Nitric Oxide 24 (2011) 1–7

Contents lists available at ScienceDirect

Nitric Oxide
journal homepage: www.elsevier.com/locate/yniox

Review

Dual role of NO donors in the reversal of tumor cell resistance and EMT:
Downregulation of the NF-jB/Snail/YY1/RKIP circuitry
Benjamin Bonavida ⇑, Stavroula Baritaki
Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, Johnson Comprehensive Cancer Center, University of California,
Los Angeles, CA 90095, United States

a r t i c l e i n f o a b s t r a c t

Article history: Several studies have implicated the role of Nitric Oxide (NO) in the regulation of tumor cell behavior
Received 18 August 2010 and have shown that NO either promotes or inhibits tumorigenesis. These conflicting findings have
Revised 29 September 2010 been resolved, in part, by the levels of NO used such that low levels promote tumor growth and high
Available online 8 October 2010
levels inhibit tumor growth. Our studies have focused on the use of high levels of NO provided pri-
marily by the NO donor, DETANONOate. We have shown that treatment of resistant tumor cells with
Keywords: DETANONOate sensitizes them to apoptosis by both chemotherapeutic drugs and cytotoxic immuno-
EMT
therapeutic ligands. The underlying mechanisms by which NO sensitizes tumor cells to apoptosis
Resistance
Therapeutic targets
were shown to be regulated, in part, by NO-mediated inhibition of the NF-jB survival/anti-apoptotic
NF-jB/Snail/YY1/RKIP circuitry pathways and downstream of NF-jB by inhibition of the transcription factor Yin Yang 1 (YY1). In
Apoptosis addition to NO-induced sensitization to apoptosis, we have also shown that NO induced the expres-
sion of the metastasis-suppressor/immunosurveillance cancer gene product, Raf-1 kinase inhibitor
protein (RKIP). Overexpression of RKIP mimics NO in tumor cells-induced sensitization to apoptosis.
The induction of RKIP by NO was the result of the inhibition of the RKIP repressor, Snail, downstream
of NF-jB. These findings established the presence of a dysregulated NF-jB/Snail/YY1/ RKIP circuitry in
resistance and that treatment with NO modifies this loop in tumor cells in favor of the inhibition of
tumor cell survival and the response to cytotoxic drugs. Noteworthy, the NF-jB/Snail/YY1/RKIP loop
consists of gene products that regulate the epithelial to mesenchymal transition (EMT) and, thus,
tumor metastasis. Hence, we have found that treatment of metastatic cancer cell lines with DETAN-
ONOate inhibited the EMT phenotype, through both the inhibition of the metastasis-inducers, NF-jB
and Snail and the induction of the metastasis-suppressor, RKIP. Altogether, the above findings estab-
lish, for the first time, the dual role of high levels of NO in the sensitization of tumor cells to apop-
totic stimuli as well as inhibition of EMT. Hence, NO donors may be considered as novel potential
therapeutic agents with dual roles in the treatment of patients with refractory cancer and in the pre-
vention of the initiation of the metastatic cascade via EMT.
Ó 2010 Elsevier Inc. All rights reserved.

Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Establishment of the NF-jB/Snail/YY1/RKIP circuitry in cancer cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Role of NF-jB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Role of Snail. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Role of YY1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Role of RKIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
The NO donor, DETANONOate, reverses tumor cell resistance for apoptosis and inhibits EMT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Abbreviations: Bcl-xL, B-cell leukemia-xL; CDDP, cis-diammine-dichloro-platinum; DETANONOate, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl) amino] diazen-1-ium-
1, 2-diolate; DHMEQ, dehydroxymethylepoxyquinomicin; DR, death receptor; ERK, extracellular signal-regulated kinase; FasL, Fas ligand; IAP, inhibitor of apoptosis protein;
LNCaP, lymph node carcinoma of prostate; MAPK, Mitogen-activated protein kinases; MEK, MAP kinase kinase; NF-jB, nuclear factor-jB; si, small interfering; TRAIL, TNF-
related apoptosis-inducing ligand; XIAP, X-linked inhibitor of apoptosis.
⇑ Corresponding author. Fax: +1 310 206 2791.
E-mail address: bbonavida@mednet.ucla.edu (B. Bonavida).

1089-8603/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.niox.2010.10.001
2 B. Bonavida, S. Baritaki / Nitric Oxide 24 (2011) 1–7

NO-mediated sensitization of tumor cells to apoptosis by chemotherapeutic drugs and immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Role of NF-jB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Role of Snail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Role of YY1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
The role of RKIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
NO-mediated inhibition of EMT in metastatic cell lines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Role of NF-jB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Role of Snail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Role of YY1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Role of RKIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
In vivo validation of in vitro findings obtained with DETANONOate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Overall conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Introduction of the resistance (through NO-mediated sensitization to apoptotic

During the last decades, several therapeutics strategies have
been developed for the treatment of patients with cancer. Such ap-
proaches resulted in significant clinical responses and, sometimes,
prolongation of survival. However, subsets of patients do not ini-
tially respond or develop resistance, thus, becoming refractory to
subsequent treatments. Further, the non-responsive patients usu-
ally develop metastasis and are also unresponsive to treatments
[1]. Hence, there is an urgent need to develop new therapeutic
interventions to overcome both the resistance and metastasis.
One of the main underlying mechanisms in the development of
resistance in cancer patients is the acquisition by the tumor cells
of strategies to inhibit the apoptotic pathways. Dysregulation of
the apoptotic signaling pathways is common in cancer and it has
been implicated in the pathogenesis and progression of tumors.
These include (a) cell accumulation caused by failure of normal
cells turnover mechanisms (programmed cell death) and, thus,
creating a permissive environment for genetic instability and
oncogene activation (b) promotion of tumor cell-resistance to
immune attack (c) contribution of tumor cell-resistance to chemo-
therapy and radiation (d) tumor cells’ ability to survive in the face
of hypoxia and (e) fostering tumor metastasis by allowing the cells
to survive in a detached state [2–6].
In metastasis, the tumor cells invade the basement membrane
and circulatory system, extravasate through the endothelial lining
of the blood vessels and form colonies in tissue sites. The migration
of the epithelial layer into the mesenchymal layer before reaching
the basement membrane is known as ‘‘epithelial to mesenchymal
transition’’ (EMT) [7]. During EMT, the polarized epithelial cells ac-
quire certain attributes of mesenchymal cells and, thus, are able to
penetrate the mesenchymal layer through the basement mem-
brane and invade neighboring tissues. Breaching of the mesenchy-
mal layers leads to infiltration into the blood vessels and migration
to other points of the body [8].
The objective of our current studies has been to delineate the
underlying biochemical, molecular, and genetic mechanisms that
are responsible for the resistance in cancer and the development
of the EMT phenotype and subsequent metastasis. These studies
may lead to the discovery of novel targets for therapeutic interven-
tions in the reversal of resistance and inhibition of EMT. We have
established in model tumor cell lines the existence of a dysregu-
lated NF-jB/Snail/YY1/RKIP circuitry. This circuitry was found to
regulate both tumor cell resistance and EMT. Treatment of the
tumor cells with the NO donor, DETANONOate, modifies this
circuitry and associated gene products and results in the reversal
stimuli) and inhibition of the EMT phenotype. This review will
briefly discuss the above circuitry and the role of each of the in-
volved gene products in the regulation of resistance and EMT.
Establishment of the NF-jB/Snail/YY1/RKIP circuitry in cancer
cells
Based on studies reported by us and others, we have estab-
lished in tumor cells the presence of a dysregulated NF-jB/
Snail/YY1/RKIP loop [9–13]. This loop was found to be pivotal in
the regulation of tumor cell sensitivity to cytotoxic apoptotic
stimuli (for example, chemotherapeutics drugs and cytotoxic lym-
phocytes/ligands). In addition, we have established that this loop
is also involved in the regulation of EMT [9]. Below, we briefly de-
scribe how the above loop was established and demonstrate that
each gene product of this loop regulates the remaining gene
products.
Role of NF-jB
Cancer cells have been shown to exhibit a constitutively hyper-
activated NF-jB survival signaling pathways [14]. This hyperacti-
vation results downstream in the transcriptional activation of
various anti-apoptotic gene products including Bcl-xL, XIAP and
Yin Yang 1 (YY1) [15,16]. In addition, NF-jB has been reported to
regulate positively EMT and metastasis [17–19]. EMT is induced,
in part, directly via NF-jB-mediated activation of the transcription
of the EMT-inducer Snail [20], and indirectly via downregulation of
the expressions of the metastasis-suppressor cancer gene products,
RKIP and E-cadherin [21,22]. We and others have shown that inhi-
bition of NF-jB activity by different agents (e.g. drugs, specific
inhibitors, siRNA silencing, etc.) results in the reversal of tumor
resistance to apoptotic stimuli and inhibition of cancer cell inva-
sion and migration [23–32]. We have reported that one of the
underlying mechanisms by which NF-jB inhibition by NO sup-
presses tumor cell resistance and metastasis is through inhibition
of the downstream targets Snail and YY1 and induction of RKIP
[9,31–33] (see scheme Fig. 1A).
Role of Snail
Snail is a member of the Snail superfamily of zinc finger tran-
scription factors. It has been shown to play a role in embryonic
development, neural differentiation, cell division and cell survival
 B. Bonavida, S. Baritaki / Nitric Oxide 24 (2011) 1–7
Fig. 1. Effect of DETANONOate on the expression and activity of each of the gene
products involved in the NF-jB/Snail/YY1/RKIP loop and their intrarelationships.
(A) The inhibition of NF-jB activity by DETANONOate results in the transcriptional
repression of its downstream targets Snail and YY1. In addition, the DETANONOate-
mediated NF-jB inhibition also induces RKIP expression via de-repression of its
promoter by Snail. (B) DETANONOate inhibits the expression and activity of Snail
through NF-jB inhibition and S-nitrosylation, respectively. Snail inhibition, in turn,
derepresses the RKIP promoter and increases the RKIP levels. The RKIP induction
results in further inhibition of NF-jB and its downstream targets YY1 and Snail. (C)
In addition to NF-jB and Snail, DETANONOate directly S-nitrosylates and deacti-
vates YY1. Since YY1 functions as a transcriptional activator of Snail, the
DETANONOate-mediated inhibition of YY1 results in Snail downregulation and
subsequent induction of RKIP. The overexpression of RKIP, in turn, further inhibits
the NF-jB activity. (D) As mentioned above, RKIP induction by DETANONOate has a
direct inhibitory effect on NF-jB activation which in turn leads to decreased
expression of Snail and YY1.
[34]. It also plays an important role in the acquisition of invasion
and migratory properties by cancer cells during tumor progression
by triggering EMT [35]. Snail transcription is activated by NF-jB
and YY1 [20,36], while it acts directly as a transcriptional repressor
of the metastasis-suppressor gene products E-cadherin and RKIP
[21,22]. Our findings demonstrate that DETANONOate-mediated
inhibition of Snail and de-repression of E-cadherin is only one
mechanism by which DETANONOate upregulates E-cadherin
expression. We do not rule out other pathways regulated by
DETANONOate in the regulation of E-cadherin. Snail silencing has
been shown to result not only in reversal of the mesenchymal can-
cer cell phenotype, but also in resensitization of resistant tumor
cells to apoptotic stimuli [9,31,37]. We have reported that inhibi-
tion of both EMT and resistance by Snail silencing is partially
attributed to induction of RKIP, which in turn results in inhibition
of NF-jB and YY1 [9,31,33] (see scheme Fig. 1B).
Role of YY1
Yin Yang 1 (YY1) is a multifunctional DNA-binding protein that
can activate, repress, or initiate transcription depending on the
context in which it binds (directly or indirectly through complex-
ing with other DNA-binding proteins) [38]. YY1 has been
documented in the repression of several genes including IFN-c,
GM-CSF and IL-3 [39–41]. We have also identified YY1 as a
transcriptional repressor of the death receptors Fas and DR5, thus
suggesting that YY1 might have a critical role in the regulation of
tumor cell resistance to cytotoxic immune ligands, such as FasL
and TRAIL [11,42,43]. The direct role of YY1 in the regulation of
3
tumor resistance to the above apoptotic stimuli has been shown
by direct silencing of YY1 using siRNA, which resensitized the
resistant tumor cells to apoptosis [43]. In contrast, YY1 has been
reported to activate the transcription of the EMT-inducer Snail
[36], therefore, suggesting that YY1 might be also involved in the
regulation of EMT during cancer metastasis (see scheme Fig. 1C).
Role of RKIP
The Raf-1 kinase inhibitory protein (RKIP) is a member of the
phosphatidylethanolamine-binding protein family (PEBP). It is a
small cytosolic protein that was initially shown to play a role in
lipid metabolism and phospholipid membrane biogenesis [44]. Al-
most a decade ago, RKIP was first reported to function as an inhib-
itor of both the Raf-1/MEK/ERK and NF-jB survival pathways
[45–47]. Later, Lorenz et al. [48] demonstrated that RKIP also
regulates negatively the activation of G-protein coupled receptors
(GPCRs), resulting in attenuation of GPCR-mediated signaling. RKIP
has been further shown to regulate tumor cell sensitivity to apop-
totic stimuli including chemotherapy [49]. In addition, our studies
have identified RKIP as an immunosurveillance gene product
based on RKIP involvement in the regulation of tumor cell resis-
tance to cytotoxic immune ligands, such as TRAIL [9,33]. RKIP
expression is significantly downregulated in several malignancies
and almost absent in metastatic tumors [50–55]. RKIP overexpres-
sion in in vitro and in vivo tumor models has shown significant
anti-metastatic effects, thus supporting the role of RKIP as a
‘‘metastasis-suppressor’’ gene product [44,50,54]. We have further
demonstrated that RKIP overexpression directly correlates with
the inhibition of EMT and reversal of tumor resistance to apoptotic
stimuli, namely, through a common mechanism which involves
inhibition of the expressions and activities of NF-jB, Snail, and
YY1 [9,31,33] (see scheme Fig. 1D).
Overall, the above findings clearly establish the tightly con-
trolled NF-jB/Snail/YY1/RKIP loop that is dysregulated in cancer
cells and that is responsible, in part, for tumor cell survival and
resistance and the initiation of EMT.
The NO donor, DETANONOate, reverses tumor cell resistance for
apoptosis and inhibits EMT
We and others have reported that treatment of tumor cells with
high levels of NO [56] derived by DETANONOate at the concentra-
tions of 500–1000 mM inhibited NF-jB activity through S-nitrosy-
lation of its p50 subunit [10,11,13,57]. Thus, we hypothesized that
treatment of tumor cells with NO will result in the modification of
the NF-jB/Snail/YY1/RKI loop by inhibiting downstream the
expressions and activities of Snail and YY1 and resulting in the
induction of RKIP. RKIP, in turn, will further potentiate the inacti-
vation of NF-jB and its downstream gene targets. Hence, such ef-
fects by NO will result in the inhibition of the anti-apoptotic
mechanisms as well as inhibition of EMT (see general scheme of
NO effects in Fig. 2).
Below, we will describe briefly the experimental data that dem-
onstrated, following treatment of resistant and metastatic prostate
tumor cell lines, that the NO donor, DETANONOate, results in the
sensitization of tumor cells to apoptosis and inhibits the EMT phe-
notype. The role of each of the gene products that is implicated in
the NF-jB/Snail/YY1/RKIP circuitry has been examined and our
data demonstrate that each gene product regulates both tumor cell
resistance and EMT.
DETANONOate is an NO donor which belongs to the family of
diazoniumdiolates (formerly NONOates). It spontaneously dissoci-
ates in a pH-dependent first order process with a half life of 20 h
and 56 h at 37 °C and 22–25 °C, pH 7.4, respectively, to liberate
4 B. Bonavida, S. Baritaki / Nitric Oxide 24 (2011) 1–7

Fig. 2. Schematic diagram of the crosstalks among the members of the NF-jB/Snail/YY1/RKIP loop in the regulation of tumor resistance to apoptotic stimuli and EMT by
DETANONOate. NO mediates its biological effect on reversal of tumor cell resistance and EMT mainly by interfering with components of the NF-jB/Snail/YY1/RKIP circuit.
Tumor cells with highly resistant and metastatic phenotypes show constitutive activation of the NF-jB pathway and its downstream transcriptional targets YY1, Snail and
several anti-apoptotic gene products such as Bcl-xL, XIAP and survivin. The above gene products have been linked directly and indirectly with acquisition of tumor cell
resistance to both chemotherapy and immunotherapy and induction of EMT. Snail and YY1 act as transcriptional repressors of RKIP and death receptor genes such as DR5 and
Fas, respectively. Snail also acts as an essential initiator of EMT via inhibiting the transcription of the metastasis-suppressor genes RKIP and E-cadherin, while it induces
directly and/or indirectly the expression of mesenchymal markers. We show that NO inhibits the expressions and activities of NF-jB, Snail and YY1 and induces RKIP.
Induction of RKIP has been shown to resensitize resistant tumors to apoptosis mediated by drugs and/or cytotoxic death ligands such as TRAIL, and to reverse EMT. RKIP
induction also further facilitates the inhibition of NF- jB and its targeted genes Snail and YY1. Overall, the NO-mediated regulation of the NF-jB/Snail/YY1/RKIP loop via the
above mechanism results in reversal of tumor cell resistance to apoptotic stimuli and inhibits the migratory and invasive properties of metastatic tumor cells, thus it inhibits
EMT. Green lines correspond to the NO-mediated effects on the indicated gene products, while red lines correspond to the constitutive basil levels in tumor cells in the
absence of NO.

two moles of NO per mole of the parent compound, DETANONOate
[58,59]. The decomposition is not catalyzed by thiols or biological
tissues, unless specifically designed to and because NO release fol-
lows simple first-order kinetics [60]. The rate of NO release can be
accurately predicted. This can be achieved via specific modification
of the NONOate structure, which can stabilize the drug in solution
and potentially engender a selective NO release in different organs,
vascular beds, or specific cell types [61,62].
At 37 degree this is equal to 28 uM NO relesaed each second, one might use this as a
comparison instead or together with SNAP. If the peak concentratrion is of importance it might
be possible to accieve similar leverls with PDNO infusions in vivo. 20 = 72 000 sek.
NO-mediated sensitization of tumor cells to apoptosis by
chemotherapeutic drugs and immunotherapy
We have selected to utilize the chemotherapeutic drug, cis-
platinum (CDDP) and immunotherapeutic ligand TRAIL as repre-
sentatives for chemotherapy and immunotherapy, respectively.
We demonstrate that treatment of the resistant cells with
DETANONOate resulted in tumor cell sensitization to apoptosis
by CDDP and TRAIL, as assessed by activation of the effector cas-
pase 3. The reversal of tumor resistance to the apoptotic stimuli
used was a function of both the concentrations of DETANONOate
and either CDDP or TRAIL. The DETANONOate-mediated cell sen-
sitization to TRAIL and CDDP was shown to be synergistic as
determined by isobologran analysis [10] (unpublished data) In
addition, the reversal of tumor resistance to TRAIL was shown
to involve both the type I and type II apoptotic pathways
[10,11]. We further analyzed the underlying mechanism of
DETANONOate-mediated cell sensitization focusing specifically
on the role of each of the gene products of the NF-jB/Snail/
YY1/RKIP loop, as described below.
Role of NF-jB
Treatment of our cell lines with DETANONOate resulted in the
inhibition of NF-jB activity as a result of its inhibition of its
DNA-binding activity [10,11]. DETANONOate inhibited the NF-jB
activity part, via DETANONOate-induced S-nitrosylation of p50
[10]. DETANONOate also resulted in significant inhibition of NF-
jB regulated anti-apoptotic gene products such as Bcl-xL and XIAP
[10,11,13]. The direct role of NF-jB in DETANONOate-mediated
tumor cell sensitization was corroborated with experiments dem-
onstrating that treatment of the tumor cell lines with the specific
NF-jB inhibitor, DHMEQ, mimicked DETANONOate in tumor cell
sensitization to both CDDP and TRAIL and inhibition of its down-
stream anti-apoptotic targets XIAP, Bcl-xL and survivin [10,11]
(unpublished data) (Fig. 1A).
Role of Snail
Treatment of resistant cells with DETANONOate had a signifi-
cant negative impact on the expression of Snail mRNA and protein
levels [unpublished data]. The DETANONOate-mediated inhibition
of Snail expression may be, in part, attributed to inhibition of its
transcriptional activator, NF-jB. In addition, DETANONOate caused
a rapid S-nitrosylation of the Snail protein which resulted in inhi-
bition of its activity as assessed by DNA-binding assays [unpub-
lished data]. The direct role of NO-mediated inhibition of Snail in
tumor cell sensitization was demonstrated by Snail silencing using
specific siRNA assays. Our findings demonstrate that, in contrast to
untreated or cells treated with control siRNA, treatment with Snail
siRNA mimicked DETANONOate in terms of inducing significant
potentiation of apoptosis following treatment with either TRAIL
or CDDP [9] (Fig. 1B).
 B. Bonavida, S. Baritaki / Nitric Oxide 24 (2011) 1–7
Role of YY1
In addition to Snail inhibition, cell treatment with DETANONO-
ate also inhibited the expression levels of another NF-jB targeted
gene product, YY1 [11,13]. In addition, DETANONOate was shown
to abolish YY1 DNA-binding activity through direct S-nitrosylation
[11,13,57]. Since YY1 is a transcriptional activator of Snail, the inhi-
bition of YY1 activity might, therefore, contribute to further sup-
pression of Snail expression. Since YY1 is a negative regulator of
the transcription of the TRAIL receptor DR5, we examined whether
the DETANONOate-mediated YY1 inhibition results in upregula-
tion of DR5 expression, thus contributing to cell sensitization to
TRAIL apoptosis. Indeed, we have confirmed that cell treatment
with DETANONOate led to selective upregulation of DR5 with no
effect on the TRAIL receptor DR4 or the decoy receptors DcR1
and DcR2 [11] (unpublished data). The direct role of YY1-induced
inhibition by DETANONOate was further demonstrated following
treatment of tumor cells with YY1 siRNA. Such treated cells, in con-
trast to untreated or treated cells with control siRNA, resulted in
significant induction of apoptosis following cell treatment with
either TRAIL or CDDP [11,32,33] (unpublished data) (Fig. 1C).
The role of RKIP
In contrast to DETANONOated-mediated inhibition of NF-jB,
Snail and YY1, cell treatment with DETANONOate had rather an
inducing effect on RKIP expression, that was attributed to DETAN-
ONOate-mediated inhibition of its direct and indirect suppressors
NF-jB, Snail and YY1 (10, 11, submitted data). The direct role of
DETANONOate-mediated RKIP induction in the regulation of tumor
cell resistance to both CDDP and TRAIL was demonstrated by treat-
ment of tumor cells with an RKIP-expression vector. In comparison
with untreated or cells treated with an empty vector, cell over-
expressing RKIP were significantly sensitized tumor cells to CDDP
and TRAIL-apoptosis, similar to the one observed after cell treat-
ment with DETANONOate. In addition, overexpression of RKIP re-
sulted in inhibition of YY1 and Snail expression which might be
possibly attributed to the suppression of NF-jB activity by RKIP
[9] (Fig. 1D).
Conclusions
The above findings demonstrate that treatment of resistant
tumor cells, which exhibit a dysregulated NF-jB/Snail/YY1/RKIP
loop, with DETANONOate resulted in the modification of this
loop, and that each gene product was shown to directly regulate
tumor cell resistance, as would have been expected from a loop.
Our findings with normal human derived peripheral blood leuko-
cytes and treated with DETANONOate, alone or in combination
with chemotherapy, did not result in any significant toxicity
however additional studies should be performed using normal
bone marrow derived cells and hematopoietic cells for analysis
of toxicity. In addition, the findings demonstrate potential novel
therapeutic targets whose modifications can reverse tumor cell
resistance.
NO-mediated inhibition of EMT in metastatic cell lines
Besides the sensitizing effects of DETANONOate for apoptosis in
resistant tumor cells, our studies also identified DETANONOate as a
potent inhibitor of cell invasion and migration, thus resulting in
reversal of the EMT cell phenotype. Specifically, treatment of the
metastatic tumor cells with DETANONOate inhibited the expres-
sion of mesenchymal gene products such as fibronectin and vimen-
tin while it upregulated the levels of the metastasis-suppressor
E-cadherin, as assessed by western blot analysis. The above find-
ings were also confirmed by immunohistochemistry [submitted
5
data]. To examine further the underlying mechanism of DETANON-
Oate-mediated EMT inhibition we investigated selectively the con-
tribution of each of the members of the NF-jB/Snail/YY1/RKIP
loop.
Role of NF-jB
The constitutive activation of NF-jB observed in various malig-
nant tumors is known to promote angiogenesis and invasion, thus,
increasing the metastatic cell potential [17]. In addition, NF-jB is
critical for the transcriptional activation of the EMT-inducer Snail
in metastatic tumors. Our studies with metastatic cell line models
were also characterized by their constitutively activated NF-jB
[63], which was significantly deactivated following cell treatment
with DETANONOate, as mentioned above. The direct role of
DETANONOate-mediated inhibition of NF-jB in the regulation of
EMT was corroborated by the NF-jB inhibitor, DHMEQ. Cell treat-
ment with DHMEQ mimicked DETANONOate in the induction of
E-cadherin and inhibition of both vimentin and fibronectin, thus
supporting the critical role of NF-jB in the regulation of EMT
[unpublished data].
Role of Snail
The direct role of DETANONOate-mediated inhibition of Snail
expression and activity in the inhibition of EMT was examined in
cells treated with Snail siRNA. In contrast to untreated or cells
treated with control siRNA, transfection with Snail siRNA re-
sulted in the reversal of EMT to MET (mesenchymal to epithelial
transition), accompanied by induction of the metastasis supres-
sors E-cadherin and RKIP and inhibition of both vimentin and
fibronectin [31]. In addition, overexpression of Snail in the
non-metastatic prostate carcinoma cell line, LnCaP, resulted in
the induction of EMT [31]. These studies demonstrate the direct
role of Snail inhibition by DETANONOate and the resulting inhi-
bition of EMT.
Role of YY1
The role of YY1 in metastasis has not been clearly elucidated in
the literature. However, given the positive transcriptional regula-
tion of the EMT-inducer Snail by YY1 [36], we examined whether
DETANONOate-mediated inhibition of YY1 expression and activity
has a direct role in the inhibition of EMT. Our preliminary findings
show that YY1 silencing by siRNA mimics DETANONOate in terms
of inhibiting the mesenchymal cell phenotype through downregu-
lation of the expressions of Snail, vimentin and fibronectin and
induction of the metastasis suppressors RKIP and E-cadherin. The
direct transcriptional regulation of RKIP by YY1 has been further
studied and our preliminary evidence supports a direct suppressive
effect of YY1 on the RKIP promoter activity.
Role of RKIP
The expression of the metastasis-suppressor RKIP has been
reported to be diminished up to barely detected in several
metastatic tumor tissues compared to their normal counterparts.
Accordingly the basal levels of RKIP expression in our studied
metastatic cell line models were low and could be induced after
cell treatment with DETANONOate. The direct role of RKIP induc-
tion in the regulation of EMT by DETANONOate was demon-
strated by RKIP overexpression assays. In contrast to untreated
or cells treated with an empty vector, treatment with the RKIP-
expression vector resulted in downregulation of Snail, vimentin
and fibronectin and induction of E-cadherin [31]. The above find-
ings suggest that one mechanism by which RKIP mediates its
anti-metastatic effects is through inhibition of EMT and its induc-
tion by DETANONOate has a pivotal role in the NO-mediated
supression of EMT.
6 B. Bonavida, S. Baritaki / Nitric Oxide 24 (2011) 1–7
Conclusions
The above findings demonstrate the direct role of the NF-jB/
Snail/YY1/RKIP circuitry in the regulation of EMT. Cell treatment
with DETANONOate modifies the loop and results in the inhibition
of EMT. Each of the gene products in the loop was shown to be di-
rectly involved in the regulation of EMT. The findings also demon-
strate novel therapeutic targets in the inhibition of EMT and
metastasis.
In vivo validation of in vitro findings obtained with
DETANONOate
The above findings demonstrate that treatment of resistant and
metastatic cell lines with DETANONOate resulted in tumor cell
sensitization to apoptosis by both chemotherapeutic drugs and
immunotherapy. In addition, the above data also show that treat-
ment with DETANONOate inhibited the EMT phenotype. We have
analyzed whether our in vitro findings may be validated in vivo
in pre-clinical models using mice bearing tumor xenografts. In-
deed, ex vivo tumor tissues derived from mice bearing the human
metastatic prostate cancer cell line, PC-3, and treated with DETAN-
ONOate, had decreased expression of YY1 expression, upregulation
of DR5, and reversal of the EMT phenotype [11] (unpublished data).
Similarly, mice bearing tumor xenografts and treated with the
combination of DETANONOate and CDDP showed significant tumor
regression in contrast to those treated with either DETANONOate
or CDDP alone [11] (unpublished data). These preliminary findings
demonstrate that the findings obtained in vitro are validated in vivo
in mice. In addition, clinical data in human cancer patients have
been reported on either the systemic administration of nitroglyc-
erin in patients with lung cancer [64] and in patches in patients
with prostate cancer [65] and both studies showed significant clin-
ical efficacy.
Overall conclusions and future directions
We have demonstrated that treatment of tumor cells with
DETANONOate simultaneously sensitizes tumor cells to apoptotic
stimuli induced by both chemotherapy and immunotherapy and
also inhibits the induction of the EMT phenotype. The findings of
the role of DETANONOate in the regulation of EMT were also
shown to be manifested in other tumors such as Non-Hodgkin’s
B-cell lymphoma cell lines. Analysis of the underlying mechanisms
by which DETANONOate mediates its activities was shown to be
due, in part, to modification of the dysregulated NF-jB/Snail/
YY1/RKIP circuitry. We have also demonstrated that each gene
product in this circuitry is directly involved in the regulation in
both the resistance and the EMT phenotype (Fig. 2).
The implications of our present findings are the demonstration
of the potential therapeutic application of NO donors in the treat-
ment of patients with refractory cancer and metastasis. In addition,
the dysregulated gene products in the circuitry are potential diag-
nostic biomarkers as well as targets for the generation of new ther-
apeutic agents.
Future directions include additional pre-clinical studies in ani-
mal models for validation of our in vitro findings as well as analysis
of different NO donors for their therapeutic effects. In addition, the
establishment of the prognostic significance of the circuitry using
patient-derived tumor tissues could also be further exploited.
Acknowledgments
The authors acknowledge the various investigators who partici-
pated in the described studies in this review, namely, Doctors Sara
Huerta-Yepez, Mario Vega, Fumiya Hongo, Kam Yeung, and Michael
Palladino. We also acknowledge the Jonsson Comprehensive Cancer
Center for their valuable assistance. The assistance of Daphne Liang
in the preparation of this manuscript is also acknowledged.
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