Research Article Cytotoxicity of Biologically Synthesized ...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 535796 10 pageshttpdxdoiorg1011552013535796

Research ArticleCytotoxicity of Biologically Synthesized Silver Nanoparticles inMDA-MB-231 Human Breast Cancer Cells

Sangiliyandi Gurunathan1 Jae Woong Han1 Vasuki Eppakayala1

Muniyandi Jeyaraj2 and Jin-Hoi Kim1

1 Department of Animal Biotechnology Konkuk University 1 Hwayang-dong Gwangjin-gu Seoul 143-701 Republic of Korea2 GS Centre for Life Sciences Sundarapuram Coimbatore Tamil Nadu 641024 India

Correspondence should be addressed to Sangiliyandi Gurunathan gsangiliyandiyahoocomand Jin-Hoi Kim jhkim541konkukackr

Received 4 January 2013 Revised 5 June 2013 Accepted 6 June 2013

Academic Editor B E Kemp

Copyright copy 2013 Sangiliyandi Gurunathan et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Silver nanoparticles (AgNPs) have been used as an antimicrobial and disinfectant agents However there is limited informationabout antitumor potential Therefore this study focused on determining cytotoxic effects of AgNPs on MDA-MB-231 breastcancer cells and its mechanism of cell death Herein we developed a green method for synthesis of AgNPs using culturesupernatant of Bacillus funiculus and synthesized AgNPs were characterized by various analytical techniques such as UV-visiblespectrophotometer particle size analyzer and transmission electron microscopy (TEM) The toxicity was evaluated using cellviability metabolic activity and oxidative stress MDA-MB-231 breast cancer cells were treated with various concentrations ofAgNPs (5 to 25120583gmL) for 24 h We found that AgNPs inhibited the growth in a dose-dependent manner usingMTT assay AgNPsshowed dose-dependent cytotoxicity against MDA-MB-231 cells through activation of the lactate dehydrogenase (LDH) caspase-3 reactive oxygen species (ROS) generation eventually leading to induction of apoptosis which was further confirmed throughresulting nuclear fragmentation The present results showed that AgNPs might be a potential alternative agent for human breastcancer therapy

1 Introduction

Breast cancer is the second most common cause of cancerdeath in women [1 2] Many cancers initially respondto chemotherapy and later they develop resistance [3ndash5]Currently available chemopreventives and chemotherapeuticagents cause undesirable side effects [6 7] therefore develop-ing a biocompatible and cost effective method of treatmentfor cancer is indispensable The development of nanotech-nology has been a boon to mankind as its significance pavedthe way for several applications in therapeutics [8] catalysis[9] microelectronics biosensing devices [10] air and waterpurifiers paints [11] and so forth Recently AgNPs havegained much interest among the emerging nanoproducts inthe field of nanomedicine due to their unique propertiesand obvious therapeutic potential in treating a variety of

diseases including retinal neovascularization [12ndash15] Thenanoparticles can be synthesized by physical chemical andbiological methods The physical methods are initially usedto give a low yield [16] Chemical methods use variouschemical agents to reduce metallic ions to nanoparticlesThis comprises certain drawbacks as there will be use oftoxic chemicals and generation of hazardous byproducts[16] In the medical aspects applications of nanoparticlesincreased tremendously only when the biological approachfor nanoparticle synthesis came into focus The variousresources available naturally for green synthesis of nanopar-ticles are plants plant products bacteria fungi algae yeastand viruses [17]Though there is a large platform for the greensynthesis of nanoparticles themost commonly preferred wayis the bacterial synthesis as they are easy to handle andgenetic manipulation is also possible [13 18 19]

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The major implication of this biological approach issimple and less time consuming In addition to this the highyield low toxicity low cost and its biocompatibility add toits value [20] An additional advantage is that the size ofthe nanoparticles synthesized can also be easily controlled byvarious controlling parameters like pH temperature [13] andthe use of stabilizers to prevent aggregation is not required asthe proteins in the system act as stabilizers [14] Nanoparticleswith smaller radius of curvature have higher catalytic activityhence angular shapes are preferable due to their smaller radiiof curvature compared to spherical particles of the same vol-ume Several research groups have successfully demonstratedthe superior antimicrobial efficacy of AgNPs either as theyare or in composites with polymer [21ndash25] In addition ourgroup and another research group demonstrated that AgNPshave potential cytotoxicity against cancer [15 26 27] andantiangiogenic property in microvascular endothelial cells[28 29]

Recently Rani et al [27] reported that AgNPs inhibitproliferation of human glioblastoma cells Franco-Molina etal [30] evaluated the effects of colloidal silver on MCF7human breast cancer cells Sanpui et al [31] demonstratedthat AgNPs not only disrupting normal cellular functionand but also affecting the membrane integrity inducedvarious apoptotic signaling genes of mammalian cells leadingto programmed cell death Hsin et al [32] reported thatAgNPs induced apoptosis in NIH3T3 cells by heighteningthe ROS generation and activated JNK pathway leadingto mitochondria-dependent apoptosis Recent studies haveshown that AgNPs accumulation in the liver could inducecytotoxicity via oxidative cell damage [32ndash34] Reactive oxy-gen species (ROS) are continually generated and eliminatedin biological systems They play an important role in avariety of normal biochemical functions and abnormalityin their function results in pathological processes Excessiveproduction of ROS in the cell is known to induce apoptosis[35 36] ROS generation has been shown to play an importantrole in apoptosis induced by treatment with AgNPs [27 3738]

A number of studies have reported that AgNPs mayinduce cytotoxicity in phagocytosing cells such as not onlymouse peritoneal macrophages but also human monocytes[38ndash40] Further studies suggested that the cytotoxic effectswere induced by reactive oxygen species (ROS) resultingin cellular apoptosis at least low concentrations and shortincubation times [37 41ndash43] The production of ROS hasalso been implicated in DNA damage caused by AgNPswhich was reported in a number of in vitro studies [2738 44] Caspase-3 is one of the key molecules in apoptosisand its activation is often considered as the point of noreturn in apoptosis [45] Activation of caspase-3 results inthe cleavage of (inhibitor of caspase-activated DNAse) ICADand translocation of (caspase activated DNAse) CAD to thenucleus ultimately resulting in DNA fragmentation Themost prominent event in the early stages of apoptosis isinternucleosomal DNA cleavage by endonuclease activities[46] Previous studies suggested that AgNPs treated cancercell and noncancer cells revealed enhanced caspase-3 activityand formation of significant DNA laddering [14 15 47]

Currently a variety of cytotoxic agents have been used inthe treatment of breast cancer such as doxorubicin cisplatinand bleomycin [30 48] Although usage of doxorubicincisplatin and bleomycin provides beneficial effect but theefficacy and demerits are uncertain [30] Therefore it isnecessary to find novel therapeutic agents against cancerwhich are biocompatible and cost effective Therefore thisstudy was designed to synthesize AgNPs using biologicalsystem and to evaluate potential toxicity and the generalmechanism of biologically synthesized AgNPs in MDA-MB-231 human breast cancer cells

2 Materials and Methods

21 Materials Penicillin-streptomycin solution trypsin-ED-TA solution RPMI-1640 medium Dulbeccorsquos modifiedEaglersquos medium (DMEMF-12) and 1 antibiotic-anti-mycotic solution were obtained from Life TechnologiesGIBCOGrand IslandNYUSA Fetal bovine serum (FBS) invitro toxicology assay kit was purchased from Sigma-Aldrich(St Louis MO USA)

22 Synthesis of AgNPs Luria-Bertani broths were preparedand used as described earlier [13] B funiculus cultures wereobtained from the GS Center for Life Sciences Coimbat-ore India The novel bacteria were isolated from industrialwastewater and sequence has been submitted at GenBankThe strain was grown aerobically at 37∘C in LB mediumSynthesis of AgNPs was carried out according to the methoddescribed previously [13] Briefly bacteria were grown in a500mL Erlenmeyer flask that contained LB broth withoutNaCl or nitrate medium The flasks were incubated for 21 hin a shaker set at 120 rpm and 37∘C After the incubationperiod the culture was centrifuged at 10000 rpmmin andthe supernatant used for the synthesis of AgNPs Three vialsthe first containing AgNO

3(Sigma USA 999 pure) with-

out the supernatant the second containing only the culturesupernatant and the third containing the supernatant andAgNO

3solution at a concentration of 1mM were incubated

for 60min at 40∘C The extracellular synthesis of AgNPs wasmonitored by visual inspection of the test tubes for a changein the color of the culture medium from a clear light yellowto brown and by measurement of the peak exhibited byAgNPs in the UV-vis spectra the synthesis of nanoparticleswas confirmed

23 Cell Culture MDA-MB-231 human breast cancer cellswere kindly provided by Professor Ssang-Goo Departmentof Animal Biotechnology Konkuk University and weremaintained in Dulbeccorsquos modified Eaglersquos medium (DMEM)supplemented with 10 fetal bovine serum (FBS) and 1antibiotic-antimycotic solution Cells were grown to conflu-ence at 37∘C and 5 CO

2atmosphere All experiments were

performed in 6-well plates unless stated otherwise Cellswere seeded onto the plates at a density of 1 times 106 cells perwell and incubated for 24 h prior to the experiments Thecells were washed with (phosphate buffered saline pH 74)

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PBS and incubated in fresh medium containing differentconcentrations of AgNPs dissolved in water

24 Characterization of AgNPs Characterization of synthe-sized AgNPs particles was carried out according to themethods described previously [13 49] The AgNPs were pri-marily characterized by UV-visible spectroscopy Ultraviolet-visible (UV-vis) spectra were obtained using aWPA (BiowaveII) Biochrom Cambridge UK The particle size of dis-persions was measured by Zetasizer Nano ZS90 (MalvernInstruments Ltd UK) Transmission electron microscopy(TEM JEM-1200EX) was used to determine the size andmorphology of AgNP

25 Cell Viability Assay The cell viability assay was mea-sured using the 3-(45-dimethylthiazol-2-yl)-25-diphenyl-tetrazolium bromide dye reduction assay which was per-formed to determine the cytotoxic effect of the AgNPs atvarious concentrations Briefly the MDA-MB-231 cells wereplated onto 96-well flat bottom culture plates with variousconcentrations of AgNPs All cultures were incubated for 24 hat 37∘C in a humidified incubator After 24 h of incubation(37∘C 5 CO

2in a humid atmosphere) 10120583L of MTT

(5mgmL in PBS) was added to each well and the plate wasincubated for a further 4 h at 37∘C The resulting formazanwas dissolved in 100120583L of DMSO with gentle shaking at37∘C and absorbance wasmeasured at 595 nmwith an ELISAreader (Spectra MAX Molecular Devices USA) The resultswere given as the mean of three independent experimentsConcentrations of AgNPs showing a 50 reduction in cellviability (ie IC50 values) were then calculated

26 Membrane Integrity Cell membrane integrity of MDA-MB-231 cells was evaluated by determining the activity oflactate dehydrogenase (LDH) leaking out of the cell accord-ing to the manufacturerrsquos instructions (in vitro toxicologyassay kit TOX7 Sigma USA) The LDH assay is based onthe release of the cytosolic enzyme LDH from cells withdamaged cellularmembranesThus in cell culture the courseof AgNPs induced cytotoxicity was followed quantitatively bymeasuring the activity of LDH in the supernatant Brieflycells were exposed to various concentrations of AgNPs for24 h then 100120583L per well of each cell-free supernatant wastransferred in triplicates into wells in a 96-well and 100 120583Lof LDH assay reaction mixture was added to each well After3-hour incubation under standard conditions the opticaldensity of the color generatedwas determined at awavelengthof 490 nm using a Microplate Reader

27 Determination of ROS Intracellular reactive oxygenspecies (ROS) were measured based on the intracellu-lar peroxide-dependent oxidation of 2101584071015840-dichlorodihydro-fluorescein diacetate (DCFH-DA Molecular Probes USA)to form the fluorescent compound 2101584071015840-dichlorofluorescein(DCF) as previously described [50] Cells were seededonto 24-well plates at a density of 5 times 104 cells per welland cultured for 24 h After washing twice with PBS freshmedium containing 87 120583g AgNPs or 1mMH

2O2was added

and the cells were incubated for 24 h For control the cellswere added 20 120583M of DCFH-DA and incubation continuedfor 30min at 37∘C The cells were rinsed with PBS 2mLof PBS was added to each well and fluorescence intensitywas determined with spectrofluorometer (Gemini EM) withexcitation at 485 and emission at 530 nm For control the cellsgrown in 24-well plates for 24 hwere added an antioxidant N-acetyl-L-cystein (NAC 5mM) for 1 h prior to exposing themto 174 to 87 120583gmL AgNPs or 1mM H

2O2for 12 h 20120583M of

DCFH-DA was then added and the cells were incubated for30min at 37∘ before measuring changes of DCF fluorescenceas described

28Measurement of Caspase-3Activity Thecellswere treatedwith 87120583g AgNPs or purified caspase-3 or inhibitor for 24 hThe activity of caspase-3 was measured inMDA-MB-231 cellsusing a kit from Sigma (St Louis MO USA) according tothe manufacturerrsquos instructions Cells were washed with ice-cold PBS and lysed with 100 120583L of lysis buffer [50mM Tris-HCl (pH 75) 150mM NaCl 1mM EGTA 1mM NaF 1Nonidet P-40 1mM PMSF and protease inhibitor cocktail]for 30min at 4∘C Protein extracts were collected after cen-trifugation at 14000 rpm for 10min Protein concentrationwas determined using the Bio-Rad protein assay kit (Bio-RadUSA) Equal amounts (50120583g) of protein extracts were mixedwith assay buffer [20mM HEPES (pH 74) 100mM NaCl01 CHAPS 10mM DTT 1mM EDTA and 10 sucrose]added to 96-well Microtiter plates and incubated with thecaspase-3 substrate (acetyl-Asp-Glu-Val-Asp p-nitroanilide(Ac-DEVD-pNA) and caspase-3 inhibitor (Ac-DEVD-CHO)for 1 h and the absorbance read at 405 nm The colorimetricassay is based on the hydrolysis of caspase-3 substrate bycaspase-3 resulting in the release of the p-nitroaniline (pNA)moiety The concentration of the pNA released from thesubstrate is calculated from the absorbance values at 405 nmThe assay was done with noninduced cells and also in thepresence of purified caspase-3 and caspase-3 inhibitor (Ac-DEVD-CHO) for a comparative analysis

29 DNA Fragmentation Assay MDA-MB-231 (106 cells mL)were seeded in 6-wellMicroplates and treated with 87 120583gmLof AgNPs After 24 h of treatment the culture medium wasremoved and the cells were harvested by scraping with 1mLof PBS and lysed with 500120583L of lysis buffer [20mM Tris-HCl (pH 80) 5mM EDTA 400mM NaCl 1 SDS and10mgmL proteinase K] for 1 h at 55∘C FragmentedDNAwasextracted with phenolchloroformisoamyl alcohol (25 24 1vvv) precipitated with ethanol and resuspended in Tris-EDTA buffer (TE pH 80) containing 20120583gmL RNase AFor quantitative analyses of DNA content an equal amountof DNA was loaded and run on a 10 agarose gel containing1 120583gmL ethidium bromide at 70V and the DNA fragmentswere visualized by exposing the gel to ultraviolet lightfollowed by photography For control cells grown in 24-well plates for 24 h were added an antioxidant N-acetyl-L-cystein (NAC 5mM) for 1 h prior to exposing them to 87120583MAgNPs

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3 Results and Discussion

31 Extracellular Synthesis of AgNPs There are several phys-ical and chemical methods that have been used for thesynthesis of metallic nanoparticles [51] The developmentof biological method is essential because it provides costeffective environmentally friendly and easy process forsynthesis and application of metallic nanoparticles Severalmicroorganisms have the potential to interact withmetal ionsreducing them into metallic nanoparticles [52] In this workwe took the advantage of using microorganism for synthesisof AgNPs using the culture supernatants of B funiculus Asshown in Figure 1 the supernatants of B funiculus wereincubated with silver nitrate (1mM) The appearance of ayellowish-brown color in the reaction vessels suggested theformation of colloidal AgNPs Thus it was evident that themetabolites excreted by the culture exposed to silver couldreduce silver ions clearly indicating that the reduction of theions occurs extracellularly through reducing agents releasedinto the solution by B funiculus The color change fromyellow to brown provides a piece of evidence to support thesynthesis of AgNPs and also it is due to the excitation ofsurface plasmon vibrations typical of AgNPs [13 53ndash56]

Further the AgNPs were characterized by UV-visiblespectroscopy The UV-visible absorption spectra of the cellfiltrates were measured in the range of 300ndash700 nm usinga UV-visible spectrophotometer This technique has beenproved to be a very valuable and useful technique for thecharacterization of nanoparticles [53ndash55] By using UV-visible spectroscopy we could measure the diameter by thespectral response of silver nanoparticles as the diameterincreases the peak plasmon resonance shifts to longer wave-lengths and broadens Additionally UV-visible spectroscopyprovides a mechanism to monitor how the nanoparticleschange over time A strong and broad surface plasmon peaklocated at 420 nm was observed for the AgNPs preparedusing supernatants of B funiculus (Figure 2) The bandaround 420 nm suggests that the particles were well dispersedwithout aggregation Observation of the strong but broadsurface plasmon peak has been well known in the case ofvarious metal nanoparticles over a wide size range of 2ndash100 nm [53 54]

32 Size Distribution Analysis by Dynamic Light Scattering(DLS) To know the size of synthesized AgNPs size distribu-tion analysis was performed using dynamic light scatteringin aqueous solution It was found that the average size ofAgNPs was 20 nm Figure 3 shows that the particles range insize from 10 to 20 nm and possess an average size of 20 nmsynthesized by B funiculus DLS results showed an averagediameter of 20 nm and a low polydispersity index of less than008 indicating that a monodisperse distribution of mostly asingle and uniform size of species is present in solution

Regarding the size of the AgNPs several studies havebeen reported using a biological system such as using culturesupernatant of K pneumonia produces the size of particlesrange from 282 to 122 nm and possess an average size of525 nm [55] Similarly both culture supernatant and biomassof B licheniformis producesabout the size of 50 nm [49 56]

Gurunathan et al [13] reported that the particles range insize from 422 to 896 nm and possess an average size of50 nm size of AgNP synthesized from culture supernatantof E coli was 50 nm using DLS and TEM Shanshouryet al [57] reported the extracellular biosynthesis of metallicAgNPs by the reduction of aqueousAg+ using Escherichia coliATCC 8739 Bacillus subtilis ATCC 6633 and Streptococcusthermophilus ESh1 and revealed the size range between 5 and25 nm

33 Size and Morphology Analysis of AgNPs by Transmis-sion Electron Microscopy (TEM) Further characterizationof AgNPs was examined using TEM to know the size andmorphology of AgNPs the representative TEM image wasshown in Figure 4 and indicates well-dispersed particleswhich are more or less spherical The TEM analysis revealedthat the particle size of silver particles shows that the particlesize ranges from 10 to 20 nm

To determine their size distribution we measuredapproximately more than 200 particles from various samplesand represented them as the size distribution analysis (Fig-ure 4(b)) The average size of the particles synthesized byB funiculus is approximately 20 nm A significant proportionof largely spherical AgNPs within the range of 20 nm wereobserved in TEM micrographs The spherical particles arereasonably uniform and range in size from 10 to 20 nm andare in agreement with DLS data

34 Dose-Dependent Cytotoxicity Effect of AgNPs in MDA-MB-231 Cells The cell viability assay is one of the importantmethods for toxicology analysis which explain the cellularresponse to a toxic materials and it can provide informationon cell death survival andmetabolic activities [27] RecentlyPiao et al [43] reported that AgNPs and AgNO

3showed

cytotoxicity in a dose-dependent manner in human Changliver cells among these materials AgNPs showed highercytotoxicity compared toAgNO

3 AgNPs treated cells showed

the decreased metabolic activity which depends on nature ofcell types and size of nanoparticles [58] Franco-Molina et al[30] reported that colloidal silver induced dose-dependentcytotoxic effect on MDA-MB-231 breast cancer cells In ourexperiment the cells were treated with various concentra-tions (0ndash25 120583gmL) of AgNPs for 24 h and the results suggestthat AgNPswere able to reduce the cell viability ofMDA-MB-231 cells in a dose dependent manner At 24 h of treatmentAgNPswas found to be cytotoxic to the cells at concentrationsof 10 120583gmL and higher (Figure 5) In agreement with ourresults other research groups have reported that cell viabilitywas significantly reduced as a function of both culture timeand AgNP concentration in human IMR-90 and U251 cellsmouse embryonic stem cells and A549 lung cells [27 44]Our results suggest that the lowest concentration of AgNPssignificantly inhibits the growth of cells

35 Impact of AgNPs onMembrane Integrity LDH is a solublecytosolic enzyme which is released into the extracellularmedium because membrane damage consequently leads toapoptosis It is widely accepted as an indicator of lytic cell

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(a) (b) (c)

Figure 1 Synthesis of AgNPs by culture supernatant of B funiculusThe figure shows flask containing samples of AgNO3

after exposure to60min (a) AgNO

3

with the extracellular culture supernatant of B funiculus (b) andAgNO3

plus supernatant ofB funiculus (c) It is observedthat the color of the solution turned from colorless to brown after 1 h of the reaction indicating the formation of AgNPs

0

05

1

15

2

25

3

35

300 400 500 600 700 800

Abso

rban

ce (a

u)

Wavelength (nm)

Figure 2 The absorption spectrum of AgNPs synthesized by Bfuniculus culture supernatant The absorption spectrum of AgNPsexhibited a strong broad peak at 420 nm and observation of such aband is assigned to surface plasmon resonance of the particles Thesamples were collected and were incubated with 1mM silver nitratesolution After the incubation period the samples were visualized inUV-vis spectra

0

5

10

15

20

01 1 10 100 1000 10000

Inte

nsity

()

Size distribution by intensity

Size (dmiddotnm)

Figure 3 Size distribution analysis by DLS The particle sizedistribution revealed that the particles range from 10ndash20 nm Theaverage particle size was found to be 20 nm

deathThe results show that cellmembrane integrity inMDA-MB-231 cells was compromised in a dose dependent mannerby AgNPs of 20 nm diameter (Figure 6) The inverse rela-tionship between the LDH and the MTT cell viability resultsadds support to the accuracy of the data In the LDH assayas the concentration of the AgNPs increased cells becameprogressively more cytotoxic leading to a higher absorbancereading in the LDH assay and a decrease in absorbance inthe MTT assay with a concurrent decrease in the percentage

of viable cells Park et al [58] observed with various sizesof AgNPs that cell membrane integrity in L929 fibroblastswas compromised by all three AgNPs with 20 nm AgNPsbeing more potent than 80 and 113 nm AgNPs However thecell membrane integrity was affected slightly in RAW 2647macrophages Song et al [59] reported that water-solublemPEG-SH-coated AgNPs decreased cell viability in dose-and time-dependent manners at dosage levels between 625and 10000120583gmL causedmembrane damage (LDH leakage)and decreased the activities of superoxide dismutase andglutathione peroxides AgNPs induced the release of LDHin a concentration- and time-dependent manner indicatingthat AgNPs reduced the membrane potential in A549 cellsLee et al [60] observed that the LDH level was increased210 when cells were cultivated for 48 h in the culturemedium containing AgNPs at 100 120583gmL Hussain et al [33]demonstrated that exposure to AgNPs for 24 h resulted ina concentration-dependent increase in LDH leakage andexhibited a significant cytotoxicity at 10ndash50120583gmL in BRL 3Arat liver cells

36 Determination of IC50 Values of AgNPs To focus on thecytotoxic effect of particular concentration the half maximalinhibitory concentration (IC50)was calculated as the concen-tration required to inhibit the growth of tumor cells in cultureby 50 compared to the untreated cells AgNPs at 87 120583gmLdecreased the viability of MDA-MB-231 cells to 50 andthis was chosen as the IC50 Longer exposures resulted inadditional toxicity to the cells These results demonstratethat AgNPs mediate a concentration-dependent increase intoxicity Because 87120583gmL concentrations of AgNPs werefound to be the IC50 further experiments were carried outusing this concentration to show the effect of AgNPs againstMDA-MB-231 cells Gopinath et al [26] investigated themolecular mechanism of 10ndash15 nm size of AgNP mediatedcytotoxicity in BHK21 (noncancer) and HT29 (cancer) cellsand they observed that 27 120583gmL seems to be IC50 Zanetteet al [61] investigated the effects of AgNPs on skin usingthe human-derived keratinocyte HaCaT cell line model andsuggested that AgNPs caused a concentration- and time-dependent decrease of cell viability with IC50 values of 68plusmn 1 120583M (MTT assay) and 12 plusmn 12 120583M (SRB assay) after 7days of contact The IC50 results obtained from our studies

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(a)

0

3

6

9

12

15

5 10 15 20Size (nm)

Inte

nsity

(au

)

(b)

Figure 4 Size and morphology of AgNPs analysis by TEM (a) Several fields were photographed and were used to determine the diameterof nanoparticles The average range of observed diameter was 20 nm (b) Particle size distributions from TEM image

are comparable with earlier reports and synthesized AgNPsshow more efficacy than earlier reports However the actionof AgNP depends on size shape conditions of media andtype of cells are and also dose and time dependent

37 Effect of AgNPs in Cellular Reactive Oxygen SpeciesOxidative stress is one of the key mechanisms of toxic-ity related to nanoparticle exposure [62] The interactionbetween AgNPs and mammalian cells can induce oxidativestress by inducing the cellular ROS production so that itexceeds the cellular antioxidant capacity [27]Oxidative stressplays important roles in a variety of normal biochemicalfunctions and abnormality in their function results in patho-logical processes Excessive production of ROS in the cellis known to induce apoptosis [63 64] ROS generation hasbeen shown to play an important role in apoptosis inducedby treatment with AgNPs [27 37 38] Our studies providedevidence for a molecular mechanism of AgNPs inducinggeneration of ROS and it could be one of the factors forapoptosis Earlier studies show that AgNPs could inducegeneration of ROS in macrophages [58] and human Changliver cells [43]

To know the effect of AgNPs in oxidative stress we mea-sured ROS generation using the H2DCF-DA assay AgNPsinduced intracellular ROS generation was evaluated usingintracellular peroxide-dependent oxidation of DCFHDA toform fluorescent DCF Cells were also treated with a char-acteristic ROS generating agent H

2O2(1mM) as a positive

control DCF fluorescence was detected in cells treated withAgNPs for 24 h As shown in Figure 7 the ROS levelsgenerated in response to AgNPs were significantly higher inAgNPs treated cells than control ROS generation in cellstreated with both AgNPs and H

2O2was decreased when

the cells were pretreated with NAC an antioxidant Takentogether all these results indicate that cell death is mediated

0

20

40

60

80

100

120

0 5 10 15 20 25

Cel

l via

bilit

y (

of c

ontro

l)

Concentrations of AgNPs (120583gmL)

Figure 5 Effect of AgNPs on Cell viability of MDA-MB-231 cellsCells were treated with AgNPs at various concentrations for 24 hand cytotoxicity was determined by the MTT method The resultsrepresent the means of three separate experiments and error barsrepresent the standard error of the mean Treated groups showedstatistically significant differences from the control group by theStudentrsquos t-test (119875 lt 005)

by ROS production which might alter the cellular redoxstatus and it is a potential reason for cell death

38 Caspase-3 Activation of AgNP-Induced Apoptosis Thecaspase-3 activation cascade plays an important role inseveral apoptotic mechanisms [65ndash67] To investigate thepotential effect of AgNP on apoptotic pathway we examinedthe activity of caspase-3 in AgNP treated MDA-MB-231 cellsFigure 8 depicts the increase in the levels of caspase-3 duringtreatment with AgNPs The IC 50 value of AgNPs 87120583gmLincreased the activity of caspase-3 to a level comparable withthat of caspase-3 activation The cellular metabolic activityseems to be affected by the AgNPs therefore the possibility

BioMed Research International 7

0

005

01

015

02

025

03

0 5 10 15 20 25

Abso

rban

ce (a

u)

Concentrations of AgNPs (120583gmL)

Figure 6 Effect of AgNPs on LDH activity in MDA-MB-231LDH activity was measured by changes in optical densities due toNAD+ reduction which were monitored at 490 nm as describedin Materials and Methods Section using the cytotoxicity detectionlactate dehydrogenase kit The results represent the means of threeseparate experiments and error bars represent the standard error ofthe mean Treated groups showed statistically significant differencesfrom the control group by the Studentrsquos t-test (119875 lt 005)

0

1

2

3

4

5

6

Con AgNPsNAC

NAC

Relat

ive fl

uore

scen

ce o

f DCF

AgNPs + H2O2H2O2

+ NAC

Figure 7 ROS generation in AgNPs treated MDA-MB-231 cellsRelative fluorescence of DCF was measured using a spectrofluo-rometer with excitation at 485 and emission at 530 nm The resultsrepresent the means of three separate experiments and error barsrepresent the standard error of the mean Treated groups showedstatistically significant differences from the control group by theStudentrsquos t-test (119875 lt 005)

of apoptosis induction by the AgNPs was assessed especiallyat the IC50 Levels of caspase-3 a molecule which plays akey role in the apoptotic pathway of cells were increasedfollowing the treatment with AgNPs The increased level ofcaspase 3 activation suggested that AgNPs caused cell deaththrough apoptosis

39 DNA Fragmentation The DNA laddering technique isused to visualize the endonuclease cleavage products of apop-tosis [46]This assay involves extraction of DNA from a lysedcell homogenate followed by agarose gel electrophoresisApoptosis of the AgNP treated cells was accompanied by areduction in the percentage of cells in G0G1 phase and an

(fold

s rela

tive t

o co

ntro

l)

0

04

08

12

16

2

Con AgNPs Inhibitor Caspase-3

Casp

ase-

3 ac

tivity

AgNPs+ inhibitor

Caspase-3+ inhibitor

Figure 8 AgNPs induce apoptosis in MDA-MB-231 cells bycaspase-3 activation MDA-MB-231 cells were treated with AgNPspurified caspase-3 and caspase-3 inhibitor for 24 h The assaywas performed as described in Materials and Methods SectionThe caspase-3 activity is based on the hydrolysis of caspase-3substrate (acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA)by caspase-3 resulting in the release of the p-nitroaniline (pNA)moiety The concentration of the pNA released from the substrateis calculated from the absorbance values at 405 nm The assay wascarried out in the presence of purified caspase-3 and caspase-3inhibitor (Ac-DEVD-CHO) for a comparative analysis The resultsrepresent the means of three separate experiments and error barsrepresent the standard error of the mean Treated groups showedstatistically significant differences from the control group by theStudentrsquos t-test (119875 lt 005)

increase in the percentage of G2M phase cells indicating cellcycle arrest atG2M [60]TheROS can act as signalmoleculespromoting cell cycle progression and can induce oxidativeDNA damage [68 69] Further we examined the impactof AgNPs in DNA fragmentation DNA fragmentation isbroadly considered as a characteristic feature of apoptosis[70] Induction of apoptosis can be confirmed by two factorssuch as irregular reduction in size of cells in which the cellsare reduced and shrunken and lastly DNA fragmentationThe DNA fragmentation in the present study was verified byextractingDNA fromMDA-MB-231 cells treatedwith variousconcentrations of AgNPs followed by detection in the agarosegel Figure 9 clearly indicates that the DNA ldquoladderingrdquopattern in MDA-MB-231 cells treated with AgNPs is one ofthe reasons for cell death Earlier studies by Gurunathan andcoworkers demonstrated that both cancer and noncancer celllines treated with silver nanoparticle exhibit the formation ofDNA ladder [14 29] The deposition of metal particles insidethe nucleus could affect the DNA and cell division Genotoxicstudies of titanium dioxide (TiO

2) nanoparticles revealed

dose-dependent DNA damage chromosomal aberrationsand errors in chromosome segregation [71] and formationof sister chromatic exchanges [72] Treatment with AgNPsinduced the production of micronuclei (MN) [27] Mroz etal [73] hypothesized that nanoparticles and reactive oxidativespecies induce DNA damage activating p53 and proteinsrelated to DNA repair and mimicking irradiation relatedcarcinogenesis

8 BioMed Research International

M 1 2 3 4

Figure 9 Effect of AgNPs on DNA fragmentation MDA-MB-231Cells were treated with AgNPs for 24 h and DNA fragmentation wasanalyzed by agarose gel electrophoresis Lane M 1 kB ladder lane1 control lane 2 AgNP (87 120583gmL) lane 3 AgNP + NAC lane 4NAC (5mM)

4 Conclusion

Recently AgNPs are used as an antimicrobial agent in wounddressings and coatings in medical devices Developing bio-compatiblemolecule as an anticancer agent is one of the novelapproaches in the field of cancer therapy using nanotechnol-ogy We have successfully synthesized and prepared stableAgNPs (20 nm) using novel bacterium B funiculus whichis green environmentally friendly cost effective and rapidmethod for synthesis of AgNPs The present study revealedthat the potential cytotoxic effect of biologically synthesizedAgNP in MDA-MB-231 cells by inhibiting growth of cellsconcentration-dependent activation of LDH increased levelof ROS generation and activation of caspase-3 which isconsidered to be the most significant of the executionercaspases resulting in cellular apoptosis Our results suggestthat oxidative stress seems to be involved in nanoparticlecytotoxicityThe overall results indicated that the biologicallysynthesized AgNPs have antiproliferative activity throughinduction of apoptosis in MDA-MB-231 breast cancer cellline suggesting that biologically synthesized AgNPs mightbe a potential alternative agent for human breast cancertherapy This study demonstrates the possibility of usingAgNPs to inhibit the growth of the tumor cells and theircytotoxicity for potential therapeutic treatments and offers anew method to develop molecule for cancer therapy Finallycost effectiveness biocompatibility and facileness to modifythese silver nanoparticles make them a viable choice in futurebiomedical applications

Acknowledgments

This paper was supported by the SMART Research ProfessorProgram of Konkuk University Dr Sangiliyandi Gurunathanwas supported by Konkuk University SMART Full-time

Professorship This work was supported by BioGreen 21Program of the RDA (Grant no PJ009625) and ARPC (Grantno 111047-5) Republic of Korea

References

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[3] S R D Johnston ldquoAcquired tamoxifen resistance in humanbreast cancermdashpotential mechanisms and clinical implica-tionsrdquo Anti-Cancer Drugs vol 8 no 10 pp 911ndash930 1997

[4] S Kato H Endoh YMasuhiro et al ldquoActivation of the estrogenreceptor through phosphorylation by mitogen-activated pro-tein kinaserdquo Science vol 270 no 5241 pp 1491ndash1494 1995

[5] R Lupu M Cardillo C Cho et al ldquoThe significance of hereg-ulin in breast cancer tumor progression and drug resistancerdquoBreast Cancer Research and Treatment vol 38 no 1 pp 57ndash661996

[6] K Brown ldquoBreast cancer chemoprevention risk-benefit effectsof the antioestrogen tamoxifenrdquo Expert Opinion on Drug Safetyvol 1 no 3 pp 253ndash267 2002

[7] L L Smith K Brown P Carthew et al ldquoChemoprevention ofbreast cancer by tamoxifen risks and opportunitiesrdquo CriticalReviews in Toxicology vol 30 no 5 pp 571ndash594 2000

[8] H Liu Y Liu Z Wang and P He ldquoFacile synthesis of mono-disperse size-tunable SnSnanoparticles potentially for solar cellenergy conversionrdquo Nanotechnology vol 21 no 10 Article ID105707 2010

[9] K S Shin J-Y Choi C S Park H J Jang and K Kim ldquoFacilesynthesis and catalytic application of silver-deposited magneticnanoparticlesrdquo Catalysis Letters vol 133 no 1-2 pp 1ndash7 2009

[10] L Zhou X He D He K Wang and D Qin ldquoBiosensing tech-nologies for Mycobacterium tuberculosis detection status andnew developmentsrdquo Clinical and Developmental Immunologyvol 2011 Article ID 193963 8 pages 2011

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2

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[12] R Bhattacharya and P Mukherjee ldquoBiological properties ofldquonakedrdquo metal nanoparticlesrdquoAdvanced Drug Delivery Reviewsvol 60 no 11 pp 1289ndash1306 2008

[13] S Gurunathan K Kalishwaralal R Vaidyanathan et alldquoBiosynthesis purification and characterization of silvernanoparticles using Escherichia colirdquo Colloids and Surfaces Bvol 74 no 1 pp 328ndash335 2009

[14] K Kalishwaralal V Deepak S R K Pandian et al ldquoBiosynthe-sis of silver and gold nanoparticles using Brevibacterium caseirdquoColloids and Surfaces B vol 77 no 2 pp 257ndash262 2010

[15] M I Sriram S B M Kanth K Kalishwaralal and SGurunathan ldquoAntitumor activity of silver nanoparticles inDaltonrsquos lymphoma ascites tumor modelrdquo International Journalof Nanomedicine vol 5 no 1 pp 753ndash762 2010

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[19] N Pugazhenthiran S Anandan G Kathiravan N K UPrakash S Crawford and M Ashokkumar ldquoMicrobial synthe-sis of silver nanoparticles byBacillus sprdquo Journal ofNanoparticleResearch vol 11 no 7 pp 1811ndash1815 2009

[20] K Kalimuthu S Vijayakumar and R Senthilkumar ldquoAntimi-crobial activity of the biodiesel plant Jatropha curcas LrdquoInternational Journal of Pharma and Bio Sciences vol 1 no 3article 29 2010

[21] I Sondi and B Salopek-Sondi ldquoSilver nanoparticles as antimi-crobial agent a case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and Interface Science vol275 no 1 pp 177ndash182 2004

[22] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

[23] S K Gogoi P Gopinath A Paul A Ramesh S S Ghosh andA Chattopadhyay ldquoGreen fluorescent protein-expressingEscherichia coli as a model system for investigating theantimicrobial activities of silver nanoparticlesrdquo Langmuir vol22 no 22 pp 9322ndash9328 2006

[24] P Sanpui A Murugadoss P V D Prasad S S Ghosh andA Chattopadhyay ldquoThe antibacterial properties of a novelchitosan-Ag-nanoparticle compositerdquo International Journal ofFood Microbiology vol 124 no 2 pp 142ndash146 2008

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[28] S Gurunathan K-J Lee K Kalishwaralal S SheikpranbabuR Vaidyanathan and S H Eom ldquoAntiangiogenic properties ofsilver nanoparticlesrdquoBiomaterials vol 30 no 31 pp 6341ndash63502009

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[31] P Sanpui A Chattopadhyay and S S Ghosh ldquoInduction ofapoptosis in cancer cells at low silver nanoparticle concentra-tions using chitosan nanocarrierrdquo ACS Applied Materials andInterfaces vol 3 no 2 pp 218ndash228 2011

[32] Y H Hsin C F Chen S Huang T S Shih P S Lai andP J Chueh ldquoThe apoptotic effect of nanosilver is mediated

by a ROS- and JNK-dependent mechanism involving themitochondrial pathway inNIH3T3 cellsrdquoToxicology Letters vol179 no 3 pp 130ndash139 2008

[33] S M Hussain K L Hess J M Gearhart K T Geiss and J JSchlager ldquoIn vitro toxicity of nanoparticles in BRL 3A rat livercellsrdquo Toxicology in Vitro vol 19 no 7 pp 975ndash983 2005

[34] S Kim J E Choi J Choi et al ldquoOxidative stress-dependenttoxicity of silver nanoparticles in human hepatoma cellsrdquoToxicology in Vitro vol 23 no 6 pp 1076ndash1084 2009

[35] J L Martindale and N J Holbrook ldquoCellular response tooxidative stress signaling for suicide and survivalrdquo Journal ofCellular Physiology vol 192 no 1 pp 1ndash15 2002

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[37] C Carlson SMHussein AM Schrand et al ldquoUnique cellularinteraction of silver nanoparticles size-dependent generation ofreactive oxygen speciesrdquo Journal of Physical Chemistry B vol112 no 43 pp 13608ndash13619 2008

[38] R Foldbjerg P Olesen M Hougaard D A Dang H JHoffmann and H Autrup ldquoPVP-coated silver nanoparticlesand silver ions induce reactive oxygen species apoptosis andnecrosis in THP-1 monocytesrdquo Toxicology Letters vol 190 no2 pp 156ndash162 2009

[39] E-J Park J Yi Y Kim K Choi and K Park ldquoSilver nanopar-ticles induce cytotoxicity by a Trojan-horse type mechanismrdquoToxicology in Vitro vol 24 no 3 pp 872ndash878 2010

[40] Z Shavandi T Ghazanfari and K N Moghaddam ldquoInvitro toxicity of silver nanoparticles on murine peritonealmacrophagesrdquo Immunopharmacology and Immunotoxicologyvol 33 no 1 pp 135ndash140 2011

[41] L Braydich-Stolle S Hussain J J Schlager and M-C Hof-mann ldquoIn vitro cytotoxicity of nanoparticles in mammaliangerm-line stem cellsrdquo Toxicological Sciences vol 88 no 2 pp412ndash419 2005

[42] R P Nishanth R G Jyotsna J J Schlager S M Hussainand P Reddanna ldquoInflammatory responses of RAW 2647 mac-rophages upon exposure to nanoparticles role of ROS-NF120581Bsignaling pathwayrdquo Nanotoxicology vol 5 no 4 pp 502ndash5162011

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[49] K Kalishwaralal V Deepak S Ramkumarpandian H Nella-iah and G Sangiliyandi ldquoExtracellular biosynthesis of silvernanoparticles by the culture supernatant of Bacillus licheni-formisrdquoMaterials Letters vol 62 no 29 pp 4411ndash4413 2008

10 BioMed Research International

[50] H-Y Shim J-H Park H-D Paik S-Y Nah D S H L Kimand Y S Han ldquoAcacetin-induced apoptosis of human breastcancer MCF-7 cells involves caspase cascade mitochondria-mediated death signaling and SAPKJNK12-c-Jun activationrdquoMolecules and Cells vol 24 no 1 pp 95ndash104 2007

[51] A D Edelstein and R C Cammarata Nanomaterials SynthesisProperties and Applications Taylor amp Francis Boca Raton FlaUSA 1996

[52] T Klaus-Joerger R Joerger E Olsson and C-G GranqvistldquoBacteria as workers in the living factory metal-accumulatingbacteria and their potential for materials sciencerdquo Trends inBiotechnology vol 19 no 1 pp 15ndash20 2001

[53] M Sastry K S Mayya and K Bandyopadhyay ldquopHDependentchanges in the optical properties of carboxylic acid derivatizedsilver colloidal particlesrdquo Colloids and Surfaces A vol 127 no1ndash3 pp 221ndash228 1997

[54] M Sastry V Patil and S R Sainkar ldquoElectrostatically controlleddiffusion of carboxylic acid derivatized silver colloidal particlesin thermally evaporated fatty amine filmsrdquo Journal of PhysicalChemistry B vol 102 no 8 pp 1404ndash1410 1998

[55] A R Shahverdi S Minaeian H R Shahverdi H Jamali-far and A-A Nohi ldquoRapid synthesis of silver nanoparticlesusing culture supernatants of Enterobacteria a novel biologicalapproachrdquoProcess Biochemistry vol 42 no 5 pp 919ndash923 2007

[56] K Kalimuthu R S Babu D Venkataraman M Bilal and SGurunathan ldquoBiosynthesis of silver nanocrystals by BacilluslicheniformisrdquoColloids and Surfaces B vol 65 no 1 pp 150ndash1532008

[57] R E L Shanshoury S E Elsilk and M E Ebeid ldquoExtracel-lular biosynthesis of silver nanoparticle using Escherichia coliATCC 8739 Bacillus subtilis ATCC 6633 and Streptococcusthermophilus ESh1 and their antimicrobial activityrdquo ISRN Nan-otechnology vol 2011 Article ID 385480 7 pages 2011

[58] MVD Z ParkAMNeigh J PVermeulen et al ldquoThe effect ofparticle size on the cytotoxicity inflammation developmentaltoxicity and genotoxicity of silver nanoparticlesrdquo Biomaterialsvol 32 no 36 pp 9810ndash9817 2011

[59] X L Song B Li K Xu et al ldquoCytotoxicity of water-solublemPEG-SH-coated silver nanoparticles in HL-7702 cellsrdquo CellBiology and Toxicology vol 28 no 4 pp 225ndash237 2012

[60] Y S Lee DW Kim Y H Lee et al ldquoSilver nanoparticles induceapoptosis and G2M arrest via PKC120577-dependent signaling inA549 lung cellsrdquoArchives of Toxicology vol 85 no 12 pp 1529ndash1540 2011

[61] C Zanette M Pelin M Crosera et al ldquoSilver nanoparticlesexert a long-lasting antiproliferative effect on human ker-atinocyte HaCaT cell linerdquo Toxicology in Vitro vol 25 no 5 pp1053ndash1060 2011

[62] A Nel T Xia LMadler andN Li ldquoToxic potential of materialsat the nanolevelrdquo Science vol 311 no 5761 pp 622ndash627 2006

[63] J L Martindale and N J Holbrook ldquoCellular response tooxidative stress signaling for suicide and survivalrdquo Journal ofCellular Physiology vol 192 no 1 pp 1ndash15 2002

[64] J Sastre F V Pallardo and J Vina ldquoMitochondrial oxidativestress plays a key role in aging and apoptosisrdquo IUBMB Life vol49 no 5 pp 427ndash435 2000

[65] C Gianinazzi D Grandgirard H Imboden et al ldquoCaspase-3mediates hippocampal apoptosis in pneumococcal meningitisrdquoActa Neuropathologica vol 105 no 5 pp 499ndash507 2003

[66] T Matsura M Kai Y Fujii H Ito and K Yamada ldquoHydrogenperoxide-induced apoptosis in HL-60 cells requires caspase-3activationrdquo Free Radical Research vol 30 no 1 pp 73ndash83 1999

[67] T S Zheng S F Schlosser T Dao et al ldquoCaspase-3 con-trols both cytoplasmic and nuclear events associated withFas-mediated apoptosis in vivordquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no23 pp 13618ndash13623 1998

[68] R Alvarez-Gonzalez H Spring M Muller and A BurkleldquoSelective loss of poly(ADP-ribose) and the 85-kDa fragmentof poly(ADP- ribose) polymerase in nucleoli during alkylation-induced apoptosis of HeLa cellsrdquo Journal of Biological Chem-istry vol 274 no 45 pp 32122ndash32126 1999

[69] R Hu K-T Yong I Roy H Ding S He and P NPrasad ldquoMetallic nanostructures as localized plasmon res-onance enhanced scattering probes for multiplex dark-fieldtargeted imaging of cancer cellsrdquo Journal of Physical ChemistryC vol 113 no 7 pp 2676ndash2684 2009

[70] R T AllenW J Hunter III and D K Agrawal ldquoMorphologicaland biochemical characterization and analysis of apoptosisrdquoJournal of Pharmacological and Toxicological Methods vol 37no 4 pp 215ndash228 1997

[71] J Wang G Zhou C Chen et al ldquoAcute toxicity and biodistri-bution of different sized titanium dioxide particles in mice afteroral administrationrdquo Toxicology Letters vol 168 no 2 pp 176ndash185 2007

[72] P-J Lu I-C Ho and T-C Lee ldquoInduction of sister chromatidexchanges and micronuclei by titanium dioxide in Chinesehamster ovary-K1 cellsrdquoMutation Research vol 414 no 1ndash3 pp15ndash20 1998

[73] R M Mroz R P F Schins H Li et al ldquoNanoparticle-driven DNA damage mimics irradiation-related carcinogenesispathwaysrdquo European Respiratory Journal vol 31 no 2 pp 241ndash251 2008

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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