Download Microtubule-active drugs: mechanism of action and resistance

Charles University in Prague, Faculty of Science
Department of Cell Biology
Special Chemical and Biological Programmes:
Molecular Biology and Biochemistry of Organisms
Vojtěch Dostál
Microtubule-active drugs: mechanism of action and resistance
Mikrotubulární jedy: mechanismus účinku a rezistence
Bachelor’s thesis
Advisor: RNDr. Lenka Libusová, Ph.D.
Faculty of Science, Charles University
Laboratory of Molecular Genetics of Development
Prague 2013
Abstract
Microtubular cytoskeleton represents a target for a myriad of diverse chemical compounds, referred to
as microtubule-active drugs. Produced by certain plants, animals or microbes, the substances often
effectively elicit cell death – especially in animals and also in plants to a certain extent, but never in
species which produce them to defend against their predators. Nowadays, several microtubule-active
substances constitute hallmarks of anti-cancer treatment and agricultural weed control. There is an
enormous sum of knowledge about the action of paclitaxel (taxol), vinca alkaloids and colchicine,
three best-known microtubule-active compounds used in medicine, and new research often challenges
the previously accepted theories. This work investigates the mechanism of action of microtubuleactive drugs from the angle of biochemistry and cell biology, as well as from the physiological
standpoint. Effects on microtubule levels and dynamics and the path towards the cell death are
reviewed. In the last chapter, attention is given to drug activity in both animal and plant bodies and,
finally, to drug-producing plant species which often show substantial resistance.
Keywords: microtubule-active drugs, microtubules, tubulin, microtubule dynamics, chemotherapy,
paclitaxel, vinca alkaloids, colchicine, drug resistance
Abstrakt
Mikrotubulární jedy tvoří různorodou skupinu chemických látek. Vznikají v tělech některých rostlin,
živočichů či mikroorganismů. Jejich společným rysem je schopnost vazby na mikrotubulární
cytoskelet. Tyto látky, reprezentované např. paclitaxelem (taxol), vinca alkaloidy a kolchicinem, jsou
schopné účinně vyvolat buněčnou smrt v živočišných a často i v rostlinných buňkách. V současnosti
jsou proto mikrotubulární jedy důležitou součástí protinádorové léčby i boje proti škůdcům v
zemědělství. Ačkoliv je k dispozici značné množství informací o mechanismu účinku těchto látek,
nejnovější poznatky jsou často v rozporu s těmi staršími. Tato práce shrnuje účinky mikrotubulárních
jedů z pohledu biochemie a buněčné biologie, ale také z fyziologického hlediska. Jsou diskutovány
účinky mikrotubulárních jedů na množství polymerovaných mikrotubulů či mikrotubulární dynamiku
a následná cesta k buněčné smrti. V poslední kapitole je pozornost přesunuta na aktivitu těchto látek v
živočišných a rostlinných tělech s důrazem na rostliny, jež tyto látky vytváří a jejichž mikrotubuly jsou
k nim tudíž značně odolné.
Klíčová slova: mikrotubulární jedy, mikrotubuly, tubulin, mikrotubulární dynamika, chemoterapie,
paclitaxel, vinca alkaloidy, kolchicin, rezistence k léčivům
Abbreviations
1KP – 1000 Plants Initiative
FRAP – fluorescence recovery after photobleaching
GDP - guanosine diphosphate
GTP - guanosine triphosphate
GTPase – guanosin triphosphate hydrolase
IMS – ion mobility spectrometer
Kd - dissociation constant
MALDI - matrix-assisted laser desorption/ionization
MAP – microtubule-associated protein
MS – mass spectrometry
MTOC - microtubule organizing center
PDB – Protein Data Bank
Pi - inorganic phosphate
QWBA - quantitative whole body autoradiography
SAC – spindle assembly checkpoint
Treg – regulatory T cell
I would like to express my dearest gratitude to my supervisor, RNDr. Lenka Libusová, Ph.D., for her
patient guidance and valuable advice.
Statement of authorship
I confirm that this bachelor's work has been written solely by me, using only the cited literature and
consultation with the supervisor.
Vojtěch Dostál
Prague, 2013
Contents
1. Introduction ..................................................................................................................................... 1
1. 1. Microtubule structure and dynamics ....................................................................................... 1
1. 2. Microtubule-active drugs......................................................................................................... 3
2. Microtubule-active drugs: mechanism of action ............................................................................. 7
2. 1. Effects on microtubule assembly............................................................................................. 7
2. 2. Effects on microtubule dynamics ............................................................................................ 8
2. 3. Towards the cell death ........................................................................................................... 11
3. Microtubule-active drugs in the context of a body ........................................................................ 14
3. 1. Action in the mammalian body ............................................................................................. 14
3. 2. Action in the plant body ........................................................................................................ 17
4. Conclusion ..................................................................................................................................... 20
5. Bibliography .................................................................................................................................. 21
1. Introduction
Cytoskeleton has been shown to participate in almost every aspect in the life of a cell and the list of its
functions by far extends the original designation of cytoskeleton as a static "scaffold". Three essential
classes of cytoskeletal structures exist, one of them being microtubules, the other two being
microfilaments and intermediate filaments.
Microtubules are intracellular filamentous structures composed of α- and β-tubulin heterodimers. They
have been associated with many different physiological processes: cell shape, division and mitosis,
transport of intracellular vesicles and organelles (trafficking), cell signalling1 as well as cilia and
flagella formation.2 First reports of fibrillar structures in flagella date back to early 20th century, but
true visualization of microtubules has been achieved thanks to the progress of electron microscopy
during the 1950s and 1960s.3 Nowadays, microtubules are known to be present in all eukaryotic
species examined to date, including all animals, fungi and plants.4
The microtubular cytoskeleton is a powerful target for many naturally occurring and also synthetic
substances. They are often produced by various plants as a part of their defense strategy against
herbivorous predators. By altering the characteristics of microtubules in the cell, they have profound
impact on the cell physiology and survival. From the practical standpoint, the substances are often
used in medicine (anticancer treatment) and agriculture (weed control). The aim of this thesis is to
evaluate the mechanisms of action of these substances and explain why the plants that specialize in
producing these compounds exhibit considerable resistance against their effects.
1. 1. Microtubule structure and dynamics
The principal building blocks of microtubules are α- and β-tubulin subunits. Their structure is crucial
to understanding the binding of microtubule-active drugs. Both α- and β-tubulin are globular proteins
(55 kDa5 and ∼450 amino acids each6) with 36-42% amino acid identity7 and their 3D structures
consist of two internal β-sheets surrounded by several α-helices.8 The otherwise very compact model
of a tubulin subunit can be dissected into several structural domains. The N-terminal domain, made of
several closely packed α-helical and β-sheet loops, binds GTP and has an intrinsic GTPase activity.
The amino acid chain continues by a smaller intermediate (or "activation") domain. This is then
followed by two antiparallel α-helices running along the two subunits and thus forming an additional
C-terminal domain (see Figure 1).9,10 11
1
Figure 1: Ribbon structure of a bovine tubulin αβ-heterodimer,11 showing (a) view from the inside
of microtubule and (b) view from the outside of microtubule. α-tubulin (bottom) is shown binding
GTP and a putative zinc ion (grey), β-subunit (top) is shown in complex with paclitaxel and GDP.
PDB code 1jff, Löwe et al (2001).11
The bottom diagram linearly depicts the general organization of tubulin domains (three black
boxes), β-sheets (blue) and α-helices (red). Both are approximately aligned to the amino acid scale
(bottom ruler). β-barrels and α-helices extracted from Nogales et al (1998).8
In the cell, α- and β-subunits assemble into stable heterodimers (Kd ∼10−11 M), forming a significant
tubulin pool: 1-8 μM concentration of free heterodimers is normally present in cytosol,12 out of
∼20 μM total tubulin concentration in cells (determined for mouse 3T3 cells).13 In favorable
conditions, heterodimers assemble into microtubules, long hollow cylinders of 24 nm in diameter.
They consist of a variable number of protofilaments (often 13 in mammals), oriented in circle and
running lengthwise from one end of the microtubule to the other. All subunits present in the
protofilament chain are arranged head-to-tail and in such way that β-subunits always point towards a
so-called plus (+) end of the microtubule and α-subunits towards a minus (-) end. This effectively
means that microtubules are polar.14 C-terminal tubulin domains face the outer microtubule surface,
2
N-terminal GTPase domain is exposed to the inner surface and guanosine nucleotides are always
bound between the neighboring subunits (see Figure 1).6,10
In the cell, minus end of microtubules is typically anchored to the microtubule-organizing centre
(MTOC) while plus end grows towards the surrounding cytoplasm by adding heterodimer-GTP
subunits.15 As more and more subunits are added to the growing microtubule "lattice", subunits get
buried inside the microtubule structure. Contact between catalytic domains of α-subunit of one
heterodimer and β-subunit of the previous heterodimer causes the GTP in the β-subunit to hydrolyze
into GDP and inorganic phosphate (Pi). Most of the β-subunits in microtubules have GDP bound to
themselves, except for a so-called "GTP cap" at the microtubular plus end. As the hydrolysis is a
rather stochastic process, the GTP cap can sometimes be lost, causing a destabilization and subsequent
massive depolymerization at the microtubular plus end (a so-called catastrophe).14 The reversal of the
process is called a rescue. Both rescue and catastrophe can also happen at minus end, albeit less
often.15 These processes represent the molecular basis of microtubular dynamics: the speed of
microtubule growth varies between 5-20 μm/min,15 but the growth is discontinuous because
catastrophes occur approximately every 50 seconds (data from human tumor cell lines).16
In vivo, tubulin-based microtubules are accompanied by a variety of cytosolic proteins that are able to
bind or associate with microtubular cytoskeleton and often affect its stability one way or the other. Tau
protein, for example, is expressed in neural tissue where it stabilizes the axonal microtubules. Other
microtubule-associated proteins (MAPs) often play similar roles.17 In contrast, several proteins shift
the equilibrium towards microtubule depolymerization, including stathmin (OP18) and XKCM1.17
Similarly, katanin, spastin and fidgetin belong to a group of microtubule-severing enzymes that cut the
microtubules in an ATP-dependent fashion.18 A group of so-called motor proteins is able to transport
material along the microtubules and includes predominantly the kinesin and dynein superfamilies. 19
Apart from proteins, many natural substances can bind microtubules and affect their properties.
1. 2. Microtubule-active drugs
The diversity of microtubule-active natural substances is enormous and still continues to grow. The
canonical drugs include colchicine, paclitaxel (and related taxanes) and vinblastine (or other vinca
alkaloids), but several hundred of other similarly acting substances have been discovered,20 such as
cryptophycins, dolastatins, epithilones, discodermolides, halichondrin and many more. The list is
expected to grow even further because these compounds often show potent pesticidal, antiparasitic and
anticancer effects. As a matter of fact, paclitaxel (also called taxol) and vinca alkaloids are routinely
3
administered against a wide range of malignant tumors20 and these agents are hot topics of
biomedicine research (paclitaxel/taxol search, the most famous drug from the group, gives more than
24 000 results in PubMed).
The majority of these drugs are naturally occurring substances. Various plants or even animals (such
as marine sponges) produce them, probably to prevent their herbivorous predators from eating them20
or to defend against the parasites and pathogens.21 Vinblastine was isolated from periwinkle
(Catharanthus roseus, formerly Vinca rosea),22 colchicine was obtained from meadow saffron
(Colchicum autumnale)23 while paclitaxel comes from the bark of Pacific yew tree (Taxus brevifolia),
although it is now suspected to originate from a bark-living endophytic fungus (genus Taxomyces).24
All in all, microtubule poison-based strategy of defense is, because of its efficiency, a relatively
widespread phenomenon and can serve as an example of evolutionary convergence.25 The substances
are generally thought to mimic endogenous microtubular regulators.20 Their actual mechanism of
action will be discussed in the next chapter.
The common and defining feature of microtubule-active drugs is their affinity to αβ-tubulin
heterodimers. The majority of the microtubule-active substances bind to one of three "binding sites",
or pockets, on the surface of tubulin. The binding sites are named after their typical ligands – the
"taxane site", "vinca domain" and the "colchicine domain" (see Table 1 for a list of characteristic
ligands and Figure 2 for their binding sites).1 As for taxane site ligands, they occupy a pocket on the
luminal side of β-subunit.10 Vinca domain is located in the inter-dimer space between β-subunit of one
dimer and α-subunit of the following dimer,26 while colchicine domain ligands bind at the
intra-dimeric space between α- and β-subunit of one heterodimer, adjacent to the GTP of α-subunit.10
(27
Figure 2: Schematic image of two aligned αβ-heterodimers of a hypothetical microtubule
protofilament. Various binding sites are shown at their approximate places: G – GTP/GDP binding
site, C – colchicine binding site, V – vinca binding site, T – taxane binding site. Redrawn from
Fojo & Menefee (2005).27
4
Table 1: Microtubule-active agents can be roughly divided into three main categories based on
their binding site. Based on Jordan & Wilson (2004)1, if not stated otherwise.
Binding site
Example drugs
Taxane site
Paclitaxel (taxol), docetaxel, epithilones, discodermolide
Vinca domain
Vinca alkaloids (vinblastine, vincristine, vinorelbine, vinflunine),
cryptophycin 52, halichondrins, hemiasterlins
Colchicine
domain
Colchicine, combrestatins, 2-methoxyestradiol,
methoxybenzenesulphonamide; nocodazole28
28
There is a considerable diversity in the differential affinity of the substances towards free heterodimers
and, on the other hand, tubulin polymers (microtubules). Paclitaxel is an example of a substance which
preferentially binds to assembled microtubules along their length and reaches its binding site inside the
microtubules by passing through small openings (nanopores) in the microtubule cylinder29,30;
vinblastine binds best to the high-affinity sites at the microtubular plus end. On the contrary,
colchicine complexes better with soluble tubulin heterodimers.1
The significance of concentration of these drugs has been known for a long time31 and it is crucial not
only to the understanding of their correct dosage but also their actual mechanism of action, which can
differ enormously along the concentration range (see Chapter 2). In cell cultures, paclitaxel is known
to gradually accumulate in the cells, following an approximately hyperbolic curve and reaching a level
of equilibrium at some time (at least when we neglect the cell culture growth – which is greatly
diminished in higher concentrations of paclitaxel). The most potent influx and efflux mechanism of
paclitaxel and vinblastine is passive diffusion.32,33 The role of Mdr1 P-glycoprotein, a multidrug
resistance transporter, in active efflux of microtubule-active drugs has been studied extensively and
proves to be very important, especially in low (and thus clinically relevant) drug concentrations.34,35
Influx of paxlitaxel may36 or may not37 be partially mediated by OATP1B3 anion-transporting protein.
In vivo, these substances are usually either promptly detoxified by cytochrome 450 enzymes38 or
sometimes excreted into bile unchanged.39
It should be noted here that the intracellular concentration of microtubule-active drugs is always much
higher than concentration of drug added to the growth medium. The reason for this effect is high
affinity of intracellular binding sites for tubulin, compared to the extracellular non-specific binding
sites. Drugs may concentrate ten, hundred or almost thousand-fold in animal cell cultures. High
exposure to paclitaxel has lead to >200 µM concentration in cells (before any wash-out), far exceeding
the total tubulin binding capacity and even the water solubility of paclitaxel.32,34,40 Indeed, taxanes
5
have been even shown to crystallize under these conditions.41 Similarly, vincristine and vinblastine
accumulate intracellularly up to 60-fold.42 Corresponding results were acquired from 3D histocultures
with paclitaxel.43 However, there are very few or no in vivo studies of paclitaxel accumulation in
tumor cells in the complex environment of a mammalian body. One of the reasons for this is the
difficulty to measure intracellular concentration in a tissue sample composed not only of actual cells
but also extracellular matrix (M. A. Jordan, University of California, Santa Barbara, personal
communication).
6
2. Microtubule-active drugs: mechanism of action
Reports of colchicine, the first microtubule-active drug to be discovered, acting as a potent mitotic
disruptor caused a small revolution in the field of cytogenetics. Gradually, more and more substances
with various effects on mitotic spindles have been found and these compounds have been designated
as "microtubule-active drugs" to underline the basic structure that they have been thought to act on –
the microtubules. Indeed, the defining feature is their binding specificity for tubulin, the building
block of microtubules. Some of these substances have gradually found an important place in anticancer therapy and the basic mechanism of function seems resolved. However, after decades of
intensive studies, there are more questions than answers. How do they affect microtubules – and do
they have other targets besides microtubules? How do their effects cause cell death and subsequent
tumor regression? Indeed, it was frequently shown that effects are strongly concentration-dependent.
However, are their true in vivo effects comparable to results from in vitro studies?
2. 1. Effects on microtubule assembly
All microtubule-active substances are routinely divided into two groups: microtubule-stabilizing and
microtubule-destabilizing. Generally, vinca domain and colchicine domain binders (such as vinca
alkaloids and colchicine) belong to the microtubule-destabilizing group, while most taxane domain
binding agents (typically paclitaxel) belong to the group of microtubule-stabilizing drugs.20 The
nomenclature itself is self-explanatory. Paclitaxel was shown to promote microtubule assembly in
vitro at 0.25 µM concentration44 and similar effects were reported in mouse fibroblast cells with
10 µM paclitaxel in medium. The microtubules in this experiment were able to stay polymerized even
at 4 °C, a temperature which normally prevents assembly and disrupts existing microtubules.45 On the
other hand, colchicine and vinblastine are traditionally called microtubule-destabilizing because they
decrease the ratio of assembled to disassembled tubulin and, as a consequence, disrupt the mitotic
spindle.46,47
The key evidence for the "stabilizing/destabilizing" hypothesis comes from studies of several mutant
cell lines. They were artificially selected for resistance in presence of low doses of microtubule-active
drugs, either by single-dose exposure to a low drug concentration, or by sequentially exposing the cell
culture to higher and higher concentration of the drug. The problem is that this kind of treatment can
cause the cells to acquire more than one mutation and their phenotypes may thus be difficult to
interpret. Nevertheless, some of the cell lines were shown to have acquired mutations which increase
or diminish microtubule assembly.48 In 1989, F. Cabral’s laboratory reported isolation of several
7
paclitaxel and colcemid (an analog of colchicine) resistant cell lines. The paclitaxel-resistant cells
were hypothesized to have intrinsically decreased levels of assembled microtubules, so as to withstand
the stabilizing effect of paclitaxel. The same principle, but in reverse, would apply for colcemidresistant cell lines. In accordance with this hypothesis, paclitaxel-resistant cell lines were in general
colcemid-sensitive and colcemid-resistant strains were almost invariantly over-sensitive to paclitaxel.48
Paclitaxel-resistant cell lines were later shown to assemble their microtubules to a lesser amount and
paclitaxel-dependent cell lines (i. e. those that have to be continuously supplied with paclitaxel to
survive) assemble only 32% of the microtubular mass present in the wild-type cells.49 Several articles
point to a cross-resistance phenomenon. In principle, this means that cell lines resistant to one
microtubule-stabilizing drug are resistant to other drugs from the same group and the same applies to
the group of microtubule-destabilizing drugs.50,51 Similarly, it was shown that vinorelbine counteracts
the microtubule-polymerizing effect of paclitaxel in wild-type KB3-1 cells when administered in
appropriate amounts;52 this indicates that the two drugs interfere with microtubule assembly in
opposite ways.
A modification of the hypothesis has recently been proposed by Ganguly et al (2010) from F. Cabral’s
lab, who noticed an unusually high number of microtubule fragments (microtubules not attached to
MTOC) in paclitaxel-dependent chinese hamster ovary (CHO) cells grown without paclitaxel.53 These
fragments are reported to be very rare in the parental CHO cell line but are also effectively prevented
by adding sufficient amount of paclitaxel to the growth medium of the paclitaxel-dependent cell line.
The article suggests that paclitaxel reduces the correct amount of microtubule detachment from
centrosomes, needed for proper cell division and microtubule turnover. This effect is presumably
achieved by strengthening the lateral interactions between protofilaments, which prevents the
microtubules from breaking apart. On the other hand, vinblastine and colcemid are hypothesized to
increase the microtubule detachment, an effect that is also thought to be toxic for cells – although no
articles dealing with drugs other than paclitaxel in relation to microtubule detachment have been
published to date.
2. 2. Effects on microtubule dynamics
Although the presented stabilizing/destabilizing dichotomy seems very plausible considering the
above arguments, there is a significant number of scientists who oppose it. This group is best
represented by M. A. Jordan’s and L. Wilson’s lab, claiming that microtubule-active drugs act by
suppressing the microtubule dynamics rather than changing the polymer levels. As reviewed in
Correia & Lobert (2001),54 microtubule-active drugs – at least at some concentrations – indeed almost
8
invariantly decrease duration and rate of microtubular growth and shortening and often increase the
time that microtubules spend in pause state, not growing or shrinking (data from in vitro and cell line
studies). The effect of these drugs on microtubule dynamics can be best seen in microtubule
life-history plots (see Figure 3) which show suppressed dynamics in the presence of drugs.
55
Figure 3: Life history plots of microtubules in artificial cell-free systems, measured by videoenhanced differential interference contrast microscopy. The growth and shortening of individual
microtubules can be seen in the control sample and in presence of 0.4 μM vinblastine, a classic
vinca alkaloid that inhibits dynamics. Reprinted from Ngan et al. (2000).55
While the stabilizing/destabilizing hypothesis fails to explain specifically why polymer levels should
cause cell death and tumor regression,48 the hallmark of dynamic hypothesis is that it recognizes the
importance of microtubule dynamics for the correct course of mitosis. Microtubules of the mitotic
spindle, more then any other microtubules, need to be very dynamic in order to attach properly to the
kinetochores, align chromosomes at the metaphase plate and then separate the chromatids correctly in
the anaphase.1 Indeed, spindle microtubules are much more dynamic then the interphase microtubules
and the half-time of incorporation of tubulin subunits is 18-fold shorter in the mitotic cells, according
to studies using FRAP (fluorescent recovery after photobleaching).56 However, cell lines differ in their
microtubular dynamic parameters (the plus end dynamicity of BSC-1 cell line is almost three times
lower then in CHO cells57) and none of them have problems undergoing mitosis.
The dynamic hypothesis is supported by data from mutant cell lines as well. Human lung cancer cell
line A549 was selected for resistance to paclitaxel and two paclitaxel-dependent cell lines (striving in
2 nM paclitaxel) were isolated. Their dynamicity was increased by 57% and 167% in comparison to
the A549 cell line, when grown without paclitaxel, and the cells had difficulties completing mitosis
(mitotic index increased from 4.7% in the parent cell line to 22.6% in the paclitaxel-dependent cell
line grown without paclitaxel). Addition of paclitaxel to the medium decreased the mitotic index back
to 6.4%, but the microtubular dynamics of these "rescued" lines was not evaluated.58 The results were
9
later challenged when a similarly designed experiment by the opponents reported isolation of a much
stronger paclitaxel-dependent cell line (needing 200 nM paclitaxel) which showed slightly decreased –
definitely not increased - microtubule dynamics in the absence of paclitaxel. However, changes in
polymer levels were reported and presented as a proof for the destabilizing/stabilizing hypothesis.53
These two articles show clearly how misleading the usage of mutant cell lines can be as resistance
proves out to be a trait with diverse causality.
If all microtubule-active drugs act by suppressing dynamics, we can expect a synergistic (mutually
additive) effect of these drugs when applied together. Indeed, there are some reports that combinations
of "stabilizing" and "destabilizing" drugs are effective in treating cancer in clinical trials – such as a
combination of microtubule-stabilizing estramustine and microtubule-destabilizing vinorelbine.59
Additionally, a strong synergism of paclitaxel and vinorelbine was reported from melanoma cell lines
at various concentrations, including the extremely low ones (3 nM paclitaxel + 0.01 nM vinorelbine).60
Undeniably, both "stabilizing/destabilizing" and "dynamic" hypotheses are backed up by hard data and
both appear plausible. Efforts have been made to create a unifying theory; for example, it was
suggested that dynamics and polymer stability are closely linked and drugs change the balance
between growing plus-end conformation and disassembling plus-end conformation, at least in
drug-dependent cell lines.61 How this might apply to the mechanism of action in wild-type cell lines is
unclear. Similarly, the aforementioned hypothesis of microtubule detachment-based mechanism of
action53 might as well mean that "microtubule-stabilizing" drug paclitaxel decreases the microtubule
dynamics by reducing the number of detached microtubules. This shows that both hypotheses are
closer to each other then previously thought. There are general tendencies to claim that both models
are valid and contribute to the effects of microtubule-active drugs.62
The most attractive idea at the moment is that, while both mechanisms are biologically relevant, they
work at different drug concentrations. This has been shown many years ago. For example, low
concentrations of vinblastine in the growth medium (even as little as 0.1-0.3 nM) suppress the
microtubule dynamics by binding to high-affinity sites at the plus ends (Kd = 1-2 µM). Vinblastine
binds to low-affinity sites along the microtubule surface (Kd = 0.25 mM) in concentrations exceeding
10 nM. These concentrations lead to massive microtubule disassembly. Additionally, extremely high
vinblastine concentrations (growth medium concentrations of 10 µM or more) cause formation of
tubulin paracrystals, massive non-physiological aggregates (see Figure 4).15 An analogous dependence
on concentration has been reported for paclitaxel15 – 30 nM concentration reduced dynamicity by 31%
in Caov-3 cell line,16 but 80 nM concentrations increased the polymer levels in HeLa cells by 50% and
330 nM concentration of paclitaxel lead to a 5-fold increase in polymer mass.63
10
Figure 4: Changes in microtubule polymer mass at various concentrations of vinblastine, a drug
which depolymerizes microtubules at concentrations higher then ∼10 nM. Lower concentrations
reduce the microtubule dynamics without altering polymer levels. Reprinted from Jordan (2002).15
2. 3. Towards the cell death
Microtubule-active drugs have been exploited because of their ability to kill cancer cells and cause
tumor regression; this means that all potential mechanisms of action must be considered in terms of
their ability to cause cell death. It was shown that the effects of many of these substances can vary,
based principally on their cellular concentration. We could elucidate the true mechanism of action
against the cancer cells if we analyzed the cellular events at the working (cytotoxic) concentrations of
the microtubule-active drugs.
However, the scientific opinion is widely divided concerning the actual mechanism of cytotoxicity that
leads to cell death. Jordan and Wilson, in their experiments from the early 1990s, claimed that both
vinca alkaloids and paclitaxel work by targeting the dynamics of mitotic spindle microtubules and
effectively arresting cells at the metaphase/anaphase boundary of mitotic division. They have
repeatedly shown that the mitotic arrest is associated with suppressed microtubular dynamics rather
than polymer levels.31,42,63 On the other hand, experiments with paclitaxel-dependent cell lines have
shown that the maximum suppression of microtubule dynamics occurs after addition of 5-10 fold
lower amounts of paclitaxel then those actually needed to rescue the cells from cell death. This would
suggest that not disruption of dynamics, but possibly altered microtubule levels, is the actual cause of
11
cell death. However, the stabilizing/destabilizing model itself, as described in the chapter 2. 1., cannot
explain why microtubule function should be compromised in situations with altered polymer levels.64
In the past, various scenarios of cell death have been discussed in terms of action of
microtubule-active drugs (mostly studied on paclitaxel). There is general agreement that, upon drug
addition to the growth medium, cells enter a state of a prolonged mitotic arrest1,65 attributed to the
disruption of mitotic spindle. This is accompanied by chronic activation of spindle assembly
checkpoint (SAC). The specific molecular pathways leading to cell death are generally unknown and
many contradicting articles have been published. Single-cell imaging approaches show a profoundly
diverse response among the treated cancer cells: some cells die in mitosis, some complete mitosis but
die in the subsequent interphase, while other cells do not die until the second mitosis. It was suggested
that dying cells exhibit two fates: they either enter apoptosis, or slip into a "postmitotic phase", often
resulting in tetraploidy and subsequent delayed-type death. Cell fates varied profoundly among cell
lines and also depended on a type of microtubule-active drugs applied: nocodazole preferentially
caused temporary mitotic arrest, followed by several cycles of incomplete mitoses (endocycling) and
death, while paclitaxel treatment, in most cases, caused temporary mitotic arrest and then death in the
following interphase.66 In case of apoptosis, a classical pathway involving caspases is often reported
and inactivation of antiapoptotic Bcl2 and Bcl-xL proteins was often mentioned as an important step.
However, single-cell imaging shows that apoptotic pathways are very variable.67 It was also reported
that paclitaxel induces autophagy in human cell lines68 and it seems that, upon addition of paclitaxel,
autophagy competes with apoptosis.69
Additionally, paclitaxel was surprisingly shown to directly bind Bcl2 protein70 and, in this manner,
mimic the receptor Nur77.71 This nuclear receptor had already been known to interact with Bcl2 and
change the (typically) antiapoptotic nature of Bcl2 into proapoptotic by binding to a Bcl2 disordered
loop between BH3 and BH4 domains and exposing the BH3 domain.72 Similarities in paclitaxel/Nur77
common binding site on both tubulin and Bcl2 proteins were presented as one of the proofs for the
hypothesis, but key evidence comes from a successful immunoprecipitation of Bcl2-Nur77 complex
with anti-Bcl2 antibodies – this complex could, however, be displaced by paclitaxel, which competes
with Nur77 for a binding site at the Bcl2 disordered loop. Moreover, far-western blotting of Nur77
with Bcl2 was successful unless paclitaxel was added to the mix.71 All in all, the cellular equilibrium
between antiapoptotic (Bcl2) and proapoptotic signals (Bcl2+Nur77 or paclitaxel) may be shifted after
paclitaxel administration and the overall effect can thus differ dramatically, based on relative levels of
the proteins and factors. These new discoveries, if true, would revolutionize the whole field of
paclitaxel research; however, no similar interactions have been presented for other microtubule-active
drugs to date.
12
Much less has been written on the lethal effects of the microtubule-active drugs in plant cells or other
organisms. Cytological observations are usually limited to microtubule-active herbicides, as reviewed
in Gunning & Hardham (1982)73 and Vaughn & Lehnen (1991).74 For instance, dinitroaniline
herbicides were shown to depolymerize spindle and cortical microtubules and cause a prometaphase
arrest in root cells of wiregrass (Eleusine indica).75 Other effects include multipolar spindle formation
and occurrence of binucleate cells (cells with two nuclei) and similar phenomena were reported to
occur after application of several plant-produced microtubule-active substances (colchicine, vinca
alkaloids).76
13
3. Microtubule-active drugs in the context of a body
While the previous chapter was a general summary of molecular mechanisms and cellular changes
upon administration of microtubule-active drugs, the aim of this passage is to evaluate the complex
interactions which occur in the environment of a multicellular body. Summary of our knowledge about
actions in mammals (usually investigated or extrapolated to the human body) and in plants is
presented to allow comparison.
3. 1. Action in the mammalian body
The lack of knowledge about behavior of microtubule-active drugs in the human body is bewildering.
Almost all information on the mechanism of action of these substances comes from in vitro studies
with cell lines. While the phenotype of final cell fate (mitotic arrest and apoptosis) remains very
comparable "in vivo" as in animal cell lines,67,77 the mechanism of cell poisoning may be a different
story.
Some researchers think that the situation in the mammalian body gives credit to the "dynamic"
hypothesis, claiming that changes in polymer levels are unlikely, given the low concentrations of drug
present in the interstitial fluid,61 or that the effects on dynamics are very profound even in minute
concentrations (8 nM paclitaxel), significantly less than is commonly achieved in the blood plasma of
cancer patients.63 The situation is unclear because, for example, fairly high paclitaxel concentrations of
0.1-10 µM have reportedly been achieved in human plasma by 2- to 24-hours infusions.78 Similarly,
vincristine is known to reach maximal serum concentrations of ∼1 µM and other vinca alkaloids just a
little less than that.79
Latest approaches of physical chemistry, such as coherent anti-Stokes Raman scattering microscopy
and mass spectrometry (MS) imaging have been used to analyze the tissue distribution of microtubule
active drugs. While Raman scattering microscopy suffers from low detection limits (29 mM for
paclitaxel80), MS imaging has a potential to shed light on drug distribution in whole-body samples.
Mass spectrometer, using MALDI (matrix-assisted laser desorption/ionization) combined with ion
mobility separation (IMS), was used to analyze vinblastine distribution in rat whole-body sections.
During the procedure, a thin sample of tissue is covered by an organic acid-based "matrix" and ionized
with laser beam, which gradually scans the whole tissue surface. High concentrations of vinblastine
were found in liver, renal cortex and tissue surrounding the guts, suggesting the predominant routes of
vinblastine detoxification and excretion. Similar results were obtained from a quantitative whole-body
14
autoradiography (QWBA) experiment using 3H-vinblastine (see Figure 5).81 However, no whole-body
drug distribution measurements of tumor-bearing mice treated with microtubule-active drugs have
been conducted.
Figure 5: Whole-body sections of rat. (A) Mass spectrometry image of rat body section;
distribution of m/z 811 ion (specific for vinblastine) in the various organs of the rat after
vinblastine administration (bright = signal). Arrow points to renal pelvis where a weak interfering
signal was located. (B) Autoradiography photograph after 3H-vinblastine administration, showing
comparable results. Adjusted contrast, dark = signal. Both images from Trim et al (2008).81
(A)
(B)
The truth is that we cannot even be sure that microtubule-active drugs primarily disrupt mitosis in
vivo. Human tumors are different from in vitro cell lines and xenograft tumors: luckily for cancer
patients, they have much longer doubling times (the median doubling time of analyzed human tumors
was 147 days compared to 1–12 days for xenografts and ∼33 hours for human cell lines).82 A tumor is
a complicated system in which only a low percentage (<1%) of cells is dividing at a time and it would
thus seem that microtubule-active drugs would be unable to eliminate the tumor only by targeting
mitosis.83 This paradox can, however, be explained by an efficient intracellular accumulation of drugs
upon their administration: once the cell eventually enters mitosis, the drug remains in concentrations
sufficient to cause cell death.84 However, this explanation seems to be in contradiction with biopsies
obtained from cancer patients treated with epithilones (ixabepilone): the microtubules are severely
stabilized but there are very few cells arrested in mitosis82 – but that could still mean that some cells
15
probably die in the subsequent interphase84 (as indeed documented by single-cell imaging in cell
lines66).
If we accept these assumptions, a question arises why administration of microtubule-active drugs
should be specific enough to cause elimination of the tumor – indeed, there are many cell types in a
human body which proliferate at an incredible rate (and sure, myelosuppression occurs commonly in
patients treated with these substances1). Some researchers claim that, in fact, tumor cells do proliferate
at an incomparable speed (with a mean cell cycle duration of only 48 hours85) but a large portion of
them dies – leading to the long doubling time of solid tumors.84 An alternative explanation, however,
can be formed: the primary target of microtubule-active drugs in tumors is not a mitotic spindle and
these substances rather act on some of the interphase functions of microtubules, namely vesicular
trafficking. According to this reasoning, the drugs affect transport and signaling with microtubuleassociated tumor suppressors such as p53, BRCA1, Rb and androgen receptor.82 For instance, p53 is
transported by dynein towards the nucleus but the nuclear accumulation of the protein is impaired
when the cells are treated with paclitaxel or vincristine.86 Cancer cells are purportedly "especially
sensitive" to the disruption of above-mentioned microtubule-associated signaling proteins82 (because
they must often resist the stressful conditions which push them towards apoptosis – hypoxia,
withdrawal of growth factors, lack of anchorage87 – and must do everything to escape it).
There is at least one real "in vivo" situation which clearly shows that microtubule-active drugs do not
exclusively target cells in mitosis: the neurons - a practically non-dividing cell type. One of the side
effects of cancer therapy associated with this class of drugs is the high prevalence of
therapy-associated neuropathy (seen in 70–90% of patients) Sensory neurons are usually affected,
frequently causing a limb pain.82 Histologically, nerve fiber loss, axonal atrophy and demyelination
have been observed in human peripheral neuronal tissue,88 suggesting a considerable tissue damage.
Microtubule bundles and aggregates have also been reported.89 Additionally, neuronal cell cultures
subjected to paclitaxel show massive microtubule reconfiguration (switches of the plus/minus
directionality) and impaired vesicular transport in the axons.90 This set of effects clearly demonstrates
how complex the body response to microtubule-active drugs is.
Similar conclusions could be drawn from the well-known effects of microtubule-active drugs on blood
capillaries and their endothelium. It is assumed that the drugs affect some of the interphase functions
of endothelial microtubules – either they might disrupt the microtubule-microfilament cooperation in
cell shaping and migration,91 or they possibly block the signaling pathway of VEGF (vascular
endothelial growth factor) receptor.92
16
Other studies link anticancer activity of microtubule-active drugs to a completely different
phenomenon: selective specificity towards regulatory T-cells (Treg). This subgroup of lymphocytes,
among other functions, potently inhibits effector T cells and thus suppresses cellular immunity against
cancer cells. Paclitaxel was shown to induce Bcl2/Bax mediated apoptosis of Treg cells and it was
suggested that this effect leads to induction of anticancer immunity.93,94
3. 2. Action in the plant body
Plants, especially weeds, are a frequent target of various microtubule-active substances. As a matter of
fact, approximately one quarter of all herbicides acts (directly or indirectly) on microtubules.74
Additionally, several plants produce their own microtubule-active substances in order to protect from
their herbivores and these plant species are thus naturally subjected to high concentrations of their
"own" poisons. The latter phenomenon can be used to compare mammalian and plant sensitivity to
microtubule-active drugs. The evolutionary solution of producing a poison on one hand and not being
poisoned on the other hand could be very inspirational for intelligent drug design. Indeed,
administration of safe, activable compounds is a hot topic of anticancer treatment.
Generally, plants are less susceptible to plant-produced microtubule-active drugs, such as colchicine,
vinblastine or podophyllotoxin, than animals.95 The substances must be administrated in higher
concentrations in order to elicit a comparable cellular response. Colchicine working concentrations in
plants are generally 10–1000× higher than in animals73 and the relatively high affinity of colchicine
towards animal tubulin has been associated with two kingdom-specific amino acid residues in the
colchicine binding region – Pro 268 and Ala 248 of β-tubulin, substituted by Val/Ile and Ser,
respectively, in non-animal species. These substitutions are suspected to cause significant
rearrangements in the tubulin molecule and impair the binding of colchicine.96 Surprisingly, paclitaxel
differs from the above-mentioned compounds because it is equally effective in plant and animal
cells.95 Some studies claim that the paclitaxel binding site is more conserved then the colchicine
binding site, which supposedly diverged among plants and animals.97 A compelling idea is that, in
fact, paclitaxel is an "unknown" molecule for both animal and plant cells because this compound is
synthesized inside the bark-living endophytic fungi.24
Still, some plant species are considered less sensitive to certain mitotic poisons, such as in the case of
paclitaxel and onion (Allium); at the current level of understanding, it can only be hypothetized that
the onion plant itself has a tubulin binding protein which competes with paclitaxel at its binding site.95
On the other hand, plants are generally more susceptible to microtubule-active herbicides; for instance,
17
oryzaline and dinitroaniline herbicides have no effects on microtubules and mitosis in the animal cells;
this has been attributed to differences in animal and plant tubulin.74
Relatively few is known about kinetics and dynamics of microtubule-active compounds inside the
plant bodies. In Levan's pre-war pioneering works, the substances were applied to plant by soaking the
roots in a solution of the drug; this method is, in fact, still in use.98 Endogenous alkaloids are produced
in a certain plant organ or structure (seeds, bark, leaves) but can sometimes be transported to an
another organ where they often accumulate in the cell vacuoles. Some alkaloids can freely pass the
vacuolar membrane (tonoplast) but, once inside this acidic compartment, are protonated and
effectively trapped.99 While this may apply to vinblastine and other alkaloids, paclitaxel has no
ionizable side groups100 and cannot be easily trapped inside the vacuole. High amounts of paclitaxel
are therefore found in the plant cell wall.101
In any case, the producer plants clearly must have a mechanism to escape the effects of microtubuleactive drugs. Considering the importance of this phenomenon (which might help us develop more
powerful or more selective drugs), it comes as a surprise that we do not know very much about it and
only a few articles on this topic have been published to date.
The first mitotic poison to be discovered – and subsequently studied – was colchicine, an alkaloid
produced by the meadow saffron (Colchicum). Very soon, scientists began to wonder why Colchicum
is unaffected even though the plant itself contains as much as 0.4% colchicine by dry weight. In 1939,
Albert F. Blakeslee compared the situation to the "snake and snake's venom" conundrum and
expressed a belief that Colchicum contains an antidote102 (which, although theoretically possible, has
never been found in the plant). Further studies have shown that the resistance to colchicine is very
specific – Colchicum is sensitive to other mitotic poisons (acenaphthene).103 The molecular mechanism
for Colchicum resistance to colchicine is not known95 and no relevant publications have been
published to date.
Several instances show that the plant resistance to its poison can be conferred by its pharmacokinetic
and pharmacodynamic characteristics of the drug inside the tissues of drug-producing plants. In the
case of Vinca rosea, which is at least 100–1000x less sensitive to vinblastine than the control species
(peppergrass – Lepidium, Brassicaceae), it is hypothesized that intracellular vinblastine is in its
inactive form, which is secreted from the cells and, during or after secretion, modified to become the
active vinblastine molecule.104 This hypothesis, however, fails to explain why the active vinblastine
would not reenter the cells. A similar, yet more sophisticated scenario has been suggested for the
resistance of Podophyllum peltatum to podophyllotoxin. This substance is present in the plant vacuoles
18
as inactive glucoside (podophyllotoxin-4-O-β-D-glucopyranoside) and thus cannot harm the plant. At
the same time, however, a highly specific β-glucosidase is present in the cytosol but is practically
inactive because its pH optimum lies in acidic values (5.0, compare with ∼ pH 7.0 in the cytosol).
Only when insects feed on the Podophyllum plant, the cells are damaged, vacuolar membrane is
disrupted and the enzyme comes into contact with its substrate. The acidic pH of the mix leads to rapid
deglucosidation and activation of the podophyllotoxin, effectively poisoning the herbivorous
insects.105
There are other solutions to being resistant to one's own poisons. One of them is to alter the molecular
target of the drug and thus escape its deleterious effects. One of the possible explanations for Vinca
rosea resistance to vinblastine is that the vinca domain in tubulin is mutated to prevent the vinblastine
from binding to it.104 This has, however, never been proved for vinblastine and, until recently, for any
other tubulin-binding compound. As a part of the 1000 Plants Initiative (1KP), European yew tree
(Taxus baccata) transcriptome was sequenced and its tubulin gene has recently been analyzed to allow
comparison with other tubulins. Results show that the yew tree tubulin is highly mutated in the
paclitaxel binding region when compared with a homologous human tubulin gene. 65% of all
substitutions in α- and β-tubulins are located on the surface of β-tubulin; moreover, 95% of these are
substitutions on the luminal side of β-tubulin (where paclitaxel binding site is found). Some of the
mutations are located adjacent to the nanopores which form in the microtubule wall and allow
diffusion of paclitaxel towards the lumen of the microtubule, but subsequent analysis has shown that
the size of these openings is not significantly altered by these substitutions.106 Massive analyses are
currently conducted on other plants such as Colchicum (J. A. Tuszynski, University of Alberta,
personal communication).
19
4. Conclusion
The constant thirst for new chemotherapeutic agents and substantial side effects of the presently
available microtubule-active drugs have provoked an intensive search for the true mechanism of action
and specificity of these substances. Altered polymer levels and microtubular dynamics are two
measurable effects of microtubule-active drugs. Latest high-throughput or in vivo experiments often
challenge the traditional views about the mechanism of action and new hypotheses have been formed
concerning the role of variable cell fate,66 Bcl2 dysregulation and molecular mimicry,71 disruption of
intracellular trafficking82 or induction of cytotoxic lymphocytes94 in the course of cancer treatment.
Many of these findings will certainly play an important role in future research. Similarly, a lot remains
unknown about pharmacokinetics and pharmacodynamics of microtubule-active drugs; a great wealth
of information can be acquired from in vivo studies using model animals or human biopsies. The
approaches of mass spectrometry imaging81, which can measure drug concentrations without a need
for fluorescent derivatives or radioactive labeling, will certainly find their place in modern drug
development and assessment.
Interestingly, important data may come from observing nature – more specifically, how it is possible
that some organisms are evidently immune towards the effects of these compounds. The plants which
produce these compounds, such as Colchicum, Podophyllum or Taxus, are the best current models of
this phenomenon. A few means of escaping the deleterious effects of microtubule disruption have
already been described in these medicinal plants and, as more information is gathered, it will open the
door to a more rational drug design and delivery.
20
5. Bibliography
1. Jordan, M. A. & Wilson, L. Microtubules as a target for anticancer drugs. Nat. Rev. Cancer 4,
253–265 (2004).
2. Silflow, C. D. & Lefebvre, P. A. Assembly and Motility of Eukaryotic Cilia and Flagella. Lessons
from Chlamydomonas reinhardtii. Plant Physiol. 127, 1500–1507 (2001).
3. Wells, W. A. Microtubules get a name. J Cell Biol 168, 852–853 (2005).
4. Dutcher, S. K. Long-lost relatives reappear: identification of new members of the tubulin
superfamily. Curr. Opin. Microbiol. 6, 634–640 (2003).
5. Bryan, J. & Wilson, L. Are Cytoplasmic Microtubules Heteropolymers? Proc Natl Acad Sci U S A
68, 1762–1766 (1971).
6. Downing, K. H. & Nogales, E. Tubulin and microtubule structure. Curr. Opin. Cell Biol. 10, 16–
22 (1998).
7. Little, M. & Seehaus, T. Comparative analysis of tubulin sequences. Comparative Biochemistry
and Physiology Part B: Comparative Biochemistry 90, 655–670 (1988).
8. Nogales, E., Wolf, S. G. & Downing, K. H. Structure of the alpha beta tubulin dimer by electron
crystallography. Nature 391, 199–203 (1998).
9. Downing, K. H. & Nogales, E. Tubulin structure: insights into microtubule properties and
functions. Curr. Opin. Struct. Biol. 8, 785–791 (1998).
10. Amos, L. A. What tubulin drugs tell us about microtubule structure and dynamics. Semin. Cell
Dev. Biol. 22, 916–926 (2011).
11. Löwe, J., Li, H., Downing, K. H. & Nogales, E. Refined structure of alpha beta-tubulin at 3.5 A
resolution. J. Mol. Biol. 313, 1045–1057 (2001).
12. Caplow, M. & Fee, L. Dissociation of the Tubulin Dimer Is Extremely Slow, Thermodynamically
Very Unfavorable, and Reversible in the Absence of an Energy Source. Mol Biol Cell 13, 2120–
2131 (2002).
13. Hiller, G. & Weber, K. Radioimmunoassay for tubulin: a quantitative comparison of the tubulin
content of different established tissue culture cells and tissues. Cell 14, 795–804 (1978).
14. Conde, C. & Cáceres, A. Microtubule assembly, organization and dynamics in axons and
dendrites. Nature Reviews Neuroscience 10, 319–332 (2009).
15. Jordan, M. A. Mechanism of action of antitumor drugs that interact with microtubules and tubulin.
Curr Med Chem Anticancer Agents 2, 1–17 (2002).
16. Yvon, A.-M. C., Wadsworth, P. & Jordan, M. A. Taxol Suppresses Dynamics of Individual
Microtubules in Living Human Tumor Cells. Mol. Biol. Cell 10, 947–959 (1999).
17. Bhat, K. M. R. & Setaluri, V. Microtubule-Associated Proteins as Targets in Cancer
Chemotherapy. Clin Cancer Res 13, 2849–2854 (2007).
18. Roll-Mecak, A. & McNally, F. J. Microtubule severing enzymes. Curr Opin Cell Biol 22, 96
(2010).
19. Caviston, J. P. & Holzbaur, E. L. F. Microtubule motors at the intersection of trafficking and
transport. Trends Cell Biol. 16, 530–537 (2006).
20. Dumontet, C. & Jordan, M. A. Microtubule-binding agents: a dynamic field of cancer
therapeutics. Nat Rev Drug Discov 9, 790–803 (2010).
21. Wagner, L. J. Effect of taxol and related compounds on growth of plant pathogenic fungi.
Phytopathology 84, 1173–1178 (1994).
22. Noble, R. L., Beer, C. T. & Cutts, J. H. Role of chance observations in chemotherapy: Vinca
rosea. Ann. N. Y. Acad. Sci. 76, 882–894 (1958).
23. Sapra, S. et al. Colchicine and its various physicochemical and biological aspects. Med Chem Res
22, 531–547 (2013).
24. Stierle, A., Strobel, G. & Stierle, D. Taxol and taxane production by Taxomyces andreanae, an
endophytic fungus of Pacific yew. Science 260, 214–216 (1993).
25. Goodin, S., Kane, M. P. & Rubin, E. H. Epothilones: Mechanism of Action and Biologic Activity.
JCO 22, 2015–2025 (2004).
26. Cormier, A., Marchand, M., Ravelli, R. B. G., Knossow, M. & Gigant, B. Structural insight into
the inhibition of tubulin by vinca domain peptide ligands. EMBO Rep 9, 1101–1106 (2008).
21
27. Fojo, A. T. & Menefee, M. Microtubule Targeting Agents: Basic Mechanisms of Multidrug
Resistance (MDR). Seminars in Oncology 32, 3–8 (2005).
28. Sharma, S. et al. Characterization of the Colchicine Binding Site on Avian Tubulin Isotype BVI.
Biochemistry 49, 2932–2942 (2010).
29. Ross, J. L. & Fygenson, D. K. Mobility of Taxol in Microtubule Bundles. Biophys J 84, 3959–
3967 (2003).
30. Freedman, H., Huzil, J. T., Luchko, T., Ludue a, R. F. & Tuszynski, J. A. Identification and
Characterization of an Intermediate Taxol Binding Site Within Microtubule Nanopores and a
Mechanism for Tubulin Isotype Binding Selectivity. J. Chem. Inf. Model. 49, 424–436 (2009).
31. Jordan, M. A., Thrower, D. & Wilson, L. Effects of vinblastine, podophyllotoxin and nocodazole
on mitotic spindles. Implications for the role of microtubule dynamics in mitosis. J. Cell. Sci. 102,
401–416 (1992).
32. Kuh, H. J., Jang, S. H., Wientjes, M. G. & Au, J. L. Computational model of intracellular
pharmacokinetics of paclitaxel. J. Pharmacol. Exp. Ther. 293, 761–770 (2000).
33. Maswadeh, H. et al. A molecular basis explanation of the dynamic and thermal effects of
vinblastine sulfate upon dipalmitoylphosphatidylcholine bilayer membranes. Biochim. Biophys.
Acta 1567, 49–55 (2002).
34. Jang, S. H., Wientjes, M. G. & Au, J. L.-S. Interdependent effect of P-glycoprotein-mediated drug
efflux and intracellular drug binding on intracellular paclitaxel pharmacokinetics: application of
computational modeling. J. Pharmacol. Exp. Ther. 304, 773–780 (2003).
35. Hunter, J., Jepson, M. A., Tsuruo, T., Simmons, N. L. & Hirst, B. H. Functional expression of Pglycoprotein in apical membranes of human intestinal Caco-2 cells. Kinetics of vinblastine
secretion and interaction with modulators. J. Biol. Chem. 268, 14991–14997 (1993).
36. Takano, M. et al. Paclitaxel-resistance conferred by altered expression of efflux and influx
transporters for paclitaxel in the human hepatoma cell line, HepG2. Drug Metab. Pharmacokinet.
24, 418–427 (2009).
37. Yamaguchi, H. et al. Rapid screening of antineoplastic candidates for the human organic anion
transporter OATP1B3 substrates using fluorescent probes. Cancer Lett. 260, 163–169 (2008).
38. Yao, D., Ding, S., Burchell, B., Wolf, C. R. & Friedberg, T. Detoxication of Vinca Alkaloids by
Human P450 CYP3A4-Mediated Metabolism: Implications for the Development of Drug
Resistance. J Pharmacol Exp Ther 294, 387–395 (2000).
39. Leamon, C. P. et al. Reducing Undesirable Hepatic Clearance of a Tumor-Targeted Vinca
Alkaloid via Novel Saccharopeptidic Modifications. J Pharmacol Exp Ther 336, 336–343 (2011).
40. Jordan, M. A. et al. Mitotic Block Induced in HeLa Cells by Low Concentrations of Paclitaxel
(Taxol) Results in Abnormal Mitotic Exit and Apoptotic Cell Death. Cancer Res 56, 816–825
(1996).
41. Foss, M., Wilcox, B. W. L., Alsop, G. B. & Zhang, D. Taxol Crystals Can Masquerade as
Stabilized Microtubules. PLoS ONE 3, e1476 (2008).
42. Jordan, M. A., Thrower, D. & Wilson, L. Mechanism of inhibition of cell proliferation by Vinca
alkaloids. Cancer Res. 51, 2212–2222 (1991).
43. Kuh, H.-J., Jang, S. H., Wientjes, M. G., Weaver, J. R. & Au, J. L.-S. Determinants of Paclitaxel
Penetration and Accumulation in Human Solid Tumor. J Pharmacol Exp Ther 290, 871–880
(1999).
44. Schiff, P. B., Fant, J. & Horwitz, S. B. Promotion of microtubule assembly in vitro by taxol.
Nature 277, 665–667 (1979).
45. Schiff, P. B. & Horwitz, S. B. Taxol stabilizes microtubules in mouse fibroblast cells. Proc. Natl.
Acad. Sci. U.S.A. 77, 1561–1565 (1980).
46. George, P., Journey, L. J. & Goldstein, M. N. Effect of vincristine on the fine structure of HeLa
cells during mitosis. J. Natl. Cancer Inst. 35, 355–375 (1965).
47. Vandecandelaere, A., Martin, S. R. & Engelborghs, Y. Response of microtubules to the addition
of colchicine and tubulin-colchicine: evaluation of models for the interaction of drugs with
microtubules. Biochem J 323, 189–196 (1997).
48. Cabral, F. & Barlow, S. B. Mechanisms by which mammalian cells acquire resistance to drugs
that affect microtubule assembly. FASEB J 3, 1593–1599 (1989).
22
49. Barlow, S. B., Gonzalez-Garay, M. L. & Cabral, F. Paclitaxel-dependent mutants have severely
reduced microtubule assembly and reduced tubulin synthesis. J. Cell. Sci. 115, 3469–3478 (2002).
50. Schibler, M. J. & Cabral, F. R. Maytansine-resistant mutants of Chinese hamster ovary cells with
an alteration in alpha-tubulin. Can. J. Biochem. Cell Biol. 63, 503–510 (1985).
51. Gupta, R. S. Podophyllotoxin-resistant Mutants of Chinese Hamster Ovary Cells: CrossResistance Studies with Various Microtubule Inhibitors and Podophyllotoxin Analogues. Cancer
Res 43, 505–512 (1983).
52. Giannakakou, P., Villalba, L., Li, H., Poruchynsky, M. & Fojo, T. Combinations of paclitaxel and
vinblastine and their effects on tubulin polymerization and cellular cytotoxicity: characterization
of a synergistic schedule. Int. J. Cancer 75, 57–63 (1998).
53. Ganguly, A., Yang, H. & Cabral, F. Paclitaxel dependent cell lines reveal a novel drug activity.
Mol Cancer Ther 9, 2914–2923 (2010).
54. Correia, J. J. & Lobert, S. Physiochemical aspects of tubulin-interacting antimitotic drugs. Curr.
Pharm. Des. 7, 1213–1228 (2001).
55. Ngan, V. K. et al. Novel Actions of the Antitumor Drugs Vinflunine and Vinorelbine on
Microtubules. Cancer Res 60, 5045–5051 (2000).
56. Saxton, W. M. et al. Tubulin dynamics in cultured mammalian cells. J. Cell Biol. 99, 2175–2186
(1984).
57. Yvon, A. M. & Wadsworth, P. Non-centrosomal microtubule formation and measurement of
minus end microtubule dynamics in A498 cells. J. Cell. Sci. 110, 2391–2401 (1997).
58. Gonçalves, A. et al. Resistance to Taxol in lung cancer cells associated with increased
microtubule dynamics. Proc Natl Acad Sci U S A 98, 11737–11742 (2001).
59. Borrega, P. et al. Phase II trial of vinorelbine and estramustine in the treatment of metastatic
hormone-resistant prostate cancer. Urologic Oncology: Seminars and Original Investigations 22,
32–35 (2004).
60. Photiou, A., Shah, P., Leong, L. K., Moss, J. & Retsas, S. In vitro synergy of paclitaxel (Taxol)
and vinorelbine (navelbine) against human melanoma cell lines. Eur. J. Cancer 33, 463–470
(1997).
61. Correia, J. J. & Lobert, S. Molecular Mechanisms of Microtubule Acting Cancer Drugs. The Role
of Microtubules in Cell Biology, Neurobiology, and Oncology 21–46 (2008).
62. Kamath, K., Wilson, L., Cabral, F. & Jordan, M. A. βIII-Tubulin Induces Paclitaxel Resistance in
Association with Reduced Effects on Microtubule Dynamic Instability. J. Biol. Chem. 280,
12902–12907 (2005).
63. Jordan, M. A., Toso, R. J., Thrower, D. & Wilson, L. Mechanism of mitotic block and inhibition
of cell proliferation by taxol at low concentrations. Proc Natl Acad Sci U S A 90, 9552–9556
(1993).
64. Ganguly, A. & Cabral, F. New insights into mechanisms of resistance to microtubule inhibitors.
Biochim Biophys Acta 1816, 164–171 (2011).
65. Rieder, C. L. & Maiato, H. Stuck in Division or Passing through: What Happens When Cells
Cannot Satisfy the Spindle Assembly Checkpoint. Developmental Cell 7, 637–651 (2004).
66. Gascoigne, K. E. & Taylor, S. S. Cancer Cells Display Profound Intra- and Interline Variation
following Prolonged Exposure to Antimitotic Drugs. Cancer Cell 14, 111–122 (2008).
67. Shi, J., Orth, J. D. & Mitchison, T. Cell type variation in responses to antimitotic drugs that target
microtubules and kinesin-5. Cancer Res. 68, 3269–3276 (2008).
68. Zhang, X., Chen, L.-X., Ouyang, L., Cheng, Y. & Liu, B. Plant natural compounds: targeting
pathways of autophagy as anti-cancer therapeutic agents. Cell Prolif. 45, 466–476 (2012).
69. Xi, G. et al. Autophagy inhibition promotes paclitaxel-induced apoptosis in cancer cells. Cancer
Lett. 307, 141–148 (2011).
70. Rodi, D. J. et al. Screening of a library of phage-displayed peptides identifies human Bcl-2 as a
taxol-binding protein. Journal of Molecular Biology 285, 197–203 (1999).
71. Ferlini, C. et al. Paclitaxel Directly Binds to Bcl-2 and Functionally Mimics Activity of Nur77.
Cancer Res 69, 6906–6914 (2009).
72. Lin, B. et al. Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan
receptor Nur77/TR3. Cell 116, 527–540 (2004).
23
73. Gunning, B. E. S. & Hardham, A. R. Microtubules. Annual Review of Plant Physiology 33, 651–
698 (1982).
74. Vaughn, K. & Lehnen, L. Mitotic Disrupter Herbicides. Weed Sci. 39, 450–457 (1991).
75. Vaughn, K. C. Cytological studies of dinitroaniline-resistant Eleusine. Pesticide Biochemistry and
Physiology 26, 66–74 (1986).
76. Vaughan, M. A. & Vaughn, K. C. Mitotic disrupters from higher plants and their potential uses as
herbicides. Weed technology : a journal of the Weed Science Society of America 2, 533–539
(1988).
77. Milross, C. G. et al. Relationship of Mitotic Arrest and Apoptosis to Antitumor Effect of
Paclitaxel. JNCI J Natl Cancer Inst 88, 1308–1314 (1996).
78. Sonnichsen, D. S. & Relling, M. V. Clinical pharmacokinetics of paclitaxel. Clin Pharmacokinet
27, 256–269 (1994).
79. Levêque, D. & Jehl, F. Molecular pharmacokinetics of catharanthus (vinca) alkaloids. J Clin
Pharmacol 47, 579–588 (2007).
80. Kang, E. et al. In situ visualization of paclitaxel distribution and release by coherent anti-Stokes
Raman scattering microscopy. Anal. Chem. 78, 8036–8043 (2006).
81. Trim, P. J. et al. Matrix-Assisted Laser Desorption/Ionization-Ion Mobility Separation-Mass
Spectrometry Imaging of Vinblastine in Whole Body Tissue Sections. Anal. Chem. 80, 8628–
8634 (2008).
82. Komlodi-Pasztor, E. Mitosis is not a key target of microtubule agents in patient tumors. Nature
reviews. Clinical oncology 8, 244–50 (2011).
83. Komlodi-Pasztor, E., Sackett, D., Wilkerson, J. & Fojo, T. (Not) too early to say, ‘no targeting of
mitosis!’ Reply. Nat. Rev. Clin. Oncol. 8, (2011).
84. Kitagawa, K. Too early to say, ‘no targeting of mitosis!’ Nat Rev Clin Oncol 8, 444; author reply
444 (2011).
85. Tubiana, M. & Malaise, E. Comparison of cell proliferation kinetics in human and experimental
tumors: response to irradiation. Cancer Treat Rep 60, 1887–1895 (1976).
86. Giannakakou, P. et al. p53 is associated with cellular microtubules and is transported to the
nucleus by dynein. Nat. Cell Biol. 2, 709–717 (2000).
87. Brown, J. M. & Wouters, B. G. Apoptosis, p53, and Tumor Cell Sensitivity to Anticancer Agents.
Cancer Res 59, 1391–1399 (1999).
88. Sahenk, Z., Barohn, R., New, P. & Mendell, J. R. Taxol neuropathy. Electrodiagnostic and sural
nerve biopsy findings. Arch. Neurol. 51, 726–729 (1994).
89. Scripture, C. D., Figg, W. D. & Sparreboom, A. Peripheral Neuropathy Induced by Paclitaxel:
Recent Insights and Future Perspectives. Curr Neuropharmacol 4, 165–172 (2006).
90. Shemesh, O. A. & Spira, M. E. Paclitaxel induces axonal microtubules polar reconfiguration and
impaired organelle transport: implications for the pathogenesis of paclitaxel-induced
polyneuropathy. Acta Neuropathol. 119, 235–248 (2010).
91. Schwartz, E. L. Antivascular actions of microtubule-binding drugs. Clin. Cancer Res. 15, 2594–
2601 (2009).
92. Lu, H., Murtagh, J. & Schwartz, E. L. The Microtubule Binding Drug Laulimalide Inhibits
Vascular Endothelial Growth Factor-Induced Human Endothelial Cell Migration and Is
Synergistic when Combined with Docetaxel (Taxotere). Mol Pharmacol 69, 1207–1215 (2006).
93. Zhang, L. et al. Differential impairment of regulatory T cells rather than effector T cells by
paclitaxel-based chemotherapy. Clinical Immunology 129, 219–229 (2008).
94. Liu, N., Zheng, Y., Zhu, Y., Xiong, S. & Chu, Y. Selective Impairment of
CD4+CD25+Foxp3+Regulatory T cells by paclitaxel is explained by Bcl-2/Bax mediated
apoptosis. Int. Immunopharmacol. 11, 212–219 (2011).
95. Vaughn, K. & Vaughan, M. Mitotic Disrupters from Higher-Plants - Effects on Plant-Cells. Acs
Symposium Series 380, 273–293 (1988).
96. Banerjee, M., Roy, D., Bhattacharyya, B. & Basu, G. Differential colchicine-binding across
eukaryotic families: the role of highly conserved Pro268beta and Ala248beta residues in animal
tubulin. FEBS Lett. 581, 5019–5023 (2007).
24
97. Morejohn, L. C. & Fosket, D. E. Taxol-induced rose microtubule polymerization in vitro and its
inhibition by colchicine. J. Cell Biol. 99, 141–147 (1984).
98. Yemets, A. I. & Blume, Y. B. Progress in plant polyploidization based on antimicrotubular drugs.
Open Hortic J 1, 15–20 (2008).
99. Shitan, N. & Yazaki, K. Accumulation and membrane transport of plant alkaloids. Curr Pharm
Biotechnol 8, 244–252 (2007).
100.Mahoney, B. P., Raghunand, N., Baggett, B. & Gillies, R. J. Tumor acidity, ion trapping and
chemotherapeutics. I. Acid pH affects the distribution of chemotherapeutic agents in vitro.
Biochem. Pharmacol. 66, 1207–1218 (2003).
101.Choi, H.-K. et al. Localization of paclitaxel in suspension culture of Taxus chinensis. Journal of
microbiology and biotechnology 11, 458–462 (2001).
102.Blakeslee, A. F. The Present and Potential Service of Chemistry to Plant Breeding. American
Journal of Botany 26, 163 (1939).
103.Levan, A. The Effect of Acenaphthene and Colchicine on Mitosis of Allium and Colchicum.
Hereditas 26, 262–276 (1940).
104.Kramers, M. R. & Stebbings, H. The insensitivity of Vinca rosea to vinblastine. Chromosoma 61,
277–287 (1977).
105.Dayan, F. E., Kuhajek, J. M., Canel, C., Watson, S. B. & Moraes, R. M. Podophyllum peltatum
possesses a beta-glucosidase with high substrate specificity for the aryltetralin lignan
podophyllotoxin. Biochim. Biophys. Acta 1646, 157–163 (2003).
106.Tuszynski, J. A. et al. Modeling the Yew Tree Tubulin and a Comparison of its Interaction with
Paclitaxel to Human Tubulin. Pharm. Res. 29, 3007–3021 (2012).
25