Download Anti-Invasive Activity of Niacin and Trigonelline against Cancer Cells

Biosci. Biotechnol. Biochem., 69 (3), 653–658, 2005
Communication
Anti-Invasive Activity of Niacin and Trigonelline against Cancer Cells
Nobuhiro H IRAKAWA, Rieko O KAUCHI, Yutaka M IURA, and Kazumi Y AGASAKIy
Department of Applied Biological Science, Tokyo Noko University, Saiwai-cho 3-5-8, Fuchu, Tokyo 183-8509, Japan
Received November 5, 2004; Accepted January 25, 2005
The effects of niacin, namely, nicotinic acid and
nicotinamide, and trigonelline on the proliferation and
invasion of cancer cells were studied using a rat ascites
hepatoma cell line of AH109A in culture. Niacin and
trigonelline inhibited the invasion of hepatoma cells at
concentrations of 2.5–40 M without affecting proliferation. Hepatoma cells previously cultured with a
reactive oxygen species (ROS)-generating system
showed increased invasive activity. Niacin and trigonelline suppressed this ROS-potentiated invasive capacity
through simultaneous treatment of AH109A cells with
the ROS-generating system. The present study indicates
for the first time the anti-invasive activities of niacin and
trigonelline against cancer cells.
Key words:
hepatoma; invasion; nicotinamide; nicotinic
acid; torigonelline
Endless proliferation and metastasis are two biological properties of cancer cells. Metastasis is the
primary cause of death in human cancer. Cancer
metastasis is attained by a sequence of steps,1) of which
invasion is a particularly complicated process and the
key step in the metastatic cascade.2,3) Furthermore,
invasion and metastasis might be correlated with
reactive oxygen species (ROS).4,5) Some food factors
with antioxidative activity inhibit the invasion of
hepatoma cells.5–8) Coffee is reported to suppress tumor
cell invasion by reducing oxidative stresses in vitro and
ex vivo.9) Trigonelline is a major constituent in coffee
and niacin-related compound. Niacin is one of the watersoluble vitamins, and reportedly has moderate radicalscavenging activity.10) The present study was attempted
to define the effects of niacin, i.e., nicotinic acid and
nicotinamide, and trigonelline on the proliferation and
invasion of hepatoma cells.
Nicotinic acid and nicotinamide were purchased from
Tokyo Kasei Kogyo (Tokyo), and trigonelline from
Sigma Chemical (St. Louis, MO). Niacin and trigonelline were dissolved in culture medium and sterilized
with filtration prior to use.
A rat ascites hepatoma cell line of AH109A was
provided by the Institute of Development, Aging, and
Cancer of Tohoku University, Sendai, Japan. AH109A
cells were maintained in the peritoneal cavities of male
y
Donryu rats, prepared from accumulated ascites, and
cultured in vitro in Eagle’s minimum essential medium
(MEM, Nissui Pharmaceutical, Tokyo) containing 10%
calf serum (CS, obtained from Gibco BRL, Grand
Island, NY) (10% CS/MEM), as described previously.5)
To eliminate contaminated macrophages and neutrophils, cells were cultured at least for 1 week after
isolation and used for the assays described below.
The effects of niacin and trigonelline on the proliferation of AH109A cells were examined by measuring
the incorporation of [methyl-3 H]thymidine (20 Ci/
mmol, New England Nuclear, Boston, MA) into the
acid-insoluble fraction of cells for 4 h, as described
previously.11)
The effects of niacin and trigonelline on the invasion
of AH109A cells were examined by the co-culture
system,12) with slight modifications as described previously.5) Briefly, mesothelial cells (M-cells) were
isolated from the mesentery of male Donryu rats (6–8
weeks old). After digestion by trypsin, 1:2 105 cells
were plated in a 60-mm culture dish with 2-mm grids
(Nunc A/S, Roskilde, Denmark), and cultured for 7–10
days to attain a confluent state in 10% CS/MEM. Then
AH109A cells (2:4 105 cells per dish) were applied on
the monolayer of M-cells in 10% CS/MEM with niacin
or trigonelline for 24 h and 48 h respectively. Invaded
cells and colonies underneath M-cells were counted with
a phase-contrast microscope. Usually ten areas were
counted, and the invasive activity of AH109A cells
was expressed as the number of invaded cells and
colonies/cm2 .
To examine the bioavailability of trigonelline, the
time-dependent effect on the proliferation and invasion
of AH109A was studied using trigonelline-loaded rat
serum. Trigonelline was suspended in a 0.3% carboxymethyl cellulose sodium salt (CMC, Wako Pure Chemicals, Osaka, Japan) aqueous solution at a concentration
of 17.4 mg (¼ 100 mmol)/ml. Trigonelline suspension
was intubated at a dose of 100 mmol/ml/100 g body
weight to male Donryu rats (6 weeks old) which had
been fasted overnight, and blood was collected at the
time points indicated in Fig. 2B. Serum was prepared by
centrifugation, sterilized by filtration, and added to the
culture media at a concentration of 10% instead of CS
for the proliferation and invasion assays. The prolifer-
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654
N. HIRAKAWA et al.
Fig. 1. Effects of Nicotinic Acid (A) and Nicotinamide (B) on the Proliferation and Invasion of AH109A Cells.
Nicotinic acid and nicotinamide were directly dissolved in culture medium at the concentrations indicated in the figure. The proliferative
activity of AH109A cells was determined by the incorporation of [methyl-3 H] thymidine and the invasive activity by invasion assay, as described
in the text. Data are the means SEM of six wells (proliferation) and ten areas (invasion). Values not sharing a common letter are significantly
different at P < 0:05 by the Tukey–Kramer multiple comparisons test.
ative and invasive activities of AH109A in the presence
of trigonelline-loaded rat serum were measured as
described above.
To study the effect of ROS on invasion, AH109A
cells were cultured for 4 h in the absence or presence of
10 mM niacin or trigonelline and/or 4 mg/ml hypoxanthine (HX, Sigma) with 7 104 U/ml xanthine oxidase
(XO, Sigma).13,14) To investigate the effect of trigonelline-loaded rat serum, the same treatment as described
above was performed with medium containing 10%
trigonelline (100 mmol/ml/100 g body weight)-loaded
rat serum or vehicle (0.3% CMC)-loaded rat serum
instead of the medium containing 10% CS. The treated
AH109A cells were then washed once with 10% CS/
MEM and seeded on the M-cell monolayer in 10% CS/
MEM without niacin, trigonelline, or trigonelline-loaded
serum and ROS. After culturing for 24 or 48 h, invaded
cells and colonies underneath M-cells were counted with
a phase-contrast microscope, as described above.
Intracellular peroxide levels in AH109A cells were
assessed by flow cytometric analysis using a fluorometric probe (20 ,70 -dichlorofluorescin diacetate, DCFH-DA,
Molecular Probes, Eugene, OR)15) with Epics Elite
EPS (Beckman-Coulter, Hialeah, FL), as described
previously.16)
Data were expressed as means SEM. Multiple
comparison was performed by one-way analysis of
variance (ANOVA) followed by the Tukey–Kramer
multiple comparisons test; P < 0:05 was considered
statistically significant. All procedures for animal experiments in this report were conducted in accordance
with guidelines established by the Animal Care and Use
Committee of Tokyo Noko University, and approved by
the committee.
First we examined the effect of niacin on the
proliferation and invasion of AH109A cells (Fig. 1).
Nicotinic acid and nicotinamide exerted no influence on
the proliferation of AH109A cells at concentrations up
to 40 mM in the medium. In contrast, nicotinic acid and
nicotinamide commenced to suppress invasion at 2.5 mM,
suppressed it approximately linearly to 20 mM, and
maintained the inhibitory effect up to 40 mM. The effect
of nicotinic acid was almost identical to that of
nicotinamide. While trigonelline suppressed AH109A
invasion at concentrations of 2.5–40 mM, it showed little
influence on hepatoma proliferation at the same concentrations (Fig. 2A). As shown in Fig. 2B, rats were
given oral administration of trigonelline, blood was
withdrawn 0, 0.5, 1, 2, 3, 6, and 12 h later, and the effect
of trigonelline-loaded rat serum on the proliferation and
invasion of AH109A cells was investigated. Serum
obtained 2 h after oral administration most strongly and
significantly inhibited the invasion of AH109A cells,
and significant inhibition was also observed at 3 and 6 h
later. These results suggest that trigonelline can be
absorbed from the gastrointestinal tract and that trigonelline itself or its metabolite(s) can retain their
effectiveness in the blood, although the possibility that
orally administered trigonelline induced an effective
factor(s) originating in the host cannot be excluded. The
trigonelline-loaded rat serum, however, had little effect
on the proliferation of AH109A cells at any time point
investigated. To examine whether niacin would inhibit
the ROS-potentiated invasion of hepatoma cells, the
invasion assay was performed with AH109A cells precultured in ROS-containing medium. As shown in
Niacin and Trigonelline Inhibit Cancer Cell Invasion
655
Fig. 2. Effects of Trigonelline (A) and Trigonelline-Loaded Rat Serum (B) on the Proliferation and Invasion of AH109A Cells.
(A) Trigonelline was directly dissolved in culture medium at the concentrations indicated in the figure. (B) Trigonelline was suspended in
0.3% CMC aqueous solution. Trigonelline (100 mmol/ml/100 g body weight)-loaded rat serum was obtained at the indicated times after oral
intubation. With the medium containing 10% of each rat serum, the proliferation and invasion assays were conducted as described in the text.
Data are the means SEM of 6 wells (proliferation) and 10 areas (invasion). Values not sharing a common letter are significantly different at
P < 0:05 by the Tukey–Kramer multiple comparisons test.
Fig. 3. Effects of Nicotinic Acid (A) and Nicotinamide (B) on the Invasion of AH109A Cells Pre-Treated with HX and XO.
AH109A cells were cultured for 4 h in the absence or presence of 10 mM of niacin and/or HX (4 mg/ml) with XO (7 104 U/ml). After
treatment, the cells were washed and applied a M-cell monolayer in 10% CS/MEM without niacin and HX–XO. The invasive activity was
determined 24 h later, as described in the text. Data are the means SEM of ten areas. Values not sharing a common letter are significantly
different at P < 0:05 by the Tukey–Kramer multiple comparisons test.
Fig. 3, the invasive activity of AH109A cells precultured in the HX–XO system which generated ROS
was significantly higher than that of AH109A cells with
no treatment. Nicotinic acid and nicotinamide at 10 mM
inhibited the ROS-potentiated invasive activity by preculturing the hepatoma cells with HX and XO. Likewise,
trigonelline itself (10 mM) (Fig. 4A) and trigonelline
(100 mmole/100 g body weight)-loaded rat serum
(Fig. 4B) suppressed the ROS-potentiated invasive
activity of the pre-treated cells. As shown in Fig. 5,
AH109A cells treated with HX–XO for 1 h contained
more intracellular peroxides than did control cells
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N. HIRAKAWA et al.
Fig. 4. Effects of Trigonelline (A) and Trigonelline-Loaded Rat Serum (B) on the Invasion of AH109A Cells Pre-Treated with HX and XO.
(A) AH109A cells were cultured for 4 h in the absence or presence of 10 mM of trigonelline and/or HX (4 mg/ml) with XO (7 104 U/ml).
After treatment, the cells were washed and applied on the M-cell monolayer in 10% CS/MEM without trigonelline and HX–XO. The invasive
activity was determined 48 h later as described in the text. (B) The same treatment was performed for 4 h with the medium containing 10%
trigonelline (100 mmol/ml/100 g body weight)-loaded rat serum or vehicle (0.3% CMC)-loaded rat serum obtained 2 h after oral administration.
After treatment, AH109A cells were washed and subjected to invasion assay, as described in (A). Data are the means SEM of 10 areas. Values
not sharing a common letter are significantly different at P < 0:05 by the Tukey–Kramer multiple comparisons test.
Fig. 5. Flowcytometric Analysis of Intracellular Peroxide in AH109A Cells Treated with Exogenous Reactive Oxygen Species (ROS) and
Trigonelline or Trigonelline-Loaded Rat Serum (RS).
AH109A cells were cultured for 1 h without or with 7 104 U/ml XO and 4 mg/ml HX in the absence or presence of 10 mM trigonelline in
CS (A) or trigonelline-loaded RS (B). After treatment for 1 h, DCFH-DA was added at a final concentration of 64 mM and the cells were incubated
for 20 min. Basal and ROS-activated intracellular peroxide levels are indicated as solid lines. A representative result is shown.
(control vs. HX–XO) when analyzed with a flow
cytometer using DCFH-DA as an indicator. Trigonelline
(10 mM) and trigonelline (100 mmol/100 g body weight)loaded rat serum did not inhibit this rise in the
intracellular peroxide levels of AH109A cells (HX–
XO vs. HX–XO + trigonelline in Fig. 5A, HX–XO vs.
HX–XO + trigonelline-loaded rat serum in Fig. 5B).
Neither nicotinic acid nor nicotinamide scavenged the
ROS-induced rise in the intracellular peroxide levels at
10 mM (data not shown).
In the present study, we found for the first time that
niacin and trigonelline can inhibit the invasive activity
Niacin and Trigonelline Inhibit Cancer Cell Invasion
of cancer cells at low concentrations at which they
exerted no influence on proliferation. Niacin, trigonelline, and trigonelline-loaded rat serum were found to
inhibit ROS-induced elevation of the invasive activity of
AH109A cells. In a separate experiment, we analyzed
the XO activity by measuring uric acid generated by
HX–XO reaction, and nicotinic acid, nicotinamide, and
trigonelline were found not to affect XO activity at
10 mM, suggesting that they were not involved in the
inhibition of the ROS-potentiated invasion through
interference with ROS generation under the experimental conditions adopted here.
Nicotinic acid and nicotinamide are reported to have
moderate radical-scavenging activities at a high concentration of 10 mM.10) Hence we measured intracellular
peroxide levels after treating AH109A cells with niacin
in the absence or presence of HX–XO. But niacin failed
to suppress the ROS-induced rise in intracellular
peroxide levels. The failure of niacin to scavenge the
intracellular peroxides in the present study might be due
to a low concentration of 10 mM. Trigonelline was also
found not to scavenge the intracellular peroxides, this
being consistent with the previous finding that trigonelline had almost no scavenging ability against OH
radical.10) These results suggest that the free-radical
scavenging activity might not be involved in the
suppressive effect of niacin and trigonelline on the
ROS-potentiated invasion of hepatoma cells, unlike
resveratrol,17) a polyphenol present in grapes. Coffee
reportedly suppresses hepatoma cell invasion by reducing oxidative stress in vitro.9) Thus it is unlikely that
trigonelline is responsible for the inhibitory action of
coffee on hepatoma invasion by way of scavenging
ROS. Other components such as chlorogenic acid and
caffeic acid18) might be responsible for the suppressive
effect of coffee on invasion through reduction of
oxidative stress.
The niacin concentration in normal human plasma is
minute: reported to be under 0.4 mM.19) If this be true of
CS or RS, then the final concentration of niacin derived
from the serum in the control medium that contains 10%
CS or RS is estimated to be less than 0.04 mM. This
concentration range is below 1/250 of 10 mM, which is
the least effective dose of niacin, suggesting that niacin
derived from serum might be negligible from the
pharmacological point of view. In contrast, the basal
medium, MEM, provides nicotinamide to the medium at
a final concentration of about 8 mM. This concentration is
comparable to the least effective dose of nicotinamide
(10 mM). We found that nicotinamide did not inhibit XO
activity up to 100 mM. Thus nicotinamide derived from
both serum and MEM in the control medium (approximately 8 mM) might not exert any influence on XO
activity, and probably not on invasive activity. As to the
latter activity, we need to confirm this using nicotinamide-free MEM.
Potentiation of invasive activity of AH109A cells by
ROS is reportedly mediated by the autocrine/paracrine
657
16)
loop of hepatocyte growth factor (HGF), which is
known as a cell motility factor.20) Hence there is a
possibility that niacin and trigonelline suppress the
ROS-induced increase in AH109A invasion by interrupting this loop through, for instance, an inhibition of
HGF receptor phosphorylation. Since prostaglandins are
known to enhance the invasion of cancer cells,21,22)
another possibility that niacin and trigonelline interrupt
prostaglandin synthesis cannot be ruled out. Further
studies including those on other possibilities are needed
to clarify the exact modes of action of niacin and
trigonelline on hepatoma cell invasion.
In conclusion, we found for the first time that niacin,
trigonelline, and trigonelline-loaded rat serum inhibited
the invasion of AH109A cells without affecting the
proliferation of the cells, and suppressed the ROSpotentiated invasive capacity of hepatoma cells. The
mechanisms by which niacin and trigonelline inhibit
invasion remain to be elucidated.
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