Molecules 2012, 17, 1307-1318; doi:10.3390/molecules17021307
Effects of the Cyclin-Dependent Kinase 10 (CDK10) on the
Tamoxifen Sensitivity of Keloid Samples
Ying Liu †, Zhibo Xiao †, Daping Yang *, Lihong Ren, Guofeng Liu and Lin Yang
Department of Plastic and Aesthetic, The Second Affiliated Hospital of Harbin Medical University,
Harbin 150086, China; E-Mails: firstname.lastname@example.org (Y.L.);
email@example.com (Z.X.); firstname.lastname@example.org (L.R.);
email@example.com (G.L.); firstname.lastname@example.org (L.Y.)
† These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +86-451-8629-7062; Fax: +86-451-8629-7062.
Received: 12 December 2011; in revised form: 14 January 2012 / Accepted: 17 January 2012 /
Published: 1 February 2012
Abstract: Cyclin-dependent kinase 10 (CDK10) is a cell cycle regulating protein kinase,
which has just been discriminated in recent years. In this paper, mRNA and protein
expression of CDK10 were first investigated by a comparative study between 23 human
keloid tissue samples and their adjacent normal skin. To further address its potential as a
therapeutic target in the treatment of keloid, a plasmid expressing the CDK10 gene was
transfected into keloid fibroblast. The effects on tamoxifen-induced apoptosis were then
investigated using Western blot assay and flow cytometry. Results showed that there is a
generally down-regulated expression of CDK10 in keloid compared to normal skin
samples. Transfection with the recombinant CDK10 plasmid significantly decreased the
viability of cells and increased the apoptosis rates. Tamoxifen sensitivity in keloid
fibroblasts was observed after treatment with the recombinant CDK10 plasmid. The results
suggested that CDK10 may play an important role in enhancement of tamoxifen efficiency,
and its expression may have a synergistic effect on keloid treatments.
Keywords: CDK10; keloid; apoptosis; tamoxifen
Molecules 2012, 17
Keloid can extend beyond the boundaries of the original wound and invade the normal surrounding
skin. The clinical appearance of keloid is a raised growth, usually accompanied by pruritus and pain.
Since the pathogenesis of keloid is still unknown, keloid healing remains impaired . Development
of keloid contains atypical fibroblasts and consists of overabundant extracellular matrix components
including collagen, fibronectin and certain proteoglycans . Treatment for keloid is problematic, with
no single modality producing uniformly satisfactory results .
Tamoxifen [1-(p-dimethylaminoethoxyphenyl)-1,2-diphenyl-1-butene], a selective estrogen receptor
(ER) modulator, has been widely used for the treatment and prevention of recurrence for patients with
hormone receptor (ER or progesterone receptor)-positive breast cancers in more than 120 countries
throughout the worldwide . Many studies have shown that the mode of action of tamoxifen is
connected with apoptosis. It was found that in vitro administration of tamoxifen induced a Bcl-2
up-regulation in breast cancer cells [5–7]. The same thing happened in human cholangiocarcinoma cell
line QBC939, where an up-regulation of Bcl-2 and a down-regulation of Bax has been found after
tamoxifen treatment . Tamoxifen is one of the most successful agents used in the management of
hormone receptor positive breast cancer. Recently, it has been suggested that tamoxifen might be a
novel option for the clinical modulation of wound healing [9–11]. Tamoxifen was originally thought to
inhibit cell growth by competitive binding to the estrogen receptor, but it has been shown to inhibit the
growth of some estrogen-negative breast cancer cell lines . The benign mesenchymal tumors
desmoids, which show low expression in estrogen receptors, have been treated successfully with
tamoxifen . It has also been indicated that tamoxifen decreases fibroblast function in Dupuytren’s
affected palmar fascia  and in retroperitoneal fibrosis . Furthermore, tamoxifen has been
approved to reduce proliferation of both keloid and normal dermal fibroblasts [15,16]. Payne 
stated that down-regulating causes of fibrosis with tamoxifen are a possible molecular approach to
treat rhinophyma. Evidence  suggests that there was a significant inhibition of keloid fibroblasts by
tamoxifen, and tamoxifen concentrations greater than 20 µM had a deadly effect on keloid cells, while
concentrations between 8 and 12 µM demonstrated significant inhibition of fibroblast cells (p < 0.01).
The mechanism of tamoxifen-decreased fibrosis is not entirely understood.
Cyclin-dependent kinases (CDKs), which belong to a large protein family, have 13 members that
have been found so far in human cells, including CDK10 . The function of CDKs10 was proven as
an important determinant of resistance to endocrine therapies (tamoxifen) for breast cancer .
CDK10 silences increases ETS2-driven transcription of c-RAF, resulting in MAPK pathway activation
and loss of tumor cell reliance upon estrogen signaling , but to the best of our knowledge, there are
still no literature reports on the roles of CDK10 in keloid pathogenesis.
In this study, first a comparative study of CDK10 mRNA and protein expression in 23 human
keloid and adjacent normal skin tissue samples by quantitative real-time PCR and Western blot assay
was undertaken. Then, whether CDK10 expression was relevant to tamoxifen sensitivity in keloid was
investigated by MTT, flow cytometry and Western blot assay. As far as we know, this is the first report
demonstrating the effects of CDK10 on keloid tamoxifen sensitivity.
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2. Results and Discussion
2.1. Expression of CDK10 in Keloid and Normal Skin Samples by Quantitative Real-time PCR and
Western Blot Assay
CDK10 mRNA expression of 23 keloid and normal skin samples was detected by real-time PCR
analysis. The mRNA level of CDK10 was noted to be differentially expressed in the keloid and normal
skin samples. As shown in Figure 1, CDK10 mRNA levels were significantly higher in the normal skin
samples (median 1.72, range 0.57 to 3.56) than in keloid (median 0.47, range 0.10 to 0.85).
Figure 1. CDK10 expression in keloid and normal skin samples (* means p-value of <0.01
compared with normal skin samples).
Then CDK10 protein expression of keloid and normal skin samples were checked by Western blot
assay (Figure 2). CDK10 protein was greatly decreased in keloid compared with normal skin samples.
This result confirmed the lower expression level of CDK10 in keloid samples.
Figure 2. The protein level of CDK10 in keliod and normal skin samples detected by
Western blot assay (1–4, normal skin samples; 5–8, keliod samples). The blots were
stripped and reprobed with anti-β-actin antibody to normalize the protein loading. Bands
were quantitated by densitometric analysis. Fold change represents the protein level of
keliod and samples to the first normal skin sample and the resulting protein levels were
then normalized to the β-actin protein.
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2.2. Results of Transfection
After 72 h transfection of CDK10 with the pCMV6-plasmid and control plasmids, the expression of
CDK10 can be detected by Western blot analysis. Western blot analysis for CDK10 revealed that there
was a remarkable increase in CDK10 protein expression in CDK10 transfected fibroblasts compared
with cells transfected with control plasmid and untransfected controls (Figure 3).
Figure 3. CDK10 protein expression increased notably after pCMV6-CDK10 transfection.
Cells were harvested 72 h after transfection; the relative density of bands was quantified by
densitometry. The transfected group of CDK10 protein (lane1) demonstrated a visible
increase relatively to the empty plasmid transfected (lane 2) or untreated (lane 3) keloid
2.3. Cytotoxicity Assays
The MTT method was used to measure the cell optical density of pCMV6-CDK10-transfected
fibroblast after treated with various concentrations of tamoxifen (4–50 µM) for 24 h, 48 h and 72 h,
respectively. Results showed that tamoxifen inhibited the growth of pCMV6-CDK10-transfected cells
in a time- and dose-dependent manner (Figure 4). The IC50 values of normal keloid fibroblast cells and
transfected keloid fibroblast cells were then compared in the following experiment. The IC50 values
significantly decreased in the pCMV6-CDK10-transfected cells compared to that of control
(p < 0.01) (Table 1). All these results showed that CDK10 transfected keloid cells were more sensitive
to tamoxifen treatment.
Figure 4. Effect of tamoxifen towards keloid fibroblast as determined by MTT assay.
4812 1620 3050
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Table 1. Inhibition concentrations 50% (IC50) values for tamoxifen towards keloid
fibroblast cells and pCMV6-CDK10-transfected cells determined by MTT assay.
The symbols * indicate significant differences (p < 0.01) with respect to control (keloid
pCMV6-CDK10-transfected cells 9.41 *
2.4. Keloid Fibroblast Cell Apoptosis as Detected by Annexin V-FITC/PI
Apoptosis plays an important role in keloid treatment. It is a highly regulated death process by
which cells undergo inducible non-necrotic cellular suicide . The Annexin V-FITC apoptosis
detection kit was employed to examine the influence of tamoxifen on keloid fibroblast apoptosis by
flow cytometry. As shown in Figure 5, only a small percentage of untreated keloid fibroblast (2.64%)
cells bound to annexin V-FITC. After treated with tamoxifen, the percentage of annexin V-FITC binding
keloid fibroblast cells increased to 12.76%. In contrast, when pCMV6-CDK10-transfected cells treated
with tamoxifen, the percentage of annexin V-FITC binding cells increased significantly to 80.18%
(p < 0.01). To sum up, dots were dispersed and shifted to the Q2 side when pCMV6-CDK10-keloid
fibroblast was treated with tamoxifen, indicating that the cells moved to the late apoptotic stage.
Figure 5. Tamoxifen-induced apoptosis in pCMV6-CDK10-keloid fibroblast using
annexinV-FITC/PI. (a) Keloid fibroblast treatment with 0 µM tamoxifen; (b) Keloid
fibroblast treatment with 8 µM tamoxifen; (c) pCMV6-CDK10-keloid fibroblast treatment
with 0 µM tamoxifen; (d) pCMV6-CDK10-keloid fibroblast treatment with 8 µM tamoxifen.
Molecules 2012, 17
2.5. Bax and Bcl-2 Expression of Keloid Fibroblast as Detected by Western Blot
A high Bax/Bcl-2 ratio was clearly correlated with increased apoptotic sensitivity to test reagents .
As shown in Figure 6, Western blot analysis revealed a significant increase in the expression of Bax in
tamoxifen treated pCMV6-CDK10-keloid fibroblast cells, while there was a significant decrease in
Bcl-2 expression, indicating that the Bax/Bcl-2 ratio increased significantly.
Figure 6. Tamoxifen-mediated up-regulation of Bax and down regulation of Bcl-2 by
Western blot assay. Keloid fibroblast cells or pCMV6-CDK10-keloid were treated with 8 µM
tamoxifen for 48 h, respectively. The blots were stripped and reprobed with anti-β-actin
antibody to normalize protein loading. Fold change was calculated as described in Figure 3.
Keloid scarring is a disease arising from dermal injury due to a number of aetiologic factors;
however, its pathogenesis and optimal clinical treatment remain poorly understood. Many treatments
have been used, including surgery, radiation, interferon, laser ablation, steroid injection, and pressure
therapy among many others, but few obtain good results and recurrence of keloid remains common [2,3].
Cdk10 is a Cdc2-related kinase, previously referred to as PISSLRE, that may play a role in
regulating the G2/M phase of the cell cycle . The CDK10 gene was located to chromosome 16q24
in humans. It was first mentioned in 1994  and was noticed to have a close relationship to many
cancers. Research showed that the expression of CDK10 was over-expression in lung adenocarcinoma 
follicular lymphoma  and seminomas , and low-expression in breast cancer patients who have
poorer prognosis after surgical procedure. Iorns et al.  have shown that CDK10 gene knockdown
reproducibly caused resistance to tamoxifen treatment, and these authors also showed that CDK10
could make tamoxifen more effective in treating breast cancer through C-RAF/MAPK kinase pathway
activity . Activation of the pathway components ERK1/2 and MEK1/2 was observed following
CDK10 silencing, while suppression of ERK pathway signaling at the same time as CDK10 inhibition
restored sensitivity to tamoxifen. So far, there is no research showing the expression of CDK10 in
keloid tissues. In this study, real-time PCR and Western blot were used to detect the level of CDK10
mRNA and protein level in 23 patients with keloid and adjacent normal skin samples. Results showed
us that the relative expressions of CDK10 mRNA and protein level are lower compared with normal
skin. Kasten and Giordano  noticed CDK10 has been shown to inhibit the transactivation capacity
Molecules 2012, 17
of ETS2, so CDK10 loss would be predicted to potentiate ETS2-mediated gene transcription.
By comparing our data with previously published work, we propose that CDK10 determines the
response to tamoxifen and other endocrine therapies by modulates intracellular signaling. CDK10
normally binds and represses the ETS2 transcription factor and has an ETS2-binding site in the c-RAF
promoter and by using ChIP it was demonstrated that both CDK10 and ETS2 bind to this site .
In the absence of CDK10 activity, c-RAF transcription is significantly unregulated due to relief of
ETS2 repression. This increase in c-RAF expression leads to activation of downstream components of
the MAPK pathway, including MEK1,2 and p42/p44 MAPK, which increase the expression of cyclin
D1 , resulting in tamoxifen resistance by circumventing the reliance upon estrogen signaling .
This evidence may indicate that in keloid the CDK10 may play a role by the same principle.
To investigate the effects of CDK10 on keloid fibroblast, we transfected keloid cells with pCMV-6
CDK10 plasmid to study how CDK10 over-expression affects the sensitivity to tamoxifen treatment,
or whether it can make the tamoxifen more effective in keloid cells. Our experimental results
demonstrate that pCMV6-CDK10-transfected cells treated with tamoxifen showed a significantly
increased apoptosis rate at both the early and late periods (p < 0.01) compared with the control group
treated with tamoxifen. This result revealed that expression of the CDK10 protein had a synergistic
effect on apoptosis in combination with tamoxifen treatment. Data obtained from flow cytometric
annexin V-FITC/PI staining showed that tamoxifen induced apoptosis in pCMV6-CDK10-keloid
fibroblast. Thus, we further evaluated the expression of the pro-apoptotic protein Bax and the
anti-apoptotic protein Bcl-2. These two proteins were crucial determinants of the apoptotic response
mediated by many agents. Bcl-2 family proteins play important roles in apoptosis regulation.
Anti-apoptotic (Bcl-2 and Bcl-xL, etc.) and pro-apoptotic (e.g., Bad, Bax and Bak) are two of the
major members in Bcl-2 family [31–33]. Anti-apoptotic Bcl-2 and Bcl-xL inhibit apoptosis by
sequestering proforms of capsases or by preventing the release of mitochondrial apoptogenic
factors [34,35], whereas Bad, Bax and Bak inhibit Bcl-2 activity and promote apoptosis . In this
study, tamoxifen treatments altered the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax)
proteins, resulting in pCMV6-CDK10-keloid fibroblast cell apoptosis. This result could explain the
lower IC50 values of tamoxifen-treated pCMV6-CDK10-keloid fibroblast. As such, these data
confirmed the synergistic affect of CDK10 expression in combination with tamoxifen treatment on
keloid apoptosis rates. Although the mechanism of CDK10 action is unclear, CDK10 can be taken as a
potential marker for sensitivity in prospective clinical trials of keloid patients treated with tamoxifen
therapies. Further studies are demanded to further discuss the mechanism of CDK10 and the
relationship with tamoxifen in keloid cells.
4. Experimental Section
4.1. Patients and Treatment
Keloid and normal skin tissue samples were obtained from 23 patients who underwent surgeries
from 2009 to 2011 at the Second Affiliated Hospital of Harbin Medical University. Informed consent
was obtained from each patient recruited, and the study was approved by the Hospital Ethics
Committee. Keloid cells were obtained at surgical release from six patients aged 18–37 years who had
Molecules 2012, 17
a non-peduncle keloid on the manitrunk, ear lobe and upper arm of at least 1-year evolution, with
clinical activity such as growth, hyperaemia, pruritus and pain. Primary cultures of fibroblasts from the
surgical specimens were then established. Cells from passages 3 to 8 were used for experiments.
Cell was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY,
USA). All cell lines were supplemented with 10% fetal bovine serum (FBS) and 5 mmol/L L-glutamine
in a 5% CO2 air incubator at 37 °C. Cells were transient transfected with pCMV6-CDK10 or a control
plasmid using GeneJuice® Transfection Reagent (Novagen) according to the manufacturer’s protocol.
4.2. RNA Extraction and Quantitative Real-time PCR
Total RNA was extracted from the tissue samples with the RNApure Tissue Kit (CWBIO, CW0584).
Quantitative real-time PCR (qPCR) was performed using the PrimeScript™ RT reagent Kit (Takara,
DRR037A). The qPCR reactions were carried out by SYBR green PCR master mix (Takara, DRR083M)
with an Multiplex Quantitative PCR System (Applied Bios stem), and β-actin was used as an internal
standard. Primers were designed for qPCR from Primer Express software (Applied Bios stems).
The primer sequences employed were: CDK10, forward: 5'-TGGACAAGGAGAAGGATG-3', reverse:
5'-CTGCTCACAGTAACCCATC-3'; β-actin, forward: 5'-AGAAGGAGATCACTGCCCTGGCACC-3'
reverse: 5'-CCTGCTTGCTGATCCACATCTGCTG-3'. The PCR cycling conditions were as follows:
10 min at 95 °C, 40 cycles of 30 s at 95 °C, 30 s at 54 °C, and 30 s at 72 °C; and finally 5 min at 72 °C.
Melting curve analysis was conducted to determine the specificity of the reaction. Probe experiments
showed that the efficiencies of amplification of the primers for the target and reference genes were
approximately equal. Each sample was tested in triplicate. A DNA dissociation curve was produced
to confirm the specificity of the amplification after the thermal cycling. Relative expression level
changes were calculated according to 2-⊿Ct(⊿Ct = Ct[CDK10] − Ct[β-actin]) method as described
4.3. Western Blot Analysis
Total protein extracts of the keloid and normal skin samples and cells were prepared by
homogenization in RIPA (Beyotime, P0013B). Briefly, for isolation of total protein fractions, cells or
tissue samples were collected, washed twice with ice-cold PBS, and lysed using cell lysis buffer
[20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA,
1% Na2CO3, 0.5 µg/mL leupeptin, 1 mM phenylmethanesulfonyl fluoride (PMSF)]. The lysates were
collected by scraping from the plates and then centrifuged at 12,000 rpm at 4 °C for 15 min.
Total protein samples (20 µg) were loaded on a 12% of SDS polyacrylamide gel for electrophoresis,
and transferred onto PVDF transfer membranes (Millipore, Billerica, MA, USA) at 0.8 mA/cm2 for
70 min. Membranes were blocked at room temperature for 2 h with blocking solution (1% BSA in
PBS plus 0.05% Tween-20). Membranes were incubated overnight at 4 °C with primary antibodies
(anti-â-actin and anti-Bax were mouse polyclonal antibodies; anti-Bcl-2 and anti-CDK10 were rabbit
polyclonal antibodies) at a 1:1,000 dilution (Biosynthesis Biotechnology Company, Beijing, China)
in blocking solution. After thrice washings in TBST for each 5 min, membranes were incubated for 1 h
at room temperature with an alkaline phosphatase peroxidase-conjugated anti-mouse secondary
antibody at a dilution of 1:500 in blocking solution. Detection was performed by the BCIP/NBT
Molecules 2012, 17
Alkaline Phosphatase Color Development Kit (Beyotime Institute of Biotechnology) according to the
manufacturer’s instructions. Bands were recorded with a digital camera (Nikon, Tokyo, Japan).
4.4. Transient Transfection
The pCMV6-CDK10 expression plasmid was acquired from Doctor Zhong Xiangyu (Department of
General Surgery, the Second Affiliated Hospital of Harbin Medical University, China). The plasmids
were sequenced from OriGene Company (Rockville, MD, USA). Plasmid DNA from Escherichia coli
cell lysates was extracted and purified using a PureLinkTM Hipure Plasmid DNA Purification Kit
(Invitrogen, Carlsbad, CA, USA). Keloid fibroblast was transfected with pCMV6-CDK10 using
GeneJuice® Transfection Reagent (Novagen) according to the manufacturer’s protocol. In brief, cells
were trypsinized and plated onto six-well plates. Then, transfection reagent was added and incubated at
room temperature for 5 min. The appropriate volume of plasmid DNA was then added and the cells
were incubated for an additional 15 min. The common complete medium was replaced by the
antibiotics and serum-free medium. Six hours after the transfection, the medium was replaced by the
common complete medium again. After 24, 48 and 72 h the transfection, the cells were then prepared
for Western blot analysis, MTT assays, or flow cytometry.
4.5. Cytotoxicity Assay
Inhibition of cell proliferation of tamoxifen (SIGMA) was measured by MTT assay . Briefly,
keloid fibroblast was plated in 96-well culture plates (1 × 105 cells/well) separately. After 24 h
incubation, normal keloid fibroblast cells was treated with tamoxifen (4, 8, 12, 16, 20, 30 and 50 µM,
eight wells per concentration) for 72 h. pCMV6-CDK10-keloid fibroblast cells was treated with
tamoxifen (4, 8, 12, 16, 20, 30 and 50 µM, eight wells per concentration) for 24, 48 h or 72 h.
MTT solution (5 mg/mL) was then added to each well. After 4 h incubation, the formazan precipitate
was dissolved in dimethyl sulfoxide (100 µL), and then the absorbance was measured in an ELISA
reader (Thermo Molecular Devices Co., Union City, NJ, USA) at 570 nm. The cell viability ratio was
calculated by the following formula: Inhibitory ratio (%) = [(ODcontrol − ODtreated)/(ODcontrol)] × 100%.
4.6. Flow Cytometric Analysis of Cell Apoptosis
The extent of apoptosis was measured through annexinV-FITC apoptosis detection kit (Beyotime
Institute of Biotechnology, Shanghai, China) as described by the manufacturer’s instructions. After
exposure to tamoxifen for 24 h, cells were collected, washed twice with PBS, gently resuspended in
annexin V binding buffer and incubated with annexin V-FITC/PI in dark for 15 min and analyzed by
flow cytometry using FloMax software. The fraction of cell population in different quadrants was
analyzed using quadrant statistics. The lower left quadrant contained intact cells; lower right quadrant
apoptotic and in the upper right quadrant necrotic or post-apoptotic cells.
4.7. Statistical Analysis
The data were expressed as mean ± S.D. All statistics were calculated using the STATISTICA
program (Stat Soft, Tulsa, OK, USA). A p-value of <0.01 was considered as significant.
Molecules 2012, 17
In the present study, a lower expression of CDK10 was found by a comparative study of mRNA and
protein expression in 23 human keloid and adjacent normal skin tissue samples by quantitative
real-time PCR and Western blot assay. To further determine the potential of CDK10 as a therapeutic
target for tamoxifen, CDK10 was over expressed in keloid fibroblast cells and the presence of
apoptosis was detected by flow cytometry and Western blot assay.
The apoptosis rate of the combination treatment (tamoxifen combined with pCMV6-CDK10)
increased when compared with pCMV6-CDK10 or tamoxifen alone. Further, the present study showed
that the increased expression of CDK10 significantly enhanced the effects of tamoxifen treatments on
Bax and Bcl2. The findings indicated that CDK10 is an attractive therapeutic target because of its
ability to suppress keloid fibroblast growth and enhance tamoxifen sensitivity in keloid.
This work was supported by the foundation of nature science fund of Heilongjiang province
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Sample Availability: Not available.
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