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Effects of Botulinum Toxin Type A on Expression of Genes in Keloid Fibroblasts

Wang Xiaoxue, Chen Xi and Xiao Zhibo
Aesthetic Surgery Journal 2014 34: 154 originally published online 24 May 2013
DOI: 10.1177/1090820X13482938
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ERN
INT ATI

IBUTION
TR

AL CON
ON

Research

Effects of Botulinum Toxin Type A on

Expression of Genes in Keloid Fibroblasts

Aesthetic Surgery Journal

2014, Vol 34(1) 154159
2013 The American Society for
Aesthetic Plastic Surgery, Inc.
Reprints and permission:
http://www. sagepub.com/
journalsPermissions.nav
DOI: 10.1177/1090820X13482938
www.aestheticsurgeryjournal.com

Wang Xiaoxue, PhD, MD; Chen Xi, PhD, MD; and Xiao Zhibo, MD

Abstract
Background: Invasive growth of fibroblast cells, which is regulated by multiple biological factors, is the key event in the pathophysiology of keloid
scars. Recent studies have suggested that botulinum toxin type A (BoNT-A) could inhibit invasive growth of keloids. However, the molecular mechanisms
are unknown.
Objective: The authors explore the effect of BoNT-A on the expression of genes relevant to invasive growth in keloid fibroblasts.
Methods: With 112 genes that were relevant to invasive growth, the authors utilized microarray analysis to study messenger RNA expression profiles
in keloid fibroblasts treated with BoNT-A. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to confirm the microarray results.
Results: Analyses from microarray and qRT-PCR revealed that the S100A4 gene was upregulated and that the TGF-1, VEGF, MMP-1, and PDGFA genes
were downregulated in fibroblasts treated with BoNT-A.
Conclusions: The BoNT-A altered expression levels of S100A4, TGF-1, VEGF, MMP-1, and PDGFA genes in keloid fibroblasts provide a useful clue
for exploring the function of BoNT-A and finding a novel treatment for keloid scarring.
Keywords
botulinum toxin type A, keloid, gene expression, invasive growth, fibroblast, research
Accepted for publication July 26, 2012.

Keloids are benign skin tumors that may appear after

wound healing in genetically predisposed patients. They are
characterized by excessive proliferation of fibroblast cells
and an overabundance of collagen at the site of a healed
injury, and they can invade skin beyond the boundaries of
the original wound with no spontaneous regression.1 High
occurrence of keloids has been well documented in Asian
and African populations, but less is known of their prevalence in other ethnicities.2 Keloids can occur on various
parts of the body such as the face, upper extremities, chest,
presternal area, neck, back, lower extremities, and breasts.
Some studies have reported that keloid fibroblasts are
the result of abnormal gene expression due to sequence
mutations.3-5 For example, suppression of apoptotic genes
and the resulting proliferation of fibroblast cells might
contribute to keloid development. Several studies have
reported that the disruption or elimination of genetically
altered cells might decrease keloid potential,6-8 but no
guaranteed treatments have been established, and the
exact etiology of keloids is still unknown.9,10 It is reasonable to believe that the lack of effective therapy is due to
an insufficient understanding of keloid pathology.

Some studies have suggested a role for botulinum toxin

type A (BoNT-A) in the treatment of keloids.11-13 Growing
evidence suggests that BoNT-A influences cell apoptosis
and proliferation and therefore may play a role in the
expression of genes relevant to abnormal fibroblast proliferation.14-16 Despite its potential applications in keloid
therapy, limited information is available on the mechanism of BoNT-A in the treatment of keloids.17,18
Here, we utilize gene microarray and quantitative realtime polymerase chain reaction (qRT-PCR) to evaluate the
differential expression patterns of genes relevant to invasive growth of keloid fibroblasts in response to BoNT-A
From the Second Affiliated Hospital of the Harbin Medical
University, Harbin City, China. Dr Xiaoxue and Dr Xi contributed
equally to this article.
Corresponding Author:
Dr Xiao Zhibo, Department of Plastic Surgery, the Second Affiliated
Hospital of Harbin Medical University, Harbin City, P.R. China,
150086.
E-mail: xiaozhibodoctor@yahoo.com.cn

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Xiaoxue et al 155

exposure, which may potentially lead to the development

of effective therapeutic treatment for keloids. To our
knowledge, this is the first study to investigate a role for
BoNT-A in affecting gene expression significant to invasive
growth of keloid-causing fibroblasts.

instructions. A DNase I treatment was applied to remove

traces of contaminating DNA. RNA quality and quantity
were checked using an Agilent 2200 Bioanalyzer Platform
(Shengxiong Biocompany of China) for fibroblasts before
proceeding to the analyses.

Methods
Cell Cultures and BoNT-A Treatment

Microarray Processing and Data Extraction

To establish primary cell cultures, we obtained fresh tissue samples from the keloid lesions of 12 patients from
Northeast China. The patients were recruited according
to the random order in which they came to the hospital.
Fibroblasts were grown to confluence in Dulbeccos
modified Eagles medium containing 10% fetal bovine
serum (FBS), 10 g/mL streptomycin, and 50 IU/mL
penicillin. Cells were cultivated in 5% carbon dioxide at
37C. After the fourth culture passage, 3 experimental
(BoNT-A treatment) and 3 control (no BoNT-A treatment)
fibroblast groups were established. Experimental groups
were treated with each of 2 different concentrations (1
L/106 cells and 2.5 L/106 cells) of BoNT-A (Hengli;
Lanzhou Biocompany, Lanzhou City, China) for 24-, 48-,
or 72-hour periods to determine cell metabolic activity
for each time course and with each concentration of
BoNT-A. We were seeking the correct combination of
BoNT-A concentration and treatment time to induce a
reduction of approximately 50% in the number of viable
keloid fibroblasts compared with untreated (control)
keloid fibroblasts. The behavior of keloid fibroblasts over
each time course experiment was monitored by phase
contrast microscopy and high enlargement photographs.

Microarray analyses in this study included all genes that

have been found that are relevant to invasive growth of
cells. We used 112 such genes, all of which can be found
in Table 1.
For microarray processing, we used 1 g of extracted
RNA. To produce Cy3-labeled complementary RNA
(cRNA), we amplified the RNA samples and labeled them
using the Agilent Low RNA Input Fluorescent Linear
Amplification Kit (SuperArray Bioscience Corp, Frederick,
Maryland) following the manufacturers protocol. Yields
of cRNA and dye incorporation rate were measured with
an ND-1000 Spectrophotometer (SuperArray Bioscience
Corp). The hybridization procedure was carried out using
the Agilent Gene Expression Hybridization Kit (SuperArray
Bioscience Corp). Briefly, 1.65 g Cy3-labeled fragmented
cRNA in hybridization buffer was hybridized overnight to
the Whole Human Genome Oligo Microarray 4x44K
(SuperArray Bioscience Corp). Microarray hybridization
and washing were performed using reagents and instruments (hybridization chambers and rotating oven) as
indicated by the manufacturer (SuperArray Bioscience
Corp). Microarrays were scanned using the Microarray
Scanner System (SuperArray Bioscience Corp).

Microarray Data Analysis

Cell Viability Analysis

To assess cell metabolic activity for each of the 3 treatment
time courses and at each BoNT-A concentration, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide
(MTT)
assays were performed. After fibroblast BoNT-A incubation
for 24, 48, and 72 hours, respectively, 3 106 cells were
briefly seeded in each well of a 96-well plate containing 100
L RPMI-1640 medium supplemented with 10% FBS. Then,
50 L MTT (Shengxiong Biocompany, Shanghai, China;
5-mg/mL stock solution) was added and the cells were incubated at 37C for 4 hours. Next, 0.2 mL dimethylsulfide was
added to stop the reactions. The absorbance of each well was
determined spectrophotometrically at 570 nm by a microplate reader (Shengxiong Biocompany of China). All results
are the mean SD of at least 3 separate experiments, measuring each parameter in triplicate. Statistical significance was
assessed by the Student t test and was assigned at P < .05.

RNA Isolation
Total RNA from BoNT-A treatment and control fibroblasts was extracted with NucleoSpinRNAII (Shengxiong
Biocompany of China) according to the manufacturers

The CEL files generated by the microarray were converted

into DCP files using dChipV1.3 software (http://www.
dchip.org). We then normalized the DCP files and generated raw gene expression data by using model-based
analysis of the dChip system. For comparison of global
gene expression profiles between 2 sample sets, we used
Significance Analysis of Microarray software (http://www.
stat.stanford.edu/tibs/SAM/index.html). In this study, we
considered genes differentially expressed if we saw at least
a 2-fold change in the level of RNA transcript expression
in 3 independent experiments with P < .05. Differences in
RNA transcript expression were calculated by dividing the
signal intensity values of genes from BoNT-Atreated
fibroblasts by those from control fibroblasts.

Quantitative Real-Time PCR

To validate the results from the microarray, we further
employed qRT-PCR to measure messenger RNA (mRNA)
abundance for genes that were found to be differently
expressed by microarray analysis. Quantitative RT-PCR was
performed using the Taq-Man miRNA assay system (Applied
Biosystems, Foster City, California). The PCR reaction for

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Aesthetic Surgery Journal 34(1)

Table 1. All 112 Genes Used for the Microarray

AP15
1

ARHC
2

BRSM1
3

CASP8
4

CASP9
5

CAV1
6

CD44
7

CDH1
8

COL4A2
9

CSF1
10

CSF1R
11

CST3
12

CTSB
13

CTSD
14

CTSL
15

DCC
16

EHM2
17

ELA2
18

ENPP2
19

ERBB2
20

ETS1
21

ETS2
22

ETV4
23

FES
24

FGF1
25

FGF2
26

FOS
27

HGF
28

HPSE
29

HRAS
30

ICAM5
31

IGF2
32

ITGA2
33

ITGA3
34

ITGA5
35

ITGA6
36

ITGB1
37

ITGB3
38

KAI1
39

KISS1
40

LAMB1
41

LAMC1
42

LIMK1
43

MAP2K4
44

MDM2
45

MGAT3
46

MGAT5
47

MGEA5
48

MICA
49

MMP1
50

MMP10
51

MMP11
52

MMP13
53

MMP14
54

MMP15
55

MMP16
56

MMP2
57

MMP3
58

MMP7
59

MMP8
60

MMP9
61

MTA1
62

MUC1
63

MYC
64

NCAM1
65

NGFB
66

NM23A
67

NME4
68

ODC1
69

PDGFA
70

PECAM1
71

PIK3C2B
72

PLAU
73

PLAUR
74

PTEN
75

PTGS2
76

RAC1
77

RAF1
78

S100A4
79

SERPINB2
80

SERPINB2
81

SERPINE1
82

SNCG
83

SPP1
84

SRC
85

TGFA
86

TGFB1
87

THBS1
88

THBS2
89

TIMP1
90

TIMP2
91

TIMP3
92

TMPRSS4
93

VEGF
94

VEGFC
95

VTN
96

PUC18
97

PUC18
98

PUC18
99

Blank
100

Blank
101

Blank
102

GAPD
103

GAPD
104

PPIA
105

PPIA
106

PPIA
107

PPIA
108

RPL13A
109

RPL13A
110

ACTB
111

ACTB
112

Arabic numbers under the name of each gene represent the position of the gene in the microarray.

each mRNAcomplementary DNA was run in quadruplicate. A negative control without template was included in
parallel to assess the specificity of the PCR reaction.
Polymerase chain reaction was carried out on an AB7900
system (Applied Biosystems) in a 20-L volume with the
following thermal cycling parameters: enzyme activation at
95C for 10 minutes, 40 cycles of denaturation at 95C for
15 seconds, and annealing/extension for 60 seconds. All
other conditions were set to the manufacturers values. Data
acquired from the PCR reactions were analyzed using
SDS2.3 software (Applied Biosystems). Comparisons were
described as log values of the ratio of mRNA expression in
BoNT-A treatment versus control fibroblasts.

Results
Treatment of Keloid Fibroblasts With
BoNT-A
Based on the MTT assay, treatment with BoNT-A 2.5
L/106 for 48 hours proved to be the ideal condition for a
50% reduction in the number of viable keloid fibroblasts

Figure 1. Viability of keloid fibroblast cells in time course

experiments as assessed by the MTT assay.

(Figure 1). These parameters ensured that a sufficient

number of viable fibroblasts could remain as a source of
mRNA for microarray analyses and also that comparisons

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Xiaoxue et al 157

Table 2. Significantly Altered Expression of 5 Genes With Botulinum

Toxin Type A Treatment
Gene Name
S100A4

Fold Change

Log2(Fold Change)

P Value

156.5500

3.96

.00457

TGF-1

0.00896

3.82

.00499

VEGF

0.02477

3.75

.00510

MMP-1

0.03959

2.97

.00567

PDGFA

0.04832

2.88

.00640

Figure 3. Messenger RNA (mRNA) expression of 5 genes

with and without BoNT-A treatment, as assessed by
quantitative real-time polymerase chain reaction.

level of S100A4 was upregulated 4.01-fold, and the expression levels of TGF-1, VEGF, MMP-1, and PDGFA were
downregulated 3.73-, 3.65-, 3.12-, and 2.68-fold, respectively (Figure 3).

Discussion
Figure 2. Gene expression profiles of (1) TGF-1, (2) VEGF,
(3) MMP-1, (4) PDGFA, and (5) S100A4 in (A) control
fibroblasts (without botulinum toxin type A [BoNT-A]
treatment) and (B) fibroblasts treated with BoNT-A, as
assessed by gene microarray. Differential signal intensities
correspond with brightness of spot.

of treated and untreated keloid fibroblasts yielded evident

morphological changes that strongly suggested alterations
in cell growth condition.

BoNT-A Microarray Data Analysis

Comparing the normalized signal intensities obtained for
the untreated versus treated fibroblasts and using a fold
change cutoff of 2 (P < .05), we found that the expression
of S100A4 genes was significantly higher in fibroblasts
treated with BoNT-A, whereas the expression of TGF-1,
VEGF, MMP-1, and PDGFA genes was significantly lower
(Figure 2). The gene sets with significantly altered expression are described in Table 2.

Validation of Microarray Data by qRT-PCR

Quantitative RT-PCR data indicated that transcriptional
levels of S100A4, TGF-1, VEGF, MMP-1, and PDGFA were
perfectly correlated to microarray results. The expression

Keloids are dermal tumors in which an overabundance of

extracellular matrix is produced during the wound-healing
process.11-13 They appear as raised, red, and inflexible scar
tissue that can be itchy and painful. The lesions expand
over the boundaries of the initial injury site through the
rapid proliferation of fibroblasts.19,20 Thus, keloids represent a derailment of the protective wound-healing process.
Invasive growth is one of the typical characteristics of
keloid lesions, which is why keloids frequently resist all
clinical therapies and often recur. This invasive growth
characteristic also means that the quality of life of many
patients is seriously compromised.21
Numerous keloid treatments are currently available,
including surgical excision, steroid injection, silicone gel
application, radiation and pressure therapy, and laser
treatment. Few of these, however, can achieve excellent
therapeutic results, perhaps due to the invasive growth
characteristics of keloid lesions. Thus, it is pertinent to
find an alternative method for treating keloids.
Many investigators are considering BoNT-A as an alternative to existing therapies.22,23 BoNT-A is a neurotoxin that
is thought to block the release of neurotransmitters such as
acetylcholine at the neuromuscular junction as well as in
autonomic neurons.24 However, recent findings have still
not clearly identified the acetylcholine-blocking mechanisms for BoNT-A; some studies have shown roles for
BoNT-A in inducing changes in expression levels of genes
such as TGF-1 and CTGF,25,26 but these findings could not
be explained by a BoNT-A action mechanism involving the
blocking of neurotransmitters. Thus, investigators have
concluded that BoNT-A is not only a neurotoxin but also a

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Aesthetic Surgery Journal 34(1)

multifunctional toxin and that it may participate in the

regulation of genes. Our results confirm that BoNT-A could
regulate the expression of some genes.
Prior to this study, the authors22 had already completed
a prospective clinical study to evaluate the effects of
BoNT-A as a therapeutic agent in the treatment of keloids,
in which 12 patients with 1 or more keloids were included.
Patients discontinued any prior keloid treatment at least 3
months before starting intralesional BoNT-A therapy.
Clinical evaluations regarding the number, size, and site of
the lesions were performed. Assessments of therapy success were performed on the basis of patient satisfaction,
photographic record, and observations by an independent
observer immediately posttreatment, at 1 and 3 months
posttreatment, and at 1 year posttreatment. Success was
graded on a 5-point scale as follows: no improvement,
poor (up to 25% improvement), fair (26%-50% improvement), good (51%-75% improvement), and excellent
(76%-100% improvement). The therapeutic outcome was
excellent in 3 patients, good in 5 patients, and fair in 4
patients. None of the patients exhibited failure of therapy.
From these trials, it was evident that BoNT-A is a promising treatment method for keloids. However, the greatest
obstacle to the widespread application of BoNT-A in controlling keloids remains the lack of understanding of its
molecular mechanisms. The present study attempts to
elucidate this by attributing a gene regulatory function to
BoNT-A.
Based on the results of this study and those of other
investigators, it is evident that BoNT-A may be an effective
and safe treatment for keloids. However, the mechanism
of action is still not entirely clear. Several factors could
contribute to clinical improvement: first, BoNT-A prevents
contraction of muscle and skin near keloid tissue, which
decreases tensile force during the course of traumatic cicatrization. Second, some research has shown that BoNT-A
can suppress the secretomotor function and trophic effects
of the cell.27 Finally, BoNT-A influences cellular apoptosis
and cellular proliferation, which are involved in maintaining a balance in cellular dynamics.28,29
We found that the S100A4 gene was highly expressed in
keloid fibroblasts. S100 proteins belong to the superfamily
of EF-hand calcium-binding proteins and have multifunctional roles in various cellular processes, including cell
growth and differentiation, cell cycle regulation, apoptosis, transcription, and cell surface receptor activities.30 To
date, 23 different S100 proteins have been identified, all of
which are characterized by high homology, low molecular
weight, 2 calcium-binding EF-hands, and tissue-specific
expression.30 S100A4 has been implicated in several
inflammatory skin conditions such as psoriasis, atopy, and
squamous cell carcinoma.31-35 S100A4 genes are constitutively expressed by neutrophils, activated monocytes, and
macrophages, and they exert their role as the chemotactic
factor for leukocyte recruitment.36,37 Apart from its expression in immune cells, including neutrophils and monocytes, S100A4 expression is also increased in cells in some
patients with chronic inflammation.38

Our findings strengthen the notion that BoNT-A treatment can significantly affect the pathogenesis of keloids,
particularly the invasive growth of keloid fibroblast cells,
by influencing the regulation of some genes involved in
cell invasion. A limitation of our study was our inability to
conclusively identify the underlying cause of the different
expression patterns of certain genes. These patterns are
often influenced by sophisticated molecular mechanisms.
Therefore, more work is needed to further explore the
molecular networks affected by BoNT-A.

Conclusions
Through gene microarray and qRT-PCR, we confirmed
modulation of expression in 5 genes (S100A4, TGF-1,
VEGF, MMP-1, and PDGFA) in keloid-causing fibroblast
cells treated with BoNT-A. These findings provide an
explanation for the possible mechanism of action behind
BoNT-As keloid prevention and further explain the effects
of BoNT-A on keloid fibroblasts.

Disclosures
The authors declared no potential conflicts of interest with
respect to the research, authorship, and publication of this
article.

Funding
The article was supported by unrestricted grants from the
National Natural Science Foundation of China (8100063) and
the Wuliande Foundation of Harbin Medical University (WLDQN1114), which funded manuscript writing and research.

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