Results and Discussion
The importance of the cytoskeleton in cancer biology is currently recognized and its
candidate role as a potential target for cancer treatment is considered today [21]. Actin, microtubule, and vimentin
cytoskeletons are retained key players that underpin cancer cellular processes such
as cancer cell migration, cell adhesion structures, and metastasis formation [22]. In vivo and in vitro studies
report antiproliferative effects of TTO in tumors and skin disorders [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17], but the molecular
mechanisms underlying this effect remain to be fully elucidated. The effect of TTO
and its main active component terpinen-4-ol on the cytoskeletal apparatus has been
poorly explored until now.
Sensitivity of drug-sensitive and drug-resistant melanoma cells to tea tree
oil and terpinen-4-ol
In our study, firstly, we wanted to confirm the higher sensitivity of human
melanoma drug-resistant M14 ADR cells to the cytotoxic action of TTO and its
main active component terpinen-4 ol when compared to their drug-sensitive
counterpart M14 WT cells [11]. Thus, an
MTT assay was performed on M14 ADR cells and their parental M14 WT cells after
treatment with TTO (0.01%) or terpinen-4-ol (0.005 or 0.01%) for
24, 48, and 72 h. In these experiments, STS was used as a positive
control of apoptosis induction. In general, a dose-dependent decrease in
viability of both cell lines occurred ([Fig.
1]). As expected, M14 ADR cells appeared to be less sensitive to STS
compared to M14 WT cells, but more sensitive to the action of terpinen-4-ol.
After exposure of M14 ADR cells to either 0.005 or 0.01% terpinen-4-ol
for 24 ([Fig. 1a]), 48 ([Fig. 1b]), and 72 h ([Fig. 1c]), a decrease in viable cell
percentage was observed, greater than that observed in the sensitive
counterparts. In particular, in drug-resistant cultures, cell viability
decreased 50% (24 h), 52% (48 h), and
64% (72 h) after treatment with 0.005% terpinen-4-ol,
and 71% (24 h), 66% (48 h), and 67%
(72 h) after treatment with 0.01% terpinen-4-ol. M14 WT cells
showed a decrease in viable cell percentage of 32% (24 h),
28% (48 h), and 4% (72 h) after exposure to
0.005% terpinen4-ol, and of 65% (24 h), 63%
(48 h), and 34% (72 h) after exposure to 0.01%
terpinen-4-ol. MTT results suggest that, contrarily to M14 ADR cells,
drug-sensitive cells were able to recover the damage induced by terpinen-4-ol.
With treatment of 0.01% TTO induced in melanoma cells, a reduction of
cell viability percentages greater than that was induced by terpinen-4-ol.
Indeed, the exposure to TTO for 24 ([Fig.
1a]), 48 ([Fig. 1b]), and
72 h ([Fig. 1c]) caused a
reduction of cell viability percentages of 86, 89, and 98%,
respectively, in M14 ADR cultures and a reduction of 78, 86, and 75%,
respectively, in M14 WT cells. These data confirm that resistant melanoma cells
were more sensitive to the treatment with TTO when compared to wild-type
cells.
Fig. 1 Effects of terpinen-4-ol on human melanoma cell viability.
The MTT test performed on M14 WT and M14 ADR cells treated with TTO
(0.01%) or terpinen-4-ol (0.005 or 0.01%) for (a)
24 h, (b) 48 h, and (c) 72 h
revealed a dose-dependent decrease in viability of both cell lines.
Drug-resistant M14 ADR cells showed to be more sensitive to the effect
of both TTO and terpinen-4-ol than their sensitive counterparts. STS
(0.25, 0.5, and 1 µM) was used as a positive control of
apoptosis induction.
In addition, ultrastructural studies were carried out by SEM in order to analyze,
at high resolution, the effect of terpinen-4-ol on the morphology of melanoma
cells. SEM analysis showed that untreated M14 WT and M14 ADR cells displayed a
characteristic spindle-shaped morphology, with the cell surface covered by
numerous and randomly distributed microvilli ([Fig. 2a, b]). Treatment with 0.01% terpinen-4-ol for
24 h ([Fig. 2c, d]) produced
profound morphological alterations. In drug-sensitive cells ([Fig. 2c]), exposure to the
Melaleuca active component induced the loss of the fusiform shape in
some cells, suggesting a reorganization of the underlying cytoskeletal
architecture.
Fig. 2 Effect of terpinen-4-ol on the morphology of melanoma
cells. Ultrastructural studies were carried out by scanning electron
microscopy. M14 WT (a, c) and M14 ADR (b, d)
treated with 0.01% terpinen-4-ol for 24 h (c,
d). The treatment induced profound morphological alterations
with the loss of fusiform morphology (c) and formation of holes
in the plasma membrane of resistant cells (d).
However, the effects of terpinen-4-ol were even more devastating in
drug-resistant M14 ADR cells: a strong redistribution and clusterization of the
microvilli ([Fig. 2d], arrows) was
visible around real holes on the plasma membrane. These results indicated a
higher sensitivity of M14 ADR cells to the action of terpinen-4-ol when compared
to the wild-type cells, yet after 24 h of interaction with the active
component.
Further, a flow cytometric cell cycle analysis was performed on sensitive and
drug-resistant M14 cells after treatment for 24, 48, and 72 h with TTO
(0.01 and 0.02%) and terpinen-4-ol (0.005 and 0.01%). In M14 WT
cells, treatments did not induce significant alterations in cell cycle
distribution. Instead, M14 ADR cells showed to be more sensitive than their
sensitive counterparts. Even at 24 h, TTO and terpinen-4-ol treatments
induced a cytotoxic effect, as a 2- to 3-fold increase of the sub-G1 apoptotic
peak was observed in respect to untreated samples, depending on the
concentrations (Figs. 1S and 2S, Supporting Information). At longer times, the
apoptotic cell population increased to 25.9 and 20.9% (after 0.01 and
0.02% TTO treatment, respectively) and to 3.4 and 12.2% (after
with 0.005 and 0.01% terpinen-4-ol treatment, respectively) ([Fig. 3]).
Fig. 3 Cell cycle analysis performed by flow cytometry on
sensitive and drug-resistant M14 cells after treatment for 24, 48, and
72 h with TTO (0.01 and 0.02%) and terpinen-4-ol (0.005
and 0.01%). .
Thus, cell cycle analysis further confirmed the higher sensitivity of M14 ADR
cells to both terpinen-4-ol and the entire mixture of TTO. The observations by
high-resolution SEM substantiated the strong interaction of terpinen-4-ol with
the plasma membrane of drug-resistant cells, which is in accordance with
previous data obtained by biophysical and ultrastructural studies [18]. Furthermore, morphological
modifications observed on both M14 WT and M14 ADR cells strongly suggested an
involvement of the cytoskeletal apparatus in the cell adaptation and response to
the main active component.
Thus, we analyzed the effects of TTO and terpinen-4-ol on the cytoskeleton
architecture of M14 melanoma cells using LSCM. The purpose of these observations
was to verify if cytoskeletal elements were involved in the mechanism of action
of Melaleuca essential oil and its active component terpinen-4-ol. We
chose the subcytotoxic concentration of terpinen-4-ol employed in the MTT test
and cell cycle analysis (0.005%) and the relative amount present in the
entire mixture of TTO (0.01%) in order to reveal the eventual early
alterations of the cytoskeletal elements. All the images are relative to a
single confocal section.
Effect of tea tree oil and terpinen-4-ol on actin and tubulin
cytoskeleton
Double labeling experiments of both F-actin and α- and
β-tubulin allowed to simultaneously highlight the effect of
TTO and terpinen-4-ol on actin microfilament and microtubule architecture.
Untreated M14 WT cells displayed an actin cytoskeleton organized in stress
fibers, ruffles, and actin connected with microvilli ([Fig. 4b, c], and insert, arrows). After
treatment with TTO ([Fig. 5b]) or
terpinen-4-ol ([Fig. 6b]) for
24 h, the stress fibers became less evident. Modifications of the
cytoskeletal architecture induced by TTO and terpinen-4-ol were also visible in
the tubulin network. In untreated M14 WT cells ([Fig. 4a]), microtubules radiate out from
the microtubule-organizing center, forming a network around the nucleus and
extending much of the length and breadth of the cell. After treatment with TTO,
alterations affecting the perinuclear cage structure were visible ([Fig. 5a, c], insert, arrows), and even
more evident in cells treated with terpinen-4-ol ([Fig. 6a, c] and [Fig. 7], inserts, arrows).
Fig. 4 LSCM observations of actin and microtubules architecture of
untreated M14 WT cells at 24 h from seeding. (a)
Microtubules labeled with anti-α- and
β-tubulin antibodies (green) and (b) F-actin
microfilaments labeled with TRITC-conjugated phalloidin (red) (c)
merge (tubulin: green; actin: red; yellow: colocalization). Microtubules
radiate out from the microtubule-organizing center, forming a network
around the nucleus and extending much of the length and breadth of the
cell (a). Actin cytoskeleton is organized in stress fibers.
F-actin connected with filopodia and microvilli was visible (b,
c and insert, arrows).
Fig. 5 LSCM observations of actin and microtubules architecture of
M14 WT after treatment with 0.01% TTO for 24 h.
(a) Microtubules labeled with anti-α- and
β-tubulin antibodies (green) and (b) F-actin
microfilaments labeled with TRITC-conjugated phalloidin (red) (c)
merge (tubulin: green; actin: red; yellow: colocalization). After
treatment with TTO, stress fibers became less evident; modification of
the tubulin network around the nucleus was visible (a, c,
insert and arrows).
Fig. 6 LSCM observations of actin and microtubules architecture of
M14 WT after treatment with 0.005% terpinen-4-ol for
24 h. a Microtubules labeled with anti-α-
and β-tubulin antibodies (green) and b F-actin
microfilaments labeled with TRITC-conjugated phalloidin (red) c
merge (tubulin: green; actin: red; yellow: colocalization). After
treatment with.005% terpinen-4-ol stress fibers became less
evident (b) and alterations of the tubulin network around the
nucleus were clearly detected (a, c, insert and
arrows).
Fig. 7 LSCM observations of microtubules architecture of M14 WT
after treatment with 0.005% terpinen-4-ol for 24 h
a Microtubules labeled with anti-α- and
β-tubulin antibodies (green). b Nuclei labeled
with Hoechst. Alterations of the tubulin network around the nuclei were
visible (a, c and insert, arrows). Arrow heads: dividing
cells. Insert: 3D maximum intensity reconstruction.
Untreated drug-resistant variant M14 ADR cells exhibited an actin cytoskeleton
organized in stress fibers ([Fig. 8b, c])
almost identical to those of the sensitive counterpart M14 WT cells. After
treatment with TTO or terpinen-4-ol for 24 h ([Figs. 9b] and [10]b, respectively), the stress fibers
were not visible anymore. The microtubule network showed alterations in
proximity of the nuclei ([Fig. 9a, c] and
[Fig. 10a, c], respectively).
Fig. 8 LSCM observations of actin and microtubules architecture of
untreated M14 ADR cells 24 h from seeding. a Microtubules
labeled with anti-α- and β-tubulin
antibodies (green) and b F-actin microfilaments labeled with
TRITC-conjugated phalloidin (red) c merge (tubulin: green; actin:
red; yellow: colocalization). Microtubules form a network around the
nucleus and extend outward to the cell periphery (a). Similar to
wild-type cells, drug-resistant cells displayed an actin cytoskeleton
organized in stress fibers (b).
Fig. 9 LSCM observations of actin and microtubules architecture of
M14 ADR cells after treatment with 0.01% TTO for 24 h.
a Microtubules labeled with anti-α- and
β-tubulin antibodies (green) and b F-actin
microfilaments labeled with TRITC-conjugated phalloidin (red) c
merge (tubulin: green; actin: red; yellow: colocalization). Treatment
with TTO induced the alteration of the perinuclear tubulin network
(a, arrows) and the disappearance of the stress fibers
(b).
Fig. 10 LSCM observations of actin and microtubules architecture
of M14 ADR cells after treatment with 0.005% terpinen-4-ol for
24 h. a Microtubules labeled with anti-α-
and β-tubulin antibodies (green) and b F-actin
microfilaments labeled with TRITC-conjugated phalloidin (red) c
merge (tubulin: green; actin: red; yellow: colocalization). Treatment
with terpinen-4-ol induced the alteration of the perinuclear tubulin
network (a, c, arrows) and the disappearance of the stress
fibers (b) .
After 48 h, the effects of TTO and terpinen-4-ol were still visible.
Indeed, in drug-sensitive cells, treatment with TTO and terpinen 4-ol induced
the disappearance of the stress fibers ([Figs.
12]
b and [13]b,
respectively), which was instead clearly evident in M14 WT control cells ([Fig. 11b, c]). Alterations of the
microtubular network around the nuclei were still visible in cells treated with
both the essential oil ([Fig. 12a, c],
inserts) and its main active component ([Fig.
13a, c], inserts).
Fig. 11 LSCM observations of actin and microtubules architecture
of untreated M14 WT cells 48 h from seeding. a
Microtubules labeled with anti-α- and
β-tubulin antibodies (red) and b F-actin
microfilaments labeled with fluorescein-conjugated phalloidin (green)
c merge (tubulin: red; actin: green; yellow: colocalization).
At 48 h, sensitive cells displayed a microtubule (a) and
microfilament (b) pattern similar to that of the 24 h
ones. Colocalization areas were visible in perinuclear regions. .
Fig. 12 LSCM observations of actin and microtubules architecture
of M14 WT cells after treatment with 0.01% TTO for 48 h.
a Microtubules labeled with anti-α- and
β-tubulin antibodies (red) and b F-actin
microfilaments labeled with fluorescein-conjugated phalloidin (green)
c merge (tubulin: red; actin: green; yellow: colocalization).
Treatment with TTO induced the collapse of the perinuclear microtubules
(a, c, inserts) and the disappearance of the stress
fibers and cell organelle-linked F-actin (b, c).
Fig. 13 LSCM observations of actin and microtubules architecture
of M14 WT cells after treatment with 0.005% terpinen-4-ol for
48 h. a Microtubules labeled with anti-α-
and β-tubulin antibodies (red) and b F-actin
microfilaments labeled with fluorescein-conjugated phalloidin (green)
c merge (tubulin: red; actin: green; yellow: colocalization).
Similar to TTO, terpinen 4-ol also induced the collapse of the
perinuclear microtubules (a, c, inserts) and the
disappearance of the stress fibers and cell organelle-linked F-actin
(b, c).
In M14 ADR cells, the damage induced by TTO and terpinen-4-ol on both actin and
microtubule networks lasted over the time ([Figs. 15] and [16],
respectively). Dramatic alterations of cell morphology and depolymerization of
F-actin were seen in drug-resistant cells treated with TTO ([Fig. 15b]) and terpinen-4-ol ([Fig. 16b]) when compared to control cells
([Fig. 14b]). Collapse and
alterations of microtubules of the nuclear cage were confirmed and clearly
visible in both TTO- ([Fig. 15a, c],
inserts) and terpinen-4-ol-treated resistant cells. In the latter, thick
microtubule bundles and strongly fluorescent accumulations were observed ([Fig. 16a, c], inserts).
Fig. 14 LSCM observations of actin and microtubules architecture
of untreated M14 ADR cells after 48 h. a Microtubules
labeled with anti-α- and β-tubulin
antibodies (red) and b F-actin microfilaments labeled with
fluorescein-conjugated phalloidin (green) c merge (tubulin: red;
actin: green; yellow: colocalization).
Fig. 15 LSCM observations of actin and microtubules architecture
of M14 ADR cells after treatment with 0.01% TTO for
48 h. a Microtubules labeled with anti-α-
and β-tubulin antibodies (red) and b F-actin
microfilaments labeled with fluorescein-conjugated phalloidin (green)
c merge (tubulin: red; actin: green; yellow: colocalization).
Collapse and alterations of the microtubules of the nuclear cage were
confirmed (a, c, inserts). Dramatic alterations of cell
morphology and depolymerization of F-actin were visible (c,
b).
Fig. 16 LSCM observations of actin and microtubules architecture
of M14 ADR cells after treatment with 0.005% terpinen-4-ol for
48 h. a Microtubules labeled with anti-α-
and β-tubulin antibodies (red) and b F-actin
microfilaments labeled with fluorescein-conjugated phalloidin (green)
c merge (tubulin: red; actin: green; yellow: colocalization).
Treatment with terpinen-4-ol induced the collapse of perinuclear
microtubules (a, c, and inserts) and the disappearance of
the stress fibers and cell organelle-linked F-actin (b,
c).
After 72 h, M14 WT cells recovered from the TTO-induced damage of both
actin and tubulin cytoskeletons In fact, TTO-treated cells showed stress fibers
([Fig. 18b, c]) similar to those
observed in the control cells ([Fig. 17b,
c]) while they still appeared less evident in cells treated with
terpinen-4-ol ([Fig. 19b, c]). This
recovery was also found at the level of the microtubular network of TTO-treated
cells ([Fig. 18a]), which showed a
tubulin architecture almost identical to that of the control cells ([Fig. 17a]).
Fig. 17 LSCM observations of actin and microtubules architecture
of untreated M14 WT cells 72 h from seeding. A
Microtubules labeled with anti-α- and
β-tubulin antibodies (red) and b F-actin
microfilaments labeled with fluorescein-conjugated phalloidin (green)
c merge (tubulin: red; actin: green; yellow: colocalization).
Cells displayed elongated shapes with microfilaments and microtubules
running parallel to the long axis of the cell (a-c).
Fig. 18 LSCM observations of actin and microtubules architecture
of M14 WT cells after treatment with 0.01% TTO for 72 h.
a Microtubules labeled with anti-α- and
β-tubulin antibodies (red) and b F-actin
microfilaments labeled with fluorescein-conjugated phalloidin (green)
c merge (tubulin: red; actin: green; yellow: colocalization).
M14 WT cells recovered TTO-induced damage of both actin and the tubulin
cytoskeleton.
Fig. 19 LSCM observations of actin and microtubules architecture
of M14 WT cells after treatment with 0.005% terpinen-4-ol for
72 h. a Microtubules labeled with anti-α-
and β-tubulin antibodies (red) and b F-actin
microfilaments labeled with fluorescein-conjugated phalloidin (green)
c merge (tubulin: red; actin: green; yellow: colocalization).
M14 WT cells recovered terpinen-4-ol-induced damage of both actin and
the tubulin cytoskeleton.
At 72 h, M14 ADR cells recovered the actin damage induced by TTO ([Fig. 21b, c]) but not that induced by
terpinen-4-ol ([Fig. 22b, c]). Quite
completely reconstituted stress fibers and the microtubular nuclear network were
indeed found in resistant TTO-treated cells, which recovered their morphology
([Fig. 21]). Conversely, damages by
terpinen-4-ol on F-actin microfilament, microtubules, and cell morphology
persisted over time ([Fig. 22]).
Fig. 20 LSCM observations of actin and microtubules architecture
of untreated M14 ADR cells 72 h from seeding. A
Microtubules labeled with anti-α- and
β-tubulin antibodies (red) and b F-actin
microfilaments labeled with fluorescein-conjugated phalloidin (green)
c merge (tubulin: red; actin: green; yellow: colocalization).
Similar to wild-type cells, resistant cells displayed elongated shapes
with microfilaments and microtubules running parallel to the long axis
of the cell (a-c).
Fig. 21 LSCM observations of actin and microtubules architecture
of M14 ADR cells after treatment with 0.01% TTO for
72 h. a Microtubules labeled with anti-α-
and β-tubulin antibodies (red) and b F-actin
microfilaments labeled with fluorescein-conjugated phalloidin (green)
c merge (tubulin: red; actin: green; yellow: colocalization).
M14 ADR cells recovered the actin damage induced by TTO. Stress fibers
and the microtubular nuclear network appeared to be quite completely
reconstituted (a-c).
Fig. 22 LSCM observations of actin and microtubules architecture
of M14 ADR cells after treatment with 0.005% terpinen-4-ol for
72 h. a Microtubules labeled with anti-α-
and β-tubulin antibodies (red) and b F-actin
microfilaments labeled with fluorescein-conjugated phalloidin (green)
c merge (tubulin: red; actin: green; yellow: colocalization).
Damages by terpinen-4-ol on the F-actin microfilament, microtubules, and
cell morphology persisted over time (a-c).
The analysis of the actin cytoskeleton by LSCM evidenced a clear action of the
essential oil and the main active component on F-actin. Indeed, the treatment
with both TTO and terpinen-4-ol induced the disappearance of the stress fibers
in both M14 WT and M14 ADR cells. Actin filaments are, in contrast to
intermediate filaments and microtubules, semi-flexible filaments, forming
dendritic or cross-linked structures [23]
[24]. Actin itself is
considered the most dynamic of the three cytoskeletal proteins capable of strong
structural changes in the time scale of minutes, thus determining the shape of a
cell.
Besides globular and filamentous actins (G- and F-actin), there are actin bundles
and networks, such as submembrane actin cortex, linked together by
cross-linkers, and molecules that connect single actin filaments either
transiently or non-transiently. In cell cultures with stiff substrates, the
actin cytoskeleton tends to organize in “stress fibers” (dorsal,
transverse arcs, ventral), which are bundles of actin filaments cross-linked by
an actin-binding protein such as fascin and tensed by myosin II molecular motors
[25]
[26]
[27]
[28].
In M14 WT and M14 ADR cells treated with essential oil and terpinen-4-ol, stress
fibers were not detectable anymore, whereas a fluorescent signal lining plasma
membrane and a diffuse weak fluorescence in the cytoplasm were revealed. These
observations suggested that in our experimental conditions, TTO and
terpinen-4-ol were able to interfere with the bundling of microfilaments in
stress fibers. Conversely, disaggregation of the actin cortex did not seem to
occur, since fluorescein phalloidin recognized F-actin in proximity of the cell
membrane. Moreover, adherent cells with lost stress fibers generally maintained
their shape, suggesting that other cytoskeletal elements contributed to
maintaining the anchoring of the cells to the substrate. Functional and
structural interconnections between actin, microtubules, and IFs are well known.
The tight association of the actin cytoskeleton with cell adhesive structures
has been largely studied [23]. Both actin
fibers and intermediate filaments can guide microtubules to the cell periphery
and may contribute to microtubule localization at the rim of FAs [29]
[30]. The physical linkage between vimentin IFs and FAs strengthens
the adhesions and promotes their dynamics, boosting the migratory potential of
cells [21]
[31]. At 72 h, stress fibers were still revealed in M14 WT
cells treated with both TTO and terpinen-4-ol and in M14 ADR cells treated with
TTO, but not in drug-resistant cells treated with terpinen-4-ol. The
irreversible damage induced by terpinen-4-ol on the plasma membrane of M14 ADR
cells might further contribute to the loss of actin cytoskeletal integrity.
As far the microtubular network, we found that both TTO and terpinen-4-ol induced
a disorganization of the perinuclear cage, which was particularly evident at
48 h, with the rupture and collapse of microtubules. Interactions
between the cytoskeleton and the nucleus are well known and affect global
cytoskeleton organization. Actin microfilaments, microtubules, and vimentin IFs
link the nucleus to the cytoplasmic cytoskeleton via the linker of
nucleoskeleton and cytoskeleton complex that is present at the nuclear membrane
[32]. Microtubules, actin filaments,
and IFs are connected by SUN (inner nuclear membrane) domain and KASH domain
(outer nuclear membrane) protein linkers to the nuclear envelope. In particular,
actin microfilaments are directly linked to the nucleus by nesprin 1 or nesprin
2, microtubules indirectly by nesprin 4 and kynesin, and IFs indirectly by
plectin and nesprin 3, with nesprin proteins being linked to the SUN adaptor
protein, localized in the inner membrane and connected to the nuclear lamina.
Cytoskeleton links to the nucleus are emerging to be pivotal in various
physiological processes, including cell migration, ability for cells to cope
with mechanical stress, and altered gene expression profiles [33]
[34].
Of note, the damage of the perinuclear cytoskeleton was easily detected in
α- and β-tubulin-labeled-treated cells, but
not in F-actin-labeled cells. However, damage to the perinuclear actin (actin
cap) cannot be excluded a priori, since three elements are strictly
interconnected by cross-linker proteins such as plectin [35]. On the basis of our observations, we
can speculate that the treatments with terpinen-4-ol and TTO induced the
detachment and collapse of microtubules from the nuclear envelope.
Effect of tea tree oil and terpinen-4-ol on vimentin intermediate
filaments
Vimentin, a major constituent of the IF family of proteins, is ubiquitously
expressed in normal mesenchymal cells and is known to maintain cellular
integrity and provide resistance against stress. Herein, we report results
obtained by LSCM observations on vimentin IFs of both M14 WT and M14 ADR cells
after treatment with 0.01% TTO and 0.005% terpinen 4-ol for 24,
48, and 72 h. Both M14 WT ([Fig.
23a]) and M14 ADR cells ([Fig.
24a]) showed a robust architecture of vimentin Ifs, with a perinuclear
network from which IFs branch out towards the cell periphery. After treatment
with TTO for 24 h, an increase of the fluorescence signal was revealed
in M14 WT cells ([Fig. 23b]) and M14 ADR
cells ([Fig. 24b]). In particular, single
IFs appeared to be less resolved and fluorescent vimentin accumulations were
revealed (arrows). Forty-eight hours of treatment with TTO and terpinen-4-ol
exerted a strong effect in drug-resistant M14 ADR cells ([Fig. 26b]) when compared to their parental
counterpart ([Fig. 25b]). Thick vimentin
bundles and strongly fluorescent accumulations (arrows) appeared to be clearly
detectable. As previously reported, at 72 h from treatment, M14 WT cells
recovered the damage induced by TTO, displaying a vimentin pattern similar to
that of untreated cells ([Fig. 27]), and
a number of mitotic cells was revealed in the cultures ([Fig. 28]). Moreover, single vimentin IFs
were more clearly visible. In terpinen-4-ol-treated samples ([Fig. 29]), a number of cells rounded in
shape was revealed. Conversely, drug-resistant M14 ADR cells ([Figs. 30]
[31]
[32]
) did not
recover the cytoskeleton damage. Thick vimentin bundles (arrows) and strongly
labeled accumulations (arrows) were still visible in TTO-treated samples ([Fig. 31]). Similarly, in
terpinen-4-ol-treated cells ([Fig. 32]),
distortion and detachment of IF bundles and strongly fluorescent vimentin
accumulations were observable. All these observations denounced that the effect
of TTO and its active component terpinen-4-ol persisted over time also on
vimentin IFs of drug-resistant cells.
Fig. 23 LSCM observations of vimentin architecture of M14 WT cells
before (a) and after treatment with 0.01% TTO (b)
for 24 h. Large vimentin bundles or strongly fluorescent
accumulations were revealed (b, arrows).
Fig. 24 LSCM observations of vimentin architecture of M14 ADR
cells before (a) and after treatment with 0.01% TTO
(b) for 24 h. Single Ifs appeared to be less resolved
and fluorescent vimentin accumulations in the proximity of nuclei were
revealed (b, arrows).
Fig. 25 LSCM observations of vimentin architecture of M14 WT cells
before (a) and after treatment with 0.01% TTO (b)
or 0.005% terpinen-4-ol for 48 h. (c) Large
vimentin bundles or strongly fluorescent accumulations were revealed
(b, c, arrows).
Fig. 26 LSCM observations of vimentin architecture of M14 ADR
cells before (a) and after treatment with 0.01% TTO
(b) or 0.005% terpinen-4-ol for 48 h.
(c) After treatment with TTO and terpinen-4-ol, thick
vimentin bundles and strongly fluorescent accumulations (arrows) were
clearly detectable.
Fig. 27 LSCM observations of vimentin architecture of untreated
M14 WT cells 72 h from seeding.
Fig. 28 LSCM observations of vimentin architecture of M14 WT cells
after treatment with 0.01% TTO for 72 h. At 72 h
from the treatment, M14 WT cells recovered the damage induced by TTO and
a number of mitotic cells was revealed in the cultures.
Fig. 29 LSCM observations of vimentin architecture of M14 WT cells
after treatment with 0.005% terpinen-4-ol for 72 h. M14
WT cells recovered the damage induced by TTO and a number of mitotic
cells was revealed in the cultures.
Fig. 30 LSCM observations of vimentin architecture of untreated
M14 ADR cells 72 h from seeding.
Fig. 31 LSCM observations of vimentin architecture of M14 ADR
cells after treatment with 0.01% TTO for 72 h. Thick
vimentin bundles (arrows) and strongly labeled accumulations (arrows)
were still visible in TTO-treated samples.
Fig. 32 LSCM observations of vimentin architecture of M14 ADR
cells after treatment with 0.005% terpinen-4-ol. Distortion and
detachment of IF bundles and strongly fluorescent vimentin accumulations
(arrows) were detected.
We found that both TTO and terpinen-4-ol noticeably changed the IFs architecture
by inducing the formation of strong fluorescent accumulations in the proximity
of the nucleus (perinuclear collapse) and vimentin large cables. This alteration
appeared still more evident in drug-resistant M14 ADR cells and differently from
damages induced on actin and microtubules, irreversible over time both in TTO-
and terpinen-4-ol-treated cells.
The vimentin IF network provides a cytoarchitecture with mechanical stability
that also enables precise spatiotemporal coordination between all three
cytoskeletal components [36]. Vimentin can
interact with actin filaments both directly through its C-terminal tail [37] and indirectly through the cytoskeletal
cross-linking proteins [35]. More
precisely, transverse arcs and ventral stress fibers interact with vimentin IFs
through plectin [38]. Vimentin IFs also
interact with microtubules through the tumor suppressor APC [39] and indirectly via cross-linking with
plectin [35].
In conclusion, results obtained in the present study clearly point out to the
cytoskeleton as a further target of M. alternifolia and its main active
component terpinen-4-ol and could account for the inhibition of proliferation
and aggressiveness of drug-resistant M14 cells. Strikingly, the cytoskeletal
alterations observed by LSCM on actin, microtubules, and vimentin appeared
localized in precise different structures of the cytoskeletal network,
i. e., actin stress fibers, tubulin perinuclear cage, and vimentin IF
architecture. In our experimental conditions, vimentin IFs appear to be the
cytoskeletal element more affected by the treatments. Cross-linker proteins
might play a role in the mechanism of action of TTO, and further studies will be
carried out in order to better understand this issue.
Materials and Methods
Tea tree oil
TTO was purchased from Variati SPA (batch no. 061220052) and was certified for
the following chemical composition: α-pinene 2.5%,
sabinene 0.2%, α-terpinene 10%, para cimene
1.9%, d-limonene 1%, 1,4-cineole 2.9%,
γ-terpinene 21.2%, α-terpinolene
3.5%, terpinen-4-ol 40.3%, α-terpineol
3.2%, aromadendrene 1.2%, δ-cadinene
0.9%, globulol 0.3%, and viridiflorol 0.4%. The analysis
was performed by using a gas chromatograph fitted with a flame ionization
detector.
Chemicals and reagents
RPMI 1640 medium, penicillin/streptomycin solution 100X, L-glutamine
solution 200 nM, and MEM nonessential amino acids 100X were purchased
from EuroClone S.p.A. FBS was purchased from Hyclone. Anti-α- and
β-tubulin antibodies, DMSO, MTT, phalloidin TRITC, PBS,
ribonuclease, STS, terpinen-4-ol (purity 100%), and Triton X-100 were
purchased from Sigma-Aldrich. Anti-vimentin antibody was purchased from Santa
Cruz Biotechnology. Secondary Alexa 488 goat anti-mouse IgG and Alexa 633 goat
anti-mouse IgG were purchased from Molecular Probes. Propidium iodide was
purchased from Applichem. Glutaraldehyde (25% acqueous solution) and
sodium cacodylate (powder) were purchased from TAAB Laboratories Equipment Ltd.
Osmium (VIII) oxide for microscopy was purchased from Merck KGaA.
Cell cultures
The established human melanoma cell lines M14 WT and M14 ADR were grown in RPMI
1640 medium supplemented with 1% L-glutamine, 1% nonessential
amino acids, 100 IU/mL penicillin/streptomycin, and 10%
FBS at 37°C in a 5% CO2 humidified atmosphere in air.
The M14 ADR cell line was selected culturing M14 WT cells in the presence of
40 µM ADR (Adriblastina; Pharmacia & Upjohn S.p.A) as
described in Calcabrini et al. [11].
MTT assays
M14 WT and M14 ADR cells were seeded in 96-well plates at a density of
1×104 cells/well in complete medium
(200 µL). On the following day, cells were treated with TTO
(0.01%) or terpinen-4-ol (0.005 or 0.01%) in triplicate. STS
(0.25, 0.5, and 1 µM) was used as a positive control of
apoptosis induction. After 24, 48, and 72 h of treatment, the cell
medium was removed, and cells were washed with PBS and incubated with
0.5 mg/mL MTT for 3 h at 37°C. After removing
the MTT solution, the samples were lysed by 100 µL DMSO and
analyzed by a microplate reader (Fusion Universal Microplate Analyzer; Packard)
at 570 nm. After normalizing the optical density (OD) of each well to
the absorbance value of the blank, the percentage of cell viability was
calculated as follow: (OD mean value of the treated sample/OD mean value
of the control sample)×100.
Flow cytometry
For cell cycle analysis, M14 WT and M14 ADR cells were seeded in complete medium
(2×105 in 6 wells). After 24 h, they were treated
with 0.01% TTO or 0.005% terpinen-4-ol. After 24, 48, and
72 h, cells were collected, washed twice with cold PBS, and centrifuged.
The pellet was fixed in 70% ethanol and kept at 4°C until the
time of staining with PBS containing 40 μg/mL propidium
iodide and 100 μg/mL ribonuclease at 37°C for
30 min. Samples were then analyzed by a BDLSRII flow cytometer (Becton,
Dickinson & Company). Percentages of cells in subG1, G1, S, and
G2/M phases were calculated using FACS (Fluorescence-Activated Cell
Sorter) Diva Software (Becton, Dickinson & Company).
Scanning electron microscopy
To analyze the effect of terpinen-4-ol on the cell morphology of melanoma
cells, 5×105 cells were seeded on porous membranes
(8.0 µm pore; Falcon) that stood in 6-well plates and were
incubated at 37°C in a 5% CO2 humidified atmosphere
in air. After 24 h cells were treated with 0.01% terpinen-4-ol.
After 18 h of incubation, samples were fixed with 2.5%
glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3), with 2%
sucrose added, for 30 min at room temperature. After post-fixation with
1% OsO4 in 0.1 M cacodylate buffer (pH 7.3), cells were
dehydrated through a graded ethanol series, critical point-dried in
CO2 (030 Balzers device), and gold coated by sputtering (SCD 040
Balzers device, Bal-Tec). The samples were then examined with a SEM-FEG (Quanta
200 Inspect, FEI Company).
Laser scanning confocal microscopy
For immunofluorescence analyses, 5×104 cells were seeded in
24-well cluster plates onto 12-mm cover glasses and incubated overnight at
37°C in a 5% CO2 humidified atmosphere in air. The next day, the
cells were treated with 0.01% TTO or 0.005% terpinen-4-ol.
At the end of treatments, for the immunostaining of tubulin and actin, samples
were washed with PBS and fixed in 3.7% paraformaldehyde with 2%
sucrose for 30 min at room temperature. Samples were then washed twice
in PBS, permeabilized with 0.5% Triton X-100 for 5 min, and
incubated with a blocking solution (10% FBS, 10% AB serum,
1% bovine serum albumin in PBS) for 30 min. Microtubule staining
was performed by incubating melanoma cells with a mouse monoclonal
anti-α/β-tubulin antibody mixture
diluted 1:50 in PBS containing 10% FBS, 10% AB serum, and
1% bovine serum albumin for 30 min at room temperature. Samples
were then washed in PBS and incubated with a mixture of secondary Alexa 488 goat
anti-mouse IgG (1:50) and phalloidin TRITC (1:100) or Alexa 546 goat anti-mouse
IgG (1:50) and phalloidin FITC (1:100) for 30 min at room temperature.
Cells were rinsed three times with PBS afterwards.
At the end of treatments, for vimentin staining, samples were washed with PBS,
fixed for 5 min in methanol (room temperature), followed by
2 min in acetone (−20°C), and allowed to air dry for
10–15 min. Then, cells were rehydrated in PBS containing
10% FBS, 10% AB serum, and 1% bovine serum albumin for
10 min and incubated with a mouse monoclonal anti-vimentin antibody
diluted 1:50 in the same solution at room temperature. After 30 min,
samples were washed in PBS and incubated with a secondary Alexa 633 goat
anti-mouse IgG (1:50) for 30 min at room temperature. Cells were then
rinsed three times with PBS.
Finally, all samples were mounted in PBS containing 50% glycerol.
Observations were performed using a Leica TCS SP2 LSCM (Leica; Microsystems)
equipped with Ar/Kr and He/Ne lasers.
Supporting information
Cell cycle analysis on sensitive and drug-resistant M14 cells after treatment
with TTO (0.01 and 0.02%) and terpinen-4-ol (0.005 and 0.01%)
for 24 and 48 h (Figs. 1S and 2S).
