Vol 3, Issue 2, 2021 (49-59)
http://journal.unpad.ac.id/idjp
*Corresponding author,
e-mail : tkyaraki@gunma-u.ac.jp (T. Araki)
https://doi.org/10.24198/idjp.v3i2.35909
© 2021 Y. Takahashi et al
Effect of Nicotine- and Tar-Removed Cigarette Smoke Extract on Cancer Metastasis
Yuta Takahashi, Ph.D.1,2,3, Takuya Araki, Ph.D.2,4*, Ayumu Nagamine, Ph.D.1,2,
Hideaki Yashima, Ph.D.4, Daisuke Nagano, Ph.D.2, Kyoko Obayashi, Ph.D.1 and
Koujirou Yamamoto, Ph.D.2,4
1Education Center for Clinical Pharmacy, Faculty of Pharmacy, Takasaki University of Health
and Welfare, 60 Nakaorui-machi, Takasaki, Gunma, 370-0033, Japan.
2Department of Clinical Pharmacology and Therapeutics, Gunma University Graduate School
of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan.
3Department of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa
Women’s University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179, Japan.
4Department of Pharmacy, Gunma University Hospital, 3-39-15 Showa-machi, Maebashi,
Gunma 371-8511, Japan.
Submitted : 3 Oct 2021, Revised : 14 Oct 2021, Accepted : 26 Oct 2021, Published : 3 Nov 2021
Abstract
Cigarette smoking is known to impact the promotion of carcinogenesis and tumor metastasis.
On the other hand, some components in smoke were found to have health-promoting effects,
and cancer suppressor effects of components in tobacco smoke have attracted attention.
Although some studies showed the cancer suppressive effect of cigarette smoke extract (CSE)
in vitro study, the effect of CSE administration on cancer is controversial. In this study, we
investigated the effect of CSE-administration on tumor metastasis in a spontaneous tumor
metastasis model using B16-BL6 cells, which is more clinical conditions. C57BL/6NCr mice
were subcutaneously inoculated B16-BL6 cells into the footpad of the right rear leg. CSE was
intraperitoneally administrated for 28 days from the day of inoculation. At 2 weeks after
inoculation, the primary focus was excised. Subsequently, survival days of the mice were
recorded to determine the effect of CSE-administration on spontaneous metastasis. The effect
of CSE, α, β-unsaturated ketones, and aldehydes on B16-BL6 cell invasiveness were confirmed
by matrigel invasion assay. Survival days of mice injected with 100% CSE was significantly
shortened than that of control. B16-BL6 cell invasiveness was accelerated by the treatment
with 0.1% CSE and 3 µM of crotonaldehyde. Intraperitoneal CSE-administration may progress
spontaneous metastasis of B16-BL6 cells via enhancement of B16-BL6 cell invasiveness. As
the cause, we found that crotonaldehyde contained in CSE may enhance the invasion ability of
cancer cells. To clarify the cancer-suppressing effect of tobacco components, the effect of
crotonaldehyde-removed CSE on tumor should be assessed in detail.
Keywords: cigarette smoke extract (CSE), metastasis, crotonaldehyde (CA), B16-BL6 mouse
melanoma cells, invasion
Y. Takahashi et al / Indo J Pharm 3 (2021) 49-59
50
1. Introduction
Cigarette smoking is widely known to
impact the promotion of carcinogenesis and
tumor metastasis in several cancer types [1-
4]. Especially in lung cancer, it is reported
that smoking rate and carcinogenic risk are
significantly correlated, and about 70% of
lung cancer were attributed to smoking in a
study of Japanese men with a relatively low
increase in cancer risk due to smoking [1].
Similarly, for the esophagus, liver, pancreas,
head and neck, cervix, and bladder cancers,
smoking has been reported to be one of the
significant carcinogenic risks [5-10].
The Mainstream of cigarette smoke is
composed of about 4,300 kinds of particulate
components and about 1,000 kinds of gas
components [11]. Of these, approximately 70
components have been reported to be
carcinogenic or may have some adverse
health effects [12]. In a study of cigarette
smoke, cigarette smoke exposure was
reported to increase lung metastasis and
tumor volume in colon and pancreatic cancer
cell lines in mice [13, 14]. Nicotine, known
as one of the causes of tobacco dependence,
is also known to impact tumor growth and
progression [15, 16], and Nguyen et al.
reported that nicotine promotes the
proliferation and migration of melanoma cell
line by regulating PD-L1 expression via α9
nicotinic acetylcholine receptors [17].
On the other hand, Sayed et al. found
health-promoting components in cigarette
smoke [18], and reported that some
components, such as cembratriene-4, 6-diol
and methyl vinyl ketone (MVK), have a
tumor-suppressing activity [19-22]. Thus,
cancer suppressor effects of components in
tobacco smoke have attracted attention, and
several studies about cancer suppression
using tobacco smoke have been conducted.
Saito et al. showed that the tumor-promoting
activity of 12-O-tetradecanoylphorbol 13-
acetate, one of the phorbol esters, was
suppressed by cembratriene-4, 6-diol,
isolated from tobacco smoke concentrate, in
vitro study [20], and Sayed et al. reported that
cembratriene-4, 6-diol could inhibit tumor
cell invasion [19]. In addition, our group has
also reported that the pretreatment of highly
metastatic B16-BL6 mouse melanoma (B16-
BL6) cells with nicotine- and tar-removed
cigarette smoke extract (CSE) could reduce
the number of lung nodules of B16-BL6 cells
in hematogenous lung metastasis model mice
injected with B16-BL6 cells through the tail
vein [21]. Furthermore, as a study to evaluate
the effect of CSE on tumor metastasis in mice,
Hatai et al. conducted an in vivo study on the
tumor-suppressive activity of intraperitoneal
(i.p.) administration of CSE in mice and
showed the possibility of i.p. administration
of CSE to suppress liver metastasis in a
model of transsplenic liver metastasis using
colon-26 [22]. They further searched for
components involved in cancer metastasis
suppression and found MVK, an α, β-
unsaturated ketone contained in tobacco
smoke, can suppress metastasis through
suppression of invasion of colon-26 cells
[22]. However, in the study by Hatai et al.,
the survival time of mice was not found to be
prolonged by CSE administration [22], and
the cancer metastasis-suppressing effect of
CSE administration on living organisms has
51
not been clarified yet. Furthermore, since
CSE was administered intraperitoneally in
their study, it cannot be ruled out that liver
metastasis may have been suppressed by
direct exposure of CSE to the primary organ
of tumor cell engraftment, spleen. Thus, the
cancer metastasis-suppressing effect of CSE
administration on living organisms remains
controversial.
In this study, we investigated the effect
of CSE on tumor metastasis a spontaneous
tumor metastasis model in which B16-BL6
cells were seeded subcutaneously in the
footpad and subsequently developed lung
metastatic nodules, which is more clinical
conditions and can rule out the effects of
direct exposure of CSE to the primary organ
of tumor cell engraftment.
2. Materials and Methods
2.1. Materials
Frontier Lights brand cigarettes
containing 1 mg of tar and 0.1 mg of nicotine
per cigarette, were purchased from Japan
Tobacco, Inc. (Tokyo, Japan). Cambridge
filters, used to remove almost all particles
and nicotine from cigarette smoke, were
obtained from Heinr. Borgwaldt GmbH
(Hamburg, Germany). Fetal bovine serum
(FBS) was from BioWest Co. (Nuaillé,
France). EDTA trypsin solution (EDTA: 2.2
mM, trypsin: 0.25%) was from Mediatech,
Inc. (Manassas, VA, USA).
Penicillin/streptomycin solution (penicillin:
50,000 U/mL, streptomycin: 50 mg/mL) was
from Cosmo Bio Co., Ltd. (Tokyo, Japan).
Dulbecco’s modified Eagle’s medium
(DMEM) with L-glutamine was from
Invitrogen Corp. (Carlsbad, CA, USA).
Dulbecco’s phosphate-buffered saline
without calcium and magnesium [DPBS (-)]
was from Nissui Pharmaceutical Co., Ltd.
(Tokyo, Japan). Growth factor-reduced
Matrigel matrix and FALCON cell culture
inserts were from Becton Dickinson Labware
(Bedford, MA, USA).
2.2. Preparation of CSE
The CSE was prepared according to the
method described in a previous report [21].
Briefly, CSE was obtained by bubbling the
filtered mainstream of smoke (gas phase)
into DPBS (-) (1 mL per 3 cigarettes). As a
filter, Cambridge filter was used to remove
the particle phase containing tar and nicotine.
The suction speed was kept constant (1
L/min) using a suction pump (Nippon
Rikagaku Kikai Co., Ltd., Tokyo, Japan), and
smoke was bubbled for 1 min. The CSE
contained DPBS (-) solution was
immediately filtrated with a 0.22 μm filter.
The filtered solution, 100% CSE, was stored
at -80°C until use and diluted to various
concentrations with DPBS (-) at the time of
use.
2.3. Animals
Specific pathogen-free male
C57BL/6NCr mice (7 weeks old) purchased
from Japan SLC, Inc. (Hamamatsu, Japan)
were used as metastatic melanoma syngeneic
animals. Mice were maintained in an air-
conditioned room (23±2°C and 60±10%
humidity) under an artificial 12-hour
light/dark cycle (7:00 a.m.- 7:00 p.m.). Food
and water were given ad libitum during the
experimental period. This study was
approved by the Animal Experiment
52
Committee of Mukogawa Women's
University (Approval No. P-11-2012-06-A),
and all procedures followed the Guidelines
for the Care and Use of Laboratory Animals
at the University.
2.4 Cells
A highly metastatic B16-BL6 mouse
melanoma cell line was kindly provided by
Dr. Futoshi Okada of Tottori University
(Yonago, Japan). Cells less than 50 passages
were used in all experiments. B16-BL6 cells
were cultured in DMEM containing 10%
FBS and 0.1% penicillin/streptomycin
solution in a humidified incubator at 37°C in
the presence of 5% CO2.
2.5. Evaluation of spontaneous metastasis
of tumor cells
Sub-confluent B16-BL6 cells were
harvested with EDTA trypsin solution and
resuspended in DPBS (+) to the appropriate
concentrations. Fifty microliters of cell
suspension (2 × 107 cells/mL) were
subcutaneously injected into the footpad of
the right hind leg of syngeneic C57BL/6NCr
mice. Two weeks after the inoculation, the
mice were anesthetized with diethyl ether
and the enlarged primary tumor was excised.
After 4 weeks from tumor inoculation, 10, 30,
and 100% CSE were administrated with i.p.
to the mice at the dose of 16 mL/kg daily. As
a control, DPBS (-) was administered. The
survival of the mice in each group was then
followed up for 100 days and the date of
death was recorded. The survival duration
was determined as the number of days after
tumor cell inoculation. Dead mice were
dissected for confirmation of tumor
metastases. Each group contained 7 animals
at the start of the experiment.
2.6. Proliferation assay
B16-BL6 cells were seeded on a 12
well plate at 1 × 105 cells/well. Then, cells
were treated with CSE (0.01, 0.03 and 0.1%
as final concentration), MVK (1, 3, 10 µM),
CA (1, 3, 10 µM), ACR (1, 3, 10 µM) or
DPBS(-). After 72 h of exposure to CES,
cells were collected and the number of cells
in each well was determined using a coulter
counter.
2.7. Matrigel invasion assay
B16-BL6 cells were resuspended in
FBS-free DMEM to obtain a concentration of
4.0 × 105 cells/mL, and 500 µL of cell
suspension was added to the upper layer
within the cell culture insert coated with
Matrigel on the filter. To the lower layer,
DMEM containing fibronectin as a
chemoattractant was added. After 24 h of
incubation with CSE (0.01, 0.03 and 0.1% as
final concentration), 3 µM of MVK, 3 µM of
CA, 3 µM of ACR or 0.1% of DPBS(-).
Uninfiltrated cells, which remain on the top
of the filter, were wiped with a swab. The
infiltrated cells on the bottom surface of the
filter were Giemsa stained and counted under
a microscope.
2.8. Statistical Analyses
Data are expressed as the mean ± S.E.
Survival data were analyzed by the log-rank
test. Data from in vitro experiments were
analyzed by Dunnett's test. Statistical
analyses were performed using the Graphpad
Prism 4 software package (Graphpad
53
Software, Inc., San Diego, CA, USA). A
difference was considered significant when
p<0.05.
3. Results
3.1. Effect of intraperitoneal CSE
administration on survival time of
spontaneous metastasis model mice
Although administration of CSE with a
concentration of under 30% did not affect the
survival time of mice, the survival time was
significantly shortened by administration of
100% CSE compared to controls (Fig. 1).
There was no significant change in the
weight and thickness of the primary tumor
collected 14 days after inoculation of B16-
BL6 cells in each group (data not shown).
Figure 1. Effect of intraperitoneal CSE
administration on survival time of
spontaneous metastasis model mice
*P<0.05 vs control (n = 5-7).
Figure 2. Effect of CSE exposure on the (a) invasion and (b) proliferation of B16-BL6 cells.
*P<0.05 vs control ((a) n = 3 and (b) n = 6, respectively).
54
3.2. Effect of CSE on invasion and
proliferation of B16-BL6 cells
Invasiveness was significantly
increased in the 0.1% CSE exposure group
compared to the control (Fig. 2a). On the
other hand, the cell proliferation rate was not
affected by CSE exposure (Fig. 2b).
3.3. Effect of MVK, ACR, and CA on B16-
BL6 cell invasion
CA increased invasiveness of B16-BL6
cells about 4-fold compared to the control,
though ACR and MVK did not show a
significant effect on the invasiveness of B16-
BL6 cells (Fig. 3).
Figure 3. Effect of MVK, ACR, and CA on
the invasion of B16-BL6 cells. *P<0.05 vs
control (n = 5-6).
Figure 4. Effect of MVK, ACR, and CA on the proliferation of B16-BL6 cells. *P<0.05 vs
control (n = 4).
3.4. Effect of MVK, ACR, and CA on B16-
BL6 cell proliferation
The exposure of 10 μM of MVK or
ACR significantly suppressed the
proliferation of B16-BL6 cells, whereas 3
μM or under of MVK and ACR did not affect
the proliferation rate of B16-BL6 cells, and
CA did not inhibit proliferation regardless of
exposure concentration (Fig. 4).
4. Discussions
In this study, we assessed the effect of
intraperitoneal administration of CSE on
tumor metastasis using a spontaneous cancer
55
metastasis model in which B16-BL6 cells are
disseminated subcutaneously in the footpad
as primary cancer. As a result, as Hatai et al.
showed in a study on intraperitoneal
administration of CSE to transsplenic liver
metastasis model mice using colon-26 cells,
CSE administration could not prolong the
survival time of mice [22], but rather
significantly shortened the survival of
C57BL/6NCr mice when 100% CSE was
used. Since Hatai et al. have reported that
intraperitoneal administration of 100% CSE
was not toxic to non-cancer-planted mice
[22], the shortening of survival time by 100%
CSE administration confirmed in this study
was considered to be the result of CSE
affecting the proliferation and metastasis of
B16-BL6 cells. In addition, because the
primary lesion was resected 14 days after
cancer cell dissemination and the thickness
or weight of the primary lesion did not
change at that point, the shortened survival time
was considered to be caused by the effect of CSE
on cancer metastasis rather than the primary
lesion. So, we assessed the effect of CSE and
its major components on the invasion and
proliferation of cancer cells to find the cause
of the shortened survival time. First, we
analyzed the effect of CSE on cancer cell
proliferation and infiltration. As a result,
although CSE exposure did not affect cancer
cell proliferation as Hatai reported, CSE
promoted cancer cell invasion, which was
different from the report by Hatai et al. These
results suggested that the decrease in survival
time by CSE may be due to an increase in
invasion ability of B16-BL6 cells.
Next, to clarify the causative agent of
this invasiveness promotion, we assessed the
effects of MVK, ACR, and CA, which are α,
β-unsaturated ketones and aldehydes and
considered as main components of CSE, on
the invasion of B16-BL6 cells. As a result,
while those substances did not affect the
proliferation of cancer cells, the invasion
ability of B16-BL6 cells was significantly
increased by CA. From these results, it was
considered that CA was involved in the
decrease in survival time of CSE-
administered mice through the promotion of
B16-BL6 cell invasion. Although CA has
been reported to have carcinogenicity and
lung cell injury [23, 24], its impact on the
invasion ability of tumors has not been
reported. To the best of our knowledge, this
is the first report that CA has significantly
improved the infiltration capacity of cancer
cells.
Despite preparing CSE by a method
shown in Hatai’s report, CSE enhanced
cancer cell invasion in our study, while CSE
showed inhibitory efficacy on cancer
invasion in Hatai’s report [22]. As the reason
for this difference, it was considered to be a
large difference in CA concentration in CSE
in addition to the difference in cell lines.
Slight changes in conditions when extracting
CSE or a difference in the lot of tobacco to
use may cause a difference in the components
contained in tobacco smoke. We need to
examine in detail the factor affecting CA
content in smoke and differences in CA
content in smoke between lots of tobacco. In
any case, in order to assess the tumor-
suppressive efficacy of CSE in detail, it was
considered important to remove CA, which
56
has an enhancing effect on cancer invasion
ability, not only nicotine and tar.
Although CSE suppressed metastasis
of cancer cells in the previous study, CSE
could not suppress metastasis in our study,
and the cause of the discrepancy in these
results is thought to be due to the difference
in the level of CSE exposure to cancer cells.
In vitro study about the effect of tobacco
smoke, CSE was exposed directly to cancer
cells, and cancer cells are exposed to CSE
with high level. Similarly, in the study of
Hatai et al., intraperitoneal administration of
CSE could expose the spleen, where cancer
cells first engrafted in the model, and cancer
cells to high levels of CSE. And this high
level of CSE exposure can have a high
metastasis-suppressing effect. Bourgeois et
al. reported that cellular response to CSE
exposure is dependent not only on the
nominal concentration of CSE, but also on
specific experimental variables, including
the total cell number, and the volume of CSE
solution used [25]. They also reported that
the effective dose of CSE is more accurately
related to the amount of bioavailable
chemicals per cell. Similarly, Lee et al.
reported that CSE-induced cytotoxicity was
reduced at high cell densities [26]. Based on
these reports, it is considered that the
metastasis of cancer cells was not suppressed
in this study because the transfer of CSE to
the subcutaneous footpad where cancer cells
engrafted was insufficient. In this study, we
planned to weigh the lung, which is the site
of metastasis, to evaluate the effect of CSE
on metastasis. However, because of severe
damage to the lung due to cancer metastasis,
lung tissue could not be collected, and the
cancer metastasis suppressing effect of CSE
could not be evaluated in detail. In addition,
since CA could not be removed from CSE,
the effect of CA-removed CSE on tumor
could not be assessed. We need to investigate
in detail whether exposure to CA-removed
CSE can suppress cancer.
5. Conclusion
In conclusion, intraperitoneal
administration of CSE significantly
shortened the survival time of spontaneous
lung metastasis model C57BL/6NCr mice
seeded with B16-BL6 cells. As the cause, we
found that CA contained in CSE may
enhance the invasion ability of cancer cells.
To the best of our knowledge, this is the first
report that CA has significantly improved the
infiltration capacity of cancer cells. In the
future, to clarify the cancer-suppressing
effect of tobacco components, it was
considered important to evaluate the cancer
suppression effect of CA-removed CSE in
detail and to establish a delivery system,
which can transfer the components in CSE to
cancer tissues efficiently.
Acknowledgement
We thank Prof. Kazuki Nakamura,
Assoc. Prof. Noriko Yoshikawa, and Lecturer
Shizuyo Horiyama of Mukogawa Women's
University for their advice on research.
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