Chemoprevention by Prunella vulgaris L. Extract of Non-Small Cell Lung Cancer Via Promoting Apoptosis and Regulating the Cell Cycle

Abstract

Chemoprevention is one feasible approach to decreasing morbidity and mortality of non-small cell lung cancer (NSCLC). The present study aimed to explore the mechanisms of chemoprevention of NSCLC by Prunella vulgaris L. (PV) using a PV extract of 60% ethanol (P-60). In an A/J mouse model benzo[a]pyrene induction of lung tumors was significantly reduced difference by P-60 group. In addition, P-60 was found to have the ability to regulate cell cycle and induce apoptosis in SPC-A-1 cells. Therefore, we propose that P-60 has potential as a lung cancer chemopreventive agent.

Full text

Asian Pacic Journal of Cancer Prevention, Vol 11, 2010 1355
Prunella vulgaris L. Chemoprevention of Non-Small Cell Lung Cancer - Apoptosis and Cell Cycle Regulation
Asian Pacic J Cancer Prev, 11, 1355-1358
Introduction
Lung cancer, including small cell lung cancer and
non-small cell lung cancer, is the leading cause of cancer
deaths in the world (Minna et al., 2002). Non-small cell
lung cancer (NSCLC) is the main common type in all
cases, accounting for about 80%. It can be caused by
carcinogenic substances such as chemicals from tobacco
smoke (Hecht, 2002), pollution such as ionizing radiation
(Shin et al., 2002), and viral infection (Kountouri et al.,
2010). Its four-year morbidity and mortality were found
to be 95-100/100 000 and 87%, respectively (Parkin et
al., 2001; Blanchon et al., 2006; Eilstein et al., 2008).
Common treatments for lung cancer include surgery,
radiotherapy and chemotherapy; however, these treatments
often result in serious side effects such as bone marrow
suppression, leukopenia, impaired immune function,
nausea and vomiting (Nico, 2001). Therefore, early
prevention and treatment need to be stressed.
Chemoprevention is one possibility (Han et al.,
2009). It has been reported that p53 gene, mediating the
cell apoptosis, is highly related to the occurrence and
development of NSCLC. It is also known that cytoplasmic
p53 may rapidly translocate to the mitochondria under
pro-apoptotic stress (Robbins et al., 2010). Cell cycle
regulation and apoptosis induction were recognized as the
underlying apoptosis mechanisms of chemoprevention of
NSCLC (Nakamura et al., 2009; Murugan et al., 2010).
Prunella vulgaris L., a Labiatae plant, is used
commonly as dietary supplements in world, which is
effective in preventing or treating diseases. It has been
reported that it had immune modulatory effects through
1Key Laboratory of Delivery Systems of Chinese Meteria Medica, Jiangsu Provincial Academy of Chinese Medicine, Jiangsu,
Nanjing, 2Biotechnology Labortory of Chinese Medicine, Macau University of Science and Technology, Macau, 3Analysis Center
of Rudong County Grain Bureau, Jiangsu, Nantong, China *For correspondence : jxiaobin2005@hotmail.com
Abstract
Chemoprevention is one feasible approach to decreasing morbidity and mortality of non-small cell lung
cancer (NSCLC). The present study aimed to explore the mechanisms of chemoprevention of NSCLC by Prunella
vulgaris L. (PV) using a PV extract of 60% ethanol (P-60). In an A/J mouse model benzo[a]pyrene induction of
lung tumors was signicantly reduced difference by P-60 group. In addition, P-60 was found to have the ability
to regulate cell cycle and induce apoptosis in SPC-A-1 cells. Therefore, we propose that P-60 has potential as a
lung cancer chemopreventive agent.
Keywords: Prunella vulgaris L.- in vivo chemoprevention - NSCLC - apoptosis - cell cycle - SPC-A-1 cells
RESEARCH COMMUNICATION
Chemoprevention by Prunella vulgaris L. Extract of Non-Small
Cell Lung Cancer Via Promoting Apoptosis and Regulating
the Cell Cycle
Liang Feng2, Xiaobin Jia1,2﹡, Maomao Zhu3, Yan Chen1, Feng Shi1
activating NF-kappaB and MAP kinase (Collins et al.,
2009), antiestrogen receptor (Choi et al., 2010), and
antitumor activity (Lee et al., 1988). An extract has
inhibited mutagenicity and carcinogenicity of benzo[a]
pyrene, 1,6-dinitropyrene and 3,9-dinitrouoranthene
(Horikawa et al., 1994; Vostálováet al., 2010). It also
prevents UVB-induced DNA damage and oxidative stress
in HaCaT keratinocytes (Cheung et al., 2008). PV is rich in
phenolic acids, avonoids, coumarins, triterpenes, volatile
oil, polysaccharides (Feng et al., 2010; Moon et al., 2010),
all demonstrating chemopreventive potential (Tanaka et
al., 1993; Lin et al., 2008; Petronelli et al., 2009).
The purpose of this study was to investigate the
chemoprevention effect of PV extract on non-small cell
lung cancer both in vitro and in vivo. There are many lung
cancer animal models (Malkinson et al., 1992) including
carcinogen-induced (Das et al., 2007) and transgenic
models (Liu et al., 2002). Here we used benzo[a]pyrene
with A/J mice. In addition, the activity of cell cycle
regulation and apoptosis induction were also studied to
explore the possible chemoprevention mechanisms.
Materials and Methods
Plant material
4 kg dried spikes of PV was ordered from medicinal
corporation of Bozhou, Anhui province, China. Herb was
authenticated as Prunella vulgaris L. by Professor D.K.
Wu, from Nanjing University of Chinese Medicine.
Chemicals
Cisplatin was ordered from Jiangsu Hengrui Medicine

Liang Feng et al
Asian Pacic Journal of Cancer Prevention, Vol 11, 2010
1356
Co., Ltd., (Lianyungang, China, batch number: 08062524).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), was purchased from Sigma (USA) and
benzo[a]pyrene (B[a]P) was purchased from Aladdin
(Shanghai, China). V-FITC Apoptosis Detection Kit was
ordered from Nanjing KeyGen Biotech. Co. Ltd (Nanjing,
China).
Preparation of PV extracts
4 kg spike of PV was weighted and refluxed
successively in vacuum reux accumulator (20 L) with
10-fold amount of 60% ethanol (v/v), 30% ethanol (v/v)
and distilled water for 2 h/time (2 times). Extracts were
merged and concentrated by rotary evaporation at 60°C
and further dried in a vacuum oven at 60°C. The nal crude
yields were obtained after volatilizing the solvent, and the
obtained extracts were 180 g, 157 g and 157g, respectively.
Cell culture
SPC-A-1 cells were routinely maintained in RPMI-
1640 medium, supplemented with 10% fetal calf serum,
100 U/mL of penicillin and 100 U/mL of streptomycin in
a humidied incubator at 5% CO2 and 37°C. The medium
was renewed every 2 days. The cells were digested by
0.25% trypsin-0.01% EDTA and used for seeding into
96 or 24-well plates.
Cell proliferation assay
Cell proliferation inhibition of different concentration
P-60 on SPC-A-1cell was determined by MTT assay.
Briey, cells were seeded in 96-well plates (4×104 cells/
mL) and treated with the crude extract (16, 80, 400 and
2000μg/mL) for 48 h. Each well was added with 100 μL
of MTT (5 mg/mL) and incubated at 37°C for 4 h. The
MTT solution was discarded and the wells were added
with 100 μL of DMSO to dissolve the formed formazan.
The samples were examined on a SPECTRAmax 190
microplate spectrophotometer (Molecular Devices, USA)
at 580 nm.
Chemoprevention activity in A/J mice
Female A/J mice were obtained from Nanjing Model
Animal Research Center. The mices were maintained
under standard conditions at 25°C ± 2°C and 50% ± 10%
relative humidity, and fed with a standard diet and water
at random. A/J mice received B[a]P in corn oil (100 mg/
kg) by intraperitoneal injection. One week after injection,
mices of the control group were given orally 0.4 mL saline
solution (0.9% NaCl) everyday for 24 weeks, while mices
of the treatment group were given orally 0.4 mL P-60
(10.0 g/kg) everyday for 24 weeks. After 24 weeks, the
mices were sacriced with CO2. Lungs were removed and
xed in Tellyesniczky’s solution (70% ethanol, methanol
and acetic acid in a ratio of 20:5:3) for at least 24 h, and
then stored in 70% ethanol (v/v). The number of tumors
was calculated to determine the tumor inhibition ratio.
Photographs of the samples were taken using a (SZX7,
OLYMPUS, Cannon camera) with auto-focus.
Apoptosis detection
The cells were seeded in 25cm2 flask (Gibco,
Invitrogen, USA) for apoptosis analysis. After being
cultured for 2 days, the cells were treated with the P-60 in
125, 250 and 500 μg crude drug/mL and then maintained
at 5% CO2 and 37°C for 48 h. These cells were detached
with 0.25% trypsin-0.01% EDTA solution and centrifuged
at 2000 × g for 5 min. After removing supernatant, the cells
were washed twice with phosphate buffered solution (PBS,
pH =7.4) and centrifuged at 2000 × g for 5 min to collect
5 × 105 cells. Cells were stained with 5μL annexin V-FITC
and 5μL propidium iodide according to the manufacturer’s
instructions of V-FITC apoptosis detection kit. Then the
cells samples were detected by using a ow cytometer
(Beeton-Diekinson, USA) with uorescence excitation
wavelength at 488 nm and emission wavelength at 530 nm.
Cell cycle analysis
SPC-A-1 cells (4×104 cells/mL) were seeded in 25cm2
ask for cell cycle distribution analysis. The cells were
treated with various concentrations of P-60 (125, 250
and 500 μg crude drug /mL) for 48 h and then detached
by using 0.25% trypsin-0.01% EDTA solution. Cell
suspension was xed with 70% ethanol (v/v) for 2 h and
washed in PBS, then added with 100 μL RNase A (1 mg/
mL) and heated in a warm bath at 37°C for 30 min. The
cells were then stained with 400 μL propidium iodide (50
μg/mL) and incubated in the dark at room temperature for
30 min. The samples were detected by ow cytometry
with uorescence excitation wavelength at 488 nm and
emission wavelength at 530 nm. Data from 10,000 cells
were collected for each data le.
Statistical analysis
All data were expressed as means ± standard deviation
(SD), and analyzed by one-way ANOVA with SPSS 16.0
software. The level of signicance was set at p < 0.05.
Results
As can be seen from Figure 1, P-60 showed the
strongest antiproliferative activity on SPC-A-1 cells
compared with P-30 and P-w (p≤0.01). The IC50 were
0.65 ± 0.15, 1.63 ± 0.25 and 4.84 ± 0.32 mg crude drug/
mL, respectively. Based on determination result of this
experiment, P-60 fraction was chosen for the further
study. As can be seen in Figure 2, P-60 showed anti-lung
cancer activity against SPC-A-1 cells in dose-dependent.
With the increase of dose, the inhibition rates of 16, 80,
400 and 2000 μg crude drug/mL were 20.1 ± 1.98%, 32.3
± 5.48%, 49.8 ± 13.4%, 97.5 ± 5.95%, respectively. The
results showed that the optimal dose was selected between
16 and 2000 μg crude drug /mL for further study.
As shown in Figure 3, after being treated with 10 mg
crude drug/mL P-60 for 24 weeks, the number of tumors
in back side and front side was lower than untreated group
(31.2 ±5.66 vs. 3.0 ± 2.16, p≤0.01). The gures and data
showed that treatment with P-60 decreased the tumor
multiplicity by 90.3%.
Apoptosis plays a crucial role for it is an important
mechanism of chemoprevention (Nakamura, 2009;
Murugan et al., 2010; Robbins et al., 2010). In the
apoptosis study, SPC-A-1 cells were stained with annexin

Asian Pacic Journal of Cancer Prevention, Vol 11, 2010 1357
Prunella vulgaris L. Chemoprevention of Non-Small Cell Lung Cancer - Apoptosis and Cell Cycle Regulation
0
25.0
50.0
75.0
100.0
Newly diagnosed without treatment
Newly diagnosed with treatment
Persistence or recurrence
Remission
None
Chemotherapy
Radiotherapy
Concurrent chemoradiation
10.3
0
12.8
30.0
25.0
20.3
10.1
6.3
51.7
75.0
51.1
30.0
31.3
54.2
46.8
56.3
27.6
25.0
33.1
30.0
31.3
23.7
38.0
31.3
V/PI and then analysed with ow cytometry. The cells in
Q3 quadrant were viable and were negative for both PI/
annexin V; the cells in Q4, early apoptotic cells, were
positive for annexin V and negative for PI; Q2, were
positive for annexin V and PI. It can be clearly seen in
Figure 4, the cells were signicantly increased in Q2+Q4
quadrant with the increase of dose (5.7% in blank group
to 37.0% in 500 μg crude drug/mL group). Moreover,
the percentage of apoptotic cells induced by 500 μg
crude drug/mL P-60 was increased remarkably after 48 h
treatment compared with the blank group (4.2%).
As shown in Figure 5, the cells were arrested in G0/
G1 phase after being treated with P-60. After being
treated with P-60 for 48 h, the percentage of G0/G1 phase
increased from 34.3 ± 2.3% in normal group to 49.9 ±
3.1% in 500 μg/mL P-60 group (p≤0.01). The percentage
of G2/M phases decreased from 16.1 ± 3.3% to 11.5 ±
2.3% with 500 μg/mL P-60 (p≤0.01). Moreover, P-60
regulated dose-dependently cell cycle.
Discussion
Chemoprevention is the potential and optimal strategy
for reversing, delaying and preventing the occurrence and
development of NSCLC (Johnson et al., 2008). Natural
dietary agents have drawn a great deal of attention for
NSCLC prevention due to its potential activity. The
different compounds in natural dietary agents can protect
normal cells against carcinogenesis (van Breda et al.,
2008). From the results of our experiments, we can nd
that the P-60 possessed chemopreventive activity on
NSCLC in vitro and in vivo. The mechanism studies on
the chemoprevention has been shown that apoptosis is
an important pathway for chemoprevention of NSCLC
(Takeshi et al., 2000; Adhami et al., 2009; Das et al., 2009;
Amin et al., 2010). The cell cycle process is regulated
to lead the cancer cell to death. Taken together, our
results, for the rst time, suggest that the P-60 extract has
inhibitory effects in SPC-A-1 cell line and promoting the
cell apoptosis via regulating cell cycle phase.
From the above results, our investigation, for the rst
time, showed that chemoprevention ability and efcacy
of PV extract against NSCLC in SPC-A-1 cells, and A/J
mice in vivo and in vitro. Moreover, its apoptosis effect
on SPC-A-1 cells was evaluated. The results showed that
PV extract could induce apoptosis of SPC-A-1 cells via
Figure 1. Cell Proliferation Inhibition of P-60, P-30
and P-w on SPC-A-1 Cells. ﹡* p ≤ 0.01, P-60 Group vs.
P-30 Group; ﹟﹟ p ≤ 0.01, P-60 Group vs. P-w Group
Figure 3. Efcacy of P-60 Against B[a]P-induced
Lung Tumorigenesis in A/J Mice. a,b untreated P-60
Group, c,d Treated P-60 Group, Tumors are indicated by the
white arrows
Figure 2. Cell Proliferation Inhibition of Different
dose P-60 on SPC-A-1 Cells. Cisplatin was Used as
Positive Control. The Data are Expressed as Mean ± SD from
Three Triplicate Experiments (n=12)
P-60 μg crude drug/mL
b
c d
ab
c d
a
b
c
d
a
b
c
d
a
Figure 4. Apoptosis Effect of P-60 on SPC-A-1 Cells
After Annexin V-FITC/propidium Iodide Staining.
(A) Blank control group; (B) 125 μg crude drug/mL P-60
group; (C) 250 μg crude drug/mL P-60 group; (D) 500 μg
crude drug/mL P-60 group. The percentage of apoptotic cells
was expressed as Q2+ Q4
(A) (B)
(C) (D)
Figure 5. Effects of Different Concentration P-60 on
Cell Cycle Progression in SPC-A-1 Cells. Percentage of
cell cycle phases of SPC-A-1 Cells. *p ≤ 0.05, 250 & 500 μg
crude drug/mL Group vs. Blank Group; ﹟p ≤ 0.05, 250 & 500
μg Crude drug/mL Group vs. Blank Group. Data are mean ±
SD from two independent experiments

Liang Feng et al
Asian Pacic Journal of Cancer Prevention, Vol 11, 2010
1358
regulating cell cycle from G0/G1 phase to S phase. The
all results from this study indicate that PV extract can
potentially be used as a lung cancer chemopreventive
agent.
Acknowledgments
Thanks for grants from Jiangsu Science Foundation
(BK2006155) and Jiangsu Chinese Medicine Leading
Talent (2006).
References
Adhami VM, Khan N, Mukhtar H (2009). Cancer
chemoprevention by pomegranate: laboratory and clinical
evidence. Nutr Cancer, 61, 811-5.
Amin AR, Wang D, Zhang H, et al (2010). Enhanced anti-
tumor activity by the combination of natural compounds,
(-)-epigallocatechin-3-gallate and luteolin: potential role of
p53. J Biol Chem.
Blanchon F, Grivaux M, Asselain B, et al (2006). 4-year mortality
in patients with non-small-cell lung cancer:development and
validation of a prognostic index. Lancet Oncol, 7, 829-36.
Cheung HY, Zhang QF (2008). Enhanced analysis of triterpenes,
avonoids and phenolic compounds in Prunella vulgaris L.
by capillary zone electrophoresis with the addition of running
buffer modiers. J Chromatogr A, 1213, 231-8.
Choi JH, Han EH, Hwang YP, et al (2010). Suppression of
PMA-induced tumor cell invasion and metastasis by aqueous
extract isolated from Prunella vulgaris via the inhibition of
NF-kappaB-dependent MMP-9 expression. Food Chem
Toxicol, 48, 564-71.
Collins NH, Lessey EC, DuSell CD, et al (2009). Characterization
of antiestrogenic activity of the Chinese herb, prunella
vulgaris, using in vitro and in vivo (Mouse Xenograft)
models. Biol Reprod, 80, 375-83.
Das A, Bortner J, Desai D, et al (2009). The selenium analog of
the chemopreventive compound S,S’-(1,4-phenylenebis[1,2-
ethanediyl])bisisothiourea is a remarkable inducer of
apoptosis and inhibitor of cell growth in human non-small
cell lung cancer. Chem Biol Interact, 180, 158-64.
Das RK, Banerjee S,Bhattacharya S (2007). Amelioration of
benzo (a) pyrene-induced lung carcinogenesis in strain a
mice by diphenylmethyl selenocyanate. Exp and Toxicol
Pathol, 58, 351-60.
Eilstein D, Uhry Z, Lim TA, et al (2008). Lung cancer mortality
in France Trend analysis and projection between 1975 and
2012, using a Bayesian age-period-cohort model. Lung
Cancer, 59, 282-90.
Feng L, Jia XB, Shi F, et al (2010). Identication of two poly-
saccharides from Prunella vulgaris L. and evaluation on their
anti-lung adenocarcinoma activity. Molecules, 15, 5093-103.
Han EH, Choi JH, Hwang YP, et al (2009). Immunostimulatory
activity of aqueous extract isolated from Prunella vulgaris.
Food Chem Toxicol, 47, 62-9.
Hecht SS (2002). Cigarette smoking and lung cancer: chemical
mechanisms and approaches to prevention. Lancet Oncol,
3, 461-69.
Horikawa K, Mohri T, Tanaka Y, et al (1994). Moderate
inhibition of mutagenicity and carcinogenicity of benzo[a]
pyrene, 1,6-dinitropyrene and 3,9-dinitrouoranthene by
Chinese medicinal herbs. Mutagenesis, 9, 523-6.
Johnson TE, Kassie F, O’Sullivan MG, et al (2008).
Chemopreventive effect of kava on 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanone plus benzo[a]pyrene- induced
lung tumorigenesis in A/J mice. Cancer Prev Res, 1, 430-8.
Kountouri MP, Mammas IN, Spandidos DA (2010). Human
papilloma virus (HPV) in lung cancer: unanswered
questions. Lung Cancer, 67, 125.
Lee H, Lin JY (1988). Antimutagenic activity of extracts from
anticancer drugs in Chinese medicine. Mutat Res, 204,
229-34.
Lin CW, Hou WC, Shen SC, et al (2008). Quercetin inhibition
of tumor invasion via suppressing PKC delta/ERK/AP-1-
dependent matrix metalloproteinase-9 activation in breast
carcinoma cells. Carcinogenesis, 29, 1807-15.
Liu J, Johnston MR (2002). Animal models for studying lung
cancer and evaluating novel intervention strategies. Surg
Oncol, 11, 217-27.
Malkinson AM (1992). Primary lung tumors in mice:an
experimentally manipulable model of human
adenocarcinoma. Cancer Res, 52, 2670-6.
Minna JD, Roth JA, Gazdar AF, (2002). Focus on lung cancer.
Cancer Cell, 1, 49-52.
Moon DO, Kim MO, Lee JD, et al (2010). Rosmarinic acid
sensitizes cell death through suppression of TNF-alpha-
induced NF-kappaB activation and ROS generation in
human leukemia U937 cells. Cancer Lett, 288, 183-91.
Murugan RS, Priyadarsini RV, Ramalingam K, et al (2010).
Intrinsic apoptosis and NF-κB signaling are potential
molecular targets for chemoprevention by black
tea polyphenols in HepG2 cells in vitro and in a rat
hepatocarcinogenesis model in vivo. Food Chem Toxicol.
Nakamura Y (2009). Chemoprevention by isothiocyanates:
molecular basis of apoptosis induction. Forum Nutr, 61,
170-81.
Nico V Z (2001). Chemoprevention of lung cancer. Lung Cancer,
34, S91-4.
Parkin DM, Bray, Ferlay J (2001). Estimating the world lung
cancer burden: Globocan 2000. Int J Cancer, 94, 153-6.
Petronelli A, Pannitteri G, Testa U (2009). Triterpenoids as new
promising anticancer drugs. Anticancer Drugs, 20, 880-92.
Robbins D, Gu X, Shi R, et al (2010). The chemopreventive
effects of Protandim: modulation of p53 mitochondrial
translocation and apoptosis during skin carcinogenesis.
PLoS One, 5, e11902.
Shin S, Cha HJ, Lee EM, et al (2002). Alteration of miRNA
proles by ionizing radiation in A549 human non-small cell
lung cancer cells. Int J Oncol, 35, 81-6.
Vostálová J, Zdarilová A, Svobodová A (2010). Prunella vulgaris
extract and rosmarinic acid prevent UVB-induced DNA
damage and oxidative stress in HaCaT keratinocytes. Arch
Dermatol Res, 302, 171-81.
Takeshi C, Kan S, Masanori S, et al (2000). Inhibition of
N-ethyl-N’-nitro-N-nitrosoguanidine- induced duodenal
tumorigenesis in mice by whole-leaf Aloe arborescens Miller
var. natalensis Berger. Asian Pac J Cancer Prev, 1, 283-8.
Tanaka T, Kojima T, Kawamori T, et al (1993). Inhibition of
4-nitroquinoline -1-oxide-induced rat tongue carcinogenesis
by the naturally occurring plant phenolics caffeic, ellagic,
chlorogenic and ferulic acids. Carcinogenesis, 14, 1321-5.
van Breda SG, de Kok TM, van Delft JH (2008). Mechanisms
of colorectal and lung cancer prevention by vegetables: a
genomic approach. J Nutr Biochem, 19, 139-57.