Table Of ContentRheeetal.BMCCancer2012,12:28
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RESEARCH ARTICLE Open Access
Inhibition of STAT3 signaling and induction of
SHP1 mediate antiangiogenic and antitumor
activities of ergosterol peroxide in U266 multiple
myeloma cells
Yun-Hee Rhee1†, Soo-Jin Jeong2†, Hyo-Jeong Lee2†, Hyo-Jung Lee2†, Wonil Koh2†, Ji Hoon Jung2†, Sun-Hee Kim2†
and Kim Sung-Hoon2*†
Abstract
Background: Ergosterol peroxide (EP) derived from edible mushroom has been shown to exert anti-tumor activity
in several cancer cells. In the present study, anti-angiogenic activity of EP was investigated with the underlying
molecular mechanisms in human multiple myeloma U266 cells.
Results: Despite weak cytotoxicity against U266 cells, EP suppressed phosphorylation, DNA binding activity and
nuclear translocalization of signal transducer and activator of transcription 3 (STAT3) in U266 cells at nontoxic
concentrations. Also, EP inhibited phosphorylation of the upstream kinases Janus kinase 2 (JAK2) and Src in a time-
dependent manner. Furthermore, EP increased the expression of protein tyrosine phosphatase SHP-1 at protein
and mRNA levels, and conversely silencing of the SHP-1 gene clearly blocked EP-mediated STAT3 inactivation. In
addition, EP significantly decreased vascular endothelial growth factor (VEGF), one of STAT3 target genes at cellular
and protein levels as well as disrupted in vitro tube formation assay. Moreover, EP significantly suppressed the
growth of U266 cells inoculated in female BALB/c athymic nude mice and immunohistochemistry revealed that EP
effectively reduced the expression of STAT3 and CD34 in tumor sections compared to untreated control.
Conclusion: These findings suggest that EP can exert antitumor activity in multiple myeloma U266 cells partly with
antiangiogenic activity targeting JAK2/STAT3 signaling pathway as a potent cancer preventive agent for treatment
of multiple myeloma cells.
Keywords: ergosterol peroxide, JAK2, STAT3, angiogenesis, multiple myeloma
Background mechanisms for anti-cancer activity of EP still remain
Ergosterol Peroxide (EP), 5a, 8a-epidioxy-22E-ergosta-6, unclear.
22-dien-3b-ol, is found in plants [1], lichens [2] and STAT proteins originally discovered as latent cytoplas-
mushrooms such as Ganoderma lucidum [3], Sporothrix mic transcription factors [11] are involved in a variety of
schenckii [4] and Cordyceps sinensis [5]. Despite various cellular processes such as cell proliferation, differentia-
biological effects of EP such as immunosuppressive tion and apoptosis [12,13]. Of the STAT proteins,
[6-8], anti-viral [9], anti-inflammatory [10] and anti- STAT3 is often constitutively activated in many human
tumor [5,10] activities, the underlying molecular cancer cells including multiple myeloma, leukemia, lym-
phoma, and solid tumors [14,15]. Upon activation,
STAT3 undergoes phosphorylation-induced homodi-
merization, leading to nuclear translocation, DNA bind-
*Correspondence:[email protected] ing and subsequent gene transcription. STAT3
†Contributedequally
2CollegeofOrientalMedicine,KyungHeeUniversity,Seoul130-701,South phosphorylation is mediated through the activation of
Korea
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Rheeetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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non-receptor protein tyrosine kinases Janus activated 0.5% Triton X-100, 1 mM aprotinin, 1 mM leupeptin, 1
kinases (JAKs) and c-Src [16]. mM pepstatin, 10 mM iodoacetamide, 2 mM phenyl-
STAT3 participates in oncogenesis through up-regula- methylsulfonyl fluoride (PMSF), and 1 mM NaVO ] and
4
tion of genes encoding apoptosis inhibitors such as bcl- centrifuged at 14, 000 × g for 10 min at 4°C. Protein
x , bcl-2, and survivin [15]. Also, recent studies reported samples were collected, separated onto 10-12% SDS-
L
the evidences that STAT3 is involved in the regulation PAGE gels and electrotransferred to a nitrocellulose
of angiogenesis through modulation of VEGF, a key reg- membrane. The membranes were blocked in 5% nonfat
ulator of angiogenesis [17-19]. In this regards, we inves- skim milk, probed with primary antibodies for STAT3,
tigated anti-angiogenic effect of EP in association with phospho-STAT3Y705, JAK2, phospho-JAK2Y1007/1008, Src,
JAK2/STAT3 pathway and STAT3 related VEGF expres- phospho-SrcY416 (Cell Signaling, Danvers, MA), SHP-1,
sion in U266 cells in vitro and mouse xenograft model. VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) and
b-actin (Sigma, St. Louis, MO) at 4°C for overnight, and
Methods then exposed to HRP-conjugated secondary antibodies
Isolation of ergosterol peroxide (EP) at room temperature for 2 h. Protein expression was
EP was isolated according to the Krzyczkowski’s method detected by using enhanced chemiluminescence (ECL)
as previously described [20]. kit (Amersham Pharmacia, Piscataway, NJ).
Cell culture Electrophoretic mobility shift assay (EMSA) for STAT3-
U266 (multiple myeloma), SCC4 (head and neck squa- DNA binding
mous cell carcinoma), DU145 (prostate cancer), and The STAT3-DNA binding was analyzed by electro-
MDA-MB-231 (breast cancer) cells were obtained from phoretic mobility shift assay (EMSA) using a 32P-labeled
American Type Culture Collection (ATCC) (Rockville, high-affinity sis-inducible element (hSIE) probe (5’-
MD) and maintained in RPMI 1640 supplemented with CTTCATTTCCCGTAAATCCCTAAAGCT-3’ and 5’-
10% FBS and antibiotics. Human umbilical vein AGCTTTAGGGATTTACGG GAAA TGA-3’) as pre-
endothelial cells (HUVECs) were isolated from fresh viously described [22]. Briefly, nuclear extracts were
human umbilical cord vein by collagenase treatment as incubated with the hSIE probe and the protein-DNA
described previously [21]. The cells were maintained in complexes were separated onto 5% native polyacryla-
in EBM-2 containing 2% FBS, 0.04% hydrocortisone, mide gels. The gels were dried, and the radioactive
0.4% hFGF-B, 0.1% VEGF, 0.1% IGF-1, 0.1% ascorbic bands were quantitated with Storm 820 and Imagequant
acid, 0.1% hEGF, 1% GA-1000, and 1% Heparin (Walk- software (Amersham Pharmacia, Piscataway, NJ).
ersville, MD)
RT-PCR analysis
Cytotoxicity assay Total RNA was extracted by using Trizol reagent (Invi-
The cytotoxic effect of EP was evaluated by 3-(4, 5- trogen) according to the manufacturer’s instructions.
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide cDNAwassynthesizedfrom1μgoftotalRNAandsub-
(MTT)assay.U266cellswereseededonto96-wellmicro- jected to PCR reaction by using Superscript One Step
plate at a density of 1 × 104 cells/well and treated with reverse transcription-PCR (RT-PCR) kit (Invitrogen).
variousconcentrationsofEP(0,6.25,12.5,25or50μM) ThePCRconditionswere30cyclesof94°Cfor15s,55°C
for24h. The culturemediumwasremovedaftercentri- for30s,and72°Cfor1min.Theprimersequenceswere
fugingtheplateandMTTsolution(1mg/ml)wasadded as follows: SHP-1 (forward primer 5’-AAT GCG TCC
to each well until formazan was constituted. MTT lysis CATACTGGCCCGA-3’;reverseprimer5’-CCCGCA
solution (20% SDS and 50% dimethylfermamide) was GTTGGTCACAGAGT-3’),andGAPDH(forwardpri-
addedtodissolveformazanandopticaldensity(OD)was mer 5’-TCA CCA TCT TCC AGG AGC GA-3’; reverse
measured using microplate reader (Tecan Austria primer5’-CACAATGCCGAAGTGGTGGT-3’).
GmbH,Grödig,Austria)at570nm.Cellviabilitywascal-
culated as a percentage of viable cells in EP-treated siRNA transfection
groupversusuntreatedcontrolbyfollowingequation. siRNA oligonucleotides for SHP-1 (SantaCruz biotech-
nology, SantaCruz, CA) were transfected by using Lipo-
(cid:2) (cid:3) (cid:2) (cid:3)
Cellviability(%)= OD(EP)−OD(Blank) / OD(Control)−OD(Blank)×100 fectAMINE transfection reagent (Invitrogen, Carlsbad,
CA) according to manufacturer’s protocols.
Western blotting
Enzyme- linked immunosorbent assay (ELISA) for VEGF
Cells lysates were prepared using lysis buffer [50 mM
U266 cells were seeded onto 6-well plates at a density of
Tris (pH 7.4), 300 mM NaCl, 5 mM EDTA (pH 8.0),
1 × 106 cells/well and treated with 25 μM EP. The
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VEGF levels in the supernatant were measured by using activation of STAT3 in U266 cells. EP significantly
a Quantikine VEGF ELISA kit (R&D systems, Minnea- inhibited the constitutive activation of STAT3 in time-
polis, MN) according to manufacturer’s protocols. and dose-dependent manners (Figure 1A and 1B,
respectively). Tyrosine phosphorylation of STATs causes
In vitro tube formation assay their dimerization and translocation to the nucleus,
Tube formation assay was performed on Matrigel (Bec- where they bind to DNA and regulate gene transcription
ton Dickinson Labware, Bedford, MA) as described pre- [22]. Furthermore, we evaluated whether EP can affect
viously [23]. In brief, human umbilical vein endothelial on constitutive STAT3 activation in other cancer cell
cells (HUVECs) (3 × 104 cells/well) were seeded onto lines. SCC4 (head and neck squamous cell carcinoma),
Matrigel-coated 24-well plates and treated with VEGF DU145 (prostate cancer), and MDA-MB-231 (breast
(20 ng/ml) in the absence or presence of EP (0, 10 or 25 cancer) cells expressing constitutive STAT3 were treated
μM) for 6 h. Cells were fixed with 2% paraformaldehyde with or without EP. As shown in Figure 1C, EP inhibited
and stained with 2% crystal violet. Tube formation was STAT3 activation without affecting total STAT3 level.
observed under an Axiovert S 100 light microscope Consistent with the results of Western blotting in Figure
(Carl Zeiss, Weimar, Germany) and counted in ran- 1A and 1B, gel shift assay showed that EP clearly inhib-
domly selected areas using NIH Scion image program. ited the STAT3 DNA-binding activity in time- and
dose-dependent manners (Figure 1D and 1E,
Mouse xenograft model respectively).
Six-week-old female athymic nude mice were purchased
from Jung Ang lab animal (Seoul, Republic of Korea) Ergosterol peroxide suppresses JAK2 and Src in U266
and maintained under conventional conditions. U266 cells
cells (2 × 106 cells) were mixed with Matrigel (Becton STAT3 is activated by soluble tyrosine kinases JAKs
Dickinson, 50%, in 100 μl) and injected subcutaneously [24]. Effect of EP was measured on the activation of
on the right flank of the mice. After 5 days of inocula- JAK2 by Western blotting. EP suppressed the phosphor-
tion, the mice (n = 5/group) were given intraperitonial ylation of JAK2 in a time-dependent manner (Figure
(i.p) injection of EP at 100 mg/Kg in 2% tween-80, or 2A), while it did not change the level of JAK1 (data not
bortezomib i.p. 0.25 mg/kg in PBS every 2 or 3 days. shown). The Src is another tyrosine kinase family to
Control mice were administered the solvent vehicle. activate STAT3 signaling [16]. EP also decreased the
Tumor volume was measured every other day with cali- phosphorylation of Src in a time-dependent manner
per and calculated according to the formula; V = (Figure 2B). These results indicate that EP inactivates
0.25a2b, where a is the smallest superficial diameter and the upstream kinases JAK2 and Src of STAT3 in U266
b is the largest superficial diameter. cells.
Immunohistochemistry Ergosterol peroxide enhances expression of SHP-1 in
The animals were sacrificed 20 days after U266 inocula- U266 cells
tion, and tumors were immediately removed, fixed in Non-transmembrane protein tyrosine phosphatases
4% paraformaldehyde, paraffin-embedded, and sectioned (PTPs) play important roles in the negative regulation of
at 4 μm. Immunohistochemical staining for phospho- the JAK/STAT signaling [25]. Thus, we investigated
STAT3(Cell Signaling Technology, Danvers, MA), CD whether PTPs are involved in EP-mediated STAT3 inac-
34 (microvessel marker, Abcam, Boston, MA) and tivation using a PTP inhibitor sodium pervanadate. As
TUNEL (Calbiochem, Darmstadt, Germany) was per- shown in Figure 3A, pervanadate reversed EP-induced
formed and detected by DAB substrate staining (brown). STAT3 inactivation in a dose-dependent manner, indi-
cating the important role of PTP(s) in EP induced
Statistical analysis STAT3 inactivation in U266 cells.
All values were expressed as means ± S.D. Data were Since SHP-1 is a PTP expressed most abundantly in
analyzed by one-way analysis of variance (ANOVA) and hematopoietic cells [26,27], the effect of EP on the
by student’s t-test (Sigma plot®, San Rafael, CA, USA). expression of SHP-1 was examined. EP enhanced SHP-1
protein expression in time- and dose-dependent man-
Results ners (Figure 3B and 3C). Consistently, EP increased the
Ergosterol peroxide suppresses STAT3 activation in U266 expression of SHP-1 at the mRNA level in a time-
cells dependent manner (Figure 3D). Conversely, EP-
STAT3 is implicated in cell survival and growth in mul- mediated SHP-1 was reduced in a time course by the
tiple myeloma cells [14]. In light of this event, we exam- removal of EP containing media, indicating that SHP-1
ined whether EP can modulate the constitutive expression by EP is reversible (Figure 3E). Furthermore,
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g
A B
EP
EP
0 4 8 16 24 (h) 0 6.25 12.5 25 (μM)
p-STAT3 p-STAT3
STAT3 STAT3
C
SCC4 DU145 MDA-MB231
EP - + - + - +
p-STAT3
STAT3
D E
EP
EP
0 6.25 12.5 25 (μM)
0 2 4 8 (h)
STAT3
STAT3
Figure1Ergosterolperoxide(EP)suppressesSTAT3activationinU266cells.(A)Cellsweretreatedwith25μMEPfor0,4,8,16or24h.
(B)CellsweretreatedwithvariousconcentrationsofEP(0,6.25,12.5or25μM)for8h.(C)SCC4,DU145andMDA-MB-231cellsweretreated
withorwithoutEP(25μM)for8h.WholecellextractswerepreparedandsubjectedtoWesternblottingtodeterminelevelofphospho-STAT3
andSTAT3.(DandE)GelshiftmobilityassaywasperformedusingnuclearextractstoexaminetheSTAT3-DNAbindingactivity.
blocking SHP-1 using its specific siRNA clearly Consistently, secretion of VEGF was significantly
abolishes the ability of EP to inhibit STAT3 while inhibited by EP treatment in time- and dose-depen-
scrambled siRNA had no effect on the expression (Fig- dent manners by ELISA (Figure 4C and 4D,
ure 3F). Taken together, these results showed evidence respectively).
that SHP-1 plays a critical role in the suppression of To further confirm the anti-angiogenic activity of EP,
STAT3 phosphorylation by EP. in vitro tube formation assay was conducted using
HUVECs treated either with or without VEGF in the
Ergosterol peroxide (EP) exerts anti-angiogenic activity in absence or presence of EP. As shown in Figure 4E, no
vitro tube formation was observed in untreated cells, while
Cytotoxicity of EP against U266 cells were determined clear tube formation was exhibited in VEGF-treated
by MTT assay. U266 cells were treated with various control. Notably, EP treatment inhibited VEGF-induced
concentrations of EP (0, 6.25, 12.5, 25 or 50 μM) for tube formation in a dose-dependent manner. Then, we
24 h. As shown in Figure 4A, EP had no significant tested whether EP can affect VEGF-mediated STAT3
cytotoxic effect in U266 cells. STAT3 contributes to activation in HUVECs. As shown in Figure 4F, VEGF
angiogenic regulation by inducing VEGF expression treatment clearly enhanced phosphorylation of STAT3
[28,29]. Thus, VEGF protein expression was analyzed in HUVECs, which was supported by Ebrahem’s evi-
at the cellular and protein levels in EP-treated cells. dences [30]. In contrast, total STAT3 levels were not
EP suppressed protein expression of VEGF in a time- significantly changed by the treatment of VEGF and/or
dependent manner by Western blotting (Figure 4B). EP (Figure 4F).
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A
EP )
e
g
0 4 8 16 24 (h) n
a1.00
h
c
p-JAK2 d
ol0.75
f
(
2
JAK2 K0.50
A
J
2/0.25
K
(cid:533)-actin
A
J
p-0.00
0 4 8 16 24 (h)
B
EP
0 4 8 16 24 (h) e)
g 1.00
n
a
p-Src h
c
d 0.75
ol
f
Src c ( 0.50
r
S
(cid:533)-actin rc/ 0.25
S
-
p
0.00
0 4 8 16 24 (h)
Figure2Ergosterolperoxide(EP)inhibitsphosphorylationofJAK2andSrcinU266cells.Cellsweretreatedwith25μMEPfor0,4,8,16
or24h.WholecellextractswerepreparedandsubjectedtoWesternblottingtodeterminelevelof(A)phospho-JAK2andJAK2,adn(B)
phospho-SrcandSrc.
Ergosterol peroxide exerts anti-tumor activity in vivo can exert antitumor effects via antiangiogenic activity
To confirm the anti-tumor efficacy of EP, U266 cells at low concentration and apoptotic activity at higher
were subcutaneously inoculated into BALB/c athymic concentration.
nude mice in the flank area, and starting after 5 days
injection, the mice were administered EP or bortezomib Discussion
(a positive control) every 2-3 day with intraperitonial (i. Multiple myeloma, also called plasma cells myeloma, is
p.) injection. Tumor growth was monitored every other characterized by accumulation of secretory plasma cells.
day for 20 days. Tumor volumes in EP- or bortezomib- For the treatment of multiple myeloma, various che-
treated groups were decreased compared with untreated motherapeutic agents such as vincristine, carmustine,
control (Figure 5A) without significant body weight loss mephalan, cyclophosphamide, doxorubicin, prednisone
in EP-treated group (Figure 5B). and dexamethasone have been used in combination or
Consistently, immunohistochemistry showed that EP either drug alone [31]. However, many multiple mye-
treatment reduced the expression of phospho-STAT3 loma patients frequently had no response to these
and angiogenesis marker CD34 compared to untreated agents and their prolonged exposure induced toxicity
control (Figure 5C), implying the antitumor effect of even in normal cells. Recently, phytochemicals are con-
EP is associated with regulation of angiogenesis and siderably advocated as rich sources of anti-cancer agents
STAT3 in vitro and in vivo. In addition, TUNEL posi- that deserve more rigorous and valuable investigations.
tive cells for apoptosis were effectively increased in Regarding this issue, several studies suggested phyto-
EP-treated tumor sections. However, considering that chemicals such as curcumin [14], resveratrol [32] and
EP treatment for 48 h or 72 h exerted significant cyto- capsaicin [33] as potent anti-cancer agents for multiple
toxicity against U266 cells, we can postulate that EP myeloma treatment. We also found that genipin [34]
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A
0 0 10 25 50 Pervanadate (μM)
- + + + + EP
p-STAT3
STAT3
B EP C
EP
0 4 8 16 24 (h) 0 6.25 12.5 25 (μM)
SHP-1 SHP-1
(cid:533)-actin (cid:533)-actin
D EP E EP Wash
0 4 8 16 24 (h) 0 4 0 4 8 (h)
SHP-1
SHP-1
GAPDH (cid:533)-actin
25 e) 2.5
Relative mRNA level ofSHP-1/GAPDH (fold)1120505 P-1/-actin (fold chang(cid:69) 0112....5050
H
0 S 0.0
0 4 8 16 24 (h) 0 4 0 4 8 (h)
EP Wash
F - + - + - + EP
- - + + - - Scrambled siRNA
- - - - + + SHP-1 siRNA
SHP-1
pSTAT-3
STAT-3
(cid:533)-actin
Figure3Ergosterolperoxide(EP)enhancesexpressionofSHP-1inU266cells.(A)Cellsweretreatedwith25μMEPintheabsenceor
presenceofpervanadate(10,25or50μM)for4h.WholecellextractswerepreparedandsubjectedtoWesternblottingtodeterminelevelof
phospho-STAT3andSTAT3.(BandC)Cellsweretreatedwith25μMEPfor0,4,8,16or24h(B)andvariousconcentrationsofEP(0,6.25,12.5
or25μM)for4h(C).WholecellextractswerepreparedandsubjectedtoWesternblottingtodetermineSHP-1expressoin.(D)Cellswere
treatedwith25μMEPfor0,4,8,16or24h.RT-PCRwasperformedtoanalyzemRNAexpressionofSHP-1.(E)Cellsweretreatedwith25μMEP
for4h(left),ortreatedwith25μMEPfor1hfollowedbywashingPBStoremoveEPandresuspensioninfreshmedium(right).Western
blottingwasperformedforSHP-1.(F)CellsweretransientlytransfectedwitheitherSHP-1orscrambledsiRNA(50nM)for48handthentreated
with25μMEPfor4h.Westernblottingwasperformedforphospho-STAT3andSTAT3.
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A B
ol)120 EP
r
nt 0 4 8 16 24 (h)
o
c
of 80 VEGF
%
y (
bilit 40 (cid:533)-actin
a
vi
ell
C 0
0 10 20 30 40 50
Ergosterol peroxide ((cid:80)M)
D
C
1250(cid:71) 1250
ml)1000(cid:71) ml)1000
F (pg/ 750(cid:71) (cid:81)(cid:81)(cid:71) (cid:81)(cid:81)(cid:71) F (pg/750 **
EG 500(cid:71) (cid:81)(cid:81)(cid:71) EG500 **
V V
250(cid:71) 250
0(cid:71) 0
0 4 8 16 24 0 6 12.5 25
Time (h)(cid:71) Concentration (μM)
E
) 2000
2
m
m
a ( 1500 Control VEGF VEGF + EP
e
r **
a 1000
e
b
u
al t 500 **
ot
T 0
VEGF - + + +
EP (μM) 12.5 25
F
EP
VEGF - + - +
p-STAT3
STAT3
(cid:69)-actin
Figure4Ergosterolperoxide(EP)exertsanti-angiogenicactivityinvitro.(A)U266cellsweretreatedwithvariousconcentrationsofEP(0,
6.25,12.5,25or50μM)for24h.CytotoxiceffectofEPwasevaluatedbyMTTassay.(B)U266cellsweretreatedwith25μMEPfor0,4,8,16or
24h.CelllysateswerepreparedandsubjectedtoWesternblottingforVEGF.(CandD)U266cellswereseededatdensityof1×106cells/well
onto6-wellplatesandtreatedwith25μMEPfor0,4,8,16or24h(C)andvariousconcentrationsofEP(0,6,12.5or25μM)for24h(D).Levels
ofVEGFinthesupernatantsweremeasuredbyusingaQuantikineVEGFELISAkit.(E)Forinvitrotubeformationassay,HUVECs(3×104cells/
well)wereseededontoMatrigelcoated24-wellplatesandtreatedwithVEGF(20ng/ml)intheabsenceorpresenceofEP(0,12.5or25μM)for
6h.Cellswerefixedwith2%paraformaldehyde,stainedwith2%crystalviolet,andthenumberoftubewasrandomlycountedinselectedareas
(left).RepresentativephotographsoftubeformationincellstreatedwithVEGF(20ng/ml)intheabsenceorpresenceof25μMEP(right).Each
experimentwasrepeatedthreetimesandalldatawereexpressedasmeans±S.D.**,p<0.01vsuntreatedcontrol.(F)HUVECsweretreated
withVEGF(20ng/ml)intheabsenceorpresenceof25μMEP.Westernblottingwasperformedforphopho-STAT3andSTAT3.
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A B
25
250
Control
)
3 100 mg/Kg EP
m
200
c 0.25 mg/Kg Velcade )
e ( (g20
m150 ht
u g
ol ei
v w
or 100 y 15 Control
m d 100 mg/Kg EP
o
u B 0.25 mg/Kg Velcade
T 50
0 10
5 10 15 20 25 0 5 10 15 20
Days Days
C
Control EP
CD34
Control EP
p-STAT3
Control EP
TUNEL
Figure5Ergosterolperoxide(EP)exertsanti-tumoractivityinvivo.Six-week-oldfemaleBALB/cathymicnudemiceweresubcutaneously
injectedintheflankareawith2×106cellsofU266in100μlofmatrigelmixedPBS.Fivedaysafterinoculation,mice(n=5/group)wereeach
givenevery2-3dayintraperitonial(i.p.)injectionofEPat100mg/kgin2%tween-80orbortezomibat0.25mg/kginPBS.Controlmicewere
administeredthesolventvehicle.Tumorvolumesweremeasuredeveryotherdaywithcaliper.(A)Bodyweightwasmeasuredeveryotherday
afteradministrationofEPorbortezomib.(B)TumorvolumewascalculatedaccordingtotheformulaV=0.25a2b,whereaisthesmallest
superficialdiameterandbisthelargestsuperficialdiameter.(C)Micewerekilledonday20aftercellinoculation,andtumorswereimmediately
removed,fixed,embeddedandsectionedat4μmforimmunostainingofbiomarkers.Representativephotomicrographsofapoptosisdetected
byimmunostainingofCD34,phospho-STAT3andTUNELat×400,200and100ofmagnification,respectively.ThesectionsweredetectedasDAB
substratestaining(brown)andcounterstainedwithMayer’shematoxylinsolution(right).GraphsshowtheCD34index(angiogenesis),TUNEL
index(apoptosis)and,pSTAT3indexintumorsections.**p<0.01and***p<0.001comparedwithvehicle-treatedcontrolmice(left).
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and icariside II [35] could be applied for multiple mye- Recently, Niu and colleagues reported that constitutive
loma therapy by inducing apoptosis and targeting the activity of STAT3 up-regulated VEGF expression and
signaling molecules such as STAT3. tumor angiogenesis [49]. Wei and colleagues also
The STAT proteins were identified for the last decade reported that overexpression of constitutively activated
as latent cytoplasmic transcription factors in response to mutant STAT3 sufficiently increased VEGF expression
all cytokine driven signaling [15]. Seven mammalian and tumor angiogenesis in vivo [50]. In contrast, domi-
STAT proteins such as STAT1, 2, 3, 4, 5a, 5b and 6 act nant negative STAT3 mutant inhibited VEGF expression
as multifunctional mediators to regulate various cellular as well as angiogenesis. Additionally, it is of interest that
processes such as cell proliferation, differentiation, STAT3 activation by its upstream Src regulates VEGF
angiogenesis, and apoptosis [36]. Especially, STAT3 is mediated angiogenesis and conversely blocking STAT3
constitutively activated in many human cancers, includ- inhibits Src-induced VEGF expression [51]. Consistently,
ing prostate cancer [37], breast cancer [38], squamous in the present study, EP significantly prevented VEGF-
cell carcinoma of the head and neck (SCCHN) [39], induced phosphorylation of STAT3 as well as VEGF-
nasopharyngeal carcinoma and multiple myelomas [40]. mediated tube formation in HUVECs, indicating anti-
Thus, STAT3 is considered as a valuable therapeutic angiogenic activity of EP by inhibiting STAT3 phos-
target molecule for cancer treatment. phorylation. Furthermore, we confirmed anti-tumor
Ergosterol peroxide (EP) is a steroid derivative isolated effect of EP in mouse xenograft tumor model. Consis-
from medicinal mushroom [41]. Several studies reported tent with the results of in vitro experiments, EP
anti-cancer activity of EP in various types of cancer decreased U266 tumor growth as well as suppressed the
cells. For instance, Russo and colleagues reported that expression levels of phospho-STAT3 and CD34 by
EP attenuated cell growth and induced apoptosis in immunohistochemistry. Although there are evidences
human prostate cancer LNCaP and DU-145 cells [42]. that EP induces apoptosis at concentrations of 12.5-50
Kobori and colleagues reported that EP suppressed μM in LNCaP and DU-145 cells for 72 h [41,42,52], in
inflammatory response in RAW264.7 macrophages and the current study, EP did not show any cytotoxicity
growth of HT29 colon adenocarcinoma cells [43]. Also, against U266 cells for 24 h up to 50 μM. Actually,
Chen and colleagues suggested that EP from the fer- immunohistochemistry revealed that TUNEL positive
mentation mycelia of Ganoderma lucidum cultivated in cells were increased in EP treated tumor sections, imply-
the medium exerted the cytotoxic effect against Hep 3B ing that EP can exert anti-angiogenic activity at non-
cells [44]. In the present study, we found that EP exerts toxic concentrations and possibly induce apoptosis only
anti-cancer activity through the inhibition of angiogen- after long term culture or at high doses. Thus, mechan-
esis by targeting the STAT3 signaling pathway in U266 ism and pharmacokinetic studies with EP are still
cells in vitro and in vivo. required in vitro and in vivo to elucidate the relationship
EP suppressed constitutive activation of STAT3 in between its anti-angiogenic and apoptotic activities at
U266, SCC4, DU145 and MDA-MB-231 cells. EP also different doses in the near future
inhibited the STAT3-DNA binding activity and the
nuclear translocation of STAT3, suggesting that EP can Conclusions
prevent STAT3 activation at the transcriptional level. Our findings demonstrate that EP can exert antitumor
The inhibitory effect of EP on STAT3 activation was activity in multiple myeloma U266 cells by anti-angio-
partly associated with the inhibition of upstream kinases genic activity targeting JAK2/STAT3 signaling pathway
JAK2 and Src by EP treatment. Furthermore, protein as a potent anti-cancer agent for multiple myeloma
tyrosine phosphatases (PTPs) are known to be impli- treatment.
cated in STAT3 signaling, including SHP-1 [45], SHP-2
[46], PTEN [47], SOCS-1 [48] and so on. Our results
Acknowledgements
revealed that EP remarkably enhanced the expression of
ThisworkwassupportedbytheKoreaScienceandEngineeringFoundation
SHP-1 at levels of protein and mRNA. In contrast, EP (KOSEF)grantfundedbytheKoreagovernment(MEST)(No.2011-0006220).
had no significant effect on other PTPs such as SHP-2
Authordetails
and PTEN (data not shown). To confirm the signifi-
1ClinicalTrialInstitute,DankookUniversity,Chenan330-714,SouthKorea.
cance of SHP-1, we utilized PTP inhibitor pervanadate 2CollegeofOrientalMedicine,KyungHeeUniversity,Seoul130-701,South
or SHP-1 siRNA to block the expression of SHP-1. As Korea.
expected, EP failed to inhibit STAT3 activation in the
Authors’contributions
presence of pervanadate or SHP-1 siRNA, supporting YHRSJJandSHKSAdesignedtheresearchstudies;YHR,HJeLandHJuL
that SHP-1 plays a critical role in dephoshophorylating carriedouttheexperiments;YHRSJJandSHKanalyzedandinterpretedthe
data;YHRSJJandSHKwrotethedraftofthemanuscript.Allauthorsread
STAT3 by EP in U266 cells.
andapprovedthefinalmanuscript.
Rheeetal.BMCCancer2012,12:28 Page10of11
http://www.biomedcentral.com/1471-2407/12/28
Competinginterests 22. YuCL,MeyerDJ,CampvellGS:EnhancedDNA-bindingactivityofastat3-
Theauthorsdeclarethattheyhavenocompetinginterests. regulatedproteinincellstransformedbythesrconcoprotein.Science
1995,269:81-83.
Received:8July2011 Accepted:20January2012 23. GrantDS,KinsellaJL,FridmanR,AuerbachR,PiaseckiBA,YamadaY,ZainM,
Published:20January2012 KleinmanHK:InteractionofendothelialcellswithalamininAchain
peptide(SIKVAV)invitroandinductionofangiogenicbehaviorinvivo.J
CellPhysiol1992,153(3):614-625.
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