Current Cancer Drug Targets, 2005, 5, 285-298 285

Matrix Metalloproteinase Inhibitors as Anticancer Therapeutics

F. Mannello*,1 , G. Tonti1 and S. Papa2

1Institute
of Histology and Laboratory Analysis; 2Institute of Morphological Sciences; Faculty of Sciences MM.
FF. NN., University of Urbino “Carlo Bo”, 61029 Urbino, Italy
Abstract: Matrix metalloproteinases (MMPs), also designated as matrixins, play a central role in many
biological processes and are involved both in physiologic cellular processes and in pathologic situations
such as tumor growth, invasion and metastasis. For more than 30 years MMPs have been considered as
promising targets for cancer therapy and a number of different synthetic and natural MMP inhibitors have been
identified as cytostatic and anti-angiogenic agents and have begun clinical testing in view of their specific
implication in malignant tissues. Although preclinical studies were so compelling to encourage several
clinical trials, the past years have seen a consistent number of disappointments and limited success. The critical
examination of previous studies shed light on new information about the cellular source, substrates and mode of
action of MMPs, focusing the attention of future research on the identification of specific MMP targets in
tumors at different stage of tumor progression, both in order to improve efficacy and to reduce the side effect
profile. In this review we discuss the current view on the feasibility of MMPs as target for therapeutic
intervention in cancer, taking into account that the perspective may be of great value for molecular medicine for
the twenty-first century, providing intriguing information about the MMPs as mediators in biology and
pathology, and as targets for disease therapies.
Keywords: Matrix metalloproteinases, gelatinases, cancer, protease inhibitors, therapeutic strategies, clinical trials.

INTRODUCTION constitute the zinc binding site and a “methionin-turn” motif

that lies beneath the active site zinc ion [3]. The ion-binding
Extracellular proteinases are involved in a wide variety of
motif reads HEBXHXHBGBXHZ, where histidine (H),
developmental and disease-associated processes proving to
glutamic acid (E) and glycine (G) are invariant, B is a bulky
be key protagonists in many physiological and pathological
hydrophobic residue, X is a variable residue and Z is a
mechanisms. The first observation made 40 years ago of an
family-specific amino acid (serine in MMPs). All MMPs
activity during metamorphosis in tadpole tails, able to
have an N-terminal hydrophobic signal sequence
degrade interstitial collagen, opened a new field of
(predomain) which leads their synthesis to the endoplasmic
unexpected biomedical research and the way to study what
reticulum and their secretion into the extracellular space; this
has now become the matrix metalloproteinase (MMP) family
amino acid stretch is thereafter removed. This predomain is
[1]. These endopeptidases belong to the wider metzincin
followed by a 77-87 amino acid-long prodomain that
group, which in turn constitutes one of several
constitutes the N-terminus of the secreted enzyme and
metalloendopeptidase families [2-4]; all the metzincins are
maintains it in its latent form until its removal or
zinc-dependent proteinases and according to their structural
disruption. The prodomain keeps the enzyme inactive
characteristics are subdivided into serralysins, adamalysins,
through a mechanism identified as “ cysteine switch” where
astacins and matrixins [3]. As our knowledge of the MMP
the unpaired cysteine in the highly conserved “Pro-Arg-Cys-
family continues to grow, we are learning that their activities
Gly-X-Pro-Asp” sequence forms a bridge with the catalytic
are not limited to matrix degradation [1]. MMPs seem to
zinc, thus preventing enzymatic activity. The enzyme
touch almost every aspect of mammalian biology and,
acquires total proteolytic capacity when the prodomain
through more than 1,000 new publications each year, certain
becomes chemically removed by cleavage [1]. The active site
exciting discoveries solidified the field, solving previous
is of great importance: it specifically binds to selective
puzzles and pointing the research in new directions (starting
substrates by means of its active site cleft, through specificity
from biochemical studies, discussing the flurry of activity
sub-site pockets that bind amino acids adjacent to the
that involved the gene regulation through molecular biology
scissile peptide bond, and through secondary substrate-
approaches, and finally focusing the interest to MMPs as
binding exosites located outside the active site [5]. These
therapeutic targets in several diseases).
domains represent the minimal structure of MMPs found in
MMP-7 (matrilysin) and MMP-26 (endometase/matrylisin-
STRUCTURAL AND FUNCTIONAL COMPLEXITY 2) which lack any other domain. All the other MMPs have a
OF MMPs hinge region varying in length and composition which also
influences substrate specificity [6] , and a four-blade β-
All MMPs share a conserved structural topology, which
propeller structure representing the hemopexin/vitronectin-
consists of a catalytic domain containing three histidines that
like domain [7]. Two metalloproteinases, Gelatinase A and
B, are further characterized by the presence of three head-to-
*Address correspondence to this author at the Istituto di Istologia ed tail cystein-rich repeats within the catalytic domain. This
Analisi di Laboratorio, Facoltà di Scienze MFN, Via E. Zeppi, Università structure resembles the collagen-binding type II repeats of
degli Studi di Urbino “Carlo Bo”, 61029 Urbino (PU), Italy; Tel: +39-
0722-351479; Fax: +39-0722-322370; E-mail: f.mannello@uniurb.it fibronectin and is necessary for the binding and cleavaging

1568-0096/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

286 Current Cancer Drug Targets, 2005, Vol. 5, No. 4 Mannello et al.

activities of these MMPs [8]. Not all MMPs are secreted
enzymes; membrane-type (MT) MMPs have been identified
to contain a single-pass transmembrane domain and a short
cytoplasmic C-terminal tail or to be anchored to the cell
membrane by a glycosyl phosphatidylinositol anchor (Fig.
(1)).
Even though MMPs were initially considered only as
destroyers of the structural integrity of the extracellular
matrix (ECM) environment, in later times this opinion was
superseded thank to numerous studies where these enzymes
were regulating numerous biological processes in a more
complete manner thus highlighting their multiple and more
complex activity [9]. MMPs are known to be able to degrade
most of the components of the ECM, often having
overlapping substrates [10]. ECM is not a static structure
but it is prone to undergo constant changes, due to the
normal turnover of its elements and to many other
physiological and pathological processes, such as
development, inflammation, wound healing, cancer and
central nervous system diseases, all initially necessitating a
proteolytic breakdown, and subsequently a new synthesis
and deposition of ECM components. MMPs play a fine
regulative role in all these processes, not only because they
modify the physical structure and conformation of the ECM,
but also because by doing so they expose biologically active
sites called “matricryptic sites” otherwise hidden and unable
to carry out their function [11]. In fact, besides cleaving
virtually all ECM components, MMPs can also cleave many
cell-surface and pericellular molecules, thus regulating

Fig. (1). Schematic representation of the domain structure of the matrix metalloproteinases.
The archetypal MMPs contain: a signal peptide (necessary for secretion); propeptide (containing a conserved sequence PRCGxPD, in
which the cysteine forms a covalent bond with the Zn2+ of the catalytic site; this cysteine switch maintains the enzyme in its
zymogenic proform); catalytic domain (with zinc binding site HExGHxxGxxHS); hinge region (a proline-rich sequence that links the
catalytic domain to hemopexin region); hemopexin carboxy-terminal domain (which mediates interactions with TIMPs, cell-surface
molecules, and proteolytic substrate specificity for individual MMPs; the first and the last of the four hemopexin repeats are linked by
a disulfide bond). The gelatine-binding MMPs contain three inserts of fibronectin type II in the catalytic domain (which improve
collagen and gelatin degradation efficiency). The furin-activated secreted MMPs contain a recognition motif (RxKR) between their
propeptide and catalytic domain for intracellular furin convertases. A vitronectin insert between propeptide and catalytic domain was
also peculiarly detected. Anchored MMPs were characterized by a carboxy-terminal single-span transmembrane domain with a short
cytoplasmic domain or by the glycosylphosphatidylinositol (GPI) anchor (which attack both these classes of MMPs to the cell
surface), or an N-terminal signal anchor (which anchors proMMP to the Golgi complex. This MMP is further characterized by its
unique cysteine array and immunoglobulin domain). [Adapted from Refs. 25 and 50].
Matrix Metalloproteinase Inhibitors as Anticancer Therapeutics
differentiated signalling pathways, permitting cell migration
and differential cell-cell and cell-matrix interactions [12].
In order to fulfil their role, MMPs need to be localized at,
or in the vicinity of, the cell surface. It has been evidenced
for instance that MT-MMPs maintain their complete
enzymatic activity when localized on invadopodia, but loose
their proteolytic capacity when found in a secreted form [13].
In addition, several MMPs may be bound to the cell
membrane often thanks to the association with adhesion
receptors or cell surface proteoglycans such as integrins,
immunoglobulin superfamily members and cell-surface
heparan sulphate proteoglycans [12]. This ability of
selectively sequestering particular MMPs in specific contact
sites between the cell and the ECM, permits to direct the
MMP proteolytic activity only where required, maintaining
a strict control on the ECM degradation and subsequent
release/activation of other functional components. The
function of the ECM is not merely that of a scaffold that
maintains the integrity of the extracellular environment, but
it embraces many aspects of cell regulation and behaviour,
resulting from its capacity of sequestering on its surface
signalling molecules such as growth factors and growth
factor-binding proteins, and from its interaction with the cell
interior via integrin mediated mechanisms [11]. Being the
ECM a reservoir for many biologically important regulative
signals, MMPs can control the availability of several bio-
active compounds released after proteolytic degradation of
their binding proteins (e.g., fibroblast- and insulin-growth
factor from perlecan and insulin-growth factor binding
protein, respectively) [12]; moreover MMPs are involved in
the activation and release from the cell surface of growth
factor precursors such as members of the epidermal growth
factor family [11]. Additionally, most MMPs interact with
the cytokine network as well, by cleaving a variety of
cytokines, cytokine receptors and chemokines, such as tumor
necrosis factor-α, Fas-ligand (a type II membrane protein
belonging to the TNF family of cytokines), interleukin-2
receptor α and interleuchin-8 [14], sometimes increasing
their functional activity, and sometimes down regulating it
[12].
Worthy of notice is another important unexpected
regulatory function controlled by MMPs; in fact, they can
induce and/or inhibit apoptosis in anchorage-dependent cells
by modifying and disrupting matrix-associated signalling
components necessary for cell survival [15]. MMP substrates
also include other proteinases, enabling them to act on serine
proteinases such as urokinase-type plasminogen activator and
plasminogen, often inactivating the enzyme but
concomitantly generating new active molecules like
angiostatin from plasminogen [16]. In spite of the amazing
differentiated roles MMPs can play, it is not surprising that
they are key components of many fundamental
physio/pathological pathways; due to their characteristics
these proteinases become involved in processes that also
necessitate cell migration and tissue remodelling [9, 17, 18].
MMPs are key elements during nervous system development
as they might be involved in the migration of precursor cells
to their final destination and also in axonal growth; this is
possible because MMPs are localized on the neuronal growth
cone and on tips of invadopodia regulating axonal guidance
and elongation. MMPs contribute to the process of
myelinogenesis; in this case MMPs could be involved in the
Current Cancer Drug Targets, 2005, Vol. 5, No. 4 287
remodelling of the brain matrix allowing the expansion of
oligodendroglial processes [18].
MMPs are assumed to be involved in various
morphogenetic processes [17], revealing in some instances
the complex and apparently dichotomic (or contradictory)
way of action of the MMPs. In fact, MMP-dependent ECM
degradation is necessary for the branching of the kidney
buds, as it enables their development by reducing
extracellular resistance [12]; on the other hand, the salivary
gland formation depends on the presence of a surrounding
structure which supports the generation and expansion of
clefts in the epithelium, whilst MMP-mediated degradation
of the mesenchyme denies this stabilizing necessity [12, 17].
Of great importance is the role MMPs play during vascular
neoformation in physio-pathological processes (such as
tissue development and repair). For angiogenesis to take
place, many structural and functional changes need to occur;
in fact, endothelial cells of outgrowing new blood vessels
need to migrate through the surrounding tissue requiring
ECM degradation and remodelling; moreover, to start off the
series of events that leads to new blood vessel generation,
several angiogenic factors must be released or activated,
while anti-angiogenic factors must be rendered inactive:
MMPs actively contribute to all these events [19]. MMPs
may contribute to angiogenesis thanks to their capacity of
binding to integrin molecules; for example, the integrin
αvβ3, capable of recognizing specific sequences present in
ECM components, is highly expressed on angiogenic
endothelial cells thus allowing MMP dependent vascular
invasion [20]. Studies on angiogenic islets revealed that
certain MMPs (MMP-2 and MMP-9) are upregulated and
that the inhibition of MMP9 suppresses angiogenesis [20,
21]. Surprisingly, MMPs can inhibit angiogenic
mechanisms through the generation of breakdown products
such as angiostatin, which derives from plasminogen, and
endostatin and tumstatin, which are fragments of collagen
XVIII and IV, respectively [16]. As revealed, MMPs regulate
and are involved in many biological pathways [18], resulting
to be fundamental elements of normal physiological
processes; as a consequence of their peculiar control of these
events they can turn into disease-associated molecules in
spite of uncontrolled activity [9].
NEW CONCEPTS OF MMP ACTION PROMOTING
TUMOR PROGRESSION
A growing body of evidence indicates that MMPs are
directly involved in almost every type of human cancer, often
evidencing a negative correlation between MMPs and
prognosis [22]. Recent studies were performed in order to
analyse the potential utility of MMP-9 as marker both in the
follow-up and in the prognosis of breast cancer patients,
evidencing its significative correlation with the clinical
status of each patient [23]. Moreover, as MMP-2 and -9
expression has been correlated with metastatic risk in several
human carcinoma, including breast cancer, their prognostic
value has been analysed, leading to the conclusion that these
MMPs and their co-expression is an unfavourable prognostic
factor for relapse-free survival breast cancer patients [24], thus
being as useful tool in identifying patients for additional
therapy. Many studies identify MMPs as key elements in
various steps of tumor development and progression as they
288 Current Cancer Drug Targets, 2005, Vol. 5, No. 4
are generally found in greater amounts and more often in and
around malignant cancers than in normal, benign or
premalignant tissues. Experimental data based on animal
studies show that MMPs contribute to cancer progression, in
particular when an altered balance between matrix
metalloproteinases and tissue inhibitors of
metalloproteinases (MMPs/TIMPs ratio) is present [25].
Surprisingly, MMPs have been found to be expressed not
only directly by malignant cells, but often by the supporting
stromal cells; it is possible that cancer cells might stimulate
tumor associated stromal cells to synthesize and release
MMPs in a paracrine manner through the secretion of
interleukins, interferons and growth factors [9].
For tumor progression to take place, several alterations in
cell physiology occur, such as self-support in growth
signals, escape from apoptosis, infinite replication,
angiogenesis, tissue invasion and metastasis [22]. MMPs
seem to take part in several of these processes, promoting
cell growth and survival, releasing some membrane-bound
precursors of growth factors (such as tumor growth factor-α
and insulin-growth factors), and indirectly promoting cell
survival by acting on integrins and other ECM components
[9]. Cell survival is not only obtained by supplying growth
factors but also by avoiding cells to undergo the
phenomenon of apoptosis which normally occurs in cases of
attack from the immune system and detachment from the
ECM; strangely enough MMPs occupy apoptotic and anti-
apoptotic roles [15]. For a tumor to survive and grow, cancer
cells need to be continuously supported by new blood vessel
formation, and several studies indicate that various MMPs
are key agonists in angiogenesis [19]. In fact, experimental
models, where the levels of these proteinases have been
down-regulated, show a consistent decrease in neovascular
formation. In addition to degrading collagen type IV and
other ECM components (thus eliminating physical barriers
to blood cell migration) MMP-9 increases the bioavailability
of pro-angiogenic factors, MMP-2 exposes cryptic binding
sites that permit endothelial cell migration, while MMP-14
facilitates the penetration of endothelial cells into the tumor
tissue leading to new blood vessel sprouting and capillary
tube formation [19, 25, 26].
Malignant cancers differ from in situ cancers and benign
neoplasms because they possess the capacity of overcoming
physical barriers and spreading to other organs of the body.
Tumor metastasis is a complex process where cell motility
is supported in parallel with proteolysis of the ECM. During
invasion, malignant cells detach from the primary tumor,
cross the epithelial basement membrane invading the
surrounding stroma, enter blood vessels or lymphatics,
extravasate and finally establish new proliferating colonies at
a distant site: MMPs are shown to take part in most of these
sequential events [4]. In order to detach from the primary
tumor and migrate, cells must loose their adhesive
characteristics and increase their cell motility, sustained by
the specific cleavage of ECM components [12, 26]. Several
studies evidenced that malignant cell migration does not
depend on a continuous and intense extracellular matrix
degrading activity, but rather on cycles of localized and
regulated MMP action [11]. In order to reach distant sites
and colonize new organs travelling through the blood flow,
malignant cells must enter, survive and exit blood vessels or
lymphatics: while the intravasation process seems to be rate-
Mannello et al.
limiting and dependent on the action of MMP-9, the
extravasation event does not necessitate proteolytic activity
but rather is a consequence of the disruption of blood vessels
owing to the proliferation of immobilized tumor cells
growing on vascular endothelium [12]. Neoplastic cells are
also recognized and attacked by the immune system, but
cancer cells have developed many strategies in order to
escape immune surveillance, and MMPs seem to be
involved in some of these mechanisms, through the
activation of neutrophils, macrophages, cytotoxic T cells and
natural killer cells [14].
Interesting evidences attribute to various MMPs the
capacity of regulating the immune system response against
cancer cells by cleaving several chemokines, influencing
(both in a positive or negative manner) their infiltration and
migration [27]. In fact, while the monocyte chemoattractant
protein-3 is cleaved and made inactive by MMP-2 and its
breakdown product in turn acts as an antagonist [28], MMP-
9 cleaves the neutrophil chemoattractant interleukin-8
increasing its activity but it also inhibits the mobilisation
and survival of leukocytes acting as an agonist in reducing
the immune response towards malignant cells [29]. It is
interesting to note that even though the immune response
fights against cancer cells trying to defeat the tumor, chronic
inflammation is in some cases a characteristic of certain
neoplastic pathologies; in contradiction to their normal role,
neutrophils and macrophages synthesize several MMPs and
can so favour cancer cell growth and invasion by releasing
them [22]. A misleading behaviour has recently been
attributed to tumor-infiltrating T-lymphocytes which seem
to contribute to the malignant phenotype through their up-
regulated MMP-9 secretion, pointing out that inflammatory
cells may contribute to tumor progression [30]. For
metastatic cancer cells to start off the formation of new
colonies far away from their origin, it is necessary to
establish physical contacts with the host tissue stroma.
MMPs might be released either from invading malignant
cells or by stromal cells, including fibroblasts, endothelial
cells and also leucocytes. The expression of MMPs from
these cells is induced by physical contact or in a paracrine
manner by tumor cells which can directly regulate tissue
remodelling [25] and indirectly enhance tumor survival and
angiogenesis by releasing ECM-linked growth factors [12].
As growing evidence underlines, it is likely that MMPs are
strongly involved in the development of malignant lesions;
so, if MMPs promote cancer growth, then their TIMPs
should block it, but things are not always so simple [1].
Several clinical studies demonstrated a negative correlation
between high TIMP levels and prognosis; this might be due
to the fact that increasing MMP levels during tumor
development are balanced with higher expression of TIMPs,
reflecting the fact that cancer progression is characterized by
augmented TIMP presence but is not caused by it [25]. Just
to complicate the situation, some experimental studies
revealed cancer-promoting properties of TIMPs [28]. In fact,
TIMP-1 and -3 may stimulate angiogenesis and have a
positive correlation with tumor growth, proliferation and
vascularization, while TIMP-2 and -4 can inhibit tumor cell
apoptosis [15, 31]. It is not known if these results depend on
proteinase inhibition, as TIMPs can act independently from
MMPs by up-regulating anti-apoptotic factors and displaying
growth-factor-like activities [16, 32].
Matrix Metalloproteinase Inhibitors as Anticancer Therapeutics
As can be seen, most studies show a negative association
between MMP expression and prognosis. However, there are
a few cases where the augmented levels of specific MMPs
positively correlate with clinical results, leading to a more
favourable prognosis. In fact, human macrophage
metalloelastase, also referred as MMP-12, plays an important
role in the inhibition of tumor progression and invasion in
patients with colo-rectal carcinoma being associated with
increased survival, probably due to its capacity of inhibiting
angiogenesis through the conversion of plasminogen to
angiostatin [33, 34]. Reduction in metastases in colorectal
carcinoma is achieved through the release of MMP-9 by
infiltrating macrophages [35]. Moreover, Gelatinase A plays
a key role during invasion and metastasis of malignant cells
and is associated with invasive phenotype and poor
prognosis in several solid tumors. However, MMP-2 seems
to hold a different roles in haematological malignancies. In
fact, in acute myeloid leukaemia the positivity of bone
marrow blast cells for MMP-2 reflects a better prognosis
[36]. Recent studies on the activity of MMP-2 in Hodgkin’s
lymphoma evidence that MMP-2 expression significantly
correlates with a favourable outcome; this positive
association between MMP-2 presence and clinical results is
unexpected and surprising in view of the role MMPs play in
solid tumors, but it may depend on the fundamental
differences between haematological malignancies and the
other (solid) tumor types [37]. Gelatinase B has a clinical
relevance in early stages of breast cancer, as an augmented
expression of this MMP by endothelial cells leads to an
increased survival thus representing a potential positive
indicator [38].
ENDOGENOUS MECHANISMS OF ACTIVATION
AND INHIBITION
Given that MMPs regulate many normal and
pathological processes, their expression and activation must
be meticulously controlled in order to obtain the correct
enzymatic activity in the right amount, at the right cell-type
and time. The first control takes place at the level of
transcription [39], as MMPs are not constitutively
expressed, (an exception in this regard comes from MMP-2
which is often constitutively expressed) but are transcribed
following cell activation induced by cytokines (including
interleukins and interferons), growth factors (such as
epidermal-, keratinocyte, nerve-, vascular endothelial-, α-
and β-tumor growth factors) and by the extracellular matrix
metalloproteinase inducer “EMMPRIN” [31]. MMP gene
expression can also be up- or down-regulated by phorbol
esters [4], integrin-derived signals [20], cell-cell or cell-
matrix interactions [9], cell stress and changes in cell shape
[40]. Most of the signals that act on MMP expression
produce their effects through the activation of proto-
oncogenes products (e.g. c-fos and c-jun), which
heterodimerize and bind the activator protein-1 sites present
in the promoter region of many MMPs [41]. According to
the cell type and to its microenvironment, MMP expression
can be stimulated or inhibited through phosphorylated
Mitogen Activated Protein Kinase pathways (e.g., pERK 1-2
and MAPK). Other nuclear factors with regulative roles are
represented by the Erythroblastosis Twenty Six (ETS)
family of oncoproteins (that bind specific sites internally to
MMP gene promoter sequences), by nuclear factor-κB and
Current Cancer Drug Targets, 2005, Vol. 5, No. 4 289
by Signal Transducers and Activators of Transcription (e.g.
STAT-1 and -3) [39]. One more factor that can account for
the variability in MMP regulation, is the presence of genetic
variations that can influence the development and
progression of various diseases. Genetic alterations such as
single-nucleotid polymorphism localize within MMP
promoter sequences and influence the rate of transcription
through the generation or abrogation of transcription-factor
binding sites [25].
MMPs are secreted under the inactive form of proenzymes
or zymogens. The zinc ion, indispensable for the catalytic
activity, is bound to three histidines in the catalytic domain,
and the fourth ligand is the unpaired cysteine sulphydryl
group present in the predomain of the enzyme. To reach total
activation of MMPs, this cysteine link must be removed and
the thiol group replaced by a water molecule capable of
attacking the peptide bonds of MMP substrates (i.e. the
cystein switch) [4]. The extracellular activation of several
MMPs results from the action of various serine proteinases
such as the plasminogen activator urokinase and plasmin
(the active breakdown product of plasminogen), which
proteolytically cleave the MMP prodomain generating the
totally active form of the enzyme [1]. In addition, many
MMPs can be activated by other already mature MMPs [9].
MMP activity is also regulated by several types of
endogenous inhibitors. The main inhibitor in tissue fluids is
α2-macroglobulin, an abundant plasma protein which binds
to MMPs and thereafter causes the removal of these
complexes through a scavenger receptor-mediated
endocytosis [4, 5]. Another mechanism operating in a
similar way is mediated by thrombospondins which bind to
MMP-2 and -9, causing their clearance by endocytosis and
blocking their proteolytic activity [42]. MMP inhibitors
present in tissues and cells are known as TIMPs [32] and
surprisingly exert not only MMP inhibitory effects, since
they are also involved in cell growth events due to their
growth-factor-like properties and mitogenic activity probably
uncorrelated to their MMP inhibitory functions [15, 31]. A
recently discovered class of inhibitors has a structural
similarity with TIMPs and is in most cases represented by
protein breakdown subdomains. For example, the proteolytic
degradation of the procollagen C-terminal proteinase
enhancer protein releases a C-terminal fragment that has
MMP inhibitory effects [43]; a potent cell surface MMP
inhibitor is the cell receptor known as reversion-inducing
cysteine-rich protein with Kazal motifs which also regulates
ECM integrity and angiogenesis [44]. Although other
proteins have been reported as novel MMP inhibitors (such
as non-collageneous domain of type IV collagen as integrin
ligand, and laminin-binding domain of agrin), the
physiological targets remain unclear as well as their
applicability in therapeutic strategies.
To avoid the detrimental effects of uncontrolled
extracellular matrix degradation, MMP activity is then
highly regulated at various levels: although under steady-
state conditions in healthy non-remodelling tissues it is
nearly undetectable (due both to low levels of constitutive
expression and to efficient homeostatic inhibitory
mechanisms), a marked expansion in technology and
knowledge showed that the expression of MMPs in cancers
by normal and tumor-associated cells was often the rule,
rather than the exception. It was then realized that inhibition
290 Current Cancer Drug Targets, 2005, Vol. 5, No. 4
of MMP activity might not necessarily prevent the escape of
tumor cells, but it could be useful in keeping small
metastatic lesions in a "dormant state", highlighting from
the 1990s the interest in drug-discovery programmes
focusing the attention to MMPs as a therapeutic target for
cancer.
THERAPEUTIC INHIBITION OF MMPs
Most of the evidence that MMPs are implicated in cancer
development and progression stems from experimental
models (in particular with knockout mice) in which
expression of selected MMPs in tumor cells has been
observed to promote tumor growth, invasion and metastasis
[25]. In virtually all human cancers, MMP expression and
activity in patient tissues, serum or urine has been found to
be increased and to correlate with invasiveness and poor
prognosis, supporting the experimental data and the notion
that MMPs play a crucial role in human cancer progression,
even though there are instances where MMP expression
appears to be associated with a more favourable prognosis
[22]. The expression of MMPs in tumors can be determined
by immunohistochemical staining, Western blotting,
Northern blotting or by reverse transcriptase-polymerase
chain reaction analysis [45, 46], while the enzymatic activity
can be determined with collagen, gelatin or casein
zymography [47-49]. In numerous works with different types
of cancer, the expression and activity of particular MMPs and
their correlation with clinico-pathologic characteristics of the
patients have been extensively studied [22, 25], and two
conceptually straightforward relationships between MMP and
cancer have been found: the association between MMP
expression and tumor grade or aggressiveness, and the
correlation of MMP activity with recurrence or metastatic
risk [15, 25, 31].
Until recently, MMP expression and activity have been
associated primarily with tumor invasion and metastasis (a
multistep process in which cell motility is coupled to
proteolysis and which involves interactions of cells with the
ECM) [22]; it is now becoming clear, partly owing to
transgenic mouse models, that MMPs may play a critical
role in early events in tumor development, directly
participating in the generation of signals that induce tumor
cell proliferation and inhibit apoptosis [15, 31, 40]. In this
respect, the perceived role of MMPs both in early cancer
development and in invasive/ metastatic progression places
them in the forefront for cancer therapy targets [12, 50].
Accordingly, numerous MMP-inhibiting strategies have
been tried both in experimental models of cancer and in
clinical trials. Several general strategies for blocking MMP
gene transcription were evaluated [41] based on: 1) targeting
extracellular factors involved in MMP synthesis or in
activating suppressors of MMP expression or blocking
signalling by cytokines or growth factors that up-regulate
MMPs (these studies emphasized the complexity of selecting
MMP targets for cancer therapy reinforcing the need to define
specific proteases involved in each peculiar tumor at each
stage); 2) targeting the signal transduction pathways that
mediate MMP induction or blocking general oncogenic
signalling pathways can hamper cancer progression by means
of MMP production and action (these approaches highlighted
the crucial step that every therapeutic MMP inhibitor must
Mannello et al.
have little or no deleterious effects on signal transduction,
important function for normal physiological processes); 3)
targeting the nuclear factors that up-regulate MMP genes
(this approach might represent a powerful way to generate
specificity in anti-MMP therapies, emphasizing the need for
more peculiar compounds to block MMP synthesis); 4)
targeting MMP transcripts using ribozymes or antisense
constructs (these approaches, that directly inhibited MMP
synthesis down-regulating in mouse models MMP
production by cancer cells resulting in reduced tumor burden
or metastasis, open the question of their translation into
clinical practice). Although there are many opportunities to
block the synthesis of MMPs, none of them has been
translated into clinical applications, due to several unsolved
problems (not all MMP genes respond in the same way to
the same stimulus; relative nonspecific nature of the
signalling that induces MMP expression; difficulties of
targeting transcription-factor activity), emphasizing the
limited knowledge of the driving elements that regulate
MMPs and suggesting cell-transfection studies to provide
relevant information about transcriptional control of MMP
genes.
Several other strategies for blocking MMP activity were
evaluated [25] based on: 1) targeting proMMP-activation
(this approach does not interfere with enzyme expression and
may be mediated by several natural products, some anti-
inflammatory cytokines and inhibitors of proteases and
convertases that regulate/inhibit the proMMP activation
process, highlighting their potential for combinatorial
treatment of cancer); 2) targeting interactions between MMPs
and crucial proteins (these approaches have been achieved
both with compounds that inhibit MMP binding to
integrins and with recombinant proteins containing toxins
fused to an MMP cleavage site). Although these strategies
have not been tested in clinical practice, it could be a means
of specifically targeting cancer-promoting functions of MMPs
without impeding their potential tumor-defying functions
and peculiarly leading to cancer cell death, emphasizing a
sufficient promising selectivity for cancer cells [50].
A crucial approach in MMP-inhibiting strategies,
especially in clinical trials, is represented by the inhibitors of
MMP active-site that have been expected to represent, over
the past 25 years, a new approach to cancer treatment in
conjunction to traditional cytotoxic drugs. Although the first
therapeutic applications of MMP active-site inhibitors
(MMPIs) involved the specific endogenous inhibitors
TIMPs [22, 32], their use in cancer therapy was extremely
limited, since protein-based treatments are difficult to
administer due to their poor pharmacokinetics as well as to
the paradoxical effects of these multifunctional proteins (i.e.
promoting tumor-cell growth and proliferation in addition to
inhibiting MMP activity) [31]. These pieces of evidence
highlighted the need for natural and/or synthetic MMPIs that
selectively target/inhibit specific MMPs.
Several synthetic MMPIs have been developed to that
end and shown to be effective in blocking tumor growth in
experimental animals, validating the concept of MMPs as
therapeutic targets for cancer [32]. These observations led to
high expectations of synthetic MMP blocking compounds in
human clinical trials; even though, unfortunately, synthetic
MMPIs have so far failed to live up to their expectations
Matrix Metalloproteinase Inhibitors as Anticancer Therapeutics
(e.g. some cancer patients had poorer survival as results of
their administration) [51], it is no longer correct to state that
MMPIs are not useful for cancer treatment. The principal
approach taken for the identification of small molecules
(synthetic and natural products) as MMPIs was the substrate-
based design of peptide derivatives from information related
to the sequence of the cleavage site. The screening of natural
products had generally not resulted in the discovery of potent
compounds, and the most active natural products are
structurally similar to certain of the peptide derivatives
obtained by the substrate-based design. The successive
discovery of non-peptide-based inhibitors had, in part, been
aided by the advent of detailed structural information of
MMPI complexes, which is enabling rational inhibitor
design. Several researchers and pharmaceutical industries
have used information derived from the three-dimensional
structures of MMPs or large scale screens both to identify (or
design de novo) compounds with MMP-inhibitory
properties and to optimize the process of structure-based
design of analogues of compounds obtained by the substrate-
based approach [52]. The requirement for a molecule to be an
effective inhibitor of the MMP class of enzymes is a
functional group (e.g., carboxylic acid, hydroxamic acid,
sulphydryls, phosphoric acid derivatives, aminocarboxylates,
tetracycline derivatives, etc.) capable of chelating the active-
site zinc 2+ ion (known as zinc binding group), at least one
functional group which provides a hydrogen bond interaction
with the enzyme backbone, and one or more side chains
which undergo effective van der Waals interactions with the
enzyme subsites. It has been revealed that this requirement
can be satisfied by a variety of different structural classes of
Table 1. Synthetic and Natural Compound Acting as MMP Inhibitors*
MMPIs Targets
Synthetic collagen peptidomimetic compounds
Batimastat (BB-94) Broad spectrum of MMPs, ADAM-17
Solimastat (BB-3644) Broad spectrum of MMPs, TACE
Marimastat (BB-2516) MMP-1, -2, -3, -7, -9, -12
Synthetic collagen nonpeptidic compounds
Prinomastat (AG3340) MMP-1, -2, -3, -7, -9, -14
Tanomastat (BAY 12-9566) MMP-2, -3, -9, -13
D2163 (BMS-275291) MMP-1, -2, -9
Synthetic tetracycline derivative
Metastat (Col-3/CMT-3) MMP-1, -2, -8, -9, -13
Synthetic biphosphonate
Alendronate MMP-2
Natural compounds
Neovastat (Æ-941) MMP-2, -9, -12, -13
Cathechins (green tea extract) MMP-2, -9
Acetylsalicylic acid MMP-2
* (reviewed from Refs . [22, 25, 41, 50, 51-53, 57, 59, 64, 65, 75-78, 83, 84])
Current Cancer Drug Targets, 2005, Vol. 5, No. 4 291
MMPIs which had been discovered through a number of
techniques, including structure-based design and
combinatorial chemistry [52]. Although on the basis of the
structural characteristics of MMPIs four broad classes had
been identified (i.e. succinyl hydroxamates; sulfonamide
hydroxamates and related structures; non-hydroxamates;
natural products and their derivatives) [51], preclinical
studies and clinical trials were performed (and some are
actually in itinere) on five main classes of molecules with
intriguing MMP-inhibition capability (i.e. synthetic
peptides, non peptidic molecules, chemically modified
tetracyclines, biphosphonates, and natural compounds), that
may inhibit tumor growth by enhancing development of
fibrotic capsule around the tumor thereby preventing tumor
invasion, by inhibiting tumor-induced angiogenesis or by
inducing apoptosis in tumor cells [12, 25, 50]. The
principal side effect of some of these drugs/compounds is
musculoskeletal pain, especially in tendons and joints,
possibly due to the inhibition of tumor necrosis factor-α
converting enzyme and shedding of tumor necrosis factor-α
receptor II [53], even though in preclinical studies they
revealed efficient against malignant tumors (Table 1). These
side effects led to the discontinuation of clinical trials for
several MMPIs and to the search for new MMPIs with less
severe side effects.
The synthetic collagen peptidomimetic MMP inhibitors
are pseudopeptides that mimic the structure (cleavage site) of
MMP substrates and function as reversible competitive
inhibitors; hydroxamate inhibitors are small (Mr <600)
peptide analogs of fibrillar collagens, which inhibit MMP
activity by specifically interacting with the Zn2+ in their
Side effects
Local tissue reaction (peritonitis)
Musculoskeletal stiffness
Mild to severe joint and muscle pain
Arthralgia, joint-related complaints
Mild joint pain, thrombocytopenia, liver enzymes elevations, nausea
Not causes joint pain
Limited systemic toxicity; mild phototoxicity; reversible sideroblastic anemia
(not described up-to-date in definite clinical trials)
Musculoskeletal pain and joint stiffness
Mild to moderate toxicity, neurologic and gastrointestinal effects
(not described up-to-date in definite clinical trials)
292 Current Cancer Drug Targets, 2005, Vol. 5, No. 4 Mannello et al.

A B C
OH
HN O
O O
H O
S N H H N
N O H N N
HOHNOC N H
H N
S O H H
HO
OH O N
H O H O

Fig. (2). Synthetic collagen peptidomimetic MMP inhibitors.

Chemical structure of Batimastat (A), Marimastat (B), and Solimastat (C).

catalytic site, and include mainly Batimastat and Marimastat
(British Biotech) (Fig. (2). Batimastat (BB-94) is a low-
molecular-weight broad-spectrum hydroxamate-based
inhibitor that inhibits MMPs and members of the
adamalysin family of metalloproteinases; it was the first
MMPI to enter clinical trials, resulting well tolerated but
with limited utility by poor water solubility, which requires
intraperitoneal administration, and by paradoxical effect of
promoting liver metastasis in mouse model [54]. Phases I
and II clinical trials have not shown any marked responses
and actually there is no further development of this
compound for cancer therapy [52]. A broad spectrum MMP
inhibitor structurally related to Batimastat is Solimastat
(BB-3644), which thanks to its capacity of actively and
efficiently inhibiting the processing of cell-bound TNF-α
potentially limits its toxicity, was evaluated for clinical
studies; however, this inhibitor failed to provide an
advantage over other MMPIs and presented side effects such
as muscolo-skeletal pain revealing its clinical inefficacy [55].
Marimastat (BB-2516) is an orally bioavailable low-
molecular-weight molecule with a broad-spectrum MMP-
inhibiting activity, showing a dose-limiting side effect from
mild to severe joint and muscle pain [50]. This compound
has been studied in different Phase III clinical trials, showing
no statistically significant advantage over conventional first-
line treatment and without improving overall survival
benefit, even though an increased survival was reported in
patients with inoperable colorectal hepatic metastases
showing side-effect symptoms [56]; based on the outcome of
Phase III trials, further clinical trials with Marimastat have
been performed only in cancer of the gastrointestinal tract,
revealing a very modest survival benefit [57], and
demonstrating the most positive results in patients with
advanced gastric cancer who had previously received
chemotherapy treatment [58].
The synthetic collagen nonpeptidic MMP inhibitors are
collagen-mimicking compounds (known as "deep-pocket
MMPIs") that have been synthesized on the basis of the 3-D
conformation of the MMP zinc-binding site. They are more
specific and have better oral bioavailability than that of
peptidomimetic MMPIs and include mainly Prinomastat
(Agouron/Pfizer), Tanomastat (Bayer) and D2163
(Chiroscience/Bristol Myers-Squibb) (Fig. (3)).
Prinomastat (AG3340) is a synthetic, low-molecular-weight
lipophilic MMPI that inhibits several MMPs with
inhibitory activity on tumor growth and angiogenesis in
different xenograft models [59]. There were no beneficial
effects (i.e., no survival benefit or difference in symptomatic
progression) of the Prinomastat trials, both in combination
with standard chemotherapy or alone; moreover, Phase II
trials were discontinued after preliminary results because of
lack of primary efficacy [57]. Tanomastat (BAY 12-9566) is
a synthetic MMPI which inhibits the activity of
stromelysins and gelatinases [59]. Preliminarily
discontinued in Phase III trials, this compound showed
worse than placebo, or at least no evidence on progression-
free or overall survival [53].
D2163 (BMS-275291) is an orally bioavailable specific
inhibitor of both MMP-2 and -9, able to inhibit
angiogenesis, not inhibiting tumor necrosis factor-α receptor
II shedding, and not causing joint pain as a side effect [50].
This compound shows reduced activity against "sheddase"
enzyme activity attributed to non-MMP metalloproteinases
(such as those of the adamalysin family), probably
implicated in the musculoskeletal side effects. In rodent

A B C
Cl
O
O O
H
N HS N
SO2 O HO2C N NHMe
H
N S H O
NHOH O
O N
S O
N

Fig. (3). Synthetic collagen nonpeptidomimetic MMP inhibitors.

Chemical structure of Prinomastat (A), Tanomastat (B) and D2163 (C).
Matrix Metalloproteinase Inhibitors as Anticancer Therapeutics
A B
NH2 OSO3H
HN
N OH
H H
Fig. (4). Natural MMP inhibitors.
Chemical structure of Squalamine (A), Epicathechin (B) and Acetylsalcylic acid (C).
models, it has shown tumor growth inhibition and
significant reduction of metastases [52]. Actually, a Phase II
trial in patients with early stage of breast cancer was
terminated [60], whereas Phase II/III trial in combination
with standard chemotherapy is ongoing in non-small cell
lung cancer patients, demonstrating a good oral bio-
availability and the toxicity endpoint for study continuation
fulfilled [61].
The natural MMP inhibitors are compounds extracted
from natural sources with broad functions and better oral
bioavailability than that of other MMPIs; they mainly
include neovastat (Aeterna), cathechins and acetylsalicylic
acid (Fig. (4)). Neovastat (Æ-941) is an orally bioavailable
extract from shark cartilage (i.e. containing bioactive
compounds, such as squalamine), with multifunctional
antiangiogenic effects (through the binding inhibition of
vascular endothelial growth factor to endothelial cells,
depending on tyrosine phosphorylation pathways), able to
inhibit only gelatinases, collagenases and elastases and
inducing apoptosis [62]. Although the most common side
effects (such as musculoskeletal pain and joint stiffness) have
been reported, this compound demonstrated antitumor
activity in different experimental mouse tumors; in
particular, in animal metastatic bone tumor models [63],
Neovastat demonstrated to retard tumorigenic cell spread and
growth, suggesting its clinical utility in preventing bone
metastases in cancer patients. Preliminary results of Phase III
trials suggest improvement in survival in a small group of
cancer patients or at least disease stabilization (currently, it
is evaluated as a monotherapy treatment) [62].
Epicathechins are natural compounds extracted from
green tea with multifunctional antiangiogenic and anti-tumor
effects, able to inhibit gelatinases and stromelysins. In
preclinical studies, they were shown in rodent models to
have the capability to inhibit cell invasion and neo-
A B
H2N
OH
C
C A
HO
CONH2
OH
OH O OH O OH
Fig. (5). Synthetic drug derivative MMP inhibitors.
Chemical structure of Metastat (A), Alendronate (B), and Periostat (C).
Current Cancer Drug Targets, 2005, Vol. 5, No. 4 293
C
OH
OOCCH3
HO OH
H
H
COOH
OH
OH
angiogenesis, and to induce apoptosis [64]. Components of
green tea flavonoids (e.g., epigallocatechin-3-gallate), which
is being tested in a Phase I trial, acts as specific gelatinase
inhibitor with promising results [65, 66]. Moreover,
acetylsalicylic acid, an old drug that possesses previously
unrecognized activity against the MMPs, reduces the risk of
colon cancer [12, 59], not only decreases inflammation,
thereby possibly affecting cancer development, but also
directly inhibits MMP-2 activity and decreases cancer-cell
invasion [67].
The fourth class of MMPIs is represented by the synthetic
tetracycline derivative MMP inhibitors (Fig. (5)).
Tetracyclines, apart from their established role as classical
antibiotics, have been shown to inhibit connective tissue
breakdown by multiple non-antimicrobial mechanisms. A
series of studies analysed the specificity of anticollagenolytic
activity of tetracyclines and identified the site on the
molecule responsible for the non-antimicrobial but anti-
MMP properties. This led to the generation of the first series
of many chemically modified tetracyclines [68], which have
several potential advantages over conventional tetracyclines
(e.g., long term systemic administration without
gastrointestinal toxicity and higher plasma levels) [50]. It
has been suggested that chemically modified tetracyclines
inhibit active or pro-MMPs due to their capacity to chelate
zinc ions at the binding site in the catalytic domain,
disrupting the normal protein conformation and causing
cleavage into enzymatically inactive fragments [69].
The main drug evaluated in clinical trials is Metastat
(Col-3, or CMT-3) (Collagenex), which possesses no anti-
microbial effects but exhibits MMP inhibitory properties. In
particular, it inhibits the synthesis and activity of MMP-2,
-9 and -14, and it down-regulates the expression of various
inflammatory cytokines. In preclinical studies, its ability to
inhibit cell invasion and neo-angiogenesis was shown in
C
O OH N
ONa3H2O H H
P OH
OH CH3 CH2OH
OH HCL
P
OH CONH2
O H2 O
OH
O OH O 2
294 Current Cancer Drug Targets, 2005, Vol. 5, No. 4
rodent models [57]. Results from preclinical pharmacological
studies suggest that Metastat can be used for cancer treatment
due to its capacity to limit tumor growth and to evoke
cytostasis [69]. Positive results have been seen in Kaposi’
sarcoma [57], and preliminary data of a Phase II trial revealed
that Metastat induced disease stabilization in conjunction
with gelatinase suppression in plasma. In this cases,
phototoxicity and sideroblastic anemia was found to be dose
limiting [59]. Further studies are been carried out in several
cancer models to identify newer analogues of chemically
modified tetracyclines that possess a strong MMP inhibitory
activity and inhibitory effects on cell growth, invasion and
tumor metastasis [70].
The synthetic biphosphonates MMP inhibitors are an
old group of pharmacologic substances that possess
previously unrecognized activity against MMPs, blocking
their proteolysis by means of their zinc-coordinating
properties [72]. Their use in treatment of disturbance of
calcium homeostasis and palliative treatment of patients with
skeletal metastases had been established [12], but only
recently has their efficacy to inhibit gelatinolytic activity
(even though with mechanism of action not completely
elucidated), been evaluated. They have a perculiar ability to
prevent the inhibitory effect of TIMP-2 on MMP-2
degradation by plasmin, thereby enhancing the inactivation
of MMP-2 [50]. Innovative studies are being carried out on
the tripeptide phosphonate MMPIs, which are analogues of
phosphotryptophan derivatives, and show high specificity to
MMP-2 and -8. Their uncommon way of binding to MMPs
characterizes them as right-hand-side inhibitors, stemming
from the presence of a proline residue in the binding site
[73]. Recent studies focus on the specificity of the actions
against MMPIs. Structure-based analyses led to the
generation of different classes of hydroxamic acid derivatives
that are specific and strong inhibitors of MMP-1, -2, -9, -13
and tumor necrosis factor-α converting enzymes. The ability
to distinguish among various MMPs, makes these inhibitors
more suitable for long term treatment of osteo-arthritic
diseases and cancer. Recent studies evaluated their efficacy,
and pointing out their potential as clinical candidates [74].
The search for potent, selective, and orally bio-available
MMPIs led to the discovery of newer classes of
hydroxamates (e.g. ABT-518 and -770), that show high
specificity towards MMP-2 and -9 and are now under
evaluation in Phase I clinical trials in cancer patients [75].
CONSIDERATIONS AND FUTURE PERSPECTIVES
Influenced in part by the success of HIV protease
inhibitors, MMPs became a prime target for drug-discovery
programmes since the 1990s [52]. The failure of synthetic
MMPIs in clinical trials in patients with advanced cancer
raises questions as to the suitability of MMPs as therapeutic
targets. Inhibition of MMP for cancer treatment has not lived
up to the expectations; nevertheless, this therapeutic
principle should not be discarded before the results of clinical
trials with early-stage cancer are known (e.g. it is worth
remembering that MMPIs did, in fact, work as well as
conventional therapy in some trials)[42, 57]. However, in
view of the current understanding of how MMPs function, a
new assessment of anti-MMP strategies is warranted and, in
this respect, a critical re-examination of disappointing results
deserves several crucial comments.
Mannello et al.
Although several chemical studies have been undertaken
with 3-D structural analyses of the enzyme active site, it
should be noted, though, that true specificity and selectivity
of action has not been achieved for all MMPIs [76]. New
classes of MMPIs that are distinct from those originally
designed by crystal structure analyses of MMP enzyme in
zymogen form (evaluating both catalytic and hemopexin C-
terminal domains) might offer additional therapeutic
possibilities. MMPIs designed as for example novel
mechanism-based gelatinase selective synthetic inhibitors
may offer unexpected and intriguing possibilities for
targeting specific tumor MMPs [77]. Moreover, generally
MMPIs do not exhibit the serious and often life-threatening
toxicities associated with traditional anticancer
chemotherapeutic drugs and many of them have even been
administered to healthy volunteers in Phase I trials [52, 59,
75].
Adverse effects encountered with MMPIs include a
musculo-skeletal syndrome (consisting of joint stiffness and
pain in hands, arms and shoulders) that, although usually
mild and reversible, has led to the discontinuation of
treatment in several cases [50]. This musculo-skeletal
syndrome is dose-dependent, and several hypotheses have
been investigated to find out its cause; it is probable that
inhibition of sheddase activity of non-MMP proteinases may
be involved in the etiology of this side effect [52]. As a
confirmation of this hypothesis, MMP inhibitor BMS-
275291 (known as D2163), which shows reduced activity
towards sheddases, doesn’t cause musculo-skeletal disorders
[78]. The identification of the exact mechanism that leads to
this side-effect has been complicated by the different
functional roles for specific MMPs in the various animal
models analysed. In conclusion, these negative effects result
from the non-selective or combined inhibition of several
important MMPs [76]. Therefore, unexpectedly, it seemed,
rather indirect but convincingly, that MMP activity is also
involved in normal joint functions.
The importance of designing selective inhibitors of
specific MMPs involved in disease states such as cancer and
arthritis rather than broad range MMPIs, has become more
appreciated, and the identification of enzymes responsible for
the joint pain induced by MMPIs remains a high priority for
drug development.
Although MMPs have been examined as therapeutic
targets for various diseases [12, 22, 25, 50-53, 59, 75, 76,
78], the unique disease in which there has been a success in
understanding the role of MMPs is periodontitis. This
pathology (in which inflammatory cytokines are elicited by
bacteria in the gingival tissues resulting in substantial
connective-tissue destruction by MMPs) is prevented by
treatment with doxycycline hydrate (Periostat, a tetracycline
analog, the only MMPI to be licensed)(Fig. (5)), which both
decreases MMP gene expression and inhibits proteolytic
activity [1], reducing the loss of connective tissue. In
particular, this modified tetracycline shows specificity
towards MMPs responsible for fibrillar collagen turn-over
(i.e., MMP-1 and -8) [79].
Periostat is effective in the treatment of adult
periodontitis because it decreases MMP activity that would
degrade the support structures that hold the teeth in place
and not because of its anti-microbial activity. The first study
Matrix Metalloproteinase Inhibitors as Anticancer Therapeutics
on the treatment of periodontitis with Periostat revealed
positive clinical results at low doses of drug [80], and further
studies showed improved clinical outcome and reduction in
pocket’s death when Periostat was administered together
with conventional treatment [81]. It has been hypothesized
that the efficacy of Periostat is due to its ability to bind to
the calcified surfaces of teeth roots, thus increasing drug
concentration as much as five-folds than that found in blood,
and reaching concentrations sufficient to directly inhibit
MMPs [78]. An unexpected use of Periostat has been in the
treatment of coronary heart diseases following the finding
that there is a correlation between periodontal inflammation
and the increased risk of heart disease and stroke [82].
Reports demonstrate that periodontitis produced endotoxins
and cytokines, which initiate atherogenesis and thrombo-
embolic events, and permit the entry of bacteria in the
systemic circulation, and increases bacteraemia. However,
the significance of, and the substantial effect of Periostat in
the treatment of cardiovascular morbidity and mortality have
yet to be analysed and fully understood [76].
Despite very promising and exciting results in preclinical
studies, the early experience with larger clinical trials has
been disappointing, and actually, hope has been dampened
considerably owing to the failure of several Phase III trials to
show any survival benefit of MMPI treatment or, in some
cases, alarming reports that patients who are treated with
MMPI fared worse than their placebo-treated counterparts
[50, 59]. This could be due the fact that under certain
circumstances, MMP inhibition may be detrimental rather
that therapeutic. For example, blocking certain MMPs may
increase tumor vascularization thus favouring tumor growth
[77]. These poor results have been attributed to the fact that
aggressive, already disseminated cancers are not likely to
respond to a monotherapy with agents that modify their
invasive potential and, thus, require prolonged and early
treatment to demonstrate clinical benefit. Accordingly, the
design of Phase III clinical trials, traditionally used for
conventional cytotoxic chemotherapeutic agents, may be
inappropriate for this novel class of inhibitors, especially in
high-risk patients (e.g., subjects with clinically metastatic
cancer or where subclinical metastatic spread has already
occurred) [83].
Several other factors probably contributed to the failure of
these clinical trials, and these must be accurately
investigated before concluding that MMP inhibition has no
therapeutic benefit. Among these are the persistent problems
of specificity for the targeted MMP (not all MMPIs used
thus far have been shown to be highly selective for any
individual MMP), and the need for assays to verify
inhibition of target MMP enzymes in vivo (the identification
of the best markers of MMPI activity). In fact, the recent
development of new procedures to assess the in vivo
inhibition of MMPs [84] may help to resolve fundamental
problems, which is a prerequisite for increasing efficacy and
reliability of drug trial design and execution. A more
selective approach that takes into account the identity of
MMPs that any given tumor relies on will be more
desirable, and one strategy that appears highly attractive is to
block MMP interaction with cell surface docking molecules.
Because it appears increasingly clear that location in general
and cell surface localization in particular are important for
MMP-mediated tumor invasion and metastasis, disruption of
Current Cancer Drug Targets, 2005, Vol. 5, No. 4 295
the mechanisms that help retain secreted MMPs on the cell
surface may provide an effective means to inhibit the function
of individual MMPs [50]. Although controversial, several
tantalizing pieces of evidence indicate that the hemopexin C-
terminal domain can simultaneously inhibit angiogenesis,
cell proliferation and cell migration, suggesting an
innovative strategy to block MMP enzymes (through
noncatalytic targeting) [75]. In the same way, the discovery
of exosites as a crucial place for proteolytic MMP function,
may offer promising and highly novel targets for new drugs
[12]. In this respect, the issue of broad-spectrum versus
selective MMPIs development and use, deserves a careful
reflection: although the selective inhibition of a limited set of
MMPs at early stages of the neoplastic disease might be
more effective than using broad-spectrum inhibitors that are
active against most MMP family members, at later stages of
cancer pathology, when many distinct MMPs have been up-
regulated, broad-spectrum inhibitors could be more effective.
Even though most recent evidence indicates that the main
roles for MMPs are in the early stages of neoplasia, and in
view of the fact that MMPs might be expressed by tumors
and the reactive stroma at different stages of the cancer, there
is an overlap between substrate specificities and common
inhibitory mechanisms. It is therefore also crucial to consider
MMP activities not just on the basis of the performance of
individual enzymes, but as an interconnected and
interdependent proteolytic system [78].
Moreover, since both natural and synthetic compounds
were typically administered at the maximal tolerated dose,
whatever selectivity they may display at lower doses is most
likely overridden. On the other hand, non-selective
inhibition of MMP activity may result in the abrogation of
potentially beneficial effects of MMPs, with a net adverse
effect on overall patient survival. Even though the inhibition
of metalloproteinase activities can result in a decreased
release of beneficial natural products (e.g. angiostatin) [25],
MMP inhibition is not always balanced by a corresponding
decrease in tumor progression [57]. Nevertheless, both
preclinical experiments and most of the clinical trials
indicate that the tumor growth restraint is a rare event and
the predominant effect is more likely to be beneficial, given
appropriate conditions of disease stage and drug efficacy.
Furthermore, in order to balance the positive and negative
aspects of these enzymes (in respect to the rationale for their
targeting) [1, 12, 15], it is crucial to emphasize that MMPIs
are unlikely to be cytotoxic (i.e. killing cells), but only
cytostatic drugs (i.e. growth-arrested but viable cells),
thereby necessitating long-term treatment modalities and
combined administration with other anticancer therapies to
achieve their maximum efficacy. Nonetheless, the intriguing
evidence for both pro- and anti-apoptotic effects of several
MMPs indicates that it might be possible to develop specific
inhibitors that regulate cancer programmed cell death (e.g.,
MMPs as switch to live on or die for) [15]. For example, the
detection of the apoptosis-inducing properties of MMPIs
may widen their use in neoplastic disorders (including
haematological malignancies, where they have been rarely
studied) [45]; since these drugs were felt to work only by
inhibiting extracellular matrix degradation, they were not
expected to benefit patients with leukaemias [15, 45, 57,
78], suggesting these diseases as excellent candidates for
future clinical evaluation of MMPI-based treatments that may
296 Current Cancer Drug Targets, 2005, Vol. 5, No. 4
have a place in the therapeutical arsenal aimed at inhibiting
growth and invasion of malignant tumors.
Although several approaches are under investigation to
inhibit tumor growth and invasion (such as catalytic and
noncatalytic targeting of MMPs, gene targeting, broad and
specific MMP inhibition, etc.) it will be interesting also to
explore the role of MMPIs as chemopreventive agents, in
patients with genetic predisposition to cancer development,
or in patients in which the primary tumor has been removed.
WHAT LIES AHEAD FOR MMPIs?
In reviewing the literature and reading between the lines
one sees optimism that the changes in trial design and
compound characteristics may lead to the discovery of
effective MMPIs as cancer fighter and/or tools for answering
questions on the many ways in which these proteases can
influence complex biological processes. As previously
suggested, "history has shown that the MMP field is
resilient and persistent and what started from observations on
frogs developed into a field that is fit for a prince" [1].
Whether MMPIs will become a standard in the cancer
arsenal is still unclear and there is much groundwork yet to
be done on after the past 45 years of intense study on these
enzymes. There is a pressing need for basic and translational
research to develop and validate assays that identify tumors
expressing target enzymes and to assess the efficacy of
specific compounds and optimal doses that reduce tumor-
associated proteolytic activity. Moreover, attention to the
stage and type of cancer that is likely to be evaluated in
clinical versus preclinical studies is crucial (i.e. to determine
the role of specific MMPs in specific stages of tumor
progression as well as to develop animal models that
recapitulate clinical trial design).
Although several factors will undoubtedly continue to
strongly influence drug development decisions in the future
(e.g., patent issues, competition and impatience), an analysis
of the expression patterns of the entire family of MMPs in
the cancer type and stage used in clinical trials (through new
technologies, such as degradomics, microarrays and imaging
approaches)[15, 84, 85], and correlation with clinical
outcome to determine prognostic significance, may allow
more rational decisions on the appropriate MMPs to target
[78]. This will accomplish a dream of molecular medicine
for the twenty-first century through the identification of
MMPIs as highly specific and efficacious agents for cancer
therapy.
ACKNOWLEDGEMENT
We would like to thank Mr. Federico Bastianelli for
skilfull tecnhical assistance.
ABBREVIATIONS
MMPs = Matrix metalloproteinases
MT-MMPs = Membrane type of matrix
Metalloproteinases
ECM = Extra-cellular matrix
TNF = Tumor necrosis factor
Mannello et al.
TIMPs = Tissue inhibitor of metalloproteinases
EMMPRIN = Extracellular matrix metalloproteinase
Inducer
MAPK = Mitogen activated protein kinase
ETS = Erythroblastosis twenty six
STAT = Signal transducers and activators of
Transcription
MMPIs = Matrix metalloproteinase inhibitors
ADAM = A disintegrin and metalloproteases
TACE = TNF-alpha converting enzyme
GPI anchor = Glycosylphosphatidylinositol anchor
Ig-like = Immunoglobulin-like domain
domain
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