Chinese Medical Journal 2011;124(8):1221-1228 1221

Original article
Berberine potentizes apoptosis induced by X-rays irradiation
probably through modulation of gap junctions
LIU Bing, WANG Qin, YUAN Dong-dong, HONG Xiao-ting and TAO Liang

Keywords: berberine; gap junctions; X-rays irradiation; apoptosis

Background Clinical combination of some traditional Chinese medical herbs, including berberine, with irradiation is
demonstrated to improve efficacy of tumor radiotherapy, yet the mechanisms for such effect remain largely unknown. The
present study investigated the effect of berberine on apoptosis induced by X-rays irradiation and the relation between this
effect and gap junction intercellular communication (GJIC).
Methods The role of gap junctions in the modulation of X-rays irradiation-induced apoptosis was explored by
manipulation of connexin (Cx) expression, and gap junction function, using oleamide, a GJIC inhibitor, and berberine.
Results In transfected HeLa cells, Cx32 expression increased apoptosis induced by X-rays irradiation, while inhibition
of gap junction by oleamide reduced the irradiation responses, indicating the dependence of X-rays irradiation-induced
apoptosis on GJIC. Berberine, at the concentrations without cytotoxicity, enhanced apoptosis induced by irradiation only
in the presence of functional gap junctions.
Conclusions These results suggest that berberine potentizes cell apoptosis induced by X-rays irradiation, probably
through enhancement of gap junction activity.
Chin Med J 2011;124(8):1221-1228

R adiotherapy has been used to treat cancer for more

than 100 years. However, the development of cancer
radioresistence and related side effects encountered
during conventional radiotherapy becomes a major
problem in clinical cancer radiotherapy. Majority of
cancer cells have been shown to acquire radioresistance
through escaping the induction of apoptotic death by
irradiation.1,2 Many approaches that improve the efficacy
of radiotherapy by preventing and/or reversing the
radioresistance have been developed, including the
combination of radiotherapy with some plant-derived
agents.3 For example, some compounds such as curcumin,
ellagic acid and resveratrol were reported to enhance the
sensitivity of tumor cells to irradiation through
modification of apoptotic pathways in different type of
tumors.4,5
Berberine, a clinically important natural isoquinoline
alkaloid derived from a plant used traditionally in
Chinese medicine has been reported to exhibit
chemopreventive and anti-inflammatory activities and is
widely used in treatments of many diseases, e.g. diabetes6
and cancer.7 Recently, berberine was reported to be used
as an adjuvant to improve radiotherapy against lung
cancer.8 However, the mechanism of such effect is largely
unknown.
Gap junctions connect the cytoplasms of adjacent cells
through the end-to-end docking of single-membrane
structures called connexons, formed by a ring of six
connexin (Cx) monomers9 and mediate direct intercellular
movement of cytoplasmic signaling molecules. Gap
junction intercellular communication (GJIC) is essential
for many physiological events, including cell
synchronization, differentiation, cell growth, and
metabolic coordination of avascular organs.10 Many
evidences have also shown the important role of GJIC in
the carcinogenic process.11
Our previous works demonstrate the GJIC-dependent
enhancements of cytotoxicity of cisplatin and
etoposide.12,13 A simple inference from these reports is
that induction of apoptotic or necrotic processes in one
cell can cause a molecular “death signal” to be
transmitted to neighboring cells via gap junctions. It has
been established that gap junctions increase the
sensitivity of prostate cancer cells to tumor necrosis
factor-α-induced apoptosis.14 Such a “bystander” effect
has also been observed for ionizing radiation, in which
cells not irradiated but coupled to irradiated cells become
damaged or die.15,16 In light of this, the reports of a gap
DOI: 10.3760/cma.j.issn.0366-6999.2011.08.019
Department of Pharmacology, Zhongshan School of Medicine, Sun
Yat-sen University, Guangzhou, Guangdong 510080, China (Liu B,
Wang Q, Yuan DD, Hong XT and Tao L)
Correspondence to: Dr. TAO Liang, Department of Pharmacology,
Zhongshan School of Medicine, Sun Yat-sen University,
Guangzhou, Guangdong 510080, China (Tel and Fax:
86-20-87332318. Email: taol@mail.sysu.edu.cn)
This work was supported by grants from the National Natural
Science Foundation of China (No. 30973434 and No. 30772577),
the Grant for Development of Science and Technology from
Department of Science and Technology of Guangzhou, China (No.
2008Z1-E241), and the Grant for Development of Important New
Drugs from Ministry of Health of China (No. 2009ZX09303-007).
Drs. LIU Bing and WANG Qin contributed equally to this paper.
Conflicts of interest: none.
1222
junction dependent contribution to cisplatin or radiation
toxicity suggest that increase of the intercellular spread of
the “death signal,” by enhancement of gap junction
formation or function which decreases commonly in
tumorigenic conversion and malignant progression,
would potentize the cytotoxic actions.
In the present study, we aimed to investigate if in stable
transfected HeLa cells, a cell line of cervical cancer that
irradiation-induced apoptosis is dependent on GJIC and
to determine the effects of berberine on the apoptosis
induced by X-rays irradiation.
METHODS
Materials
Berberine and oleamide were from Sigma (St. Louis, MO,
USA); G418, hygromycin and doxycycline were from
Calbiochem (San Diego, CA, USA).
Calcein-acetoxymethyl ester (calcein-AM) and CM-DiI
were from Molecular Probes (Carlsbad, CA, USA). Cell
culture reagents were obtained from Invitrogen (USA).
All other reagents were from Sigma unless stated
otherwise.
Cell lines and cell culture
The HeLa cell line expressing Cx32 under the control of a
bidirectional tetracycline-inducible promoter was
described previously.17 In this stably transfected cell line,
the Cx32 coding sequence is followed by a sequence
coding for a thrombincleavable COOH-terminal epitope
tag consisting of a hemagglutinin (HA) epitope followed
by a 6× (His-Asn) sequence. Connexin expression was
induced with 1 μg/ml doxycycline for 48 hours. Cells
were cultured at 37°C 5% CO 2 in Dulbecco’s modified
Eagle’s medium supplemented with 10% fetal bovine
serum, 100 μg/ml G418 sulfate, and 200 μg/ml
hygromycin B (all from Life Technologies; Rockville,
MD, USA).
Western blotting
Cx32 expression was evaluated on total cellular lysate by
mouse anti-HA clone HA-7 IgG (Zymed, USA) diluted
1:1000 in 5% (w/V) skimmed dry milk in TBST (0.01
mol/L phosphate buffered saline (PBS), pH 7.4, and
0.05% (V/V) Tween-20) and incubated overnight at 4°C.
The blot was then incubated with alkaline phosphatase-
conjugated goat anti-mouse IgG as secondary antibody at
1:2000 dilution for 1 hour at room temperature.
Peroxidase reaction was executed by enhanced
chemiluminescence (ECL) detection kit (Amersham
International plc, UK). Equal loading of proteins was
confirmed by probing with β-tubulin diluted 1:10000 in
TBST/5% milk for 1 hour at room temperature and then
revealed with secondary antibody used at the same
conditions.
“Parachute” dye-coupling assay
The assay for gap junction function was performed
Chin Med J 2011;124(8):1221-1228
essentially as described before.17,18 Donor and receiver
cells were grown to confluence. The donor cells were
double-labeled with 5 μmol/L CM-DiI, a gap
junction-impermeable membrane dye, and 5 μmol/L
calcein-AM, which is converted into the gap
junction-permeable dye calcein intracellularly. The donor
cells were then trypsinized and seeded onto the receiver
cells at a 1:150 donor/receiver ratio. The donor cells were
allowed to attach to the receiver cells for 4 hours at 37°C
and then examined with a fluorescence microscope and
photographed.
X-rays irradiation treatment
The clinical 8-MV X-rays beam produced by a PRECISE
PRO linear accelerator (Elekta, Sweden) was used for the
irradiation treatment. The cell monolayer in the 25-cm2
flasks was always positioned at isocentre at the distance
of 100 cm from the focus. Irradiation dosimetry was
performed at an output rate of 140 MU/min and the dose
rate is 1.09 Gy/min. Since 8 MeV protons have a buildup
of approximate 2 cm, irradiation is done with a 1.5 cm
thick build-up layer of polystyrene to confirm a stable
dose distribution at the bottom surface of the flasks with
the medium depth of 0.5 cm. Oleamide, a direct inhibitor
of gap junction channels,19 was dissolved in DMSO at 7
mg/ml, diluted to final concentration of 7 μg/ml in culture
medium, and added to the cells to inhibit GJIC 4 hours
before X-rays irradiation treatment. Berberine was
dissolved in DMSO at 0.1 mmol/L, and the final
concentration in culture medium is 0.1 μmol/L. Before
X-rays irradiation exposure, cells were pretreated with
berberine for 4 hours.
Sulforhodamine B (SRB) assay
The toxic effect of berberine on cell line was measured
using the SRB colorimetric assay.20 Briefly, cells were
grown in 96-well plates for 48 hours. Culture medium
was aspirated, and the cells were fixed with 1 ml of 10%
cold TCA/ well at 4°C for 30 minutes, washed five times
with deionized water and dried at room temperature
overnight. 1 ml of 0.4% SRB in 1% acetic acid solution
was added to each well and the plate was dried again at
room temperature, then SRB was removed and the plate
was washed five times with 1% acetic acid. SRB bound
to the cells was solubilized with 150 μl Tris-buffer (10
μmol/L, pH 10.5) per well and the plate was left on a
plate shaker for 10 minutes. Absorbance was quantitated
using a 96-well plate reader, at a wavelength of 570 nm.
Hoechst 33258 staining
Hoechst 33258 staining was performed as described
previously with a few modifications.21 Briefly,
immediately after exposure to X-rays irradiation,
confluent cells were diluted to 8×104/ml and incubated
for 48 hours at 37°C in 6-well plates. Then these cells
were fixed with 4% paraformaldehyde for 30 minutes at
room temperature and washed once with PBS. Fixed cells
were stained with Hoechst 33258 of 50 ng/ml and
incubated for 30 minutes at room temperature and washed
Chinese Medical Journal 2011;124(8):1221-1228
with PBS. Apoptotic cells were identified by
condensation and fragmentation of nuclei examined by
fluorescence microscopy. The apoptotic rate of cell
population was calculated as the ratio of apoptotic cells to
total cells counted ×100. A minimum of 500 cells were
counted for each treatment.
Flow cytometry
After exposure to X-rays irradiation, confluent cells were
diluted to 8×104/ml and incubated for 24 hours at 37°C in
6-well plates. The samples were collected, washed in cold
PBS, and resuspended in 100 μl 1 ×binding buffer with
2×105 cells containing 5 μl of Annexin V-FITC
(Pharmingen, San Diego, CA, USA) and 5 μl of PI
(Pharmingen). Samples were mixed gently and incubated
at room temperature in the dark for 20 minutes. A
minimum of 10 000 cells within the gated region were
analyzed. Four hundred μl of 1 × binding buffer was
added to each sample tube, and the samples were
analyzed by FACS (Beckman Coulter, CA, USA).
Apoptotic rate is represented as the number of early
apoptotic cells (Annexin V+/PI–) and late apoptotic cells
(Annexin V+/PI+) divided by total cell number.
Statistical analysis
Data were statistically analyzed using unpaired Student’s
t test at a significance level of P <0.05 and presented as
means ± standard error (SE), unless otherwise indicated,
using Sigma Plot (Jandel Scientific, San Rafael, CA,
USA).
RESULTS
Apoptosis induced by X-rays irradiation is gap
junction dependent
The cells were 90% to 100% confluent at the time of
X-rays exposure and had substantial opportunity for
formation of gap junctions. The connexin expression
induced by doxycycline and gap junction function
revealed by dye coupling was shown in Figure 1,
respectively. Exposure of the transfected cells to 1 μg/ml
of doxycycline for 48 hours induced connexin expression.
The inhibitory effect of oleamide (7 μg/ml) on HeLa cells
expressing Cx32 was shown in Figure 1B.
Figure 2 shows the effects of doxycycline (1 μg/ml) and
X-rays irradiation (6 Gy) on apoptosis of wild type HeLa
cells, which were not transfected with the
Cx32-containing plasmid. Hoechst staining assay
indicated that doxycycline did not affect the cell
apoptosis. X-rays irradiation at its clinical dose increased
the apoptotic rate of cell population. Enhanced apoptosis
by irradiation was not affected by addition of doxycycline.
Flow cytometry assay also showed no influence of
doxycycline on cell apoptosis and irradiation response
(Figure 2). These results demonstrated that doxycycline
itself had no unexpected effects on HeLa cells or cellular
response to X-rays irradiation.
1223
Figure 1. Doxycycline (Dox) induction of Cx32 expression and
pharmacologic inhibition of junctional coupling. Western
blotting for Cx32 expression following 1 μg/ml doxycycline
treatment (A). Parachute assay of dye coupling through gap
junctions composed of Cx32 in HeLa cells treated with 7 μg/ml
oleamide (olea). The upper panel is the fluorescence image; the
lower panel overlays the corresponding phase-contrast images (B).`
Figure 3 illustrates the effect of gap junction on apoptosis
induced by X-ray irradiation in HeLa cells transfected
with Cx32 cDNA. As shown in Figure 3, exposure of
Cx32-expressing HeLa cells (doxycycline-induced) to
X-ray irradiation (6 Gy) significantly increased the
apoptosis rate compared to that of doxycycline-uninduced
cells (no connexin expressing), indicating that cells with
functional gap junctions were much more sensitive to
X-rays irradiation than the cells without gap junction.
Pretreatment of Cx32-expressing cells with oleamide at
the concentration verified to inhibit dye coupling among
these cells (Figure 1B) decreased apoptosis rate to
approximately 40% (Figure 3). There was no significant
difference in apoptosis rate between the oleamide-treated
and untreated doxycycline-uninduced HeLa cells (no
connexin expression; data not shown). Overall, these
results indicate the gap junction-dependent X-rays
irradiation induced apoptosis.
Effect of berberine on apoptosis induced by X-rays
irradiation
In HeLa cells expressing Cx32, we examined the effects
of berberine on apoptosis induced by X-rays irradiation.
Cells were confluent and treated with berberine for 4
hours, followed by exposure to X-ray irradiation (6 Gy).
The cytotoxic effect of berberine was firstly detected by
SRB assay. Figure 4 showed that berberine itself had no
cytotoxicity in HeLa cells at concentrations below 0.1
μmol/L.
Figure 5 indicated that berberine, over the range 0.001 to
0.100 μmol/L, enhanced X-rays irradiation-induced
apoptosis in HeLa cells expressing Cx32 in the
concentration-dependent way. Pretreatment of cells with
0.1 μmol/L of berberine increased the apoptosis rate by
approximately 57.3% compared to berberine-untreated
cells. Thus, these results suggest that berberine, without
1224 Chin Med J 2011;124(8):1221-1228

Figure 2. Effects of doxycycline (Dox) and X-rays irradiation on apoptosis of HeLa cells not transfected with the plasmid coding for
Cx32. The apoptosis of cells after doxycycline and irradiation treatment was shown in 2A by Hoechst staining assay and 2B panel by flow
cytometry assay. Arrow, apoptotic cells (Original magnification ×200). Bars are mean ± SE from four to five independent experiments.
Figure 3. Effects of gap junctions on X-rays irradiation-induced HeLa cell apoptosis. The apoptotic rate of non- Cx32-expressing cells
and Cx32-expressing cells treated with 6 Gy X-rays irradiation and the effect of inhibition of GJIC on apoptosis by oleamide (olea) was
assayed by Hoechst staining assay (3A) and flow cytometry assay (3B) (Original magnification ×200). Apoptotic cells (Arrow, original
magnification ×200). Bars are mean ± SE from five independent experiments.*Significantly different from control, †significantly different
from Cx32-expressing cells with irradiation group, P <0.05.

cytotoxicity itself, increases apoptosis induced by X-ray
irradiation in HeLa cells with gap junction.
Enhancement of X-ray irradiation-induced apoptosis
by berberine depends on GJIC
Figure 6A showed that 0.1 μmol/L of berberine enhanced
X-ray irradiation-induced apoptosis determined by
Hoechst staining assay in HeLa cells expressing Cx32
induced by doxycycline, but it had no effect on apoptosis
in doxycycline-uninduced cells which were without gap
junction. The enhancement effect of berberine on X-ray
irradiation-induced apoptosis was inhibited by
pretreatment of Cx32-expressing HeLa cells with
oleamide (right panel). Consistent with the results
obtained by Hoechst staining assay, flow cytometry assay
showed the enhancement effect of berberine on apoptosis
by X-ray irradiation only in the presence of functional
gap junctions (Figure 6B).
DISCUSSION
The present study demonstrates that there is a
GJIC-dependent component of the apoptosis induced by
X-rays irradiation. This component is absent when
preventing connexin expression, as well as inhibiting gap
junction function by use of the connexin channel inhibitor,
oleamide. The fact that tumor cell is widely accepted to
be linked with loss of GJIC and/or connexins11 and inhibi-
tion of GJIC is usually seen in ultrasoft X-rays radiation
at higher doses20 may partly contribute to development of
Chinese Medical Journal 2011;124(8):1221-1228
Figure 4. The effect of berberine on HeLa cells growth shown
by SRB assay. Bars are mean ± SE from four independent
experiments.*Significantly different from control, P <0.05.
radioresistence. For tumor cells with reduced GJIC,
development of drugs and methods that can recover or
increase GJIC provide a new and potent way to enhance
effect of radiotherapy on these tumors.
Specifically, we investigate the influence of berberine on
the apoptosis induced by X-rays irradiation in HeLa cells
expressing Cx32. At 0.01 to 0.1 μmol/L, much lower than
its effective concentration for antineoplastic action,
berberine significantly increases the apoptosis induced by
X-ray irradiation. The berberine-induced enhancement of
apoptosis caused by irradiation is seen only in cells with
functional gap junctions and not in either not expressing
connexin or where the gap junctions are
pharmacologically inhibited. This establishes a
GJIC-dependent mechanism for the effect of berberine on
irradiation-induced apoptosis. The present data lead to the
idea that berberine, at lower concentration than that
needed for anticancer effects, may improve efficacy of
radiotherapy for tumors with GJIC.
The present study makes clear that there is a
1225
Cx32-composed gap junction-dependent component of
X-ray irradiation toxicity, presumably mediated by gap
junction-mediated intercellular diffusion of toxic factors.
In addition to Cx32, it has been shown that increasing
cell-cell communication in Cx43 overexpressing tumor
cells enhances irradiation sensitivity.22 Such transfer
through gap junction channels has been widely reported
for several connexins.23 To date, there is no reported
study on the effect of berberine on GJIC mediated by
Cx32 or other connexin channels. To obtain information
about exact mechanism underlying the effect of berberine
on irradiation toxicity and precisely which tumors may be
affected by berberine, it is necessary to investigate the
effects of berberine on GJIC mediated by Cx32 and other
connexin channels, and in different tumor cell types.
Apoptosis is currently recognized as a major mode of
radiation-induced cell death.24,25 In all apoptosis
including that induced by X-rays irradiation,26 varying
proportions of cells undergoing apoptosis occur in
distinct stages of apoptotic process, from very early stage
continuously through to secondary necrosis.27,28 Though
intercellular coupling of dying cells by gap junctions
gradually decreases throughout the apoptotic process, the
early apoptotic cells are still well-coupled with adjacent
viable cells.29 Thus, gap junctions can induce the
synchronous cell death behavior of coupled cells. It is
suggested from our data that gap junctions-mediated
transmission of the “death signal” from apoptotic cells
initially damaged by irradiation to neighboring viable
cells contributes to clusters of coordinately apoptotic cells
and secondary expansion of cell death.
Some plant-derived agents have been shown the
synergistic tumor-killing effect when combination of
irradiation treatment. For example, genistein significantly
enhanced radiosensitivity by suppressing the activation of
the survival signals, Akt and p42/p44 by radiation in
human esophageal cancer cell line.30 Inhibition of nuclear
Figure 5. Effects of berberine on apoptosis
of Cx32-expressing cells by irradiation were
determined by Hoechst staining assay (A)
and flow cytometry assay (B) respectively.
Bars are mean ± SE from four-five
independent experiments. *Significantly
different from non berberine-treated group,
P <0.05.
1226 Chin Med J 2011;124(8):1221-1228

Figure 6. Effects of berberine (BBR) on X-rays irradiation-induced apoptosis. Hoechst staining assay showed the effect of berberine (0.1
μmol/L) on apoptosis by irradiation in both non Cx32-expressing cells and Cx32-expressing cells (left panel) and the effect of inhibition
of GJIC by oleamide (olea) on berberine-enhanced apoptosis by irradiation (right panel) (A). Bars are mean ± SE from six independent
experiments. *Significantly different from control, †significantly different from Cx32-expressing cells without berberine treatment group,
P <0.05. Flow cytometry assay showed the effect of GJIC on berberine-enhanced apoptosis by irradiation (B) as in A. Bars are mean ± SE
from six to eight independent experiments. *Significantly different Cx32-expressing cells with irradiation group, P <0.05.

factor-κB, COX-2, and 5-LOX by resveratrol sensitizes
HeLa and SiHa cells to ionizing radiation.29 But up to
now, there is no report concerning the role of GJIC in
alteration of radiosensitivity induced by plant-derived
agents.
Berberine has been demonstrated to induce apoptosis
through a mitochondria/caspases pathway in human
hepatoma cells,31 and in human promonocytic U937 cells
via activation of caspase-9 and caspase-332 in a
concentration range from 10 to 100 μmol/L. Many
unexpected side effects of berberine were identified at
such blood concentrations. Therefore, the present results
that berberine at 0.1 μmol/L, which is much lower than
that mentioned above, enhances the sensitivity of HeLa
cells to irradiation only in the presence of functional gap
junctions suggest a clinical significance that berberine
can improve the efficiency of radiotherapy through
enhancement of GJIC with less side effects.
A previous study has suggested the vital involvement of
p38 MAPK pathway and ROS generation in
Chinese Medical Journal 2011;124(8):1221-1228
berberine-enhanced irradiation responses.33 In the present
study, the cytotoxicity of X-rays irradiation was explored
on transfected HeLa cells divided into two groups: the not
induced group (no Cx32 expression) and the
doxycycline-induced group (Cx32 expression). Activation
of p38 MAPK pathway and induction of ROS generation
by berberine should occur in both groups. However, the
cytotoxicity of X-rays irradiation in Cx32 expression
group is greater than no Cx32 expression group.
Furthermore, inhibition of GJIC by oleamide could
reverse berberine-enhanced apoptosis by irradiation. Thus,
our results suggest that apart from other probable
pathways, there also exists a GJIC-dependent component
in berberine-enhanced apoptosis by irradiation.
The mechanism by which berberine enhances the activity
of gap junction in HeLa cells is not detected in this study.
It has been demonstrated that berberine increases the
COX2 expression through Runx2 and p38 MAPK
signaling pathway,34 which is correlated with induction of
Cx43 expression.35 Thus, upregulation of COX2
expression by berberine may also lead to increased level
of Cx32 expression and corresponding enhanced activity
of gap junctions.
The present work demonstrates that berberine enhances
apoptosis induced by irradiation only in the presence of
functional gap junctions, which provides a novel basis for
improvement of clinical cancer radiosensitivity.
Upregulation or maintenance of gap junction function by
use of some plant-derived agents may be an effective and
safe way to improve the potency of cancer radiotherapy .
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(Received October 22, 2010)
Edited by HAO Xiu-yuan