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Introduction
Topoisomerase II (Topo II) is essential for DNA
metabo-lism, where it acts to adjust the topology of DNA during
transcription, replication, recombination, repair and mitosis[1]. Topo II have been validated as clinically important
targets for cancer chemotherapy, and their inhibitors are
central components in many therapeutic
regimens[2,3]. These Topo II-targeting agents can be divided into two categories
according to their mechanisms of action: Topo II poisons
and catalytic inhibitors. Topo II poisons are able to stabilize
the reversible covalent Topo II-DNA complex termed the
cleavage complex, while catalytic inhibitors act on the other
steps in the catalytic cycle without trapping the covalent
complex. Although Topo II poisons are among the frequently
used regimens in the clinical treatment of human
malign-ancies, there are still some limitations such as dose-limiting
toxicities and drug resistance leading to treatment failure
after initial effective therapy. Moreover, drugs originating
from different chemical families, which share a common
cellular target, generally exhibit different spectra of anticancer
activity. Consequently there is an increasing interest
focusing on searching and developing anticancer agents
targeting human Topo II.
Salvicine is a novel diterpenoid quinone compound
obtained by structural modification of a natural product lead
isolated from Salvia prionitis Hance (Labiatae) with potent
growth inhibitory activity against a wide spectrum of human
tumor cells in vitro[4] and in mice bearing human tumor
xenograft[5] (Yuan S, et al, unpublished data). Salvicine has
also been found to have a profound cytotoxic effect on
multidrug-resisitant (MDR) cells. Moreover, Salvicine
significantly reduced the lung metastatic foci of MDA-MB-435
orthotopic xenograft with no obvious inhibition on primary
tumor growth. These results demonstrate that salvicine is a
promising anticancer drug candidate and is in phase II
clinical trials in China.
Salvicine has emerged as a novel Topo II inhibitor with
distinct modes of action, which are dependent on ROS
generation. This review will focus on the effects and
mechanisms of salvicine as a novel Topo II inhibitor as well as its
distinguished anticancer activity in different systems.
Salvicine is a novel Topo II poison with distinct
mode of action
Salvicine was found to inhibit the activity of Topo II in a
routine screen with an approximate IC50 value of 3 µmol/L in
kDNA decatenation assays. Similar results were attained by
Topo II-mediated supercoiled DNA relaxation
assay[7]. Salvicine acted as a Topo II poison through its marked
enhancement effect on Topo II-mediated DNA double-strand
breaks as observed in the DNA cleavage assay without
intercalating into DNA[6]. In contrast, no inhibitory activity
was observed against the catalytic activity of
TopoI[6]. Consistently, cytotoxicities of salvicine to parent (JN394)
and TOP1 deleted (JN394top1-) yeast cells are at the same
level[7]. Salvicine displays high activity against JN394t2-1
cells at 25°C where the top2-1 protein shows wild type
activity, while no growth inhibition was observed at the
semi-permissive temperature of 30°C in the concentration range
of interest[7]. Furthermore, JN394t2-5 cells, which express
top2-5 mutant allele are highly resistant to salvicine and
etoposide (VP16)[7]. These results indicate that Topo II is
the primary cellular target of salvicine and confirm that
salvicine kills yeast cells mainly by trapping the DNA-Topo
II cleavage complex.
Salvicine elicites ROS and acts on multi-steps in the
catalytic cycle of Topo II By dissecting individual steps of
the catalytic cycle of Topo II, the mechanism by which
salvicine inactivates Topo II was found to be different to
that of other anti-Topo II agents. Salvicine greatly promotes
Topo II-DNA binding and inhibits pre- and post-strand Topo
II-mediated DNA relegation without interference with the
forward cleavage steps[6]. Moreover, molecular modeling
studies predicted that salvicine binds to the ATP pocket in
the ATPase domain and superimposes on the phosphate and
ribose group[8]. Consistently, salvicine exhibits higher
affinity for the ATPase domain of human Topo IIa than ADP and
ATP in the surface plasmon resonance binding assays and
inhibits ATP hydrolysis catalyzed by this
domain[8]. ATP competitively and dose-dependently blocks the interactions
between salvicine and the ATPase domain of Topo
IIα, indicating that salvicine shares a common binding site with ATP
and functions as an ATP competitor[8]. Thus, salvicine is the
first reported non-intercalative eukayotic Topo II poison that
binds to the ATP-binding pocket of Topo IIα. It is
noteworthy that inhibition of Topo II activity was abrogated by
GSH(Cai Y, et al, unpublished data), which is a ROS scavenger,
suggesting that the inhibitory effect of salcivine on Topo II
might due to ROS generation. Together, salvicine emerged
as a novel Topo II poison with a distinct mode of action with
ROS generation, competitively binding to ATP pocket,
promoting Topo II-DNA binding and inhibiting Topo
II-mediated DNA relegation (Figure 1).
ROS-dependent and Topo II-mediated DNA damage response induced by salvicine
It is well accepted that Topo II poisons exert
antipro-liferative activity by inducing DNA double stand beaks, which
may in turn trigger DNA damage response cascades and
ultimately apoptosis. It is notable that salvicine stimulates
intracellular ROS production and subsequently elicits
DSBs[9]. N-acetyl cysteine (NAC), an antioxidant, effectively
attenuates the salvicine-induced ROS enhancement and also
DNA DSBs. Moreover, NAC abrogates salvine-induced
Topo II-DNA cleavable complexes formation and the growth
inhibition of salvicine-treated JN394top2-4 yeast cells,
collectively indicating that Topo II is a target of the
salvicine-induced ROS[9]. Heat treatment that reversed the
salvicine-trapped DNA-Topo II cleavage complex reversed the
accumulation of DNA DSB as well, demonstrating that
salvicine-induced DNA damage is triggered by ROS and mediated by
Topo II. The breakage and/or reunion reaction of DNA Topo
II can be interrupted by DNA intercalators (eg doxorubicin),
enzyme binders (eg etoposide), DNA lesions (eg abasic
sites), or oxidative stress to produce Topo-mediated DNA
damage[10,11]. Salvicine structurally contains quinone, a
chemically active moiety. Most of the quinone-containing
anticancer drugs are believed to stimulate
ROS as part of their anti-tumor activities or
toxicities[10,12]. Accordingly, NAC attenuated salvicine-induced apoptosis and cytotoxicity in
MCF-7 cells[9]. Thus, salvicine generates ROS that
modulates Topo II-mediated DNA damage, contributing to the
comprehensive biological consequences of salvicine treatment,
such as DNA DSBs, apoptosis, and cytotoxicity in tumor
cells.
Salvicine selectively induces DNA damage in the c-myc
p2 promoter region Salvicine induces DNA strand breaks in
human promyelocytic leukemia HL-60 cells and breast
cancer MCF-7 cells [13,14]. DNA damage correlates well with cell
growth inhibition, suggesting that Topo II is the primary
cellular target of salvicine, which is also demonstrated by
the study using a yeast genetic
system[7]. DNA damage induced by brief exposure to salvicine could be partially
reversed, but early DNA breaks triggered the process of
apoptosis[13]. Preferential damage in the P2 promoter region
of the oncogene c-myc was detected, whereas no obvious
DNA damage was found in the 3' region of the same gene in
both HL-60 and MCF-7 cells[13,14]. Salvicine induces a
dose-dependent decrease in c-myc gene transcription,
concomitant with an increase in c-jun
expression[13,14]. It appears possible that DNA damage within such genomic regions is
an early event, which could lead to growth inhibition
mediated by alterations of the expression of selected
proliferation regulatory genes, such as c-myc, c-jun, and ultimately
cell death.
Salvicine disrupted the catalytic subunit of DNA-dependent protein kinase DNA DSBs induced by salvicine
activates Ataxia-telangiectasia-mutated (ATM) and ATM-
and Rad3-related (ATR) kinases and phosphorylastion of
histone H2AX in lung carcinoma A549 cells (Zhang Y,
et al, unpublished data), which are well documented in DSB-
induced cellular responses[15]. Unexpectedly, salvicine
selectively downregulates the protein levels of the catalytic
subunit of DNA-dependent protein kinase (DNA-PK[CS])
but not the Ku70 and Ku86 subunit[9]. Salvicine treatment
also reduces the kinase activity of DNA-PK in MCF-7 cells,
which might be due to the reduction of the (DNA-PK[CS])
protein[9]. DNA-PK is composed of DNA-PKcs
of approximately 450 kDa and two smaller Ku subunits (Ku70
and Ku86), is a critical component of non-homologous end joining
(NHMJ) pathway[16], which is the predominant pathway for
DSB repair (including Topo II-mediated DNA damage repair)
in mammals[17]. Thus, salvicine simultaneously damages DNA
and disrupts the DNA repair pathway, which could enhance
its therapeutic effectiveness and overcome the resistance
caused by DNA repair[18]. NAC pretreatment abrogates the
effects of salvicine on the protein level and the activity of
DNA-PK, indicating that ROS generation is involved in the
salvicine-induced DNA damage and repair (Figure 2). The
effects of salvicine-induced ROS on Topo II and DNA-PK
give new insights into the diverse biological activities of
ROS.
Salvicine induced telomere erosion and down-regulated
Telomere repeat binding factor 2 Except for genomic DNA
damage, salvicine has been shown to induce telomere
erosion and to downregulate the activity of
telomerase[19,20]. Salvicine treatment resulted in apoptosis and
down-regulation of telomerase activity in a time- and concentration-dependent manner in HL-60 cell[19]. Further study proved
that telomerase inhibition by salvicine is an early event in
human lung carcinoma A549 cells[20]. Though salvicine,
VP-16, mitomycin C, cisplatin and adriamycin induce telomere
erosion in A549 cells, only salvicine downregulates the
activity of telomerase after high concentration and short
exposure[20]. Telomere repeat binding factor 2 (TRF2) has been
increasingly recognized to be involved in DNA damage
response and telomere maintenance[21]. Salvicine led to
disruption of TRF2, independently of either its transcription or
proteasome-mediated degradation (Zhang Y, et
al, unpublished data). TRF2 protein was found to protect both
telomeric and non-telomeric DNA from salvicine-induced
damage by overexpressing the full-length
trf2 gene (Zhang Y, et al, unpublished data). Together, salvicine could
induce DNA damage both in genomic DNA and telomere and,
at the same time, disrupt TRF2, which maintains the integrity
of DNA (Figure 2).
Salvicine displays potent anticancer activity
Salvicime possesses potent antitumor activity
in vitro and in vivo Salvicine displayes potent growth inhibitory
activity against a panel of human tumor cells in
vitro and in mice bearing human tumor
xenografts[4,5]. Salvicine is as cytotoxic as VP-16 and weaker than VCR in three leukemia
cell lines measured by microculture tetrazolium (MTT)
assays after 72-h treatment[4]. Salvicine is over 5.41- and
4.15-fold more potent than VCR and VP-16 against 12 lines of
solid tumor cells[4]. Particularly, Salvicine presents better
activities against gastric and lung carcinoma cells. Moreover,
the antitumor effect of salvicine was found to be associated
with its ability to induce apoptosis in K-562 and SGC-7901
cells[22]. The anticancer activity of salvicine was also
evaluated in animal models. Salvicine possesses a significant
antineoplastic activity against murine S-180 sarcoma and
Lewis lung cancer, and human lung adenocarcinoma
xenografts A-549 and LAX-83[5]. Consistent with the results
from the in vitro study, salvicine displays significant
inhibition on lung and gastric adeocarcinoma including A-549,
SPC-A4, SGC-7901, MKN-28 and MKN-45 xenografts with
the optimal T/C value of 39.4%, 48.5%, 40.0%. 58.6% and
55.7%, respectively, while salvicine has no growth
inhibitory effects on IBC, BEL-7402, HO8910 and HCT116 nude
mice xenografts (Yuan S, et al, unpublished data). Phase 2
clinical trials demonstrated that salvicine is well-tolerated
by the patients with little side effects and toxicities
(unpublished data).
Salvicine overcomes multidrug resistance and
down-regulates P-glycoprotein by JNK activation
MDR is considered to be an important impediment to the effective
chemotherapy of cancer. Therefore, it is noteworthy that
salvicine is able to overcome the MDR caused by P-gp
overexpression. Salvicine effectively kills tumor cells
overexpressing P-gp with IC50
values of 1.55 µmol/L for K562/A02 cells, 4.50 µmol/L
for KB/VCR cells, and 1.40 µmol/L for
MCF-7/ADM cells, close to those for their corresponding
parental cell lines: 0.87 µmol/L for K562 cells, 2.26 µmol/L for
KB cells, and 2.61 µmol/L for MCF-7
cells[23]. The mean resistance factor for salvicine is 1.42, which is much lower than
that of vincristine (344.35), doxorubicin (233.19) and etoposide
(71.22)[23]. Salvicine induces similar
levels of apoptosis in MDR K562/A02 and parental K562 cells, promising its
activity against MDR. Unlike other MDR modulators that inhibit
the drug efflux by P-gp, salvicine downregulates
mdr-1 and P-gp expression in K-562/A02 MDR
cells[23]. Further study indicated that the transcription
factor c-Jun activation
induced by salvicine repressed mdr-1 gene
expression[24]. Salvicine suppresses
mdr-1 expression in MDR cells and promotes c-jun expression in both MDR and parental K562
cells. Moreover, enhanced c-jun expression precedes
reduction of mdr-1 expression after salvicine treatment in
K562/A02 cells. In contrast, c-jun antisense
oligodeoxy-nucleotides prevents salvicine-stimulated enhancement of
c-Jun protein and reduction of mdr-1 gene
expression[24]. Salvicine promotes phosphorylation of c-Jun-N-terminal
kinase and c-Jun protein in MDR K562/A02 and parental
K562 cells. Accordingly, salvicine enhances DNA binding
activity of transcription factor activator protein 1 in the
electrophoretic mobility shift assays. Moreover, c-Jun antisense
oligodeoxynucleotides also inhibited salvicine-induced
apoptosis and cytotoxicity in MDR and parental K562
cells[24]. A recent study found that salvicine induced equal
ROS generation and glutathione depletion in both MDR and
parental K562 cells (Cai Y, et al, unpublished data).
Pretreatment of K562/A02 cells with NAC eliminated JNK
phosphorylation, c-jun activation and P-gp downregulation
induced by salvicine (Cai Y, et al, unpublished data).
Together, salvicine-triggered oxidative stress stimulates
c-Jun-N-terminal kinase phosphorylation and activation,
resulting in c-Jun phosphorylation and activation. Activated
c-Jun promotes expression of c-jun itself, represses mdr-1
transcription, and triggers pro-apoptotic signals, resulting
in low mdr-1 expression and cell death (Figure 2).
Salvicine inhibits tumor metastasis activity related to
Rho-dependent pathway and ROS-triggered p38MAPK
pathway Except for its antiproliferative activity, salvicine has
also been reported to possess antimetastatic
effects[25]. Metastasis refers to the dissemination of cancer cells
from initial tumor to distant sites and involves a series of
processes, including loss of adhesion, acquisition of cell motility,
extracellular proteolysis, and
angiogenesis[26]. Salvicine significantly reduces the lung metastatic foci of MDA-MB-435
orthotopic xenograft, without obviously affecting primary
tumor growth[25]. A comparison of gene expression profiles
of primary tumors and lung metastatic focus between
salvicine-treated and untreated groups using the CLOTECH
Atlas Human Cancer 1.2 cDNA microarray revealed that genes
involved in tumor metastasis, particularly those closely
related to cell adhesion and motility, were obviously
down-regulated, including fibronectin, integrin alpha3, integrin
beta3, integrin beta5, FAK, paxillin, and
RhoC[25]. Consis-tently, salvicine significantly downregulated RhoC at both
mRNA and protein levels, greatly inhibited stress fiber
formation and invasiveness of MDA-MB-435 cells, and
markedly blocked translocation of both RhoA and RhoC from
cytosol to membrane, indicating that the unique
antimetas-tatic action of salvicine is closely related to Rho-dependent
signaling pathway[25] (Figure 2). However, the mechanism
that leads to downregulation of RhoC needs to be further
clarified.
In an effort to explore the relationship between the
antimetastatic activity of salvicine and ROS generation,
salvicine was found to be capable of generating ROS in
human breast cancer MDA-MB-435 cells that was significantly
reversed by a ROS scavenger NAC( Zhou JC, et
al, unpublished data). Salvicine also inhibits cell adhesion to
fibronectin and collagen by disrupting the formation of both
focal adhesions and actin stress fibers, leading to the
rounding up of cells (Zhou JC, et al, unpublished data). In addition,
salvicine downregulates β1 integrin affinity, clustering, and
signaling via FAK and paxillin, and by contrast activated
ERK and p38 MAPK (Zhou JC, et al, unpublished data).
The specific inhibitor of MEK1/2 (U0126) or p38 MAPK
(SB203580) abrogates the inhibitory effects of salvicine on
β1 function and cell adhesion. Notably, NAC also abolishes
the activation of ERK and p38 MAPK, thereby protecting
β1 integrin affinity and restoring cell adhesion and spreading
(Zhou JC, et al, unpublished data). Therefore, salvicine
activates ERK and p38MAPK via triggering ROS generation,
which drives inactivation of β1 integrin function and results
in cell adhesion inhibition. Collectively, the antimetastatic
activity of salvicine is related to the Rho-dependent
signaling pathway and ROS-triggered p38MARK pathway (Figure
2). The relationship between these two pathways deserves
further study.
Concluding remarks
Salvicine is a novel Topo II poison that binds to the
ATPase domain of Topo II, promoting DNA-Topo II binding,
inhibiting Topo II mediated DNA relegation and ATP
hydrolysis. Salvicine displayed multi cellular effects
including inducing Topo II inhibition, DNA damage,
circumventing P-gp-mediated MDR and inhibiting tumor cell adhesion.
All of these effects at least partially are dependent on ROS
generation, indicating salvicine-elicited ROS plays a central
role in the anticancer activity of salvicine (Figure 2). Thus,
as an antitumor drug candidate, salvicine can also be used
as a tool to study the complicated roles of ROS in different
physiological processes in tumor cells, which will provide
useful information for future clinical studies.
Acknowledgements
We are grateful to Dr Jin-sheng ZHANG for providing
salvicine.
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