# Nuclear Factor-kappa B  as a Resistance Factor to Platinum-Based Antineoplasic Drugs

Authors: Vilma Maldonado Lagunas; Jorge Meléndez-Zajgla
Journal: Metal-Based Drugs
 (2008)

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## Abstract

Platinum drugs continue to be major chemotherapy drugs for cancer treatment. Nevertheless, acquired or intrinsic resistance to these compounds is common in human tumors. One important mechanism for this resistance is the avoidance of cells entering the apoptotic pathway. Nuclear factor-kappa B (NF-kappa B, NF-κB) is a pleiotropic transcription factor key in determining the death threshold of human cells. This factor is important in the final response of cells to platinum drugs, as exemplified by in vitro and in vivo models showing that inhibition of NF-κB sensitizes cancer cells to the effects of these drugs. New approaches focusing on the inhibition of NF-κB could help to minimize or even eliminate intrinsic or acquired resistance to platinum drugs.


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## Body

## 1. Introduction

Cis-diaminedichloroplatinum (II), first known as Peyrone' salt, was synthesized in 1844 by the Italian
doctor, Michele Peyrone [1]. Fifty years later,
Alfred Werner “the Father of Coordination Chemistry” elucidated its
structure [2]. This inorganic
compound now known as cisplatin or CDDP is a neutral complex, Pt (NH3)2Cl2,
with a central platinum atom (Pt), two chloride atoms (Cl-), and two molecules
of ammonia (see Figure 1). In 1965, American chemist Rosenberg et al., in Michigan
State University, found that electrolysis with platinum electrodes inhibits the
growth of Escherichia coli bacteria. This
research group determined that platinum oxidized by electrolysis to Pt+2 reacts with sodium chloride and ammonium salts in the bacterial growth media,
forming cisplatin [3]. Due to the ability
of cisplatin to inhibit cell division, Rosenberg
analyzed its possible anticancer properties and found that, indeed, this
compound  inhibited  the growth of sarcomas transplanted into rats.
Nowadays, cisplatin has become one of the major chemotherapy drugs [4].Figure 1
Structure of cisplatin:cis-diamminedichloroplatinum (II).


## 2. Cisplatin Mechanism of Action

Cisplatin enters the cell mainly by passive diffusion, although its efflux and uptake have been
linked to copper metabolic pathways, implicating the high-affinity cooper
transporter (CTR1) and the copper-transporting P-type adenosine triphosphate
(ATP-7B) [5, 6]. Once inside the cell, cisplatin forms
adducts with DNA with a preference for nucleosomal regions. In this process,
cisplatin losses one of its chloride ions and binds a molecule of water in
order to attach to the nitrogen-7 position of a DNA purine. Subsequently, the
other chloride is replaced by another molecule of water, thereby binding to DNA
in a covalent form to produce 1, 2 or 1, 3 intrastrand or interstrand cross-links.
Cisplatin also forms simple monoadducts with DNA, or monoadducts that bind also
to proteins or glutathione molecules (see Figure 2) [7, 8]. The importance of this molecular
mechanism is highlighted by the reports showing that the level of platinum-DNA
adducts correlates with clinical response of cisplatin [9, 10].Cisplatin bound to DNA (a) intrastrand crosslink (b),  interstrand crosslink (c), and monoadducts (d).
(a)(b)(c)(d)DNA damage produced by cisplatin is detected and repaired by the nucleotide
excision pathway (NER) [11, 12]. This pathway involves two subpathways;
transcription-coupled NER and global genomic NER. Furuta et al. [12] reported
that transcription-coupled NER-deficient cells are hypersensitive to cisplatin,
irrespective of their global genomic NER status, showing that the former
pathway could be responsible for resistance to the platinum drug. If the damage
produced by cisplatin is not totally repaired, cells emit signals to initiate
cellular death through apoptosis or necrosis, depending on the particular
cisplatin concentration and specific tissue involved [13].Several signal transduction pathways are activated in the cell after exposure of
cisplatin, including the 3 main subfamilies of MAPK kinases, namely, extracellular
signal-regulated kinase (ERK) [14], c-Jun NH2-terminal
kinase (JNK) [15, 16], and p38 mitogen-activated protein
kinase (p38 MAPK) [17, 18]. Cisplatin also activates v-akt murine
thymoma viral oncogene homologue (AKT) [19, 20] and nuclear factor-kappa B (NF-κB)
pathways [21, 22].


## 3. NF-Kappa B Transduction Pathway

NF-kappa B is a family of transcription factors constituted by 15 dimers that result
from different combinations of 5 proteins (Rel (cRel), Rel A (p65), Rel B,
NFkB-1 (p105/p50), and NFkB-2 (p100/p52)). Each of these subunits contains a 300-amino
acid Rel homology (RH) domain, which has the ability to bind to a defined DNA
sequence (see Figure3) [23]. These dimers
regulate the expression of hundreds of genes involved in immune and
inflammatory response, proliferation, differentiation, and cell survival. However,
examples are also known where NF-κB functions as a proapoptotic
factor. The control over cell survival is achieved mainly through upregulation
of the antiapoptotic proteins, cIAP1, cIAP2, XIAP, Blf/A1, BCL-xL, and FLIP,
whereas the proapoptotic activity is mediated by FAS, FASL, DR4, and DR5 genes [24]. Although not
universal, it seems that the antiapoptotic functions of NF-κB are
mediated by dimers containing the relA subunit of this transcriptional factor.NF-κB family proteins.
(a)(b)One critical step in the control of NF-κB activity is the
association of these dimers with members of the inhibitor of kappa B family (Ikappa-B
alpha, Ikappa-B beta, Ikappa-B epsilon, p105/gamma, p100/delta, and BCL3). The
union of a particular dimer with one Ikappa-B molecule prevents its nuclear
translocation. NF-κB subunits can be released from
its inhibitor by specific posttranslational processes, such as phosphorylation or
ubiquitination followed by proteosome-mediated proteolysis (see Figure 4).Figure 4
Molecular mechanisms of NF-κB activation.Structure of two analogs of cisplatin (a) carboplatin (b) oxaliplatin.
(a)(b)The most studied upstream activator of NF-κB is the inhibitor of kappa B kinase (IKK) complex. This complex contains
two kinase catalytic subunits, IKK alpha and IKK beta, as well as a helical
subunit termed IKK gamma (NEMO) which plays a critical role in the
assembly of the IKK complex. Both catalytic kinase subunits are
highly homologous, but are activated by different stimuli. Once activated with
proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α)
or interleukin-1β (IL-1β), IKK β inactivates Ikappa-B-α,
Ikappa-B-β, and Ikappa-B-ε, inducing
the so-called canonic NF-κB pathway, described
previously. IKK-α is activated by more diverse stimuli, such as CD40, lymphotoxin β, or
lipopolysaccharide, which induce processing of the p52 precursor protein, p100,
forming homo- or heterodimers with p50 to constitute the noncanonical NF-κB pathway
[25].


## 4. NF-Kappa B and Cisplatin Resistance

Platinum drug resistance can be mediated by several mechanisms, such as drug inactivation,
cellular drug efflux, alterations in drug target, modulation of DNA repair, and
evasion from apoptotic cell death [13, 26].Due to the importance of NF-κB in determining the final outcome of
an apoptotic insult and the fact that most cancer cells present a constitutive
activation of this transcription factor, it is not unexpected that it could be
involved in resistance to platinum drugs. Earlier reports showed that cisplatin
is able to induce activation of NF-κB [22, 27], thereby providing a mechanism of intrinsic
resistance. Furthermore, low-dose gamma irradiation induces a crossresistant
phenotype in HeLa cells, which is associated with NF-κB activation by a deregulation of silencer
of death domain (SODD) protein expression [28]. NF-κB activation after cisplatin exposure
seems to be a widespread phenomenon in cancer [29] and normal cells  [30]. However, cisplatin exposure results in
downregulation of NF-κB activity in hepatoma cells [31] although the reason
for this remains unclear. Tissue-specific differences could play a role since
mice lacking p65 subunit die at 15 days of gestation by massive liver cell
apoptosis, showing a particular and specific requirement for the NF-κB antiapoptotic
function, specifically in liver homeostasis [32]. Alternatively, the
well-known negative feedback mediated by Ikappa B synthesis, which
downregulates NF-κB activity after an initial
stimulus, could explain this contradiction [33]. Further complicating
this situation, different combinations of NF-κ subunits are known to have
opposing transcriptional activities, which could help explain the contradictory
results. In addition, cancer cells with cisplatin-resistant phenotypes have
elevated NF-κB activity [21, 34] although the
molecular reason behind this activation remains obscure.Supporting the relevance of NF-κB importance in the control of apoptosis induced by cisplatin is that its inhibition by different methods
sensitizes cancer cells to the drug. Genistein, a soy isoflavonoid with NF-κB-inhibiting
properties, potentates cisplatin effects on pancreatic cancer cells [35]. Similarly, inhibition
of NF-κB translocation or activation increased the efficacy of cisplatin on an in vivo model of ovarian cancer [21] and on cultured head
and neck [36], prostate [37], and esophageal [38] cancer cell lines.
In this regard, it is noteworthy that patients with esophageal tumors resistant
to chemotherapy fail to downregulate NF-κB after therapy [39].In addition, it has been shown that NF-κB may be important in
acquired chemoresistance since even a transient exposure to small doses of an
antineoplasic agent or radiation induces cross-resistance to cisplatin by the
activation of this transcription factor [28, 40, 41].Also of note is that a recent phase I trial showed that inhibition of NF-κB with
bortezomib, a proteasome inhibitor, made ovarian cancer patients more sensitive
to carboplatin [42]. Similarly, preclinical
studies demonstrated that the newly synthesized NF-κB
inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), enhanced the sensitivity of
YCU-H and KB cells to cisplatin [36]. Furthermore, the
importance of NF-κB in resistance can be found
in the blocking of its activation by an adenovirus carrying a “superrepressor”
form of I-κB (ad-IkappaBalpha) in cisplatin-resistant lung cancer cells, which restored
their sensitivity to control levels found in sensitive cell lines [43]. These results
warrant further exploration of the possible clinical use of NF-κB inhibitors
in patients with intrinsic or acquired platinum drug-resistant cancers.


## 5. Mechanisms of NF-Kappa B Activation by Cisplatin

After DNA damage, several transduction cascades are activated, among them JNK and p38
[16]. Activation of JNK
takes place via the MEKK1/SEK1 cascade required for cell death after
platinum drug exposure [44]. MEKK1 activation
drives the activation of NF-κB, seen after cisplatin
treatment [45], providing a basis
for a possible mechanism of acquired resistance. On the other hand, Yeh et al.
[46] demonstrated that the MEK/ERK pathway is one of the NF-κB inhibitory
circuits activated after exposure of cervical cancer cells to cisplatin. This
mechanism relies on the alteration of the phosphorylation of p65 by protein
phosphatase-4 [46]. These cascades
activate the phosphorylation, ubiquitination, and degradation of NF-κB
inhibitor I-κB, allowing translocation of active NF-κB dimers into the nucleus [22], providing a plausible
basis for intrinsic or acquired resistance, as previously discussed.


## 6. Downstream Targets of NF-Kappa B

As mentioned above, NF-κB is a pleiotropic transcription
factor with target genes involved in several cellular processes. At least 20
proteins involved in the regulation of apoptosis present kappa-B consensus
sites in their promoters and are actively regulated by this transcription
factor [47]. Although no comprehensive study of the NF-kappa
B-responsive genes involved in cisplatin resistance has been published, recent
reports indicate that Bfl-1/A1 [48] and c-Myc [49] could be 2 of these genes, but clearly more
investigations are needed.


## 7. New Platinum Compounds

After the initial discovery of cisplatin, several analogs have been synthesized with
the purpose of improving their antineoplastic activity and reducing adverse
effects such as nephrotoxicity. One of the successful analogs is carboplatin,
which contains a platinum atom surrounded with two ammonia groups and two other
ligands in a ring structure. Cisplatin appears to be superior to carboplatin in
terms of therapeutic effectiveness for some tumors such as germ cell tumors,
bladder cancer, as well as head and neck cancer, while in others (e.g., lung  and ovarian cancer), their efficacies are
comparable [50]. Carboplatin
treatment downregulates constitutive NF-κB activity and prevents
nuclear retention of p65 in liver cancer [51] and glioma cell
lines [52].Oxaliplatin is another cisplatin analog that contains a platinum atom complexed with
1,2-diaminocyclohexane that has an oxalate ligand. Its spectrum of activity
and mechanism of action and resistance are different from cisplatin
and carboplatin [53]. Downregulation of NF-κB transactivation
by pharmacological inhibitors enhances oxaliplatin cytotoxicity in a panel of 4
colon adenocarcinoma cell lines [54].Recently, a new approach has been the synthesis of  water-soluble platinum complexes that can be
absorbed after oral administration, such as JM216 and its metabolite JM118 [55], which have
demonstrable oral antitumor activity in mice broadly equivalent to
intravenously administered cisplatin and a toxicological profile similar to
that of carboplatin. To date, there are no studies focusing on the activity of NF-κB in
relation to these compounds.A new promising approach is the encapsulation of cisplatin in sterically stabilized,
long circulating, PEGylated liposomes, such as SPI-77, which show more
stability in plasma and have a longer circulation time, greater efficacy, and
lower toxicity than free cisplatin. Similar to this compound is lipoplatin,
which is formed from cisplatin and liposomes composed of dipalmitoyl
phosphatidyl glycerol (DPPG), soy phosphatidyl choline (SPC-3), cholesterol,
and methoxy-polyethylene glycol-distearoyl phosphatidylethanolamine
(mPEG2000-DSPE) [56, 57]. There are no studies to date on the
routes that activate these new drugs.


## 8. Conclusion

In order to increase the benefit of current platinum-based drugs and to direct effort to
obtain improved agents, it is of great importance to understand the molecular
basis of acquired and intrinsic resistance. NF-κB is a key to this
understanding due to its importance in determining the final cell response to
platinum drugs. New approaches focusing in the inhibition of this factor could
help to minimize or even eliminate resistance to platinum drugs or to provide
drugs with less systemic toxicity.



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*Source: 576104-2008-04-08.xml*