Effects of the cyclin-dependent kinase 10 (CDK10) on the tamoxifen sensitivity of keloid samples.
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.
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ABSTRACT: The insect steroid hormone 20E-hydroxyecdysone (20E) regulates gene transcription via a genomic pathway by forming a transcription complex that binds to DNA with the help of the chaperone proteins Hsc70 and Hsp90. However, the non-genomic mechanisms by which 20E regulates gene expression remain unclear. In this study, we found that 20E regulated the phosphorylation of serine/threonine protein kinase cyclin-dependent kinase 10 (CDK10) through a non-genomic pathway to mediate gene transcription in the lepidopteran Helicoverpa armigera. The downregulation of CDK10 by RNA interference in larvae and the epidermal cell line delayed development and suppressed 20E-induced gene transcription. CDK10 was localized to the nucleus via its KKRR motif, and this nuclear localization and the ATPase motif were necessary for the efficient expression of 20E-inducible gene. The rapid phosphorylation of CDK10 was induced by 20E, whereas it was repressed by the inhibitors of G-protein-coupled receptors, phospholipase C, and Ca(2+) channels. Phosphorylated CDK10 exhibited increased interactions with heat shock proteins (Hsps) Hsc70 and Hsp90 and then promoted the interactions between Hsps and ecdysone receptor EcRB1 and the binding of Hsps-EcRB1 complex to the 20E response element for the regulation of gene transcription. CDK10 depletion suppressed the formation of the Hsps-EcRB1 complex at HR3 promoter. These results suggest that 20E induces CDK10 phosphorylation via a non-genomic pathway to regulate gene transcription in the nucleus.Endocrinology 02/2014; · 4.72 Impact Factor
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.
Molecules 2012, 17
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.
Molecules 2012, 17
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.
48 1216 20 30 50
Molecules 2012, 17
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.