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Received: 3 January 2018    Accepted: 27 March 2018

DOI: 10.1111/pim.12531

ORIGINAL ARTICLE

Proliferation of prostate epithelia induced by IL-­6 from stroma

reacted with Trichomonas vaginalis

J.-H. Kim1,2 | I.-H. Han1 | Y.-S. Kim3 | C.-S. Noh4 | J.-S. Ryu1,2

1
Department of Environmental Biology and
Medical Parasitology, Hanyang University Summary
College of Medicine, Seoul, Korea Benign prostatic hyperplasia (BPH) is characterized by the proliferation of stromal
2
Department of Biomedical
and epithelial cell types in the prostate, and interactions between the two types of
Science, Graduate School of Biomedical
Science & Engineering, Seoul, Korea cells. We demonstrated previously that proliferation of prostate stromal cells was
3
Department of Biochemistry and Molecular induced by BPH epithelial cells in response to Trichomonas vaginalis (Tv) infection via
Biology, Hanyang University College of
crosstalk with mast cells. In this study, we investigated whether IL-­6 released by the
Medicine, Seoul, Korea
4
Department of Internal Medicine, Hallym proliferating stromal cells in turn induce the BPH epithelial cells to multiply. When
University Han River Seongshim Hospital, culture supernatants of the proliferating prostate stromal cells were added to BPH
Seoul, Korea
epithelial cells, the latter multiplied, and expression of cyclin D1, FGF2 and Bcl-­2 in-
Correspondence creased. Blocking the IL-­6 signalling pathway with anti-­IL-­6R antibody or JAK1/2 in-
Jae-Sook Ryu, Department of Environmental
Biology and Medical Parasitology, Hanyang hibitor inhibited the proliferation of the BPH epithelial cells and reduced the
University College of Medicine, Seoul, expression of IL-­6, IL-­6R and STAT3. Also, epithelial-­mesenchymal transition was de-
Korea.
Email: jsryu@hanyang.ac.kr tected in the proliferating BPH epithelial cells. In conclusion, IL-­6 released from pro-
liferating prostate stromal cells induced by BPH epithelial cells infected with Tv in
Funding information
Basic Science Program through the National turn induces multiplication of the BPH epithelial cells. This result provides first evi-
Research Foundation of Korea (NRF), Grant/ dence that the inflammatory microenvironment of prostate stromal cells resulting
Award Number: NRF-2017R1A2B4002072;
Ministry of Science; ICT from Tv infection induces the proliferation of prostate epithelial cells by stromal-­
epithelial interaction.

KEYWORDS
cytokines, inflammation, prostatic hyperplasia, protozoan infections

1 |  I NTRO D U C TI O N
Trichomonas vaginalis (Tv), the cause of trichomoniasis, a common
infection of the urogenital system, is the most prevalent sexu-
1
ally transmitted protozoan. The World Health Organization esti-
mates that more than half of the new 276.4 million Tv infections
each year are male. 2 The majority of infections are nonsymp-
tomatic in men and remain undiagnosed and untreated, which
has been hypothesized to result in chronic persistent prostatic
infection. 3 Tv has been observed in the prostate tissue of some
patients with benign prostatic hyperplasia (BPH) and prostate
cancer, and in the urine of patients with prostatitis.4-6 Also, Tv
elicits acute and chronic inflammation within the parenchyma of
the prostate gland.7
BPH is the most common age-­related disease of the male, oc-
curring clinically in about half of all men at 70 years of age, but the
cause is not known.8 Prostatic inflammation, including activation
of inflammatory cells and production of cytokines, appears to be a
major cause of BPH and prostate cancer.9,10 Inflammatory mediators
are secreted by the prostatic stroma consequent upon ageing, and
the levels of these mediators are sufficient to increase the prolif-
erative rate of both epithelial and stromal fibroblasts.11 Although
inflammation may play a role in the development of benign prolif-
erative diseases of the prostate, for example BPH, the connection
between inflammation, Tv infection and prostate enlargement is not
fully understood.
Stromal-­
epithelial interactions play key regulatory roles in
prostate development and in homeostasis in the adult prostate.12

Parasite Immunology. 2018;40:e12531. wileyonlinelibrary.com/journal/pim © 2018 John Wiley & Sons Ltd  |  1 of 9
https://doi.org/10.1111/pim.12531
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Proliferation induced by interactions between prostatic epithelial
and stromal cells is related to the development of BPH, and bidi-
rectional stromal-­epithelial interactions have been reported in the
prostate gland.13 Similar interactions have been investigated in cor-
neal disease where soluble cytokines and growth factors secreted
by corneal stromal cells contribute to the proliferation and differen-
tiation of corneal epithelial cells.14
IL-­6 is a potent inflammatory cytokine with diverse func-
tions in numerous cell types and one of the mediators of chronic
15,16
inflammation in prostate cancer. It is known to regulate a
variety of complex cellular processes such as proliferation, dif-
ferentiation and gene activation.17 Expression of IL-­6 and the
IL-­6 receptor has been detected in BPH and/or prostate cancer
tissue.18,19 Sutcliffe et al have hypothesized that IL-­6 induction
by Tv in normal prostate epithelial cells triggers a signalling cas-
cade through the proto-­o ncogenes, PIM1, c-­M YC and HMGA1,
which ultimately leads to prostate carcinogenesis. 3 We reported
previously that IL-­6 produced by Tv-­infected prostate epithelial
cells induced epithelial-­m esenchymal transition (EMT) via NF-­k B
and JAK/STAT3. 20 Although the role of IL-­6 in the chronic in-
flammation in prostate cancer has been described, the involve-
ment of IL-­6 in epithelial cell proliferation in BPH has not been
examined.
We reported previously that BPH epithelial cells stimulated by
Tv caused inflammation and induced the proliferation of prostate
stromal cells by activating mast cells. 21 In this study, we show that
inflammatory mediator(s) (especially IL-­6) released from such prolif-
erating stromal cells induced by Tv infection induce the proliferation
of BPH epithelial cells. This result is the first evidence that stromal-­
epithelial interaction plays a role in prostatic epithelial cell prolifer-
ation induced by Tv.
2 |  M ATE R I A L S A N D M E TH O DS
2.1 | Parasites and host cells
Trichomonas vaginalis (Tv) isolate T016, human BPH epithelial cell line
(BPH-­1), human mast cell line (HMC-­1) and human prostate stromal
cell line WPMY-­1 were cultured as described in our previous study.21
All the cells were incubated in 5% CO2 at 37°C.
2.2 | Preparation of conditioned media
2.2.1 | Production of BPH-­1 cell
conditioned medium
BPH-­1 cells were seeded at 1 × 105 cells/well in BPH-­1 medium in
the wells of 12-­well plates (Corning, NY, USA). After 24 hours, the
medium was changed to serum-­free medium and the cells were
cultured for another 24 hours to stabilize. The cells were then in-
cubated with or without live Tv in a ratio of 1:10 for 6 hours, and
supernatants were collected, filtered through 0.2-­μm filters (GVS,
IN, USA) and stored at frozen. The supernatants of the BPH-­1 cells
incubated with and without Tv were named TCM (Tv-­conditioned
medium) and CM (conditioned medium), respectively.
2.2.2 | Other conditioned media
HMC-­1 mast cells were incubated with TCM and CM, in the same
way, yielding conditioned media MTCM and MCM, and prostate
stromal cells (WPMY-­1) were incubated with MTCM, MCM, TCM
and fresh medium, yielding W-­MTCM, W-­MCM, W-­TCM and W-­CM,
respectively. Final concentrations of the conditioned media used for
various assays were adjusted to 10% with cell culture medium.
2.3 | Measurement of cytokine level
Cytokine concentrations of conditioned media (MTCM, MCM, TCM,
CM) containing inflammatory mediators were measured by ELISAs
(BD Biosciences, San Diego, CA, USA) according to the manufac-
turer’s instructions.
2.4 | Proliferation assays
The proliferation assays were performed using CCK-­8 assay
and wound-­h ealing assay as previously decribed. 21 To measure
the proliferation of cells, CCK-­8 assay and wound-­h ealing assay
were used. The CCK-­8 assay is a sensitive colorimetric assay to
determine the number of viable cells. CCK-­8 reagent (Enzo Life
Sciences, NY, USA) was added to each well 2 hours before obtain-
ing spectrophotometric readings according to the manufacturer’s
directions. The amount of the dye generated is directly propor-
tional to the number of living cells. For wound-­
h ealing assay,
BPH epithelial cells were seeded at 2 × 105 cells/well on 24-­well
plates and cultured in BPH-­1 medium for 24 hours. The medium
was changed to serum-­f ree medium for another 24 hours. The cul-
tured BPH epithelial cells reached confluence and were wounded
by scraping across the surface of the well with a sterile micropi-
pette tip. The cells were washed immediately, and the wells were
filled with 1 ml of control (serum-­f ree) medium or 10% conditioned
medium (W-­MTCM, W-­M CM or W-­CM) for 48 hours. Before and
after incubation, at least ten different fields of the wounded area
of each sample were photographed with an inverted microscope
(Leica, Las software). Wound areas were calculated using ImageJ
software.
2.5 | Immunofluorescence for localization of IL-­
6 receptor
To observe the expression of IL-­6 receptor on the surface of BPH-­1
cells, we used an immunofluorescence assay. IL-­6R expression was
identified by detecting glycoprotein 130 (gp130), which forms one
subunit of the type I cytokine receptor in the IL-­6 receptor fam-
ily. BPH-­1 cells were seeded at 1 × 105 cells/well on sterile cover
glasses in 24-­well plates and cultured in BPH-­1 medium. After over-
night incubation, the medium was changed to control (serum-­free)
KIM et al.
medium or 10% conditioned media (W-­MTCM, W-­MCM, W-­CM)
and cultured at 37°C for 24 hours. The cells were washed immedi-
ately, fixed with 4% paraformaldehyde for 10 minutes at −20°C and
blocked with 0.1% normal goat serum for 1 hour. The cover glasses
were then incubated with anti-­gp130 antibody (1:500, rabbit poly-
clonal, Cell Signaling Technology; 2 μg/mL) overnight at 4°C, and
then washed and stained with Alexa 594-­labelled goat anti-­rabbit
lgG (#A11012, 1:500, Invitrogen, CA, USA) at 37°C for 1 hour.
The cover glasses were mounted in Vectashield mounting medium
(Vector Laboratories, Burlingame, CA) with DAPI to visualize nuclei.
Fluorescence was measured with a confocal microscope (Leica, Las
software).
2.6 | Reverse transcription PCR (RT-­PCR)
Levels of mRNA in W-­MTCM-­treated BPH epithelial cells were
measured by RT-­PCR. The cells were seeded at 2 × 105 cells/well
on 24-­well plates (Corning) with BPH-­1 medium. After overnight in-
cubation, the medium was changed to conditioned medium (10%).
After incubation for another 1 hour, the cells were collected and
mRNA was extracted. Total RNA was extracted using Tri-­RNA rea-
gent (Favorgen Biotech Corp, Kaohsiung, Taiwan) according to the
manufacturer’s instructions. RNA samples were reverse-­transcribed
to cDNA using Maxime RT preMix (Oligo (dt) 15 Primer) (iNtRON
Biotechnology, Sungnam, Korea). The cDNA was used as a template
for subsequent PCR amplification using gene-­specific primers. The
following primers were used: human IL-­6R forward 5′-­ACC AGA
CCA GAC CAG ACA CC-­3′, reverse 5′-­A AG GCC AGG GTC ACA
TAC AG-­3′; human IL-­6 forward 5′-­TGG CTG CAG GAC ATG ACA
ACT-­3′, reverse 5′-­ATC TGA GGT GCC CAT GCT ACA-­3′; human N-­
cadherin forward 5′-­T TT GAG CTT GGC ACT CCT TT-­3′, reverse 5′-­
GGC TAC TGG CTC TCA GTT GG-­3′; human fibronectin1 forward
5′-­TGT GGT TTG GAA CTG GTT GA-­3′, reverse 5′-­CCA GGA ACA
TGT GAC AGT GG-­3′; human cyclin D1 forward 5′-­GAG GAA CAG
AAG TGC GAG GAG-­3′, reverse 5′-­TGG AGT TGT CGG TGT AGA
TGC-­3′; human Bcl-­2 forward 5′-­TCC ATG TCT TTG GAC AAC CA-­3′,
reverse 5′-­C TC CAC CAG TGT TCC CAT CT-­3′; human FGF2 for-
ward 5′-­ CCG TTA CCT GGC TAT GAA GG-­3′, reverse 5′-­ACT GCC
CAG TTC GTT TCA GT-­3′; GAPDH forward 5′-­GTC AGT GGT GGA
CCT GAC CT-­3′, reverse 5′-­AGG GGT CTA CAT GGC AAC TG-­3′.
Target mRNA was amplified using Maxime PCR PreMix (i-­StarTaq)
(iNtRON Biotechnology) by Mastercycler (Eppendorf, Germany) for
RT-­PCR, and the PCR products were separated by agarose gel elec-
trophoresis. The bands were visualized with StaySafe Nucleic Acid
Gel stain (RSS01, RBC, Banqiao City, Taiwan) and identified using an
ExcelBand™ 100 bp+3K DNA Ladder100 bp DNA Ladder (DM2300,
SMOBIO, Hsinchu city, Taiwan). GAPDH was used as an internal
control.
2.7 | Western blot analysis
Cells seeded at 2 × 10 5 cells/well on 24-­
w ell plates with
BPH-­1 medium were harvested and lysed in PRO-­P REP protein
|
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extraction solution (iNtRON Biotechnology). Cell lysates were
scraped, and protein concentrations of the lysates were meas-
ured with the Bradford assay. Equal amounts of protein from
the lysates were denatured in 5 x protein sample buffer (ELPIS
biotech, Daejeon, Korea), separated by SDS-­PAGE on 12% po-
lyacrylamide gel and transferred to Immun-­B lot® PVDF mem-
branes (Bio-­R ad, Quarry Bay, Hong Kong). The membranes were
probed with the following primary antibodies overnight at 4°C:
anti-­I L-­6 R antibody (1:1000; Bioss, Boston, MA, USA), p-­S TAT3
antibody (1:1000; Abcam, Cambridge, MA, USA), anti-­b cl-­2 an-
tibody (1:100; Oncogene Research Products, CA), anti-­
F GF2
antibody (1:500; Abcam), anticyclin D1 (phospho S90) antibody
(1:500; Abcam), antivimentin antibody (1:1000; Cell Signaling),
anti-­E-­c adherin antibody (1:1000; Cell Signaling) or β-­a ctin poly-
clonal antibody (1:10 000; Abcam). The plates were incubated
with goat anti-­rabbit IgG polyclonal antibody (1:10 000; ADI-­
SAB-­3 00-­J, Enzo) for 1 hour at room temperature. The blots
were visualized using Chemiluminescent Sensitive Plus HRP
Microwell and/or Membrane Substrate (SurModics, MN, USA),
and signals were measured with a Chemi-­D ocⓇ (Bio-­R ad, CA,
USA).
2.8 | Statistical analysis
Statistical analyses were performed using SPSS statistical
software, version 21 (IBM, Chicago, IL, USA). The Mann-­
Whitney U test was used to compare results, and P val-
ues < .05 were considered statistically significant. The data
are expressed as means ± SEMs of three to four independent
experiments.
3 | R E S U LT S
3.1 | Growth of prostate stromal cells induced by
BPH epithelial cells infected with Tv via crosstalk
involving mast cells
We first tested that cytokine production by BPH epithelial cell
line (BPH-­1) incubated with Tv for 6 hours. IL-­6 , CXCL8, CCL2
and IL-­1β were higher than when incubated in the absence of Tv
(Figure 1A). Similarly, CXCL8 levels in supernatants of the human
mast cells (HMC-­1) incubated with TCM were higher than in those
incubated in control epithelial cell supernatant (Figure 1B). The
data in Figure 1C show that when prostate stromal cells (WPMY-­1
cells) were incubated with the various supernatants described in
Methods, only MTCM (mast cells incubated with TCM) induced
them to multiply. This indicates that mast cells exposed to some
factor(s) released by Tv-­s timulated epithelial cells in turn release
some factor(s) including cytokines that induces the growth of stro-
mal cells. This confirms our previous finding 21 that the inflamma-
tory microenvironment induced by Tv-­infected BPH epithelial cells
caused proliferation of prostate stromal cell via the intermediary
of mast cells.
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F I G U R E   1   Growth of prostate stromal cells induced by BPH epithelial cells infected with Tv via crosstalk with mast cells. A, Cytokine
levels in the supernatants of cells of the BPH epithelial cell line (BPH-­1) incubated with and without Tv for 6 h. The supernatants of BPH-­1
cell incubated with and without Tv were designated TCM (Tv-­conditioned medium) and CM (conditioned medium), respectively. B, CXCL8
levels in the supernatants of cells of the human mast cell line (HMC-­1) incubated with TCM and CM. The supernatants were named MTCM
(mast cells incubated with TCM) and MCM (mast cells incubated with CM, respectively). C, Growth of prostate stromal cells (WPMY-­1 cells)
incubated with the indicated supernatants was measured by CCK-­8 assay after 72 h. The left axis refers to IL-­6, CXCL8 and CCL2, and the
right axis to IL-­1β. *P < .05

by measuring glycoprotein 130 (gp130), which forms one subu-

3.2 | Proliferation of BPH epithelial cells in
response to conditioned media from the multiplying
stromal cells
To determine whether the culture supernatant (W-­MTCM) of pros-
tate stromal cells proliferated with MTCM could activate BPH epi-
thelial cells, we first measured cytokine levels. When BPH-­1 cells
were stimulated with W-­MTCM, various cytokines (IL-­6, CXCL8,
CCL2 and IL-­1β) and their mRNA was increased, and levels of these
cytokines were significantly higher than those induced by W-­MCM,
W-­CM or medium alone (Figure 2A,B). Multiplication of the BPH-­1
cells was also stimulated more by treatment with W-­MTCM than
with other conditioned media such as W-­CM, W-­TCM and W-­MCM
(Figure 2C,D).
Several factors, including growth factors, cell cycle regulators
and apoptotic factors, are involved in the regulation of cell prolifer-
ation. 22-24 In this study, the transcript levels of FGF2, Bcl-­2 and cy-
clin D1 in the proliferating BPH-­1 were determined by RT-­PCR. The
mRNA levels of these factors were significantly higher in the pres-
ence of W-­MTCM than those in the presence of W-­MCM, W-­CM
or fresh medium (Figure 2E). This suggests that cyclin D1, Bcl-­2 and
FGF2 are involved in the proliferation of BPH-­1 cells. In summary,
these findings suggest that some inflammatory mediator(s) from the
proliferating stromal cells induce the proliferation of the BPH epi-
thelial cells.
3.3 | Expressions of interleukin-­6 signalling
molecules in the proliferating BPH epithelial cells
IL-­6 expression has been reported to increase in inflamed BPH
tissue, and increased expression of IL-­6 receptor was found in
BPH and prostate carcinoma patients.18,19 In order to examine
the involvement of IL-­6 signalling in the proliferation of BPH-­1
in response to W-­MTCM, we investigated the expression of the
interleukin-­6 receptor (IL-­6R). IL-­6R expression can be assessed
nit of the type I cytokine receptor in the IL-­6 receptor family.17
When gp130 was visualized by immunofluorescence, expres-
sion was stronger in the W-­MTCM-­t reated cells than in the cells
treated with W-­M CM or W-­CM (Figure 3A). The same result was
obtained when IL-­6 receptor levels were assessed by Western
blotting and RT-­P CR (Figure 3B,C). Binding of IL-­6 to IL-­6R/gp130
dimer induces phosphorylation of JAK, which in turn phosphoryl-
ates STAT3, and p-­S TAT3 and IL-­6 levels were also higher in the
W-­MTCM-­t reated cells than those in W-­M CM-­ or W-­CM-­t reated
cells(Figure 3B,C). Taken together, these data indicate that IL-­6
signalling molecules are strongly induced in the proliferating BPH
epithelial cells by treatment with W-­MTCM.
3.4 | Blocking of IL-­6 signalling reduces the
proliferation of BPH epithelial cells
When IL-­6 binds to IL-­6R, JAK1/2 recruits signal transducers and
activators of transcription (STATs) to the cytokine receptors. 25 To
investigate whether interleukin-­6 is involved in the proliferation of
BPH epithelial cells, IL-­6 signalling was blocked using anti-­gp130
antibody or a JAK1/2 inhibitor (ruxolitinib). The proliferation of
the W-­MTCM-­t reated BPH-­1 was significantly reduced by these
agents (Figure 4A,B). Moreover, levels of p-­S TAT3, bcl-­2 , FGF2, p-­
cyclin D1 and IL-­6 were also reduced (Figure 4C,D). This demon-
strates that IL-­6 signalling is involved in the proliferation of BPH-­1
cells.
3.5 | Involvement of IL-­6 in EMT induction in the
proliferating BPH epithelial cell
Epithelial-­mesenchymal transition (EMT) is a complex process that
refers to the transformation of epithelial cells into stromal cells. It is
integral to development, wound healing and stem cell behaviour and
contributes to fibrosis and cancer progression. 26 The upregulation of
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F I G U R E   2   Proliferation of BPH epithelial cell line (BPH-­1) stimulated with conditioned media of the proliferated stromal cells (W-­MTCM).
A, Production of cytokines by BPH-­1 stimulated with W-­MTCM (supernatant of WPMY-­1 prostate stromal cells incubated with MTCM). The
left axis refers to IL-­6, CXCL8 and CCL2 and the right axis to IL-­1β. B, mRNA levels of cytokines released by BPH-­1 incubated with W-­MTCM
were detected by RT-­PCR. Proliferation of BPH-­1 cells incubated with W-­MTCM was measured by CCK-­8 assay (C) and wound healing (D).
E, Cyclin D1 (regulator of cell cycle progression), bcl-­2 (anti-­apoptotic factor) and FGF2 (fibroblast growth factor-­2) were measured by
Western blotting. Media, BPH-­1 cells alone; W-­CM, culture supernatant from prostate stromal cells; W-­TCM, culture supernatant from
prostate stromal cells cultured with TCM; W-­MCM, culture supernatant from prostate stromal cells cultured with MCM (culture supernatant
from mast cells); W-­MTCM, culture supernatant from prostate stromal cells cultured with MTCM (culture supernatant from mast cells
activated with TCM); TCM, culture supernatant from BPH-­1 incubated with Tv. *P < .05

mesenchymal markers and downregulation of epithelial markers are
features of EMT. To examine whether EMT was detectible in the BPH
epithelial cells exposed to W-­MTCM including IL-­6, the expression of
EMT markers was examined. Expression of mesenchymal markers
(vimentin, N-­cadherin, fibronectin) increased and that of epithelial
marker (E-­cadherin) decreased in the BPH epithelial cells, and this
was prevented by pretreatment with IL-­6R antibody or ruxolitinib
(Figure 5). These observations indicate that EMT occurs in the W-­
MTCM-­treated BPH-­1 cells and that IL-­6 is involved in the EMT.
4 | D I S CU S S I O N
Clinical BPH is an extremely common disease in older men. 27,28 It
is an immune inflammatory disease, 28 and although the causes are
unclear, 29 chronic inflammation and an abnormal wound-­
healing
30
procedures are known to play major roles.
Trichomonas vaginalis (Tv) is generally known as a surface-­
dwelling parasite, but their capability to invade in submucosa and
stromal tissues has shown.5,6 Mitteregger et al hypothesized that
benign prostatic hyperplasia may be associated with chronic reten-
tion of Tv in the prostate gland.5 Prostate growth associated with
Tv infection has been studied by two groups; Twu et al found that
Tv macrophage migration inhibitory factor caused inflammatory re-
sponses, prostate cell growth and invasiveness in both a BPH-­1 cell
line and a prostate cancer cell line,31 and in our previous study, we
reported that BPH epithelial cells stimulated with Tv induced the
proliferation of prostate stromal cells via crosstalk involving mast
cells. 21 In the current study, BPH-­1 cells incubated with Tv were
shown to induce the proliferation of prostate epithelial cells by
acting on prostate stromal cell via mast cells. Therefore, this study
provides the first experimental evidence that stromal-­epithelial in-
teractions play a role in the prostatic epithelial proliferation induced
by Tv infection.
The prostate stroma composed of smooth muscle cells, fi-
broblasts, endothelial cells, macrophages, mast cells and undif-
ferentiated cells. 32 Human stromal prostate cells participate in
the development of BPH by secreting several pro-­i nflammatory
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F I G U R E   3   Expressions of interleukin-­6 signalling molecules in the proliferating BPH epithelial cells. A, Expression of IL-­6 receptor
(gp130) on the surface of BPH epithelial cells stimulated with W-­MTCM was observed by fluorescence microscopy. B, Expression of IL-­6R
and p-­STAT3 in BPH epithelial cells incubated with W-­MTCM was measured by Western blotting. C, IL-­6R and IL-­6 transcript levels in BPH
epithelial cells incubated with W-­MTCM were measured by RT-­PCR. Media, BPH-­1 cells alone; W-­CM, culture supernatant from prostate
stromal cells; W-­MCM, culture supernatant from prostate stromal cells cultured with MCM (culture supernatant from mast cells); W-­MTCM,
culture supernatant from prostate stromal cells cultured with MTCM (culture supernatant from mast cells activated with TCM)

F I G U R E   4   Blocking of IL-­6 signalling

reduces proliferation of BPH epithelial
cells (BPH-­1). Pretreatment of BPH-­
1 cells with anti-­gp130 antibody and
JAK1/2 inhibitor (ruxolitinib) reduces their
proliferation compared with incubation
with W-­MTCM alone (A and B). C, The
production of p-­STAT3, bcl-­2, FGF2 and
p-­cyclin D1 by the BPH epithelial cells
was also reduced by the IL-­6R antibody
and JAK1/2 inhibitor. D, Levels of IL-­6
transcripts in the BPH-­1 cells were also
lowered by anti-­gp130 antibody or
ruxolitinib. Media, BPH-­1 cells alone;
W-­MTCM, culture supernatant from
prostate stromal cells cultured with
MTCM (culture supernatant from mast
cells activated with TCM). *P < .05 vs
media. †P < .05 vs W-­MTCM

cytokines and chemokines, which recruiting immune cells to
the prostate and inducing their own proliferation. 8 The stro-
mal microenvironment is a critical determinant of benign vs
malignant growth. Microenvironmental factors such as infec-
tion and wounding may alter cellular equilibria leading to BPH
or prostate cancer. 29 In this context, our data indicate that the
inflammatory response induced by BPH epithelial cells stimu-
lated by Tv induces the proliferation of prostate stromal cells
via mast cells, and these inflamed stromal cells then liberate
factors that stimulate the proliferation of BPH epithelial cells
via IL-­6 signalling.
Stromal-­epithelial interactions play a key regulatory role in pros-
tate development and in the maintenance of the adult prostate in
health and disease.12 Siejka et al reported that bidirectional stromal-­
epithelial interactions occur in the prostate gland; when superna-
tants of prostate stromal cells (or BPH epithelial cells) were added
to BPH epithelial cells (or prostate stromal cells), proliferation was
in both cases increased.13 In addition, interactions between stromal
and epithelial cells contribute to the regulation of proliferation and
differentiation of corneal epithelial cells.14
IL-­6 is a well-­known biomarker not only in prostatic inflamma-
tion but also in other types of inflammation.33,34 IL-­6 is expressed
KIM et al. |
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F I G U R E   5   Involvement of IL-­6 in
EMT induction in the proliferating BPH
epithelial cell. EMT induction in BPH
epithelial cells treated with W-­MTCM
(A and B). Inhibition of IL-­6 receptor
and JAK signalling reduced EMT (C and
D). Mesenchymal markers (vimentin,
N-­cadherin, fibronectin) increased and
epithelial marker (E-­cadherin) decreased in
the proliferating BPH epithelial cells, and
this was prevented by pretreatment with
IL-­6R antibody or ruxolitinib

in both stromal and epithelial cells in BPH, and is produced in re-
sponse to infections of prostate epithelial cells by pathogens such as
35-37
Propionibacterium, Mycoplasma and T. vaginalis. Recently, Tv in-
fection of prostate epithelial cells was suggested to trigger prostate
carcinogenesis by inducing IL-­6.3 Also, IL-­6 induced EMT in normal
prostate and BPH epithelial cells. 20,38,39 Upon IL-­6 binding, the IL-­
6R/gp130 dimer induces phosphorylation of JAK1,3, which in turn
phosphorylates STAT.40 In this study, expression of IL-­6 was found
to increase in proliferating BPH epithelial cells, so IL-­6 may play a
critical role in the proliferation of these cells.
EMT may be classified into 3 subtypes. Type 1 EMT involves
the transition of primordial epithelial cells into motile mesenchy-
mal cells and is associated with the generation of diverse cell types
during embryonic development and organogenesis.41 Type 3 EMT
42
occurs in carcinoma cells. Type 2 EMT involves the transition of
secondary epithelial cells to resident tissue fibroblasts and is associ-
ated with wound healing, tissue regeneration, and organ fibrosis.43
Also, type 2 EMT is induced in response to inflammation, especially
during wound healing and tissue regeneration, but stops once in-
flammation is reduced.43 McNeal has suggested that the initial le-
sion in BPH is not in the stroma but is due to abnormal growth of
the epithelium caused by EMT.44 In our previous studies, EMT of
normal prostate epithelial cells was found to be induced by Tv in-
fection, and IL-­6 signalling via NF-­kB and JAK/STAT3 was shown to
be involved in the EMT. 20,45 In the present study, IL-­6 signalling was
implicated in EMT as well as the proliferation of BPH epithelial cells.
Thus, our results indicate that the EMT may be classified as type 2,
which suggests that a healing process occurs in inflamed prostate
epithelial cells. These results hint that the EMT is involved in the
abnormal growth in BPH, but this idea needs further investigation.
Cyclin D1, bcl-­2 and FGF2 have been reported to link to BPH
or prostate cancer. 22,46-48 Also, IL-­6/STAT3 signalling has been
involved in expression of each factor in pancreatic ductal ade-
nocarcinoma, autophagy and melanocyte proliferation, respec-
tively.49-51 In this study, increased expression of these factors was
identified, and blocking of IL-­6 signalling was reduced levels of the
factors. Therefore, it is indicated that cyclin D1, bcl-­2 and FGF2
were involved in the proliferation of BPH epithelial cells through
IL-­6 signalling.
In conclusion, IL-­6 released from proliferating prostate stromal
cells induced by BPH epithelial cells infected with Tv in turn in-
duces multiplication of the BPH epithelial cells. This result for the
first time provides evidence that the inflammatory microenviron-
ment of prostate stromal cells resulting from Tv infection induces
the proliferation of prostate epithelial cells by stromal-­e pithelial
interaction (Supplementary Figure 1).
AC K N OW L E D G E M E N T S
This research was supported by Basic Science Program through
the National Research Foundation of Korea (NRF) funded by the
Ministry of Science and ICT (NRF-­2017R1A2B4002072).
C O N FL I C T O F I N T E R E S T
None.
ORCID
J.-S. Ryu  http://orcid.org/0000-0001-9145-7900
REFERENCES
1. Abdolrasouli A, Amin A, Baharsefat M, Roushan A, Hemmati
Y. Moraxella catarrhalis associated with acute urethritis imitat-
ing gonorrhoea acquired by oral-­genital contact. Int J STD AIDS.
2007;18:579‐580.
2. World Health Organization. Global Incidence and Prevalence of
Selected Curable Sexually Transmitted Infections – 2008. Geneva,
Switzerland: WHO; 2012.
3. Sutcliffe S, Neace C, Magnuson NS, Reeves R, Alderete JF.
Trichomonosis, a common curable STI, and prostate carcinogenesis-­a
proposed molecular mechanism. PLoS Pathog. 2012;8:e1002801.
4. Lee JJ, Moon HS, Lee TY, Hwang HS, Ahn MH, Ryu JS. PCR for diag-
nosis of male Trichomonas vaginalis infection with chronic prostatitis
and urethritis. Korean J Parasitol. 2012;50:157‐159.
 |
8 of 9       KIM et al.
5. Mitteregger D, Aberle SW, Makristathis A, et al. High detection rate
of Trichomonas vaginalis in benign hyperplastic prostatic tissue. Med
Microbiol Immunol. 2012;201:113‐116.
6. Gardner WA Jr, Culberson DE, Bennett BD. Trichomonas vaginalis in
the prostate gland. Arch Pathol Lab Med. 1986;110:430‐432.
7. De Marzo AM, Platz EA, Sutcliffe S, et al. Inflammation in prostate
carcinogenesis. Nat Rev Cancer. 2007;7:256‐269.
8. Penna G, Fibbi B, Amuchastegui S, et al. Human benign prostatic hy-
perplasia stromal cells as inducers and targets of chronic immuno-­
mediated inflammation. J Immunol. 2009;182:4056‐4064.
9. St Sauver JL, Jacobsen SJ. Inflammatory mechanisms associated
with prostatic inflammation and lower urinary tract symptoms. Curr
Prostate Rep. 2008;6:67‐73.
10. Orsted DD, Bojesen SE. The link between benign prostatic hyper-
plasia and prostate cancer. Nat Rev Urol. 2013;10:49‐54.
11. Begley LA, Kasina S, MacDonald J, Macoska JA. The inflammatory
microenvironment of the aging prostate facilitates cellular prolifer-
ation and hypertrophy. Cytokine. 2008;43:194‐199.
12. Kogan-Sakin I, Cohen M, Paland N, et  al. Prostate stromal cells
produce CXCL-­1, CXCL-­2, CXCL-­3 and IL-­8 in response to epithelia-­
secreted IL-­1. Carcinogenesis. 2009;30:698‐705.
13. Siejka A, Schally AV, Barabutis N. The effect of LHRH antagonist ce-
trorelix in crossover conditioned media from epithelial (BPH-­1) and
stromal (WPMY-­1) prostate cells. Horm Metab Res. 2014;46:21‐26.
14. Kobayashi T, Shiraishi A, Hara Y, et al. Stromal-­epithelial interaction
study: the effect of corneal epithelial cells on growth factor expres-
sion in stromal cells using organotypic culture model. Exp Eye Res.
2015;135:109‐117.
15. Nguyen DP, Li J, Tewari AK. Inflammation and prostate cancer: the
role of interleukin 6 (IL-­6). BJU Int. 2014;113:986‐992.
16. Neurath MF, Finotto S. IL-­6 signaling in autoimmunity, chronic in-
flammation and inflammation-­associated cancer. Cytokine Growth
Factor Rev. 2011;22:83‐89.
17. Heinrich PC, Behrmann I, Muller-Newen G, Schaper F, Graeve L.
Interleukin-­6-­t ype cytokine signalling through the gp130/Jak/STAT
pathway. Biochem J. 1998;334(Pt2):297‐314.
18. Engelhardt PF, Seklehner S, Brustmann H, Lusuardi L, Riedl CR.
Immunohistochemical expression of interleukin-­2 receptor and in-
terleukin-­6 in patients with prostate cancer and benign prostatic
hyperplasia: association with asymptomatic inflammatory prostati-
tis NIH category IV. Scand J Urol. 2015;49:120‐126.
19. Siegsmund MJ, Yamazaki H, Pastan I. Interleukin 6 receptor mRNA
in prostate carcinomas and benign prostate hyperplasia. J Urol.
1994;151:1396‐1399.
20. Han IH, Kim JH, Kim SS, Ahn MH, Ryu JS. Signalling pathways asso-
ciated with IL-­6 production and epithelial-­mesenchymal transition
induction in prostate epithelial cells stimulated with Trichomonas
vaginalis. Parasite Immunol. 2016;38:678‐687.
21. Kim JH, Kim SS, Han IH, Sim S, Ahn MH, Ryu JS. Proliferation of
prostate stromal cell induced by benign prostatic hyperplasia epi-
thelial cell stimulated with Trichomonas vaginalis via crosstalk with
mast cell. Prostate. 2016;76:1431‐1444.
22. Giri D, Ropiquet F, Ittmann M. Alterations in expression of basic
fibroblast growth factor (FGF) 2 and its receptor FGFR-­1 in human
prostate cancer. Clin Cancer Res. 1999;5:1063‐1071.
23. Alao JP. The regulation of cyclin D1 degradation: roles in cancer de-
velopment and the potential for therapeutic invention. Mol Cancer.
2007;6:24.
24. Elmore S. Apoptosis: a review of programmed cell death. Toxicol
Pathol. 2007;35:495‐516.
25. Wang SW, Sun YM. The IL-­6/JAK/STAT3 pathway: potential thera-
peutic strategies in treating colorectal cancer (Review). Int J Oncol.
2014;44:1032‐1040.
26. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-­
mesenchymal transition. Nat Rev Mol Cell Biol. 2014;15:178‐196.
27. Bianchi-Frias D, Vakar-Lopez F, Coleman IM, et  al. The effects of
aging on the molecular and cellular composition of the prostate mi-
croenvironment. PLoS ONE. 2010;5:e12501.
28. Kramer G, Mitteregger D, Marberger M. Is benign prostatic hy-
perplasia (BPH) an immune inflammatory disease? Eur Urol.
2007;51:1202‐1216.
29. Cunha GR, Hayward SW, Wang YZ, Ricke WA. Role of the stromal
microenvironment in carcinogenesis of the prostate. Int J Cancer.
2003;107:1‐10.
3 0. Schauer IG, Rowley DR. The functional role of reactive stroma in
benign prostatic hyperplasia. Differentiation. 2011;82:200‐210.
31. Twu O, Dessi D, Vu A, et al. Trichomonas vaginalis homolog of mac-
rophage migration inhibitory factor induces prostate cell growth,
invasiveness, and inflammatory responses. Proc Natl Acad Sci USA.
2014;111:8179‐8184.
32. Flickinger CJ. The fine structure of the interstitial tissue of the rat
prostate. Am J Anat. 1972;134:107‐125.
33. Bardan R, Dumache R, Dema A, Cumpanas A, Bucuras V. The role
of prostatic inflammation biomarkers in the diagnosis of prostate
diseases. Clin Biochem. 2014;47:909‐915.
3 4. Tanaka T, Narazaki M, Kishimoto T. IL-­6 in inflammation, immunity,
and disease. Cold Spring Harb Perspect Biol. 2014;6:a016295.
35. Hobisch A, Rogatsch H, Hittmair A, et al. Immunohistochemical lo-
calization of interleukin-­6 and its receptor in benign, premalignant
and malignant prostate tissue. J Pathol. 2000;191:239‐244.
36. Drott JB, Alexeyev O, Bergstrom P, et al. Propionibacterium
acnes infection induces upregulation of inflammatory genes and
cytokine secretion in prostate epithelial cells. BMC Microbiol.
2010;10:126.
37. Kim SS, Kim JH, Han IH, Ahn MH, Ryu JS. Inflammatory responses
in a benign prostatic hyperplasia epithelial cell Line (BPH-­1) infected
with Trichomonas vaginalis. Korean J Parasitol. 2016;54:123‐132.
38. Xu H, Chen Y, Chen Q, et al. DNMT1 regulates IL-­6-­ and TGF-­beta1-­
induced epithelial mesenchymal transition in prostate epithelial
cells. Eur J Histochem. 2017;61:2775.
39. Rojas A, Liu G, Coleman I, et al. IL-­6 promotes prostate tumorigen-
esis and progression through autocrine cross-­activation of IGF-­IR.
Oncogene. 2011;30:2345‐2355.
4 0. Yang XY, Wang LH, Farrar WL. A role for PPARgamma in the
regulation of cytokines in immune cells and cancer. Ppar Res.
2008;2008:961753.
41. Kalluri R, Weinberg RA. The basics of epithelial-­mesenchymal tran-
sition. J Clin Invest. 2009;119:1420‐1428.
42. Zeisberg M, Neilson EG. Biomarkers for epithelial-­mesenchymal
transitions. J Clin Invest. 2009;119:1429‐1437.
43. Lee K, Nelson CM. New insights into the regulation of epithelial-­
mesenchymal transition and tissue fibrosis. Int Rev Cell Mol Biol.
2012;294:171‐221.
4 4. McNeal JE. Origin and evolution of benign prostatic enlargement.
Invest Urol. 1978;15:340‐345.
45. Kim JH, Han IH, Kim SS, et  al. Interaction between Trichomonas
vaginalis and the prostate epithelium. Korean J Parasitol.
2017;55:213‐218.
46. MacManus CF, Pettigrew J, Seaton A, et  al. Interleukin-­8 signal-
ing promotes translational regulation of cyclin D in androgen-­
independent prostate cancer cells. Mol Cancer Res. 2007;5:737‐748.
47. Chung KS, Cheon SY, An HJ. Effects of resveratrol on benign pros-
tatic hyperplasia by the regulation of inflammatory and apoptotic
proteins. J Nat Prod. 2015;78:689‐694.
48. Ropiquet F, Giri D, Lamb DJ, Ittmann M. FGF7 and FGF2 are in-
creased in benign prostatic hyperplasia and are associated with in-
creased proliferation. J Urol. 1999;162:595‐599.
49. Hu B, Zhang KD, Li SB, et al. HIC1 attenuates invasion and metas-
tasis by inhibiting the IL-­6/STAT3 signalling pathway in human pan-
creatic cancer. Cancer Lett. 2016;376:387‐398.
KIM et al. |
      9 of 9

50. Qin B, Zhou Z, He J, Yan C, Ding S. IL-­6 inhibits starvation-­

induced autophagy via the STAT3/Bcl-­2 signaling pathway. Sci Rep.
2015;5:15701.
51. Dong L, Li Y, Cao J, et  al. FGF2 regulates melanocytes viability
through the STAT3-­transactivated PAX3 transcription. Cell Death
Differ. 2012;19:616‐622.
How to cite this article: Kim J-H, Han I-H, Kim Y-S, Noh C-S,
Ryu J-S. Proliferation of prostate epithelia induced by IL-­6
from stroma reacted with Trichomonas vaginalis. Parasite
Immunol. 2018;40:e12531. https://doi.org/10.1111/
pim.12531

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