Rubratoxin A specifically and potently inhibits protein phosphatase 2A and suppresses cancer metastasis.
ABSTRACT Although cytostatin analog protein phosphatase 2A (PP2A)-specific inhibitors are promising candidates of a new type of anticancer drug, their development has been hindered because of their liability. To find new classes of PP2A-specific inhibitors, we conducted a screening with microbial metabolites and found that rubratoxin A, a classical mycotoxin, is a highly specific and potent inhibitor of the enzyme. While rubratoxin A inhibits PP2A at Ki = 28.7 nm, it hardly inhibited any other phosphatases examined. Rubratoxin B, a close analog, also specifically but weakly inhibits PP2A at Ki = 3.1 microM. The inhibition of intracellular PP2A in cultured cells is obviously observed with 20 microM rubratoxin A treatment for 3 h, inducing the overphosphorylation in PP2A substrate proteins. Although rubratoxins and cytostatin differ in the apparent structures, these compounds share similarities in the structures in detail and PP2A-binding manners. Rubratoxin A showed higher suppression of tumor metastasis and reduction of the primary tumor volume than cytostatin in mouse experiments. As a successor of cytostatin analogs, rubratoxin A should be a good compound leading to the development of antitumor drugs targeting PP2A.
- SourceAvailable from: Russell J Cox[Show abstract] [Hide abstract]
ABSTRACT: A series of directed knockout experiments, combined with an in vitro assay of pathway components, has elucidated for the first time the chemical steps involved in the biosynthesis of the tropolone class of fungal maleic anhydrides. The pathway involves the stepwise oxidation of aldehyde and methyl carbon atoms to form a 1,2-dicarboxylate. A hydrolase-catalyzed interconversion of this and the corresponding maleic anhydride, followed by decarboxylation of the diacid leads to the pathway's final product of stipitatic acid.Angewandte Chemie International Edition in English 05/2014; · 13.45 Impact Factor
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ABSTRACT: Taxa of the Talaromyces purpurogenus complex were studied using a polyphasic approach. ITS barcodes were used to show relationships between species of the T. purpurogenus complex and other Talaromyces species. RPB1, RPB2, β-tubulin and calmodulin sequences were used to delimit phylogenetic species in the complex. These data, combined with phenotypic characters, showed that the complex contains four species: T. purpurogenus, T. ruber comb. nov. and two new species T. amestolkiae sp. nov. and T. stollii sp. nov. The latter three species belong to the same clade and T. purpurogenus is located in a phylogenetic distant clade. The four species all share similar conidiophore morphologies, but can be distinguished by macromorphological characters. Talaromyces ruber has a very distinct colony texture on malt extract agar (MEA), produces bright yellow and red mycelium on yeast extract sucrose agar (YES) and does not produce acid on creatine sucrose agar (CREA). In contrast, T. amestolkiae and T. stollii produce acid on CREA. These two species can be differentiated by the slower growth rate of T. amestolkiae on CYA incubated at 36 °C. Furthermore, T. stollii produces soft synnemata-like structures in the centre of colonies on most media. Extrolite analysis confirms the distinction of four species in the T. purpurogenus complex. The red diffusing pigment in T. purpurogenus is a mixture of the azaphilone extrolites also found in Monascus species, including N-glutarylrubropunctamine and rubropunctatin. Talaromyces purpurogenus produced four different kinds of mycotoxins: rubratoxins, luteoskyrin, spiculisporic acid and rugulovasins and these mycotoxins were not detected in the other three species.Persoonia - Molecular Phylogeny and Evolution of Fungi 12/2012; 29:39-54. · 3.49 Impact Factor
Rubratoxin A specifically and potently inhibits
protein phosphatase 2A and suppresses cancer
Shun-ichi Wada,1Ihomi Usami,1Yoji Umezawa,2Hiroyuki Inoue,1Shun-ichi Ohba,1Tetsuya Someno,1
Manabu Kawada1,3and Daishiro Ikeda1
1Numazu Bio-Medical Research Institute, Microbial Chemistry Research Foundation, Shizuoka;2Microbial Chemistry Research Foundation, Tokyo, Japan
(Received October 20, 2009 ⁄Revised November 6, 2009 ⁄ Accepted November 8, 2009 ⁄ Online publication December 20, 2009)
Although cytostatin analog protein phosphatase 2A (PP2A)-spe-
cific inhibitors are promising candidates of a new type of antican-
cer drug, their development has been hindered because of their
liability. To find new classes of PP2A-specific inhibitors, we con-
ducted a screening with microbial metabolites and found that
rubratoxin A, a classical mycotoxin, is a highly specific and potent
inhibitor of the enzyme. While rubratoxin A inhibits PP2A at Ki =
28.7 nM, it hardly inhibited any other phosphatases examined. Ru-
bratoxin B, a close analog, also specifically but weakly inhibits
PP2A at Ki = 3.1 lM. The inhibition of intracellular PP2A in cultured
cells is obviously observed with 20 lM rubratoxin A treatment for
3 h, inducing the overphosphorylation in PP2A substrate proteins.
Although rubratoxins and cytostatin differ in the apparent struc-
tures, these compounds share similarities in the structures in detail
and PP2A-binding manners. Rubratoxin A showed higher suppres-
sion of tumor metastasis and reduction of the primary tumor vol-
ume than cytostatin in mouse experiments. As a successor of
cytostatin analogs, rubratoxin A should be a good compound
leading to the development of antitumor drugs targeting PP2A.
(Cancer Sci 2010; 101: 743–750)
bolism. Protein phosphatase (PP)2A is a ubiquitous serine⁄thre-
onine phosphatase in eukaryotic cells, which regulates the
phosphorylation state and function of a broad range of pro-
teins.(1,2)We previously isolated a PP2A-specific inhibitor,
cytostatin, from an actinomycete culture.(3–5)Its analog, fostrie-
cin, and other PP2A-specific inhibitors have also been reported
since mid 1990s.(6–11)These new inhibitors have higher specific-
ities to PP2A than okadaic acid, a previously identified inhibitor
(PP1 IC50⁄PP2A IC50: fostriecin, 104–105; okadaic acid,
100),(6,12)and have shown promising antitumor activities in ani-
mal experiments.(6,9,13)A phase I clinical study of fostriecin was
reported, but the result was ambiguous because of the instability
of the compound and its short supply.(14)Therefore, to further
investigate the feasibility of PP2A-specific inhibitors as an anti-
cancer drug, we have been searching for new stable PP2A inhib-
itors from microbial metabolites.
In the present study, we found rubratoxin A as a new PP2A-
specific inhibitor. Rubratoxins are one of the oldest mycotoxins,
whose existence as a hepatotoxic agent to livestock was first
implied in 1957.(15)Isolated from Penicillium rubrum, the struc-
tures of rubratoxins were determined in approximately the
1960s–1970s.(16,17)Since then, numerous studies, usually using
rubratoxin B, have reported the toxic effects of rubratoxins,
including a case of rubratoxicosis in humans, and investigations
are still being pursued.(16–19)However, the toxicity mechanism
remains ill-defined because the target molecule of rubratoxins
has not been elucidated. This is the first report to unveil the pri-
he phosphorylation of intracellular protein plays a central
role in the regulation of eukaryotic cell function and meta-
mary target molecule of rubratoxins, showing their specific and
potent inhibition of PP2A activity and the convincing binding
model. The antitumor and antimetastatic effects of rubratoxin A
were also examined in several mouse models comparing the
effects of cytostatin to evaluate the utility of PP2A as a novel
target of cancer treatment.
Materials and Methods
Screening of the PP2A inhibitor. The screening assay was per-
formed on 96-well plates using 100% acetone or 70% ethanol
extract (24 mg⁄mL) libraries of microbial cultures consisting of
fungi, actinomycetes, and other bacteria. Each extract was inoc-
ulated at 1 lL⁄well. The substrate, p-nitrophenyl phosphate
(pNPP), was dissolved at 200 mM in a reaction buffer composed
of 50 mM Tris-HCl (pH 8.5), 20 mM MgCl2, and 0.5 mM dith-
iothreitol (DTT) at 10 lL⁄well on the plate. A human blood cell
PP2A (Millipore, Billerica, MA, USA) dissolved in a buffer
composed of 20 mM MOPS (pH 7.5), 150 mM NaCl, 60 mM
2-mercaptoethanol, 1 mM MgCl2, 1 mM EGTA, 0.1 mM MnCl2,
1 mM DTT, 10% glycerol, and 0.1 mg⁄mL bovine serum albu-
min at 0.5 units⁄9 mL was added at 90 lL⁄well to start the reac-
tion. After incubation at 37?C for 30 min, the reaction product
p-nitrophenol was measured at 405 nm with a Multiskan MS
microplate reader (Labsystems, Pitesti, Romania).
Purification of rubratoxins. A rubratoxin-producing Penicil-
lium sp. strain was cultured in the autoclaved polished rice grain
medium containing 2.5% soybean meal (Ajinomoto Takara,
Tokyo, Japan) and 25% water at 26?C for 2 weeks. The 67%
acetone extract of the culture was partitioned with BuOH⁄H2O.
Rubratoxins were purified from the BuOH fraction by sequential
chromatographies with an YMC*GEL ODS-A column (6 nm,
S-150 lm; YMC, Kyoto, Japan), Inertsil ODS-3 column
(20 mm·250 mm; GL Sciences, Torrance, CA, USA), and
Sephadex LH-20 column (GE Healthcare, Chalfont St Giles,
UK). For the octa decyl silyl (ODS) chromatography, isocratic
35% acetonitrile containing 0.1% trifluoroacetic acid was used
as a solvent, while 35% acetonitrile was used for the Sephadex
LH-20 chromatography. Rubratoxins were identified by nuclear
magnetic resonance (NMR) and mass spectrometry (MS) analy-
ses with JEOL JNM-A400 (JEOL, Tokyo, Japan), PE SCIEX
API165 (Perkin Elmer, Waltham, MA, USA), and JEOL JMS-
T100LC (JEOL, Tokyo, Japan) spectrometers. The purity of ru-
bratoxins A and B isolated and used in this study were estimated
by HPLC (UV215nm) with a minimum of 96.6% and 93.2%,
Phosphatase inhibition assays. Rabbit skeletal muscle PP1,
recombinant PP2B with calmodulin, recombinant protein–tyro-
sine phosphatase-1B (PTP-1B) (Millipore, USA), and calf intes-
3To whom correspondence should be addressed.
ª ª 2009 Japanese Cancer Association
|vol. 101|no. 3|
tine alkaline phosphatase (CIP; New England Biolabs, Ipswich,
MA, USA) were employed for the other phosphatase inhibition
assays. The pNPP assays were performed as per PP2A in the
buffers recommended by each enzyme’s manufacturers.
Immunoblotting. The cells were cultured at 2 · 105cells⁄mL
as previously described(5)for 24 h, and treated with the com-
pounds for 3 h. The cell lysates were separated by sodium dode-
acrylamide gels, followed by the immunoblotting(20,21)using
Tris-buffered saline (10 mM Tris-HCl [pH 7.5] ad 150 mM
NaCl). Horseradish peroxidase-conjugated antirabbit immuno-
globulin G (IgG) and antimouse IgG antibodies (GE Healthcare,
UK) were used as the secondary antibody at 1⁄2000 and
1⁄5000, respectively. Antiphosphotyrosine mouse monoclonal
antibody (Millipore, USA) was used at 1⁄1000. The other anti-
bodies were rabbit polyclonal antibodies (Cell Signaling Tech-
nology, Beverly, MA, USA), which was used at 1⁄1000 or
Molecular modeling. The rubratoxin A-PP2A binding model,
PP2A–RUB_AH, was created with Discovery Studio v2.1
(Accelrys Software, San Diego, CA, USA) based on the crystal
structures of microcystin–LR and okadaic acid–PP2A (PDB
code 2ie3 and 2ie4).(22)
The stereo structure of the rubratoxin A–dicarboxylic acid
form, RUB_AH, was deduced from the structure of rubratoxin
B(23,24)converting the C26 carbonyl to the hydroxyl group and
conjugating a water molecule to C23 and C24. RUB_AH was
superimposed on microcystin–LR in the structure 2ie3 consider-
ing the direction of the inhibitors in the binding model of cytost-
atin analogs to PP2A.(22,25)The carbonyl oxygens in the
dicarboxylic acid (-O-CO-C = C-COO-) of RUB_AH over-
lapped with the position of the oxygens of microcystin–LR
(-CO-NH-CH-COO), thus it was fixed at the coordinate. The
lactone ring positioned in the close vicinity of Cys269 was also
fixed at the residue by Michael addition. The preliminary struc-
ture was energy minimized with the simulation module of Dis-
covery Studio using the chemical force feedback force field, and
the resultant model PP2A–RUB_AH was obtained.
Mouse experiments. The mouse experiments were conducted
in accordance with a code of practice established by the ethical
committee of the Microbial Chemistry Research Foundation
(Shizuoka, Japan). The mice were purchased from Charles River
(Yokohama, Japan) and maintained in a pathogen-free barrier
facility for 1 week before the experiments.
Identification of rubratoxins as PP2A-specific inhibitors. To
discover new PP2A-specific inhibitors, we screened more than
20 000 microbial culture extracts and found a modest PP2A
inhibitor, harzianic acid.(10)Further investigations revealed a
Penicillium sp. extract showing an extremely strong PP2A inhi-
bition and containing a more potent PP2A inhibitor than harzi-
anic acid. Two active compounds were purified and identified as
rubratoxins A and B (Fig. 1a) by spectroscopic analyses, includ-
ing various NMR and MS experiments (Appendix S1). Despite
the absence of any record to date that rubratoxins are PP2A
inhibitors, it is quite feasible that rubratoxins share some struc-
tural similarities to cytostatin and cantharidin (Fig. S1). In liquid
chromatography–MS experiments using aqueous solvents, each
rubratoxin showed an equilibrium between the molecules conju-
gated with (70–95% in 35% acetonitrile) or without a water
molecule, indicating the majority of rubratoxins in aqueous solu-
tion existed as dicarboxylic acid forms opening a maleic anhy-
dride moiety. The dicarboxylic acid form is considered to be the
active form, like cantharidin and tautomycin.(12)
The inhibitory effects of rubratoxins A and B on isolated
PP2A, PP1, PP2B, PTP-1B, and CIP were examined by pNPP
dephosphorylation assays. Rubratoxin A potently inhibited
PP2A with an IC50of 170 nM, while rubratoxin B modestly
inhibited the enzyme with an IC50of 2.95 lM (Fig. 1b). Both
rubratoxins, up to 200 lM, failed to significantly inhibit any
other phosphatases tested (Fig. 1b). The Ki values, as objective
potency, of rubratoxins A and B against PP2A were 28.7 and
3.1 lM, respectively, and the plot patterns showed typical com-
petitive inhibition (Fig. 1c).
cells. The accumulation of phosphorylated proteins is typically
Protein phosphorylation states in rubratoxin-treated cells were
(a) (b) (c)
the activities of isolated PP2A, PP1, PP2B, recombinant protein–tyrosine phosphatase-1B (PTP-1B), and calf intestine alkaline phosphatase (CIP) in
the p-nitrophenyl phosphate dephosphorylation assays are shown. Mean ± SD. (n = 3). (c) Lineweaver–Burk plot of PP2A inhibition by
rubratoxins A and B. PP2A inhibitory activity of rubratoxins A and B were assessed using varying substrate concentrations. Ki values of
rubratoxins A and B to PP2A were calculated as 28.7 nM and 3.1 lM from the regression lines, respectively.
Protein phosphatase (PP)2A-specific inhibition by rubratoxins. (a) Structures of rubratoxins A and B. (b) Effects of rubratoxins A and B on
ª ª 2009 Japanese Cancer Association
monitored by immunoblotting using antibodies recognizing
phosphorylated substrate peptides of kinases and phosphotyro-
sine. The protein phosphorylation was clearly increased in
mouse B16-BL6 melanoma, human HEK293 embryonic kidney,
DU-145 prostate cancer, and HepG2 hepatocellular carcinoma
cells treated with 20 lM rubratoxin A for 3 h (Fig. 2). The over-
phosphorylation was obvious in AGC kinase substrates (phos-
pho cyclic AMP-dependent protien kinase (pPKA), phospho
protein kinase C (pPKC),and pAkt substrates), while it was an
ambiguous or cell- or substrate-dependent manner in other
kinase substrates (phospho cyclin-dependent kinase (pCDK) and
phospho ataxia telangiectasia mutated/ataxia telangiectasia and
rad3-related protein (pATM/ATR) substrates, and pTyrosine.
We then investigated the phosphorylation states of individual
PP2A-substrate proteins. Rubratoxin A generally and obviously
augmented the phosphorylation of PP2A-specific substrate
cyclic AMP response element binding protein (CREB),(26)its
related protein activating transcription factor-1 (ATF-1), and
c-Myc,(27)although the phosphorylated c-Myc signal was too
weak to be detected in B16–BL6 and HEK293 (Fig. 3a). Rubra-
toxin A did not generally induced the increase of phosphoryla-
tion in a PP1 substrate cdc25C(28)and pleckstrin homology
domain leucine-rich repeat protein phosphatase (PHLPP)
substrate Akt(29)(Fig. 3b). Mitogen-activated protein (MAP)
kinases extracellular signal-regulated kinase (ERK)1⁄2, Jun-N-
terminal kinase (JNK), and p38 are known to be dephosphoryl-
ated by PP2A and MAP kinase phosphatases.(30,31)While the
phosphorylation of JNK and p38 specifically increased by rubra-
toxin A treatment (Fig. 3a), such effects of ERK1⁄2 were only
observed in HepG2 cells (Fig. 3b). This could reflect the ratio of
the expression levels of PP2A and MAP kinase phosphatases, or
the preference of each MAP kinase for the certain phosphatase.
The protein phosphorylation patterns in 20 lM rubratoxin
A-treated cells were similar to those in 20–50 nM okadaic acid-
treated cells (Fig. S2a), suggesting that rubratoxin A inhibits
PP2A in cells with at least the same specificity of okadaic acid,
and its effect on the other proteins, including other phosphata-
ses, was negligible. Despite the failure at 20 lM (Figs 2, 3), a
Logarithmically-growing B16–BL6, HEK293, DU-145, and HepG2 cells
were treated with 20 lM rubratoxins (rubratoxin A [RA] or rubratoxin
B [RB]) or vehicle (control; 0.07% MeCN) for 3 h. Cell lysates
containing 10 lg protein were separated by sodium dodecylsulfate–
polyacrylamide gel electrophoresis, and the phosphorylation states of
proteinswere analyzedby immunoblotting
recognizing the phosphorylated substrates of protein kinase A (pPKA),
protein kinase (pPKC), Akt, cyclin-dependent kinase (pCDK), ataxia
telangiectasia mutated ataxia telangiectasia mutated and rad3-related
Expression level of the protein phosphatase (PP)2A catalytic subunit
and b-tubulin were detected as the controls.
Overphosphorylation of proteins in rubratoxin A-treated cells.
specifically overphosphorylated by rubratoxin A treatment (a) and
those independent of the treatment (b) are shown. Immunoblotting
were performed using the protein- or phosphorylated protein-specific
antibodies. Recognition sites of phosphorylated proteins are cyclic AMP
response element binding protein (CREB) Ser133, c-Myc Thr58⁄Ser62,
Jun-N-terminalkinase (JNK) Thr183⁄Tyr185,
cdc25C Ser216, Akt Ser473, and ERK1⁄2 extracellular signal-regulated
kinase (ERK)1⁄2 Thr180⁄Tyr204.
transcription factor-1 (ATF-1) was concomitantly detected by the
phosphorylated CREB antibody. Phosphorylated cdc25C
recognizes only the human protein, while the other antibodies
recognize both human and mouse proteins.
Overphosphorylation of protein phosphatase (PP)2A substrate
by rubratoxin(RA) treatment. Intracellularproteins
Wada et al.Cancer Sci|
ª ª 2009 Japanese Cancer Association
| vol. 101|no. 3|
higher concentration of rubratoxin B induced a similar over-
phosphorylation of proteins (Fig. S2b). The effects of rubratox-
ins were enhanced by increasing the concentration of the vehicle
Binding simulation of rubratoxin A and PP2A. To consolidate
the PP2A-specific inhibition by rubratoxin A, binding simulation
was conducted. Since okadaic acid and microcystin–LR bind to
the same catalytic site interacting with surrounding amino acids
in a highly overlapping manner(22)and the fact that rubratoxins
are also considered to bind to the site (Fig. 1c), we constructed
the rubratoxin A-PP2A binding model based on the crystal
structures of the inhibitor–PP2A complex (Fig. 4a–c).
There are three characteristics in the binding model. The first
is the interactions around the a,b-unsaturated d-lactone ring.
Besides the covalent bond of Cys269 to C3, Arg268 and the car-
bonyl oxygen on the ring form a hydrogen bond (Fig. 4b–d).
These interactions are also indicated in the binding of cytostatin
analogs to PP2A,(25,32)and a similar covalent bond was reported
in the binding of N-methyldehydroalanine of microcystin–LR to
Cys269.(22)Ceramidastin, a close analog of rubratoxin B(33)
whose lactone ring is opened and reduced, hardly inhibited
PP2A (IC50> 200 lM; Hiroyuki Inoue, personal communica-
tion, 2009). Cys269 and Arg268 are conserved only in PP2A
and its related proteins PP4 and PP6, whereas they are absent
from other protein phosphatases, such as PP1, PP2B, PP5, and
PP7. Those interactions are regarded as a significant determinant
for the high specificity of rubratoxins and cytostatin analogs to
The second characteristic is the insertion of the hydrophobic
chain into the hydrophobic pocket around Trp200, as seen in the
crystal structures or binding models of other compound–PP2A
binding complexes (Fig. 4b–d). CH⁄p interaction(34)
implied between the methyl terminal of rubratoxin A and
The third is the intermolecular and intramolecular hydrogen
and ionic bond networks at the center of the molecule, as shown
in Figure 4(c,d). When rubratoxin A was simply replaced with
rubratoxin B in this model, the distances of C23–C26, C24-C26,
and C16–C25 were enlarged (Fig. S3). Therefore, to tighten the
structure of rubratoxin A to adapt for the appropriate interaction
with the surrounding amino acid residues, the intramolecular
hydrogen bonds could play a major role in the difference of the
inhibitory potency of rubratoxins A and B. Similar interactions
with the surrounding amino acids were observed in our binding
model of cytostatin–PP2A, although the ligand was slightly sep-
arated from Arg214 (Fig. S4), confirming that rubratoxins and
cytostatin analogs inhibit the enzyme in a similar manner and
that dicarboxylic acid in rubratoxin A could be a substitute for
phosphate in cytostatin analogs.
Antitumor effect of rubratoxin A. The antitumor and antimet-
astatic effects of rubratoxin A were examined in several mouse
experiments. In an experimental metastatic model using
C57BL⁄6N mice intravenously inoculated with B16–BL6 cells
(Table 1), only 3 days of rubratoxin A pre-administration before
cellular inoculation clearly suppressed the formation of lung
metastatic foci. Although cytostatin also suppressed metastasis
in this model, the same dose of rubratoxin A showed 15%
increased inhibitory activity.
In a spontaneous metastatic model using C57BL⁄6N mice
subcutaneously inoculated with Lewis lung carcinoma (3LL)
cells, the intraperitoneal administration of 1.6 mg⁄kg⁄day
rubratoxin A significantly reduced the size of the primary
tumor (Fig. 5a,b). The spontaneous lung metastasis showed a
distinct decrease in the number and size of foci in the rubratox-
in A-treated mice (Fig. 5c,d). Toxic effects, including body
weight loss (Fig. S5a) and hepatotoxicity with a partially
whitened liver (3⁄5 mice, <3% of liver surface), were observed
with the administration of 1.6 mg⁄kg⁄day rubratoxin A.
Although cytostatin at 3.2 mg⁄kg⁄2 days apparently induced a
similar growth suppression of the primary tumor size (Fig. 5a),
the final tumor weights and lung metastasis at day 21 showed
no significant reduction (Fig. 5b,c). The toxic effect of
3.2 mg⁄kg⁄2 days cytostatin resulted in partially whitened liver
(3⁄5 mice) and was comparable with 1.6 mg⁄kg⁄day rubratoxin
A, although no body weight loss was observed in this treatment
(Fig. S5a). In a follow-up experiment with almost the same
condition, similar effects of rubratoxin A were reproduced
(Fig. S5b–e). The antitumor and antimetastatic effects of rubra-
toxin A were better than or comparable to those of doxorubicin
and lentinan, while the weight loss by rubratoxin A was milder
PP2A catalytic subunit a (dark gray), PP2A–RUB_AH, are shown from two angles. (b) Binding site of PP2A–RUB_AH is magnified. Rubratoxin A
and amino acid residues of PP2A involved in the interactions are depicted with thick and thinner lines, respectively. Atoms in the structure C, O, N,
and S are gray, red, blue, and yellow, respectively. Red sphere represents oxygen atom of the crystallization water. Hydrogen or ionic bonds are
shown with green dashed lines, and the CH⁄p interaction is shown with a brown line. (c) Binding site of PP2A–RUB_AH is shown from a different
angle. To clarify the hydrogen bonds, rubratoxin A is highlighted in yellow. (d) Interactions in the binding complex are illustrated. Amino acid
residues forming covalent, hydrogen, and ionic bonds, and CH⁄p interaction with rubratoxin A, are shown beside the interacting parts.
Binding model of rubratoxin A to protein phosphatase (PP)2A. (a) Binding model of rubratoxin A–dicarboxylic acid form (yellow) and
ª ª 2009 Japanese Cancer Association
than that by doxorubicin in the treatments. Considering the sus-
tained use of doxorubicin for cancer treatments, the toxicity of
rubratoxin A appears manageable with further investigation and
We also conducted an experiment using immunodeficient
SCID (CB17⁄Icr–Prkdcscid⁄CrlCrlj) and NOD–SCID (non-obese
diabetic [NOD].CB17-Prkdcscid⁄J) mice bearing 3LL cells
(Fig. S6). Although the primary tumor weight in SCID mice
was not significantly reduced, the antimetastatic effects of rubra-
toxin A were observed in both mouse models. The numbers of
lung metastatic foci decreased to 13–22% of the control in SCID
and C57BL⁄6N mice and to 35% in NOD–SCID mice by rubra-
toxin A treatment.
In the present study, we found that rubratoxin A specifically and
potently inhibited PP2A. The enzyme inhibition assays and
phosphorylation patterns of cellular proteins ensured its high
specificity to PP2A, and the existence of any other target mole-
cules was not implied. Although it is still difficult to exclude the
possibility that rubratoxin A has other target molecules, as in the
case of other inhibitors in general, one can consider it as a
highly specific and potent PP2A inhibitor comparable with
cytostatin analogs. Rubratoxin B also specifically inhibited
PP2A in our assays, albeit weaker than rubratoxin A. Consider-
ing the ubiquity of PP2A, the inhibition of the enzyme would be
the main cause for most of the previously reported biological
effects of rubratoxin B.
Taking advantage of their high specificity to PP2A, cytosta-
tin analog compounds were expected to function as good
molecular probes. However, because of their liability, their
applications have been limited, while less specific PP2A inhibi-
tors, such as okadaic acid and calyculin A, have been broadly
used in biochemical and cell biological protocols. Rubratoxin A
is stable, as no degradation was observed in normal stocks for
2 years and in 0.2 N HCl treatment at 60?C for 30 min. With
the higher stability and comparable high specificity, rubratoxin
A will be a successor of cytostatin analogs as a PP2A-specific
molecular probe and a lead compound of clinical drugs for
Less specific PP2A inhibitors, such as okadaic acid and
microcystin–LR, are known as tumor promoters, whereas it is
Table 1. Antimetastatic effect of rubratoxin A and cytostatin
No. lung foci
0.1 mg⁄kg 0.4 mg⁄kg 1.6 mg⁄kg
— NS<0.001 <0.001 <0.005
Six-week-old female C57BL⁄6N mice were divided into five groups
(n = 7 or 14) and intraperitoneally injected with 0.1, 0.4, or 1.6 mg⁄kg
rubratoxin A, 1.6 mg⁄kg cytostatin, or vehicle (10% dimethylsulfoxide
and 0.5% Tween-80 in saline) every 24 h for 3 days. Mice were
intravenously inoculated with 2.5 · 104cells⁄100 lL of B16–BL6
melanoma cells at 24 h after the last injection of the agent. Fourteen
days after the cellular inoculation, the mice were killed and the
number of lung metastatic foci was counted under a microscope. NS,
subcutaneously inoculated with 1 · 106cells⁄100 lL
saline of Lewis lung carcinoma cells in the left flank
1.6 mg⁄kg⁄day RA(n = 5),
cytostatin (CST, n = 5), or vehicle (control; 250 lL of
Primary tumor sizes were measured with a caliper
twice per week (a). Mice were killed at day 21, and
the weights of excised primary tumors (b) and
number of each size of lung metastatic foci (c)
were measured. Some of the representative lung
lobes from RA-treated or non-treated mice are
shown in (d). (*) Difference between control and
RA treatment was significant (P < 0.01). Graph data
are mean ± SE. For clarification, only the results of
the representative three groups are shown among
the total 13 groups of mice that were treated with
various conditions of RA or CST at the same time.
Antitumor and antimetastatic effects of
A (RA).C57BL⁄6N micewere
3.2 mg⁄kg⁄2 days
Tween-80,n = 7).
Wada et al.Cancer Sci|
ª ª 2009 Japanese Cancer Association
|vol. 101|no. 3|
unclear whether rubratoxin A has such a function. Rubratoxin
A, as well as okadaic acid, induces the increase of c-Myc
oncogene phosphorylation and accumulation in cultured cells,
which might lead to some tumor-promotion effects. Side-by-side
experiments for these compounds using appropriate mouse
models might be required. However, we observed promising
anticancer effects of rubratoxin A instead of any tumor promo-
tion in mouse experiments, and the tumor-promoting effects of
okadaic acid class compounds are experimentally induced under
the presence of initiator mutagens.(35)Even if rubratoxin A has
some tumor-promoting effects, they should be negligible or
manageable. Although another problem, hepatotoxicity, also
warrants a solution, the dramatic antimetastatic effects of rubra-
toxin A and cytostatin are regarded as a common effect of
PP2A-specific inhibitors, which should be considered in cancer
treatments. Clarifying the difference in the molecular mecha-
nisms in hepatotoxicity and the anticancer activity of rubratoxin
A will lead to less toxic and potent anticancer drugs.
The activation of natural killer (NK) cells is considered to be
a cause of the antimetastatic effects of cytostatin,(9)and rubra-
toxin A was also found to activate NK cells in some experiments
(Fig. S7). The phosphorylation of CREB, as shown in this study,
might be involved in NK cell activation via the stimulation of
interleukin (IL)-2 release from T cells.(36)However, weaker but
still obvious antimetastatic effects of rubratoxin A have been
observed in NK cell-deficient NOD–SCID mice. The activity of
other leukocytes existing in NOD–SCID mice, the impairment
of cell adhesion via focal adhesion kinase phosphorylation and
paxillin phosphorylation, and the secretion of tissue inhibitor of
metalloproteinases-1 (TIMP-1) could also contribute to the anti-
The systemic administration of rubratoxins and other PP2A
inhibitors with higher hydrophilicity, such as microcystins and
cytostatin analogs, induces hepatic disorder in mice, whereas
okadaic acid and calyculin A, which are hydrophobic inhibitors,
have not been reported to induce such toxicity.(12,35)Hepatotox-
icity is a common effect of hydrophilic PP2A inhibitors. Since
rubratoxin B enhances the secretion of several cytokines, such
as IL-8, tumor necrosis factor-a, macrophage colony stimulating
factor (M-CSF), and granulocyte macrophage colony stimulat-
ing factor (GM-CSF) from liver-resident cells, it was suggested
that macrophages and neutrophils are recruited and activated in
the liver, which attack liver tissue and cause the hepatic disor-
der.(38,39)Microcystins also induce IL-8 secretion in neutrophils,
and the infiltration of neutrophils into the liver tissue is regarded
as an important factor for microcystin-induced liver injury.(40)
Although the detailed mechanism of the hepatotoxicity is still
obscure, some common principles, including the inhibition of
the enzyme, increment of cytokine secretion, and infiltration of
leukocytes into liver, can contribute to the hydrophilic PP2A
In conclusion, in the present study, we report that a classical
mycotoxin, rubratoxin A, discovered approximately 50 years
ago without an identity of its initial target molecule, is a highly
specific and potent PP2A inhibitor. Rubratoxin A has broad and
efficient potential for use in biochemical and cell biological
areas as an excellent molecular probe. With its dramatic anti-
metastatic effects, rubratoxin A should become a lead compound
among a new class of anticancer drugs. Further studies to eluci-
date the molecular mechanism of how PP2A inhibition leads to
antimetastatic effects and hepatotoxicity are in progress.
We thank Professor Maurice Moss (University of Surrey, UK), Mr That
Nguyen, and Dr Ryuichi Sawa (Microbial Chemistry Research Founda-
tion, Japan) for providing information about rubratoxins and experimen-
tal techniques and for the assistance with the mass spectrometric
analysis. We also thank Professor Sachi Sri Kantha (Gifu Pharmaceutical
University, Japan) for critically reading this manuscript. This work was
supported by a Grant-in-Aid for Cancer Research from the Ministry of
Education, Science, Sports, and Culture of Japan.
1 Janssens V, Goris J. Protein phosphatase 2A: a highly regulated family of
serine⁄threonine phosphatases implicated in cell growth and signalling.
Biochem J 2001; 353: 417–39.
2 Eichhorn PJ, Creyghton MP, Bernards R. Protein phosphatase 2A regulatory
subunits and cancer. Biochim Biophys Acta 2009; 1795: 1–15.
3 Amemiya M, Ueno M, Osono M et al. Cytostatin, a novel inhibitor of cell
adhesion to components of extracellular matrix produced by Streptomyces sp.
MJ654-NF4. I. Taxonomy, fermentation, isolation and biological activities.
J Antibiot 1994; 47: 536–40.
4 Amemiya M, Someno T, Sawa R, Naganawa H, Ishizuka M, Takeuchi T.
Cytostatin, a novel inhibitor of cell adhesion to components of extracellular
matrix produced by Streptomyces sp. MJ654-NF4. II. Physico-chemical
properties and structure determination. J Antibiot 1994; 47: 541–4.
5 Kawada M, Amemiya M, Ishizuka M, Takeuchi T. Cytostatin, an inhibitor of
cell adhesion to extracellular matrix, selectively inhibits protein phosphatase
2A. Biochim Biophys Acta 1999; 1452: 209–17.
6 Lewy DS, Gauss CM, Soenen DR, Boger DL. Fostriecin: chemistry and
biology. Curr Med Chem 2002; 9: 2005–32.
7 Matsuzawa S, Suzuki T, Suzuki M et al. Thyrsiferyl 23-acetate is a novel
specific inhibitor of protein phosphatase PP2A. FEBS Lett 1994; 356: 272–4.
8 Usui T, Marriott G, Inagaki M, Swarup G, Osada H. Protein phosphatase 2A
inhibitors, phoslactomycins. Effects on the cytoskeleton in NIH⁄3T3 cells.
J Biochem 1999; 125: 960–5.
9 Kawada M, Kawatsu M, Masuda T et al. Specific inhibitors of protein
phosphatase 2A inhibit tumor metastasis through augmentation of natural
killer cells. Int Immunopharmacol 2003; 3: 179–88.
10 Kawada M, Yoshimoto Y, Kumagai H et al. PP2A inhibitors, harzianic acid
and related compounds produced by fungus strain F-1531. J Antibiot 2004; 57:
11 Yamaoka M, Sato K, Kobayashi M et al. FR177391, a new anti-
hyperlipidemic agent from Serratia. IV. Target identification and validation by
chemical genetic approaches. J Antibiot 2005; 58: 654–62.
12 Swingle M, Ni L, Honkanen RE. Small-molecule inhibitors of ser⁄thr protein
phosphatases: specificity, use and common forms of abuse. Methods Mol Biol
2007; 365: 23–38.
13 Masuda T, Watanabe S, Amemiya M, Ishizuka M, Takeuchi T. Inhibitory
effect of cytostatin on spontaneous lung metastases of B16-BL6 melanoma
cells. J Antibiot 1995; 48: 528–9.
14 de Jong RS, Mulder NH, Uges DR et al. Phase I and pharmacokinetic study of
thetopoisomerase IIcatalytic inhibitor fostriecin. Br JCancer 1999; 79:882–7.
15 Burnside JE, Sippel WL, Forgacs J, Carll WT, Atwood MB, Doll ER. A
disease of swine and cattle caused by eating moldy corn. II. Experimental
production with pure cultures of molds. Am J Vet Res 1957; 18: 817–24.
16 Moss MO. The rubratoxins, toxic metabolites of Penicillium rubrum Stoll. In:
Ciegler A, Kadis S, Ajl SJ, eds. Microbial Toxins. New York and London:
Academic Press, 1971; 381–407.
17 Moss MO. Microbial food toxicants: rubratoxins. In: Recheigl M, ed.
Handbook of Foodborne Diseases of Biological Origin. Florida: CRC Press,
18 Abbott SP. Mycotoxins and indoor molds. Indoor Environment Connections
2002; 3: 14–24.
19 Iwashita K, Nagashima H. Rubratoxin B induces interleukin-6 secretion in
mouse white adipose tissues and 3T3-L1 adipocytes. Toxicol Lett 2008; 182:
20 Wada S, Niimi M, Niimi K et al. Candida glabrata ATP-binding cassette
transporters Cdr1p and Pdh1p expressed in a Saccharomyces cerevisiae strain
deficient in membrane transporters show phosphorylation-dependent pumping
properties. J Biol Chem 2002; 277: 46809–21.
21 Wada S, Tanabe K, Yamazaki A et al. Phosphorylation of candida glabrata
ATP-binding cassette transporter Cdr1p regulates drug efflux activity and
ATPase stability. J Biol Chem 2005; 280: 94–103.
22 Xing Y, Xu Y, Chen Y et al. Structure of protein phosphatase 2A core
enzyme bound to tumor-inducing toxins. Cell 2006; 127: 341–53.
23 Paul IC, Sim GA, Hamor TA, Robertson JM. Fungal metabolites. Part II. The
structure of byssochlamic acid: X-ray analysis of byssochlamic acid bis-
p-bromphenylhydrazide. J Chem Soc 1963; 5502–12.
ª ª 2009 Japanese Cancer Association
24 Bu ¨chi G, Snader KM, White JD, Gougoutas JZ, Singh S. Structures of
rubratoxins A and B. J Am Chem Soc 1970; 92: 6638–41.
25 Lawhorn BG, Boga SB, Wolkenberg SE et al. Total synthesis and evaluation
of cytostatin, its C10-C11 diastereomers, and additional key analogues: impact
on PP2A inhibition. J Am Chem Soc 2006; 128: 16720–32.
26 Wadzinski BE, Wheat WH, Jaspers S et al. Nuclear protein phosphatase 2A
dephosphorylates protein kinase A-phosphorylated CREB and regulates
CREB transcriptional stimulation. Mol Cell Biol 1993; 13: 2822–34.
27 Yeh E, Cunningham M, Arnold H et al. A signalling pathway controlling
c-Myc degradation that impacts oncogenic transformation of human cells. Nat
Cell Biol 2004; 6: 308–18.
28 Margolis SS, Walsh S, Weiser DC, Yoshida M, Shenolikar S, Kornbluth S.
PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25
dephosphorylation. EMBO J 2003; 22: 5734–45.
29 Brognard J, Sierecki E, Gao T, Newton AC. PHLPP and a second isoform,
PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating
distinct Akt isoforms. Mol Cell 2007; 25: 917–31.
30 Keyse SM. Protein phosphatases and the regulation of mitogen-activated
protein kinase signalling. Curr Opin Cell Biol 2000; 12: 186–92.
31 Dickinson RJ, Keyse SM. Diverse physiological functions for dual-specificity
MAP kinase phosphatases. J Cell Sci 2006; 119: 4607–15.
32 Teruya T, Simizu S, Kanoh N, Osada H. Phoslactomycin targets cysteine-269
of the protein phosphatase 2A catalytic subunit in cells. FEBS Lett 2005; 579:
33 Inoue H, Someno T, Kato T, Kumagai H, Kawada M, Ikeda D. Ceramidastin,
a novel bacterial ceramidase inhibitor, produced by Penicillium sp. Mer-
f17067. J Antibiot 2009; 62: 63–7.
34 Nishio M, Hirota M, Umezawa Y. The CH⁄p Interaction. Evidence, Nature,
and Consequences. New York: Wiley-VCH, 1998.
35 FujikiH, Suganuma M. Tumor promotion byinhibitors ofprotein phosphatases 1
36 Katsiari CG, Kyttaris VC, Juang YT, Tsokos GC. Protein phosphatase 2A is a
negative regulator of IL-2 production in patients with systemic lupus
erythematosus. J Clin Invest 2005; 115: 3193–204.
37 Nagashima H, Maeda-Nakamura K, Iwashita K, Goto T. Induced secretion of
tissue inhibitor of metalloproteinases-1 (TIMP-1) in vivo and in vitro by
hepatotoxin rubratoxin B. Food Chem Toxicol 2006; 44: 1138–43.
38 Nagashima H, Nakamura K, Goto T. Hepatotoxin rubratoxin B induced the
secretion of TNF-alpha, IL-8, and MCP-1 in HL60 cells. Biochem Biophys
Res Commun 2001; 287: 829–32.
39 Nagashima H, Nakamura K, Goto T. Rubratoxin B induced the secretion of
hepatic injury-related colony stimulating factors in human hepatoma cells.
Toxicol Lett 2003; 145: 153–9.
40 Kujbida P, Hatanaka E, Campa A, Curi R, Poliselli Farsky SH, Pinto E.
Analysis of chemokines and reactive oxygen species formation by rat and
human neutrophils induced by microcystin-LA, -YR and -LR. Toxicon 2008;
Additional Supporting Information may be found in the online version of this article:
Fig. S1. Chemical structures of rubratoxins A and B, cantharidin and its dicarboxylic acid form cantharidic acid, and cytostatin analogs.
Fig. S2. Effects of okadaic acid and rubratoxin B (RB) on the phosphorylation of intracellular proteins. (a) Phosphorylation patterns of proteins
in rubratoxins- or okadaic acid (OA)-treated cells were detected by immunoblotting. Cells were treated with 20 lM rubratoxin A (RA), 20 or
50 nM OA, or vehicle (control) for 3 h. Since the rubratoxins and okadaic acid were stocked in 35% acetonitrile and dimethylsulfoxide
(DMSO), respectively, final 0.07% acetonitrile and 0.25% DMSO were contained in all of the treatments to exclude the influence of using dif-
ferent vehicles. (b) Phosphorylation states of the proteins were detected as above, increasing the concentration of RB. Final 0.7% acetonitrile
was used as the vehicle in all of the treatments. p-ATF-1, phosphorylated-activating transcription factor-1; p-CREB, phosphorylated-cyclicAMP
response element binding protein; pPKA, phosphorylated substrates of protein kinase A (pPKA), pPKC phosphorylated substrates of protein
Fig. S3. Differences in intramolecular hydrogen bonds between rubratoxins A and B. Structures of rubratoxin A (gray) and B (yellow in the struc-
turally different part) in the binding model protein phosphatase 2A–RUB_AH are shown with intramolecular hydrogen bonds represented as green
(rubratoxin A) and red (rubratoxin B) dashed lines with distances.
Fig. S4. Binding model of cytostatin to protein phosphatase (PP)2A. (a) Magnified view of the binding site. Cytostatin and amino acid residues of
PP2A involved in the interactions are depicted with thick and thinner lines, respectively. Atoms in the structure C, O, N, S, and P are gray, red,
blue, yellow, and orange. Red sphere represents oxygen atom of the crystallization water. Hydrogen or ionic bonds are shown with green dashed
lines, and CH/p interaction is shown with a brown line. (b) Binding site is shown from a different angle. Cytostatin is highlighted in yellow.
(c) Illustration of the interactions between cytostatin and PP2A catalytic subunit. The cytostatin–PP2A binding model was constructed based on
the structure protein data bank (PDB) code 2ie3. Although the absolute configuration of the lactone ring of cytostatin differ from that of rubratoxin
A, it is still in the close vicinity of Cys269 and Arg268 and similar interactions are expected. In unrefined positioning, the phosphate of cytostatin
was positioned, overlapping with the carbonyl of microcystin–LR interacting with Arg89. Therefore, the lactone ring was fixed to Cys269 by
Michael addition, and an oxygen atom in the phosphate was fixed at the coordinate of the corresponding oxygen in the carboxyl of microcystin–
LR. This preliminary model was energy minimized and successively refined as follows with energy minimization in each step: the carbonyl on the
lactone ring set the distance restraint with an imino group of Arg268, and the restraint was released. Fixation of the oxygen in the phosphate was
relaxed to the harmonic restraint, and the olefin tail of cytostatin was directed towards the hydrophobic pocket around Try200. Resultant
cytostatin–PP2A binding model was obtained after the final energy minimization.
Fig. S5. Supplementary data for C57BL⁄6N mouse experiments. (a) Body weight changes of the mice employed in the first experiment (Mean ±
SE). (b–d) Results of the follow-up experiment. The second experiment was carried out almost identically as the first experiment using doxorubi-
cin and lentinan as the controls instead of cytostatin (CST). After the subcutaneous inoculation of Lewis lung carcinoma cells (day 0), 0.1, 0.4,
and 1.6 mg⁄kg rubratoxin A (RA; days 1–21, excluding days 14 and 18), 2.5 mg⁄kg doxorubicin (days 1–21, excluding days 11, 18, and 20), and
2 mg⁄kg lentinan (days 1–11) were intraperitoneally injected. Primary tumor sizes (b) and body weights (e) were measured during the experiment.
Mice were killed on day 21; the excised primary tumor weight was measured (c) and the number of lung metastases was counted (d). Nine mice
were used as the control and seven were used for other treatments. Data shown are mean ± SE. (*) Differences were significant between the
control and drug treatment (P < 0.01) in (c) and (d).
Fig. S6. Antitumor and antimetastatic effects of rubratoxin A (RA) on immunodeficient mice. Five-week-old female SCID (CB17⁄
Icr–Prkdcscid⁄CrlCrlj) and non-obese diabetic (NOD)–SCID (NOD.CB17-Prkdcscid⁄J) mice were subcutaneously inoculated with 1 · 106cells⁄100
lL saline of Lewis lung carcinoma cells in the left flank (day 0). Rubratoxin A (RA, 1.6 mg⁄kg) or vehicle (control, 5% dimethylsulfoxide, 0.5%
Tween-80) were intraperitoneally injected on days 1–12, 13–15, 18, and 20 to SCID mice, and on days 1–12, 13–15, and 20 to NOD mice. Pri-
mary tumor volumes (a) and body weights (d) were measured during the experiment. Mice were killed on day 21; excised primary tumor weight
was measured (b) and the number of lung metastatic foci in each size was counted (c). Data shown are mean ± SE. (*) Differences were signifi-
cant between the control and RA treatment (P < 0.01) in (b) and (c).
Wada et al.Cancer Sci|
ª ª 2009 Japanese Cancer Association
| vol. 101| no. 3|
Fig. S7. Effects of rubratoxins A and B on natural killer (NK) cell activity. Six-week-old female BDF1mice were intraperitoneally injected
with saline (control), 0.4 or 1.6 mg⁄kg rubratoxin A (RA), 1.6 mg⁄kg rubratoxin B (RB), 1.6 mg⁄kg cytostatin (CST), or a cytostatin analog,
leustroducsin H (LH) daily for 3 days. One day after the last injection, cytolytic activity in splenocytes was assessed against B16–BL6 (a) and
YAC-1 (b) target cells. Data presented are mean ± SE. E⁄T, effector⁄target ratio.
Appendix S1. Mass spectrometry and nuclear magnetic resonance rubratoxin spectrometric data.
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ª ª 2009 Japanese Cancer Association