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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
Glucose Regulated Protein
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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. .
Masalu et al
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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
Glucose Regulated Protein
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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.
Masalu et al
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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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Glucose Regulated Protein
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