Anti-proliferative and anti-metastasis activity of Chinese medicinal
plants and the investigation on the anti-lung cancer metastasis
activity of the essential oil of Cangzhu
Name of dispatched researcher（派遣研究者名）

Na HanInstructor（指導教官，研究指導者）

Na HanAffiliation of Instructor（所属）

School of Traditional Chinese Meteria
Medica, Shenyang Pharmaceutical
University

Host Collaborator（受入研究者）

Ikuo Saiki

Abstract:
75 herbal extracts from the most commonly used herbs in the clinic in China werescreened the anti-proliferative activity against A549 cells, and the most effective extract (PC)among them was fatherly screened the anti-proliferative activity against A549, A2508, UACC
257, MDA-M231 in multiple dosages. As a result, PC displayed the better selectivity against melanoma cell line A2508 and UACC 257. We next evaluated the potential of PC on inhibiting themetastasis of melanoma cells by wound healing experiment and transwell chamber assay andresults showed that PC had obvious in vitro anti-metastasis activity of UACC 257 cells. In thein vivo experimental lung metastasis model, PC could inhibit the metastatic lung colonization of
UACC 257-luc cells significantly, which supplied a solid proof for the utilization of PC in treating
melanoma and its metastasis.
Background:
Cancer has become the most common disease in the world, for example, individuals bornin the US today have a 41% lifetime risk of being diagnosed with cancer. Even though we havefound out lots of chemical agents like platinum, CPT-11, taxol, vincristine, ginsenoside Rg3 andetc in the recent centuries, the emergence of new problems seems to be always accompanied, likethe drug-resistant problem, ignorable severe adverse effects and etc. Especially, cancer metastasis is largely incurable because of the resistance of disseminated tumor cells to existing therapeutic agents, which makes the development of new anticancer or anti-metastasis agents longterm
and also urgent [1-3].
China has accumulated precious experiences in a long history by using the herbal treatment.
We believed that these experiences provided us the best sources to treat the incurable disease
like cancer and metastasis. In previous study, 600 prescriptions from five Eminent and Experience Chinese Medicine Practitioners in Beijing were analyzed to get the frequency of each drug

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used in lung cancer metastasis treatment. All the applications of the herbal drugs or their combinations with the chemical therapy were reported to either improve the long-term survival rateof patients with malignant tumor, or change the bad status of their life [4]. In order to find thepotential anti-cancer or metastasis materials, we investigated the anti-proliferative and anti-metastasis activities of these most common used herbs in cancer patients in clinic in present study
and hope to get the candidates for treating cancers in further study.
Thus, in this study, we concerned about two questions: (1) We expect to screen new potential
anticancer or metastasis medicinal plants from the mostly commonly used herbs; (2) We expect
to choose one candidate to investigate the in vivo anti-metastasis activity and hope to evaluate
its real anti-metastasis activity.
Materials and Methods:
Cell culture and cytotoxicity assay
Four cell lines including A549 (human lung adenocarcinoma), A2508 (human melanoma),
UACC 257 (human melanoma cell line) and MDA-M231 (human breast cancer) were obtained
from the Japanese Cancer Research Resources Bank (Tokyo, Japan). 25 Medicinal herbs
were purchased from Guoda Pharmacy in Shenyang and numbered from 1 to 25. Each one
was extracted with methanol, methanol-water and water ultrasonically to get three kinds of extracts so that 75 tested samples were gotten.
These cell lines were maintained as monolayer cultures in RPMI-1640 supplemented with 10%
fetal bovine serum (FBS) in an atmosphere containing 5% CO2/air at 37 ℃. The quantification ofcell viability was performed using the cell proliferation reagent WST-1 (4-[3-(4-iodophenyl)-2-(4nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate) (DOJINDO, Kuma-moto, Japan). All the celllines were plated in 96-well microplates in 5000 cells/wells and then incubated for 24 h. Samplecontaining medium was added into each well, and cells were incubated for another 24 h. As last, 10µl WST-1 reagents were added into each well, and the absorbance was measured at 450 nm.
Wound healing assay
UACC 257 cells were plated in a 24-well plate at a concentration of 5 × 105 and allowed toform a conﬂuent monolayer for 24 h. The monolayer was then scratched with a sterile pipette tip(1000 μ l), washed with medium to remove ﬂoated and detached cells with PBS, and photographed(time 0h). Cells were successively treated in medium in the presence of different concentrationsof PC (40 μ g/ml) along with the vehicle DMSO for 24 h. Scratched areas were photographed(magnification,× 40) at 0 h and then again 24 h later to assess the degree of wound healing.

The percentage of wound closure was estimated by the following equation: wound closure % = 1–
(wound area at t 24/ wound area at t0) × 100%, where t24 is the time after wounding and t0 is thetime immediately after wounding.
Migration and invasion assay
The filters of a Transwell cell culture insert (8 μ m pore size; Whatman Japan KK, Tokyo, Japan) were pre-coated with fibronectin (Iwaki, Tokyo, Japan, 1 μ g/filter) on the lower surfaces. For− 120 −

the invasion assay, the upper surface of the filter was coated with Matrigel (Becton-Dickinson,
Bedford, MA, 2 μg/filter). Cells were pre-incubated with or without PC (40 μg/ml) for 24 h. Aftertrypsinization, cells in 0.1% (v/v) BSA medium (3 × 105) were placed in the upper chamber ofTranswells. After the subsequent incubation at 37 ℃, the residual cells were removed from theupper surface of the membrance and the migrated cells on the lower surface of the membrane
were fixed in 100% methanol and stained with hematoxylin and eosin. Migration was determinedby counting the cells with a microscope at 100 × magnification. Five visual fields were chosenrandomly and the average number of migrating cells in the five fields was taken for each group.
Experimental lung metastasis model
Female scid mice (six weeks, 14-19 g) were purchaced from Japan SLC, Inc. (Hamamatsu,
Japan) and maintained in a temperature- controlled, pathogen-free room. All animals werehandled according to the approved protocols and guidelines of the Animal Committee of Toyama
University. For the experimental metastasis model, UACC 257-luc cells were inoculated intravenously (i.v., 1× 106) with or without pre-treatment with PC (40 μg/ml, 24 h) for each mouse. For
the lung metastasis imaging, mice were injected with D-luciferin the day after tumor inoculation,then the lungs were removed to measure luminescence using in vivo imaging system (IVIS Lumina Ⅱ , Caliper Life Sciences, MA, USA).
Statistical analysis
All the data are expressed as the mean ± SD of at least two or three independent experiments unless otherwise stated. Statistical significance was analyzed using Student’s t-test. P<
0.05 was considered significant.
Result and Discussion
1. The screening of 75 herbal extracts from the mostly commonly used herbs in clinic
Due to lung cancer is one of the most lethal cancers with the highest incidence in the worldwide, we referred to the literatures to get the most commonly used traditional Chinese medicines
in treating lung cancers. As a result, 25 herbal medicines were selected and extracted with water,
methanol-water and methanol to get 75 kinds of extracts. And then, they were screened the antilung cancer activity by using A549 cells at the concentration of 100 μg/mL. As shown in Table 1,among the 75 medicinal herbal extracts, the methanol extract and methanol- water extract of thesample NO. 15 (named PC for short) showed the best anti proliferative activity against A549 cellswith the inhibition rate over 90%. To further confirm its effects on other cell lines, we evaluatedthe cytotoxicity with more cell lines at multiple concentrations. The results were shown in Fig.
1. It could be found out that PC inhibited the proliferation of A549, A2508, UACC 257 and MDAM231 cells in dose dependent manner. Especially for the melanoma cells A2508 and UACC 257,
PC displayed obviously better inhibition activity compared with other cell lines at 80μg/ml, which
illustrated that PC might be more sensitive to the melanoma cells. However, at 40μg/ml, the selectivity disappeared and PC displayed mild inhibition activity against all the cell lines. PC is a

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traditional kidney enriching agent and its anti metastasis activity haven’t been reproted before.
In further study, we focused on the anti-metastasis potential of PC against melanoma cells at the
non-cytotoxic dosage 40μg/ml.
Table 1 The anti-proliferative activities of 75 herbal extracts against A549 cell line
NO.

yields1
74.78%
2
16.05%
3
46.18%
4
68.63%
5
31.68%
6
12.17%
7
18.23%
835.88%
9
11.65%
10
14.48%
Form 2-2（様式２－２）
1113.66%
12
18.90%
13
5.39%
17
11.40%
14
7.57%
15
8.90%
18
40.38%
16
6.32%
19
21.07%
17
11.40%
20
29.67%
18
40.38%
21
11.75%
19
21.07%
22
18.73%
20
29.67%
21
11.75%
23
25.74%
22
18.73%
24
17.60%
23
25.74%
250.36%
24
17.60%
Taxol
25
0.36%

Inhibition rate (%)

Taxol

Water
Methanol
Inhibition rateyieldsInhibition rate
20.85%
7.34%
23.59%-1.83%
4.89%-29.09%-2.05%
17.98%
6.885%-12.05%
5.88%-4.17%
7.45%
2.96%
7.75%
10.53%
2.09%
7.15%
22.68%
0.82%
23.5%
2.01%
10.62%-18.17%10.90%
5.04%
6.98%
11.29%
7.69%
3.52%
14.67%
3.22%-24.76%14.40%
4.83%
6.91%-13.69%21.79%-30.5%
5.16%2.97%
2.72%3.62%-5.07%-11.23%-8.27%
18.03%
92.69%
11.46%
1.20%
22.2%
19.29%
12.16%
17.52%
5.56%3.91%
14.45%
5.16%
2.72%-5.07%
5.42%
16.85%
7.48%
11.46%
1.20%
22.2%-39.20%1.24%5.56%-23.26%17.52%
14.45%
21.34%
3.90%-4%7.48%
5.42%
16.85%-39.20%-23.26%15.49%
5.16%1.24%
19.59%-11%21.34%
6.17%3.90%-4.90%-4%
15.49%
19.59%
21.6%
0.51%5.16%
20%
-11%
6.17%
-4.90%
Inhibition rate
21.6%
0.51%
20%
44.75% rate
Inhibition
44.75%

Water-Methanol (1:1)
yieldsInhibition rate
15.02%
21.55%
19.90%-26.40%48.06%
7.53%
51.31%-14.15%16.56%
4.99%
5.59%
16.09%
9.34%
28.77%
35.19%-13.03%15.43%
0.35%
23.07%
7.25%
10.42%-15.42%42.34%
2.64%
6.97%-13.61%19.50%-17.84%12.25%
0.335%
10.51%
93.46%
35.91%
34.46%
10.58%
13.82%
22.41%
5.77%
19.50%-17.84%22.43%-11.29%35.91%
34.46%
12.21%-31.46%22.41%
5.77%
15.70%-31.59%22.43%-11.29%12.21%-31.46%24.66%
16.16%
15.70%-31.59%27.08%-5.69%
24.66%
16.16%
3.51%
25.2%
27.08%-5.69%
3.51%
25.2%

100

A2508

90

UACC257

80

M231

70

A549

60
50
40
30
20
10
0

20 μg/mL

40 μg/mL

80 μg/mL

1 The anti-proliferative
activities
PC against
A2508,UACC
MDA-M231
cell
Fig.Figure
1 The anti-proliferative
activitiesof PC ofagainstA549, A549,
A2508,
UACC
257 257
andandMDA-M231celllines atlines at three different dosages

three different dosages(Cells were seeded into 96 well plate and incubated for 24 h. Then sample in different concentrations were
(Cellswereinto
seeded96andwellincubate
plate andfor
incubatedfor 24h. Thensamplein different
concentrations
were
added
into eachadded
eachinto
well
24 h. After
that,
WST-1
reagent
was added
into each wellandthe inhibition
rates were
calculated
accordingthe formula:1-(opticaldensitythethesamplewell rates/ optical
well
and incubate
for 24h. After that,
WST-1toreagent
was added
into eachwellofandinhibition
weredensity
calculatedof the blank control well)according to the formula: 1-(optical density of the sample well / optical density of the blank control well)− 122 −

2. The po tentia l eva lu ation of PC in inh ib iting the meta stasis of melano ma ce lls in vitro

In order to evaluate the anti-metastasis potential of PC, wound healing experiment, migration and invasion

2. The potential evaluation of PC in inhibiting the metastasis of melanoma cells in vitro
In order to evaluate the anti-metastasis potential of PC, wound healing experiment, migration and invasion assay were performed in our research. As shown in Fig. 2, PC could inhibit thecell migration of UACC257 cells, with the inhibition rate 29%, as determined by the wound clo-

2-2（様式２－２）
FormForm
2-2（様式２－２）

0h

24 h

SampleSample0h

24 h

100 100
90 90

Relative migration (% of control)

Blank
Blank

Relative migration (% of control)sure assay. In addition, PC pre-treatment showed significant inhibition of migration activity (Fig.

80 80
70 70
60 60
50 50
40 40
30 30

** **

20 20
10 10
0

0

Control
Control

SampleSampleB B

A A

Figure2 Effectsof PCofonPC
cellularmigrationof UACC
cells in cells
wound
healing healing
experiment
Figure2 Effects
on cellularmigrationof UACCin woundexperiment

Figure 2 Effects of PC on cellular migration of UACC cells in wound healing experiment(Cells treated
were treated
PC for24hs inwound
healing
assay.
Briefly,cell
UACC
257 cellwere
monolayers
werea sterile(CellsPCwithfor 24hs
in woundhealing
assay.
Briefly,
UACC
monolayers
scraped(Cellswereweretreated withwith
PC for
24hs
in woundhealing
assay.
Briefly,
UACC
257257cell monolayers
were scraped
withwitha sterile
scrapedwithasterilemicropipette
tip,
and
the
cells
were
treated
for
24hs
with
PC
at
the
dosageof
40μg/mL
micropipettetip,
andthe cells
weretreatedfor 24hs
withPCthe
at dosage
the dosageof 40μg/mL
or withverhicle.
Scratchedareas
micropipette
tip,
and
the
cells
were
treated
for
24hs
with
PC
at
of
40μg/mLorwithverhicle.
Scratchedareas
werewereor with verhicle. Scratched areas were photographed (magnification, × 40) at 0h and then again 24h later
photographed(magnification,
×40)
at
0h
and
then
again
24h
later
to
assessthedegreeofwound
healing.
The
data
are
photographed(magnification,
at 0h healing.
and thenTheagain
24harelater
to assess the degree
healing. Theexperiments.
data areto assessthe degree ×40)
of wounddata
representative
resultsofofwound
two independent
representative
resultsof twoindependent
experiments.
**p<0.01
comparedthe blank
controlgroup.)
representative
resultsof twoindependent
experiments.
**p<0.01
compared
withwiththe blank
controlgroup.)
200 200
180 18090 90

160 160
140 140

70 70*** ***

100 100
80 80
60 60
40 40
20 20
0 0

Invasion cell number

120 120

Invasion cell number

80 80

Migration cell number

Migration cell number**p<0.01 compared with the blank control group.)

60 60
50 50

*** ***

40 40
30 30
20 20
10 10

Control
Control

SampleSampleA A

00

Control
Control

SampleSampleB B

FigureEffect
3 Effectof PC
on migration
or invasionof UACC257
cells
in Transwellchamberassay
Figureofon migration
migration
orinvasion
invasionofUACC257
UACC257
cells
Transwellchamberassay
Figure33 Effect
of PC
PC on
or
of
cells
inin
Transwellchamber
assay

5
the
assay
invasion
assay
pretreatedfor h.
12
h.for
Then
anThenaliquot
of
cells
(3×10
5
(For(Forthe migration
assay(A) (A)and andinvasion(B),(B),cellscells
werepretreatedwithwith
PC PC
forwith12
Thenanofancells(3×10) )(Formigration
the migration
assay
(A)
andassayinvasion
assay(B),were
cells
were pretreatedPC
12 aliquoth.
aliquot
5transferred
to
upper
well.
After
incubation,
migratory
andinvasiveon bottom
the bottom
of insert
the insert
of cellsto(3each× each10) wastransferredtoeach upper
well.andAfter
incubation,andinvasivecellsmembraneon thewerewere
waswastransferred
upper
well.
After
incubation,
migratory
invasivecellscells
onmigratory
theof themembrane
fixed
in
100%
methanolandstainedwithhematoxylin
anderosin.
Migrationandinvasion
weredeterminedbycountingbottomof the insert
membranewere fixed inmethanolandwithhematoxylin
erosin.
fixed in 100%
methanoland stainedwith hematoxylin
and100%erosin.
Migration
andstained
invasion
weredetermined andby counting
the the
cells
with
a
microscopeat×100
magnification.
Fivevisualfieldswerechosenrandomly
andtheaveragenumberofinvasiveand atinvasionwere determinedby counting
the cells
witha microscope× 100 number
magnification.
cells withMigrationa microscope×100 magnification.
Five visual
fields werechosenrandomly
and theataverageof invasive
in
the
five
fieldstaken
for eachgroup.
experiment
performed
in triplicate.
presented
as theFivefieldswerechosenrandomly
andtheaveragenumberof ininvasive
cells
thedata
fivearefieldswascellscells
in thefivevisual
fieldswaswas
taken
for
each
group.
EachEachexperimentwaswasperformed
triplicate.
TheinThedata
arepresented
as thetaken
foreach group.
Eachexperiment
was performedin triplicate.
The datablankpresented
as the mean ±mean±SDof threereplicateexperiments.
***p<0.001
comparedthe untreatedcontrolgroup.)
mean±SDof threereplicateexperiments.
***p<0.001
compared
withwiththe untreatedblankarecontrolgroup.)

SD of three replicate experiments. ***p<0.001 compared with the untreated blank control group.)

3. The
poten
evalu
ation
of PC
in inhib itingmetastasisof melano
ce llsin viv
3. Thepoten
tialtial
evalu
ation
of PC
in inhib iting
thethemetastasisof melanomama
ce llsin vivo o− 123 −
Basedon theresultsof thein vitro
experiments,
performed
in vivo
activity
evaluationof PC
fatherly.
Basedon theresultsof thein vitro
experiments,
we we
performed
the thein vivo
activity
evaluationof PC
fatherly.

UACC
257-luc
were
inoculated
intravenously(i.v.,
or without
pre-treatment
UACC
257-luc
cellscells
were
inoculated
intravenously(i.v.,
1× 1×106)106)
withwithor without
pre-treatment
withwith
PC PC(40 (40

3A) and invasion activity of UACC 257 cells (Fig. 3B) in the Transwell chamber assay by comparing the cell numbers on the lower side of the membrane between the control group and thesample group. Collectively, these results clearly indicated that PC could reduce the metastatic
potential of UACC 257 cells, which provided the proofs for the further evaluation of its in vivoanti-metastasis activity.
3. The potential evaluation of PC in inhibiting the metastasis of melanoma cells in vivo
Based on the results of the in vitro experiments, we performed the in vivo activity evaluation
Form of2-2（様式２－２）
PC fatherly. UACC 257-luc cells were inoculated intravenously (i.v., 1 × 106) with or without

pre-treatment with PC (40 μ g/ml, 24 h) for each mouse. One day after, the mice were sacrificed

metastaticlungcolonizationof UACC
257-luc cells
in this experimentalof UACC257
and thelungs
were removed
to measureluminescence
using inlung
vivometastasis
imaging model
system.
Results
showedthat pre-treatment with PC could inhibit the metastatic lung colonization of UACC
melanoma(Fig.4).
257-luc cells in this experimental lung metastasis model of UACC 257 melanoma (Fig.4).

***

Figure 4 Anti-metastasis of PC in experimental melanoma metastasis model.

FigureAnti-metastasisof PC
melanomametastasis model.
cells pre-incubatedwith or without 40(Mice were given
an4intravenous
injectionof 1in×experimental
106 UACC 257-luc
6
(Mice
were
given
an
intravenous
injection
of
1×10
UACC
257-luc
cells
pre-incubatedwithmetastasis
or withoutwere
40 μg/ml
for 24h.
μ g/ml for 24h. One days after tumor inoculation, mice were sacrificed and lungmeasured
One days
after
tumor
inoculation,
mice
were
sacrificedandlungmetastasis
weremeasured
by
the
IVIS
system.
The
data areby the IVIS system. The data are representative results of two independent experiments and are presented
representative
resultsoftwo
independent
experiments
andarepresented
as
the
mean
luminescence±SD(n=4).
***p<0.001.)
as the mean luminescence ± SD (n=4). ***p<0.001.)
In our research, we found a promising candidate owning both the anticancer and antimetastasis activity from
In our research, we found a promising candidate owning both the anticancer and antime75 herbal extracts in China. Even though the route of administration in our experimental animal model is different
tastasis activity from 75 herbal extracts in China. Even though the route of administration inwith the clinic ones such as intravenous injection or oral administration and the pharmacokinetics factors were
our experimental animal model is different with the clinic ones such as intravenous injection or
excludedin this experiments,
still
could get the conclusion
PCexcluded
might be invery
potential
becausewe
thestill
microoral administration
andwe
the
pharmacokinetics
factorsthat
were
this
experiments,
metastasis
was mimicthat
completely,whichmeanspotentialthat if only
the activeof PC could
act oncould experiment
get the conclusion
PC might
be very
becausethecomponents
micro metastasis
experithe melanoma
it would
definitely
reducethe migration
andtheinvasionof cells,
a solid
ment wascells,
mimic
completely,
which
means
that if only
activecharacterscomponentsof PCsupplying
could acton
prooftheformelanoma
the ultilizationof PC
in treating
melanomaand itsthe
metastasis.
cells,
it would
definitely
reducemigration and invasion characters of cells,
Besides, inaour
experiment,
we alsohave some
negative
results:melanoma(1) In the screeningof the anti-metastasis
supplyingsolid
proof for theultilizationof PC
in treating
and its metastasis.
Besides,in our experiment,
we of
also
some negative
results:(1) In theof theactivity, we
also investigatedthe potential
PChaveon inhibiting
lung cancer
metastasis,
butscreening
PC displayedpoor
anti-metastasis
activity,
also investigatedthealsopotential
PC on inhibiting
lung Therefore,cancer me-we
activity
not only in thewoundwehealingexperiment but
in the ofTranswellchamber assay.
tastasis,but PC
displayed
activity(2)notonlythough
in thePCwound
healingexperimentbutanti-metastasis
also in theterminatedfurtherin vivo
activitypoor
evaluation.
Even
displayed
the obvious
in vivo

activity, the molecular mechanism is undiscovered, we tried to detect the key protein in the metastasis pathway ofmelanoma cells including ERK, AKT, MMP-9 and−SLUG
124 −by western blotting, but all the proteins have nochanges except AKT after PC pretreatment. Unexpectedly, AKT displayed obvious down regulation. Thus, thespecific mechanism of PC is still unclear and should be clarified fatherly in the future.

Transwell chamber assay. Therefore, we terminated further in vivo activity evaluation. (2) Even
though PC displayed the obvious in vivo anti-metastasis activity, the molecular mechanism isundiscovered, we tried to detect the key protein in the metastasis pathway of melanoma cells including ERK, AKT, MMP-9 and SLUG by western blotting, but all the proteins have no changes
except AKT after PC pretreatment. Unexpectedly, AKT displayed obvious down regulation. Thus,
the specific mechanism of PC is still unclear and should be clarified fatherly in the future.

Reference:[1] Scott V, Robert A. Tumor Metastasis: Molecular Insights and Evolving Paradigms, cell, 2011,
147, 275-292.
[2] Pan SY, Zhou SF. New Perspectives on How to Discover Drugs from Herbal Medicines: CAM's Outstanding Contribution to Modern Therapeutics, Evidence-Based Complementary and Alternative Medicine, 2013, Article ID: http://dx.doi.org/10.1155/2013/627375[3] Heather G., Natural Products for Cancer Prevention, Semin Oncol Nurs. 2012, 28(1): 29-44.
[4] Rong ZH, The anti-lung cancer experience and clinical research results from famous TCM doctors in Beijing hospitals. 2013, Doctoral Dissertation of Beijing University of Chinese medicine.

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