AR-A 014418 Used against GSK3beta Downregulates Expression of hnRNPA1 and SF2/ASF Splicing Factors

Research ArticleAR-A 014418 Used against GSK3beta Downregulates Expressionof hnRNPA1 and SF2ASF Splicing Factors

Ajay K Yadav1 Vidhi Vashishta1 Nidhi Joshi1 and Pankaj Taneja2

1 Dr B R Ambedkar Center for Biomedical Research Delhi University Delhi 110007 India2 Institute of Nuclear Medicine and Allied Sciences Timarpur Delhi 110007 India

Correspondence should be addressed to Ajay K Yadav ajay9774gmailcom

Received 5 August 2013 Revised 23 October 2013 Accepted 2 November 2013 Published 2 January 2014

Academic Editor Minesh P Mehta

Copyright copy 2014 Ajay K Yadav et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Glioblastoma is one of the most aggressive forms of primary brain tumors of glial cells including aberrant regulation of glycogensynthase kinase 3120573 (GSK3120573) and splicing factors deregulation Here we investigate the role of small molecule AR-A014418 andManzamine A against GSK3 kinase with factual control on splicing regulators AR-A 014418 48 hrs posttreatment caused dose(25ndash100120583M) dependent inhibition in U373 and U87 cell viability with also inhibition in activating tyrosine phosphorylation ofGSK3alpha (Tyr 279) and beta (Tyr 216) Furthermore inhibition of GSK3 kinase resulted in significant downregulation of splicingfactors (SRSF1 SRSF5 PTPB1 and hnRNP) in U87 cells with downregulation of antiapoptotic genes such as BCL2 BCL-xLSurvivin MCL1 and BMI1 Similarly downregulation of splicing factors was also observed in U373 glioma cell after using SiRNAagainst AKT and GSK3beta kinase In addition potential roles of AR-A014418 in downregulation of splicing factors were reflectedwith decrease in Anxa7 (VA) variant and increase in Anxa7WT tumor suppressor transcript and proteinThe above results suggestthat inhibition of GSK3beta kinase activation could be the beneficial strategy to inhibit the occurrence of alternative cancer escapepathway via downregulating the expression of splicing regulators as well as apoptosis

1 Introduction

Glioblastoma remains challenging to us being a devastatingaggressive brain tumor Patients median survival is found tobe around 15 months and 10 less will survive 5 years afterdiagnosis [1 2] GBMrsquos heterogeneous genome wide land-scape and resistance against multimodal therapy make thiseven more challenging to treat or to at least extend the sur-vival of patient beyond expectancy Although a lot of progressis being made to revive the multigene targeted approaches torestrict the tumor growth it is going to be limited due to itsbiological complexity and tumor heterogeneity [3] Moreoverfinding a better target for GBM could be possible by study-ing the regulation of splicing factors involved in evolvingalternative splicing event with diversified encoded proteinproduct in cancer to escape apoptotic pathway Cooperativeand competitive association between splicing factor enhancerand silencer is under exploration area its functional con-sequences lead to misregulated alternative splice variantexpressionwhich could affect biology of survival signalling in

various scales of function such as mRNA stability oncogenicgain and loss in tumor suppressor function However few ofsplicing factors such as PTBP1 (polypyrimidine tract bindingprotein) SRSF1 (SR protein) and hnRNPs are found to beaberrantly overexpressed in breast cancer gliomas and soforth [4 5] Out of these in detail SRSF1 found directlymodulates the expression of tumourigenic the tyrosine kinasereceptor (RON) is associated with shorter survival andpoorPrognosis in GBMrsquos patient [6]

Since splicing factors such as SF2ASF1 and hnRNPs areoverexpressed in various cancers with constitutively activegrowth survival signals the connecting link between initiat-ing EGFR (epidermal growth factor receptor)mdashAkt kinasemdashGSK3beta kinase survival signaling pathways with regulationof splicing factors is barely been studied However few ofthe earlier reports showed p38 kinase mediated upregulationof nuclear hnRNPA1 [7] in senescent fibroblast with inhibi-tion of nucleocytoplasmic shuttling and recently [8] a linkbetweenAkt kinase and hnRNPs regulation has been hypoth-esized

Hindawi Publishing CorporationJournal of OncologyVolume 2014 Article ID 695325 9 pageshttpdxdoiorg1011552014695325

2 Journal of Oncology

0

05

1

15

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25

3

U87 CNTU87 AR

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05

1

15

2

25

U373 CNTU373 AR

Cel

l via

bilit

y (O

D595

nm)

Cel

l via

bilit

y (O

D595

nm)

50120583

M(A

R)

25120583

M(A

R)

100120583

M(A

R)

50120583

M(A

R)

25120583

M(A

R)

100120583

M(A

R)(a)

0

2

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16

U87CNT 24

U87W 24

U87 U87 U87 U87AR 24

SRSF1

0

5

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35

U87CNT 24

U87W 24

U87 U87 U87 U87AR 24 CNT 24 W 24 AR 24 CNT W 24 AR 24

SRSF5

0

2

4

6

8

10

12

14

16 PTBP1

02468

101214161820 hnRNPH3

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(b)

Figure 1 Cell viability assay and qRTPCR assay for splicing factors (a) cell viability assay for dose dependent response of U87 and U373treated with AR-A 014418 for 48 hrs (b) qRT-PCR of splicing factors (SRSF1 SRSF5 PTBP1 and hnRNPH3) in U87 glioma cell line aftertreating cells with Wortmannin and AR-A 014418 in different experiments for 24 hrs Means plusmn SE 119899 = 5 119875 le 005

In the current set of investigations we are studying theexpression of splicing factors which are themajor deregulatedfamily proteins in maneuvering the alternative splicing eventto escape apoptotic pathway potentially could be explainedafter using small molecular inhibitors against Akt kinase andGSK3beta with ability to restrict the occurrence of alternativepathway either by reversing or decreases the occurrence ofalternative splicing and will possibly help in restricting thetumor genetic heterogeneity

2 Results

21 Cell Viability Assay after Inhibiting GSK3beta and AktKinase Tumor reoccurrence and resistance are of wide con-cern to study in detail the major deregulated multitude sig-nalling pathways Assessing cell viability is one of the param-eters to study mitochondrial function and its attenuationusing small molecule against GSK3beta kinase eventually

inducing apoptotic signalling pathway Hence to study therole of GSK3beta in cell viability we have chosen dosedependent temporal kinetics of small molecule AR-A 014418andManzamineA against GSK3 kinaseWe found that 50120583MAR-A 014418 is required to inhibit the (gt50) glioma cellviability (Figure 1(a))

22 Regulation of Splicing Factor Using GSK3beta and AktKinase Inhibitors Since deregulated expressions of splicingfactors such as PTBP1 SF2ASF (SRSF1 SRSF5) and hnRNPsare the major cause that interfere with the homeostatic sig-naling proteins by inducing translational expression of diver-sified alternative splice variant with antiapoptotic gain orapoptotic loss in advancement of tumorigenicity Functionalsignificance of GSK3beta in arresting gliomagenesis thoughdownregulating the overexpression of splicing factor usinginhibitors against GSK3beta were evaluated (Figure 1(b))compared with Wortmanin (Akt kinase inhibitor) Similarly

Journal of Oncology 3

CNT

TDZ

Man

z

Man

z

Ly LyCNT

TDZ

hnRNP A1

Cytoplasm Nuclear

U87

120572-Tubulin

(a)

hnRNPA1

SF2ASF

U373

CNT-

i

Akt

-i

120572-Tubulin

GSK

3120573

-i

(b)

Figure 2 Splicing factor regulation (a) U87 glioma cell lines were treated with TDZ8 (20120583M)Manzamine A (2120583M) and LY294002 (10 120583M)for 24 hrs followed by cytoplasmic and nuclear extraction studied hnRNPA1 downregulation Results were evaluated frommultiple differentexperiments (b) U373 glioma cells were transfected with Akt RNAi and GSK3beta RNAi compared with CNT RNAi studied SF2ASF1 andhnRNPA1 downregulation

in one of the experiment downregulation of hnRNPA1 innuclear compartment of U87 glioma cells after treating withsmall molecule against GSK3beta and Akt kinase Moreoverdownregulation of hnRNPA1 and SF2ASF1 in U373 gliomacell were also observed after Akt RNAi and GSK3beta RNAi(Figure 2) this further opening the avenue ofmultifunctionalrole of GSK3beta including regulation of splicing factorexpression by which could restrict the origination of alter-native genetic splice variant or alternative apoptotic escapepathway

23 Regulation of Apoptotic Regulators Similarly geneticexpressions of antiapoptotic regulators such as BCL-xL Sur-vivin and MCL1 are significantly downregulated using spe-cific small molecule inhibitors against GSK3beta kinase asearlier shown by the other group using small molecule usedagainst Akt kinase [9] In addition BMI1 (polycomb proteinfamilymember) involved in stem cell self-renewal and cancerstem cellmaintenance [10] is also significantly downregulated(Figure 3(a)) which seems to reflect in tumorigenic potentialof U87 cells in the presence of AR-A 014418 (GSK3betainhibitor) but not with Akt inhibitor studied using protocolof soft agar assay (Figure 3(b)) suggesting that the tyrosinephosphorylation of GSK3beta kinase is critical in regulatingthe colony formation (Figure 5(c))

24 Glioma Cell Colonies Formation GSK3beta inhibitionusing AR-A 014418 and Akt kinase inhibitor Ly294002 com-pared with control (untreated) showed decrease in evolutionofU87 glioma cells originated colonies on soft agar plate assaysuggesting potential involvement of GSK3beta compared toAkt kinase (Figure 3(b))

25 Regulation of GSK3beta Mediated Signaling PathwayInhi bition of activating tyrosine phosphorylation of

GSK3alpha (Tyr 279) and -beta (Tyr 216) observed after usingGSK3beta inhibitor AR-A 014418 (50120583M) and ManzamineA (2120583M) but not by using TDZD-8 (20120583M) and Ly294002(10 120583M) suggests comparative high antitumorigenic potentialof AR-A 014418 and Manzamine A (Figures 4(a) and 5(c))Similarly translocations of GSK3 kinase beta catenin andpERK12 to the nucleus were significantly affected after usingsmall molecule Manzamine A Ly294002 and TDZD-8(Figure 4(b)) Downregulation of GSK3beta activating tyro-sine phosphorylation without change in Ser9-GSK3betaphosphorylation negates the involvement of Akt kinasemediated downstream GSK3beta inactivation Howeverinvolvement of potential Akt kinase independent of EGFRligand induced GSK3beta mediated MAP kinase regulationhas been observed (Figure 4(a)) In addition upregulation ofAnnexin 7 protein on GSK3beta inhibition (Figure 5(c)) wasfound very interesting to explore epidermal growth factorreceptor (EGFR) regulation [11]

26 GSK3beta Inhibition Induced Reversal in Anxa7 Alterna-tive Splicing Anxa7 is a Ca2+phospholipid binding proteininvolved in membrane fusion and tumor suppressor protein[12]

Earlier we have shown that Anxa7 is a EGFR negativeregulator its allelic loss has been depicted with enhancedglioma survival signaling pathway [11 13]

Here we have observed splicing deregulation with ele-vated Anxa7 VA (variant) transcript with loss in Anxa7 VB(WT) in comparison with Normal Brain cDNA Clontechcompany in U87 and U373 glioma cells (Figure 5(a)) How-ever inhibition of GSK3beta activation in presence of AR-A014418 treated for different time points (0 6 12 and 24 hrs)showed gradual inhibition in alternative splicing of Anxa7with increase in Anxa7 VB (WT) in between 6 hrs and 12 hrswith increase in Anxa7 VB transcript in time dependent


0

05

1

15

2

25

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BCL2

0 0

1

2

3

4

5

6

U87 CNT U87 ARU87 AR

Survivin

100

200

300

400

500

600

700

800

900 BCL-xL

0

02

04

06

08

1

12

14

16

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MCL1

0

05

1

15

2

25

U87 CNT U87 AR

BMI1

U87 CNT

n-fo

ld ex

pres

sion

n-fo

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pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(a)

Control

AR-A014418

LY 294002

U87

20days10days

(b)

Figure 3 (a) qRTPCR analysis of BCL2 BCL-xL Survivin MCL1 and BMI1 in glioma cell lines treated or untreated with AR-A 014418(50120583M) for 24 hrs Means plusmn SE 119899 = 5 119875 le 005 (b) Soft agar assay to estimate the tumorigenic propensity of U87 cells in presence of AR-A014418 (50 120583M) and LY294002 (10 120583M) observed for 10 days and 20 days


pTyr-GSK3

p-Akt (ser-473)

T-GSK3beta

p-Erk12

T-Akt

CNT AR

pSer9-GSK3

U87

EGF (50ngmL)

120573

120572

120573

120572

0 30 0 30

(a)

U373

T-GSK3

pErk12

Cytoplasm Nuclear

120572-Tubulin

120572-Tubulin

120573-Catenin

CNT

TDZ8

Man

z A

Man

z A

Ly LyCNT

TDZ8

(b)

Figure 4 U87 and U373 glioma cell lines were grown and plated day before incubated with small molecule inhibitors against GSK3betakinase (AR-A 014418 Manzamine A and TDZ-8) Akt kinase inhibitor (LY294002 and Wortmanin) and Bortezomib (BR) for 24 hrs (a)EGFR ligand dependent regulation of activating tyrosine phosphorylation of GSK3 kinase and downregulation of ERK12 pathway afterinhibiting the tyrosine phosphorylation of GSK3 kinase using AR-A 014418 for 24 hrs (b) Differential localization of GSK3 kinase betacatenin and p-ERK12 was studied in cytoplasmic and nuclear fraction isolated from treated U373 glioma cell line All experiments wererepeated multiple times

manner (Figure 5(b)) In addition restoration of Annexin7 protein on GSK3beta inhibition (Figure 5(c)) was foundvery interesting to explore epidermal growth factor receptor(EGFR) regulation

3 Discussion

GSK3 was been accepted and viewed as nodal regulator invaried multiple cellular signaling pathway moreover its roleis widely dispersed including cell structure metabolism andcell survival Inhibition of GSK3beta has been demonstratedlargely in treating neurodegenerative diseases [14] howeverinvolvement of GSK3beta in cancer progression varies amongdifferent cancers

Differential expression and regulation of GS3120573 in cancerappear to be tissue specific and involved in inducing molec-ular genetic heterogeneity as certain forms of cancer showedhigh expression of active GSK3120573 [15] however other cancertissues harbor low levels It was suggested that high levelsof nuclear GSK3120573 in pancreatic cells were associated withdedifferentiation and NF-kB mediated survival [16] GSK3120573activity also appears to be important for leukemic cell growthas the inhibition of GSK3120573 led to an induction of apoptosis inleukemic cells and also showed that GSK3120573 has proapoptoticrole in lung cancer by downregulating Survivin activity [17]whereas GSK3beta kinase inhibition in glioma cells inducesproapoptotic affect [18 19]

Additionally the membrane associated growth factorreceptor (GFR) induced survival signaling pathway andrecruitment of oncogenic signals for nuclear transcriptionalregulation could be aberrant due to aberrant activity ofGSK3beta as demonstrated earlier [20] while involvementof intermediary survival kinases with apparent deregulationof splicing factors is sparsely understood However dereg-ulatory behavior of splicing factors with apparent rise inalternative splicing is now the causing hallmark to investigatefurther the alternative apoptotic escape pathways Nonethe-less possibly by aligning the functional role of Growth factorsurvival kinases with deregulation of splicing regulatorsMoreover activated ERK12 kinase drivenGSK3beta primingis one of the essential molecular modulating events toregulate [21] the downstream signaling event Hence toexplore the inhibition of GSK3beta kinase with significantrepression of tumorigenic potential in glioma cell line [22]our study showed that inhibition of GSK3beta Tyrosine-216phosphorylation activity without change in inactivated Ser9phosphorylation leads to downregulation of the expressionof GSK3beta with loss of ERK12 phosphorylation To extendGSK3beta kinase activity forward in term of regulation ofsplicing factors such as PTBP1 SF2ASF and hnRNPs whichare the leading cause for the origination of alternative spliceencoded proteins are of wide concern such as caspase 9variant origination involve in occurrence of alternative escapeapoptotic pathways [23] found Akt kinase dependent [8]


0

05

1

15

2

U87 U373 NB

Anxa7 VB

minus05 0

01

02

03

04

05

06

U87 U373 NB

Anxa7 VA

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(a)

0

05

1

15

2

25

3

35

4

45

U87 AR 6hr

Anxa7 VAAnxa7 VB

U87AR 12hr U87AR 24hr

n-fo

ld ex

pres

sion

(b)

Anxa7

pErk12

U87

p (tyr)-GSK3

T-GSK3

120572-Tubulin

120572

120573

CNT

AR

Man

z

TDZ

(c)

Figure 5 Anxa7 splicing in U87 glioma (a) Estimation of Anxa7 VA (variant) and Anxa7 VB (WT) in U373 and U87 glioma cells comparedwithNormal Brain cDNA (b) Temporal kinetics of Anxa7 splicing (6 12 and 24 hrs) in presence of AR-A 014418 (GSK3beta kinase inhibitor)Means plusmn SE 119899 = 5 119875 le 005 (c) Small molecule against GSK3 kinase used to inhibit activated Tyrosine phosphorylated GSK3 kinase in U87cells incubated for 24 hrs observed restoration of Annexin 7 tumor suppressor protein


Table 1 List of primers used to estimate the quantitative genetic transcription after incubating with different kinase specific inhibitors

S number Gene name Forward primer (51015840 to 31015840) Reverse primer (51015840 to 31015840)1 GAPDH 51015840ACCACAGTCCATGCCATCAC 31015840 51015840TCCACCACCCTGTTGCTGTA 31015840

2 SF2ASF1 51015840CACTGGTGTCGTGGAGTTTG 31015840 51015840TCCTGCTGTTGCTTCTGCTA 31015840

3 hnRNPH3 51015840 TGAGGCTAGTGATGGGACAG 31015840 51015840 TGTTTCCCCAGAGCATTTTC 31015840

4 PTBP1 51015840 ACGGACCGTTTATCATGAGC 31015840 51015840AGCATCAGGAGGTTGGTGAC 31015840

5 Survivin 51015840 TTGCTCCTGCACCCCAGAGC 31015840 51015840 AGGCTCAGCGTAAGGCAGCC 31015840

6 BCL-xL 51015840 TCTCCTTTGGCGGGGCACTG 31015840 51015840 TCCACAAAAGTGTCCCAGCCGCC 31015840

7 BCL2 51015840CTCTCGTCGCTACCGTCGCG31015840 51015840AGGCATCCCAGCCTCCGTTATCC 31015840

8 MCL1 51015840 TGGAGGTGAACCCGACT TCCATG 31015840 51015840 TGGGGCTGGCTTGAGGTTCTCAA 31015840

9 BMI1 51015840GGAGACCAGCAAGTATTGTCCTATTTG31015840 51015840CTTACGATGCCCAGCAGCAATG 31015840

10 Anxa7 VB (WT) 51015840TCTCCTGTTTCTTTGGATTATAG 31015840 51015840ACCCTTCATTGCCTTACG31015840

11 Anxa7 VA 51015840CCCTAGTCAGCCTGCCACAGT31015840 51015840ACCCTTCATTGCCTTACG31015840

Interestingly small molecule against GSK3beta mediatedadditional downregulation of splicing factors such as PTBP1hnRNPA1 and SF2ASF which have all been observed firsttime by us along with decrease in antiapoptotic regulatorssuch as MCL1 Survivin and BMI1 as shown earlier [9]Moreover small molecule mediated intervention appears tobe supportive experimental observation using knock-downapproach where knocking down Akt or GSK3beta kinaseleads to downregulation of hnRNPA1 and SF2ASF (SRSF1)suggesting the involvement of partial Akt kinase andGSK3beta kinase in splicing factor downregulation which isdemonstrated further using small molecule (therapeutic)approach against GSK3beta such as Manzamine A and AR-A014418 Multiple roles of hnRNPs complex on Anxa7 tumorsuppressor protein have been demonstrated as potentialinducer of Anxa7 alternative splice variants [24] in prostatecancer Moreover loss of Anxa7 tumor suppressor expres-sion with gain in epidermal growth factor receptor proteininduced survival signaling pathway and reported earlierdemonstrated to be a high risk factor in evaluating thesurvival of glioblastoma patient [11 13]We therefore investi-gated Anxa7 alternative splicing using small molecule againstGSK3beta and invariably demonstrated inhibition in Anxa7variant with increase in transcript of Anxa7 WT gene Simi-larly Anxa7 protein restoration using small molecule againstGSK3betawas found to be an additional advantage alongwithdownregulation of MCL1 and Survivin in glioma cells whichis supported by the earlier group the relevance of radiationinduced gliomarsquos sensitization after downregulation of MCL1and Survivin using Akt kinase inhibitor in EGFR context[25 26]

In conclusion GSK3beta inhibition results in decreasein splicing factors such as PTBP1 SF2ASF and hnRNPA1decrease in antiapoptotic regulators and increase in Anxa7Small molecules such as Manzamine A and AR-A 014418[27] used against GSK3beta kinase should be further checkedto study widely the alternative splicing event These resultsclearly indicate the functional significance of Gs3kbeta inarresting gliomagenesis by these inhibitors Moreover futuretargeted drug therapeutic strategies can be designed basedon the findings of these nodal GSK3beta kinase regulatedpathways

4 Materials and Methods

41 Cell Culture and Reagents U87 and U373 human glioma(astrocytoma) cells were propagated by Dulbeccorsquos modifiedEaglersquos medium (DMEM) containing 10 fetal bovine serum(Invitrogen Carlsbad CA USA) 100UnitsmL penicillinand 100 120583gmL streptomycin Cells were seeded in 100mmplates and incubated overnight followed by treatment withLY294002 orWortmanin (Akt kinase inhibitor) AR-A 014418or Manzamine A or TDZD8 (GSK3beta inhibitor) and pro-teasome inhibitor (Bortezomib) at various concentrations for48 hrs Normal Brain RNA was purchased from Clonotechcompany

42 Quantitative Real-Time PCR Quantitative real-time pol-ymerase chain reaction (PCR) was performed using standardprocedures to determine relative mRNA transcript levelsTotal RNA was extracted using TRIzol Reagent (InvitrogenCarlsbad CA USA) and first strand cDNA was synthesizedusing random hexamer and superscript II (Invitrogen Carls-bad CA USA) Quantitative real-time PCR was performedusing MESA Green qPCR MasterMix Plus for SYBR Assayin 7300 thermocycler (Applied Biosystems) Thermal cyclingconsisted of a warm-up step of 2min at 50∘C and initialdenaturation at 95∘C for 10m followed by 40 cycles of eachpolymerase chain reaction (PCR) step 95∘C for 15 s and 60∘Cfor 1m All primers used for real-time PCR analysis weresynthesized by IDT (Integrated DNA Technologies) Thespecificity of each primer pair was confirmed by meltingcurve analysis and running amplified sample in agarose-gelelectrophoresis GAPDH was used as internal control List ofvarious gene primers (51015840 to 31015840) was enlisted in Table 1

43 Cell Viability Assay Cell viability was assessed using theMTT colorimetric assay based on the ability of viable cellsto reduce yellow MTT (3-(4-5-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide a tetrazole) to purple for-mazan product which is largely impermeable to cell mem-branes thus resulting in its accumulation within living cellsSolubilization of the cells results in the liberation of the purpleproduct which can be detected using a colorimetric mea-surement Glioma cells were cultured in 96-well microlitre


plates for various time periods in the presence of inhibitorsAfter incubation cells were observed under a contrast phasemicroscope before adding MTT solution prepared fresh as5mgmL in DMEM filtered through a 022-120583m filter andkept for 5min at 37∘C MTT solution (100120583L) was added toeach well and the plates were incubated in the dark for 4 hat 37∘C and subsequently solubilized in DMSO the extentof reduction of MTT was quantified by absorbance measure-ment at 595 nm Experimental groups were performed in atleast triplicate

44 Soft Agar Assay U87 glioma cells were grown and tryp-sinized counted suspended in 02 agar in 20 fetal bovineserum DMEM and equally (105 cell number) plated induplicate on 60mm dishes containing 05 agarose bedGrowing DMEM medium containing LY294002 (Akt kinaseinhibitor) AR-A 014418 or Manzamine A was added nextday Colonies were observed and counted at 3 different sitesat various time points (days 10 and 20) stained for viabilitywith 3mgmL 3-(45-dimethylthiazol-2-yl)-25-diphenyltet-razolium bromide (MTT) for 4 hours and visualized understandard light microscope Colony formation indicates thatindividual cells develop into cell clones that are identified assingle colonies

45 SiRNA Transfection Glioma cells were transfected a dayafter plating of glioma cells with SiRNA against Akt andGSK3beta kinase compared with CNT-i (Santa Cruz Inc) asper Santa Cruz Inc protocol Lysates were made after 48 hrsof transfections in Triton X-100 buffer

46 Protein Extraction and Immunoblotting U87 and U373glioma cell lysates were prepared in 01 Triton buffer con-taining protease and phosphatase inhibitors (Sigma-Alrich)and also were prepared cytoplasmic and nuclear extractsusing cell compartment isolation kit (Millipore) Further totalprotein was quantitated using the BCA Protein Assay Kit(Thermo Scientific Rockford IL) Blots were exposed to anti-GSK3 (Millipore) anti-phosphotyrosine GSK3 (Millipore)anti-phospho-ERK12 (Thr202Tyr204) (Millipore) anti-120572-tubulin and anti-beta-catenin (Millipore) and recognized byan HRP-conjugated horse anti-mouse secondary antibody(Santa Cruz Inc) antibodies to detect phospho-AKT123(Ser473) total Akt1 hnRNPA1 and Anxa7 (Santa cruz Inc)SF2ASF1 (Santa Cruz Inc) were recognized by an HRP-conjugated goat anti-rabbit secondary antibody (Santa CruzInc) Enhanced chemiluminescent reagent (GE Healthcare)was added to themembranes according to themanufacturerrsquosprotocol and visualized by autoradiography

47 Statistical Analysis Experimental results have beenexpressed as the mean plusmn standard error (SE) Statisticaldifferences of data were assessed by Studentrsquos-119905-test 119875 valueslower than 005 were considered significant

Abbreviations

hnRNP Heterogeneous ribonucleoproteinparticle

SRSF or SF2ASF Serinearginine-rich splicing factor 1

EGFR Epidermal growth factor receptorBCL-xL B-cell lymphoma-extra largeBMI-1 B lymphoma Mo-MLV insertion region 1

homologMCL1 Myeloid cell leukemia sequence 1

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This work was supported by research support from AKY-Professor Ramalingaswami fellowship (DBT) NewDelhi andUniversity of Delhi RampD grant

References

[1] N S Chaudhry A H Shah N Ferraro et al ldquoPredictors oflong-term survival in patients with glioblastoma multiformeadvancements from the last quarter centuryrdquo Cancer Investiga-tion vol 31 no 5 pp 287ndash308 2013

[2] J Chen and T Xu ldquoRecent therapeutic advances and insights ofrecurrentglioblastoma multiformerdquo Frontiers in Bioscience vol18 no 1 pp 676ndash684 2013

[3] F E Bleeker R J Molenaar and S Leenstra ldquoRecent advancesin the molecular understanding of glioblastomardquo Journal ofNeuro-Oncology vol 108 no 1 pp 11ndash27 2012

[4] J L Manley and A R Krainer ldquoA rational nomenclature forserinearginine-rich protein splicing factors (SR proteins)rdquoGenes and Development vol 24 no 11 pp 1073ndash1074 2010

[5] C V LeFave M Squatrito S Vorlova et al ldquoSplicing factorhnRNPH drives an oncogenic splicing switch in gliomasrdquo TheEMBO Journal vol 30 no 19 pp 4084ndash4097 2011

[6] PM Comoglio M R Green S Riva et al ldquoCell motility is con-trolled by SF2ASF through alternative splicing of the Ron pro-tooncogenerdquoMolecular Cell vol 20 no 6 pp 881ndash890 2005

[7] N Shimada I Rios H Moran B Sayers and K Hubbard ldquop38MAP kinase-dependent regulation of the expression level andsubcellular distribution of heterogeneous nuclear ribonucleo-protein A1 and its involvement in cellular senescence in normalhuman fibroblastsrdquo RNA Biology vol 6 no 3 pp 293ndash3042009

[8] N T Vu M A Park J C Shultz et al ldquohnRNP U enhancescaspase-9 splicing and is modulated by AKT-dependent phos-phorylation of hnRNP Lrdquo The Journal of Biological Chemistryvol 288 no 12 pp 8575ndash8584 2013

[9] C Li C Zhou S Wang et al ldquoSensitization of glioma cells totamoxifen-induced apoptosis by Pl3-kinase inhibitor throughthe GSK-3120573120573-catenin signaling pathwayrdquo Plos One vol 6 no10 Article ID e27053 2011

[10] J Godlewski M O Nowicki A Bronisz et al ldquoTargeting of theBmi-1 oncogenestem cell renewal factor by MicroRNA-128inhibits gliomaproliferation and self-renewalrdquoCancer Researchvol 68 no 22 pp 9125ndash9130 2008

[11] A K Yadav J J Renfrow D M Scholtens et al ldquoMonosomyof chromosome 10 associated with dysregulation of epidermalgrowth factor signaling in glioblastomasrdquo Journal of the Ameri-can Medical Association vol 302 no 3 pp 276ndash289 2009


[12] M Srivastava C Montagna X Leighton et al ldquoHaploinsuffi-ciency of Anx7 tumor suppressor gene and consequent genomicinstability promotes tumorigenesis in the Anx7(+minus) mouserdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 100 no 2 pp 14287ndash14292 2003

[13] M Bredel DM Scholtens G R Harsh et al ldquoA networkmodelof a cooperative genetic landscape in brain tumorsrdquo Journal ofthe American Medical Association vol 302 no 3 pp 261ndash2752009

[14] R V Bhat S L B Haeberlein and J Avila ldquoGlycogen synthasekinase 3 a drug target forCNS therapiesrdquo Journal ofNeurochem-istry vol 89 no 6 pp 1313ndash1317 2004

[15] A Kitano T Shimasaki Y Chikano et al ldquoAberrant glycogensynthase kinase 3120573 is involved in pancreatic cancer cell invasionand resistance to therapyrdquo Plos One vol 8 no 2 Article IDe55289 2013

[16] A V Ougolkov M E Fernandez-Zapico D N Savoy R AUrrutia andDD Billadeau ldquoGlycogen synthase kinase-3120573 par-ticipates in nuclear factor 120581B-mediated gene transcription andcell survival in pancreatic cancer cellsrdquoCancer Research vol 65no 6 pp 2076ndash2081 2005

[17] J Li M Xing M Zhu et al ldquoGlycogen synthase kinase 3120573induces apoptosis in cancer cells through increase of survivinnuclear localizationrdquo Cancer Letters vol 272 no 1 pp 91ndash1012008

[18] BM Ku Y K Lee J Y Jeong et al ldquoCaffeine inhibits cell prolif-eration and regulates PKAGSK3120573 pathways in U87MG humanglioma cellsrdquo Molecules and Cells vol 31 no 3 pp 275ndash2792011

[19] C-H Chou A-K Chou C-C Lin et al ldquoGSK3120573 regulatesBCL2L12 and BCL2L12A anti-apoptosis signaling in glioblas-toma and is inhibited by LiClrdquo Cell Cycle vol 11 no 3 pp 532ndash542 2012

[20] S Kotliarova S Pastorino L CKovell et al ldquoGlycogen synthasekinase-3 inhibition induces glioma cell death through c-MYCnuclear factor-120581B and glucose regulationrdquoCancer Research vol68 no 16 pp 6643ndash6651 2008

[21] Q Ding W Xia J-C Liu et al ldquoErk associates with and primesGSK-3120573 for its inactivation resulting in upregulation of 120573-cateninrdquoMolecular Cell vol 19 no 2 pp 159ndash170 2005

[22] S Korur R M Huber B Sivasankaran et al ldquoGSK3beta regu-lates differentiation and growth arrest in glioblastomardquoPlos onevol 4 no 10 Article ID e7443 2009

[23] R W Goehe J C Shultz C Murudkar et al ldquohnRNP L reg-ulates the tumorigenic capacity of lung cancer xenografts inmice via caspase-9 pre-mRNA processingrdquo Journal of ClinicalInvestigation vol 120 no 11 pp 3923ndash3939 2010

[24] Y Torosyan A Dobi M Glasman et al ldquoRole of multi-hnRNPnuclear complex in regulation of tumor suppressor ANXA7 inprostate cancer cellsrdquo Oncogene vol 29 no 17 pp 2457ndash24662010

[25] E P Jane D R Premkumar J D DiDomenico B Hu S-YCheng and I F Pollack ldquoYM-155 potentiates the effect of ABT-737 in malignant human glioma cells via survivin and Mcl-1 downregulation in an EGFR-dependent contextrdquo MolecularCancer Therapeutics vol 12 pp 326ndash338 2013

[26] Z Sheng L Li L J Zhu et al ldquoA genome-wide RNA interfer-ence screen reveals an essential CREB3L2-ATF5-MCL1 survivalpathway in malignant glioma with therapeutic implicationsrdquoNature Medicine vol 16 no 6 pp 671ndash677 2010

[27] K Miyashita K Kawakami M Nakada et al ldquoPotential thera-peutic effect of glycogen synthase kinase 3120573 inhibition against

human glioblastomardquoClinical Cancer Research vol 15 no 3 pp887ndash897 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


0

05

1

15

2

25

3

U87 CNTU87 AR

0

05

1

15

2

25

U373 CNTU373 AR

Cel

l via

bilit

y (O

D595

nm)

Cel

l via

bilit

y (O

D595

nm)

50120583

M(A

R)

25120583

M(A

R)

100120583

M(A

R)

50120583

M(A

R)

25120583

M(A

R)

100120583

M(A

R)(a)

0

2

4

6

8

10

12

14

16

U87CNT 24

U87W 24

U87 U87 U87 U87AR 24

SRSF1

0

5

10

15

20

25

30

35

U87CNT 24

U87W 24

U87 U87 U87 U87AR 24 CNT 24 W 24 AR 24 CNT W 24 AR 24

SRSF5

0

2

4

6

8

10

12

14

16 PTBP1

02468

101214161820 hnRNPH3

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(b)

Figure 1 Cell viability assay and qRTPCR assay for splicing factors (a) cell viability assay for dose dependent response of U87 and U373treated with AR-A 014418 for 48 hrs (b) qRT-PCR of splicing factors (SRSF1 SRSF5 PTBP1 and hnRNPH3) in U87 glioma cell line aftertreating cells with Wortmannin and AR-A 014418 in different experiments for 24 hrs Means plusmn SE 119899 = 5 119875 le 005

In the current set of investigations we are studying theexpression of splicing factors which are themajor deregulatedfamily proteins in maneuvering the alternative splicing eventto escape apoptotic pathway potentially could be explainedafter using small molecular inhibitors against Akt kinase andGSK3beta with ability to restrict the occurrence of alternativepathway either by reversing or decreases the occurrence ofalternative splicing and will possibly help in restricting thetumor genetic heterogeneity

2 Results

21 Cell Viability Assay after Inhibiting GSK3beta and AktKinase Tumor reoccurrence and resistance are of wide con-cern to study in detail the major deregulated multitude sig-nalling pathways Assessing cell viability is one of the param-eters to study mitochondrial function and its attenuationusing small molecule against GSK3beta kinase eventually

inducing apoptotic signalling pathway Hence to study therole of GSK3beta in cell viability we have chosen dosedependent temporal kinetics of small molecule AR-A 014418andManzamineA against GSK3 kinaseWe found that 50120583MAR-A 014418 is required to inhibit the (gt50) glioma cellviability (Figure 1(a))

22 Regulation of Splicing Factor Using GSK3beta and AktKinase Inhibitors Since deregulated expressions of splicingfactors such as PTBP1 SF2ASF (SRSF1 SRSF5) and hnRNPsare the major cause that interfere with the homeostatic sig-naling proteins by inducing translational expression of diver-sified alternative splice variant with antiapoptotic gain orapoptotic loss in advancement of tumorigenicity Functionalsignificance of GSK3beta in arresting gliomagenesis thoughdownregulating the overexpression of splicing factor usinginhibitors against GSK3beta were evaluated (Figure 1(b))compared with Wortmanin (Akt kinase inhibitor) Similarly


CNT

TDZ

Man

z

Man

z

Ly LyCNT

TDZ

hnRNP A1

Cytoplasm Nuclear

U87

120572-Tubulin

(a)

hnRNPA1

SF2ASF

U373

CNT-

i

Akt

-i

120572-Tubulin

GSK

3120573

-i

(b)











0

05

1

15

2

25

U87 CNT U87 AR

BCL2

0 0

1

2

3

4

5

6


Survivin

100

200

300

400

500

600

700

800

900 BCL-xL

0

02

04

06

08

1

12

14

16

U87 CNT U87 AR

MCL1

0

05

1

15

2

25

U87 CNT U87 AR

BMI1

U87 CNT

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(a)

Control

AR-A014418

LY 294002

U87

20days10days

(b)



pTyr-GSK3

p-Akt (ser-473)

T-GSK3beta

p-Erk12

T-Akt

CNT AR

pSer9-GSK3

U87

EGF (50ngmL)

120573

120572

120573

120572

0 30 0 30

(a)

U373

T-GSK3

pErk12

Cytoplasm Nuclear

120572-Tubulin

120572-Tubulin

120573-Catenin

CNT

TDZ8

Man

z A

Man

z A

Ly LyCNT

TDZ8

(b)



3 Discussion





0

05

1

15

2

U87 U373 NB

Anxa7 VB

minus05 0

01

02

03

04

05

06

U87 U373 NB

Anxa7 VA

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(a)

0

05

1

15

2

25

3

35

4

45

U87 AR 6hr

Anxa7 VAAnxa7 VB


n-fo

ld ex

pres

sion

(b)

Anxa7

pErk12

U87

p (tyr)-GSK3

T-GSK3

120572-Tubulin

120572

120573

CNT

AR

Man

z

TDZ

(c)



























Abbreviations







Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CNT

TDZ

Man

z

Man

z

Ly LyCNT

TDZ

hnRNP A1

Cytoplasm Nuclear

U87

120572-Tubulin

(a)

hnRNPA1

SF2ASF

U373

CNT-

i

Akt

-i

120572-Tubulin

GSK

3120573

-i

(b)











0

05

1

15

2

25

U87 CNT U87 AR

BCL2

0 0

1

2

3

4

5

6


Survivin

100

200

300

400

500

600

700

800

900 BCL-xL

0

02

04

06

08

1

12

14

16

U87 CNT U87 AR

MCL1

0

05

1

15

2

25

U87 CNT U87 AR

BMI1

U87 CNT

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(a)

Control

AR-A014418

LY 294002

U87

20days10days

(b)



pTyr-GSK3

p-Akt (ser-473)

T-GSK3beta

p-Erk12

T-Akt

CNT AR

pSer9-GSK3

U87

EGF (50ngmL)

120573

120572

120573

120572

0 30 0 30

(a)

U373

T-GSK3

pErk12

Cytoplasm Nuclear

120572-Tubulin

120572-Tubulin

120573-Catenin

CNT

TDZ8

Man

z A

Man

z A

Ly LyCNT

TDZ8

(b)



3 Discussion





0

05

1

15

2

U87 U373 NB

Anxa7 VB

minus05 0

01

02

03

04

05

06

U87 U373 NB

Anxa7 VA

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(a)

0

05

1

15

2

25

3

35

4

45

U87 AR 6hr

Anxa7 VAAnxa7 VB


n-fo

ld ex

pres

sion

(b)

Anxa7

pErk12

U87

p (tyr)-GSK3

T-GSK3

120572-Tubulin

120572

120573

CNT

AR

Man

z

TDZ

(c)



























Abbreviations







Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

25

U87 CNT U87 AR

BCL2

0 0

1

2

3

4

5

6


Survivin

100

200

300

400

500

600

700

800

900 BCL-xL

0

02

04

06

08

1

12

14

16

U87 CNT U87 AR

MCL1

0

05

1

15

2

25

U87 CNT U87 AR

BMI1

U87 CNT

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(a)

Control

AR-A014418

LY 294002

U87

20days10days

(b)



pTyr-GSK3

p-Akt (ser-473)

T-GSK3beta

p-Erk12

T-Akt

CNT AR

pSer9-GSK3

U87

EGF (50ngmL)

120573

120572

120573

120572

0 30 0 30

(a)

U373

T-GSK3

pErk12

Cytoplasm Nuclear

120572-Tubulin

120572-Tubulin

120573-Catenin

CNT

TDZ8

Man

z A

Man

z A

Ly LyCNT

TDZ8

(b)



3 Discussion





0

05

1

15

2

U87 U373 NB

Anxa7 VB

minus05 0

01

02

03

04

05

06

U87 U373 NB

Anxa7 VA

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(a)

0

05

1

15

2

25

3

35

4

45

U87 AR 6hr

Anxa7 VAAnxa7 VB


n-fo

ld ex

pres

sion

(b)

Anxa7

pErk12

U87

p (tyr)-GSK3

T-GSK3

120572-Tubulin

120572

120573

CNT

AR

Man

z

TDZ

(c)



























Abbreviations







Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















pTyr-GSK3

p-Akt (ser-473)

T-GSK3beta

p-Erk12

T-Akt

CNT AR

pSer9-GSK3

U87

EGF (50ngmL)

120573

120572

120573

120572

0 30 0 30

(a)

U373

T-GSK3

pErk12

Cytoplasm Nuclear

120572-Tubulin

120572-Tubulin

120573-Catenin

CNT

TDZ8

Man

z A

Man

z A

Ly LyCNT

TDZ8

(b)



3 Discussion





0

05

1

15

2

U87 U373 NB

Anxa7 VB

minus05 0

01

02

03

04

05

06

U87 U373 NB

Anxa7 VA

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(a)

0

05

1

15

2

25

3

35

4

45

U87 AR 6hr

Anxa7 VAAnxa7 VB


n-fo

ld ex

pres

sion

(b)

Anxa7

pErk12

U87

p (tyr)-GSK3

T-GSK3

120572-Tubulin

120572

120573

CNT

AR

Man

z

TDZ

(c)



























Abbreviations







Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

U87 U373 NB

Anxa7 VB

minus05 0

01

02

03

04

05

06

U87 U373 NB

Anxa7 VA

n-fo

ld ex

pres

sion

n-fo

ld ex

pres

sion

(a)

0

05

1

15

2

25

3

35

4

45

U87 AR 6hr

Anxa7 VAAnxa7 VB


n-fo

ld ex

pres

sion

(b)

Anxa7

pErk12

U87

p (tyr)-GSK3

T-GSK3

120572-Tubulin

120572

120573

CNT

AR

Man

z

TDZ

(c)



























Abbreviations







Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































Abbreviations







Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Abbreviations







Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

AR-A 014418 Used against GSK3beta Downregulates Expression of hnRNPA1 and SF2/ASF Splicing Factors