role of erbb2 mediated akt and mapk pathway in gall bladder cell proliferation induced by argemone...

ORIGINAL RESEARCH

Role of ErbB2 mediated AKT and MAPK pathway in gallbladder cell proliferation induced by argemone oiland butter yellow

Argemone oil and butter yellow induced gall bladder cell proliferation

Vivek Mishra & Kausar M. Ansari & Raj Khanna &

Mukul Das

Received: 4 August 2011 /Accepted: 11 December 2011 /Published online: 13 March 2012# Springer Science+Business Media B.V. 2012

Abstract The effect of noncytotoxic doses of argemoneoil (AO) and butter yellow (BY), the common adulter-ants in edible oil, on free radical generation and signalingpathway for cell proliferation in primary cells of gallbladder (GB) was undertaken. AO and BY showed nocytotoxicity at 0.1 μl/ml and 0.1 μg/ml concentration,respectively. AO caused significant increase in ROS after30 min and RNS after 24 h in GB cells while no changewas observed following BY treatment. Enhanced levelof COX-2 was observed following AO (0.1 μl/ml) andBY (0.1 μg/ml) treatment to cells for 24 h. AO treatmentcaused phosphorylation of ErbB2, AKT, ERK, and JNKalong with increased thymidine uptake indicating cellproliferation ability in GB cells. BY treatment alsoshowed significant expression of these proteins withthe exception of phosphorylated JNK. These resultssuggest that AO and BY have cell proliferative potentialin GB cells following up-regulation of COX-2 andErbB2; however, their downstream signaling molecules

and free radical generation have differential response,indicating that the mechanism of proliferation is differentfor both compounds and may have relevance in gallbladder cancer.

Keywords Gall bladder primary culture .

Cytotoxicity . Cell proliferation .Mice

AbbreviationsAO Argemone oilBY Butter yellowEMEM Eagle's minimum essential mediumMEM Minimum essential mediumHBSS Hank's balanced salt solutionFBS Fetal bovine serumLDH Lactate dehydrogenaseMTT 3-(4,5-dimethyl-2-yl)-2,5-dipehyl

tetrazolium bromideDMSO Dimethyl sulphoxidePBS Phosphate buffer salineErbB2 Neu proto oncogeneCOX-2 Cyclo oxigenase-2AKT Serine threonine kinaseERK Extracellular signal regulated kinaseJNK Jun-amino terminal kinase

Introduction

Primary carcinoma of the gall bladder represents ahighly lethal and aggressive malignant tumor because

Cell Biol Toxicol (2012) 28:149–159DOI 10.1007/s10565-011-9207-5

V. Mishra :K. M. Ansari :M. Das (*)Food Drug & Chemical Toxicology Group, CSIR-IndianInstitute of Toxicology Research,Council of Scientific and Industrial Research,Post Box#80, Lucknow 226 001, Indiae-mail: [email protected]

M. Dase-mail: [email protected]

V. Mishra :R. KhannaDepartment of Biochemistry, University of Lucknow,Lucknow, India

of its dormancy course, difficult diagnosis, early me-tastasis, strong invasion, and poor prognosis (Misra etal. 2003). The occurrence of gall bladder cancer(GBC) is common among all gastrointestinal malig-nancy in the Asia-Pacific region (Batra et al. 2005;Dhir and Mohandas 1999). The incidence of GBC isrelatively higher in the northern parts of India ascompared to other regions of the country (ICMR1993). Several dietary risk factors and environmentalexposure to chemical carcinogens have been speculatedfor gall bladder cancer (Obara et al. 1999; Fortner 1955).

Edible oils and fats serve as the only source of essen-tial fatty acids, add special flavors to food, and maintaincell membrane integrity (Wharton 1980). Mustard oilfrom Brassica nigra seeds is the predominant cookingmedium used in the northern parts of India, especially theGangetic basin (Khan 2001). This commodity hasattractedmaximum scope for adulterationwith argemoneoil (AO) and butter yellow (BY) (Das 2008). AO con-taminated mustard oil even for a short duration leads to aclinical condition collectively referred to as EpidemicDropsy (Das and Khanna 1997, 1998). Colorless cheapoils are mixed with a fat soluble synthetic dye butteryellow, 3′-methyl-4-dimethyl-aminoazobenzene andsold under the pretext of mustard oil. Butter yellowinteracts with macromolecules like DNA and proteinsto initiate genotoxic and mutagenic responses (Khannaand Das 1991). It has been found to produce hepatic andskin tumors as well as cancer in respiratory tract (Ashbyet al. 1983; Zabezhinskii et al. 1984).

It is hypothesized that the possible adulterants AOand BY in mustard oil could be one of the causativefactors of GBC. Since enhanced proliferation is one ofthe primary events in carcinogenesis, it was thoughtrelevant to study the proliferation of primary cell cul-ture of gall bladder by AO and BY and the involvedmechanism there in.

Materials and methods

Chemicals

Collagenase (type I), trypsin-EDTA (0.25%), collagen(type I) from calf skin, Eagle's minimum essential me-dium (EMEM or MEM), Hank's balanced salt solution(HBSS), fetal bovine serum (FBS), trypan blue, 3-(4,5-dimethyl-2-yl)-2,5-dipehyl tetrazolium bromide (MTT),2′,7′-dichlorofluroscein diacetate (DCFH-DA), and 4-

methylaminoazobenzene (butter yellow, purity>97%)were procured from Sigma-Aldrich Chemical Co. (St.Louis, MO). Alamar blue assay kit and methyl-3Hthymidine were purchased from Krishgen Biosystem(Mumbai, India,) and Amersham Biosciences (LittleChalfont, Buckinghamshire, UK), respectively.Primary antibodies against p-ErbB2 (Tyr 1248),ErbB2, p-AKT (Ser 473), p-ERK (Tyr 240), p-JNK(Thr183/Tyr 185), COX-2, β Actin and secondary anti-bodies were purchased from Santa Cruz Biotechnology(Santa Cruz, CA). Tissue culture dishes were obtainedfrom Tarson Products Private Limited, (Kolkata, India).Argemone mexicana seeds were procured from the out-skirt of Lucknow city, Uttar Pradesh, India. The seedswere crushed and the oil was extracted with the help ofSoxhlet apparatus using n-hexane (Upreti et al. 1991).The hexane containing argemone oil was filtered undervacuum through a Buchner funnel containing glasswool, and the solvent was distilled at 30°C under vacu-um in Buchii Rotavapor-R. The oil obtained was storedin amber glass bottle under nitrogen atmosphere. Theyield of argemone oil from its seeds was 35% (v/w).Since both argemone oil and butter yellow were solubleinDMSO, it was used as vehicle in experimental studies.

Isolation and culture of cells from gall bladderof Swiss albino mice

Gall bladder cells were isolated from six Swiss Albinomice of 8 to 12 week old essentially by the method ofKuver et al. (1997). Gall bladder were excised andwashed with cold phosphate-buffered saline (PBS).The tissue was chopped with a sharp scissor inEagle's Minimum Essential Medium (EMEM) andcentrifuged at 1,500 rpm for 2 min to obtain pellet.Pellet was treated with 15 ml of 0.1% collagenase for20 min and centrifuged at 1,500 rpm for 2 min. Afterwashing with PBS twice, pellet was mixed with 5 mlof 0.25% trypsin/ 0.1% EDTA, followed by incubationfor 20 min at 37°C. The samples were centrifugedagain and supernatant was discarded. The pellet wassuspended in 5 ml of EMEM containing 10% FBS,2 mM L-glutamine, 100 IU penicillin/ml, 100 μgstreptomycin/ml, 20 mM HEPES, 1.0 g glucose/l,supplemented with 5 μg insulin/ml from bovine pan-creas, 5 μg human transferrin/ml, 5 ng sodium sele-nite/ml (ITS supplement), 1× vitamins solution, and1× nonessential amino acid solution (Sigma-AldrichChemical Co, St. Louis, MO) and plated on collagen

150 Cell Biol Toxicol (2012) 28:149–159

coated plates. The seeding density of gall bladderepithelial cells was 1×106. The viable gall bladdercells (95% by Trypan blue exclusion method) werekept at 37°C humidified incubator (5% CO2/95% air).Twenty-four hours after plating, the unattached epithe-lial cells were removed, and the fresh medium wasreplaced (Kuver et al. 1997). The gall bladder cellsafter 24 h of plating were treated with different con-centrations of AO (0.1, 1, 2.5 and 5 μl/ml) and BY(0.1, 1, 10, and 100 μg/ml) for 24, 48, and 72 h.

Measurement of MTT assay

The cytotoxic effects of AO and BYon gall bladder cellswere determined by the MTT dye uptake method(Darzynkiewicz et al. 1992). Briefly, the cells wereincubated at different time periods in the presence andabsence of test sample (triplicate, in a final volume of0.1 ml) in a 96-well plate at 37°C. A 10-μl MTT (5 mg/ml PBS) was then added to the wells, and the plateswere further incubated for 4 h. The plate was centrifugedat 1,200 rpm for 10 min, and 100 μl of DMSO wasadded after removing the supernatant to dissolve theformazan formed. The absorbance was read at 530 nmafter 5 min in a microplate reader (Synergy HT of BIO-TEK International, Winooski, VT).

Measurement of lactate dehydrogenase in the medium

The leakage of the cytosolic enzyme, lactate dehydro-genase (LDH), into the medium following exposure ofAO and BY to primary culture of gallbladder cells wasassayed according to the method of Kornberg (1955)to study the cytotoxicity. Media from treated and con-trol plates were collected and centrifuged to pellet thecells. The clear medium was used for measurement ofLDH activity by calculating the reduction of pyruvateto lactate. The reduction is coupled to the oxidation ofnicotinamide adenine dinucleotide, reduced form(NADH) to nicotinamide adenine dinucleotide, oxi-dized form (NAD+), which is read at 340 nm. Thepercent LDH release was calculated by the formula:

% LDH release ¼ Test Sample � Low Controlð ÞHigh Control� Low Controlð Þ � 100

Low Control: Normal cells in 200 μl assay mediumHigh Control: Normal cells in 200 μl assay medium

containing 1% Triton X-100

Test Sample: Normal cells in 200 μl assay mediumcontaining test substance.

Alamar blue assay

The Alamar blue assay was performed to further vali-date the cytotoxic response of AO and BY in gallbladder cells. In this technique, Alamar blue detects cellviability by utilizing a blue and non-fluorescent dye,resazurin. Control or treated gall bladder cells wereincubated with 10 μl of Alamar blue solution overnightat 37°C. The fluorescent intensity was measured atrespective excitation and emission wavelength of 530and 590 nm, on a microplate reader (Synergy HT ofBIO-TEK International, Winooski, VT).

Measurement of reactive oxygen species

The generation of reactive oxygen species (ROS) wasdetected by the method of Wang et al. (1996) using 2′,7′-dichlorofluroscein (DCF) as a fluorescent probe. Thecells were incubated with 2′, 7′-dichlorofluroscein diac-etate (DCFH-DA; 100 μM final concentration) for60 min in dark at 37°C. The cells were harvested,suspended in PBS, and ROS generation was measuredin terms of fluorescence intensity (FL-1, 530 nm) of10,000 cells on flow cytometer (BD FACS Canto IIFlow cytometer, BD Biosciences, San Jose, CA).

Measurement of reactive nitrogen species

The concentration of stable nitrite, the end product fromNO generation, was determined by the method of Dinget al. (1988) using Griess reaction. Griess reagent isbased on a chemical reaction that uses sulfanilamideand naphthylethylenediamine dihydrochloride (NED)under acidic (phosphoric acid) conditions. This systemdetects NO2− in a variety of biological and experimentalliquid matrices such as plasma, serum, urine, and tissueculture medium. Nitrite content was determined in con-trol, positive control (PC; lipo polysaccharide), AO(0.1μl/ml), and BY (0.1μg/ml) treated cells on a micro-plate reader (Synergy HT of BIO-TEK International,Winooski, VT).

Western blot analysis

Gall bladder cell lysates were prepared from each group(Control, AO and BY) and were electrophoresed

Cell Biol Toxicol (2012) 28:149–159 151

through 6% SDS-polyacrylamide gels for Neu protooncogene (p-ErbB2 and ErbB2). Other proteins includ-ing cyclooxigenase-2 (COX-2), phosphorylated serinethreonine kinase (p-AKT), phosphorylated extracellularsignal regulated kinase (p-ERK), and phosphorylatedjun-amino terminal kinase (p-JNK) in cell lysates wereelectrophoresed on 10% SDS-polyacrylamide gels.Separated proteins were electrophoretically transferredonto polyvinylidene difluoride (PVDF) membranes.

After blocking the membranes with 1% BSA inTBS-T [20 mmol/L Tris, 150 mmol/L NaCl(pH 7.5), 0.02% Tween 20] for 1 h at room tempera-ture, the membrane was incubated with primary anti-bodies of ErbB-2, p-ErbB-2 (1:500), phosphorylatedAKT (1:1,000), phosphorylated JNK (1:1,000), phos-phorylated ERK 1/2 (1:1,000), and COX-2 (1:1,000)and kept in refrigerator overnight. Each blot waswashed with buffer for 25 min and incubated with

a

AO (0.1 ul/ml) 48 hrControl

b

AO (1µl/ml)Control

Fig. 1 Effect of AO on themorphology of primary gallbladder cell culture. a AO(0.1 μl/ml) showing well-adhered gall bladder cells onplate up to 48 h. b AO(1 μl/ml) showing detach-ment and formation of clus-ters of dead gall bladdercells at 48 h


1:2,000 dilutions of anti-mouse horseradish peroxi-dase conjugated secondary antibody for 1 h at roomtemperature. The blots were detected by the enhancedchemiluminescence substrates (Sebolt-Leopold et al.1999). Densitometric values of specific protein bandswere calculated after normalizing the values of β-actin bands in corresponding samples. All experi-ments were done in triplicate.

[3H] thymidine incorporation measurement

To measure cell proliferative rate, 1.0×106 cells wereseeded in culture flask in 5 ml of complete mediumwith AO (0.1 μl/ml) or BY (0.1 μg/ml) and incubatedor 72 h at 37°C in a CO2 incubator. [3H] thymidine(2 μCi) was added to the flasks, 18 h prior to thecompletion of 72 h of incubation time. The cells were

a

Control BY (0.1 ug/ml) 48 hr

BY (1 µg/ml ) Control

b

Fig. 2 Effect of BY on themorphology of primary gallbladder cell culture. a BY(0.1 μg/ml) showing well-adhered gall bladder cells onplate up to 48 h. b BY(1 μg/ml) showing detach-ment and formation of clus-ters of dead gall bladdercells at 48 h


collected with the help of harvester and incorporatedradioactivity was measured in a liquid scintillation

counter (Packard Bioscience Company, Meriden,CT).

120 24 hr 48 hr 72 hrMTT assaya

80

100

60

40

20

0MT

T u

pta

ke r

edu

ctio

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0.1 1 2.5 5 0.1 1 10 100%

Argemone oil Butter yellow(µl/ml)

7080

24 hr 48 hr 72 hrb LDH assay

6070 b y

4050

3040

1020%

LD

H r

elea

se

010

0.1 1 2.5 5 0.1 1 10 100

Argemone oil Butter Yellow

9000 24 hr 48 hr 72 hrAlamar blue assayc70008000 c

ce

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5000

uore

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ity

30004000

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en

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el

0Control 0.1 1 2.5 5 0.1 1 10 100

Argemone oil Butter yellow)lm/gµ()lm/lµ(

(µl/ml) (µg/ml)

(µg/ml)

Fig. 3 Effect of AO and BY on cytotoxicity in gall bladdercells, aMTTassay, b LDH assay, and c Alamar blue assay. Cellswere incubated with different concentrations of AO and BY for24–72 h. Different assays were performed, the details of which

are mentioned in “Material and methods”. Each value representsmean±S.E. (n03). *p<0.05, **p<0.01. Significant when com-pared to respective control


Statistical analysis

All results were expressed as the mean±standard error(SE), as indicated in the Figures. Statistical analysis ofvariance was carried out using one-way ANOVA(Snedecar and Cochran 1967). A value of p<0.05was used as the level of significance.

Results

The gall bladder cells tend to flatten out to form amonolayer on collagen coated plates during first 24 h.The cells viability was found to be 90–95% by trypanblue exclusion method after 24 h of plating.

Effect of AO and BY on the morphology of gallbladder cells

Incubation of gall bladder cells with AO (0.1 μl/ml)for 12–48 h of incubation showed no changes inmorphology of cells (Fig. 1a), while detachment ofcells along with clusters of dead cells were observed athigher concentrations of AO (1 μl/ml) incubated for

48 h (Fig. 1b). Similarly, BY (0.1 μg/ml) treatmentshowed no change in morphology during 12–48 h ofincubation (Fig. 2a). However, morphological changesin gall bladder cells in terms of detachment and clusterformation were observed when BY concentration wereincreased (1 μg/ml) (Fig. 2b). These results indicate thephenotypic changes in gall bladder cells by AO and BYat concentration of 1 μl/ml and 1 μg/ml, respectively.

600ol

) ROS estimationa500

ity

ontr

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ier

c

300nt in

te

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200

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nt c

100

Flu

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15min 30min 1hr 6hr 12hr

600 b RNS estimation

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400duct

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300

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6hr 18hr 24hr 48hr

Fig. 4 Generation of free radicals at non-cytotoxic concentra-tions of argemone oil (0.1 μl/ml) and butter yellow (0.1 μg/ml)in gall bladder cells, a ROS and b RNS. MnCl2 (100 μM) andLPS (1 μg/ml) were used as positive control (PC) for ROS andRNS generators, respectively. Data is shown as percent changeover control. Each value represents mean±S.E. (n03). *p<0.05,Significant when compared to respective control

a1 1.7 2.6 3.9 5 5.2 5.1Fold change

COX-2AO (0.1µl/ ml

Actin

Con 3 6 12 24 48 72Time (hours)

F ld hCOX-2

1 2 3 4 6 5.8 6o c ange

BY (0.1µg/ml)Actin

Con 3 6 12 24 48 72Time (hours)

b1 2.1 1.8Fold change

p-ErbB-2

ErbB-2

P-AKT1 3.0 3.2

1 4.4 2.1P-ERK

1 2 2 0 8P-JNK

. .

ActinControl AO BY

1600 c1400

1600

tio

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1200

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800

1000

inco

g p

rot

600idin

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/mg

400

Thym

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0

200

[3H

]T

0

Control AO BY

Fig. 5 Effect of AO and BYon cell signaling proteins and [3H]thymidine uptake in gall bladder cells. a Expression of COX-2in AO (0.1 μl/ml) and BY (0.1 μg/ml) exposed gall bladder cellsat different time points (3–72 h).b Expression of signalingproteins, p-ErbB2 (Tyr 1248), p-Akt (Ser 473), p-ERK (Tyr240), p-JNK (Thr 183/Tyr 185) in AO (0.1 μl/ml) and BY(0.1 μg/ml) exposed gall bladder cells at 24 h. c [3H] Thymidineuptake in AO (0.1 μl/ml) and BY (0.1 μg/ml) exposed gallbladder cells at 24 h


Effect of AO and BY on cytotoxicity in gall bladdercells

The phenotypic changes observed by AO and BY werefurther confirmed by cytotoxic assays in gall bladdercells. The effect of AO and BY on MTT assay in gallbladder cells is shown in Fig. 3a. Significant MTTuptake reduction was observed following treatment withAO and BY at 1 μl/ml and 1 μg/ml concentrations andabove, respectively, during 24–72 h of incubation(Fig. 3a). However, 0.1 μl/ml AO and 1 μg/ml BY didnot show significant MTT reduction in gall bladder cellsindicating to be noncytotoxic doses of AO and BY. AOand BY exposure to gall bladder cells showed a dose-dependent release of LDH to medium. AO treatment at1 μl/ml for 48 h onwards showed significant increase inLDH. Similarly, BY treatment (10 μg/ml) for 48 h on-wards showed significant LDH leakage (Fig. 3b).However, AO (0.1 μl/ml) and BY (0.1 μg/ml) showedno significant effect on LDH release.

Alamar blue assay is one of themost sensitive tests forcytotoxicity because of fluorescent end point. Exposureof gall bladder cells to AO and BY showed significantdecrease in fluorescent intensity at 1 μl/ml and 1 μg/mlconcentrations onwards, respectively (Fig. 3c).However, noncytotoxic concentrations of AO and BYwere found to be 0.1 μl/ml and 0.1 μg/ml, respectively,which were similar to that derived byMTTassay. Hence,further experiments in gall bladder cells were performedat non-cytotoxic concentrations of AO and BY.

Measurement of reactive oxygen species

Generation of reactive oxygen species (ROS) by non-cytotoxic concentrations of AO and BY in gall bladdercells is shown in Fig. 4a. MnCl2 (100 μM), used aspositive control, showed significantly enhanced gener-ation of ROS (20–30%) up to 1 h, which was furtherenhanced (five- to sixfold) at 6–12 h. Significant en-hancement of ROS generation (2.7-fold) was observedat early time point (30 min) in AO-treated gall bladdercells, whereas no significant change in ROS productionwas observed in gall bladder cells when treated with BY.

Measurement of reactive nitrogen species

Generation of reactive nitrogen species (RNS) by thetreatment of non-cytotoxic concentration of AO and

BY is shown in Fig. 4b. LPS (10 μg/ml; final con-centration), which is a known enhancer of RNS pro-duction was used as a positive control. AO showedsignificant enhancement of RNS generation (fivefold)at 24 h; however, no significant change in RNSproduction was observed in BY treated gall bladdercells.

Effect of AO and BY on western blot of signalingproteins

To evaluate the expression of proteins involved in cellproliferation at different time intervals, western blot ofCOX-2 protein in AO (0.1 μl/ml) and BY (0.1 μg/ml)treated gall bladder cells was carried out at 3–72 h(Fig. 5a). AO (0.1 μl/ml) and BY (0.1 μg/ml) treatmentshowed a time-dependent enhancement in the levels ofCOX-2, with a maximum response from 24 to 72 h.From these experiments, 24 h was decided as the opti-mum time point for western blot analysis of differentother proteins involved in signaling pathways in gallbladder cells treated with AO (0.1 μl/ml) and BY(0.1 μg/ml) (Fig. 5b). Over-expression of COX-2 maylead to up-regulation of its downstream signalingmolecules via ErbB2 phosphorylation. Significant en-hancement in the level of phosphorylated ErbB2 (p-ErbB2) in AO (2.1-fold) and BY (1.8-fold)-treatedcells was observed, when compared to control cells.Further, overexpression of p-AKT (3-fold), p-ERK(4.4-fold) and p-JNK (2.2-fold) in case of AO-treated gall bladder cells, indicate their roles in cellproliferation. Significant overexpression of p-AKT(3.2-fold), p-ERK (2.1-fold) was observed in BYtreated cells, while no significant change in p-JNK(0.8-fold) expression was observed, suggesting theweaker response of BY for gall bladder cell prolif-eration compared to AO (Fig. 5b).

Effect of AO and BY on [3H] thymidine incorporationto DNA of gall bladder cells

The [3H] thymidine incorporation into DNA is a mea-sure of the proliferative capacity of the cells in re-sponse to chemical agents. A significant increase in[3H] thymidine uptake was observed in AO (70%) andBY (30%) treated gall bladder cells. These resultssuggest that AO and BY plays significant role in cellproliferation of gall bladder cells (Fig. 5c).


Discussion

Carcinoma of the gall bladder is the sixth most com-mon cancer with poor prognosis (Levy et al. 2001;Jemal et al. 2007; Ito et al. 2004). The potentialcurative therapy for the disease is still surgical resec-tion. However, no specific chemo-radiotherapy pro-gram for carcinoma of the gall bladder has emergedas the definitive acceptable standard of care and thereis much room for improvement (Misra et al. 2003;Taner et al. 2004; Jarnagin et al. 2003; Ishii et al.2004; Kresl et al. 2002). This is basically due to lackof etiological factors and understanding of the molec-ular mechanism of GBC. It is speculated that dietaryfactors could be responsible for gall bladder carcinoma(Obara et al. 1999). Since, our recent studies haveshown that AO and BY, the common oil adulterantsencountered in mustard oil, which is consumed inIndian sub-continent, population, induces GBC inmice (Mishra et al. 2011). Therefore, the present studywas aimed to investigate the proliferative potential ofAO and BY in primary culture of gall bladder cellsfrom Swiss albino mice.

The present study showed the concentration depen-dent cytotoxicity of gall bladder cells treated with AO(1–5 μl/ml) and BY (1–100 μg/ml). Similar doses ofAO were found to be cytotoxic in Sertoli germ cells(Mishra et al. 2009); and for BY in primary hepatocytes(Miyazaki et al. 1982). Significant loss of membraneintegrity and leakage of LDH in the medium was ob-served by AO and BY in a dose- and time-dependentmanner. These results were further validated by a moresensitive fluorescent-based method; Alamar blue assay,indicating that the non cytotoxic concentration of AOand BY, are 0.1 μl/ml and 0.1 μg/ml, respectively.

COX-2, one of the marker for proliferation, wasfound to be induced in AO (0.1 μl/ml) and BY(0.1 μg/ml) treated gall bladder cells during 12–72 hwith a maximum response at 24 h, thus the optimumtime point for further experiments was decided to be24 h. Up-regulation of COX-2, ErbB2 and its down-stream phosphorylated proteins (AKT, ERK and JNK)was found to be induced by AO. However, BY causesup-regulation of COX-2 and ErbB2 along with itsdownstream AKT and ERK phosphorylated proteins.It has been well documented that COX-2, an inducibleenzyme responsible for conversion of arachidonic acidto prostaglandins, is expressed at low concentration in

normal tissues but can be induced by a variety of cyto-kines, mutagens, hormones, growth factors and tumorpromoters (Taketo 1998). Cox-2 has also been found tobe overexpressed in a variety of gastrointestinal tumorsincluding colorectal, gastric, esophageal, and pancreaticcancers (Eberhart et al. 1994). Association betweenCOX-2 and ErbB-2 expression has also been reportedin several human cancer types, such as cholangiocarci-noma, colon cancer, and breast cancer (Endo et al. 2002;Ristimaki et al. 2002). Recent studies have shown thathigh expression of ErbB-2 contributes to cholangiocar-cinoma cell invasion and proliferation through AKTpathway (Treekitkarnmongkol and Suthipongchai2010). Overexpression of COX-2 and ErbB-2 alongwith AKT, ERK, and JNK as well as significant increasein the thymidine uptake byAO exposure suggest the cellproliferation in gall bladder cells. On the other hand,overexpression of all the tested signaling proteins ex-cluding p-JNK protein in BY treated gall bladder cellssuggests that the proliferation response by BY may beweaker than AO, which is further validated by uptake of[3H] thymidine in cells.

ROS, which include hydroxyl radical, superoxideanion, hydrogen peroxide, and singlet oxygen, aregenerated during early time period in response tocertain stimulants while RNS (NO) is mainly generat-ed by induction of iNOS in later stage and are ofteninvolved in inflammation (Fubini and Hubbard 2003).Studies have shown that ROS induces cell prolifera-tion either by mitogen-activated protein kinase(MAPK) or by inducing a specific protein, Romo1,in normal and cancer cells (Han et al. 2003; Na et al.2008). In the present study, it was observed that non-cytotoxic dose of AO (0.1 μl/ml) resulted in freeradical generation; ROS at 30 min while RNS at24 h. Studies of Hussain et al. (2007) also observedROS generation at early time point (2, 4 h) exposure tosanguinarine (main constituent of AO) in primaryeffusion lymphoma cells. Further, production of RNSis also enhanced by AO in dropsy patients (Babu et al.2010). It has been observed that BY does not showany production of ROS and RNS. This may be due tothe fact that electrophilic metabolites of BY directlyinteract with macromolecules (Steele and Boone1992). Our surveillance studies showed that the extentof AO adulteration in edible oil is up to 30%, whileBY has been encountered to a level of 0.01% (Das2008). Based on 33 ml oil consumption per day per


capita, the exposure to AO and BY will be 9.9 ml and3.3 mg per day per capita, respectively. Thus, the doseof adulterants used in the present study assumes agreat significance in exerting their proliferating re-sponse in gall bladder cells.

These results suggest that AO and BY have cellproliferative potential in primary cultures of gall blad-der following up-regulation of COX-2 and ErbB2;however, their downstream signaling molecules andROS and RNS generation have differential response,suggesting that the mechanism of proliferation may bedifferent for both compounds.

Acknowledgements We are grateful to the Director of ourCentre for his keen interest in the study. One of us (V M) isthankful to Council of Scientific and Industrial Research (CSIR)/University Grant Commission (UGC), NewDelhi for the award ofSenior Research Fellowship. Financial assistance of CSIR SupraInstitutional Project (SIP-08) is gratefully acknowledged. Themanuscript is IITR communication #2901.

Conflict of interest The authors declare that there are noconflicts of interest.

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role of erbb2 mediated akt and mapk pathway in gall bladder cell proliferation induced by argemone...

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