Angelmarin, a novel anti-cancer agent able to eliminate the tolerance of cancer cells to nutrient starvation

Article abstract of DOI:10.1016/j.bmcl.2005.10.046

The CH₂Cl₂-soluble extract of Angelica pubescens was found to kill PANC-1 cancer cells preferentially under nutrition starvation at a concentration of 50 μg/ml, with virtually no cytotoxicity under nutrient-rich conditions. Further bioassay-guided fractionation and isolation led to the isolation of a novel compound named angelmarin as the primary compound responsible for the preferential cytotoxicity; the compound exhibited 100% preferential cytotoxicity against PANC-1 cells at a concentration of 0.01 μg/ml.

Full text of DOI:10.1016/j.bmcl.2005.10.046

Bioorganic & Medicinal Chemistry Letters 16 (2006) 581–583
Angelmarin, a novel anti-cancer agent able to eliminate
the tolerance of cancer cells to nutrient starvation
Suresh Awale,a Eduardo M. N. Nakashima,a Surya K. Kalauni,a Yasuhiro Tezuka,a
Yukiko Kurashima,b Jie Lu,b Hiroyasu Esumib and Shigetoshi Kadotaa,*
aInstitute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan
bInvestigative Treatment Division, National Cancer Center Research Institute East, Chiba 277-8577, Japan
Received 7 September 2005; revised 28 September 2005; accepted 15 October 2005
Available online 8 November 2005
Abstract—The CH2Cl2-soluble extract of Angelica pubescens was found to kill PANC-1 cancer cells preferentially under nutrition
starvation at a concentration of 50 lg/ml, with virtually no cytotoxicity under nutrient-rich conditions. Further bioassay-guided
fractionation and isolation led to the isolation of a novel compound named angelmarin as the primary compound responsible
for the preferential cytotoxicity; the compound exhibited 100% preferential cytotoxicity against PANC-1 cells at a concentration
of 0.01 lg/ml.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The physiology of solid tumors differs from that of nor-
mal tissues in a number of important aspects. Compared
to normal cells, tumor cells proliferate extremely rapid-
ly, and the demand for essential nutrients as well as oxy-
gen always exceeds the supply, due to the unregulated
growth caused by genetic and epigenetic alterations of
the cells and the insufficient and inappropriate vascular
supply.1–3 However, cancer cells show an inherent abili-
ty to tolerate extreme conditions, such as that character-
ized by low nutrient and oxygen supply, by modulating
their energy metabolism.4 Previously, we reported that
certain pancreatic cancer cell lines, such as PANC-1,
AsPC-1, BxPC-1, and KP-3, exhibit marked tolerance
and survive for prolonged periods of time even under
extremely nutrient-starved conditions.4 Thus, we
hypothesized that tolerance to nutrient starvation might
be a part of the biological response to insufficient blood
supply, and the development of drugs aimed at counter-
ing this resistance of the cells to nutrient deprivation
may serve as a novel biochemical approach to cancer
therapy.4–6 In this regard, we developed a novel screen-
ing methodology based on this anti-austerity strategy6
and screened 500 medicinal plant extracts used in
Japanese Kampo medicine. We found from this screen-
ing that a CH2Cl2-soluble extract of Angelica pubescens
exhibited preferential cytotoxicity against nutrient-de-
prived cancer cells at the concentration of 50 lg/ml.
We thus carried out further bioassay-guided fraction-
ation and isolation, which led to the isolation of a new
compound named angelmarin (1) as the primary
compound responsible for exhibiting preferential
cytotoxicity at a concentration of 0.01 lg/ml. In this
paper, we report the structure elucidation of angelmarin
(1) together with its potent in vitro preferential cytotoxic
activity.
5
4
4a
2
7
8a
O
O
O
O
O
O
10
9
H
H
11
O
OH
2
9'
8'
O
2'
3'
4'
1'
7'
OH
6'
5'
1
Keywords: PANC-1; Anti-austerity; Screening strategy; Angelmarin;
Angelica pubescens; Anti-cancer agent.
Angelmarin (1)7 was isolated as a colorless amorphous
solid and showed ½aꢀ +218.7ꢁ (c 0.025, CHCl3). Its
25
D
molecular formula was determined by HR-FABMS to
*
Corresponding authors. Tel.: +81 76 434 7625; fax: +81 76 434
5059; e-mail: kadota@ms.toyama-mpu.ac.jp
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.046

582
S. Awale et al. / Bioorg. Med. Chem. Lett. 16 (2006) 581–583
be C23H20O6 [m/z 393.1295 (M+H)+]. The IR spectrum
centration of 10 lg/ml, at which 100% cell death was
observed within 6 h. On the other hand, preferential
cytotoxicity test for columbianetin (2) and p-hydroxy-
cinnamic acid indicated that they are inactive in both
nutrient-rich and nutrient-deprived condition at a con-
centration of 100 lg/ml. This suggests the requirement
of unique structural feature of angelmarin (1) to expose
preferential cytotoxic activity.
of
1 showed the absorptions due to hydroxyl
(3280 cmꢁ1), ester carbonyl (1705 cmꢁ1), and phenyl
(1610, 1460 cmꢁ1) groups. The H NMR spectrum of
1
1 revealed signals due to two tertiary methyls (dH 1.61,
1.65), an oxygen-substituted methine (dH 5.21), an ali-
phatic methylene (dH 3.38), two sets of ortho-coupled
aromatic protons (dH 6.24, 7.68; 6.79, 7.31), two trans
olefinic protons (dH 6.14, 7.35), together with two pairs
of p-substituted phenyl protons (dH 6.83, 7.28). The 13C
NMR spectrum showed the signals of 21 carbons
including those of two ester carbonyls and four sp2
and two oxygenated sp3 carbons, one aliphatic carbon,
and two phenyl groups. These data resembled those of
columbianetin8 (2) isolated from the same extract, with
the exception of the presence of additional signals
ascribable to a p-hydroxycinnamoyl group. The COSY
and HMQC spectra revealed the partial connectivities
Pancreatic cancer is one of the most serious forms of
cancer associated with the lowest 5-year survival rates
known for cancers,13,14 and continues to be one of the
major health problems that remain unresolved at the
start of the 21st century. Currently, surgery is the only
treatment modality that offers any prospect of potential
cure.14,15 It shows resistance to almost all known chemo-
therapeutic agents such as 5-fluorouracil, taxol, doxoru-
bicin, cisplatin, and camptothecin, and exhibits only
minimal activity as a single agent in pancreatic cancer.14
Moreover, these agents were found to be virtually inac-
tive under nutrient-deprived conditions, while they
exhibit only weak toxicity under normoxic conditions.6
This indicates a different mechanism that may underlie
the anti-tumor activity of angelmarin (1) against tumor
cells growing in a low-nutrient and low-oxygen environ-
ment. Further studies on its mechanism of action and
in vivo anti-tumor activity are underway and will be
reported as a full paper.
0
(bold line) between C3–C4, C5–C6, C9–C10, C7 –C8
0
0
0
0
0
and C2 ð6 Þ–C3 ð5 Þ. These partial structures were connect-
ed based on the long-range correlations observed in
the HMBC spectrum (Fig. 1a), leading to a planar
structure of p-hydroxycinnamoyl ester of 2. Further-
more, saponification of angelmarin (1)8 afforded 2
25
D
having the optical rotation value (½aꢀ +198ꢁ) identical
22
D
to the reported value (½aꢀ +209ꢁ),9 suggesting the
absolute stereochemistry of 1 to be 10S. In addition,
the CD spectrum of 1 showed a positive Cotton
effect ([h]338 +34314, [h]298 ꢁ16932) due to an exciton
chirality (Fig. 1b),10,11 suggesting the spatial orientation
of two chromophores to be in a clockwise fashion
(Fig. 1b).9,10 Thus, the structure of 1 was concluded as
11-O-(p-hydroxycinnamoyl)columbianetin.
Angelmarin (1) was tested for its preferential cytotoxic-
ity using PANC-1 cancer cell line.12 As shown in Figure
2, PANC-1 is highly resistant to even extreme nutrient
deprivation and can survive under this condition for
more than 48 h.5 In this study, this tolerance to nutrient
deprivation was remarkably eliminated by angelmarin
(1) in a concentration- as well as time-dependent man-
ner. Cells exposed to angelmarin (1) at a concentration
of 0.01 lg/ml showed 100% cell death within 24 h of
starvation, and this sensitivity to starvation was more
pronounced when angelmarin (1) was added at a con-
Time (h)
Figure 2. Effect of angelmarin (1) on survival of PANC-1 cells under
nutrient-deprived conditions.
a
b
CD (EtOH)
3
2
O
O
1
0
O
−1
−2
UV
O
A
3
2
1
O
0
400
OH
200
300
λ (nm)
Figure 1. (a) 1H–1H COSY (bold line) and key HMBC correlations (arrows) observed for 1 and (b) CD and UV spectra of 1.

S. Awale et al. / Bioorg. Med. Chem. Lett. 16 (2006) 581–583
583
113.0 (C-4a), 113.6 (C-8), 114.5 (C-70), 115.9 (C-30,50),
116.1 (C-80), 126.6 (C-10), 128.9 (C-5), 129.8 (C-20,60),
144.3 (C-4), 151.1 (C-8a), 158.3 (C-40), 161.5, (C-2), 164.1
(C-7), 166.5 (C-90).
Acknowledgments
This work was supported in part by a grant from the
Ministry of Health and Welfare for the Second-Term
Comprehensive 10-year Strategy for Cancer Control
and Grants-in-Aid for Cancer Research from the Minis-
try of Health and Welfare and the Ministry of Educa-
tion, Science, Sports and Culture.
8. Angelmarin (1, 30 mg) was treated with LiOH in MeOH–
H2O (1 N, 2 ml) with stirring at room temperature for
28 h. The reaction mixture was then neutralized by adding
1 N HCl (2 ml) and extracted with EtOAc (2 ml · 3). The
combined organic layer was dried over MgSO4, filtered,
concentrated, and purified by preparative TLC (acetone–
25
CHCl3, 2:8) to afford 2 (8 mg) with ½aꢀD +198ꢁ (c 0.025,
References and notes
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25
7. Angelmarin (1). Colorless amorphous solid. ½aꢀD +218.7ꢁ
(c 0.025, CHCl3). IR mmax (CHCl3) cmꢁ1 3280, 1910, 1705,
1610, 1585, 1506, 1490, 1460, 1405, 1388, 1370, 1325, 1260,
1168, 1140, 1065, 980, 835. CD kmax (EtOH, 0.255 mM)
nm: 338 ([h] +34314), 298 ([h] ꢁ16932). HR-FABMS
393.1295 [calcd for C23H21O6 (M+H)+ 393.1338]. 1H
NMR (400 MHz, CDCl3) d: 1.61 (3H, s, H3-13), 1.65 (3H,
s, H3-12), 3.38 (2H, ddd, J = 16.3, 9.5, 8.0 Hz, H2-9), 5.21
(1H, dd, J = 9.5, 8.0 Hz, H-10), 6.14 (1H, d, J = 15.9, H-
80), 6.24 (1H, d, J = 9.5 Hz, H-4), 6.79 (1H, d, J = 8.3 Hz,
H-6), 6.83 (2H, d, J = 8.5 Hz, H-30,50), 7.28 (2H, d,
J = 8.5 Hz, H-20,60), 7.31 (1H, d, J = 8.3 Hz, H-5), 7.35
(1H, d, J = 15.9, H-70), 7.68 (1H, d, J = 9.5 Hz, H-3). 13C
NMR (100 MHz, CDCl3) d: 21.1 (C-13), 22.1 (C-12), 27.5
(C-9), 82.2 (C-11), 89.1 (C-10), 106.9 (C-6), 111.98 (C-3),
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