Download Cytoskeleton structure and dynamic behaviour: quick excursus from

European Review for Medical and Pharmacological Sciences
2009; 13: 13-21
Cytoskeleton structure and dynamic
behaviour: quick excursus from basic
molecular mechanisms to some implications
in cancer chemotherapy
C. ALBERTI
L.D. of Surgical Semeiotics, University of Parma (Italy)
Abstract. – Novel nanoscale microscopic
technologies are driving dramatic advances in
the knowledge of cytoskeleton structure and dynamics. Cytoskeleton, that is organized into microtubules, actin meshwork and intermediate filaments, besides providing cells with important
mechanical properties, allows, within the cell,
not only the molecule cargo transport, but also
the charged particle/biophoton transmission, so
that the cell signaling might be considered as
consisting of both molecule/chemically- and
charged particle/physically-addressed systems.
Molecular motors that drive molecule cargo
translocation along the cytoskeletal highway, either through endocytic or secretory-exocytic
mechanisms, include kinesin and cytoplasmic
dynein, traveling on microtubule, and myosin
family members, traveling along actin meshwork. The membrane-bound organelles and protein complexes are sorted with high specificity
to their various destinations. In the field of highly structured cell signaling machinery, the endocytosis appears to play an important role with
following specific changes in gene expression.
In the opposite direction, the exocytosis involves many intracellular steps toward the vesicle fusion with the plasma membrane. Insights
into cytoskeletal structure and dynamics are
providing important progress in identifying
proper targets for cancer therapy. Taxane and
Vinca alkaloids, by stabilizing the polymerized
microtubules, are able to suppress their dynamic behaviour with subsequent cell death. Epithelones, by acting in same way, are emerging as a
new class of anticancer drugs, moreover their
toxicity resulting unaffected also towards taxane-resistant cancer cells. Even the alkylating
agent nitrogen mustard exerts some cytotoxic
effects at the level of the microtubule, whereas
azaspiracid-1 induces cytoskeletal actin disorganization without affecting microtubule architecture. Regarding the influences of extracellular
mechanical forces on changes in cell adhesion
gene expression, the iatrogenic pressure-in-
duced tumor cell implantation within surgical
wounds may be prevented by perioperative administration of microtubule/actin inhibitors.
Even though, among the different cancer therapy strategies, the chemotherapy could appear to
be conceptually outclassed because of its low
cancer cell-selectivity in comparison with novel
molecular mechanism-based agents. However
"combo-strategies", that combine the chemotherapeutic high killing potential with new molecule targeted agents, may be an effective curative measure. Some anticytoskeleton agents are
under evaluation for their applications in tumor
chemotherapy; benomyl, griseofulvin, sulfonamides, that are used as antimycotic and antimicrobial drugs, appear to have a powerful antitumor potential by targeting microtubule assembly
dinamics, together with exhibiting, in comparison with taxane and Vinca alkaloids, a more limited toxicity. An exciting challenge for the next
future will be to properly define the cytoskeleton
structure and dynamic behaviour to design more
effective drugs for cancer chemotherapy.
Key Words:
Cytoskeleton, Intracellular signal trafficomics, Cytoskeleton inhibitors, Chemotherapy, Prostate cancer.
Introduction
Recent developments of microscopic technologies (atomic force microscopy, confocal
laser-scanning microscopy, fluorescence resonance energy transfer microscopy, small angle
X-ray diffraction microscopy, etc), that are able
to explore cytoskeletal structure and dynamics at
nanometer scale, have induced tremendous
progress in understanding single-molecular
Corresponding Author: Contardo Alberti, MD; mobile: +39.331.9823032
13
C. Alberti
events which promote the cytoskeleton dynamic
behaviour, thus also providing idea-of-principle
that targeting the microtubule/actin elements
may be a fruitful chance for an effective cancer
therapy.
actin meshwork (Figure 1). From here, in the reverse direction, microtubules, with their minus
ends, converge on their organizing center1-4.
Cortical actin meshwork is composed of actin
plus end filaments that are oriented toward the
plasma membrane and, in the opposite direction,
of actin minus end filaments reaching to intersect
with microtubule cytoskeleton4,5.
Intermediate filaments assemble into extensive cytoskeletal nanofibrillar complex which is
able to connect both cell surface/desmosomes
and membranous organelles with the nuclear
surface and nucleoplasm, together with providing cells with important elasto-mechanical properties. In fact, intermediate filaments are very
flexible polymers (vimentin, desmin, keratinepiplakin) that, on the one hand, act as tracks for
the transduction of mechanical perturbation (mechanical stress) from the cell periphery to the
nucleus and, on the other hand, interact with
membranous organelles, such as Golgi apparatus, lysosomes, mitochondria and vesicles, as
well as with other cytoskeleton components such
as microtubules, actin meshwork and their associate motor proteins, thus playing important key
roles in organizing organelles in the cytoplasm,
providing them with proper motility and anchorage sites1,6-8.
Cytoskeleton Structure and Dynamics
Three different cytoskeletal structures – microtubules, actin meshwork and intermediate filaments – besides providing cells with proper mechanical forces, contribute to ensure intracellular
signaling efficiency and specificity.
Microtubules are composed of several individual polarized protofilaments consisting of α/β
tubulin dimers, whose addition or loss can induce
growing or shortening of their ends. This dynamic behaviour – microtubule dynamic instability –
is important for mediating the movements of cargo-carrying motors to reach their final destination at intracellular specific sites as well as for
promoting the alignment of sister chromatids,
through tension at the kinetochores, on the mitotic spindle during metaphase, and, subsequently,
during anaphase, their segregation into the
nascent daughter cells. Micro-tubules originate
from an organizing center (MTOC) near the nucleus, faning out, with their plus ends, toward the
cell cortex, where they intersect with cortical
A
A
M
km
A
M
dm
M
A
M
mtoc
M
Figure 1. Cartoon illustration of cell cortex
actin filaments (A)/cell interior microtubules
(M) together with their plus-end(+)minus-end(-)
and kinesin (km)/cytoplasmic dynein (dm) microtubule-based motors. Cell nucleus (N), microtubule-organizing center (mtoc).
14
N
A
Cytoskeleton targeted chemotherapy
Molecular Cargo Transport and Delivery
Intracellular cargo transport – transport of organelles, secretory vesicles and protein complexes – needs specific microtubule- and actin-based
molecular motors together with polarized microtubule/actin rail-tracks along which the cargoes
go on. Molecular motors that drive cargo translocation along the cytoskeleton highway include
kinesin and cytoplasmic dynein, traveling on microtubule, and myosin, traveling along actin
meshwork, both of them managing the transit
manoeuvres through a complex cytoskeletal network8-11. Cargoes carried by kinesin motors are
transferred to the plus ends of microtubules, that
are oriented toward the cell cortex and, subsequently, upon reaching the peripheral actin meshwork, they are translocated, by myosin Va motor,
to the plus ends of F-actin filaments toward the
plasma membrane. In the opposite direction (e.g.,
in endocytosis process), cargoes are transported
by myosin Vl motors, that walk toward the minus
ends of F-actin filaments, into the cell interior,
and, subsequently, upon reaching actin-microtubule intersections, they are transferred, by cytoplasmic dynein motors, toward the minus ends of
microtubules, which are located, near the cell nucleus, in the MTOC11,12.
All motor proteins – kinesin, cytoplasmic
dynein and myosin – are able to hydrolyze ATP,
adenosine triphosphate, to ADP, hence converting energy into motion. Anyway, cargo shipping
is carried out through a well-defined three-dimensional array of actin filaments and microtubules. Dynactin, electrostatically tethering the
motors to the microtubules, prevents the free motors from diffusing away, thus facilitating their
further use for running again11. Interestingly, microtubule-associated τ (tau) protein can increase
protein motor switching at microtubule-microtubule intersections, by inhibiting kinesin motor
binding to microtubules, hence avoiding that
many kinesin molecules might give rise to a reciprocal “tug of war” at level of intersections11,13.
Within the cell, both membrane-bound organelles and protein complexes are sorted with
high specificity to their destinations. In the secretory pathway, that connect, in sequence, endoplasmic reticulum, Golgi apparatus and plasma membrane, newly synthesized proteins are
strictly screneed, in the first sorting station – endoplasmic reticulum – for misfolding or mutation (quality control), by means of different
chaperones, misfolded proteins resulting targeted for locally degradation while correctly folded
proteins are exported. This process requires specific endoplasmic reticulum-derived vesicles and
proper cytoskeletal structures for various cargo
proteins sorted from endoplasmic reticulum14-19.
The Golgi apparatus activity is directed, on the
one hand, to post-translationally modify both
protein and lipid cargoes, by modulating,
through a glycosilation process, their stability
and function, and, on the other hand, to send
them to the plasma membrane or toward other
cell compartments20-24.
In the field of highly structured cell signal
transmission machinery, that is able to translate
cell responses to external agents into specific
changes in gene expression, the endocytosis –
endocytic organelle-mediated process – appears
to play an active role in signal amplification,
propagation and spatial-temporal transduction
control. By the way, some downstream signal
cascades are carried-out by receptor-ligand complex internalization and endosome specific compartmentalization, the molecular signal trafficking among various endocytic organelles requiring, anyhow, a suitable actin/microtubule-dependent motility25-27.
In the opposite direction, the exocytosis is mediated by exosomes, that are endosomal-derived
membrane vesicles shipping extracellular molecule-messages. Exocytic process involves multiple intracell steps, including the transport of exosomes along different cytoskeletal structures toward the fusion with the plasma membrane. Particularly, exosomes display a regulatory function
in the immune system (maior histocompatibility
complex, MHC, class -I and -II), so that, in cancer immunotherapy (tumor vaccines), an important interest is emerging in their use, either as exosomes derived from cancer antigen-loaded dendritic cells or as exosomes directly derived from
tumor cell, to obtain anti-tumoral immune responses in vivo28,29. Moreover, the recent discovery of argosomes and prostasomes, leads to suggest that the exosomal pathway not only displays
regulatory functions in the immune system but
also, regarding the argosomes, might partecipate
in tissue-developmental process, and, in the case
of prostatosomes, it might be involved in promoting the fertility30,31. Otherwise, in the pathological field, certain exosomes, that transport angiogenic factors, can interact with the environment,
thus facilitating tumoral growth29.
In both the endocytic and exocytic pathways,
vesicular containers carry the cargoes with high
specificity dependently on their coat components
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C. Alberti
(coat protein complexes, COP-I and -II; clathrin
protein) and adaptors (proteins binding cargoes
to the coat-pits). While clathrin coats are used in
the endocytic and recycling pathways between
plasma membrane and lysosomes or in transGolgi membrane network, COP-I and -II coats,
instead, are used in molecule trafficking between
Golgi apparatus and endoplasmic reticulum30-32.
During clathrin-mediated endocytosis of signaling receptors (EGFR, epithelial growth factor receptor; FGFR, fibroblast growth factor receptor;
GPCR, G protein coupled receptor; etc), cargo
recognition by coated-pit proteins confers specificity on this process32-34.
Furthermore, as far as vesicular trafficking is
concerned, membrane lipids, besides playing the
role of sealing the transport containers, allow,
through their various composition, the specific
recruitment of protein cargoes from the cytosol,
by acting as raft – lipid rafts – for protein sorting
at sequential phases of both endocytic and secretory pathways35-37. Intriguingly, many signaling
molecules, such as tyrosine kinase receptor and
some Ras family members, appear to display
high affinity for these lipid rafts. Tyrosine kinase
receptor, through endocytic lipid rafts, is carried
to the endoplasmic reticulum where PI3P (phosphatidylinositol-3,4,5-triphosphate) is synthesized, whereas Ras family members are localized
to the Golgi apparatus where they may be activated by specific exchange factors such as phospholipase Cγ27,38,39.
Last but not least, just considering the tight,
even though dynamic, connections among the cytoskeleton components – microtubules, actin
structures, intermediate filaments – and between
these and membranous organelles, the mechanical perturbations on cell surface can produce organelle displacements by force transmission
through cytoskeleton. Anti-cytoskeletal drugs,
such as cytochalasin towards actin filaments and
nocodazole towards microtubules, by disrupting
either of two structures, induce a dramatic decrease in organelle spatial rearrangement40-42.
Electron Transport Along Cytoskeleton
Small electronic currents may be carried by
semiconductive electron transport along biopolymers such as cytoskeletal components .These microcurrents begin by electrons released from mitochondrial respiratory chain and NADPH
(nicotinamide adenine dinucleotide phosphate H+)-oxidase complex that, in plasma membrane,
catalyzes the reduction of oxygen to superoxide
16
−
−•
anion 0−•
2 (02 + e → 02 ), an electron donor which
spontaneously dismutases to hydrogen peroxide
•
(0−•
2 + H2 → H2O2 ). Electrons leaking from mitochondrial respiratory chain should diffuse either
along the actin filaments toward the plasma
membrane or along the microtubules toward the
nucleus; electrons leaking from plasma membrane NADPH-oxidase complex would diffuse
directly along actin/microtubule cytoskeletal
structures to various cell compartments like mitochondria or to the nucleus, where they can influence the transcriptional activity43,44. ROS, reactive oxygen species, either free radical superoxide anion or non radical hydrogen peroxide,
are important mediators of damage to cell structure, including cytoskeleton, organelle and vesicle lipid membranes, nuclear matrix proteins and
DNA, where, by means of free NF-kB (nuclear
factor-kB), are able to activate proto-oncogene
expression together with changes in tumor suppressor genes, thus promoting DNA → mRNA
transcription. Moreover, microtubules are electrical polar structures with energy supply that results from hydrolysis of their tip-bound GTP
(guanosine triphosphate) to GDP (guanosine
diphosphate) and a part of this energy can excite
cytoskeleton vibrations with subsequent generation of electromagnetic fields, so that some
mechanisms of malignity (detachment of cancer
cells, invasion, local invasion and metastatic
spread) may be assumed as dependent on the cell
electromagnetic field perturbations45,46. In addition to charged particles (electrons, protons,
ions), cytoskeletal microtubules may transmit, on
a part with glass optical fibres, optical and infraoptical radiations as well as quantized vibrational-mechanical waves47,48.
Just considering these cytoskeletal properties,
the biochemical model of intra-cell and cell-cell
signal transduction (molecule- or chemically addressed system) is more and more integrated with
a biophysical model (charged particle/biophoton
or physically addressed system)43-48.
Cytoskeleton as Attractive Target for
Anticancer Drugs
In the run of the last decades, thorough insights into cytoskeletal structure and dynamics
have provided important progress not only in the
knowledge of intracellular signaling but also in
identifying new targets for anticancer therapy.
The main classes of microtubule targeted
agents, the Vinca alkaloids (vinblastine, vinorelbine, vinflumine) and taxanes (paclitaxel, doc-
Cytoskeleton targeted chemotherapy
etaxel) are able to stabilize the polymerized microtubules, thus effectively suppressing their
dynamic behaviour at the mitotic spindle, hence
causing, in cancer cells, mitotic block with subsequent cell death49-51. Improvements in cytotoxic effects on cancer cells may be achieved by
associating taxanes or Vinca alkaloids with
Vif11, a kinesin-5 motor protein inhibitor52.
Significant anticancer activity of taxanes has
been shown in patients with pre-treated nonsmall-cell-lung, ovarian, breast carcinoma, as
well as in lymphoma53. Moreover, recent studies
of docetaxel-based chemotherapy in men with
hormone refractory prostate cancer (HRPC)
have shown a survival benefit, so lifting the burden of HRPC as a chemoresistant malignancy54.
Epothilones (ixabepilone, patupilone) –
macrolides extracted from a variety of myxobacteria such as Myxococcus xanthus, Sorangium cellulosum – are emerging as a new class of antineoplastic drugs because of their stabilizing effects,
like those taxane-induced, on the polymerized microtubules, with following suppression of microtubule dynamics54,55. However, recent research
findings reveal significant differences between
epothilones and taxanes in drug-resistance mechanisms, epothilone cytotoxicity resulting unaffected
by alanine-to-threonine substitution at reside 364
in microtubule β-tubulin, that, instead, is responsible for chemoresistance to taxanes56. Furthermore,
epothilones are more cytotoxic than taxanes in
cancer cell culture, particularly in tumors characterized by P-glycoprotein overexpression54,56,57. It
suggests that taxane-resistant cancer cells might
retain sensitivity to epothilones, which could play,
in such case, a rescue drug role54-59.
Also nitrogen mustard (component of the estramustine phosphate) exerts some cytotoxic effects at the level of the microtubule, besides
binding to nuclear matrix60.
On the other hand, cell growth inhibition
through actin filament targeting, has been recently identified in vitro as the effect of AZA-l, azaspiracid-1 (a marine toxin), which is able to induce cytoskeletal actin disorganization, without
affecting the amount of polymerized actin and
the microtubule architecture. Irreversible actin
rearrangements are completely caspase activation
indipendent events57. In the same way should act
mPTXS, macrolide pectenotoxins (PTX-1 ,-2 and
-11), that are potently cytotoxic in human cancer
cells, even though no clear explanation of their
mechanism of action at molecular level is until
now available61,62.
Regarding that extracellular mechanical forces
can induce, by cytoskeleton dependent signaling,
changes in cell adhesion gene expression, recent
experimental research data show that tumor cell
adhesion may be stimulated by exposure to increased extracellular pressure, following the activated FAK (focal adhesion kinase)- and Src gene
expression. In this regard, iatrogenic pressure-induced tumor cell implantation within surgical
wounds, may be prevented by pretreatment
or/and perioperative administration of either
actin polymerization inhibitors, such as cytochalasin D, phalloidin and latrunculin B, or microtubule polymerization inhibitors, such as
colchicine, thus enhancing tumor-free survival.
Instead, the effectiveness of taxanes in inhibiting
pressure-induced tumor cell adhesion is slightly
significant63,64.
Since microcurrent cytoskeletal network and
ROS can activate several oncogenetic signaling
pathways, the antioxidant and redox agents (electron trapping or quenching agents) – isoflavonoids,
thyocyanates, polyphenols, carotenoids, trace elements such as selenium and zinc – play an antitumoral role, by inducing a downregulation of
deleterious effects of electronic charges and
ROS43,44.
Conclusions and Perspectives
The oncological research is highly dynamic
field and recent insights into tumor signaling
pathways by cell- and molecular biology, besides
promoting the development of novel anticancer
agents, have lead to suggest new therapeutic paradigms. So-called molecular mechanism-based
drugs or smart drugs, that have been designed to
target selectively such signaling pathways, may
provide an effective antitumor activity. Therefore, among the various cancer therapies (Table
I), the chemotherapy could appear to be conceptually outclassed because of its low cancer cellselectivity and association with serious side effects. However, single-agent anticancer therapy,
even though carried out with a “smart drugs”, is
often inadequate to eradicate the disease, hence
combo strategies – regimens that combine the
chemotherapeutic high killing potential with
molecular cause-targeting selectivity of new anticancer agents – may be a rational synergic treatment, together with allowing the drug dose lowering with subsequent lower drug-induced toxici17
C. Alberti
Table I. Principle approaches to cancer therapy.
Antiproliferative
chemotherapy
Hormone therapy
(in hormone-dependent tumors)
Biomolecular selectively
targeted therapy (smart drugs)
Specific growth (a)/apoptosis
(b) pathway targeted therapy
Immunotherapy
Gene-therapy
Epigene-therapy
Cancer stem cell eradicating therapy
• Antimetabolites. Platin derivatives. Alkaloids among which several
microtubule/actin inhibitors such as taxanes and Vinca-derivatives. Antibiotics
like microtubule inhibitor macrolide epothilones. Alkylating agents such as
microtubule inhibitor nitrogen mustard
• Hormones: Androgens, Estrogens, Progestogens. Corticosteroids
• Antihormones: Gn-RH (gonadotropin releasing hormone) analogues or
antagonists. Antiandrogens. Antiestrogens. Aromatase inhibitors.
• Monoclonal antibodies against growth factor signaling
• (a) antiproliferative: Tyrosinekinase- and multikinase inhibitors. mTOR
(mammalian target of rapamicin) inhibitors; (b) proapoptogenic:
Recombinant TRAIL (tumor necrosis factor-related apoptosis inducing
ligand). Poly (ADP-ribose) polymerase (PARP) inhibitors
• Tumor vaccines. Immunomodulators
• Replacement or inactivation of defective genes. Cytoreductive gene therapy.
Gene-silencing by interfering short RNAs
• Histone-deacetylase inhibitors. DNA-methyltransferase inhibitors
• Exciting field of current research, especially with regard to a long-term
success of cancer treatment strategy
ty. Different smart drug combinations with
chemotherapeutics are currently explored in
various clinical trials to promote re-expression
of genes involved in cell differentiation and
apoptotic process, so leading to malignant disease eradication by endogenous surveillance
pathways65-67.
As far as chemotherapeutic agents are concerned, either microtubule or actin cytoskeletal
inhibitors – taxanes and Vinca alkaloids,
epothilones, pectenotoxins – can provide some
favorable results in advanced/recurrent cancer
disease, either alone or in combination with
“smart drugs”, such as tyrosine- or multikinase
inhibitors, antibodies targeting growth factor signaling, mTor (mammalian target of rapamicin)
inhibitors, recombinant TRAIL (tumor necrosis
factor-related apoptosis-inducing ligand). In this
regard, microtubule inhibitors, such as taxol,
nocodazole and colcemid, as well as cytochalasin
D, an inhibitor of actin meshwork, are able to enhance the effects of the recombinant TRAIL by
increasing caspase-3, -8, -9 activation, even in
cancer cells overexpressing antiapoptotic protein
Bcl-2, thus showing that such “combo-therapy”
can be efficient in the therapeutic management of
cancers resistant to traditional chemotherapy
(Table I)67-70.
Particularly, taxanes are the most active drugs
for the therapy of HRPC, either as single agents
or in combination with other compounds, furthermore representing the critical component of
18
the “combo-therapies”. In such prostate malignancy state also epithelones are on trial, with significant results as far as effectiveness54,57.
As above described, the machinery of intra-cell
signal trafficking and transduction – one of the
most active fields in the current biological studies
so that the related area of research could be named
as cell trafficomics like other neologisms such as
genomics, proteomics, transcriptomics – includes
both chemically/molecule- and physically/charged
particle or biophoton addressed systems, the last
one especially involving the cytoskeletal
structures43-48. In this regard, looking to the future,
an intriguing chance of cancer therapy will be accomplished, in the emerging area of nanomedicine, by making use of cytoskeletal rail-tracks and
their associated electromagnetic fields, to drive
and deliver nanosized materials (nanoparticles,
nanospiders, nanorobots, either quantum dotbound or not) as “means of carriage” of therapeutic nucleic acids or anticancer agents, respectively
into the nucleus (nanoparticle vector system for
nucleic acid delivery; nanorobot-performed in vivo
DNA-cytosurgery) or at other cell target level (targeted nanoscale drug delivery)71-76.
Moreover, considering that extracellular mechanical forces can promote, through cytoskeletondependent transduction, several changes in cell adhesion gene expression with cell implantation
within surgical wounds, the pre- and/or perioperative administration of microtubule/actin inhibitors
appears to prevent this deleterious event63,64.
Cytoskeleton targeted chemotherapy
At present, several anticytoskeleton agents are
under evaluation for their application in tumor
chemotherapy. Benomyl, griseofulvin and some
sulfonamides, that are used as antimycotic and
antimicrobial drugs, appear to have a powerful
antitumor potential by targeting microtubule assembly dinamics, together with exhibiting, in
comparison with taxane and Vinca alkaloids, a
more limited toxicity75.
An exciting challenge for the next years will
be to better define the microtubule/actin structure
and dynamics in order to develop novel cytoskeleton targeted drugs for a more effective
cancer therapy.
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