全 文 ：Chinese-German Journal of Clinical Oncology December 2007, Vol. 6, No. 6, P537–P541
DOI 10.1007/s10330-007-0105-5
Gap junction (GJ) is the essential channel of intercel-
lular exchange of information. Gap junction intercellu-
lar communication, which is mediated by gap junction,
plays an important role in regulation of cell growth and
cell differentiation, including maintenance of tissue ho-
meostasis mediating metabolic cooperation between cells
comprising tissues. The expression of connexin43 (Cx43),
which is one of the major gap junction proteins, decreases
in many tumor suppressor genes [1]. Epithelial cadherin
(E-cad) which pertains to classic CD subgroup depending
on ecto-cyte Ca2+, mediates cell-all adhesion of the same
affined type cell, and takes part in construction and main-
tenance of gap junction. It was proved that decreased ex-
pression or loss of E-cad and interruption gap junction
intercellular communication, existed in many tumors [2,
3]. So many scholars are keen on study the medicine that
can increase the expressions of Cx43 and E-cad, in order
to inhibit the growth, differentiation and metastasis of
tumor cell. Now, there are many researches on up-regu-
lating expression of Cx43 with cAMP [4], ratinoic acid [5]
and β-carotin. Researches on extractive of Chinese crude
drug, such as curcurnine, tea polyphenols have also been
reported [6], but research on compound recipe of Chinese
crude drug has seldom been reported. In this trail, the ef-
fects on regulating the growth of humor lung tumor cell
with high metastasis activity and the expression of E-cad
with FuFangTengLiGen (FFTLG) in vitro were observed,
and then the mechanism of action was approached.
Materials and methods
Chemical reagents and medicines
RPMI-1640 cell culture medium, 0.01 M pH 7.2–7.4
phosphate buffered solution, and 0.25% trypsinase (TPS)
were all bought from SIGMA corporation of America;
10% heat-inactivated fetal borine serum from Sino-Amer-
ica Biotechnology company; rabbit antibody to human
Cx43, E-cad MAB and ABC kit from Zymed corporation
of American specific procedure of immunohistochemical
ABC method acts on the kit manuals.
FFTLG composed of Dangshen, Fuling, Baizhu, Tengli-
gen, Shuiyangmeigen, and some other pure herb medicine,
1 mL of that contained 1 g crude drug. It was obtained
from Zhejiang Chinese Medical University, China.
Cell culture
Human lung tumor cell line (PG) with high metastatic
potential was obtained from Cancer Research Institute of
Zhejiang Province, China. It was serial subcultivated in
RPMI-1640 culture solution with 10% heat inactivated
The effects of FuFangTengLiGen preparation
regulating the expressions of Cx43 and E-cad of
transplantation tumor of PG cell
Yong Guo, Qinghua Yao, Weihong Yang, Weichun Dai
Department of Oncology, The First Affiliated Hospital of Zhejiang T.C.M. University, Hangzhou 310006, China
Received: 4 June 2007 / Revised: 15 July 2007 / Accepted: 15 August 2007
Abstract Objective: To study the anticancer effect of Chinese compound recipe FuFangTengLiGen (FFTLG) against trans-
plantation tumor of PG cell and preliminarily explore the connection with connexin43 (Cx43) and epithelial cadherin (E-cad).
Methods: Model rats of transplantation tumor of human large cell cancer PG were established. The low, medium and high
dose groups of FFTLG, the blank control group were set up. After 21 days medication of FFTLG through gastrogavage, the
antitumor effect of each group was compared. The gene expressions of Cx43 and E-cad in each group were quantitatively
detected and compared by the application of immunohistochemistry technique. Results: FFTLG could significantly inhibit
the growth of transplantation tumor. FFTLG could obviously up-regulate the expressions of Cx43 and E-cad, which in the
low/medium and high dose groups were separately higher than that in the blank group. Conclusion: FFTLG has definite
antitumor effect against human large cell cancer and can obviously up-regulate the expressions of Cx43 and E-cad, which
may be correlated with its effect of anti-tumor.
Key words FuFangTengLiGen (FFTLG); lung cancer cell; anticancer; connexin43 (Cx43); epithelial cadherin (E-cad)
Correspondence to: Yong Guo. Email: guoyong1047@yahoo.com.cn
538 www. springerlink. com/content/1613-9089
fetal borine serum.
Experimental animal
BALB/C-nu/nu male mice (18–22 g) 6 weeks old were
obtained from the Animal Institute of Chinese Academy
of Science in Shanghai (China), and housed in a SPF envi-
ronment at Zhejiang Chinese Medical University, China.
Recovery and passage of tumor cell lines
The cryopreserved human lung tumor cells PG gotten
from cell bank were lysed at 37 ℃. Cell suspension was
incubated in 5 mL cell culture medium, then was trans-
ferred into a conical tube and centrifuged at 1000 rpm
for 7 minutes, then pelleted cells were resuspended into
RPMI-1640 culture medium and subcultured in T-25 cul-
ture flask. They were cultured and passaged in incubator
with 5% CO2. When the cells were full of culture flask,
culture medium was abandoned. The cells were digested
with 0.25% TPS, and stopped to digested with culture
medium containing borine serum, when the cells became
round and the distance became large.
Making the model of nude mouse with human
lung tumor cell PG
According to many researches [7], after staining with
Try pan blue, viability were determined by counting the
number of Try pan blue - positive cell and the total cell
number. For subcutaneous xenografts 2 × 106 /mL PG cells
in 0.2 mL normal saline were injected into the right arm-
pit back in each mouse, which were housed in SPF envi-
ronment.
Grouping and medication
After model establishment, node mice were randomly
numbered and then were divided into four groups, and
each group was consisted of 7 mice. Control group: 0.6 mL
normal saline were given for 20 mg node mice in propor-
tion by intragastric administration one time a day; Low-
dose group: 0.2 mL FFTLG and 0.4 mL normal saline were
given for 20 mg node mice in proportion by intragastric
administer one time a day. Medium-dose group: 0.4 mL
FFTLG and 0.2 mL NS were given. High-dose group: 0.6
mL FFTLG was given. Low-dose, medium-dose, high-
dose drug consumption respectively correspond to 5
times, 10 times, and 20 times of adults. After the models
were established, medications were given one time a day
for 21 days.
The targets and method of observation
Achievement ratio of tumor transplantation, incuba-
tion period food-intake, activity and excrements were
observed after inoculation. The day after last medication,
all the mice were put to death through breaking the neck.
The tumor was dislodged intact and weighed. Then tu-
mor inhibited rate (I) was calculated as below: I = (weight
of control group – weight of treatment group) / weight of
control group × 100%.
Detection on Cx43 and E-cad of tumor tissue
Explanted tumors were treated with 10% formalin fix-
ation, gradient alcoholic dehydration and embedded by
paraffin in proper order.
At last, section preparation were stained with H＆E.
E-cad MAB specific procedures of immunohistochemical
ABC method acted on kit manuals [8, 9].
Judgment on results of immunohistochemistry
Cx43 protein like yellowish brown pallet distributed
on the cell membrane between the neighboring cells. The
intensity and scope of staining for Cx43 in tumor cells
were subjectively scored as follows cases with more than
10% membrane staining cell were considered to be posi-
tive for Cx43 expression. Otherwise, it was considered to
be negative.
E-cad was stained to be yellowish brown, the cases
with more than 60% membrane staining were considered
to be positive. Otherwise it was negative.
Statistical analysis
Measurement data were showed by the mean ± stan-
dard deviation, and enumeration count data by the per-
centage expression. Comparison between the two groups
enumeration count data was done by chi-square test. If
P was less than 0.05, it was considered significant differ-
ence. If P was less than 0.01, it was considered extremely
significant difference. Statistical package for the social
science 9.0 was used.
Results
Observation of common state
After 7 days of inoculation, tumor pallet was found on
the right axilla and back of one mouse in NS group. In the
later 3 days, tumor pallets were found in each treatment
group. The transplanted tumors in NS group grew much
faster. On the contrary, the ones in high-dose FFTLG
group appeared relatively later and grew the lowest. In
late stage, mice in NS group became more emaciated, but
there were no obvious differences about food-intake and
activity. During the trial, each one mouse in low-dose
and medium-dose groups were dead because of improper
intragastric administration.
Inhibitory action on human lung tumor cell
with different medication (Table 1)
As shown in Table 1, FFTLG with three different doses
had inhibit action on node mice with PG tumor, all the
rates of inhibit tumor were more than 25%. The inhibit
539Chinese-German J Clin Oncol, December 2007, Vol. 6, No. 6
tumor rates of high-dose, medium dose and low dose
groups were 33.64%, 27.64% and 25.57% respectively.
Compared with NS group, the anticancer effects in low-
dose and medium-dose groups were considered signifi-
cantly (P < 0.05), and in high-dose group it was extremely
significantly (P < 0.01). It was concluded that the inhibit-
tumor rate depended on the dosage of FFTLG.
Effect on Cx43 expression of human lung
tumor with different medication (Table 2
and Fig. 1–4)
As shown in Table 2 and Fig. 1–4, Cx43 protein mainly
distributed in intercellular substance and cellular mem-
brane in tumor cell. Few of Cx43 protein in cytoplasm
presented as fine grana with strong staining Cx43 pro-
tein expressed fewer in NS group. Positive quantity in
each treatment group was more than that in NS group.
The positive quantity in low-dose group was significantly
more than that in NS group (P < 0.01), with statistical dif-
ference. It was proved that FFTLG could up-regulate the
expression of Cx43 of human lung cancer, especially the
high-dose FFTLG.
Effect on E-cad expression of human lung
tumor cell with different medication (Table 3
Table 1 Comparison of groups about the weight of tumor and the
rate of anticancer
Group Cases of animal Weight of tumor (χ ± SD, g)
Rate of
anticancer (%)
NS 7 1.15 ± 0.22 –
Low-dose 6 0.85 ± 0.16* 25.57
Medium-dose 6 0.83 ± 0.11* 27.63
High-dose 7 0.76 ± 0.13** 33.64
Note: compared with the NS group, *, P < 0.05; **, P < 0.01
Table 2 Comparison of Cx43 expression of transplanted tumor in
each group
Group Cases of animal
Cx43+
χ2 valuePositive cases Positive rate (%)
NS 7 2 28.6
Low-dose 6 4 66.7 1.887*
Medium-dose 6 5 83.3 3.899**
High-dose 7 6 85.7 4.667**
Note: compared with the NS group, *, P < 0.05; **, P < 0.01
Fig. 1 NS-group Cx43 (× 400) Fig. 2 Low-dose group Cx43
(× 400)
Fig. 3 Medium-dose group
Cx43 (× 400)
Fig. 4 High-dose group Cx43
(× 400)
Fig. 5  NS-group E-cad (× 400) Fig. 6 Low-dose group E-cad
(× 400)
Fig. 8 High-dose group E-cad
(× 400)
Fig. 7 Mediun-dose group
E-cad (× 400)
540 www. springerlink. com/content/1613-9089
and Fig. 5–8)
As shown in Table 3 and Fig. 5–8, E-cad protein mainly
distributing in intercellular substance and cellular mem-
brane was stained to be yellowish brown, and some cy-
toplasms near the cellular membrane were also stained.
The E-cad protein expressions were more in high-dose
and medium-dose groups, fewer or no in NS group. There
were statistical differences between the treatment groups
and NS group. The positive quantity in low-dose group
was significantly more than that in NS group (P < 0.05),
and in medium-dose and high-dose groups they were
extremely significantly more than that in NS group (P <
0.01).
It was suggested that FFTLG could up-regulate the ex-
pression of E-cad in human lung tumor cells, especially
in high-dose group. It was found (in the trial) that as
long as E-cad was expressed in sample, where Cx43 was
expressed too, and the higher the E-cad expressed, the
higher Cx43 level.
Discussion
FFTLG is a effect Chinese compound recipe which is
found by predecessors’ experience testified by clinical
practice which can cure lots of transplantation-tumor
and conform to the theory about medicine. FFTLG has
extensive drug-effect mechanism [10]. It has affection on
inhibition of tumor growth, anti-metastasis transferring
and the multi-drugs resistance of tumor cell.
Gap junctions are intercellular channels which are
formed from members of a family of proteins. They has
conservative in the evolution of species and specificity in
tissue distribution the tumors formation. In the latter ex-
periment, the wild type connexin genes were transfixed
into the tumor cell. Inhibition of the growth of tumor
was observed. Meanwhile, the gap junction intercellu-
lar communication has been found up-regulation, so we
consider that the gap junction genes are a family of anti-
tumor genes [11, 12]. In abnormal human-embryo lung cell,
we can see high expression of Cx43 in mRNA and protein
level [13]. Cx43 protein can be seen in the gap junction.
The affection of gap junction intercellular communica-
tion is clear, and Cx43 has effect. In the other hand, Cx43
can not express in the level of mRNA or protein in hu-
man lung cancer PG cell, and has deficiency in gap junc-
tion intercellular communication. So the up-regulation of
GJIC connect growth suppression.
As shown in this trial, lung cancer PG cell had low
affection about GJIC, and low expression about Cx43, we
applied different dose of FFTLG into lung cancer PG cell,
the cell growth could be suppressed manifestly. Mean-
while, the function of GJIC had up-regulated, the expres-
sion of Cx43 had increased. The affection of anti-tumor of
FFTLG had close connection with the regulating function
of GJIC and the inducing the expression of Cx43. It could
promote the sign transmission between cells. So the pro-
liferation, differentiation and metabolism of cells could
be regulated.
E-cad gene which generally exists all kinds of endothe-
lial cells, encodes for a 120 kDa polypeptide of cell adhe-
sion transmembrane glycoprotein. It is an adhesion mol-
ecule which medicates cell junction of the same type cell.
It plays an important role on embryonic development
morphogenesis and inducing maintaining the integrity
of endothelial all [14]. Lots of researches proved that low-
expression or lose of E-cad can make tumor lose all of its
contact inhibition. Moreover it can result in proliferation
of cell without limitation and differentiation, result in
loosing the conjunction between cells and make the cell
drop from location more easily, which stronger invasive
ability is prone to the diffusion and metastasis [15].
Our study showed, that stained Cx43 proteins locat-
ed on intercellular substance and cellular membrane.
There were more stained Cx43 proteins in three treat-
ment groups, and few in NS group (Fig. 2–4). It could
be observed that the function on gap junction intercel-
lular communication of human lung cancer PG without
medication was the most weak. The expressions of Cx43
proteins in FFTLG treatment group were increased. The
expression of Cx43 in high-dose group was significantly
more than that in NS group (P < 0.01), with statistical
difference. It showed that FFTLG could up-regulate the
expression of Cx43 of human lung cancer. The anti-tumor
rate in each treatment group with FFTLG was more than
25%. Compared with the NS group, the anti-tumor rate
in the low-dose and medium-dose groups were consid-
ered significantly (P < 0.05), and in high-dose group it
was extremely significantly (P < 0.01). It suggested that
FFTLG had a significant effect on inhibiting tumor. It also
suggested indirectly that gap junction intercellular com-
munication was one of the largest parts which FFTLG af-
fected on.
In the trail, E-cad proteins were stained to be yellow-
ish brown and the cellular membrane with stained E-cad
proteins also turned to yellowish brown, the same as the
intercellular substance. E-cad proteins expressed more in
the high-dose and medium-dose groups, fewer or no in
the NS group. There were statistical differences between
Table 3 Comparison of E-cad expression of transplanted tumor in
each group
Group Cases of animal
E-cad+
P valuePositive cases Positive rate (%)
NS 7 1 16.7
Low-dose 6 4 66.7 > 0.05
Medium-dose 6 5 83.3 < 0.05
High-dose 7 6 85.7 < 0.05
541Chinese-German J Clin Oncol, December 2007, Vol. 6, No. 6
the treatment groups and NS group. The low-dose group
and NS group were compared with P < 0.05, and the medi-
um-dose, high-dose groups and NS group were compared
with P < 0.01. It suggested that FFTLG could up-regulate
the expression of E-cad of human lung cancer cell.
It was found that as long as E-cad was expressed in
samples where Cx43 was expressed too, and the higher
E-cad expressed, the higher Cx43. The expression of Cx43
was related parallel to the ones of E-cad. Maybe FFTLG
could promote the expression of Cx43 through up-regula-
tion of expression of E-cad. That was consistent with the
results of our study [16]. But few samples without expres-
sion of E-cad still have expression of Cx43. It showed that
FFTLG worked with other adhesion cells or something
else, except for E-cad which medicated cell adhesion and
created communication [17]. The special mechanisms need
to be deeply approached.
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