Two indolocarbazole alkaloids with apoptosis activity from a marine-derived actinomycete Z2039-2
ABSTRACT Bioassay-guided fractionation of the EtOAc extract from the fermentation broth of a marine-derived actinomycete Z2039-2 led to the isolation of two known indolocarbazole alkaloids, K252c (1) and arcyriaflavin A (2).1 and2 exhibited moderate cytotoxic activities against the K562 cell line, and induced apoptotic activities at 10 and 100 μM, respectively.
This is the first report on the significant apoptosis inducing effect of indolocarbazole alkaloids against K562 cancer cells.
Arch Pharm Res Vol 30, No 3, 270-274, 2007
Two Indolocarbazole Alkaloids with Apoptosis Activity from a
Marine-derived Actinomycete Z2039-2
Rui Liu, Tianjiao Zhu, Dehai Li, Jingyan Gu, Wei Xia, Yuchun Fang, HongbingLiu, Weiming Zhu, and
Key laboratory of Marine Drugs, Chinese Ministry of Education; Institute of Marine Drugs and Food, Ocean Univer-
sity of China, Qingdao 266003, PR China
(Received May 15, 2006)
Bioassay-guided fractionation of the EtOAc extract from the fermentation broth of a marine-
derived actinomycete Z2039-2 led to the isolation of two known indolocarbazole alkaloids,
K252c (1) and arcyriaflavin A (2). 1 and 2 exhibited moderate cytotoxic activities against the
K562 cell line, and induced apoptotic activities at 10 and 100 µM, respectively. This is the first
report on the significant apoptosis inducing effect of indolocarbazole alkaloids against K562
Key words: Marine-derived actinomycete, Indolocarbazole alkaloids, K252c, Arcyriaflavin A,
Cytotoxicity, Apoptosis inducer
During our studies in the search for anticancer bioactive
natural products from actinomycetes (Liu et al., 2005), a
marine-derived actinomycete strain, Z2039-2, collected on
the coast of Qingdao, was investigated. The EtOAc extract
of this strain showed significant cytotoxic activity against
the K562 cell line (the human chronic myelogenous
leukemia cell line) at a concentration of 5 µg/mL. Con-
sequently, mass fermentation and activity-guided isolation
of anticancer compounds from this strain have been
undertaken, which led to the isolation of two known
indolocarbazole alkaloids, K252c (1) and arcyriaflavin A
(2). Both of these compounds exhibited cytotoxic and
apoptotic activities against the K562 cancer cell line.
K252c (1) and arcyriaflavin A (2) are natural products,
possessing anticancer properties, which belong to the
family of indolocarbazole alkaloids. Indolocarbazole
alkaloids constitute a group of natural products that have
attracted great attention due to their original structural
features and potential therapeutic applications. Most of
these compounds are characterized by the presence of
an indolo[2, 3-a]pyrrolo[3, 4-c]carbazole core, with some
having a sugar attached. Various biological activities have
been reported for indolocarbazole alkaloids, but the greatest
interest has focused on their antitumor and neuroprotective
properties (Akinaga, et al., 2000; Prudhomme, et al.,
2003; Sanchez, et al., 2005). In this paper, the isolation
and anticancer activities of two indolocarbazole alkaloids,
K252c (1) and arcyriaflavin (2), from a marine-derived
actinomycete, Z2039-2, are reported.
MATERIALS AND METHODS
General experimental procedures
Melting points were measured using a Yanaco MP-
500D micro-melting point apparatus, and are reported
uncorrected. Optical rotations were obtained on a JASCO
P-1020 digital polarimeter. UV spectra were recorded on
Beckman DU® 640 spectrophotometer. IR spectra were
run as KBr disks on a NICOLET NEXUS 470 spectro-
photometer. 1H-, 13C-NMR and DEPT spectra, as well as
2D-NMR were recorded on a JEOL Eclips-600 spectro-
meter, using TMS as an internal standard, with the chemical
shifts recorded as values. ESI-MS was measured on a Q-
TOF ULTIMA GLOBAL GAA076 LC mass spectrometer.
Fermentation and extraction
An actinomycete strain, Z2039-2, was isolated from the
sea earth collected on the coast of Qingdao, China. The
strain was grown on a rotary shaker at 120 rpm and 28oC
Correspondence to: Weiming Zhu, and Qianqun Gu, Key labora-
tory of Marine Drugs, Chinese Ministry of Education; Institute of
Marine Drugs and Food, Ocean University of China, Qingdao
266003, PR China
E-mail: email@example.com and firstname.lastname@example.org
Two Indolocarbazole Alkaloids with Apoptosis Activity from a Marine-derived Actinomycete Z2039-2 271
for 5 days in two hundred 500 mL conical flasks, contain-
ing a liquid medium (150 mL/flask) composed of glucose
2.0%, starch 1.0%, soybean powder 0.5%, peptone 2%,
yeast extract 0.4%, beef extract 0.1%, K2HPO4 0.05%,
CaCO3 0.2% and sea-water, pH 7.0. The fermented whole
broth (30 liters) was filtered through cheese cloth to
separate the supernatant and mycelia. The former was
concentrated under reduced pressure to about a quarter
of its original volume, and then extracted three times with
EtOAc, while the latter was extracted three times with
acetone. The acetone solution was concentrated under
reduced pressure to afford an aqueous solution. The
aqueous solution was extracted three times with EtOAc,
yielding another EtOAc solution. Both EtOAc solutions
were combined and concentrated under reduced pressure
to give the EtOAc extract (3.0 g).
The EtOAc extract (3.0 g) was initially subjected to
column chromatography on silica gel, under gradient
elution with petroleum ether:acetone (from 100:0 to 0:100),
to yield 13 fractions. Of these, fraction 6 (petroleum
ether:acetone, 70:30) was further purified using a RP-18
column, under gradient elution with methanol:water, to
yield compound 1 (8 mg, methanol:water, 60:40) as yellow
crystals. Fraction 7 (petroleum ether:acetone, 65:35) was
further purified using Sephadex LH20, eluted with 100%
methanol, to give compound 2 (18 mg) as orange crystals.
Yellow crystals; positive ESI-MS m/z 312 [M + H]+; 13C-
NMR (DMSO-d6, 150 MHz) : δC = 127.9 (s, C-2), 114.0 (s,
C-3), 122.5 (s, C-3a), 121.1 (d, C-4), 119.8 (d, C-5), 124.9
(d, C-6), 111.9 (d, C-7), 139.2 (s, C-7a), 132.9 (s, C-8),
45.3 (t, C-9), 125.4 (s, C-2'), 115.5 (s, C-3'), 122.8 (s, C-
3a'), 125.2 (d, C-4'), 118.8 (d, C-5'), 124.9 (d, C-6'), 114.3
(d, C-7'), 139.1 (s, C-7a'), 119.8 (s, C-8'), 172.4 (s, C-9');
1H-NMR (DMSO-d6, 600 MHz) : δH = 11.81 (1H, brs, NH-
1), 8.04 (1H, d, J = 7.4 Hz, H-4), 7.30 (1H, td, J = 7.4, 1.0
Hz, H-5), 7.47 (1H, td, J = 7.4, 1.0 Hz, H-6), 7.77 (1H, brd,
J = 7.4 Hz, H-7), 4.96 (2H, brs, H-9), 8.49 (1H, brs, NH-
10), 11.63 (1H, brs, NH-1'), 9.21 (1H, d, J = 7.6 Hz, H-4'),
7.22 (1H, td, J = 7.6, 1.1 Hz, H-5'), 7.42 (1H, td, J = 7.6,
1.1 Hz, H-6'), 7.70 (1H, brd, J = 7.6 Hz, H-7').
Arcyriaflavin A (2)
Orange crystals; negative ESI-MS m/z 324 [M-H]-; 13C-
NMR (pyridine-d5, 150 MHz) : δC = 121.6 (s, C-2, 2'), 117.7
(s, C-3, 3'), 123.7 (s, C-3a, 3a'), 125.7 (d, C-4, 4'), 120.8
(d, C-5, 5'), 127.1 (d, C-6, 6'), 112.1 (d, C-7, 7'), 142.1 (s,
C-7a, 7a'), 130.5 (s, C-8, 8'), 172.6 (s, C-9, 9'); 1H-NMR
(pyridine-d5, 600 MHz) : δH = 12.98 (2H, brs, NH-1, 1'),
9.81 (2H, brd, J = 7.3 Hz, H-4, 4'), 7.48 (2H, brt, J = 7.3
Hz, H-5, 5'), 7.52 (2H, td, J = 7.3, 1.0 Hz, H-6, 6'), 7.61
(2H, brd, J = 7.3 Hz, H-7, 7'), 12.53 (1H, brs, NH-10).
In vitro cytotoxicity assays
The human chronic myelogenous leukemia cell line,
K562, was grown in RPMI 1640 culture medium, supple-
mented with 10% fetal calf serum, 100 U/ml penicillin and
100 µg/mL streptomycin, at 37°C in 5% CO2/100% hu-
The cytotoxic activity was evaluated by the MTT method
(Mosmann et al., 1983) using the K562 cell line. 200 µL of
cell suspensions, at a density of 5 × 103 cell mL−1, were
plated in 96 well microtiter plates under the above
conditions. Then, 2 µL of the test compound solutions (in
DMSO), at different concentrations, were individually
added to each well, and then incubated for a further 72 h
under the same conditions. 20 µL of MTT solution (5 mg/
mL in PBS) was added to each well, and incubated for a
further 4 h. 150 µL of old medium, containing MTT, was
then gently replaced with DMSO, which was then pipetted
to dissolve any formazan crystals formed. The absorbance
was then measured at 570 nm on a SPECTRA MAX
PLUS plate reader.
To assess the degree of apoptosis, cells were plated in
6-well cell culture plates (5 × 104 cells/well), in triplicate,
and then treated with DMSO (Control) or the compounds
(1 and 2) for 72 h at the indicated concentrations (Table I).
Cells were harvested, washed twice with PBS, and the
cellular DNA stained with 200 µL propidium iodide (PI, 50
µg/mL, RNase 1 µg/mL, Triton X-100 0.1%). After incubation
at 4oC for 20 min, the cells were analyzed using flow
cytometry (Becton-Dickinson, Vantage, San Diego, CA).
Detection of apoptosis by Annexin V-EGFP/PI
The assay was performed according to the instructions
of the manufacture (Keygen Biotech. Co. Ltd., Nanjing,
China). 1-5 × 105 cells were sedimentated, washed twice
with PBS, and resuspended in 500 µL binding buffer.
EGFP conjugated Annexin V (1 µL) and PI (5 µL) were
added to each sample, and the mixture incubated at room
temperature in the dark for 5 min. The cells were then
immediately subjected to fluorescence-activated cell sorter
(FACS) analysis using a Becton-Dickenson FACScan flow
cytometer and the Cell Quest software version 1.2 (Becton-
Dickenson, Mountain View, CA, U.S.A.) and the percentage
RESULTS AND DISCUSSION
The two known compounds, K252c (1) and arcyriaflavin
A (2), were isolated from the EtOAc extract of actinomycete
Z2039-2 by bioassay-guided fractionation using the cyto-
272R. Liu et al.
toxicity against K562 cells. The structures of 1 and 2 were
identified by analysis of their NMR data, as well as by
comparison of their physical and spectral data with
literature values (Kase et al., 1987; Horton et al., 1994).
Compound 1 was obtained as yellow crystals. Its
molecular formula was established as C20H13N3O by the
positive ESI-MS m/z 312 [M + H]+, in combination with
both the 1H- and 13C-NMR data. Its 1H-NMR spectrum
showed twelve signals for three OH or NH protons (δH
11.81, 11.63 and 8.49), eight aromatic protons (δH 9.21,
8.04, 7.77, 7.70, 7.47, 7.42, 7.30, and 7.22), and one SP3
methylene proton (δH 4.96). The 13C-NMR data consisted
of twenty carbon signals, including eighteen aromatic
carbons, one methylene carbon (δc 45.3) and one
carbonyl carbon (δc 172.4). These
signals of compound 1, by comparison with the known
compounds, suggested the presence of an indolo[2,3-
a]pyrrolo[3,4-c]carbazole skeleton. From the combination
of the 2D NMR (1H-1H COSY and HMBC) and comparison
with the literature (Kase et al., 1987), compound 1 was
identified as K252c.
Compound 2 was obtained as orange crystals. Its
molecular formula was established as C20H11N3O2 by the
negative ESI-MS m/z 314 [M - H]−, in combination with
both the 1H- and 13C-NMR data. Its 1H-NMR spectrum
showed six signals, two OH or NH protons (δH 12.98 and
12.53) and four aromatic protons (δH 9.81, 7.61, 7.52 and
7.48). The 13C-NMR data consisted of ten carbon signals,
including nine aromatic carbons and one carbonyl carbon
(δc 172.6). Comparison of the 1H- and 13C-NMR data of 2
with 1, and by considering the obtained molecular
formula, showed that 2 was a symmetrical molecule. The
only difference between 2 and 1 was that the two protons
at the C-9 position of compound 1 were replaced by a
carbonyl group in compound 2. Thus, compound 2 was
identified as arcyriaflavin A (Horton et al., 1994), with this
structure also verified by the 2D NMR.
The quantification of cellular growth, including the
viability, apoptosis and different phases of the cell cycle,
has become an essential parameter in the evaluation of
the anticancer activity of compounds. To evaluate the
potential anticancer effect of compounds 1 and 2, the cell
viability and cellular DNA were assessed using K562
cells. A decrease in the reduced MTT concentrations was
generally observed in the presence of both compounds 1
(IC50 = 7.8 µM) and 2 (IC50 = 66.0 µM).
Several bisindole alkaloids have previously been iso-
lated from Myxomycetes (Hashimoto et al., 1994; Frode
et al., 1994), with some that contain the maleimide group,
such as staurosporine (Omura et al., 1997), UCN-01
(Takahashi et al., 1987) and rebeccamycin (Nettleton et
al., 1985), produced by the family of Streptomyces,
Actinomycetes and Saccharothrixes. K252c and a new
bisindole alkaloid, ZHD-0501, were previously isolated
from a marine-derived Actinomadura sp. 007 in our
laboratory (Han et al., 2005). Most reports on the
antitumor bioactivity of these bisindole alkaloids have
focused on topoisomerase I mediated DNA cleavage,
potent inhibition of protein kinase C and cell-cycle check-
point inhibition. Here, the cell cycle and apoptotic effects
of K252c (1) and arcyriaflavin A (2) were examined by
flow cytometry studies on K562 cells. Compound 1 induced
significant apoptosis at 10 µM, from 0.74 (Control) to
57.30%, and to nearly 100% at 100 µM (Fig. 1 and Table
I). Compound 2 induced considerable apoptosis at 10 µM,
from 0.74 (Control) to 25.90% (Fig. 1 and Table I), and
induced significant apoptosis, upto 68.93%, at 100 µM.
The apoptosis effect of compound 2 was weaker than 1.
Fig. 1. Chemical structures of K252c (1) and arcyriaflavin A (2)
Fig. 2. Effects of compounds 1 and 2 on K562 cell cycle and apoptosis. Cells (5 × 104 cells/well) were treated with DMSO (Control) or compounds
1 and 2, at the indicated concentrations, for 72 h. Cellular DNA was stained with 200 µL propidium iodide, and then analyzed using flow cytometry.
The presented results are representative of three independent experiments.
Two Indolocarbazole Alkaloids with Apoptosis Activity from a Marine-derived Actinomycete Z2039-2273
This finding suggested that the presence of another C-9
carbonyl group appears to weaken the bioactivity of
compound 1. This result was in accordance with previous
reports (Kase et al., 1987; Horton et al., 1994). The
apoptotic effect of compound 1, which was greater than
compound 2, was also been borne out by the Annexin-V
FITC experiment (Fig. 3). To the best of our knowledge, this
is the first report on the apoptotic effects of compounds 1
and 2, while in our experiment no cell cycle inhibition
effects on K562 cells of the two compounds were observed.
This work was funded by the Chinese National Natural
Science Fund (No. 30572246) and the Chinese Ocean
Mineral Resource R & D Association (DY105-2-04). The
anti-tumor assay was performed at the Shanghai Institute
of Materia Medica, Chinese Academy of Sciences.
Akinaga, S., Sugiyama, K., and Akiyama, T., UCN-01 (7-
hydroxystaurosporine) and other indolocarbazole compounds:
a new generation of anti-cancer agents for the new century.
Anti-cancer Drug Design, 15, 43-52 (2000).
Frode, R., Hinze, C., Josten, I., Schmidt, B., Steffan, B., and
Steglich, W., Isolation and synthesis of 3,4-bis(indol-3-
yl)pyrrole-2,5-dicarboxylic acid derivatives from the slime
mould Lycogala epidendrum. Tetrahedron Lett., 35, 1689-
Hashimoto, T., Yasuda, A., Akazawa, K., Takaoka, S., Tori, M.,
and Asakawa, Y., Three novel dimethyl pyrroledicarboxylate,
lycogarubins A-C, from the myxomycetes lycogala epidendrum.
Terahedron Lett., 35, 2559-2560 (1994).
Han, X. X., Cui, C. B., Gu, Q. Q., Zhu, W. M., Liu, H. B., Gu, J.
Y., and Osada, H., ZHD-0501, a novel naturally occurring
staurosporine analog from Actinomadura sp. 007, Tetrahedron
Lett., 46, 6137-6140 (2005).
Horton, P. A., Longley, R. E., Mcconnell, O. J., and Ballas, L. M.,
Staurosporine aglycone (K252-c) andarcyriaflavin A from the
marine ascidian, Eudistoma sp. Experientia, 50, 843-845
Kase, H., Iwahashi, K., Nakanishi, S., Matsuda, Y., Yamada, K.,
Takahashi, M., Murakata, C., Sato, A., and Kaneko. M., K-
252 compounds, novel and potent inhibitors of protein kinase
C and cyclic nucleotide-dependent protein kinases., Biochem.
Biophys. Res. Commum., 142, 436-440 (1987).
Liu, R., Cui, C. B., Duan, L., Gu, Q. Q., and Zhu, W. M., Potent
in vitro anticancer activity of metacycloprodigiosin and
undecylprodigiosin from a sponge-derived actinomycete
saccharopolyspora sp. nov. Arch. Pharm. Res., 28, 1341-
Mosmann, T. J., Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity
assays. Immunol. Methods., 65, 55-63 (1983).
Nettleton, D. E., Doyle, T. W., Krishnan, B., Matsumoto, G. K.,
and Clardy, J., Isolation and structure of rebeccamycin-a
new antitumor antibiotic from nocardia aerocoligenes.,
Tetrahedron Lett., 26, 4011-4014 (1985).
Omura, S., Iwai, Y., Hirano, A., Nakagawa, A., Awaya. J.,
Table I. Distribution of the K562 cells within the cell cycle and
Distribution of the cells (%)
*Data were obtained from histograms. The numbers in the Apo.
column represent the relative percentage of apoptotic cells counted in
the sub-G0/G1 peak region to the total cell numbers. The proportions
of cells in each phase of the cell cycle (G0/G1, S, and G2/M) were
analyzed using the CellQuest computer software.
Fig. 3. The result of the annexin-v FITC experiment
274R. Liu et al.
Tsuchya, H., Takahashi, Y., and Masuma, R., A new alkaloid
AM-2282 of streptomyces origin. Taxonomy, fermentation,
isolation and preliminary characterization. J. Antibiot., 30,
Prudhomme, M., Rebeccamycin analogus as anti-cancer
agents. Eur. J. Med. Chem., 38, 123-140 (2003).
Sanchez, C., Zhu, L., Brana, A. F., Salas, A. P., Rohr, J.,
Mendez, C., and Salas, J. A., Combinatorial biosynthesis of
antitumor indolocarbazole compounds. Proc. Natl Acad. Sci.
U.S.A., 102, 461-466 (2005).
Takahashi, I., Kobayashi, E., Asano, K., Yoshida, and M.,
Nakano, H., UCN-01, a selective inhibitor of protein kinase C
from Streptomyces. J. Antibiot., 40, 1782-1784 (1987).