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Anisomycin is a Multifunctional Drug: More than

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124 Current Chemical Biology, 2010, 4, 124-132

Anisomycin is a Multifunctional Drug: More than Just a Tool to Inhibit

Protein Synthesis

Marina Macías-Silva*, Genaro Vázquez-Victorio and Jacqueline Hernández-Damián

Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de
México, México, D.F. 04510, México

Abstract: Anisomycin is a bacterial antibiotic isolated from Streptomyces griseolus. Anisomycin is mainly known as a
potent and reversible inhibitor of protein synthesis in eukaryotic organisms that acts by binding and inhibiting peptidyl
transferase activity of 60S ribosomal subunit. Interestingly, anisomycin has been widely used as an extremely potent acti-
vator of mitogen-activated protein kinase (MAPK) cascades in mammalian cells, especially of JNK, p38, and ERK1/2,
and it can also modulate other signal transduction pathways. Regulation of gene expression is another intriguing effect of
anisomycin given that it is able to superinduce the expression of certain genes, or cause degradation of some proteins. Fur-
thermore, it also affects both pro- and anti-apoptotic mechanisms. Recently, a potential therapeutic use for anisomycin has
been proposed as it can sensitize malignant cells to death, either alone or in combination with certain drugs; anisomycin
may also function as an immunosuppressant by inhibiting T cells and transplant rejection in mice. Anisomycin has been
applied in the study of memory in animals, and it has been shown that it inhibits the consolidation of new memories and
cause amnesia; however, it is necessary to carry out more studies in order for anisomycin to be considered as a potential
psychiatric drug in humans. Notably, the multifunctional feature of anisomycin has yielded great benefits for biochemical
research even though some of its mechanisms of action remain unknown.

Keywords: Anisomycin, protein synthesis inhibition, ribotoxic stress, apoptosis, signaling, cancer, MAPKs.

INTRODUCTION ribosomal subunit from the 80S ribosome system of eukary-

Anisomycin, also known by its trade name flagecidin, is
a bacterial pyrrolidine antibiotic mostly isolated from Strep-
tomyces griseolus, but it can also be produced from various
Streptomyces species [1]. It is primarily an antiprotozoal
agent with little antibacterial or antifungal activity. Aniso-
mycin is known mainly as a potent and reversible inhibitor
of protein synthesis in eukaryotic organisms but is inactive
against bacteria [2]. It also produces a ribotoxic effect that
causes activation of stress-activated protein kinases
(SAPKs), and also gene superinduction [3-6].
The chemical name for anisomycin is 2-p-
methoxyphenylmethyl-3-acetoxy-4-hydroxypyrrolidine, and
its chemical structure is shown in Fig. (1). Anisomycin re-
quires its pyrrolidine ring for its translational inhibitory ac-
tivity; any change such as an acetylation of the nitrogen or a
deacetylation at the 3’ position renders the molecule inactive
[2, 7, 8]. The acetyl group at the 3’ position of pyrrolidine
ring is crucial for signaling, since deacetyl-anisomycin loses
the ability to activate SAPKs and induce immediate-early
genes [9].
PROTEIN SYNTHESIS INHIBITION
Anisomycin is one of the most potent protein synthesis
inhibitors (PSIs). It inhibits translation by binding to 60S
*Address correspondence to this author at the Departamento de Biología
Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional
Autónoma de México. México, D.F. 04510, México; Tel: 52 (55) 56-22-
5729; Fax: 52 (55) 56-22-5611; E-mail: mmacias@ifc.unam.mx
otic cells; thereby it blocks the peptide bond formation, pre-
vents elongation and causes polyribosome stabilization by
suppressing the peptidyl transferase reaction [2, 7, 8]. The
binding site for anisomycin in the ribosome is located in the
28S rRNA (domain V), which is part of the peptidyl trans-
ferase center. Certain structural regions of some antibiotics,
like anisomycin, resemble the 3’-end of the aminoacyl-tRNA
in the transition state for peptide bond formation. Anisomy-
cin binds competitively to the ribosome and interferes with
aminoacyl-tRNA binding and movement on the ribosome
[10, 11]. Anisomycin seems to act as a mixed noncompeti-
tive inhibitor on eukaryotic peptidyltransferase activity of
ribosomes with a Ki value of 6.5 X 10
7 M [8]. It has been
suggested that anisomycin may also bind other sites in the
ribosome different to the peptidyl transferase center [8, 12].
Anisomycin inhibits protein synthesis in some protozoa,
HeLa cells, rabbit reticulocytes, yeast, and cell-free extracts
prepared from these sources, but it is inactive against ex-
tracts from E. coli. In HeLa cells, partial inhibition of DNA
synthesis but none on RNA synthesis was observed at ani-
somycin concentrations (~10 μM) that produce 95% inhibi-
tion of protein synthesis. These effects on protein and DNA
synthesis are rapid in onset and are also reversible [2, 7].
There are some reports, using mammalian cells infected with
different virus, that show anisomycin and other PSIs are able
to cause inhibition of viral RNA replication (in poliovirus) or
RNA synthesis (in adenovirus type 2), and in some cases it
causes partial viral DNA transcription (in adenovirus type 2)
[13-15].

1872-3136/10 $55.00+.00 ©2010 Bentham Science Publishers Ltd.

Anisomycin is a Multifunctional Drug
H and induction of MAPKs signaling cascades remains unclear
N
OCH3
HO O
O kinases (SAPKs) including c-jun NH2-terminal kinase
Fig. (1). Anisomycin general structure.
The chemical name for anisomycin is 2-p-methoxyphenylmethyl-3-
acetoxy-4-hydroxypyrrolidine. The pyrrolidine ring is important for
the translational inhibitory activity of anisomycin. The acetyl group
of the molecule is crucial for signaling and gene superinduction;
any acetylation at the nitrogen atom or deacetylation at the 3’ posi-
tion of the pyrrolidine ring result in an inactive molecule.
In addition to anisomycin, Streptomyces produces some
other antibiotics like cycloheximide (CHX), puromycin (Pur)
and hygromycin (Hyg). Pur is an aminonucleoside that in-
hibits protein synthesis in both prokaryote and eukaryote
cells causing premature chain termination during translation,
whereas CHX inhibits protein synthesis by blocking transla-
tional elongation only in eukaryotes [10, 11]. Pur and CHX
are also able to activate mitogen-activated protein kinases
(MAPKs) but to a lesser extent than anisomycin. In contrast,
antibiotics like Hyg and others like neomycin, kanamycin
and G418, which are all similar compounds known as ami-
noglycosides (AGAs), act by blocking protein synthesis in
both prokaryotes and eukaryotes cells by binding the small-
est ribosome subunit and inhibiting the initiation phase dur-
ing translation. Interestingly, most AGAs do not induce
MAPK activation through damaging ribosome, although
some of them like gentamicin can activate MAPKs through
other mechanisms as described below [25, 26]. Noteworthy,
anisomycin is the most potent agonist to activate MAPKs
compared with the antibiotics above mentioned, although all
of them block translation equally well.
RIBOTOXIC STRESS AND MAPK ACTIVATION
Ribotoxic stress response is a cellular reaction to ribotox-
ins, which are cytotoxic proteins characterized by their site-
directed action on eukaryotic ribosomes that lead to inhibi-
tion of protein synthesis. Ribotoxins interact with the 3’-end
of the large 28S rRNA, altering the peptidyl transferase ac-
tivity of actively translating eukaryotic ribosomes. During
protein synthesis, this region of the ribosome functions in
aminoacyl-tRNA binding, peptidyl transferase activity, and
ribosomal translocation. Thus, the ribotoxic stress response
is mainly originated from the blockage of the ribosome cycle
[5, 16-20].
Xenobiotics are a group of chemical agents with toxic
effects. They cause toxicity as the result of modulating cell
signaling that in turn modifies many cellular processes, and
interestingly, some of them act as RIPs (ribosome inactivat-
ing proteins). RIPs are the most studied ribotoxins obtained
from higher plants, or from fungi and bacteria, however, the
details of their mechanism of action as ribotoxic stressors
remain unclear [5]. One of the main effects of ribotoxic
stressors is the MAPK signaling pathway activation, al-
though the precise linkage between ribosomal RNA damage
Current Chemical Biology, 2010, Vol. 4, No. 2 125
[5, 20].
MAPKs are a family of Pro-directed Ser/Thr protein
kinases, which include the extracellular signal-regulated
kinase (ERK) 1 and 2, and the stress-activated protein
(SAPK1/JNK) and p38 kinase (SAPK2/p38) [20, 21]. The
activity of JNK and p38 is regulated through their phos-
phorylation on both Thr and Tyr residues in the motif TPY
by the upstream dual-specificity kinases MKK4/7 and
MKK3/6, respectively [22, 23]. The protein kinase MEKK1,
in turn, activates MKK4 and MKK7 through phosphoryla-
tion of Ser and Thr residues. Upon activation, SAPKs phos-
phorylate and activate transcription factors such as cJun,
ATF-2, and Elk-1, leading ultimately to the transcriptional
activation of some immediate-early genes like c-fos and c-
jun. Major cellular stresses able to stimulate SAPKs activa-
tion include exposure to UV irradiation, heat and osmotic
shock, oxidative stresses, inflammatory cytokines, withdraw
of growth factors, DNA damage agents, etc [5, 20]. SAPKs
activity can also be modulated through the ribotoxic stress
caused by agents that target the ribosome such as ultraviolet
light, certain tumor promoters, antibiotics, xenobiotics, and
some toxins [5, 20].
The mode of regulation of MAPK cascades by receptors
for growth factors or cytokine occurs through activation of
small GTP-binding proteins, including Ras, Cdc42, Rac1,
and Rho [21]. In contrast, MAPKs activation by any stress
including ribotoxic stress seems not to involve small
GTPases. One example is the case of anisomycin, as its ef-
fects on SAPKs activation seem to be independent of small
G proteins [24]. Nevertheless, some antibiotics like AGAs
seem to use small G proteins for SAPKs activation, for ex-
ample, gentamicin activates JNK pathway and causes death
in HC cells from the cochlea causing sensorineural hearing
loss, but the use of toxin B, an inhibitor of small G proteins,
decreases HC cells death. Thus, small GTPases seem to play
a role in aminoglycoside toxicity signaling as upstream
activators of the JNK signaling pathway [25]. Intriguingly,
AGAs like gentamicin and neomycin may also function as
calcium-sensing receptor (CaR) agonists in kidney cells.
CaR is a G protein coupled receptor (GPCR) able to signal
through several signal transduction pathways, and in kidney
cells, gentamicin and neomycin stimulate different signaling
pathways through CaR activation, such as intracellular Ca+2
mobilization and ERK phosphorylation, as well as elicit the
phosphorylation of Akt, GSK3
, and p38 in a PI3K depend-
ent manner [26].
The abilities of anisomycin to inhibit protein synthesis
and activate SAPKs may be dissociable. Some evidence
supports that its ability to inhibit protein synthesis per se
does not seem to activate SAPKs [5]. For instance, anisomy-
cin is a potent agonist of SAPKs at concentrations (< 3 μM)
that inhibited protein synthesis by less than 50%, suggesting
that anisomycin may activate protein kinases independently
of its ability to inhibit protein synthesis [17]. However, Ior-
danov et al. also provide evidence that anisomycin requires
the presence of actively translating ribosomes in order to
activate SAPKs. In addition, some PSIs do not activate
SAPKs like emetine (an inhibitor of the elongation cycle) or
126 Current Chemical Biology, 2010, Vol. 4, No. 2
pactamycin and harringtonine (promoters of the breakdown
the polysomes to monosomes), but they are able to suppress
the SAPK activation induced by anisomycin [9, 17]. In these
conditions, when the ribosome is blocked with some PSIs,
anisomycin loses its ability to stimulate signal transduction
from the 28S rRNA to SAPKs. This suggests that the dam-
age to the ribosome is involved in both effects of anisomy-
cin: the inhibition of protein synthesis and the activation of
SAPKs [5, 17].
The ribotoxic stress is a highly specific event considering
the finding that not all protein synthesis inhibitors have the
capacity to activate the MAP kinase pathways. It is not well
understood why some PSIs (e.g. emetine) that damage ribo-
some do not cause MAPK activation. This could be ex-
plained by the fact that different PSIs may target distinct
sites on the ribosome or bind ribosomes at different stages of
ribosomal cycle, in order to cause either translational arrest
or both MAPKs activation and protein synthesis inhibition
[5]. Ribotoxic stressors appear to be restricted to those toxi-
cants (e.g. anisomycin) that interact with or damage the R/S
loop near the 3’-end of the 28S rRNA, although the precise
linkage between ribosomal RNA damage and induction of
MAP kinase signaling cascades is unknown [5]. Iordanov et
al. have suggested that there must be as yet unidentified sig-
naling steps, presumably mediated by proteins, between
toxicant-damaged 28S rRNA and the MAPKs. Further, since
active ribosomes are required, it was suggested that this ac-
tivity of some PSIs was restricted to selected stages of the
ribosomal cycle [5, 9, 17].
So far, it is not yet clear which molecules, besides the
ribosome, are involved in the mechanism of action of aniso-
mycin to induce SAPK activation upstream of MKK4/7 and
MKK3/6 [22, 23, 27]. Coso et al. using dominant negative
mutants for Ras, RhoA, Rac1 and Cdc42 determined that
small G proteins are not required by anisomycin to activate
MAPKs [24]. Another report also showed, using toxin B,
that anisomycin acts independently of small GTPases to ac-
tivate p38 and CREB, in this case to induce Cox-2 mRNA
expression in intestinal epithelial cells [29]. In addition,
mouse embryonic stem cells lacking MEKK1 have normal
activation of JNK in response to anisomycin, heat shock, or
ultraviolet irradiation, showing that MEKK1, usually acti-
vated by small G proteins, is not required for the signaling of
these stressors [30].
Intriguingly, the pretreatment of mammalian cells with
low doses of anisomycin (~0.1 μM) that do not block trans-
lation induces homologous desensitization of intracellular
MAPKs activation and expression of some genes (c-fos, c-
jun, fos-B, junB, junD), but it does not interfere with the ex-
pression of these genes in response to growth factors like
EGF, FGF and TNF-
. Therefore, it was concluded that ani-
somycin behaves like an agonist to activate SAPKs, and also
that the desensitized component is not involved in JNK or
p38 activation by the growth factors but it appears to play a
role in responses stimulated by anisomycin, UV and hy-
perosmolarity [31].
Anisomycin is also able to regulate many different sig-
naling pathway components such as PI3K, AKT, IRS,
NFB, ROS, S6 and p70/p85S6K, eE2F kinase, PPAR,
HSF1, HDAC1, and caspases (see Table 1). In these cases,
Macías-Silva et al.
the mechanisms used by anisomycin to regulate these signal
transduction components are unknown, although some of
them may depend on the MAPK activation and/or on the
translational arrest induced by anisomycin, see Fig. (2).
REGULATION OF GENE EXPRESSION
Regulation of gene expression is another intriguing effect
of anisomycin, in view of the fact that it is able to superin-
duce the expression of certain genes, or to cause the degrada-
tion of some proteins. Gene superinduction is a phenomenon
characterized by augmented and prolonged expression of
immediate-early genes that are usually induced transiently,
and it is conventionally regarded as a secondary consequence
of translational arrest. Several mechanisms may contribute to
gene superinduction such as: increased mRNA stability,
augmented gene transcription, decreased synthesis of gene
repressors, and stimulation of nuclear signaling responses
[6].
Most protein synthesis inhibitors can trigger an abundant
accumulation of specific gene transcripts either alone or in
the presence of stimulating agents such as growth factors.
Gene superinduction induced by PSIs seems to be specific
for some genes and may depend on cell type (Table 1), and
typically but not always requires co-stimulation by specific
growth factors, or co-treatments with some stress inducers
like phorbol esters, UV irradiation or osmotic shock [3, 6,
32]. Concerning the potency of different PSIs to superinduce
immediate-early genes such as c-fos and c-jun, anisomycin
has proved to be particularly more potent as an inducer of
gene expression than CHX, Pur and emetine [28, 33].
Moreover, anisomycin superinduces immediate-early genes
at much lower doses (0.1-1 μM) than those required for pro-
tein synthesis inhibition (1-10 μM) [31]. It is important to
underline that some of the genes induced by anisomycin de-
pend on MAPK activation and/or on ribotoxic stress, see
Table 1 [33, 34].
PROTEIN DEGRADATION MODULATION
Anisomycin is also able to cause the degradation of cer-
tain proteins via ubiquitin-proteasome system (UPS), and
through mechanisms dependent on and independent of
MAPKs activation (Table 1). For instance, activation of p38
by anisomycin leads to reduction in the levels of phosphory-
lated Cx43 (connexin 43) possibly via degradation, which
might be responsible for the reduction in number of gap
junctions and levels of GJIC (gap junctional intracellular
communication) protein in liver epithelial cells [35]. Aniso-
mycin can also cause degradation via proteasome of the
M3/6 protein phosphatase that inactivates JNK in HEK293
cells, probably via JNK activation [36], whereas in MCF10A
(human breast epithelial cell line) cells, anisomycin and
CHX along with AhR (aryl hydrocarbon receptor) agonist
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can cause AhR
proteasome-mediated degradation [37]. Intriguingly, aniso-
mycin causes the degradation via UPS of Ski and SnoN on-
coproteins, important corepressors of TGF-/Smad pathway,
through an unknown mechanism that does not require
MAPK activation or protein synthesis inhibition in different
cell lines such as A549 and HeLa cells [38]. We have also
obtained some data showing that the Ski and SnoN down-
Anisomycin is a Multifunctional Drug Current Chemical Biology, 2010, Vol. 4, No. 2 127

Fig. (2). Cellular processes modulated by Anisomycin.

Anisomycin signals mainly by ribotoxic stress and activate the intracellular signaling pathways indicated. Anisomycin uses some unknown
effectors (*) to activate MAPKs and stimulate gene superinduction that have been described as sensitive to homologous desensitization. Ani-
somycin can induce cell death by either ribotoxic stress, or sensitizing resistant cells through an unknown mechanism that involves death
receptors. Anisomycin can also induce mitochondrial membrane breakdown independently of MAPK pathway. Anisomycin-dependent acti-
vation is indicated with solid arrows; unknown mechanisms of activation are indicated with dash arrows. See article text for further explana-
tions.

regulation induced by anisomycin can be blocked with spe-
cific inhibitors of TGF-
type I receptor [39].
POTENTIAL CLINICAL APPLICATIONS
Anisomycin was originally identified as an antibiotic
against certain protozoa and fungi, which led to proposed
clinical uses as an anticandidal and antiamoebic drug in hu-
mans. Later, due to its ability to arrest translation it has been
commonly used in studying neuronal processes related to
memory and learning in animals, although more studies need
to be done in order for anisomycin to be considered as a po-
tential psychiatric drug in humans. Nevertheless, anisomycin
may have potential clinical significance since it exhibits
many biological and pharmacological properties including
antitumoral, antiviral, cancer cell death sensitizer and immu-
nosuppressive activities:
1) Anisomycin Sensitizes Resistant Cells to Death
Anisomycin has been used by cancer researchers to study
some cell death processes like apoptosis and anoikis. Apop-
tosis is a genetically programmed cell death triggered by
both extracellular and intracellular events that can initiate
several molecular cascades that lead to DNA fragmentation
in the nucleus. Anoikis is a type of cell death similar to
apoptosis except that it is induced in response to the absence
of cell-matrix interactions; for instance, anoikis is relevant
for invading tumor cells because it is easier for anoikis-
resistant cells to participate in tumor metastasis.
It is known that anisomycin can affect both pro- and anti-
apoptotic mechanisms depending on the concentration used.
For example, in some cells like PC12, low doses of aniso-
mycin (<0.1 μM) seem to activate cell surviving signals such
as induction of anti-apoptotic proteins (for example Akt and
Bcl2), whereas high doses of anisomycin (>0.1 μM) block
Akt activation by NGF [40]. In some cell types, high doses
of anisomycin (>0.1 μM) also cause apoptosis by activating
SAPKs pathway, preventing the survival-promoting event,
modulating the expression of pro-apoptotic proteins (for ex-
ample, enhancing Bim and decreasing FLIP), and activating
the mitochondrial apoptotic machinery such as the activation
of Caspases-3, -8 and -12 and cytocrome c release [41-51].
In lymphoma U937 cells, anisomycin induces apoptosis by
caspase-8 activation, mitochondrial membrane potential col-
lapse, Bid activation, caspase-3 cleavage and cytochrome c
release. In addition, six gene clusters that were up- or down-
regulated by anisomycin were also detected by using mi-
croarrays. One of these gene clusters is related with a genetic
network containing genes associated with cell death, whereas
another gene cluster contains genes related with protein syn
128 Current Chemical Biology, 2010, Vol. 4, No. 2 Macías-Silva et al.

Table 1.

Anisomycin’s Biological Effects Concentration Model Ref.

GENE SUPERINDUCTION

MAPK/SAPK dependent

c-fos, c-myc and actin. 100 μM 3T3 cells [3]

c-fos/c-jun (EGF) 10 μg/ml C3H10T1/2 [33]

c-fos,c-jun, fosB, junB, junD 10 μg/ml C3H10T1/2 [68]

c.fos, c-jun, junB and junD. 25 ng/ml, 10 μg/ml HeLa, C3H 10T1/2 [31]

c-fos 25 ng/ml, 10 μg/ml HeLa, KB [69]

LDL-receptor (EGF) 50 ng/ml HepG2 [70]

c-jun, c-fos and zif268 10 ng/ml PC12 [40]

cox-2 10 μg/ml IEC-18, A549 [29]

Annexin V 1-10 μg/ml MCAS [71]

Egr-1 10 ng/ml U87MG [72]

Unknown mechanism

IL-6 (in the presence of IL-1) 20 μg/ml Hela, MDA-MB-231 [73]

CYP1A1 (cotreatment with TCDD) 0.5-10 μM MCF10A [37]

MAPK ACTIVATION

JNK/SAPK 10 μg/ml COS-7 [79]

JNK/SAPK 1 μg/ml FLS [27]

p45,p55,p38 25 ng/ml, 10 μg/ml C3H10T1/2 [28]

Gal4-Elk-1/JNK/ERK1/2 and p38 10 ng/ml U87MG, A549 [72]

p38 0.1-10 μg/ml WB-F344 [35]

p38 10 μg/ml IEC-18 [29]

p42, p44, JNK, p38 50 ng/ml HepG2 [70]

p38 0.4 μM J774A.1 [50]

p38 100 nM NIH3T3, HeLa [46]

JNK 250 ng/ml DU-145, Jurkat cells [44]

ERK1/2, p38 1-10
g/ml MCAS [71]

JNK/ERK1/2 and p38 10 ng/ml PC12 [40]

JNK/ERK1/2 and p38 25 ng/ml, 10 μg/ml C3H10T1/2 [31]

OTHER SIGNALING PATHWAYS REGU-

LATION

MAPK/SAPK dependent

Phosphorylation HSF1 (JNK) 1 μg/ml HeLa [74]

ROS induction 10 ng/ml PC12 [40]

IRS, ErbB2/ErbB3 activation 50 ng/ml Fao cells [75]

Anisomycin is a Multifunctional Drug Current Chemical Biology, 2010, Vol. 4, No. 2 129

Table 1. Contd…

Anisomycin’s Biological Effects Concentration Model Ref.

PPAR alpha phosphorylation (p38). 200 nM Cos-7 [76]

Inhibition eE2F kinase by SAPK4/p38. 40
M KB cells [77]

Tau hiperphosphorylation (GSK3) 0.4
g/ml N2a [78]

Akt kinase activator 10 μg/ml 293T, HeLa [83]

HDAC1 phosphoacetylation (with TSA ). 50 ng/ml 3T3 [80]

Unknown mechanism

S6 phosphorylation,p70/ p85S6K 10 μg/ml C3H 10T1/2 [81]

NFkB induction 20
g/ml HeLa [73]

PROTEIN DEGRADATION MODULATION

MAPK/SAPK dependent

M3/6 (JNK and SEK-1). 10 ng/ml HEK293 [36]

EGF-Receptor by p38 30 μM SW480 [82]

Phospho-Cx43 in gap-junctions 10
g/ml WB-F344 [35]

Unknown mechanism

AhR via proteasome 0.5-1 μM MCF10A [37]

Ski and SnoN via proteasome 1
M, 10 μM A549, HeLa [38]

APOPTOSIS ACTIVATION

MAPK/SAPK dependent apoptosis

Mitochondrial-caspase pathway. 1 μM U937 [49]

p38 MAPK activation (but not ERK ). 4
M, 20 μM J774A.1 [50]

Bim induction (JNK activation) 0.1
g/ml U87 [48]

TRAIL induced apoptosis 25 ng/ml M28 and REN [47]

Oncogenic Ha-Ras (JNK) 0.5 μg/ml AKR-2B [45]

Fas-mediated apoptosis 250 ng/ml DU-145 [44]

MAPK/SAPK independent apoptosis

Cytochrome c release. Caspase-9 activation 10
g/ml A549-S [42]

Blocks PI3K pathway. Inhibit Bcl-2 and Akt. 10 ng/ml, 1 μg/ml PC12 [83]

thesis [49]. Further, anisomycin and death receptor ligands
such as anti-Fas antibody (CH-11) or TRAIL (TNF–related
apoptosis-inducing ligand) can act synergistically to induce
apoptosis in multiple human glioblastoma cell lines that
show resistance to apoptosis. Anisomycin-induced ribotoxic
stress sensitizes glioblastoma cells to death receptor–induced
apoptosis via a specific mechanism requiring both JNK acti-
vation and Bim induction [48].
Anisomycin alone is a potent inducer of anoikis in some
malignant cells resistant to death such as PPC-1, PC-3, DU-
145, LNCaP, MB-MDA468 and OVCAR-3. In these cells,
anisomycin induces anoikis due to its ability to cause activa-
tion of caspase-8 and inhibition of FLIP protein synthesis but
independently of its ability to activate JNK and p38; aniso-
mycin as an anoikis sensitizer was also able to reduce the
metastatic potential of human prostate cancer cells seeded in
the mouse circulation [52]. Rüller et al. also described a pro-
cedure applied in vitro and in nude mouse transplants that
sensitizes human tumor cells (LT123), usually resistant to
chemotherapy and TNF, to ribotoxic stress-induced apop-
totic cell death. Sensitization is effected by successive appli-
cation of a combination of anisomycin, one inhibitor of his-
tone deacetylation (trichostatin A or butyrate) and flavopiri-
dol (an inhibitor of cyclin-dependent kinases) [53]. In the
future, anisomycin might possibly be employed in a particu-
lar therapeutic strategy for controlling human carcinomas.
2) Anisomycin´s Immune System Effects
Recently it was reported that a quite low dose of aniso-
mycin (<0.1 μM) was sufficient to block proliferation of T
130 Current Chemical Biology, 2010, Vol. 4, No. 2
CD4+ cells induced by Concanavalin A in mice, whereas the
levels of the immunosupressor TGF-1 fell in vivo; interest-
ingly, the blockage of proliferation can be partly restored by
adding exogenous IL-2 [54]. The treatment with anisomycin
also prolonged the survival of the transplanted skin, and de-
pressed the hypersensitivity development and the T-cell re-
sponse in the skin-transplanted mice. These results indicate
anisomycin may function as an immunosuppressant [50]. On
the other hand, anisomycin caused p38 and JNK activation to
modulate the antigen-presenting activity of dendritic cells, as
well as it synergized with LPS in driving release of IL-12
and TNF-
from dendritic cells [55].
3) Anisomycin´s Neuronal Effects
Several groups in the field of brain research have exten-
sively used anisomycin since the 1960’s. During all these
years they have been providing important information in
order to hypothesize that protein synthesis is intimately im-
plicated in memory and learning processes. A few years ago,
two interesting reports by Nader’s group stated that reacti-
vated memories require new protein synthesis to be stored
[56, 57]. Amygdala and hippocampus are the major regions
in the brain to storage and mediate memories, and a shot of
anisomycin injected directly in these areas has been pro-
posed for removal of memories by causing amnesia in ani-
mals. However, these findings do not show the mechanism
used by anisomycin, as either blocking protein synthesis or
disrupting neural circuits. In addition, the hypothesis that
memory requires protein synthesis is supported by some evi-
dence showing that rapamycin (an inhibitor of the mTOR
kinase that regulates protein synthesis) can also produce a
mild impairment in long-term memory formation [58].
The stand that memory formation is mainly attributed to
inhibition of de novo protein synthesis is now questioned due
to: lasting memories can be made even though protein syn-
thesis is completely inhibited, the memory impairments in-
duced by PSIs can be rescued by treatments with hormones
like vasopressin or corticosteroids, besides PSIs have some
side effects that might be the source of amnesia [59-62].
Moreover, several reports in the 1970s and ‘80s demon-
strated that amnesic effects caused by anisomycin could be
reversed by amphetamine, noradrenergic agonists, nicotine
and caffeine, even though protein synthesis inhibition was
greater than 90% [61].
The unknown side effects of pharmacological agents
used to study memory formation may also be involved in the
associated memory impairment. Given the plentiful molecu-
lar effects exerted by PSIs in different cell systems, it is pos-
sible that some actions other than translational arrest are in-
volved as mechanistic alternatives to explain the effects of
PSIs in learning and memory processes. Some of these ac-
tions of PSIs (e.g. anisomycin) can be gene superinduction,
post-translational modifications of proteins, synaptic altera-
tions, hyperproduction of specific proteins, and neuronal
death [6, 63].
In general, there are some technical problems related
with verifying of the affected brain regions by PSIs. Interest-
ingly Wanish et al. found differences in the time course and
the efficiency of anisomycin to inhibit protein synthesis fol-
lowing intracerebral versus systemic treatments [64]. In ad-
Macías-Silva et al.
dition, Tronson et al. also proposed that the studies of mem-
ory reconsolidation using PSIs have many limitations as they
provide little information on the specific mechanism under-
lying neuronal plasticity after retrieval [65]. Notwithstanding
the limitations mentioned previously, the studies with PSIs
have clearly shown that various cell signaling and transcrip-
tional events might be required for memory reconsolidation.
Therefore, now the side effects of PSIs not related with
translational arrest are becoming important participants in
those processes such as memory and learning. In the future,
it would be important to identify the mechanism involved in
the modulation of mRNA and protein levels, and to under-
stand the contributions of such modifications on gene ex-
pression. Another major research direction is the identifica-
tion of specific neuronal subtypes that participate in these
neuronal processes. For these studies, it would be very help-
ful to make use of the recently developed photo-released
anisomycin compound able to inhibit protein synthesis in a
spatially restricted manner, which will enable the specific
inhibition of protein synthesis in subsets of cells with tempo-
ral and spatial precision [66].
In summary, anisomycin has been a very useful tool to
study the memory and learning processes in animals; how-
ever, besides protein synthesis inhibition there are some
other side effects of PSIs involved in damaging memory
consolidation. Furthermore, more studies are needed in order
for anisomycin to be considered as a potential psychiatric
drug that could help to get rid of certain memories in hu-
mans.
NON-MAMMALIAN TARGET CELLS
In addition to anisomycin’s well known actions on
mammalian cells, there are some studies addressing its ac-
tions on several microorganisms. For instance, the presence
of antibiotics like anisomycin, CHX and Hyg B is toxic for
S. cerevisiae. In these cells, PSIs cause downregulation of
ubiquitin (UB) levels; hence UB levels are critical for the
survival of these cells. UB overexpression rescued cells from
death induced by the translational inhibitors, suggesting that
UB depletion may constitute a widespread mechanism for
the toxicity of translational inhibitors [67]. Anisomycin is
also toxic for other microorganisms such as T. vaginalis, T.
foetus, E. histolytica, T. brucei, L. donovani, P. chabaudi
and C. reinhardtii.
In conclusion, biochemical research has obtained great
benefits from the multifunctional facet of anisomycin even
though some of its mechanisms of action remain unknown.
Nevertheless, anisomycin has proved to be a useful tool for
scientific research in the fields of signal transduction and
gene expression, and it has not only been used as a protein
synthesis inhibitor. It is important to investigate its mecha-
nisms of actions in order to explain many of its effects ob-
served in vitro and in vivo. On the other hand, anisomycin
used in low doses has shown to have some intriguing effects
as an immunosuppressant or antitumoral agent, which may
be considered important for clinical applications in the fu-
ture. However, it is necessary to carry out more studies fo-
cused to authenticate these actions in other models and also
to distinguish its potential side effects.
Anisomycin is a Multifunctional Drug
ACKNOWLEDGEMENTS
Our work is supported by grants from CONACyT (No.
49493-Q) and PAPIIT/DGAPA/UNAM (No. IN222909).
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Accepted: February 15, 2010