Cytotoxic and apoptosis-inducing activities of triterpene acids from Poria cocos

Article abstract of DOI:10.1021/np100402b

Six lanostane-type triterpene acids (1a-6a), isolated from Poria cocos, and their methyl ester (1b-6b) and hydroxy derivatives (1c-6c) were prepared. Upon evaluation of the cytotoxic activity of these compounds against leukemia (HL60), lung (A549), melanoma (CRL1579), ovary (NIH:OVCAR-3), breast (SK-BR-3), prostate (DU145), stomach (AZ521), and pancreas (PANC-1) cancer cell lines, 11 compounds (5a, 6a, 2b-5b, 1c, and 3c-6c) exhibited activity with single-digit micromolar IC₅₀ values against one or more cell lines. Poricotriol A (1c), a hydroxy derivative of poricoic acid A (1a), exhibited potent cytotoxicities against six cell lines with IC₅₀ values of 1.2-5.5 μM. Poricotriol A induced typical apoptotic cell death in HL60 and A549 cells on evaluation of the apoptosis-inducing activity by flow cytometric analysis. Western blot analysis in HL60 cells showed that poricotriol A activated caspases-3, -8, and -9, while increasing the ratio of Bax/Bcl-2. This suggested that poricotriol A induced apoptosis via both mitochondrial and death receptor pathways in HL60. On the other hand, poricotriol A did not activate caspases-3, -8, and -9, but induced translocation of apoptosis-inducing factor (AIF) from mitochondria and increased the ratio of Bax/Bcl-2 in A549. This suggested that poricotriol A induced apoptosis via the mitochondrial pathway mostly by translocation of AIF, independent from the caspase pathway in A549. Furthermore, poricotriol A was shown to possess high selective toxicity in lung cancer cells since it exhibited only weak cytotoxicity against a normal lung cell line (WI-38).

Full text of DOI:10.1021/np100402b

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pubs.acs.org/jnp
Cytotoxic and Apoptosis-Inducing Activities of Triterpene Acids from
Poria cocos
Takashi Kikuchi,† Emiko Uchiyama,† Motohiko Ukiya,† Keiichi Tabata,‡ Yumiko Kimura,‡ Takashi Suzuki,‡
and Toshihiro Akihisa*,†
†College of Science and Technology, Nihon University, 1-8 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan
‡School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan
ABSTRACT: Six lanostane-type triterpene acids (1a-6a), isolated from Poria cocos, and
their methyl ester (1b-6b) and hydroxy derivatives (1c-6c) were prepared. Upon evalua-
tion of the cytotoxic activity of these compounds against leukemia (HL60), lung (A549),
melanoma (CRL1579), ovary (NIH:OVCAR-3), breast (SK-BR-3), prostate (DU145),
stomach (AZ521), and pancreas (PANC-1) cancer cell lines, 11 compounds (5a, 6a, 2b-
5b, 1c, and 3c-6c) exhibited activity with single-digit micromolar IC50 values against one
or more cell lines. Poricotriol A (1c), a hydroxy derivative of poricoic acid A (1a), exhibited
potent cytotoxicities against six cell lines with IC50 values of 1.2-5.5 μM. Poricotriol A
induced typical apoptotic cell death in HL60 and A549 cells on evaluation of the apoptosis-inducing activity by flow cytometric
analysis. Western blot analysis in HL60 cells showed that poricotriol A activated caspases-3, -8, and -9, while increasing the ratio of
Bax/Bcl-2. This suggested that poricotriol A induced apoptosis via both mitochondrial and death receptor pathways in HL60. On
the other hand, poricotriol A did not activate caspases-3, -8, and -9, but induced translocation of apoptosis-inducing factor (AIF)
from mitochondria and increased the ratio of Bax/Bcl-2 in A549. This suggested that poricotriol A induced apoptosis via the
mitochondrial pathway mostly by translocation of AIF, independent from the caspase pathway in A549. Furthermore, poricotriol A
was shown to possess high selective toxicity in lung cancer cells since it exhibited only weak cytotoxicity against a normal lung cell
line (WI-38).
he dried sclerotia of Poria cocos Wolf (Polyporaceae) are
used traditionally in Chinese herbal prescriptions as a
’ RESULTS AND DISCUSSION
T
Six lanostane-type triterpene acids, poricoic acid A (1a), por-
icoic acid C (2a), poricoic acid G (3a), poricoic acid H (4a), 25-
hydroxy-3-epidehydrotumulosic acid (5a), and dehydroeburico-
nic acid (6a), were isolated from the sclerotia of P. cocos.6,9,10,15
Dimethyl poricoate A (1b) was prepared from poricoic acid A by
methylation with trimethylsilyl (TMS)-diazomethane, and por-
icotriol A (1c) from 1b by reduction with LiAlH4.
diuretic, sedative, and analgesic.1,2 Investigations by us and by
other workers have shown that both the inner parts2-6 and the
epidermis7-10 of the sclerotia of P. cocos contain lanostane-type
triterpene acids, which were suggested to be one of the major
medicinal components of the fungus.2 Several of these acids have
potent inhibitory effects on 12-O-tetradecanoylphorbol-13-acet-
ate (TPA)-induced inflammation in mice,11,12 and on Epstein-Barr
virus early antigen (EBV-EA) activation induced by TPA.6,9,10 In
addition, in a skin tumor promotion assay using an in vivo two-
stage carcinogenesis test employing 7,12-dimethylbenz[a]-
anthracene (DMBA) as an initiator and TPA as a promoter,
16-deoxyporicoic acid B, poricoic acid C, and 25-methoxypor-
icoic acid A showed activity.9,10 Poricoic acid A showed moderate
cytotoxicities against some human cancer cells,6 and pachymic
acid and dehydrotrametenolic acid exhibited inductive effects of
apoptosis in prostate cancer cells13 and in H-ras-tranformed rat2
cells.14 In addition, we have reported that some triterpene acids
from P. cocos possess inhibitory effects on DNA polymerases.15,16
In this paper, we report the conversion of six lanostane-type
triterpene acids (1a-6a), isolated from P. cocos,6,9,10,15 into their
methyl ester (1b-6b) and hydroxy derivatives (1c-6c) and
evaluation of the cytotoxic activities of these compounds (1a-
6a, 1b-6b, and 1c-6c) against eight human cancer cell lines. In
addition, we report the induction of apoptosis in HL60 and A549
cells by poricotriol A (1c), a hydroxy derivative of poricoic acid A
(1a), and the mechanisms of the apoptotic cell death.
Compound 1b gave a [M þ Na]þ ion in the HRESIMS at m/z
549.3629, consistent with a molecular formula of C33H50O5. The
13C and 1H NMR data (Table 1) of 1b were very similar to those
of poricoic acid A,7 except for the presence of additional signals of
two methoxy groups [δC 51.2 (q) and 51.4 (q); δH 3.61 (3H, s)
and 3.78 (3H, s)]. The above evidence, coupled with analyses of
1H-1H COSY, HMQC, and HMBC (Table 1) spectra, indi-
cated that 1b was dimethyl (20R)-16R-hydroxy-3,4-seco-24-
methyllanosta-4(28),7,9(11),24(241)-tetraene-3,21-dioate. Sig-
nificant NOE correlations (Figure 1)17 were observed in the
NOESY experiment of 1b for H-18 (13β-Me) with H-16β, H-19
(10β-Me), and H-20 and for H-30 (14R-Me) with H-17R. These
NOE correlations suggested that 1b possesses the 20R-stereo-
chemistry at C-20, and the structure was established as dimethyl
(20R)-16R-hydroxy-3,4-seco-24-methyllanosta-4(28),7,9(11),24-
(241)-tetraene-3,21-dioate (dimethyl poricoate A).
Received: June 16, 2010
Published: January 20, 2011
r
Copyright
2011 American Chemical Society and
137
dx.doi.org/10.1021/np100402b J. Nat. Prod. 2011, 74, 137–144
American Society of Pharmacognosy
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Chart 1
The molecular formula of compound 1c was determined to be
C31H50O3 from its HRESIMS ([M þ Na]þ, m/z 493.3674). The
appearance of the signals for two primary hydroxy groups [δC
63.0 (t) and 63.6 (t); δH 3.82 (2H, m), 3.90 (1H, dd, J = 4.0, 10.6
Hz), and 4.13 (1H, br d, J = 8.3 Hz)] in the 13C and 1H NMR
spectra and the disappearance of two carboxy signals in the 13C
NMR spectrum of 1c when compared with those of poricoic acid
A7 and 1b suggested that 1c is the 3,21-dihydroxy derivative. This
evidence, coupled with the analysis of 1H-1H COSY, HMQC,
HMBC (Table 1), and NOESY (Figure 1)17 spectra, surpports
the conclusion that 1c is (20R)-3,4-seco-24-methyllanosta-4-
(28),7,9(11),24(241)-tetraene-3,16R,21-triol (poricotriol A).
The other five methyl ester (2b-6b) and five hydroxy (2c-
6c) derivatives were prepared from the corresponding triterpene
acids (2a-6a), respectively, in the same way as described above.
The cytotoxic activities of compounds 1a-6c along with two
anticancer drugs, cisplatin and 5-fluorouracil, were evaluated
against the following human cancer cell lines: HL60 (leukemia),
A549 (lung), CRL1579 (melanoma), NIH:OVCAR-3 (ovary),
SK-BR-3 (breast), DU145 (prostate), AZ521 (stomach), and
PANC-1 (pancreas), by means of a thiazolyl blue tetrazolium
bromide (MTT) assay, and the results are summarizedin Table 2.
Compounds 5a, 6a, 2b-5b, 1c, and 3c-6c exhibited single-digit
micromolar cytotoxicities with IC50 values of 1.2-9.5 μM
against one or more cancer cell lines. Thus, compound 5a was
active against A549 and NIH:OVCAR-3 cells, 6a against PANC-
1 cells, 2b, 4b, 5b, 3c, and 5c against HL60 cells, 3b against SK-
BR-3 cells, 1c against HL60, A549, CRL1579, NIH:OVCAR-3,
SK-BR-3, and AZ521 cells, 4c against HL60 and CRL1579 cells,
and 6c was against HL60, A549, CRL1579, and NIH:OVCAR-3
cells. In particular, the cytotoxicities of compound 5a against
A549 and NIH:OVCAR-3 cells, 6a against PANC-1 cells, 1c
against A549, CRL1579, NIH:OVCAR-3, SK-BR-3, and AZ521
cells, 4c against CRL1579 cells, and 6c against A549, CRL1579,
and NIH:OVCAR-3 cells were superior to those of the reference
compounds, cisplatin and/or 5-fluorourasil, tested in the same
assay. The prostate cancer cells (DU145) were less sensitive to
the compounds tested in this study, and 2a, 4b, 5b, 1c, and 2c
showed only moderate cytotoxicities (IC50 14.5-28.9 μM),
being less active than cisplatin (IC50 9.2 μM).
Poricotriol A (1c) exhibited potent cytotoxicities against most
of the cell lines tested (Table 2) and was evaluated for its apo-
ptosis-inducing activity against HL60 and A549 cells. HL60 and
A549 cells were incubated with 1c for 24 and 48 h, and then the
cells were analyzed in terms of cell cycle distribution by means
of flow cytometry.18 The sub-G1 peak, i.e., fragmented DNA,
was quantified as the apoptosis index. At 48 h after treatment, the
sub-G1 peak increased [HL60 (Figure 2A): from 3.0% to 38.7%;
A549 (Figure 2B): from 3.1% to 17.4%]. The cell cycle analysis of
HL60 and A549 cells also demonstrated that 1c induced a
depletion of cells in the S phase [HL60 (Figure 2A): from
42.6% to 28.9% after 24 h; A549 (Figure 2B): from 27.7% to 7.8%
after 24 h and 6.0% after 48 h] and accumulation of cells in the
G0/G1 phase (HL60: from 51.4% to 62.3% after 24 h; A549:
from 64.3% to 77.2% after 24 h). These results suggest that the
cells had undergone cell cycle arrest in the G0/G1 phase and
apoptosis after treatment with 1c.
Exposure of the membrane phospholipid, phosphatidylserine,
to the external cellular environment is one of the earliest markers
of apoptotic cell death.19 Annexin V is a calcium-dependent
phospholipid-binding protein with high affinity for phosphati-
dylserine expressed on the cell surface. Propidium iodide (PI)
does not enter whole cells with intact membranes and was used
to differentiate between early apoptotic (annexin V positive, PI
negative), late apoptotic (annexin V, PI double positive), and
necrotic cell death (annexin V negative, PI positive). The ratio of
early apoptotic cells (Figure 3A, lower right) was increased after
treatment with 1c in HL60 for 24 h (from 7.6% to 48.1%) and
48 h (52.5%), and that of late apoptotic cells (upper right) was
increased after 48 h (from 3.3% to 30.4%) (Figure 3A). On the
other hand, the proportion of early apoptotic cells did not increase
after treatment with 1c for 24 h in A549 cells (Figure 3B). This
was, however, increased after 48 h (from 4.3% for 0 h to 24.1%)
along with the increase of late apoptotic cells (from 1.1% for 0 h
138
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Table 1. 13C (125 MHz), 1H (500 MHz), and HMBC Spectroscopic Data (δ values; C5D5N) for Dimethyl Poricoate A (1b) and
Poricotriol A (1c)a
1b
1c
carbon
δC
δH
HMBC (H to C)
δC
δH
HMBC (H to C)
1
2
35.9
t
t
1.71 m
1.94 m
2.29 m
2.37 m
5
37.9
t
t
1.55 dt (5.1, 12.3)
1.66 dt (4.0, 12.3)
1.67 m
2, 5, 9, 10
2, 19
2, 5, 10, 19
1, 3
29.6
28.5
1, 3
1, 3
1.75 m
1, 3, 10
3
4
5
6
174.5
149.6
50.7
s
s
d
t
63.0
149.7
50.6
28.7
t
3.82 m (2H)
s
d
t
2.27 br d (5.7)
R, 2.51 m
1, 4, 6, 7, 9, 10, 28
4, 5, 7, 8, 10
4, 5, 7, 8
2.36 dd (8.6, 14.6)
R, 2.55 m
4, 6, 7, 9, 10, 28
4, 7, 8
28.5
β, 2.05 dd (3.6, 19.0)
5.25 t (3.0)
β, 2.08 dd (4.0, 19.0)
5.25 br s
5, 7, 8, 10
5, 6, 9, 14
7
118.2
141.5
137.3
38.8
d
s
5, 9
117.6
142.2
138.5
39.0
d
s
8
9
s
s
10
11
12
s
s
120.1
36.7
d
t
5.30 br d (3.0)
R, 2.37 m
8, 10, 12
119.5
37.7
d
t
5.43 br d (2.8)
R, 2.51 m
8, 10, 13
9, 11, 13, 14, 18
9, 11, 13, 18
9, 11, 13, 18
9, 11, 13, 14, 18
β, 1.91 dd (5.1, 17.5)
β, 2.27 dd (5.1, 17.4)
13
14
15
45.3
49.3
43.7
s
s
t
46.0
49.4
43.3
s
s
t
R, 1.72 br d (10.9)
β, 2.32 m
13, 14, 30
R, 1.77 br d (12.6)
β, 2.34 m
13, 16, 17, 30
14, 16, 30
14, 16, 30
16
17
18
19
20
21
76.1
57.5
18.2
22.2
47.6
176.4
d
d
q
q
d
s
4.37 t-like (7.3)
2.60 dd (6.1, 11.2)
0.87 s
14, 20
74.6
56.7
18.7
22.8
41.3
63.6
d
d
q
q
d
t
4.54 t-like (7.5)
2.52 dd (6.3, 13.2)
0.84 s
14, 20
12, 13, 18, 20, 22
12, 13, 14, 17
1, 5, 9, 10
12, 13, 16, 20, 21
12, 13, 14, 17
1, 5, 9, 10
1.00 s
1.10 s
2.77 dt (3.1, 10.8)
1.87 m
3.90 dd (4.0, 10.6)
4.13 br d (8.3)
2.18 m (2H)
22
17, 22
21, 23
22
23
31.1
32.9
t
t
2.16 m
20, 23
20, 23
22, 241
27.8
32.9
t
t
2.49 m
2.15 m (2H)
2.20 m
2.53 m
20, 22, 24, 25, 241
20, 22, 24
24
25
26
27
28
155.5
34.0
s
157.0
34.1
s
d
q
q
t
2.24 m
26, 27, 241
24, 25, 27
24, 25, 26
4, 5, 29
4, 29
d
q
q
t
2.33 m
24, 26, 27, 241
24, 25, 27
24, 25, 26
4, 5, 29
22.0
0.99 d (7.0)
0.98 d (7.0)
4.75 s
22.2
1.04 d (6.9)
1.03 d (6.9)
4.78 br s
4.84 s
21.9
22.0
112.3
112.0
4.79 d (2.2)
1.70 s
4, 5, 29
29
30
241
22.2
24.7
107.3
q
q
t
4, 5, 28
8, 13, 14, 15
23, 24, 25
23, 24, 25
3
22.4
25.3
106.7
q
q
t
1.77 s
4, 5, 28
1.32 s
1.38 s
13, 14, 15
23, 24, 25
23, 24, 25
4.82 br s
4.87 s
4.83 s
4.92 s
3-COOMe
51.4
51.2
q
q
3.61 s
21-COOMe
3.78 s
21
a J values (Hz) determined are shown in parentheses.
to 20.2%). A small number of necrotic cells (upper left) also were
detected in A549 cells at 48 h (from 3.4% for 0 h to 6.3%). These
results revealed that most of the cytotoxicty of 1c to HL60 and
A549 cells is due to inducing apoptotic cell death.
evaluated by western blot analysis. After treatment of HL60 cells
with 1c (30 μM) for 8 h, the level of procaspases-3, -8, and -9
diminished and cleaved caspases-3, -8, and -9 were detected
(Figure 4A). In contrast, diminution of procaspases-3, -8, and -9
and expression of cleaved caspases-3, -8, and -9 were not deteced
after treatment with 1c in A549 (Figure 4B). These results
suggest that compound 1c-induced cell death occurs through
Caspases are known to mediate the apoptotic pathway.20,21 In
order to clarify the mechanism by which compound 1c induces
apoptotic cell death, activation of caspases-3, -8, and -9 was
139
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activation of caspases-3, -8, and -9 in HL60, although not in the
case of A549.
Next, we investigated the effect of compound 1c on Bax and
Bcl-2. The proapoptotic proteins Bax and Bid and the antiapop-
totic mitochondrial protein Bcl-2 are important regulators of
cytochrome c release from mitochondria.22,23 Expression of these
proteins was examined by western blot analysis. Treatment of
HL60 cells by compound 1c (30 μM) decreased the level of Bcl-2
and increased the level of Bax in a time-dependent manner
(Figure 4A). In A549 cells, compound 1c had no effect on the
expression of Bax, but diminished Bcl-2, though to a small extent,
after 24 h of treatment (Figure 4B).24 The Bax/Bcl-2 ratio is one
of the indices of the intrinsic mechanism of apoptosis in mito-
chondria.25 Since compound 1c increased this ratio in HL60 and
A549 cells, it seems that compound 1c-induced apoptosis involved
Bax/Bcl-2 signal transduction. Compound 1c is, therefore, sug-
gested to induce apoptosis in HL60 and A549 cells by involving
the mitochondrial signal transduction pathway.
Translocation of AIF (apoptosis-inducing factor) was investi-
gated in A549 cells treated with compound 1c. AIF and endo-
nuclease G (EndoG) are capable of inducing apoptotic DNA
fragmentation in a caspase-independent pathway.26 AIF, released
Figure 1. Major NOE correlations (T) for dimethyl poricoate A (1b)
and poricotriol A (1c).17
Table 2. Cytotoxic Activity on Eight Human Cancer Cell Lines of Triterpene Acids from Poria cocos and Their Methyl Ester and
Hydroxy Derivativesa
IC50 (μM)b
HL60
A549
CRL1579
NIH:OVCAR-3
(ovary)
SK-BR-3
(breast)
DU145
AZ521
PANC-1
compound
(leukemia)
(lung)
(melanoma)
(prostate)
(stomach)
(pancreas)
triterpene acid
1a
38.0
25.9
32.6
28.4
29.4
29.2
>100
27.0
>100
>100
4.0
>100
>100
>100
>100
84.0
>100
53.1
>100
>100
9.5
>100
23.9
>100
14.5
>100
45.9
>100
68.9
>100
>100
67.4
5.5
2a
3a
>100
>100
62.3
>100
>100
>100
>100
>100
>100
51.5
4a
5a
6a
10.0
89.7
37.7
12.2
>100
methyl ester
derivative
1b
18.2
2.4
79.6
83.8
66.0
13.5
41.4
96.5
>100
27.8
42.4
97.6
76.5
20.9
91.8
23.6
26.3
49.5
67.5
>100
32.2
23.9
4.3
>100
>100
>100
28.9
32.1
19.8
34.5
14.6
14.4
53.4
>100
>100
>100
44.9
2b
3b
8.8
4b
3.6
15.9
27.5
27.5
5b
6.9
28.9
71.3
6b
23.5
>100
>100
hydroxy
derivative
1c
1.9
12.3
7.5
4.2
5.5
2.2
1.2
14.5
1.4
26.3
91.3
17.9
>100
>100
22.6
2c
80.0
11.0
41.2
>100
4.0
76.4
11.6
9.9
70.3
43.5
>100
>100
6.4
11.0
13.0
51.3
>100
11.7
20.3
13.3
13.4
>100
>100
12.5
3c
>100
>100
>100
>100
4c
2.9
5c
2.5
14.0
2.8
6c
6.7
reference
compound
cisplatin
5-fluorouracil
1.9
9.5
24.9
11.3
21.1
17.9
9.8
9.2
2.7
79.0
54.1
>100
>100
>100
>100
>100
a Cells were treated with compounds (1 ꢀ 10-4 to 1 ꢀ 10-6 M) for 48 h, and cell viability was analyzed by the MTT assay. b IC50 value is the
concentration of compound required to inhibit the growth of the cells by 50%. This was obtained on the basis of triplicate assay results.
140
dx.doi.org/10.1021/np100402b |J. Nat. Prod. 2011, 74, 137–144