New Glucose Regulated Protein 78 ( 78) as a Cytoprotection against …abap.co.in/sites/default/files/Paper-9_15.pdf · 2014. 4. 28. · Glucose Regulated Protein 78 (GRP 78) as a

Current Trends in Biotechnology and PharmacyVol. 8 (2) 185-191 April 2014, ISSN 0973-8916 (Print), 2230-7303 (Online)

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AbstractIn a previous study, has been found that

mushroom crude extract (Cantharellus sp) is ableto induce apoptosis in human cervical cancer cellline (HeLa) and the resistance to Small Cell LungCarcinoma (H157). To clarify the mechanism, wecarried out comparative proteomics analysisbetween H157 and HeLa cell lines. Differentiallyexpressed proteins were separatedelectrophoretically and identified by MatrixAssisted Laser Desorption Time of Flight MassSpectrometry (MALDI-TOF-MS). Peptide tandemmass spectra were searched against acomprehensive data base containing knownproteins derived from data bases. There wereprofound changes in 14 proteins related tomitochondrial function and oxidative stress. Theresults also showed up regulation of GRP78 intreated H157 cell line. Up regulation of GRP78was further confirmed by western blotting analysisand it was suggested that GRP78 is responsiblefor apoptosis resistance of H157 cell line whentreated with Cantharellus extract at lowconcentration.

Keywords: Glucose Regulated protein 78,Apoptosis, Cantharellus, Small Cell LungCarcinoma cell line.

IntroductionIn a stress conditions, cells have to

undergo a defense mechanism one of which is

Glucose Regulated Protein 78 (GRP 78) as aCytoprotection against Apoptosis in Small Cell Lung

Carcinoma

R. Masalu1*, K.M.M. Hosea1, M. Meyer2 , S.L. Lyantagaye1 and B. Ndimba21Department of Mol Biology and Biotechnology, University of Dar es Salaam, P.O Box 35179,

Dar es Salaam, Tanzania2 Department of biotechnology, University of the Western Cape, Private Bag X17,

Bellville 7535, Cape Town, South Africa *For Correspondence - [email protected]

the activation of unfolded protein response (UPR)(1). The function of UPR is the induction of 78-kDa glucose regulated protein (GRP78) alsoknown as immunoglobulin binding protein (BiP)that belong to the Hsp70 family (2). GRP78composed of three domains; the peptide bindingdomain, ATPase domain and a C-terminusdomain (3).

GRP78 is a multifunction endoplasmicreticulum (ER) protein involved in many cellularprocesses. As a molecular chaperone, it preventsthe formation of malfolded proteins by binding tounfolded polypeptides using energy derived fromATP hydrolysis to promote proper folding andprevent aggregation (2). It is targeting misfoldedprotein for proteosome degradation, translocatingnewly synthesized polypeptides across the ERmembrane, it possesses the capacity to bind Ca2+

which helps to immobilize Ca2+ and maintain ERcalcium homeostasis, serving as a sensor of ERstress and also it plays a role in antibody synthesistherefore it may be crucial for B-cell maturation(4).

The expression of GRP78 can be enhancedby various drugs that are capable of generatingunfolded protein load, by means of interruptionof glucose metabolism and disruption of proteinglycosylation (5). Over expression of GRP78

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results in cytoprotection in a wide variety of tumorsand protects tumor cells against apoptosis causednot only by disturbance of ER homeostasis butalso by various cancer therapeutic agents (6). Thepotential mechanisms responsible for thisprotection include preventing protein misfolding,binding of ER Ca2+ and blocking activation ofcaspase and proapoptotic proteins associatedwith the ER (7).

In recent years, proteomic technologieshave started to be used for investigating cellfunction and disease mechanisms, as they offeropportunity to answer biological questions thatwere never thought possible (8). Among manydifferent strategies for proteomic analysis, two-dimensional electrophoresis (2-DE) is a powerfuland widely used method for the complex proteinmixtures extracted from cells, tissues or otherbiological samples (9). Through proteomicstechnologies, various level of proteins that areinvolved in the signaling pathways targeted by thedrugs have been identified, (4, 10, 3).

In a previous study, has been found thatcrude extract of Cantharellus strain is able toinduce apoptosis in human cervical cancer cellline (HeLa) and the resistance to Small Cell LungCarcinoma (H157) (11). In this study, we carriedout comparative proteome analysis of HeLa andH157 cells treated with crude extract ofCantharellus with the aim of identify andfunctionally characterize crucial proteins that lieupstream of the actual event of Cantharellusinduced apoptosis. This paper reports thecytoprotection effects of glucose regulated protein78 upon apoptosis induced by Cantharellusextract.

Materials and MethodsPreparation of crude extract: Fruiting bodiesof Cantharellus sp were collected from miombowoodlands located in Tabora region, WesternTanzania. The collection was done by hand andthe fungi were put into small basket and broughtto the laboratory for identification by a mushroomtaxonomist. Thereafter, specimens were cut intosmall pieces and sun dried for period of three

days. The dried materials were further pulverizedto powder using a national super mixer grinder(MX-119) (Emerging planet India Ltd., Coimbatore641011, India). About 200g of the powdermaterials were extracted by soaking in ethylacetate. The ethyl acetate crude extracts wereconcentrated in vacuo using a rotary evaporator(HEIDOLPH® Ltd) Essex Scientific laboratorysuppliers Ltd with the bath temperaturemaintained at 40

oC. The organic extracts obtained

were kept at 4oC until further use.

Preparation of protein and protein separation:H157 and HeLa cell lines obtained from cell linescollection at the Biochemistry laboratory,Department of Biotechnology, University of theWestern Cape, Cape Town, South Africa. Culturemedia composed of Dulbecco’s Modified EagleMedium (DMEM), supplemented with 10% FetalBovine Serum (FBS) and penicillin (1 µg/ml). Cellswere seeded at a density of 2.5x104 cells per mlin 24-well culture plates and incubated at 37 oCin a humidified atmosphere of 5% CO2 for 24hours. When reached 80% confluences, weretreated with 2mg/ml of Cantharellus extract andincubated for 24 hr. Other flasks were not treated,act as control. After incubation, proteins wereextracted from the cells using 9M urea solution,the obtained protein pellet were re-suspended inurea solution stored at -20 ÚC for further use.This was done in four different experiments.Protein concentration was estimated by theBradford assay (BIO-RAD, Alfred Nobel Drive,Hercules, CA, USA) (12). Protein sample (20 µg)were run in 12% resolving and 5% stacking one-dimensional Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis (1D SDS-PAGE) using the Mini-Proteam 3 Electrophoresiscell system (BIO-RAD) and visualised usingCoomassie Brilliant Blue (CBB) staining solution.Protein samples (150 µg) from both cell linestreated and untreated were run in two-dimensionalSodium Dodecyl Sulfate-Polyacrylamide gelelectrophoresis (2D SDS-PAGE). The proteinswere focused using the 7 cm ReadyStripTM IPGstrips at a pH range of 4 – 7. .

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PDQuestTM Basic software image analysis andMALDI-TOF Mass Spectrometry: Afterseparation of proteins spots by 2D-PAGE andstaining with CBB stain. The gels were scannedusing a PharosFXTM Plus Molecular Imagerscanner (BIO-RAD) using QuantityOne software.The scanned gel images were analysed usingPDQuest Basic version 8.0 for differential proteinexpression profiles. The spot viewer was thenused to identify the differentially expressed spotsbetween the control and experimental triplicategroups in the analysis set (either quantitative orT-test). The quantitative test was used with anupper limit of 1.5 and lower limit of 0.5 folddifference and a T-test with the significance levelof 95%. A total of 107 and 87 spots from HeLaand H157 untreated and 95 and 112 spots fromHeLa and H157 treated were analyzed.

Western blot analysis: Proteins isolated fromboth HeLa and H157 were separated by 12%SDS-PAGE and then electro blotted topolyvinylidene difluoride (PVDF) transfermembranes. The membranes were washed withTBS tween (TBST) 0.1 % (v/v) Tween-20, 1x TBSand placed in 5 % blocking solution fat free milkprepared in TBS tween-TBST. GRP78 (E-4)primary antibodies (1

o Ab) Santa Cruz

biotechnology was diluted at 1:500 in 3 % bovineserum albumin (BSA) and added onto themembranes then incubated overnight at 4

oC on

with shaking to detect GRP78. Then membranewas washed with TBST and incubated with goatanti-mouse IgG-HRP: sc-2031 as secondaryantibody on a shaker for 30 minutes at roomtemperature. The SuperSignal West Pico(Thermo Fisher Scientific, USA)chemiluminescence detection substrate wasadded onto the surface of the blot containingproteins followed by incubation in a dark placefor 5 minutes. After incubation excess detectionsolution was drained off and detection was doneusing autoradiography. The blots were exposedto X-ray film for 20 minutes in a dark room. Afterexposure the film was developed using an x-rayprocessor (13).

ResultsPreparation of protein and protein separation:The standard approach for resolving proteinsinvolves two dimensional gel electrophoresis (14).Protein spots from untreated and treated H157and HeLa cells separated on narrow 4-7 pH range7 cm IPG strips in 12% SDS-PAGE and visualizedby CBB staining solution (Fig. 1). Spots indicatedby arrows were selected for MALD-TOF MSanalysis.

PDQuestTM Basic software image analysis andMALDI-TOF Mass Spectrometry: The analysisset of differentially expressed proteins in HeLaand H157 cell lines present in the T-test andQuantitative test obtained from 2D SDS-PAGE(Table 1). Total of 14 spots from both cell lineswere selected and excised automatically usingExQuestTM Spot Cutter. Out of the 14 selectedprotein spots, 6 spots were up regulated, while 8were down regulated (Table 2). This was followedby protein identification using MALDI-TOF MS incombination with database searching or peptidemass fingerprinting.

Fig.1. 12 % 2D SDS-PAGE showing proteomes of (a)untreated and treated H157 cells and (b) untreatedand treated HeLa cells



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Protein identification by MALDI-TOF MS:Peptide masses obtained from MALDI-TOF MSwere correlated with protein sequence from aprotein database technique known as peptidemass fingerprinting (PMF). The peptide massesobtained from MALDI-TOF MS were thensubmitted to MASCOT and searched usingNational Center for Biotechnology Information(NCBInr) database search. Out of the 14 proteinspots selected for MS analysis 5 spots werepositively identified (Table 3).

Western blot analysis: The GRP78 polyclonalantibody was used to recognize the inducibleexpression of GRP78 in time dependent mannerupon treated H157 cell lines. The expressionlevels were increased with treatment time (Fig.2) whereas the GRP78 expression was notobserved in HeLa.

DiscussionOur earlier studies on the investigation

on pro-apoptotic effects of Cantharellus extractshowed that H157 cell line is resistant to apoptosisinduced by the extract. This study employedcomparative proteomic analysis to evaluate theresponsible protein. The standard approach for

resolving proteins involves two dimensional gelelectrophoresis (15). This report describes theevaluation using 2-De MALDI-TOF-MS to profiledifferential protein expression in H157 and HeLatreated and untreated using cantharellus extractinduced cell death. There were profound changesin 14 protein spots, out of 14 spots; six were upregulated while eight were down regulated. Usingdatabase searches with high confidence basedupon high scores and sequences coverage, weidentified 5 proteins related to mitochondrialfunction, oxidative stress and cell motility. Theresults indicated that mitochondrial dysfunction

Table 1. Number of different expressed proteins in HeLa and H157 cell lines

Cell line Total number of spots T-test Quantitative test

Untreated TreatedHeLa 107 95 40 13H157 87 112 50 17

Table 2. The expression levels of protein spots identified by PDQuest

HeLa cell line H157 cell line

Spot number Expression level Spot number Expression level2502 downregulated 1101 downregulated3402 down regulated 1102 downregulated3501 upregulated 1107 downregulated3601 downregulated 3601 upregulated3701 upregulated 3902 upregulated7004 upregulated 5502 downregulated7604 downregulated 5505 upregulated

Fig. 2. Expression of Grp78 in H157 cell line in threedifferent time points incubation with Cantharellusextract.

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might play a role in HeLa induced apoptosis. Thiswas also confirmed by decrease in themitochondrial transmembrane potential andincrease reactive oxygen species (ROS)generation in HeLa treated cells (data not shown).

Results identified spot S 3902 (Fig. 1 andTable 3) as GRP78_Human, 78kDa glucose-regulated protein and was found overexpressedin H157 and nothing observed in HeLa treatedcell lines. GRP78 overexpression is linked withthe resistance of H157 cell upon treatment withCantharellus extract. This observation supportsthe observation reported elsewhere thatoverexpression of GRP78 renders cell moreresistance to a wide variety of severe insultsincluding apoptosis inducing chemical substancesthrough their chaperoning and anti-apoptoticeffects (16). Further the observation wasconfirmed on transformed HeLa cell with GRP78,the results revealed 50 % protection on thetransformed cell (data not shown)

GRP78 as inhibitors of apoptosis, it tendsto form complex with BCL-2 interacting killer

(BIK). BIK is a proapoptotic tumor suppressor inseveral human tissues and has been used as atherapeutic target for anti-cancer drugs andmediates apoptosis through the mitochondrialpathway (17). It has been shown that, GRP78over expression blocked BIK-induced apoptosis,suppressed estrogen starvation-induced BAXactivation and mitochondrial permeabilitytransition (18, 4). GRP78 is able to suppress bothBIK and NOXA – mediated apoptosis eitherindividually or in combination. Thusoverexpression of GRP78 could negate apoptosisresulting from BIK induction even if it is assistedby another BH3-only protein such as NOXA.Recently it was observed that BCL2 is stronglyimplicated in the survival pathway activated byGRP78 overexpression (19, 4).

GRP78 over expression binds and inhibitsthe activation of caspase -7 activated by both ERstress and genotoxic drugs. As executorcaspases, caspases-3, -6 and -7 can act in aconcert to facilitate the apoptotic process.Therefore, another mechanism for the protective

Table 3. Spots identified by MASCOT search engine

Cell line Spot number Database search results

H157 3902 SwissProt 2010_11 (522019 sequences; 184241293GRP78_HUMAN Mass: 72288 Score: 113 Expect: 1e-07Matches: 1378 kDa glucose-regulated protein OS=Homo sapiensGN=HSPA5 PE=1 SV=2

3601 NCBInr 20101113 (12273660 sequences; 4194265988gi|89574029Mass: 48083 Score: 72 Expect: 0.014 Matches: 8, mitochondrialATP synthase, H+ transporting F1 complex beta subunit [Homosapiens]

HeLa 7004 NCBInr 20101113 (12273660 sequences; 4194265988gi|4504517Mass: 22768 Score: 124 Expect: 9.2e-08 Matches: 8heat shockprotein beta-1 [Homo sapiens]

3501 NCBInr 20101113 (12273660 sequences; 4194265988gi|89574029Mass: 48083 Score: 116 Expect: 5.8e-07 Matches:11mitochondrial ATP synthase, H+ transporting F1 complex betasubunit [Homo sapiens]

3402 NCBInr 20101113 (12273660 sequences; 4194265988gi|297702014Mass: 39774 Score: 154 Expect: 9.2e-11 Matches:

13PREDICTED: actin, cytoplasmic 2-like isoform 3 [Pongo abelii]



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effect of GRP78 is the suppression of the fullactivation of the multiple caspase-mediated celldeath pathways in drug-treated cells contributingto the development of drug resistance (20). It hasbeen shown that GRP78 blocks caspase -7activation by facilitating the formation of aninhibitory complex suppressing its activation.Recent reports have shown that the ATPasedomain of GRP78 binds to procaspase -7,blocking its activation and decrease cellularapoptosis (20, 21). It can also form more inhibitorycomplexes as well as preserving the integrity ofthe existing inhibitory complex. GRP78 resemblesthe X-linked inhibitor of apoptosis (XIAP) suchthat both genes contain an internal ribosome entrysite element that allows efficient translation underphysiological stress conditions (1). The resultsreported here indicated that, 78kDa glucose-regulated protein might responsible for H157resistance to apoptosis when H157 treated withCantharellus extract. Western blot analysis wasused for validation of GRP78 proteinoverexpression at three different time points inH157 and HeLa cell lines after and beforetreatment with Cantharellus extracts. The resultsshowed that GRP78 overexpressed with time(FIg. 2). It has been observed from previousstudies that overexpression of GRP78 promotestumor proliferation and metastasis, that reducingGRP78 expression resulted in an increase intumor cell apoptosis (17, 6). This implies thatdrugs that targeted GRP78 activity mightcomplement conventional cancer therapy

Human cancer cells are derived from dissimilartissues therefore they can use different diversesignaling and defense mechanisms to acquireresistance to specific drugs. Thus, thecytoprotection of GRP78 to variety of drug is likelyto vary among cancer cells. More number of celllines is needed to be evaluated in order toestablish the mechanism of action among cancercells for the development of possible drug.

ConclusionThe results suggested that,

overexpression of GRP78 protein in H157 cell is

responsible for the apoptosis resistance of H157upon Cantharellus treatment. This was alsoconfirmed by western blotting analysis. Therefore,successful suppression of GRP78 protein activitycan offer a new therapeutic approach to H157

AcknowledgementsWe thank University of Dar es Salaam

through World Bank project component C1B1 forfinancial support. We would also like to expressour gratitude to the Third World Organization forWomen in Science (TWOWS) for a fellowshipthat enabled Rose Masalu to conduct the studyin the department of Biotechnology, University ofthe Western Cape South Africa. Finally we wishto recognize the valuable technical assistance byRoya Ndimba of the Department of Biotechnology,University of the Western Cape.

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R.C., Kaufman, R.J. and Lees, A.S. (2003).Endoplasmic reticulum chaperone proteinGRP78 protects cells from apoptosisinduced by topoisomerase inhibitors: role ofATP binding site in suppression of caspase-7activation. J. Biol. Chem, 278(23): 20915-20924

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New Glucose Regulated Protein 78 ( 78) as a Cytoprotection against …abap.co.in/sites/default/files/Paper-9_15.pdf · 2014. 4. 28. · Glucose Regulated Protein 78 (GRP 78) as a