When multiple metastases are found at the time of clinical diagnosis, surgical operations
are usually not performed, and the patients are treated mainly by radiotherapy or
chemotherapy with various anticancer drugs. To investigate the effects of anticancer
drugs on tumour cells and their lung metastasis, the experimental lung metastasis
model by Fidler and Kripke (1977) has been used (Miyoshi et al, 2000). Although anticancer
drugs are widely used for cancer therapy, a sufficient quantity of the drug for treatment
cannot be administered in many cases due to severe side effects.
Malignant melanoma is difficult to cure. Since melanoma cells have the ability to
spread to distal organs, various anticancer drugs have been used in combination to
try to improve the therapeutic effect on malignant melanoma. For example, lovastatin
has been shown to potentiate the antitumour activity of doxorubicin in murine melanoma
(Feleszko et al, 2002), and exisulind in combination with docetaxel has been shown
to inhibit the growth and metastasis of human lung cancer (Chan et al, 2002). Recently,
the antitumour effect of combinational therapy using an anticancer drug and a phytochemical
has been studied (Darzynkiewicz, 1995; Christensen and LeBlanc, 1996; Singh et al,
2002). Phytochemicals such as flavonoid have relatively low levels of toxicity (Singh
et al, 2002) and can induce apoptosis or cell cycle arrest in tumour-derived cells
(Darzynkiewicz, 1995). We previously reported that an extract of a medicinal plant
lemon grass containing various phytochemicals inhibited the formation of azoxymethane-induced
DNA adducts and aberrant crypt foci in the rat colon (Suaeyun et al, 1997).
Betulinic acid (BA), a pentacyclic triterpene isolated from the root bark of Morus
australis or Clerodendrum mandarinorum and many other medicinal plants (Zhu et al,
1996; Ko et al, 1997), has recently been reported to have cytotoxic activity against
several tumour cell lines (Pisha et al, 1995; Fulda et al, 1997, 1998; Wick et al,
1999; Kwon et al, 2002; Wachsberger et al, 2002). Betulinic acid is a novel experimental
antineoplastic agent for human melanoma cells in vitro (Pisha et al, 1995; Wachsberger
et al, 2002). Betulinic acid also induces apoptosis in neuroectodermal tumour and
glioma cells (Fulda et al, 1997, 1998; Wick et al, 1999). Furthermore, BA has been
shown to inhibit growth factor-induced angiogenesis in vitro (Kwon et al, 2002).
Vincristine (VCR) is one of the major chemotherapeutic agents used for the treatment
for malignant melanoma. Vincristine showed antitumour effects on B16 melanoma cells,
but a high dose of VCR induces severe bone marrow toxicity (Morrow et al, 1987). In
this study, we evaluated the effectiveness of combination therapy with VCR and BA
for malignant melanoma by examining the synergistic cytotoxic activity against B16F10
melanoma cells in vitro and in vivo.
MATERIALS AND METHODS
Chemicals
Betulinic acid, VCR, annexin V–FITC and propidium iodide (PI) solution was obtained
from Wako Pure Chemical Industries, Ltd, Osaka, Japan. Triton X-100, proteinase K,
agarose and boric acid were obtained from Sigma Chemical Co., St Louis, MO, USA. Betulinic
acid and VCR were dissolved in dimethyl sulphoxide (DMSO) and saline, respectively.
They were diluted with saline to appropriate concentrations and stored in a refrigerator
at 4°C until use.
Animal model
Female C57BL/6 mice (9–10 weeks old) obtained from SLC Japan (Hamamatsu, Japan) were
housed in plastic cages with sawdust bedding and given food and water ad libitum.
The room in which the mice were kept, in the Institute of Animal Experimentation,
School of Medicine, The University of Tokushima, Japan, was environmentally controlled
at a temperature of 23±2°C and humidity of 55±10% and with a 13-h light/11-h dark
cycle. All animal experiments were carried out with approval by the Institutional
Animal Care and Use Committee of the University of Tokushima School of Medicine, and
met the standards required by the United Kingdom Coordinating Committee on Cancer
Research (UKCCCR) guidelines (Workman et al, 1998).
Cell culture conditions
B16F10 melanoma cells were purchased from the American Type Culture Collection (Virginia,
USA) and were maintained in DMEM medium (Nissui Pharmaceutical Co., Japan) supplemented
with 5% calf serum (Gibco Co., NY, USA), 5% horse serum (Gibco), penicillin–streptomycin
mix (Gibco) and gentamycin (Gibco). B16F10 melanoma cells were passaged every 3–4
days.
Annexin V-based apoptosis analysis
B16F10 melanoma cells (1 × 105) were cultured in 25 cm2 tissue culture plates for
24 h before the treatment with test reagents. Betulinic acid at concentrations of
1, 2.5 and 5 μ
M or VCR at a concentration of 1 nM was added, and the cells were incubated for a
further 24 h. After the incubation, the cells were trypsinised, washed with phosphate-buffered
saline (PBS), and suspended in 1 ml of a calcium-containing binding buffer. Then 5 μl
of annexin V–FITC and 5 μl of PI were added to the cell suspensions. After 10-min
incubation at room temperature, early apoptotic cells (annexin V positive and PI negative)
and late apoptotic/necrotic cells (annexin V positive and PI positive) as well as
living cells (annexin V negative and PI negative) were detected by a flow cytometer
(Coulter Epics XL-MCL, Beckman Coulter, Tokyo, Japan). The data were analysed using
CellQuest software. The early and late apoptotic cells (annexin V positive) were defined
as the apoptotic population.
DNA fragmentation-based apoptosis analysis
Total DNA was extracted according to the method of Lee and Shacter (1999). Briefly,
B16F10 melanoma cells (2 × 106) were cultured in 60 cm2 tissue culture plates for
24 h before the treatment with test reagents. Betulinic acid at concentrations of
1, 2.5 and 5 μ
M was added, and the cells were incubated for a further 24 h. Cells were harvested,
washed with PBS and lysed in buffer containing 10 mM Tris-HCl (pH 7.8), 10 mM EDTA,
0.5% Triton X-100, 200 μg ml−1 RNaseA and 200 μg ml−1 proteinase K. After DNA was
precipitated with isopropanol, it was then resuspended in a Tris-EDTA solution. Samples
were resolved by electrophoresis on 2% agarose gels and visualised by ethidium bromide
staining and UV transillumination.
Cell cycle distribution analysis
The effect of BA or VCR on cell cycle distribution in vitro was determined by using
a flow cytometer. B16F10 melanoma cells (1 × 106) were cultured in 25 cm2 tissue culture
plates for 24 h before the treatment with test reagents. After 24 or 36 h of incubation
with BA at concentrations of 1, 2.5 and 5 μ
M or VCR at a concentration of 1 nM, B16F10 melanoma cells were fixed with 99% ethanol,
treated with RNaseA and stained with PI solution containing RNase A. A flow cytometer
was used to determine the percentages of cells in G0/G1, S and G2/M phases of the
cell cycle.
MTS assay
The standard MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium,
innersalt solution) (Takara Co., Kusatsu, Japan) assay was performed to examine cytostatic/cytotoxic
effects of BA and/or VCR on B16F10 melanoma cells according to the manufacturer's
instructions. Briefly, tumour cells (5 × 103 cells) were seeded in 96-well microtiter
plates (Becton Dickinson Co., Franklin Lakes, NJ, USA). After an overnight incubation
at 37°C in 5% CO2, serially diluted solutions of BA, VCR or both were added in triplicate
to a final volume of 200 μl. After incubation for 24 h, the medium was removed, and
the cells were washed twice with culture medium and resuspended in 200 μl of culture
medium. Then, 20 μl of MTS was added to each well, and the plates were incubated at
37°C for 1 h. The absorbance of each well was read at 450 nm on a microplate reader
(microplate reader: MTP-120, Corona Co., Katsuta, Japan). The relative viability was
calculated as follows: relative viability=[(experimental absorbance−background absorbance)÷(absorbance
of untreated controls−background absorbance)] × 100%.
Drug interaction analysis
Synergistic interaction between BA and VCR was determined by using isobologram analysis
as described in detail previously (Berenbaum, 1981; Sora et al, 1994). Briefly, inhibition
of cell proliferation was determined as described above. Equieffective concentrations
were used for the analysis. The interaction index for the two-drug combination was
computed by the following equation: interaction index=BAc/BAe+VCRc/VCRe, where BAe
and VCRe are concentrations of BA and VCR, respectively, that inhibit cell proliferation
to 40% of the control level when used alone, and BAc and VCRc are concentrations of
BA and VCR, respectively, that produce the same effect when used in combination. According
to this method, an interaction index of less than 1.0 indicates synergistic interaction
between two drugs. An interaction index of more than 1.0 indicates antagonism, and
an index of 1.0 indicates additive interaction.
Effects of BA and VCR and their combination on tumour development in C57BL/6 mice
injected with B16F10 melanoma cells
On day 0, B16F10 melanoma cells (1 × 105 cells/0.1 ml medium) were injected into the
tail veins of anaesthetised 9- to 10-week-old female C57BL/6 mice. Tumour-bearing
mice were divided into four groups (five mice in each group) and treated intraperitoneally
(i.p.) with saline (0.1 ml), BA (10 mg kg−1 day−1), VCR (0.065 mg kg−1 day−1) or a
combination of both drugs (BA, 10 mg kg−1 day−1; VCR, 0.065 mg kg−1 day−1) (Figure
1
Figure 1
Experimental protocol for animal experiment. Arrows indicate the series of injection
of saline, BA (BA, i.p., 10 mg kg−1) or VCR (VCR, i.p., 0.065 mg kg−1). Mice in groups
1, 2 and 3 were administered saline, VCR and BA, respectively. Mice in group 4 were
administered both VCR and BA.
). The dose of BA for the treatment of mice was decided by referring to a previous
report (Pisha et al, 1995). Body weights of mice were recorded every 4 days to determine
whether the treatment influenced health status. The mice were killed by cervical dislocation
under anaesthesia with diethyl ether 17 days after inoculation of tumour cells. The
lungs were removed, and two independent observers determined the numbers and sizes
of lung metastatic nodules.
Data analysis
All data were statistically analysed by analysis of variance. P<0.05 was considered
to be statistically significant.
RESULTS
Effects of VCR and/or BA on the growth of B16F10 melanoma cells in vitro
Vincristine is one of the major chemotherapeutic agents for patients with malignant
melanoma. To determine whether treatment with a combination of VCR and BA has a greater
inhibitory effect than does treatment with VCR or BA alone, we examined the inhibitory
effects of VCR alone, BA alone and the combination of them using MTS assays. When
the growth level of untreated cells was regarded as 100%, the growth levels of cells
treated with 0.1, 1 and 10 nM VCR were 83.4, 71.2 and 56.2%, respectively (Figure
2
Figure 2
Effects of VCR and/or BA on the growth of B16F10 cells. Concentrations of VCR were
0 nM (open box), 0.1 nM (hatched box), 1.0 nM (latticed box) and 10 nM (closed box).
Cytostatic/cytotoxic effects were tested by MTS assays and expressed as the percentage
of the control growth (% of untreated control). Bars, mean±s.d.
). Vincristine inhibited the growth of B16F10 cells in a dose-dependent manner. In
addition, BA also inhibited the growth of B16F10 cells in a dose-dependent manner
(1 μ
M, 92.5%; 2.5 μ
M, 91.9%; 5 μ
M, 62.0%). Combinational treatment with 1 or 10 nM VCR and BA resulted in the inhibition
of the growth of B16F10 cells in a dose-dependent manner. The combination of 10 nM
VCR and 5 μ
M BA greatly inhibited the growth of cells (12.9%).
Induction of apoptosis in B16F10 cells by VCR and BA
B16F10 melanoma cells were incubated in DMEM medium with VCR at 1 nM or BA at three
different concentrations (1, 2.5 and 5 μ
M) for 24 h. The percentage of apoptotic cells was determined as described in Materials
and methods. The apoptotic population of untreated cells was 6.1%, while that of cells
treated with 1 nM VCR was 25.2% (Figure 3
Figure 3
Apoptosis assay of B16F10 cells treated with VCR or BA. Flow cytometric analysis of
apoptosis in B16F10 cells was performed by treatment with VCR (1 nM) and BA (1, 2.5
or 5 μ
M) for 24 h and by staining with annexin V–FITC and PI.
). The apoptotic populations of cells treated with 1, 2.5 and 5 μ
M BA alone were 8.9, 15.2 and 19.5%, respectively. Moreover, the induction of apoptosis
was evaluated by DNA fragmentation (Wyllie, 1980). DNA ladder formation was detected
(Figure 4)
Figure 4
DNA fragmentation analysis of apoptosis in B16F10 cells treated with BA.
, and the fragments were stained in a dose-dependent manner. These findings indicate
that BA induced apoptosis in a dose-dependent manner.
Determination of the synergistic effect of VCR and BA using isobologram analysis
To clarify the synergistic effect of VCR and BA on inhibition of the growth of B16F10
cells, we used isobologram analysis by the method of Berenbaum (1981). Treatment with
VCR or BA resulted in inhibition of the growth of B16F10 cells in a dose-dependent
manner (Figure 5a and b
Figure 5
Effects of VCR and/or BA on the growth of B16F10 cells. Cytostatic/cytotoxic effects
were tested by MTS assays and expressed as the percentage of the control growth (A–C).
Solid and dotted lines show 1 and 10 nM VCR, respectively (C). The isobologram depicts
the interaction between BA and VCR in inhibiting the growth of B16F10 cells (D). The
solid line shows concentrations of both drugs required to inhibit cell growth to 40%
(IC40). The dotted line shows concentrations of drugs required to produce the same
degree of growth inhibition as that in the case of interactions being additive. The
numbers in boxes are interaction indices.
). The inhibitory concentration (IC40) of VCR alone and that of BA alone showing 40%
of the control growth level were 100 nM and 10 μ
M, respectively (Figure 5a and b). When IC40 was determined by combinational use of
both drugs, a combination of 10 nM VCR and 1.8 μ
M BA, and a combination of 1 nM VCR and 3 μ
M BA showed 40% growth of the control (Figure 5c). The interaction indices calculated
by the equation shown in Materials and methods were 0.28 and 0.31, which are less
than 1.0 (Figure 5d). The results of isobologram analysis showed that treatment with
a combination of VCR and BA synergistically inhibited the growth of B16F10 cells.
Induction of G2/M arrest and G1 arrest by VCR and BA, respectively
Cell cycle distribution analysis was performed to clarify the mechanism of synergic
inhibitory action on B16F10 melanoma cells. B16F10 cells were incubated with VCR and
BA for 24 or 36 h in the same manner as in the cell apoptosis assay. The proportions
of cells in the G2/M phase that had been treated with 1 nM VCR for 24 and 36 h were
29.3 and 79.8%, respectively (untreated group: 14.5% after 24 h and 10.0% after 36 h)
(Table 1
Table 1
Cell cycle distribution of B16F10 cells treated with VCR or BA
Cell cycle distribution (%)
G0/G1a
Sa
G2/Ma
Treatment
24 hb
36 hb
24 hb
36 hb
24 hb
36 hb
None
74.1
80.7
11.4
9.3
14.5
10.0
VCR (1 nM)
62.7
20.2
8.0
0.0
29.3
79.8
BA (1 μ
M)
78.8
93.3
2.0
5.6
19.2
1.1
BA (2.5 μ
M)
86.1
93.1
3.0
2.1
10.9
4.8
BA (5 μ
M)
95.5
93.4
0.0
2.2
4.4
4.4
VCR=vincristine; BA=betulinic acid.
a
Cell cycle phase.
b
Incubation time.
), indicating that VCR induces cell cycle arrest at the G2/M phase. The population
of G0/G1 cells treated with BA for 24 h increased in a dose-dependent manner (1 μ
M, 78.8%; 2.5 μ
M, 86.1%; 5 μ
M, 95.5%). Moreover, the cell population in the G0/G1 phase after 36 h of treatment
was constant (93%) regardless of the concentration (untreated group: 80%), indicating
that BA induces cell cycle arrest at the G1 phase. These results demonstrated that
VCR and BA induce cell cycle arrest at different stages.
Antimetastatic effects of VCR, BA and their combination in C57BL/6 mice treated with
B16F10 melanoma cells
To determine whether the augmenting effect of BA on the cytotoxicity of VCR against
B16F10 melanoma cells can be reproduced in vivo, animal experiments were performed
according to the schedule shown in Figure 1. B16F10 cells formed 50.2±20.7 nodules
in the lung in the nontreatment group, whereas they formed 28.0±5.4, 35.6±18.8 and
12.4±9.5 nodules in the VCR, BA and combinational administration groups, respectively
(Figure 6
Figure 6
Effects of VCR and/or BA on experimental metastasis in mice. Mice were treated with
a solvent (open box), 0.065 mg kg−1 VCR (hatched box), 10 mg kg−1 BA (latticed box),
and both drugs (closed box) as described in Materials and methods. The numbers of
tumour nodules were evaluated with classification by size (all sizes and more than
1 mm in diameter). *,**Significantly different from the control group (P<0.05 and
P<0.0001). ***Significantly different from the VCR group and the BA group (P<0.05).
). Treatment with VCR (0.065 mg kg−1 day−1) alone or in combination with BA (10 mg kg−1 day−1)
significantly suppressed the experimental metastasis. B16F10 melanoma cells formed
36.0±12.2 large nodules of more than 1 mm in diameter in the lung in the nontreatment
group, whereas they formed 14.0±2.0, 16.8±9.0 and 3.6±5.5 large nodules in the VCR,
BA and combinational administration groups, respectively. The ratios of the number
of large nodules to total metastatic nodules in the lung in the nontreatment group
and the VCR, BA and combinational administration groups were 71.7, 50.0, 47.2 and
29.0%, respectively, indicating that combinational treatment with VCR and BA significantly
suppresses the growth of B16F10 melanoma cells after their colonisation in the lung.
No weight loss was observed in any mice treated with VCR, BA or both reagents (data
not shown).
DISCUSSION
In most cases of malignant melanoma, distant metastasis is found at the time of clinical
diagnosis. It has been reported that the median survival time of stage IV melanoma
patients is about 6–7.5 months, the 5-year survival rate being approximately 6% (Bajetta
et al, 2002). Lung metastasis of melanoma cells from primary lesions is an especially
important factor influencing the prognosis of patients. Therefore, the main treatment
for malignant melanoma is various chemotherapeutic agents. Various anticancer drugs,
including bleomycin, VCR, lomustine and dacarbazine, have been used in recent years
for treating patients with melanoma (Wöll et al, 1999; Kivela et al, 2003). However,
severe side effects of the chemotherapeutic agents have limited the application of
high-dose chemotherapy or combination therapy with different anticancer drugs for
many cases of malignant melanoma (Bajetta et al, 2002).
Combinational treatment with different drugs that induce different cell cycle arrest
results in apoptosis in tumour cells (Darzynkiewicz, 1986; Bhuyan and Groppi, 1989;
Ko et al, 1997). It was thought that treatment with the combination of BA and VCR
had a synergistic cytotoxic effect on tumour cells because treatment with BA alone
and treatment with VCR alone induced cell cycle arrest in different phases. Although
BA did not induce cell cycle arrest in glioma cells (Wick et al, 1999), we found that
BA induced the accumulation of B16F10 melanoma cells in the G1 phase after 24 h of
coincubation in a dose-dependent manner. Moreover, BA induced the accumulation of
93% of B16F10 cells in the G1 phase after 36 h of coincubation. It is expected that
elucidation of the mechanism by which BA inhibits the growth of B16F10 melanoma cells
will lead to the establishment of effective combinational regimens with other antitumour
drugs with different cytotoxic mechanisms for the treatment of malignant melanoma.
It has been reported that BA enhanced the levels of BAX and BCL-2 proteins in glioma
cells and induced apoptosis in those cells (Wick et al, 1999). BA induces apoptosis
via the activation of caspases in neuroectodermal cells (Fulda et al, 1997). However,
the mechanism by which apoptosis is induced in B16F10 cells by BA remains to be determined.
Cell arrest, damage to DNA or stress to the cytoplasm or cell membrane may be the
cause of apoptosis (Aller et al, 1992; Gohen et al, 1992; Ko et al, 1997). We believe
that results of analysis of cell cycle distributions or apoptotic pathways to determine
mechanisms of cytotoxicity of other chemotherapeutic agents used in the treatment
for malignant melanoma will open the gate for the establishment of effective combinational
therapeutic regimens against the still untreatable malignant disease using BA.
We performed animal experiments to determine whether BA and VCR show a suppressive
effect on the growth or metastatic state of B16F10 melanoma cells in vivo. Antimetastatic
activity was investigated by counting lung metastatic nodules as reported previously
(Jani et al, 1993; Kang et al, 2000; Feleszko et al, 2002), because B16F10 melanoma
cells preferentially induce lung metastasis (Jani et al, 1993; Gude et al, 2001; Malafa
et al, 2002). Although treatment with VCR alone resulted in a significant suppression
of metastasis of melanoma cells to the lung, the additional use of BA resulted in
greater suppression than that in the case of treatment with VCR alone. Treatment with
a combination of VCR and BA resulted in a significant reduction in the numbers of
lung nodules of more than 1 mm in diameter in mice compared with that in the case
of treatment with VCR or BA alone. These results suggest that BA potentiates the antitumour
activity of VCR also in vivo. For the establishment of metastasis, tumour cells must
leave the original tumour mass, invade the surrounding tissue and then enter blood
vessels. The next steps are tumour cell attachment to endothelial cells in the blood
vessel, invasion and growth in the target organ (Liotta, 1986; Nicolson, 1988). We
could not determine which metastatic steps were mainly inhibited by the combinational
treatment with BA and VCR; however, the significant reduction in the number of lung
nodules of more than 1 mm in diameter in the combinational treatment group indicates
their inhibitory effect on tumour growth after attachment to target metastatic sites.
Furthermore, we observed no significant difference between weight losses in the groups
treated with VCR, BA and both, indicating that the combination of BA and VCR might
be an effective regime with side effects of relatively small magnitude for the treatment
of malignant melanoma.
In summary, treatment with a combination of VCR and BA resulted in synergistic suppression
of cell growth in vitro and in the prevention of B16F10 melanoma experimental metastasis
by direct suppression of cell growth in vivo. It is expected that BA can augment antitumour
activities of various drugs showing different types of cytotoxic mechanisms.