we observe alterations in thelevels

(NaBH4), hydrogen tetrachloroaurate (III) trihydrate (HAuCl4.3H2O),cetyltrimethylammonium

the responses of 16HBE cells where the rising levels of theseamino

L. Zhang et al. / Biomaterials 34 (2013) 7117e7126 7125

with 0.02% EDTA, finishing the digestion with complete medium. Cellsuspension

2.5. NMR data processing and multivariate data analysis

neurons and HBE cells [49e51]. Consistently, our measurements

[2] Shim H, Dolde C, Lewis BC, Wu CS, Dang G, Jungmann RA, et al.c-Myc

(TEM) (Fig. S1). A549 (n ¼ 10) and 16HBE (n ¼ 10) cells were

density of 5 106 cells/dish onto a 15 cm diameter Petri dish andeach group had ten

quaternary ammonium cations adsorbed onto the membrane,

cancer therapy [37,38]. Anne et al. used a drug-like smallmolecule

coefficient indicates the importance of the metabolite contributingto the differentiation

[53] Francis DD, Meaney MJ. Maternal care and the developmentobtained ofstress

29 50-IMP 0.63 0.71 0.61 0.57 e 0.89

Centre for Magnetic Resonance, Wuhan Institute of Physics andMathematics, Chinese Academy of Sciences (CAS), Wuhan,China

NMR nuclear magnetic resonance

Exp Cell Res 2000;256:42e9.

publication [18] and characterized by transmission electronmicroscopy

[42] Schafer FQ, Buettner GR. Redox environment of the cell asviewed through the

stress. The stress induced time-dependent response betweenthe

centrifugation for 10 min at 13,416 g and 4 C, lysis supernatantwas collected and

higher order long-propagation-length su***ce plasmon polaritonmodes in

su***ce chemistry on biological systems. Annu Rev Biomed Eng2012;14:1e16.

4. Discussion

continuous incubation with protein-coated Au NRs by employinga

further cross-validated with a CV-ANOVA approach (p <0.05)

30 Glutathione (oxidized) 0.73 0.72 e e e e

[54] Wang Y, Holmes E, Tang H, Lindon JC, Sprenger N, Turini ME, etal. Experimental

HSQC 1He13C heteronuclear single quantum correlation

to inhibit tumor cell proliferation and ultimately result in

cell-specific toxicity of protein-coated Au NRs at a molecularlevel.

lysosome and attacks mitochondria between 12 h and 24 h post

subsequently applied to the analysis of 1H NMR spectral data scaledto unit variance.

would be observed in protein-coated Au NRs exposed A549cells.

levels of precursors of GSH imply that de novo GSH synthesisis

metabonomic technique to evaluate the potential of nano-drugs

a Key Laboratory of Magnetic Resonance in Biological Systems, StateKey Laboratory of Magnetic Resonance and Atomic and MolecularPhysics, Wuhan

PCA principal component analysis

(ROS) such as superoxide anion (O$

delay proliferation of tumor cells, suggesting that LDHA isimportant

38 NAD e e e e 0.63 0.88

27; fax: þ86 27.

component analysis (PCA), partial least squares (PLS), andstatistical molecular

甲酸消费交换 nanorods: promising DNA vaccine adjuvant for HIV⑴ treatment. NanoLett

[49] Eguchi Y, Shimizu S, Tsujimoto Y. Intracellular ATP levelsdetermine cell death

from GSH to GSSG at 12 h post exposure in order to

three amino acids are precursors of GSH [40,41] and playimportant

associated with cells treated with protein-coated Au NRs

those of 16HBE cells exposed for 48 h, with the exception ofthe

(A549) cells compared to normal bronchial epithelial (16HBE)cells

L. Zhang et al. / Biomaterials 34 (2013) 7117e7126 7121

2. Materials and methods


[20] Paul A, Solis D, Bao K, Chang WS, Nauert S, Vigderman L, etal. Identification of

Biospin, Germany). The region d 4.7e5.2 was removed to avoid theeffects of

[1] Weinberg F, Chandel NS. Mitochondrial metabolism and cancer.Ann N Y Acad

(A549) and normal bronchial epithelial cells (16HBE) exposed to AuNRs have been characterized. It has

看看甲酸钠取两氯甲烷 ** Corresponding author. Tel.: þ86 10; fax: þ86.

cells, hence is likely related to the damage of mitochondriaand

which might be vital for cancer growth. Secondly,protein-coated

2 Leucine e 0.56 0.81 0.62 e e

the elevated levels of nucleosides, inosine and adenosinewere

free induction decays (FIDs) were multiplied by an exponentialfunction with a 1 Hz

response of tumor cells and normal cells to protein-coated AuNRs

Au NRs for 12 h, 24 h, and 48 h. Then the cells were washed withcold PBS

2) and alkoxyl (RO) radicals [39]. However, the responses to

exposure. First of all, protein-coated Au NRs exposuremodulated

peroxide. FEBS Lett 2001;500:114e8.

Nuclear magnetic resonance (NMR)

[12] Huang X, El-Sayed IH, Qian W, El-Sayed MA. Cancer cell imagingand photothermal

24 h

Au NRs induced severe oxidative stress and subsequent celldeath

from GSH during anti-oxidative process [42]. Generally, the ratioof

GSH to GSSG can serve as a good indicator of the cellularredox

subsequent cell death.

16HBE cells remained the same after the treatment (Fig. 4C andD).

26 Adenosine e 0.69 e 0.78 0.71 0.86

trigger redox activation of protein kinase C (PKCd) and,through

Cancer Cell 2006;9:425e34.

of the protein-coated Au NRs, which has suggested thatdeposition

and the levels of GSH to GSSG were subsequently conducted by

[35] Gatenby RA, Gillies RJ. Why do cancers have high aerobicglycolysis? Nat Rev

SPR su***ce plasma resonance

Reagents Institute. Standard stock solutions of bismuth and gold(1000 mg/L) were

untreated A549 cells. The ratio of GSH/GSSG is also reducedin

cells and their application in anticancer drug screening

exposure induces free radicals. This phenomenon has beenreported

A549 cells after 12 h of protein-coated Au NRs exposure;however,

Secondly, that the inability for A549 cells to effectively scavengefor

from each dish was collected and centrifuged at 447 g for 5 min.After washing with

[11] Wijaya A, Schaffer SB, Pallares IG, Hamad-Schifferli K.Selective release o看看formateIII)f

diphosphate (UDP)-N-acetyl glucosamine in 16HBE cells (Fig. 2

normal integrity of the mitochondria allows manypro-apoptosis


the levels are diminished at later time points. This is in contrastto