Umbilical cord tissue-derived mesenchymal stem … I Stem Cell Res Ther. 2014 .pdf · Umbilical...

Han et al. Stem Cell Research & Therapy 2014, 5:54http://stemcellres.com/content/5/2/54

RESEARCH Open Access

Umbilical cord tissue-derived mesenchymal stemcells induce apoptosis in PC-3 prostate cancercells through activation of JNK anddownregulation of PI3K/AKT signalingIhn Han1†, Miyong Yun1†, Eun-Ok Kim1, Bonglee Kim1, Min-Hyung Jung2 and Sung-Hoon Kim1*

Abstract

Introduction: Although mesenchymal stem cells (MSCs) have antitumor potential in hepatocellular carcinoma andbreast cancer cells, the antitumor mechanism of human umbilical cord mesenchymal stem cells (hUCMSCs) inprostate cancer cells still remains unclear. Thus, in the present study, we elucidated the antitumor activity ofhUCMSCs in PC-3 prostate cancer cells in vitro and in vivo.

Methods: hUCMSCs were isolated from Wharton jelly of umbilical cord and characterized via induction ofdifferentiations, osteogenesis, and adipogenesis. Antitumor effects of UCMSCs on tumor growth were evaluated ina co-culture condition with PC-3 prostate cancer cells. PC-3 cells were subcutaneously (sc) injected into the left flankof nude mice, and UCMSCs were sc injected into the right flank of the same mouse.

Results: We found that hUCMSCs inhibited the proliferation of PC-3 cells in the co-culture condition. Furthermore,co-culture of hUCMSCs induced the cleavage of caspase 9/3 and PARP, activated c-jun NH2-terminal kinase (JNK), andBax, and attenuated the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/ AKT, extracellular signal-regulated kinase(ERK), and the expression of survival genes such as Bcl-2, Bcl-xL, Survivin, Mcl-1, and cIAP-1 in PC-3 cells in Western blottingassay. Conversely, we found that treatment of specific JNK inhibitor SP600125 suppressed the cleavages of caspase 9/3and PARP induced by hUCMSCs in PC-3 cells by Western blotting and immunofluorescence assay. The homing ofhUCMSCs to, and TUNEL-positive cells on, the K562 xenograft tumor region were detected in Nu/nu-BALB/c mouse.

Conclusions: These results suggest that UCMSCs inhibit tumor growth and have the antitumor potential for PC-3prostate cancer treatment.

IntroductionAlthough clinical use of stem cells has been applied to vari-ous diseases, such as leukemia [1,2], Parkinson disease [3,4],diabetes [5], stroke [6], and cardiac disease [7-10], still limi-tations of their clinical use exist because of tumor-formationrisk, host immune rejection, and ethical issues. However,mesenchymal stem cells (MSCs) are attractive comparedwith embryonic stem cells as a substitute resource for clin-ical use [11]. MSCs, also known as stromal progenitor

* Correspondence: [email protected]†Equal contributors1Cancer Preventive Material Development Research Center, College ofOriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu,Seoul 130-701, Republic of KoreaFull list of author information is available at the end of the article

© 2014 Han et al.; licensee BioMed Central LtdCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.

cells, are found in several places in the human body, suchas bone marrow, umbilical cord, umbilical cord blood,placenta, and muscle synovial membrane [12]. Under ap-propriate culture conditions, MSCs have the potential forself-renewal and differentiation into various cell lineages forosteocytes, adipocytes, and chondrocytes [13].Recently, human umbilical cord blood (UCB) or human

umbilical cord tissue mesenchymal cells (hUCMSCs), iso-lated from fetal origins, have been studied for clinical usebecause UCMSCs are considered to be a more-primitiveprecursor than MSCs [14,15]. Also, the umbilical cordmatrix is suggested as a better source for the MSCs thanumbilical cord blood in respect of higher expansion po-tential [16]. The hUCMSCs were known to express spe-cific surface markers, such as CD44, CD105 (adhesion

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Han et al. Stem Cell Research & Therapy 2014, 5:54 of 9http://stemcellres.com/content/5/2/54

molecules), CD29, CD51 (integrin markers), SH2, andCD105 (mesenchymal stem cell markers), but nothematopoietic lineage markers, such as CD34, CD45, andHLA-class II [17-19]. Also, hUCMSCs have an immune-suppressive effect or reduced immunogenicity [20] andexpress vascular endothelial growth factor (VEGF) andinterleukin (IL)-6 [18,21]. Recently, UCB-derived MSCsshowed cytotoxicity against glioma [22] and Kaposi sar-coma [23], and umbilical cord mesenchymal stem cellssuppressed the growth of breast cancer cells [24-26].Based on previous evidence, in the present study, we in-vestigated the antitumor mechanism of hUCMSCs in PC-3 prostate cancer cells and report that hUCMSCs induceantiproliferative and apoptotic effects in PC-3 cells via ac-tivation of JNK and inhibition of the PI3K/AKT pathwayin either direct or indirect culture conditions.

Materials and methodsCulture for PC-3 prostate cancer cells and hUCMSCsPC-3 prostate cancer cells were obtained from theAmerican Type Culture Collection (ATCC, Rockville,MD, USA) and maintained in RPMI1640 containing 10%heat-inactivated fetal bovine serum (FBS) (Invitrogen,Carlsbad, CA, USA) and standard antibiotics (Invitro-gen). In contrast, umbilical cord (UC) specimens wereobtained within an hour of surgical resection underKyung Hee Medical Center IRB-approved (KMC IRB1125–03) just after appropriate written consent for theuse of the human umbilical cord tissues.Human UCMSCs were isolated from UCs of full-term

delivery patients, as previously described [27]. In brief,UCs were washed in calcium, magnesium-free phosphate-buffered saline (DPBS), and cut into 1- to 2-mm3 pieces.Samples were enzymatically digested for 1 hour at 37°Cwith 3 mg/ml of collagenase type I (Sigma-Aldrich, St.Louis, MO, USA). Cells were filtered through a 40-μmnylon cell strainer and centrifuged at 1,500 rpm for 5 mi-nutes, and pellets were collected as hUCMSCs. The cellswere plated in 100-mm tissue-culture dishes at a densityof 1 × 104 cells/cm2 for growth at 37°C in a humidified5% CO2 atmosphere in low-glucose Dulbecco modifiedEagle medium (Invitrogen) with fibroblast growth factor(FGF)-2 (Sigma), insulin (Invitrogen), antibiotic solution(100 μg/ml penicillin, and 100 μg/ml streptomycin; Invi-trogen), 1% gentamycin (Sigma), and heat-inactivated FBS(Invitrogen). Adherent cells were detached by incubationfor 5 minutes with trypLE-Express (Invitrogen) and thenreplated at the same density.

Osteogenic and adipogenic differentiation assaysDifferentiation was induced according to established proto-cols [28]. In brief, for osteogenic differentiation, hUCMSCswere cultured to 80% to 90% confluency for 14 daysin DMEM-LG supplemented with 10% FBS, 100 nM

dexamethasone, 200 μM ascorbic acid 2-phosphate, and10 mM β-glycerophosphate. Alizarin Red staining was per-formed in subconfluent hUCMSCs for the visualization ofcalcium deposition. Cells were fixed with 4% paraformalde-hyde for 10 minutes at room temperature, washed, stainedwith Alizarin Red staining solution for 1 hour in the dark,washed with 1 ml distilled water, and added by PBS. For in-duction of adipogenic differentiation, hUCMSCs werecultured to 80% to 90% confluence. Adipogenic differenti-ation media consisting of DMEM high glucose (Lonza)supplemented with 10% FCS, PSG, 10−6M dexamethasone,0.2 mM indomethacin, 0.1 mg/ml insulin, and 1 mM3-isobutylmethylxanthine (Sigma-Aldrich) were changedtwice a week for 14 days. The differentiated cells were fixedwith 4% formaldehyde and stained with Oil Red O (Sigma-Aldrich) to visualize lipid vacuoles. The red lipid imageswere observed under phase-contrast microscope.

Cytotoxicity assayCytotoxic effects of hUCMSCs against PC-3 cells wereevaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. We cocultured PC-3cells by using Transwell assay system along with severaldensities of hUCMSCs for 24 hours in the same culturecondition as hUCMSCs. The cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium brom-ide (1 mg/ml) (Sigma Chemical Co., St. Louis, MO, USA)for 2 hours and then with MTT lysis solution overnight.Optical density (OD) was measured by using a microplatereader (Molecular Devices Co., Sunnyvale, CA, USA) at570 nm. Cell viability was calculated as a percentageof viable cells cocultured with hUCMSCs versus singlecultured control.

Proliferation assayDNA synthesis was detected by using a colorimetric bro-modeoxyuridine (BrdU)-based Cell Proliferation ELISAkit (Roche Molecular Biochemicals, Mannheim, Germany)by following manufacturer’s instructions. In brief, we culti-vated PC-3 cells by using Transwell assay system alongwith several densities of hUCMSCs in the same culturecondition as hUCMSCs. For growing purposes, they werelabeled with BrdU for 48 hours, as previously described[29]. The absorbance was measured at 450 nm by micro-plate reader (Tecan, Austria). Culture medium was usedas a control for nonspecific binding.

Immunoblotting analysisImmunoblotting was done according to our standard pro-tocols, as described previously [29]. The protein sampleswere extracted, quantified, and separated on SDS-PAGEgels and electro-transferred to nitrocellulose membranes.Nitrocellulose membranes were blocked in 5% nonfat milkand incubated with primary antibodies for PARP, BAX,


Survivin (Santa Cruz Biotechnology Inc., Santa Cruz, CA,USA), cleaved caspase 9, Bcl-2, Bcl-xL, p-ERK, p-AKT,p-JNK (Cell Signaling Technology, Danvers, MA, USA)and β-Actin (Sigma). The blots were then exposed toHRP-conjugated secondary mouse or rabbit antibodiesand analyzed by using enhanced chemiluminescence (ECL)Western blotting detection system (GE HealthCareBio-Sciences, Piscataway, NJ, USA).

Inoculation of PC-3 cells and hUCMSCs in miceNu/nu-BALB/c mice (4 to 5 weeks old) were purchasedfrom the Shizuoka Laboratory Center (Kotoh, Japan)and maintained under classic conditions (55% relative

Figure 1 Characterization of mesenchymal stem cells. (A) Beta-galacto(0, 1, 3, and 5) of hUCMSCs. Cell morphology was observed by phase-contraccording to the passages of hUCMSCs. (C) Characterization of isolated hUand NANOG (red). Nuclei were stained with DAPI (blue). Scale bar, 50 μm. (osteogenic differentiation media for 14 days was performed by identificatioin Materials and Methods. The cells were stained with Oil Red-O and Alizar

humidity and 22°C ± 2°C). PC-3 cells and hUCMSCswere harvested and washed with 0.1 ml PBS. The cellsgently were mixed with equal amount of growth factor-reduced Matrigel (BD BioSciences, San Jose, CA, USA)on ice. PC-3 cells (2 × 106) were subcutaneously trans-planted into the left flank of mice, and, 2 weeks later,hUCMSCs (5 × 106) stained with PKH26 dye (Sigma)were transplanted into the right flank of mice. Eightweeks after PC-3 cell inoculation, Matrigel plugs wereisolated from mice for H&E, immunohistochemistry,and TUNEL assay. The immunofluorescence stainingimage for PKH26 dye stained hUCMSCs in PC-3 celltumor section was visualized under an Axio vision 4.0

sidase staining was used to check SA-β-gal activity in early passagesast microscopy. Scale bar, 50 μm. (B) Growth kinetics of the hUCMSCsCMSCs was performed by identification of MSC markers, OCT4 (green)D) Characterization of isolated hUCMSCs cultured in adipogenic andn of the adipogenic and osteogenic differentiation assays, as describedin Red dye staining, respectively. Scale bar, 100 μm.


fluorescence microscope (Carl Zeiss Inc., Weimar,Germany). This study was approved by and conductedin accordance with the policies set forth by the AnimalCare and Use Committee of Kyung Hee University (RefIRB; KHUASP(SE)-11–005).

Terminal deoxynucleotidyltransferase dUTP nick-endlabeling (TUNEL) assayDNA fragmentation was analyzed by using Dead Endfluorometric TUNEL assay kit (Promega, Madison, WI,USA). The tissues were fixed in 4% methanol-free formal-dehyde solution in PBS for 35 minutes at 4°C and treatedwith terminal deoxyribonucleotidyltransferase (TdT) en-zyme buffer containing fluorescein-12-dUTP for 1 hour at37°C in the dark. The slides were mounted with mountingmedium containing 4′,6-diamidino-2-phenylindole (DAPI)(Vector, Burlingame, CA, USA) and visualized under anAxio vision 4.0 fluorescence microscope (Carl Zeiss).

Figure 2 The inhibitory effect of hUCMSCs on prostate cancer cell growere cocultured for 24 hours with or without hUCMSCs at ratio of 1:10, 1:5Transwell chamber, and different numbers of hUCMSCs (hUCMSCs:PC-3s, 1on the viability of PC-3 cells by MTT assay. (B) Effect of hUCMSCs on the pPC-3 cells cocultured with hUCMSCs by phase-contrast microscopy. **P < 0

Statistical analysisStatistical analysis was performed by using MicrosoftExcel analysis tools and SigmaPlot 2001 software. Alldata values are shown as means ± standard deviation(SD). The statistical significance was analyzed by usingthe Student t test and analysis of variance. P valuesof <0.05 were considered statistically significant.

ResultsCharacterizations of MSCs isolated from umbilical cord tissuesRegular morphology of isolated MSCs from umbilicalcord (UC) was observed under phase-contrast micros-copy, and very rare SA-β-gal-positive senescent cellswere found in passages 0, 1, 3, and 5 of hUCMSCs byβ-galactosidase assay (Figure 1A) [30]. As shown inFigure 1B, the normal proliferation rate of isolated MSCswas also confirmed (Figure 1B).Taken together, early passages of hUCMSCs are appro-

priate to use in this study. Porcine umbilical cord matrix

wth in direct or indirect coculture condition. PC-3 cells (5 × 104), and 1:3 (hUCMSCs:PC-3s). PC-3 cells were placed in the lower:10, 1:5, 1:3) were seeded in the upper chamber. (A) Effect of hUCMSCsroliferation of PC-3 cells by BrdU assay. (C) Analysis of morphology of.01, ***P < 0.001 versus untreated control.


cells express mesenchymal stromal markers and tran-scription markers such as OCT4, NANOG, and Sox[31]. Therefore, the expression of these MSC markerswas evaluated in isolated hUCMSCs by immunostainingassay. As shown in Figure 1C, OCT4 and NANOG,which represent the pluripotent embryonic stem cellphenotype, were expressed in hUCMSCs. UCMSCs havemultiple lineages potential to adipogenic and osteogenicdifferentiation [13].To characterize the isolated hUCMSCs in our system,

they were cultured in the adipogenic and osteogeniccomplete media. Ten days after induction, osteogenicdifferentiation of hUCMSCs was verified as brownishorange red for extracellular calcium deposits by AlizarinRed staining (Figure 1D, upper). In addition, accumulation

Figure 3 Effect of JNK SP60015 on PARP, cleaved caspase 3, cleavedof hUCMSCs on PARP, Bax, and cleaved caspase 9 in PC-3 cells. (B) Effect op-JNK induced by hUCMSCs in PC-3 cells was detected in immunoblottingcaspase, cleaved PARP, and p-JNK were analyzed by immunofluorescence acleaved caspase 3, cleaved PARP, and p-JNK. DAPI (Blue).

of lipid vacuoles from the hUCMSCs as the indicator ofadipogenic differentiation of MSCs was detected asbright red color by Oil-red staining (Figure 1D, lower),implying that isolated hUCMSCs in this study had stemcell potential.

hUCMSCs inhibited the proliferation of PC-3 cancer cellsTo determine the antitumor effect of hUCMSCs on hu-man prostate cancer cells, PC-3 prostate cancer cells(1 × 105) were cocultured with the densities of 3.33 ×104, 2 × 104, and 1 × 104 of UCMSCs (hUCMSCs:PC-3 sratio, 1:10, 1:5, 1:3). First, we determined the viability ofPC-3 cells by MTT assay. The viability of PC-3 cellscocultured with UCMSCs (hUCMSCs:PC-3, 1:5, 1:3) wassignificantly decreased, whereas UCMSCs:PC-3 (1:10)

caspase 9, and p-JNK induced by hUCMSCs in PC-3 cells. (A) Effectf JNK SP60015 on PARP, cleaved caspase 3, cleaved caspase 9, andassay. (C) In the same condition as in (B), expression levels of cleavedssay. Each primary antibody was diluted 1/300. Light green indicates


did not show the difference compared with PC-3 cellscultured without hUCMSCs (Figure 2A). In addition, wedetermined the proliferation of PC-3 cells coculturedwith hUCMSCs by BrdU assay. The growth of PC-3 cellscocultured with hUCMSCs was decreased to 44%, 49%,and 69% of control in the presence of UCMSCs withvarious numbers of 3.33 × 104, 2 × 104, and 1 × 104, re-spectively, compared with untreated control (Figure 2B).As shown in Figure 2C, when PC-3 cells were cocul-tured in the presence of hUCMSCs (UCMSCs:PC-3,1:3), the number of PC-3 cells was rarely observed com-pared with untreated control.

hUCMSCs induced apoptosis and attenuated survivalgenes in PC-3 cellsTo determine whether apoptosis is induced in PC-3 cellscocultured with hUCMSCs, Western blotting was per-formed. PARP cleavage, cleaved caspase 3, Bax, andphosphorylation of JNK were detected in the lysatesof PC-3 cocultured with hUCMSCs (Figure 3A). Toverify whether this apoptotic event is dependent onJNK pathway, the JNK-specific inhibitor SP600125 wastreated in PC-3 cells cocultured with hUCMSCs. Con-versely, the apoptotic features such as PARP cleavage,cleaved caspase 3, and phosphorylation of JNK in PC-3cells by hUCMSCs were efficiently masked by JNK in-hibitor SP600125 with Western blotting (Figure 3B) andimmunofluorescence assay (Figure 3C). Also, as shownin Figure 4A, PI3K and phosphorylation of AKT andERK were attenuated in PC-3 cells by hUCMSC cells.

Figure 4 Effect of hUCMSCs on survival genes and JNK in PC-3 cells. PhUCMSCs at the ratio (hUCMSCs:PC-3, 1:3). Cells lysates were immunoblottecaspase 9, and β-actin antibodies. (A) Effect of hUCMSCs on PI3K, AKT, ERKcIAP-1, and β-actin in PC-3 cells.

Furthermore, the expression of survival genes such asBcl-2, Bcl-xL, Survivin, Mcl-1, and cIAP-1 was attenu-ated in PC-3 cells by Western blotting (Figure 4B).

The homing of hUCMSCs and apoptosis induction in PC-3cells in nude mouseNext, we investigated the homing of hUCMSCs to PC-3cells in mice. PC-3 cells were injected subcutaneously intothe left flank of the Balb-c/nu-nude mice. Two weeks later,PKH26-labeled hUCMSCs were transplanted into the rightflank of the mice. Mice were killed 7 days after injection.Immunohistochemistry revealed that hUCMSCs were de-tected on the PC-3 tumor region with red color by con-focal microscope (Figure 5A). In addition, TUNEL assayshowed some TUNEL-positive cells in the PC-3 cancer cellregion in mice treated with PKH26-labeled hUCMSCs(Figure 5B). However, we could not find a significantinhibitory effect of hUCMSCs on the growth of PC-3cells for tumor weight and volume in mice comparedwith untreated control 3 weeks after PC-3 cell inocula-tion (data not shown). Thus, we must perform anotheranimal study with a different number of hUCMSCs viadirect or indirect injection of hUCMSCs into the PC-3tumor region and check the possibility of teratoma inmice in the near future.

DiscussionMesenchymal stem cells (MSCs) are fibroblast-like multi-potent stem cells that can be differentiated into several celltypes, such as adipocytes, osteocytes, and chondrocytes

C-3 cells were cultured for 24 hours alone or in the presence ofd with PI3K, p-AKT, AKT, p-ERK, ERK, p-JNK, JNK and PARP, Bax, cleaved, and JNK in PC-3 cells. (B) Effect of hUCMSCs on Bcl-2, Bcl-xL, survivin,

Figure 5 Homing of hUCMSCs to PC-3 tumor site in Balb-c/nu-nude mice and their effect on TUNEL-positive cells in PC-3 tumor section.(A) Paraffin sections for H&E and IHC staining with PKH26 dye (red). The PKH26-labeled cells tracking toward the PC-3 tumor region from theopposite-side flank. White arrows indicate the labeled PKH26 (red). (B) Representative photographs of TUNEL/PI staining. Red fluorescence(PKH26) marks transplanted hUCMSCs, and green indicates TUNEL-positive cells. DAPI (blue).


[32,33]. MSCs are usually isolated from umbilical cordblood or tissues and adipocytes [34-36]. Although muchevidence suggests that MSCs can be applied to several dis-eases, such as cancers [1,22,24], cardiac disease [8,37],stroke [6], and Parkinson and Huntington diseases [3], theunderlying antitumor mechanism of MSCs was not fullyunderstood until now. Thus, in the current study, theantitumor signaling of hUCMSCs was elucidated inPC-3 prostate cancer cells. We isolated hUCMSCs fromumbilical cord tissues and confirmed positive stem cell

markers, such as OCT4 and NANOG, and successfully in-duced osteogenesis by Alizarin Red staining and adipogen-esis by Oil Red O staining, implying that hUCMSCs stillhave pluripotency of stem cells to be differentiated intoadipocytes and osteocytes.In addition, hUCMSCs treatment exhibited cytotoxic

and antiproliferative effects in PC-3 cells by MTT andBrdU assays, indicating that hUCMSCs target the growthof PC-3 cells. Similarly, Khakoo et al. [23] supported thatintravenously (i.v.) injected human MSCs home to sites of


tumorigenesis and potently inhibit the growth of Kaposisarcoma, and Chao et al. [24] reported that apoptosiswas noted during coculture of MDA-MB-231 breast can-cer cells with hUCMSCs. Furthermore, other groupsreported that Z3-MSCs have an inhibitory effect ontumor growth by secretion of Wnt-inhibitor Dkk1, leadingto downregulation of genes related to the cell cyclethrough inhibition of Wnt/β-catenin signaling [38,39].Our results and other group reports mean that hUCMSCcan be a potential therapeutic approach for the treatmentof cancer. However, the ethical issues should be alsoconsidered, before we use hUCMSC as a therapeuticapproach for tumor treatment.In general, apoptosis, called programmed cell death,

includes the intrinsic mitochondrial pathway and theextrinsic cell death pathway [40,41], and the activationof the JNK pathway is also related to apoptosis [42].Here, hUCMSCs treatment resulted in the cleavages ofcaspase 9/3 and PARP, increased phosphorylation ofJNK and upregulation of Bax as apoptotic protein, anddecreased phosphorylation of PI3K/AKT and ERK inPC-3 cells by Western blotting, demonstrating the apop-totic effect of hUCMSCs via mitochondrial and JNK-dependent pathways. Consistently, hUCMSCs treatmentattenuated the expression of survival genes, such as Bcl-2,Bcl-xL, Survivin, Mcl-1, and cIAP-1 in PC-3 cells, imply-ing an inhibitory effect of hUCMSCs on antiapoptoticproteins.To confirm the role of JNK in hUCMSCs-induced

apoptosis in PC-3 cells, JNK inhibitor study was carriedout. Conversely, treatment of JNK inhibitor SP600125reversed the apoptotic ability of hUCMSCs to cleavecaspase 9/3 and PARP in PC-3 cells by Western blottingand immunofluorescence assay, indicating that the JNKpathway mediates hUCMSCs-induced apoptosis in PC-3cells. Consistent with our data, Aikin et al. [43] claimedthat PI3K inhibition led to increased JNK phosphoryl-ation and pancreas islet cell death, which could be re-versed by the specific JNK inhibitor SP600125.Of note, the homing of hUCMSCs to PC-3 cells and

TUNEL-positive cells as an apoptotic feature was de-tected in the tumor section of PC-3 cells, implying thathUCMSCs on the left flank can move to PC-3 cells onthe right flank, as the homing of hUCMSCs to PC-3cells, possibly for cell death. Likewise, Liang et al. [44]reported that systemically infused hUCMSCs couldhome to the inflamed colon and effectively amelioratecolitis via modulation of IL-23/IL-17 by live in vivo im-aging and immunofluorescent microscopy.Overall, our findings demonstrate the antitumor po-

tential of hUCMSCs for PC-3 prostate cancer treat-ment, but further study is required for animal tumorstudy via direct or indirect injection of hUCMSCs inthe near future.

ConclusionsBased on our results, UCMSCs inhibit the tumor growthand have an antitumor potential for PC-3 prostate can-cer treatment.

AbbreviationsERK: Extracellular signal-regulated kinase; JNK: C-jun NH2-terminal kinase;PI3K: phosphatidylinositol 3-kinase; UCMSC: umbilical code mesenchymalstem cell.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsIH: experimental design, data collection and participation in manuscriptdrafting. MY: design and conception for project, data analysis, manuscriptdrafting, and final approval of the manuscript. EK: acquisition and analysis ofdata and revision of manuscript. BK: partial data collection, revision ofmanuscript, and material support. MJ: revision of manuscript, experimentaldesign, and material support. SK: design and concept, manuscript writing,and final approval of manuscript. All authors read and approved the finalmanuscript.

AcknowledgementThis work was supported by the Korea Science and Engineering Foundation(KOSEF) grant funded by the Korea government (MEST) (No. 2012–0005755).

Author details1Cancer Preventive Material Development Research Center, College ofOriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu,Seoul 130-701, Republic of Korea. 2School of Medicine, Kyung Hee University,Seoul 130-701, Republic of Korea.

Received: 11 September 2013 Revised: 25 November 2013Accepted: 21 March 2014 Published: 16 April 2014

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doi:10.1186/scrt443Cite this article as: Han et al.: Umbilical cord tissue-derived mesenchymalstem cells induce apoptosis in PC-3 prostate cancer cells through activationof JNK and downregulation of PI3K/AKT signaling. Stem Cell Research &Therapy 2014 5:54.

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