research article apoptosis induced by tanshinone iia and...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 805639 10 pageshttpdxdoiorg1011552013805639

Research ArticleApoptosis Induced by Tanshinone IIA andCryptotanshinone Is Mediated by Distinct JAKSTAT35 andSHP12 Signaling in Chronic Myeloid Leukemia K562 Cells

Ji Hoon Jung1 Tae-Rin Kwon1 Soo-Jin Jeong12 Eun-Ok Kim1

Eun Jung Sohn1 Miyong Yun1 and Sung-Hoon Kim1

1 College of Oriental Medicine Kyung Hee University 1 Hoegi-dong Dongdaemun-gu Seoul 130-701 Republic of Korea2 Basic Herbal Medicine Research Group Herbal Medicine Research Division Korea Institute of Oriental MedicineDaejeon 305-811 Republic of Korea

Correspondence should be addressed to Sung-Hoon Kim sungkim7khuackr

Received 4 April 2013 Accepted 3 June 2013

Academic Editor Khalid Rahman

Copyright copy 2013 Ji Hoon Jung 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

Though tanshinone IIA and cryptotanshinone possess a variety of biological effects such as anti-inflammatory antioxidativeantimetabolic and anticancer effects the precise molecular targets or pathways responsible for anticancer activities of tanshinoneIIA and cryptotanshinone in chronicmyeloid leukemia (CML) still remain unclear In the present study we investigated the effect oftanshinone IIA and cryptotanshinone on the Janus activated kinase (JAK)signal transducer and activator of transcription (STAT)signaling during apoptotic process We found that both tanshinone IIA and cryptotanshinone induced apoptosis by activation ofcaspase-93 and Sub-G1 accumulation in K562 cells However they have the distinct JAKSTAT pathway in which tanshinoneIIA inhibits JAK2STAT5 signaling whereas cryptotanshinone targets the JAK2STAT3 In addition tanshinone IIA enhancedthe expression of both SHP-1 and -2 while cryptotanshinone regulated the expression of only SHP-1 Both tanshinone IIA andcryptotanshinone attenuated the expression of bcl-xL survivin and cyclin D1 Furthermore tanshinone IIA augmented synergywith imatinib a CML chemotherapeutic drug better than cryptotanshinone in K562 cells Overall our findings suggest that theanticancer activity of tanshinone IIA and cryptotanshinone is mediated by the distinct the JAKSTAT35 and SHP12 signaling andtanshinone IIA has the potential for combination therapy with imatinib in K562 CML cells

1 Introduction

Salvia miltiorrhiza Bunge (Danshen) is a traditional medic-inal herb widely used for treating cardiovascular diseasein Korea China and Japan [1] To date over 90 kinds ofchemical constituents from Smiltiorrhizahave been reported[2 3] Of the phytochemicals tanshinones are a group oflipophilic abietane diterpene compounds including tanshi-none I tanshinone IIA-B cryptotanshinone dihydrotanshi-none I isotanshinone I and isocryptotanshinone I-II andhave been extensively investigated [1 4ndash7] In particular tan-shinone IIA and cryptotanshinone have been presented thepotential as anticancer drugs by targeting themultiple signal-ing pathways [8ndash18]

STAT family is transcriptional factors that play key rolesin cytokine signaling [19] STAT proteins are constitutivelyactivated in cancer cells or tissues and thus have beensuggested as attractive molecular target(s) for cancer therapyIn light of these events numerous groups reported the inhi-bitory effects of plant polyphenols such as curcumin resver-atrol piceatannol and EGCG on STAT activation in variouscancer cells [19 20] Tanshinone IIA and cryptotanshinonewere also shown to have the inhibitory effects on the STATactivation in C6 glioma [21] and DU145 prostate cancer cells[22] respectively However there is no report on the molec-ular mechanisms leading to anticancer activity of tanshi-none IIA and cryptotanshinone through the STAT signalingpathway in leukemia cells

2 Evidence-Based Complementary and Alternative Medicine

In the current study we investigated the inhibitory effectsof tanshinone IIA and cryptotanshinone on the activation ofSTAT3 or 5 linked to apoptosis in chronic myeloid leukemia(CML) K562 cells Additionally the synergistic effects of tan-shinone IIA or cryptotanshinonewith imatinib a chemother-apeutic agent for CML were examined by calculating combi-nation index (CI)

2 Materials and Methods

21 Isolation of Tanshinone IIA and Cryptotanshinone Tan-shinone IIA [23] and cryptotanshinone [24] (Figure 1(a))were isolated as previously described

22 Cell Culture Human chronic myeloid leukemia K562cells were purchased fromAmerican Type Culture Collection(ATCC Rockville MD USA) and maintained in RPMI 1640medium supplemented 10 fetal bovine serum (FBS) 2 120583ML-glutamine and penicillinstreptomycin

23 Cytotoxicity Assay Cytotoxic effects of tanshinone IIAor cryptotanshinone against K562 cells were evaluated by 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) assay Cells were seeded onto 96-well microplates ata density of 2 times 104 cells per well and exposed to variousconcentrations of tanshinone IIA or cryptotanshinone (0 1020 40 or 80120583M) for 24 h The cells were incubated with 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazoliumbromide(1mgmL) (Sigma Chemical Co St Louis MO USA) for 2 hand then with MTT lysis solution overnight Optical density(OD) was measured using a microplate reader (MolecularDevices Co Sunnyvale CA USA) at 570 nm Cell viabilitywas calculated as a percentage of viable cells in drug-treatedgroup versus untreated control by the following equation

Cell viability ()

=[OD (Drug) minusOD (Blank)][OD (Control) minusOD (Blank)]

times 100

(1)

24 Western Blot Analysis K562 cells were lysed in lysisbuffer (50mM Tris-HCl pH 74 150mM NaCl 1 Triton X-100 01 SDS 1mM EDTA 1mM Na

3VO4 1 mM NaF and

protease inhibitors cocktail) The extracts were incubated onice for 30min and supernatants were collected by centrifuga-tion at 14000 g at 4∘CTheprotein contents in the supernatantwere measured by using a Bio-Rad DC protein assay kit IIProteins were separated by electrophoresis on 125 SDS-PAGE gel and electrotransferred onto a Hybond ECL transfermembrane with transfer buffer (25mMTris 250mMglycineand 20methanol) at 300mA for 90minThemembranewasblocked in 5 nonfat skim milk and probed with primaryantibodies for p-STAT3 p-STAT5 STAT3 STAT5 p-JAK2JAK2 (Cell Signaling Tech Danvers MA USA) SHP-1SHP-2 bcl-xL mcl-1 survivin cyclin D1 cleaved caspase-9 cleaved caspase-3 poly(ADP-ribose) polymerase (PARP)and tubulin (Santa Cruz Biotechnologies Santa Cruz CAUSA) followed by incubating with horseradish peroxidase-(HRP-) conjugated secondary antibodies Protein expression

was detected by using enhanced chemiluminescence (ECL)system (Amersham Pharmacia Piscataway NJ USA)

25 Electrophoretic Mobility Shift Assay (EMSA) The STAT3or STAT5DNA binding activity was analyzed by EMSAusing gel shift chemiluminescent EMSA kit (Active motifCarlsbad CA USA) Nuclear extracts were incubated withSTAT3 (51015840-GAT CCT TCT GGG AAT TCC TAG ATC-31015840)or STAT5 (51015840-AGA TTT CTA ATT CAA TCC-31015840) consensusoligonucleotides (Santa Cruz Biotechnology Santa Cruz CAUSA) The DNAprotein complex formed was separatedfrom free oligonucleotides on 5 native polyacrylamidegels Chemiluminescent detection was performed using ECLreagents according to the vendorrsquos protocols (GEHealth CareBio-Sciences Piscataway NJ USA)

26 Cell Cycle Analysis Cell cycle analysis was performedby PI staining K562 cells were treated with tanshinone IIAor cryptotanshinone for 24 h collected and fixed in 70ethanol The cells were then incubated at 37∘C with 01RNase A in PBS for 30min and suspended in PBS containing25 120583gmL PI for 30min at room temperature The stainedcells were analyzed for DNA content in FACSCalibur (BectonDickinson Franklin Lakes NJ USA) using the Cell Questprogram (Becton Dickinson Franklin Lakes NJ USA)

27 Apoptosis Analysis by Annexin V-PI Double StainingApoptosis of the cryptotanshinone or tanshinone IIA-treatedcells was quantitated by double staining with Annexin V-FITC and PI using the Annexin V-Apoptosis Detection kit(Biovision Milpitas CA USA) according to the manufactur-errsquos instructions Apoptotic cells were analyzed by FACSCal-ibur (Becton Dickinson San Jose CA USA) to be defined asthose positive for Annexin V with or without PI staining

28 Propidium Iodide (PI) Staining K562 cells were exposedto tanshinone IIA or cryptotanshinone and plated onto poly-L-lysine-coated slide glassThe cells were fixed in 70 ethanoland stained with PI solution (50 120583gmL) (BD BiosciencesBedford MA USA) containing 100 120583gmL RNase for 10minThe slides were mounted with 70 glycerol in PBS and vis-ualized under an Axio vision 40 fluorescence microscope(Carl Zeiss Inc Weimar Germany)

29 Combination Index (CI) Calculation The CI was deter-mined by Chou-Talalay method and CalcuSyn software (Bio-soft Ferguson MO USA) A CI of less than 1 was consideredsynergistic [25]

210 Statistical Analyses All data were presented as means plusmnstandard deviation (SD) Statistical significance was verifiedby Studentrsquos t-test using SigmaPlot software (Systat SoftwareInc San Jose CA USA)

3 Results

31 Tanshinone IIA and Cryptotanshinone Exert Cytotoxicityagainst Chronic Myeloid Leukemia K562 Cells To comparethe cytotoxicity of tanshinone IIA and cryptotanshinone inK562 cells MTT assay was performed Cells were treated

Evidence-Based Complementary and Alternative Medicine 3

O O

O O

OO

CH3

CH3 CH3

H3C CH3H3CTanshinone IIA Cryptotanshinone

(a)

Tanshinone IIACryptotanshinone

Concentration (120583M)

140

120

100

80

60

40

20

00 10 20 40 80

IC50

Cel

l via

bilit

y (

of c

ontro

l)

(b)

Figure 1 Tanshinone IIA and cryptotanshinone exert cytotoxicity in K562 cells (a) Chemical structures of tanshinone IIA (left) andcryptotanshinone (right) (b) Cells were treated with various concentrations of tanshinone IIA or cryptotanshinone (0 10 20 40 or 80 120583M)for 24 h MTT assay was performed to evaluate the cytotoxicity

with various concentrations (0 10 20 40 or 80 120583M) for24 h Both tanshinone IIA and cryptotanshinone substantiallyreduced the cell viability in a dose-dependentmanner (Figure1(b)) There was no significant difference in the cytotoxicitybetween two chemicals in the cells (IC

50= sim20120583M)

32 Tanshinone IIA Inhibits STAT5 but Not STAT3 Signalingin K562 Cells Effects of tanshinone IIA on STAT3 and5 activation were examined by Western blot analysis Asshown in Figure 2(a) tanshinone IIA treatment significantlyinhibited the phosphorylation of STAT5 but not STAT3in a dose- and time-dependent manner We further con-firmed the inhibitory effect of tanshinone IIA on STAT5by gel shift mobility assay Consistent with the results ofimmunoblotting tanshinone IIA prevented the STAT5DNAbinding in a dose-dependent manner (Figure 2(b)) To findout whether tyrosine kinases mediate the tanshinone IIA-initiated STAT5 inactivation the effects of tanshinone IIAon the phosphorylation of JAK1 2 and c-Src in K562 cells

were examined The results revealed that tanshinone IIA ledto dephosphorylation of JAK2 (Figure 2(c)) but not JAK1and c-Src (data not shown) Furthermore we observed thattanshinone IIA enhanced expression of tyrosine phosphataseSHP-1 and -2 in a time-dependent manner (Figure 2(d))

33 Cryptotanshinone Inhibits STAT3 but Not STAT5 Sig-naling in K562 Cells Parallel assays were carried out incryptotanshinone-treated K562 cells Different from tanshi-none IIA cryptotanshinone reduced the phosphorylationlevel of STAT3 but not STAT5 in a dose- and time-dependent manner (Figure 3(a)) In addition cryptotan-shinone suppressed the binding of STAT3 to DNA in adose-dependent manner (Figure 3(b)) However cryptotan-shinone also inhibited the phosphorylation of JAK2 anupstream kinase of STAT3 or 5 in the cells (Figure 3(c))Besides cryptotanshinone led to increased expression ofSHP-1 but no effect on the expression of SHP-2 (Figure 3(d))


Tanshinone IIA

0 10 20 (120583M)

p-STAT3

STAT3

p-STAT5

STAT5

p-STAT3

STAT3

p-STAT5

STAT5

Tanshinone IIA (20 120583M)

0 6 12 24 (h)

(a)

Tanshinone IIA

0 10 20 (120583M)

STAT5STAT5STAT5 monomer

Free probe

(b)

Tanshinone IIA

0 10 20 (120583M)

p-JAK2

JAK2

(c)


0 3 6 12 24 36SHP-1

SHP-2

Tubulin

(h)

(d)

Figure 2 Tanshinone IIA inactivates STAT5 but not STAT3 in K562 cells (a) Cells were treated with tanshinone IIA (0 10 or 20 120583M)for 24 h (left) or 20120583M for 0 6 12 or 24 h (right) Cell lysates were prepared and subjected to Western blotting for phospho-STAT3 andphospho-STAT5 (b) Cells were treated with tanshinone IIA (0 10 or 20 120583M) for 24 h Gel shift mobility assay was performed to determinethe STAT5DNA binding activity (c) Cells were treated with tanshinone IIA (0 10 or 20120583M) for 24 h Western blotting was performed todetect phosphorylation of JAK2 (d) Cells were treated with 20120583M tanshinone IIA for 0 3 6 12 24 or 36 h Western blotting was conductedto determine the expression of SHP-1 and SHP-2

34 Tanshinone IIA and Cryptotanshinone Induce Apoptosisin K562 Cells JAKSTAT signaling regulates gene productsinvolved in various cellular processes such as survival pro-liferation and cell cycle progression [20 26 27] Both tan-shinone IIA and cryptotanshinone significantly attenuatedthe expression of STAT-related survival genes such as bcl-xL surviving and cyclin D1 in a dose-dependent manner(Figure 4(a)) However only tanshinone IIA but not crypto-tanshinone suppressed the expression of antiapoptotic mcl-1L in K562 cells (Figure 4(a) left panel) To confirm thattanshinone IIA or cryptotanshinone can induce apoptosisactivation of caspase-9 and -3 key molecules in intrinsicapoptosis pathway [28] was evaluated by immunoblottingAs expected both tanshinone IIA and cryptotanshinoneclearly induced the cleavages of caspase-9 and -3 as well as

PARP in a dose-dependent manner (Figure 4(b)) Consis-tently cell cycle analysis showed increased accumulation ofthe sub-G1 cell from 022 to 1719 or 1760 by tanshinoneIIA or cryptotanshinone in K562 cells respectively (Figure4(c)) Moreover we found that treatment of 20120583M tanshi-none IIA or cryptotanshinone dramatically increased theapoptotic cell population by Annexin V-PI double stainingto 2396 and 1801 respectively (Figure 4(d))

35 Tanshinone IIA and Cryptotanshinone SynergisticallyPromote Anticancer Effects with Imatinib in K562 Cells Bcr-abl is an abnormal gene formed by the reciprocal translo-cation between chromosomes 9 and 22 in CML [29] Weexamined whether tanshinone IIA or cryptotanshinone canaffect activation of bcr-abl by Western blotting As shown


Cryptotanshinone

0 10 20 (120583M)

p-STAT3

STAT3

p-STAT5

STAT5

p-STAT3

STAT3

p-STAT5

STAT5

Cryptotanshinone (20 120583M)

0 6 12 24 (h)

(a)

Cryptotanshinone

0 10 20 (120583M)


Free probe

(b)

Cryptotanshinone

0 10 20 (120583M)

p-JAK2

JAK2

(c)


(h)0 3 6 12 24 36SHP-1

SHP-2

Tubulin

(d)

Figure 3 Cryptotanshinone inactivates STAT3 but not STAT5 in K562 cells (a) Cells were treated with cryptotanshinone (0 10 or 20120583M)for 24 h (left) or 20120583M for 0 6 12 or 24 h (right) Cell lysates were prepared and subjected to Western blotting for phospho-STAT3 andphospho-STAT5 (b) Cells were treated with cryptotanshinone (0 10 or 20 120583M) for 24 h Gel shift mobility assay was performed to determinethe STAT3DNA binding activity (c) Cells were treated with cryptotanshinone (0 10 or 20120583M) for 24 h Western blotting was performed todetect phosphorylation of JAK2 (d) Cells were treatedwith 20 120583Mcryptotanshinone for 0 3 6 12 24 or 36 hWestern blottingwas conductedto determine the expression of SHP-1 and SHP-2

in Figure 5(a) both tanshinone IIA and cryptotanshinonereduced phosphorylation of bcr-abl in a dose-dependentmanner Then to test the synergy between tanshinone IIA orcryptotanshinone and imatinib a competitive tyrosine kinaseinhibitor used in the treatment of CML [30] K562 cells werecotreated with tanshinone IIA or cryptotanshinone (0 25 or5 120583M) in the absence or presence of imatinib (025120583M) for24 hThe cell viabilitywas significantly decreased in combina-tion of tanshinone IIA or cryptotanshinone with imatinibin a dose-dependent manner compared to untreated control(Figure 5(b)) Tanshinone IIA remarkably showed the syner-gistic effect on the imatinib-induced apoptosis with CI value= 0315 and 0628 at 25 and 5 120583M respectively (Figure 5(c))In contrast cryptotanshinone treatment with imatinib hadthe synergistic effect only at 25120583M (CI = 0776) while

showing the additional effect at 5120583M (CI = 1048) (Figure5(c)) Furthermore combination treatment of imatinib andtanshinone IIA synergistically increased the apoptotic popu-lation of Annexin V-PI double positive stained cells to 16while single treatment of imatinib or tanshinone IIA induced496 and 918 apoptosis in K562 respectively (Figure5(d))

4 Discussion

Phytochemicals are natural compounds in plants such asfruits vegetables beans grains and others In the AmericanCancer Society (ACS) report in 2008 some phytochemicalsmay account for the beneficial effects in humans to preventand treat many health conditions For this reason these


Tanshinone IIA Cryptotanshinone

0 10 20 0 10 20(120583M) (120583M)

Survivin

Cyclin D1

Tubulin

mcl-1L mcl-1L

Survivin

Cyclin D1

Tubulin

bcl-xLbcl-xL

(a)


0 10 20 0 10 20(120583M) (120583M)

Cleaved caspase-3 Cleaved caspase-3

Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Tubulin Tubulin

(b)

Cou

nts

022

Control

0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600

Tanshinone IIA

Tanshinone IIA

Cryptotanshinone

Cryptotanshinone

1091 1719 906 176

20 120583M

20 120583M

20 120583M

Sub-

G1

()

20

15

10

5

0

Control10120583M

10120583M10120583M

10120583M 20120583M

(c)

Figure 4 Continued


PI

0 10 20 (120583M)

Cryp

tota

nshi

none

Tans

hino

ne II

A

058

034

366

331

718

452

925

876

1572

824

Annexin-V

(d)

Figure 4 Tanshinone IIA and cryptotanshinone induce apoptosis in K562 cells (a and b) Cells were treated with tanshinone IIA (left) orcryptotanshinone (right) for 24 h Cell lysates were prepared and subjected to Western blotting to detect expression of apoptosis-relatedproteins bcl-xL mcl-1L surviving and cyclin D1 (a) and caspase-3 caspase-9 and PARP (b) (c) Flow cytometry analysis of sub-G1 apoptoticDNA fraction of the cells treated with tanshinone IIA or cryptotanshinone (0 10 or 20120583M) for 24 h After fixation in 75 ethanol cellswere stained with PI and analyzed by flow cytometry (d) Cells were treated with tanshinone IIA or cryptotanshinone (10 or 20120583M) for 24 hPercentage of apoptotic cells in tanshinone IIA or cryptotanshinone-treated cells by Annexin V-PI staining Each experiment was repeatedthree times

phytochemicals have been thought as valuable materialsto develop new therapeutic drug or dietary supplementRecently numerous papers have reported the potential ofphytochemicals to ameliorate the various diseases such ascancer inflammation metabolic syndrome and cardiovas-cular disease In the present study we comparatively inves-tigated the anticancer mechanism of tanshinone IIA andcryptotanshinone from S miltiorrhiza in CML a form ofleukemia characterized by the increased and unregulatedgrowth of predominantly myeloid cells in the bone marrow[31] Our group recently reported that tanshinone IIA inducesapoptosis through activation of c-jun N-terminal kinase inKBM-5 cells [18] Ge et al reported that cryptotanshinonemediates cell cycle arrest and apoptosis ofmultidrug-resistantK562ADM cells by inactivating eukaryotic initiation factor4E [10] Additionally we also reported that cryptotanshinoneenhances TNF-120572-induced apoptosis in KBM-5 cells [11]Nonetheless themolecularmechanisms leading to anti-CMLproperties of tanshinone IIA and cryptotanshinone are notfully understood yet

STAT is one of the important transcriptional factor fami-lies and plays crucial roles as a molecular target for cancerprevention and therapy [32] STAT family consists of 7 differ-ent subfamilies STAT1 2 3 4 5a 5b and 6 and STAT3and 5 are constitutively activated in cancer cells STAT 3and 5 are activated by nonreceptor tyrosine kinases of theJanus family (JAK) and c-Src [33 34] and protein tyrosinephosphatases (PTPs) such as Src homology 2 domain-containing phosphatases (SHPs) phosphatase and tensinhomolog (PTEN) and suppressor of cytokine signalingproteins (SOCS) are also linked to STAT signaling [35]Therefore the JAKSTAT3 or 5 signaling has been thoughtas a valuable molecular target for cancer therapy [34 36]

In our study we found that both tanshinone IIA andcryptotanshinone reduced the phosphorylation of JAK2 anupstream kinase of STATs in K562 CML cells Howeverthe effects of tanshinone IIA and cryptotanshinone on STATactivation were clearly different in K562 cells TanshinoneIIA reduced the phosphorylation of STAT5 but not STAT3and consistently prevented the STAT5DNA binding in thecells In contrast cryptotanshinone inactivated STAT3 butnot STAT5 at posttranslational and transcriptional levels Inaddition tanshinone IIA induced the expression of SHP-1and -2 whereas cryptotanshinone increased the expression ofSHP-1 but not SHP-2 in K562 cells (Figure 6)

The JAKSTAT signaling is involved in oncogenesis andcancer progression through upregulation of antiapoptoticgenes [37] Tanshinone IIA and cryptotanshinone commonlyrepressed the expression of bcl-xL survivin and cyclin D1in K562 cells In contrast only tanshinone IIA but notcryptotanshinone decreased the mcl-1L expression Apop-tosis induction by tanshinone IIA or cryptotanshinone wasconfirmed by activation of caspase-9 and -3 cell cycle analysisand nuclear staining using PI (Figure 6) Although tanshi-none IIA and cryptotanshinone exerted anti-CML activitiesin a different way by targeting the distinct STAT signalingthere was no significant difference in the induction of apopto-sis by them Further studies are necessary such as gene silenc-ing for SHP-2 or mcl-1L to verify the precise mechanismsresponsible for the different regulation between tanshinoneIIA and cryptotanshinone against CML cells in the nearfuture Using stable cells overexpressed STAT3 or 5 will bealso beneficial tools to prove the anti-CML mechanisms

Bcr-Abl selective tyrosine kinase inhibitor imatinib (mar-keted by Novartis as Gleevec) has been extensively used forCML therapy [30] However despite of its specific therapeutic


Tanshinone IIA Cryptotanshinone Imatinib

0 25 5 10 20 25 5 10 20 1 (120583M)

p-Bcr-abl

c-abl

120573-Actin

(a)

Cel

l via

bilit

y (c

ontro

l (

))

Cel

l via

bilit

y (c

ontro

l (

))

100

80

60

40

20

0

100

80

60

40

20

0

minus

minus minus minus

minus

+ + +Imatinib (025 120583M) minus minus minus+ + +Imatinib (025 120583M)25 5 25 5

lowastlowastlowast

lowastlowast

minus minus 25 5 25 5Tanshinone IIA (120583M) Cryptotanshinone (120583M)

(b)

Com

bina

tion

inde

x

2

1

00 02 04 06 08 1

Effect

CI = 1

CI = 0472 plusmn 02225025 (CI = 0315)5025 (CI = 0628)

25025 (CI = 0776)5025 (CI = 1048)

Tanshinone IIAimatinib Cryptotanshinoneimatinib

CI = 0812 plusmn 036

(c)

0

0

0015

1555

146

094

343

153

728

19

137

227

PI

Annexin-V

Imatinib (120583M)Tanshinone IIA (120583M)


(d)

Figure 5 Tanshinone IIA and cryptotanshinone with imatinib synergistically inhibit the viability of K562 cells (a) Cells were treated withvarious concentrations of tanshinone IIA or cryptotanshinone (0 25 5 10 or 20120583M) for 24 h Cell lysates were prepared and subjected toWestern blotting for phospho-bcr-abl (b) Cells were treated with tanshinone IIA (left) or cryptotanshinone (right) andor imatinib for 24 hCell viability was measured by MTT assay (c) The combination index (CI) between two drugs was determined by Chou-Talalay methodand CalcuSyn software (Biosoft Ferguson MO USA) (d) Cells were treated with tanshinone IIA and imatinib for 24 h at the same timePercentage of apoptotic cells in cotreated cells by Annexin V-PI double staining


SHP-1SHP-2

JAK2 SHP-1


STAT5 STAT3

mcl-1L SurvivinCyclin D1Survivin

Cyclin D1Caspase-93

bcl-xL bcl-xL

Figure 6 Schematic diagram indicating the effect of tanshinoneIIA and cryptotanshinone on JAKSTAT signaling and apoptosispathways in CML cells

effect for CML serious adverse effects and cost problem canlimit the use of imatinib In the current study we testedthe possibility that tanshinone IIA or cryptotanshinone canstimulate anti-CML effect induced by imatinib by loweringdosage in K562 cells Our data revealed that tanshinoneIIA enhanced imatinib-induced cell death more effectivelythan cryptotanshinone with CI value lt1 even at 25 120583Mdetermined by Chou-Talalay method and CalcuSyn softwareimplying significant synergy between tanshinone IIA andimatinib as a potent combination therapy for CML Howeveradditional experiments are required using in vivo mousexenograft model to validate the in vitro studies

In summary tanshinone IIA inhibited JAK2STAT5 sig-naling whereas cryptotanshinone targets the JAK2STAT3in K562 cells Furthermore tanshinone IIA enhanced theexpression of both SHP-1 and -2 while cryptotanshinoneregulated the expression of only SHP-1 Also both tanshinoneIIA and cryptotanshinone attenuated the expression of STAT-related genes such as bcl-xL survivin and cyclin D1

5 Conclusion

Our findings clearly demonstrate that anticancer activity oftanshinone IIA and cryptotanshinone is mediated by thedistinct JAKSTAT35 and SHP12 signaling in K562 cells Ofnote tanshinone IIA showed more potential for the synergywith imatinib compared with cryptotanshinone as a potentcandidate for combination therapy

Authorsrsquo Contribution

Ji Hoon Jung Tae-Rin Kwon and Soo-Jin Jeong contributedequally to this work

Acknowledgment

This work was supported by the Korea Science and Engi-neering Foundation (KOSEF) Grant funded by the Koreagovernment (MEST) (no 2011-0063466)

References

[1] L Zhou Z Zuo andM S S Chow ldquoDanshen an overview of itschemistry pharmacology pharmacokinetics and clinical userdquoJournal of Clinical Pharmacology vol 45 no 12 pp 1345ndash13592005

[2] A-H Liu L Li M Xu Y-H Lin H-Z Guo and D-A GuoldquoSimultaneous quantification of six major phenolic acids in theroots of Salvia miltiorrhiza and four related traditional Chinesemedicinal preparations by HPLC-DAD methodrdquo Journal ofPharmaceutical and Biomedical Analysis vol 41 no 1 pp 48ndash56 2006

[3] L Ma X Zhang H Guo and Y Gan ldquoDetermination offour water-soluble compounds in Salvia miltiorrhiza Bunge byhigh-performance liquid chromatography with a coulometricelectrode array systemrdquo Journal of Chromatography B vol 833no 2 pp 260ndash263 2006

[4] X Wang S L Morris-Natschke and K-H Lee ldquoNew develop-ments in the chemistry and biology of the bioactive constituentsof TanshenrdquoMedicinal Research Reviews vol 27 no 1 pp 133ndash148 2007

[5] H-C Bi Z Zuo X Chen et al ldquoPreclinical factors affectingthe pharmacokinetic behaviour of tanshinone IIA an inves-tigational new drug isolated from Salvia miltiorrhiza for thetreatment of ischaemic heart diseasesrdquo Xenobiotica vol 38 no2 pp 185ndash222 2008

[6] M-J Don C-C Shen W-J Syu Y-H Ding and C-M SunldquoCytotoxic and aromatic constituents from Salvia miltiorrhizardquoPhytochemistry vol 67 no 5 pp 497ndash503 2005

[7] M Gu G Zhang Z Su and F Ouyang ldquoIdentification of majoractive constituents in the fingerprint of Salvia miltiorrhizaBunge developed by high-speed counter-current chromatogra-phyrdquo Journal of Chromatography A vol 1041 no 1-2 pp 239ndash243 2004

[8] W Chen L Liu Y Luo et al ldquoCryptotanshinone activates p38JNK and inhibits Erk12 leading to caspase-independent celldeath in tumor cellsrdquo Cancer Prevention Research vol 5 pp778ndash787 2012

[9] W Chen Y Lu G Chen and S Huang ldquoMolecular evidenceof cryptotanshinone for treatment and prevention of humancancerrdquo Anti-cancer Agents in Medicinal Chemistry In press

[10] Y Ge R Cheng Y Zhou et al ldquoCryptotanshinone induces cellcycle arrest and apoptosis ofmultidrug resistant human chronicmyeloid leukemia cells by inhibiting the activity of eukaryoticinitiation factor 4Erdquo Molecular and Cellular Biochemistry vol368 pp 17ndash25 2012

[11] J-H Kim S-J Jeong T-R Kwon et al ldquoCryptotanshinoneenhances TNF-120572-induced apoptosis in chronic myeloid leuke-mia KBM-5 cellsrdquo Apoptosis vol 16 no 7 pp 696ndash707 2011

[12] H J Lee D B Jung E J Sohn et al ldquoInhibition of hypoxiainducible factor alpha and astrocyte-elevated gene-1 medi-ates cryptotanshinone exerted antitumor activity in hypoxicPC-3 cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2012 Article ID 390957 13 pages 2012

[13] C-Y Cheng and C-C Su ldquoTanshinone IIA may inhibit thegrowth of small cell lung cancer H146 cells by up-regulatingthe BaxBcl-2 ratio and decreasing mitochondrial membranepotentialrdquo Molecular Medicine Reports vol 3 no 4 pp 645ndash650 2010

[14] C-C Su ldquoTanshinone IIA potentiates the efficacy of 5-FU inColo205 colon cancer cells in vivo through downregulation of


P-gp and LC3-IIrdquo Experimental and Therapeutic Medicine vol3 no 3 pp 555ndash559 2012

[15] S-H Won H-J Lee S-J Jeong et al ldquoTanshinone IIa inducesmitochondria dependent apoptosis in prostate cancer cells inassociation with an inhibition of phosphoinositide 3-kinaseAKT pathwayrdquo Biological and Pharmaceutical Bulletin vol 33no 11 pp 1828ndash1834 2010

[16] S-H Won H-J Lee S-J Jeong J Lu and S-H Kim ldquoActi-vation of p53 signaling and inhibition of androgen receptormediate tanshinone IIA induced G1 arrest in LNCaP prostatecancer cellsrdquo Phytotherapy Research vol 26 no 5 pp 669ndash6742012

[17] S Xu and P Liu ldquoTanshinone II-A new perspectives for oldremediesrdquo Expert Opinion on Therapeutic Patents vol 23 pp149ndash153 2013

[18] S M Yun S J Jeong J H Kim et al ldquoActivation of C-Jun N-terminal kinase mediates tanshinone IIA-induced apoptosis inKBM-5 chronic myeloid leukemia cellsrdquo Biological amp Pharma-ceutical Bulletin vol 36 pp 208ndash214 2013

[19] R Buettner L B Mora and R Jove ldquoActivated STAT signalingin human tumors provides novel molecular targets for thera-peutic interventionrdquo Clinical Cancer Research vol 8 no 4 pp945ndash954 2002

[20] J Vera K Rateitschak F Lange C Kossow O Wolkenhauerand R Jaster ldquoSystems biology of JAK-STAT signalling inhuman malignanciesrdquo Progress in Biophysics and MolecularBiology vol 106 no 2 pp 426ndash434 2011

[21] C Tang H-L Xue H-B Huang andX-GWang ldquoTanshinoneIIA inhibits constitutive STAT3 activation suppresses prolifera-tion and induces apoptosis in rat C6 glioma cellsrdquoNeuroscienceLetters vol 470 no 2 pp 126ndash129 2010

[22] D-S Shin H-N Kim K D Shin et al ldquoCryptotanshinoneinhibits constitutive signal transducer and activator of tran-scription 3 function through blocking the dimerization inDU145 prostate cancer cellsrdquo Cancer Research vol 69 no 1 pp193ndash202 2009

[23] H-S Choi D-I Cho H-K Choi S Y Im S-Y Ryu and K-MKim ldquoMolecular mechanisms of inhibitory activities of tanshi-nones on lipopolysaccharide-induced nitric oxide generation inRAW2647 cellsrdquoArchives of Pharmacal Research vol 27 no 12pp 1233ndash1237 2004

[24] J M Hur J S Shim H J Jung and H J Kwon ldquoCryptotan-shinone but not tanshinone IIA inhibits angiogenesis in vitrordquoExperimental andMolecular Medicine vol 37 no 2 pp 133ndash1372005

[25] T-C Chou ldquoDrug combination studies and their synergy quan-tification using the chou-talalay methodrdquo Cancer Research vol70 no 2 pp 440ndash446 2010

[26] W X Li ldquoCanonical and non-canonical JAK-STAT signalingrdquoTrends in Cell Biology vol 18 no 11 pp 545ndash551 2008

[27] P Sansone and J Bromberg ldquoTargeting the interleukin-6Jakstat pathway in humanmalignanciesrdquo Journal of Clinical Oncol-ogy vol 30 no 9 pp 1005ndash1014 2012

[28] H Duan K Orth A M Chinnaiyan et al ldquoICE-LAP6 a novelmember of the ICECed-3 gene family is activated by the cyto-toxic T cell protease granzyme Brdquo Journal of Biological Chem-istry vol 271 no 28 pp 16720ndash16724 1996

[29] C L Sawyers C T Denny and O N Witte ldquoLeukemia andthe disruption of normal hematopoiesisrdquo Cell vol 64 no 2 pp337ndash350 1991

[30] B J Druker S Tamura E Buchdunger et al ldquoEffects of a sel-ective inhibitor of the Ab1 tyrosine kinase on the growth of Bcr-Ab1 positive cellsrdquo Nature Medicine vol 2 no 5 pp 561ndash5661996

[31] S Faderl M Talpaz Z Estrov and H M Kantarjian ldquoChronicmyelogenous leukemia biology and therapyrdquoAnnals of InternalMedicine vol 131 no 3 pp 207ndash219 1999

[32] B B Aggarwal G Sethi K S Ahn et al ldquoTargeting signal-transducer-and-activator-of-transcription-3 for prevention andtherapy of cancer Modern target but ancient solutionrdquo Annalsof the New York Academy of Sciences vol 1091 pp 151ndash169 2006

[33] CM Silva ldquoRole of STATs as downstream signal transducers inSrc family kinase-mediated tumorigenesisrdquo Oncogene vol 23no 48 pp 8017ndash8023 2004

[34] O V Smirnova T Y Ostroukhova and R L Bogorad ldquoJAK-STAT pathway in carcinogenesis is it relevant to cholangiocar-cinoma progressionrdquoWorld Journal of Gastroenterology vol 13no 48 pp 6478ndash6491 2007

[35] Y Han H M Amin B Franko C Frantz X Shi and R LaildquoLoss of SHP1 enhances JAK3STAT3 signaling and decreasesproteosome degradation of JAK3 and NPM-ALK in ALK+anaplastic large-cell lymphomardquoBlood vol 108 no 8 pp 2796ndash2803 2006

[36] T Hirano K Ishihara andM Hibi ldquoRoles of STAT3 in mediat-ing the cell growth differentiation and survival signals relayedthrough the IL-6 family of cytokine receptorsrdquo Oncogene vol19 no 21 pp 2548ndash2556 2000

[37] J Turkson ldquoSTAT proteins as novel targets for cancer drug dis-coveryrdquo Expert Opinion onTherapeutic Targets vol 8 no 5 pp409ndash422 2004

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


In the current study we investigated the inhibitory effectsof tanshinone IIA and cryptotanshinone on the activation ofSTAT3 or 5 linked to apoptosis in chronic myeloid leukemia(CML) K562 cells Additionally the synergistic effects of tan-shinone IIA or cryptotanshinonewith imatinib a chemother-apeutic agent for CML were examined by calculating combi-nation index (CI)

2 Materials and Methods

21 Isolation of Tanshinone IIA and Cryptotanshinone Tan-shinone IIA [23] and cryptotanshinone [24] (Figure 1(a))were isolated as previously described

22 Cell Culture Human chronic myeloid leukemia K562cells were purchased fromAmerican Type Culture Collection(ATCC Rockville MD USA) and maintained in RPMI 1640medium supplemented 10 fetal bovine serum (FBS) 2 120583ML-glutamine and penicillinstreptomycin

23 Cytotoxicity Assay Cytotoxic effects of tanshinone IIAor cryptotanshinone against K562 cells were evaluated by 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) assay Cells were seeded onto 96-well microplates ata density of 2 times 104 cells per well and exposed to variousconcentrations of tanshinone IIA or cryptotanshinone (0 1020 40 or 80120583M) for 24 h The cells were incubated with 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazoliumbromide(1mgmL) (Sigma Chemical Co St Louis MO USA) for 2 hand then with MTT lysis solution overnight Optical density(OD) was measured using a microplate reader (MolecularDevices Co Sunnyvale CA USA) at 570 nm Cell viabilitywas calculated as a percentage of viable cells in drug-treatedgroup versus untreated control by the following equation

Cell viability ()

=[OD (Drug) minusOD (Blank)][OD (Control) minusOD (Blank)]

times 100

(1)

24 Western Blot Analysis K562 cells were lysed in lysisbuffer (50mM Tris-HCl pH 74 150mM NaCl 1 Triton X-100 01 SDS 1mM EDTA 1mM Na

3VO4 1 mM NaF and

protease inhibitors cocktail) The extracts were incubated onice for 30min and supernatants were collected by centrifuga-tion at 14000 g at 4∘CTheprotein contents in the supernatantwere measured by using a Bio-Rad DC protein assay kit IIProteins were separated by electrophoresis on 125 SDS-PAGE gel and electrotransferred onto a Hybond ECL transfermembrane with transfer buffer (25mMTris 250mMglycineand 20methanol) at 300mA for 90minThemembranewasblocked in 5 nonfat skim milk and probed with primaryantibodies for p-STAT3 p-STAT5 STAT3 STAT5 p-JAK2JAK2 (Cell Signaling Tech Danvers MA USA) SHP-1SHP-2 bcl-xL mcl-1 survivin cyclin D1 cleaved caspase-9 cleaved caspase-3 poly(ADP-ribose) polymerase (PARP)and tubulin (Santa Cruz Biotechnologies Santa Cruz CAUSA) followed by incubating with horseradish peroxidase-(HRP-) conjugated secondary antibodies Protein expression

was detected by using enhanced chemiluminescence (ECL)system (Amersham Pharmacia Piscataway NJ USA)

25 Electrophoretic Mobility Shift Assay (EMSA) The STAT3or STAT5DNA binding activity was analyzed by EMSAusing gel shift chemiluminescent EMSA kit (Active motifCarlsbad CA USA) Nuclear extracts were incubated withSTAT3 (51015840-GAT CCT TCT GGG AAT TCC TAG ATC-31015840)or STAT5 (51015840-AGA TTT CTA ATT CAA TCC-31015840) consensusoligonucleotides (Santa Cruz Biotechnology Santa Cruz CAUSA) The DNAprotein complex formed was separatedfrom free oligonucleotides on 5 native polyacrylamidegels Chemiluminescent detection was performed using ECLreagents according to the vendorrsquos protocols (GEHealth CareBio-Sciences Piscataway NJ USA)

26 Cell Cycle Analysis Cell cycle analysis was performedby PI staining K562 cells were treated with tanshinone IIAor cryptotanshinone for 24 h collected and fixed in 70ethanol The cells were then incubated at 37∘C with 01RNase A in PBS for 30min and suspended in PBS containing25 120583gmL PI for 30min at room temperature The stainedcells were analyzed for DNA content in FACSCalibur (BectonDickinson Franklin Lakes NJ USA) using the Cell Questprogram (Becton Dickinson Franklin Lakes NJ USA)

27 Apoptosis Analysis by Annexin V-PI Double StainingApoptosis of the cryptotanshinone or tanshinone IIA-treatedcells was quantitated by double staining with Annexin V-FITC and PI using the Annexin V-Apoptosis Detection kit(Biovision Milpitas CA USA) according to the manufactur-errsquos instructions Apoptotic cells were analyzed by FACSCal-ibur (Becton Dickinson San Jose CA USA) to be defined asthose positive for Annexin V with or without PI staining

28 Propidium Iodide (PI) Staining K562 cells were exposedto tanshinone IIA or cryptotanshinone and plated onto poly-L-lysine-coated slide glassThe cells were fixed in 70 ethanoland stained with PI solution (50 120583gmL) (BD BiosciencesBedford MA USA) containing 100 120583gmL RNase for 10minThe slides were mounted with 70 glycerol in PBS and vis-ualized under an Axio vision 40 fluorescence microscope(Carl Zeiss Inc Weimar Germany)

29 Combination Index (CI) Calculation The CI was deter-mined by Chou-Talalay method and CalcuSyn software (Bio-soft Ferguson MO USA) A CI of less than 1 was consideredsynergistic [25]

210 Statistical Analyses All data were presented as means plusmnstandard deviation (SD) Statistical significance was verifiedby Studentrsquos t-test using SigmaPlot software (Systat SoftwareInc San Jose CA USA)

3 Results

31 Tanshinone IIA and Cryptotanshinone Exert Cytotoxicityagainst Chronic Myeloid Leukemia K562 Cells To comparethe cytotoxicity of tanshinone IIA and cryptotanshinone inK562 cells MTT assay was performed Cells were treated


O O

O O

OO

CH3

CH3 CH3


(a)



140

120

100

80

60

40

20

00 10 20 40 80

IC50

Cel

l via

bilit

y (

of c

ontro

l)

(b)



50= sim20120583M)





Tanshinone IIA

0 10 20 (120583M)

p-STAT3

STAT3

p-STAT5

STAT5

p-STAT3

STAT3

p-STAT5

STAT5


0 6 12 24 (h)

(a)

Tanshinone IIA

0 10 20 (120583M)


Free probe

(b)

Tanshinone IIA

0 10 20 (120583M)

p-JAK2

JAK2

(c)


0 3 6 12 24 36SHP-1

SHP-2

Tubulin

(h)

(d)






Cryptotanshinone

0 10 20 (120583M)

p-STAT3

STAT3

p-STAT5

STAT5

p-STAT3

STAT3

p-STAT5

STAT5


0 6 12 24 (h)

(a)

Cryptotanshinone

0 10 20 (120583M)


Free probe

(b)

Cryptotanshinone

0 10 20 (120583M)

p-JAK2

JAK2

(c)


(h)0 3 6 12 24 36SHP-1

SHP-2

Tubulin

(d)




4 Discussion




0 10 20 0 10 20(120583M) (120583M)

Survivin

Cyclin D1

Tubulin

mcl-1L mcl-1L

Survivin

Cyclin D1

Tubulin

bcl-xLbcl-xL

(a)


0 10 20 0 10 20(120583M) (120583M)


Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Tubulin Tubulin

(b)

Cou

nts

022

Control

0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600

Tanshinone IIA

Tanshinone IIA

Cryptotanshinone

Cryptotanshinone

1091 1719 906 176

20 120583M

20 120583M

20 120583M

Sub-

G1

()

20

15

10

5

0

Control10120583M

10120583M10120583M

10120583M 20120583M

(c)

Figure 4 Continued


PI

0 10 20 (120583M)

Cryp

tota

nshi

none

Tans

hino

ne II

A

058

034

366

331

718

452

925

876

1572

824

Annexin-V

(d)









0 25 5 10 20 25 5 10 20 1 (120583M)

p-Bcr-abl

c-abl

120573-Actin

(a)

Cel

l via

bilit

y (c

ontro

l (

))

Cel

l via

bilit

y (c

ontro

l (

))

100

80

60

40

20

0

100

80

60

40

20

0

minus

minus minus minus

minus


lowastlowastlowast

lowastlowast


(b)

Com

bina

tion

inde

x

2

1

00 02 04 06 08 1

Effect

CI = 1

CI = 0472 plusmn 02225025 (CI = 0315)5025 (CI = 0628)

25025 (CI = 0776)5025 (CI = 1048)



(c)

0

0

0015

1555

146

094

343

153

728

19

137

227

PI

Annexin-V



(d)



SHP-1SHP-2

JAK2 SHP-1


STAT5 STAT3


Cyclin D1Caspase-93

bcl-xL bcl-xL




5 Conclusion




Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O O

O O

OO

CH3

CH3 CH3


(a)



140

120

100

80

60

40

20

00 10 20 40 80

IC50

Cel

l via

bilit

y (

of c

ontro

l)

(b)



50= sim20120583M)





Tanshinone IIA

0 10 20 (120583M)

p-STAT3

STAT3

p-STAT5

STAT5

p-STAT3

STAT3

p-STAT5

STAT5


0 6 12 24 (h)

(a)

Tanshinone IIA

0 10 20 (120583M)


Free probe

(b)

Tanshinone IIA

0 10 20 (120583M)

p-JAK2

JAK2

(c)


0 3 6 12 24 36SHP-1

SHP-2

Tubulin

(h)

(d)






Cryptotanshinone

0 10 20 (120583M)

p-STAT3

STAT3

p-STAT5

STAT5

p-STAT3

STAT3

p-STAT5

STAT5


0 6 12 24 (h)

(a)

Cryptotanshinone

0 10 20 (120583M)


Free probe

(b)

Cryptotanshinone

0 10 20 (120583M)

p-JAK2

JAK2

(c)


(h)0 3 6 12 24 36SHP-1

SHP-2

Tubulin

(d)




4 Discussion




0 10 20 0 10 20(120583M) (120583M)

Survivin

Cyclin D1

Tubulin

mcl-1L mcl-1L

Survivin

Cyclin D1

Tubulin

bcl-xLbcl-xL

(a)


0 10 20 0 10 20(120583M) (120583M)


Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Tubulin Tubulin

(b)

Cou

nts

022

Control

0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600

Tanshinone IIA

Tanshinone IIA

Cryptotanshinone

Cryptotanshinone

1091 1719 906 176

20 120583M

20 120583M

20 120583M

Sub-

G1

()

20

15

10

5

0

Control10120583M

10120583M10120583M

10120583M 20120583M

(c)

Figure 4 Continued


PI

0 10 20 (120583M)

Cryp

tota

nshi

none

Tans

hino

ne II

A

058

034

366

331

718

452

925

876

1572

824

Annexin-V

(d)









0 25 5 10 20 25 5 10 20 1 (120583M)

p-Bcr-abl

c-abl

120573-Actin

(a)

Cel

l via

bilit

y (c

ontro

l (

))

Cel

l via

bilit

y (c

ontro

l (

))

100

80

60

40

20

0

100

80

60

40

20

0

minus

minus minus minus

minus


lowastlowastlowast

lowastlowast


(b)

Com

bina

tion

inde

x

2

1

00 02 04 06 08 1

Effect

CI = 1

CI = 0472 plusmn 02225025 (CI = 0315)5025 (CI = 0628)

25025 (CI = 0776)5025 (CI = 1048)



(c)

0

0

0015

1555

146

094

343

153

728

19

137

227

PI

Annexin-V



(d)



SHP-1SHP-2

JAK2 SHP-1


STAT5 STAT3


Cyclin D1Caspase-93

bcl-xL bcl-xL




5 Conclusion




Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Tanshinone IIA

0 10 20 (120583M)

p-STAT3

STAT3

p-STAT5

STAT5

p-STAT3

STAT3

p-STAT5

STAT5


0 6 12 24 (h)

(a)

Tanshinone IIA

0 10 20 (120583M)


Free probe

(b)

Tanshinone IIA

0 10 20 (120583M)

p-JAK2

JAK2

(c)


0 3 6 12 24 36SHP-1

SHP-2

Tubulin

(h)

(d)






Cryptotanshinone

0 10 20 (120583M)

p-STAT3

STAT3

p-STAT5

STAT5

p-STAT3

STAT3

p-STAT5

STAT5


0 6 12 24 (h)

(a)

Cryptotanshinone

0 10 20 (120583M)


Free probe

(b)

Cryptotanshinone

0 10 20 (120583M)

p-JAK2

JAK2

(c)


(h)0 3 6 12 24 36SHP-1

SHP-2

Tubulin

(d)




4 Discussion




0 10 20 0 10 20(120583M) (120583M)

Survivin

Cyclin D1

Tubulin

mcl-1L mcl-1L

Survivin

Cyclin D1

Tubulin

bcl-xLbcl-xL

(a)


0 10 20 0 10 20(120583M) (120583M)


Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Tubulin Tubulin

(b)

Cou

nts

022

Control

0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600

Tanshinone IIA

Tanshinone IIA

Cryptotanshinone

Cryptotanshinone

1091 1719 906 176

20 120583M

20 120583M

20 120583M

Sub-

G1

()

20

15

10

5

0

Control10120583M

10120583M10120583M

10120583M 20120583M

(c)

Figure 4 Continued


PI

0 10 20 (120583M)

Cryp

tota

nshi

none

Tans

hino

ne II

A

058

034

366

331

718

452

925

876

1572

824

Annexin-V

(d)









0 25 5 10 20 25 5 10 20 1 (120583M)

p-Bcr-abl

c-abl

120573-Actin

(a)

Cel

l via

bilit

y (c

ontro

l (

))

Cel

l via

bilit

y (c

ontro

l (

))

100

80

60

40

20

0

100

80

60

40

20

0

minus

minus minus minus

minus


lowastlowastlowast

lowastlowast


(b)

Com

bina

tion

inde

x

2

1

00 02 04 06 08 1

Effect

CI = 1

CI = 0472 plusmn 02225025 (CI = 0315)5025 (CI = 0628)

25025 (CI = 0776)5025 (CI = 1048)



(c)

0

0

0015

1555

146

094

343

153

728

19

137

227

PI

Annexin-V



(d)



SHP-1SHP-2

JAK2 SHP-1


STAT5 STAT3


Cyclin D1Caspase-93

bcl-xL bcl-xL




5 Conclusion




Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cryptotanshinone

0 10 20 (120583M)

p-STAT3

STAT3

p-STAT5

STAT5

p-STAT3

STAT3

p-STAT5

STAT5


0 6 12 24 (h)

(a)

Cryptotanshinone

0 10 20 (120583M)


Free probe

(b)

Cryptotanshinone

0 10 20 (120583M)

p-JAK2

JAK2

(c)


(h)0 3 6 12 24 36SHP-1

SHP-2

Tubulin

(d)




4 Discussion




0 10 20 0 10 20(120583M) (120583M)

Survivin

Cyclin D1

Tubulin

mcl-1L mcl-1L

Survivin

Cyclin D1

Tubulin

bcl-xLbcl-xL

(a)


0 10 20 0 10 20(120583M) (120583M)


Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Tubulin Tubulin

(b)

Cou

nts

022

Control

0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600

Tanshinone IIA

Tanshinone IIA

Cryptotanshinone

Cryptotanshinone

1091 1719 906 176

20 120583M

20 120583M

20 120583M

Sub-

G1

()

20

15

10

5

0

Control10120583M

10120583M10120583M

10120583M 20120583M

(c)

Figure 4 Continued


PI

0 10 20 (120583M)

Cryp

tota

nshi

none

Tans

hino

ne II

A

058

034

366

331

718

452

925

876

1572

824

Annexin-V

(d)









0 25 5 10 20 25 5 10 20 1 (120583M)

p-Bcr-abl

c-abl

120573-Actin

(a)

Cel

l via

bilit

y (c

ontro

l (

))

Cel

l via

bilit

y (c

ontro

l (

))

100

80

60

40

20

0

100

80

60

40

20

0

minus

minus minus minus

minus


lowastlowastlowast

lowastlowast


(b)

Com

bina

tion

inde

x

2

1

00 02 04 06 08 1

Effect

CI = 1

CI = 0472 plusmn 02225025 (CI = 0315)5025 (CI = 0628)

25025 (CI = 0776)5025 (CI = 1048)



(c)

0

0

0015

1555

146

094

343

153

728

19

137

227

PI

Annexin-V



(d)



SHP-1SHP-2

JAK2 SHP-1


STAT5 STAT3


Cyclin D1Caspase-93

bcl-xL bcl-xL




5 Conclusion




Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0 10 20 0 10 20(120583M) (120583M)

Survivin

Cyclin D1

Tubulin

mcl-1L mcl-1L

Survivin

Cyclin D1

Tubulin

bcl-xLbcl-xL

(a)


0 10 20 0 10 20(120583M) (120583M)


Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Pro

Cleaved

Pro

Cleaved

Caspase-9

PARP

Tubulin Tubulin

(b)

Cou

nts

022

Control

0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600 0

200

400

600

Tanshinone IIA

Tanshinone IIA

Cryptotanshinone

Cryptotanshinone

1091 1719 906 176

20 120583M

20 120583M

20 120583M

Sub-

G1

()

20

15

10

5

0

Control10120583M

10120583M10120583M

10120583M 20120583M

(c)

Figure 4 Continued


PI

0 10 20 (120583M)

Cryp

tota

nshi

none

Tans

hino

ne II

A

058

034

366

331

718

452

925

876

1572

824

Annexin-V

(d)









0 25 5 10 20 25 5 10 20 1 (120583M)

p-Bcr-abl

c-abl

120573-Actin

(a)

Cel

l via

bilit

y (c

ontro

l (

))

Cel

l via

bilit

y (c

ontro

l (

))

100

80

60

40

20

0

100

80

60

40

20

0

minus

minus minus minus

minus


lowastlowastlowast

lowastlowast


(b)

Com

bina

tion

inde

x

2

1

00 02 04 06 08 1

Effect

CI = 1

CI = 0472 plusmn 02225025 (CI = 0315)5025 (CI = 0628)

25025 (CI = 0776)5025 (CI = 1048)



(c)

0

0

0015

1555

146

094

343

153

728

19

137

227

PI

Annexin-V



(d)



SHP-1SHP-2

JAK2 SHP-1


STAT5 STAT3


Cyclin D1Caspase-93

bcl-xL bcl-xL




5 Conclusion




Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PI

0 10 20 (120583M)

Cryp

tota

nshi

none

Tans

hino

ne II

A

058

034

366

331

718

452

925

876

1572

824

Annexin-V

(d)









0 25 5 10 20 25 5 10 20 1 (120583M)

p-Bcr-abl

c-abl

120573-Actin

(a)

Cel

l via

bilit

y (c

ontro

l (

))

Cel

l via

bilit

y (c

ontro

l (

))

100

80

60

40

20

0

100

80

60

40

20

0

minus

minus minus minus

minus


lowastlowastlowast

lowastlowast


(b)

Com

bina

tion

inde

x

2

1

00 02 04 06 08 1

Effect

CI = 1

CI = 0472 plusmn 02225025 (CI = 0315)5025 (CI = 0628)

25025 (CI = 0776)5025 (CI = 1048)



(c)

0

0

0015

1555

146

094

343

153

728

19

137

227

PI

Annexin-V



(d)



SHP-1SHP-2

JAK2 SHP-1


STAT5 STAT3


Cyclin D1Caspase-93

bcl-xL bcl-xL




5 Conclusion




Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0 25 5 10 20 25 5 10 20 1 (120583M)

p-Bcr-abl

c-abl

120573-Actin

(a)

Cel

l via

bilit

y (c

ontro

l (

))

Cel

l via

bilit

y (c

ontro

l (

))

100

80

60

40

20

0

100

80

60

40

20

0

minus

minus minus minus

minus


lowastlowastlowast

lowastlowast


(b)

Com

bina

tion

inde

x

2

1

00 02 04 06 08 1

Effect

CI = 1

CI = 0472 plusmn 02225025 (CI = 0315)5025 (CI = 0628)

25025 (CI = 0776)5025 (CI = 1048)



(c)

0

0

0015

1555

146

094

343

153

728

19

137

227

PI

Annexin-V



(d)



SHP-1SHP-2

JAK2 SHP-1


STAT5 STAT3


Cyclin D1Caspase-93

bcl-xL bcl-xL




5 Conclusion




Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SHP-1SHP-2

JAK2 SHP-1


STAT5 STAT3


Cyclin D1Caspase-93

bcl-xL bcl-xL




5 Conclusion




Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article apoptosis induced by tanshinone iia and...

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