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Research paper

7, 8-diacetoxy-4-methylcoumarin induced cell death in humantumor cells is influenced by calreticulin

Amit Verma a, Anant Narayan Bhatt a, Abdullah Farooque a, Suchit Khanna a, Divya Khaitan a,Mohan B. Arya b, Anu Arya e, Ashish Dhawan e, Hanumantharao G. Raj c, Daman Saluja d,Ashok K. Prasad e, Virinder S. Parmar e, Bilikere S. Dwarakanath a,*

a Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Majumdar Marg, Timarpur, Delhi-110054, Indiab SSVPG College, Hapur, U.P, IndiacV.P. Chest Institute, University of Delhi, Delhi-110007, IndiadAmbedkar Centre for Biomedical Research, University of Delhi, Delhi-110007, IndiaeBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi-110007, India

a r t i c l e i n f o

Article history:Received 15 May 2010Accepted 30 October 2010Available online 12 November 2010

Keywords:CalreticulinHistone acetyl transferasesPolyphenolic acetatesNADPH-cytochrome c reductase

a b s t r a c t

Calreticulin (CRT), an endoplasmic reticulum resident protein demonstrates transacetylase activity inpresence of 7, 8 diacetoxy-4-methyl coumarin (DAMC) in vitro. To investigate the possible role of CRT andDAMC mediated protein acetylation in cells, we investigated the effects of DAMC in tumor cells withdifferent levels of CRT. DAMC was more toxic (clonogenicity, metabolic viability and proliferation) tohuman glioma cells (BMG-1) expressing low endogenous CRT level as compared to head and neckcarcinoma cells (KB) with a high CRT level. The cytotoxicity was accompanied by loss of mitochondrialmembrane potential in both the cells, which correlated with corresponding changes in the levels of pro-apoptotic (Bax) and anti-apoptotic (NFkB) regulators. Manipulation of CRT protein level in KB cells byapplication of small RNA interference enhanced the sensitivity by four folds while over expression of CRTin BMG-1 cells reduced their sensitivity to DAMC by ∼20% strongly suggesting the influence of CRT onDAMC induced cytotoxicity. The partial rescue of CROE cells from DAMC induced toxicity was accom-panied by changes in NFkB levels and over all protein acetylation status, besides increase in theNADPH-cytochrome c reductase activity related to its well known antioxidant property. Since CRT isover-expressed in cancer cells, which are generally resistant to radio- and chemotherapy; targeting CRTtransacetylase system, may be an attractive approach for increasing the efficacy of anticancer therapies.

� 2010 Published by Elsevier Masson SAS.

1. Introduction

Calreticulin (CRT) is calcium binding chaperone protein; presentin the lumen of endoplasmic reticulum, which regulates manycellular processes under normal and stress conditions [1,2]. CRT isassociated with diverse cellular functions such as secretion,contraction-relaxation, cell motility, cytoplasmic and mitochondrialmetabolism, folding of proteins, gene expression, cell cycle

progression, and apoptosis [3,4]. CRT is known to influence tumori-genesis and it’s over expression enhances angiogenesis facilitatingproliferation aswell asmigration of cancer cells [5,6]; however, it hasalso been found to play a role in anti tumor immunity [7,8]. Availableevidences suggest that the multifunctional activity of CRT hasemerged due to its multicompartmental presence in the cellincluding cytoplasm and membrane [9]. Therefore, detailed under-standing of CRT function, its role in determining cellular responses totherapeutic agents still remains an area of investigation.

Recent studies suggest a newer function of CRT, in the form oftransacetylase activity, where acetylation of certain target proteinsviz.NADPH-cyto c reductase, glutathione S transferase and nitricoxide synthase have been shown in vitrowith polyphenolic acetatesas an acetyl group donormolecule [10e14], The sites of acetylation ofglutathione S transferase analyzed by MALDI TOFMS and LC\MS\MSare Lys-51, -82,-124,-181,-191 and 210 and the N-terminal proline[15]. Investigations with individual domains (N, P and C) of CRT from

Abbreviation: DAMC, 7,8-diacetoxy-4-methylcoumarin; CRT, calreticulin; HAT,histone acetyltransferases; DCFH-DA, 2,7-dichlorofluorescin diacetate; HDACs,histone deacetylases; ROS, reactive oxygen species; DiOC6, 3,3'dihexyloxa-carbocyaniniodode; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide); NFkB, Nuclear factor kappa B.* Corresponding author. Fax: þ91 11 2391 9509.

E-mail addresses: dwarakanathdrbs@gmail.com, bsd@inmas.drdo.in (B.S.Dwarakanath).

Contents lists available at ScienceDirect

Biochimie

journal homepage: www.elsevier .com/locate/biochi

0300-9084/$ e see front matter � 2010 Published by Elsevier Masson SAS.doi:10.1016/j.biochi.2010.10.023

Biochimie 93 (2011) 497e505

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a nematode Haemonchus contortus have revealed that the P-domainalone possesses the CRTAase activity [16]. Studies with differentacetyl group donors have revealed that the acetylation of targetproteins is efficient with 7, 8, di acetoxy, 4 methyl coumarin (DAMC)as compared to 7-acetoxy 4methyl coumarin (7-AMC) and 3 acetoxy4 methyl coumarin (3-AMC) and is shown to be comparable with 3-oxo-2,3-dihydrobenzofuran-6,7-diyl diacetate [17,18]. Acetyl CoA, thesubstrate for histone acetyl transferase (HATs) appears to be a poordonor [19]. This transacetylase activity is found to be calciumdependent and shows autoacetylation [20]. DAMC has been found toreduce the clastogenic affect of aflatoxin B1 (AFB1) in mouse bonemarrowcells and rat lung cells, suggesting that the CRT: polyphenolicacetates systemmodifies the cellular response to damage induced bycertain clastogens [21]. Since polyphenols show pro-oxidant activityand generate oxidative stress in the cells, the polyphenolic acetateshave also been investigated for their cytotoxic effects in comparisonto the parental molecules [22,23]. DAMC and quercetin pentaacetates (QPA) have been found to be more toxic compared to theirparental compounds (7, 8, di hydroxy, 4 methyl coumarin and quer-cetin) in a breast carcinoma cell line (MDA-MB-468), which could notbe entirely attributed to the oxidative stress generated, suggestingthereby that the acetyl groups also contribute to the cytotoxic effects.Although these circumstantial evidences suggest a role for CRT in thecytotoxicity but a casual relationship between CRT mediated proteinacetylation and cytotoxicity has not been established. Therefore, thepresent studies were undertaken to investigate the cytotoxic effectsof DAMC in human tumor cell lines of different origin that vary intheir CRT levels as well as isogenic cells varying CRT levels, besidesknocking down CRT using siRNA. We found that the head and neckcarcinoma cell line (KB) having high CRT level was more resistant toDAMC as compared to the glioma cell line (BMG-1) having low CRTlevel. This relationship between CRT level and sensitivity to DAMCwas confirmed by both over expression of CRT in BMG-1 cells (CROEcells) and siRNA knock down of CRT, which resulted in proportionalchanges in sensitivities. Further, the lower degree of sensitivity toDAMC in KB and CROE cells was related to an overall increase inprotein acetylation status, with NFkB being one of the target proteinsfor modulation by CRT: DAMC transacetylase system.

2. Material and methods

2.1. Material

Dulbecco’s Minimum Essential Medium (DMEM), Penicillin G50,000 unit/l. Streptomycin 38850 unit/l and Nystatin 9078 unit/l,Blastidine S, 3-4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), 1, 4 Dithiothreitol (DTT), Dimethylsulfoxide(DMSO), 3,3’dihexyloxacarbocyaniniodode (DiOC6), Dichlorodihy-drofluorescein diacetate (DCFH-DA), Potassium chloride (KCl),Magnesium chloride (MgCl2), Ethylene-di-amine-tetra acetate(EDTA), Protease inhibitor cocktail, HEPES, Igepal, Annexin V-FITCkit and Foetal bovine serum were procured from Sigma chemicalsCo. (St Louis, USA). Bicinchoninic acid (BCA from Thermo FisherScientific Rockford USA), primary antibody (anti-NFkB, anti-baxand anti-b-Actin) were procured from BD Biosciences Pharmingen,San Diego, CA, USA and secondary antibody (IgG1 whole moleculewith HRP conjugate) was procured from Bangalore Genei India,anti-CRT PANacetylase antibody and CRT siRNA was procured fromSanta Cruz biotechnology CA USA, DAMC was synthesized bydepartment of chemistry University of Delhi, India.

2.2. Sources of cell lines

The cerebral glioma cell line (BMG-1; diploid, wild type p53;passage no: 18) was established in Bangalore, India [24]. CRT gene

was stably transfected in BMG-1 cells (CROE) and both these celllines were cultured in DMEM (1 gm Glucose/lt) with 5%foetalbovine serum containing antibiotics i.e. penicillin (100 units/ml),streptomycin (50 units/ml) and nystatin (2 mg/ml) in humidified 5%CO2 incubator at 37 �C. Head and neck squamous carcinoma celllines (KB; diploid wild type p53 passage no: 302) were procuredfrom NCCS, Pune, India and were cultured in DMEM(4.5 gm Glucose/lt) containing 10% foetal bovine serum and anti-biotics same as mentioned above. Stock culture were maintained inthe exponential growth phase by passaging them every 3 days withtheir respective growth medium in 25 cm2 plastic flask (Tarson,Calcutta, India).

2.3. Construction of plasmid containing calreticulin gene

The plasmid construct containing the CRT gene was used toamplify the CRT gene in PCR using following forward 50-CCG CCCCCT CGC TCG AGC ATG CTG CTA TCC GTG CCG -30 and reverseprimers RP: 50-CCC TGG AGG CAG AAG CTT CTA CAG CTC GTC CTTGGC-30 respectively. Amplified PCR product was further ligatedwith pEF6/V5-His-Topo (Invitrogen life technologies), mammalianexpression vector according to manufacturer’s instruction. Theligation product was transformed in DH5a strain of E. coli andplasmids were isolated from positive colonies. Final construct,pEF6/V5-His-Topo-CRT was further verified with XhoI digestion forthe right orientation of the gene and the sequence was confirmedby DNA sequencing before using the construct for transfection intothe cells.

2.4. Establishment of CROE cells

Briefly, 60e70% confluent BMG-1 cells werewashed with serumfree media. Plasmid (3e5 mg) and 6 ml of transfection reagent (lip-ofectamine, Invitrogen life technologies) were incubated in 250 mlof serum free medium for 15 min at room temperature. This reac-tion mixture was added to the washed cells and incubated for 3 h at37 �C in CO2 incubator. After 3 h, serum free media was removedand replenished with 5% serum containing medium. To establishcalreticulin over-expressing stable cell lines, transfected cells wereselected in a selection media containing blasticidine (20 mg/ml) for4 weeks. After few passages in selection medium, selection anti-biotic was removed from the medium and cells were grown in 5%serum containing growth medium.

2.5. CRT siRNA transfection

BMG-1 and KB cells were plated in 6 well plates (2.5 � 105 perwell). Following overnight incubation, the mediawas replaced withsiRNA transfection media (Santa Cruz, biotechnology) and cellswere transfected with 80 pmol of CRT siRNA and scramble(80 pmol) using the transfection reagent. Six hours after trans-fection, the media was changed to DMEM supplemented with 2%FBS and cells were incubated for 24 h. Cells were then used forwestern blot analysis to measure the CRT protein levels and DAMCinduced cytotoxicity.

2.6. Drug treatment

DAMC was dissolved in DMSO (5.5 mg/ml) and diluted in HanksBasal salt solution, HBSS (1:1) before filter sterilizing and added tothe cells in the required concentration after serial dilution ingrowth media for 24 h.

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2.7. Macrocolony assay

Cells ranging from 150 to 4000 (depending on the treatment)were plated 24 h before treating with varying concentrations ofDAMC in DMEM at 37 �C for 24 h. After the treatment, cells werereplaced with fresh growth media and incubated in CO2 incubatorfor 7e8 days with BMG-1 cells and 14 days for KB cells to allowcolony formation. Colonies (more than 50 cells) were fixed withmethanol and stained with 1% crystal violet and were counted tocalculate the plating efficiency (PE) and the surviving fraction (SF).

PE ¼ (No. of colonies counted/No of cells plated) � 100

SF ¼ PET=PEC

where PET is the plating efficiency of the treated group and PEC isthe value of the control

2.8. Cell proliferation

Following different treatments as described above, cells wereharvested at every 24 h time interval. Both floating and attachedcells were counted and fixed in 70% chilled ethanol for the analysisof cell cycle distribution. Cell proliferation was calculated bycomputing the increase in cell number and an index of proliferationP, was calculated as:

P ¼ Nt=N0

where, Nt ¼ number of cells at time “t”. N0 ¼ number of cells at thetime of treatment.

2.9. Apoptosis detection

Flow cytometric measurement of cellular DNA was performedwith ethanol fixed cells as described above. The presence of subG0 � G1 (hypodiploid) population is indicative of apoptotic celldeath. The percentage of apoptotic cells and/or sub G0 � G1 wascalculated using Modfit software (Becton and Dickinson) andreconfirmed from regional statistics in bivariate plots of DNA vsforward scatter or Side scatter. Surface exposure of phosphati-dylserine in apoptotic cells was measured by Annexin V-FITC/PIapoptosis detection kit I. FITC fluorescence was analyzed by Cell-Quest software (Becton Dickinson, CA, USA).

2.10. Metabolic viability by MTT assay

Following treatment, cells were plated in 96 well plates (3000cells/200 ml/well). After different time intervals (24e72 h) ofseeding the cells, stock MTT solution (20 ml) was added and cellswere incubated at 37 �C in the dark for 2 h. The medium wasremoved and formazan crystals formed by the cells was dissolvedusing 150 ml of DMSO. The absorbance was read at 570 nm using630 nm as reference wavelength on an ELISA reader (biotechinstruments).

2.11. Determination of intracellular redox levels and mitochondrialpotential (Djm)

DAMC induced changes in the intracellular ROS levels weremeasured by DCF fluorescence as described earlier [25]. The cellsafter DAMC (100 mM for 24 h) incubationwere washed twice in PBSand held in PBS with Ca2þ, Mg2þ, H2DCF-DA (10 mg/ml) and 5 mMglucose and subsequently incubated for half an hour at 37 �C. Afterincubation cells were washed with PBS and analyzed by flowcytometry.

In order to measure Djm, 0.1 � 105 cells were labeled with thefluorochrome DiOC6, 40 nM for 30 min prior to harvesting andincubated at 37 �C for 30 min in the dark. After incubation with thefluorochrome, cells were immediately analyzed by flow cytometry.

2.12. Immunohistochemistry

Cells were plated with density of 0.2 � 106 on glass cover slipsand incubated overnight at 37 �C in 5% CO2. Next day the cells weretreated with 100 mM DAMC for 24 h. The cells were fixed in 4%paraformaldehyde and then permeabilized with 0.2% Triton-X-100/PBS. After blocking for 1 h in 2% BSA/0.1% Triton-X-100/PBS slideswere incubated with anti-RelA/p65 antibody (1:100 dilution) fol-lowed by FITC labeled anti-mouse secondary antibody (1:500dilution, sigma) [26]. The samples were washed and mountingsolution was applied to prevent bleaching. Cells were visualizedusing fluorescence microscope (Zeiss, Germany).

2.13. SDS-PAGE and western blotting

Preparation of cell lysates and immunoblot analysis vehicletreated and DAMC treated cells were harvested after 24 h and lysedin ice cold RIPA lysis buffer (TriseHCl: 50 mM, pH 7.4, NP-40: 1%,

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Fig. 1. Cytotoxic effects of DAMC in human tumor cells lines with varying calreticulinlevels. (A) Western blot analysis of calreticulin levels in BMG-1 and KB cells (B)Concentration dependent cytotoxicity evaluated by macrocolony assay after exposureof BMG-1 and KB cells with DAMC (5, 20 and 100 mM) for 24 h. Each point is the meanof three replicates; bars represent the SEM. *P < 0.01. (C) Metabolic viability measuredafter 24 h DAMC treatment in both the cells *P < 0.05.

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Na-deoxycholate: 0.25%, NaCl: 150 mM, EDTA: 1 mM, PMSF: 1 mM,aprotinin 1 mg/ml, leupeptin 1 mg/ml and pepstatin: 1 mg/ml,Na3VO4: 1 mM, NaF: 1 mM). The protein content in the lysates wasmeasured by BCA protein assay. Protein (50e60 mg) was resolved on12e15% SDS-PAGE and electroblotted onto PVDF membrane(Amersham). The membrane was then incubated in 4% skimmedmilk for 1 h followed by primary antibody incubation (anti-NFkB,anti-bax and Pan acetylase) for 2 h. After washing, correspondingsecondary antibody (horseradish peroxidase conjugate) was addedand incubated for 1 h. After washing, the blots were developedusing ECL chemiluminescence detection reagent (Pierce).Membranes were stripped in stripping buffer (25 mM Glycine, 1%SDS, pH 2 adjusted with HCl) for 1 h washed twice in TTBS for10 min each and re-probed with b-Actin (cytoplasm) and lamininb2 (nucleus) as loading control.

2.14. Preparation of nuclear and cytoplasmic extract

The BMG-1 and KB cells were incubated with DAMC (100 mM;24 h) and treated cells were collected and centrifuged at 1500 � gfor 15min. The pellet was resuspended by gentle pipetting in 100 mlof ice cold cell lysis buffer (10 mMHepes pH 7.9, 10 mM KCl, 1.5 mMMgCl2, 1 mM DTT and protease inhibitor cocktail). The mixture wasincubated on ice for 10 min, followed by addition of Igepal at a finalconc. of 0.6% and vortexed vigorously for 10 s, and then centrifugedimmediately at 12,000 � g for 1 min at 4 �C. Following collection ofsupernatant which contained cytoplasmic fraction, the pellet wasresuspended in 20 ml of nuclear extraction buffer (20 mMHepes pH

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Fig. 2. Effects of DAMC (100 mM for 24 h) on growth inhibition in BMG-1 cells (A) and KB cells (B). Data presented here are the mean observations from three independentexperiments. (C) Induction of apoptosis by DAMC measured by flow cytometer showing the presence of sub G0 � G1 (hypodiploid) population is indicative of high apoptotic celldeath in BMG-1 cells than KB *P < 0.01. (D) AnnexinV-FITC binding in BMG-1 and KB cells showing vehicle treated control and DAMC treated cells (100 mM DAMC for 24 h).

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Fig. 3. Effects of DAMC on; (A) ROS generation and (B) mitochondrial membranepotential (Djm) in BMG-1 and KB cells. Cells were vehicle treated (0.1% DMSO) ortreated with 100 mM DAMC for 24 h before measurements by flow cytometry. Thirtyminutes prior to harvesting, cells were incubated with 10 mg/ml DCFH-DA for ROS and40 nM DiOC6 for Djm. After incubation, cells were harvested, and change in fluores-cence was measured using flow cytometry. The data shown are the representatives ofthree independent experiments.

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7.9, 0.42 M NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 1 mM DTT, 25%glycerol and protease inhibitor cocktail) and vortexed vigorously at4 �C for 30 min. The suspension was centrifuged at 14,000 � g for20 min at 4 �C. The supernatant (nuclear fraction) was collected.Protein content in both fractions was determined using BCA proteinassay kit [27].

2.15. Activation kinetics of NADPH-cytochrome c reductase assay

For the reductase assay, the cells were sonicated in presence ofprotease inhibitor cocktail. 50e70 mg protein was mixed withDAMC (10 mM), 0.05 M phosphate buffer (pH 7.7), and water tomake 0.5 ml volume. The contents were reincubated at 37 �C for10 min. The aliquots were removed periodically into a spectropho-tometer cuvette containing 0.1 mM EDTA, 36 mM cytochrome c,and 1 mM NADPH in a total volume of 1 ml. The progress of thereaction was monitored by following the absorption at 550 nm. Inthe control, DAMC was replaced by DMSO. The increment inreductase activity due to DAMC over the control was expressed aspercent activation [28,29].

3. Results

3.1. Cytotoxic effects

The cytotoxic effect of DAMC was first investigated by analyzingthe clonogenicity using macrocolony assay in BMG-1 cells (low CRT

level) and KB cells (high CRT level) (Fig. 1A, B). Exposure of cells toDAMC for 24 h induced concentration dependent cytotoxicity inboth the cell lines with more than 4 fold difference in the toxicity at100 mM, where the survivability of sensitive BMG-1 cells was 20%compared to 80% in the relatively resistant KB cells (Fig. 1B). Themetabolic viability corroborated the changes in clonogenicity, withnearly 20% viability in BMG-1 cells as compared to 45% in case of KBcells under these conditions (Fig. 1C). These results show that thedifferential cytotoxicity of DAMC in both the cell lines is inverselycorrelated with the CRT levels.

3.2. Cell proliferation and cell death

Treatment of cells with DAMC for 24 h inhibited the growth,with extent of growth inhibitionwas higher (80%) in BMG-1 cells ascompared to KB (13%), similar to the effects on metabolic viabilityand clonogenic survival; BMG-1 to be more sensitive than KB(Fig. 2A, B). The drug exposure also induced a fraction of cellssusceptible to death as indicated by the presence of hypodiploidDNA content suggesting degeneration of cells due to apoptosis and/or necrosis (Fig. 2C). On the other hand, KB cells were relativelyresistant to DAMC as insignificant level of hypodiploid DNA contentwas observed. Analysis of cells under these conditions withannexin V revealed a higher fraction of DAMC treated BMG-1 cellstesting positive to annexin V suggesting induction of apoptosis,while the fraction of annexin V positive KB cells was found to benegligible (Fig. 2D). These results suggest that the DAMC has

Fig. 4. (A) Effects of DAMC on the protein expression of calreticulin, NFkB and Bax in BMG-1 and KB cells. 2.5 � 105 cells/well of PD 35 were treated with vehicle (0.1% DMSO) orwith 100 mM DAMC for 24 h. Total cell lysates were prepared and protein (60 mg) was subjected to SDS-PAGE followed by immunoblot analysis using specific antibodies andsecondary horseradish peroxidase-conjugated antibodies. b-Actin was used as loading control. (B) Immunocytochemistry of NFkB (immunostaining with anti-p65 antibody) invehicle and DAMC treated BMG-1 and KB cells. (C) Western blot analysis for nucleo-cytoplasmic localization of NFkB in BMG-1 and KB cells after exposure to DAMC (100 mM; 24 h).Laminin b2 and b-Actin are used as loading control for nucleus and cytoplasm respectively.

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a potential of inducing apoptosis in tumor cells (BMG-1) whereasthe cytotoxicity could differ among tumor cells.

3.3. Pro-oxidant effects

Since the parental molecule, coumarin that can induce pro-oxidant effects could be responsible for the cell death; we inves-tigated the oxidative stress induced by DAMC in these cells. TheROS measurement using DCF-DA staining showed a decrease withDAMC in both the cells under present experimental conditions(Fig. 3A). Interestingly, the earlier reports suggest that DAMCinduces ROS production in MDA-MB and A549 cells suggestingthereby that the pro-oxidant effect of DAMC could be cell typedependent and experimental conditions [22,27]. We also investi-gated the mitochondrial status following exposure to DAMC byDiOC6 staining, and found a significant decrease in the meanfluorescence intensity of BMG-1 cells suggesting that DAMCinduced damage leads to loss of mitochondrial membrane potentialFig. 3B, under these conditions. On the other hand only a marginalchange was observed in KB cells thereby correlating with extent ofdamage and cell death Fig. 3B.

3.4. Alterations in protein levels

We next examined the effects of DAMC on CRT as well as NFkBand Bax (proteins known to mediate pro-survival and pro-deathresponses in cells). Significant changes were not observed in thecalreticulin levels in both the cells following DAMC treatment(100 mM; for 24 h). On the other hand, the levels of NFkB (a pro-survival protein) were significantly reduced in BMG-1 cells, whilean increase was observed in KB cells (Fig. 4A). The immuno-fluo-rescence and western blotting studies in cells revealed a high levelof nucleo-cytoplasmic accumulation of NFkB following exposure toDAMC in KB cells as compared to BMG-1 cells Fig. 4B, C. Further,DAMC enhanced the level of Bax (pro-apoptotic protein) in BMG-1cells co-relating well with the cytotoxicity data whereas a decreasewas observed in KB cells. These results suggest that presence ofDAMC in cells with different CRT levels can specifically alter theexpression and localization of pro-survival proteins like NFkB,whose function is reported to be tightly regulated by acetylation.

3.5. Effects of CRT knock down (using siRNA) on DAMCinduced cytotoxicity

To validate the role of CRT in the resistance of KB cells to DAMCinduced cytotoxicity, we used CRT knockdown by siRNA tomanipulate CRT expression in these cells. Contrary to earlierobservation (Fig. 1B) DAMC induces a significant cytotoxicity afterCRT knockdown (using siRNA) in KB cells, measured by macro-colony assay (Fig. 5A, B). However, no significant change in thesurvival was observed in DAMC treated BMG-1 cells (having lowendogenous CRT levels) on incubation with CRT siRNA. Theseresults suggest that CRT level strongly influences DAMC inducedcytotoxicity.

3.6. Effects of CRT over expression on DAMC induced cytotoxicity

To investigate explicitly the influence of CRT in cytotoxic effectsof DAMC, we studied its effects in isogenic clone of BMG-1 cellsover-expressing CRT (CROE) (Fig. 6A). BMG-1 was chosen becausethey expressed relatively low endogenous levels of CRT comparedto KB cells Fig. 1A. Metabolic viability studies revealed that theCROE cells were relatively resistant to DAMC (IC50∼55 mM)compared to the parental cells (IC50∼38 mM; Fig. 6B). Changes in theclonogenic survival and growth observed under these conditions

corroborated with the results of MTT assay, with a higher level ofsurvival in CROE cells at all DAMC concentrations (Fig. 6C, D).Moreover, the fraction of annexin V positive cells (4%) (Fig. 6E) issuggestive of apoptosis which is significantly lower than theparental BMG-1 cells (20%) Fig. 2C.

3.7. Alterations in protein acetylation and activity

We further investigated the CRT and DAMCmediated changes inprotein acetylation by comparing the total protein acetylationstatus in CROE and wild type cells using PAN acetylase antibody.Immunoblots showed a marginal increase in the gross endogenousprotein acetylation of CROE cells compared to the wild type cells(Fig. 7A). A decrease in the acetylation status of proteins wasobserved in the parental BMG-1 following exposure to DAMC,which was very evident in few proteins shown at positions indi-cated (Fig. 7A). Interestingly, the degree of acetylation of proteinswas higher in CROE cells at the same positions (Fig. 7A). Moreover,the levels of pro-survival protein NFkB remained unaltered in CROEcells (Fig. 7B) which was in contrast to the parental BMG-1 cells, butsimilar to KB cells (Fig. 4A, B) with high endogenous CRT. Theseresults strongly support the proposition that CRT has the ability tochange the acetylation status of cells (in presence of DAMC) thatcan be partially attributed to its transacetylase function. Tounequivocally establish the role of CRT in modifying the acetylationof proteins by using DAMC as an acetyl group donor, in vitro analysiswas carried out by measuring the activity of NADPH-cytochrome creductase (an ER marker enzyme localized in the membrane of ER).Acetylation of this enzyme (NADPH-cytochrome c reductase) isknown to be primarily responsible for its activity, which ismeasured colorimetrically (absorption measured at 550 nm) by theconversion of oxidized form of cytochrome c to reduced form. A

Fig. 5. Effect of calreticulin knock down on DAMC induced cytotoxicity in KB andBMG-1 cells; (A) Identification of the efficiency of CRT small interfering RNA (siRNA) bywestern blot analysis. Scrambled double-stranded RNA (dsRNA) is used as a control ofCRT siRNA. (B) Effect of calreticulin knockdown by siRNA in DAMC (100 mM; 24 h)treated BMG-1 and KB cells measured by macrocolony assay *p value<0.05, #pvalue<0.01.

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significant increase in the activity of cyto c reductase was observedfrom the cell lysate of CROE cells in presence of DAMC, showinghigh cyto c reduction as compared to wild type cells (Fig. 7C).Collectively these results clearly demonstrate that CRT can modu-late the acetylation levels of cells and alter the activity of substrateproteins (NADPH-cytochrome c reductase) which is accentuated inpresence of DAMC that can be attributed to CRT: polyphenolicacetates mediated acetylation.

4. Discussion

The acetylation of proteins in cells is catalyzed by histone ace-tylase transferases (HATs) using acetyl CoA as the acetyl groupdonor [30]. The present study explores the existence of a novelacetyl CoA independent protein acetylation system in situ (i.e. incells) mediated by CRT and DAMC. Since CRT is over expressed incertain cancers [31] understanding the role of CRT transacetylasefunction may delineate its physiological importance and serve asa target for developing therapeutics and/or adjuvant.

The results clearly show that the cytotoxic effects of DAMC isindeed related to the CRT levels, with BMG-1 cells having low CRTlevel being more sensitive than KB cells with a higher CRT level(Figs. 1 and 2) substantiating the earlier in vitro findings showingacetylation of proteins by CRT. Further, the induction of cytogenetic

damage in the form of micronuclei (data not shown) and apoptosis(Fig. 2D) observed in BMG-1 cells indicates the anticancer potentialof DAMC [22,27]. Together with relatively lesser normal tissuetoxicity (by way of damage to PBMCs) observed earlier [14,27]DAMC has the potential to be developed either as an anticancertherapeutic or adjuvant to radiation and/or chemotherapy.However, a lower degree of cytotoxicity in KB cells (high CRT level)as compared to BMG-1 cells (low CRT levels) suggests that high CRTlevels can indeed compromise the cytotoxic effects of DAMC incells. This could either be due to differences in the pro-oxidantactivity of DAMC or due to the modulation of cell signaling path-ways that may differ among tumor cells [32]. However, DAMC didnot significantly enhance ROS levels in both these cells (BMG-1 andKB; Fig. 3A) suggesting that changes in ROS levels did notcontribute to the differential cytotoxicity. Interestingly, DAMClowered the mitochondrial potential that was more prominent inBMG-1 cells compared to KB cells, which correlated with differ-ences in the cytotoxicity between these two cell lines. These resultssuggest that DAMC induced cell death is mitochondrial dependentand ROS independent similar to that observed in A549 cells earlier[27]. Alternatively, the differential cytotoxicity could also be relatedto altered cell signaling due to disturbances in the calciumhomeostasis and induction of unfolded protein response (UPR)which is intimately linked to CRT levels [4,33].

Fig. 6. A comparative study between BMG-1 and isogenic clone of BMG-1 cells over-expressing calreticulin (CROE cells) to cytotoxic effects of DAMC (24 h). (A) Western blotanalysis of expression of CRT in BMG-1 and CROE cells. Dose dependent response as evaluated by (B) MTT assay and (C) Clonogenecity. (D) DAMC (100 mM for 24 h) induced relativegrowth inhibition (%) at 48 h after drug exposure in BMG-1 and CROE cells. (E) AnnexinV-FITC binding in BMG-1 and CROE cells showing vehicle treated control and cells treatedwith 100 mM DAMC for 24 h. The data shown are the representatives of three independent experiments.

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The activation of inducible NFkB transcription factor (a pro-survival protein) complex by acetylation plays a central role inregulating the inflammatory, immune, and anti-apoptoticresponses inmammals [25,27,34e40]. The results demonstrate thatDAMC increases NFkB level including its nucleo-cytoplasmic accu-mulation and perhaps initiates a pro-survival response in KB cellswith high CRT levels resulting in a better survival as compared toBMG-1 cells with low CRT (Fig. 4A, B, C). Our results on the levels ofBax observed in presence of DAMC supports this proposition asa reduction in the level of NFkB was accompanied by a higher levelof Bax in BMG-1 cells, whereas in KB cells an increase in NFkB levelsdid not significantly alter Bax levels. This suggests that variations inthe levels of CRT can alter the overall cellular response to DAMC.NFkB appears to be at least one of the target proteins likely to playa role in regulating either a pro-survival or pro-death response [41].

Since KB and BMG-1 cells are of different origin and may havedifferences in the biological behavior, we used different approachesto establish the casual relationship between CRTand DAMC toxicity.Firstly, weknockeddownCRTusing siRNA in KB cells (with high CRTlevels) and on the other generated an isogenic clone of BMG-1 cells

(having low endogenous CRT) by ectopically over-expressing CRT(CROE cells) and examined the cytotoxic effects of DAMC. Unlike inthe parental cells, CRT siRNA significantly enhanced the DAMCinduced cytotoxicity in KB cells where CRTover expression in BMG-1 cells (CROE) reduced DAMC induced cell death as compared to theparental BMG-1 cells (Fig. 5B). Further, the increase in resistance ofCROE cells to DAMC was accompanied by a decrease in NFkB inhi-bition compared to parental cells (Fig. 6AeD). This strongly impli-cates a role for CRT in modulating the cytotoxicity of DAMC in cellsand suggests NFkB as a target protein likely to be activated by CRTand DAMC mediated acetylation.

Calreticulin has been shown to acetylate proteins using bothacetyl CoA and DAMC in vitro and share some of the properties oftype B acetyl transferases [42]. Marginal increase in the endoge-nous protein acetylation status of CROE cells (Fig. 7A) and higherlevel of acetylation of the low molecular weight proteins (<25 kD;which includes histones) in DAMC treated CROE cells (Fig. 7B)suggests that the CRT-DAMC mediated protein acetylation hasfunctional implications in cells including possible effects on chro-matin remodeling, a vital nuclear process involved in proliferation,differentiation and DNA repair. HDAC inhibitors that also createa hyperacetylation condition in the tumor cells promoting cell cyclearrest, apoptosis and cellular differentiation are currently underclinical trials as therapeutics as well as adjuvant in cancer therapy[43,44]. Therefore, CRT mediated protein acetylation that simulateeffects similar to HDAC inhibitors offers yet another target fordeveloping anticancer therapeutics as well as adjuvant that maysynergize with the effects of conventional anticancer treatmentslike radiation and chemotherapeutic agents. The in vitro resultsshowing increase in the cytochrome c reductase activity in theextracts of calreticulin over-expressing cells (before and aftertreatment with DAMC), similar to earlier studies with recombinantenzyme lends further support to this proposition (Fig. 7C) [19].Studies related to specificity of this new acetylation system and itsrelationship with histone acetyl transferase (HAT) and calciumlevels in situ are in progress.

Taken together, the circumstantial evidences presented heresuggest a relationship between DAMC induced cytotoxicity and CRTlevels in cells that may influence acetylation status of proteinssupporting the earlier suggestions of transacetylase function forCRT based on in vitro studies [10]. The study also defines the abilityof DAMC to act as a signal transduction modulator apart from itsantioxidant function. Further understanding of the mechanismsunderlying CRT and DAMC mediated regulation of cell signaling islikely to help in developing approaches that target CRT, whose overexpression in tumors partly contributes to resistance against radi-ation and chemotherapy.

Acknowledgements

This work was supported by grants from by the project INM 301,311/1.4 funded by Defence Research and Development Organisa-tion (DRDO), Government of India. A. V. was supported withfellowship from Indian Council of Medical Research (ICMR).We alsoacknowledge Yogesh Tyagi and Rohit Mathur for their help.

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Fig. 7. Effects of DAMC on; (A) Over all protein acetylation status and (B) Expression ofNFkB in BMG-1 and CRT over-expressing CROE cells. 2.5 � 105 cells/well of PD 35 weretreated with vehicle (0.1% DMSO) or with 100 mM DAMC for 24 h. Total cell lysateswere prepared and protein (80 mg/well) was subjected to SDS-PAGE (15%) followed byincubation with PAN acetylase antibody (1:200) and NFkB (1:1000). PAN acetylaseantibody recognizes variety of acetylated proteins including histones. b-Actin was usedas loading control. (C) Irreversible activation of NADPH-cytochrome c reductase byDAMC (10 mM for 10 min) in BMG-1 and CROE cells. The control received DMSO inplace of DAMC. The increament in the activity of NADPH-cytochrome c reductase dueto pre-incubation with DAMC over the control is expressed as O.D. at 550 nm. Valuesare means of three observations.

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