Fascin has recently emerged as a potential therapeutic target, as its expression in cancer cells is closely associated with tumor progression and metastasis. Following the initial discovery of a series of thiazole derivatives that demonstrated potent antimigration and antiinvasion activities via possible inhibition of fascin function, we report here the design and synthesis of 63 new thiazole derivatives by further structural modifications in search of more potent fascin inhibitors. The 5 series of analogues with longer alkyl chain substitutions on the thiazole nitrogen exhibited greater antimigration activities than those with other structural motifs. The most potent analogue, 5p, inhibited 50% of cell migration at 24 nM. Moreover, the thiazole analogues showed strong antiangiogenesis activity, blocking new blood vessel formation in a chicken embryo membrane assay. Finally, a functional study was conducted to investigate the mechanism of action via interaction with the F-actin bundling protein fascin.
Fascin has recently emerged as a potential therapeutic target, as its expression in cancer cells is closely associated with tumor progression and metastasis. Following the initial discovery of a series of thiazole derivatives that demonstrated potent antimigration and antiinvasion activities via possible inhibition of fascin function, we report here the design and synthesis of 63 new thiazole derivatives by further structural modifications in search of more potent fascin inhibitors. The 5 series of analogues with longer alkyl chain substitutions on the thiazole nitrogen exhibited greater antimigration activities than those with other structural motifs. The most potent analogue, 5p, inhibited 50% of cell migration at 24 nM. Moreover, the thiazole analogues showed strong antiangiogenesis activity, blocking new blood vessel formation in a chicken embryo membrane assay. Finally, a functional study was conducted to investigate the mechanism of action via interaction with the F-actin bundling protein fascin.
The actin-bundling
protein fascin has been linked to tumor progression,
invasion, and metastasis, a fatal development of the disease.[1−5] Fascin has recently emerged as a novel therapeutic target for treatment
of cancer metastasis.[6−8] A cell-based fascin bioassay identified compounds
with potential antimetastasis functions, with diverse chemical structural
features.[9] However, no follow-up studies
of biological activities were reported to confirm that the compounds
indeed target fascin to inhibit migration, invasion, and metastasis.[9]From shape-based molecular modeling and
subsequent rational design
and synthesis, we have recently identified a thiazole lead compound 1 (Figure 1) that effectively blocked
cell migration and invasion via interactions with the protein fascin.[10] With potent activities (IC50 in the
100 nM range) in inhibiting migration and invasion of several metastatic
humanbreast cancer cell lines such as MDA-MB-231, HeLa, and A549,
this compound exhibited no cytotoxicity at concentrations exceeding
100 μM. The finding of thiazole compounds as antimigration and
antiinvasion agents opened up new possibilities of fascin-targeting
molecules that can be further optimized for greater potency and bioavailability
while maintaining minimal cytotoxicity. To further explore and optimize
the structure–activity relationships, we have designed and
synthesized 63 additional thiazole derivatives where we sought to
(1) homologize the two lead structures by varying the substitution
on the thiazole nitrogen, (2) change the linker structure between
thiazole and phenyl groups, and (3) modify other substitution groups
on both the thiazole ring and the phenyl rings (Figure 1). These thiazole analogues were then biologically evaluated
to determine their cytotoxicity, antimigration, and antiinvasion activities.
Further, molecular modeling was performed to assist in the elucidation
of observed structure–activity-relationships. For the most
potent thiazole derivatives, an in vivo assay utilizing chick embryo
chorioallantoic membrane (CAM) was performed
to assess their in vivo antimetastasis potential by inhibiting angiogenesis.
Finally we investigated their mode of action by overexpressing the
protein fascin in cancer cell lines to determine if the activities
of the compounds can abrogate the enhanced migration and invasion
of the transfected cell lines.
Figure 1
Structures of lead compounds 1 and 2 and
their analogues from lead modifications.
Structures of lead compounds 1 and 2 and
their analogues from lead modifications.
Results and Discussion
Chemistry
As shown in Scheme 1, compounds 5 and 7 and
their isomers 6 and 8 were respectively
obtained from the N-alkylation
of the amides 3 and 4 which were prepared
following the literature procedure.[10] 4-(2,4-Dimethylphenyl)thiazol-2-amine
(9) was treated with benzenesulfonic chloride to give 10 which was transformed to the analogue 11 and
its isomer 12 by the N-methylation reaction in THF. The
acylation of the 2-aminothiazoles 13 by acyl chloride
provided the amides 14 at room temperature in dichloromethane,
and further methylation of 14 led to the desired analogues 15 and their corresponding isomers 16.
Scheme 1
Synthesis
of Analogues of Lead Compounds
Antimigration Activity and Cytotoxicity
A Transwell
migration assay was used to determine the effects of the synthesized
thiazole derivatives on the migratory capacity of MDA-MB-231, an invasive
and metastatic breast cancer cell line. The cancer cells were seeded
at a density of 2.5 × 104 per well in the upper chamber
in serum-free media. In the presence of varying concentrations of
the thiazole derivatives, the cells’ ability to migrate to
the lower chamber with media containing 5% FBS was measured by counting
the total number of cells in the lower chamber after 24 h. The concentration
of individual thiazole compounds at which inhibition of migration
is observed at 50% is defined as the IC50 value listed
in Table 1. To evaluate possible contributions
of cell viability loss to reduced migration, the thiazole derivatives
were also subjected to cell survival assays to determine their cytotoxicities.
These were conducted by treating the MDA-MB-231 cells with individual
thiazole compounds for 5 days and counting the surviving cells.
Table 1
Antimigration Efficacy
and Cytotoxicity
of the Synthetic Thiazole Derivatives
The 63 newly synthesized thiazole derivatives displayed a wide range
of antimigration activities as reflected in the IC50 values
going from a low of 24 nM to greater than 50 μM or no apparent
activity. Such variations in activity appears to be dependent on minor
structural modifications, revealing some interesting trends that may
help our understanding of the structure–activity-relationships
for further optimization of pharmacological index of thiazole derivatives.Compounds 5l–r are homologues
from the lead structure 1 that were designed and prepared
to investigate the effect of thiazole-N substitution on gain or loss
of the antimigration activity. For comparison, 1, the
most potent thiazole derivative from our previous study,[10] was also included in the assay. When the thiazole N-methyl is changed to an ethyl group (5l),
there was a marked decrease of activity from 0.218 to 1.945 μM.
Interestingly, an n-propyl substitution at the same
position (5m) confers similar antimigration activity
with an IC50 of 0.292 μM. This trend continues as
the alkyl chain length increased to n-butyl (5n, IC50 = 0.196 μM), n-hexyl
(5o, IC50 = 0.045 μM), and n-dodecyl (5p, IC50 = 0.024 μM). In
addition, allyl substitution at the thiazole nitrogen (5q) yielded activity comparable to n-propyl (5m), but changing the substitution (5r) to propynyl
leads to a significant loss of the activity, resulting in an IC50 value of 3.89 μM. On another note, putting the propynyl
group on the amidenitrogen (6) appears to restore the
activity.Attempt to modify the amide linker leads to mixed
results. If the
amide linker is replaced by a sulfamide structure, antimigration activity
was largely lost in 12 but not in 11. The
single structural difference between 11 and 12 is the methyl substitution. Thiazole N-methyl substitution
confers, whereas the amide N-methyl substitution
deprives, antimigration activity.The lead compound 2 was a potent thiazole derivative
discovered in a previous study, which also displayed significant cytotoxicity
in several cancer cell lines at 10 μM.[10] In the current study we further investigated if modifications on N-alkyl substitutions could improve the activity and toxicity
profile. Thus, when the alkyl substitution at the thiazole nitrogen
varied from methyl (5j), ethyl (7a), propyl
(7b) to allyl (7c) and propynyl (7d), the cytotoxicity of the compounds remained significant, with relative
survival ratio after 5-day treatment at 10 μM ranging from 20.4%
to 70.9%. However, the cytotoxicity is largely abrogated when the
methyl and ethyl substitution is on the amidenitrogen (4e, 8a, 8b) while strong antimigration activity
was retained. It was also noted that these compounds (7a–d) exhibited moderate to strong inhibition of
cell growth, suggesting that, in addition to blocking fascin-mediated
migration, these compounds may have other molecular targets that regulate
cell survival and growth.Next we examine how modifications
on the thiazole ring affect antimigration
activity. A methyl substitution on the 5-position of the thiazole
ring (14h) results in a near complete loss of activity.
Adding a fluorine atom at the para-position of the 1-thiazole phenyl
brings back cytotoxicity without a significant gain of antimigration
activity (14i). An attempt to move the thiazole phenyl
ring to the 5-position results in a more linear analogue that has
stronger growth inhibitory effect but also more antimigratory (14j). Interestingly, adding a thiazole N-methyl
group to the above three analogues gives a more potent 15h (compared to 14h) and 15i (compared to 15i) but an inactive 15j (compared to 14j).In comparison of 15j (thiazole N-methyl)
to 16f (amide N-methyl), it was noted
that the analogue with amide N-methyl substitution
displayed significantly greater toxicity and antimigration activity
than that with a thiazole N-methyl group.Modifications
on the thiazole phenyl ring by halogen and trifluoromethyl
substitutions afforded six analogues 14b, 14c, 14d, 14e, 14f, 14g, of which only 14e and 14f showed strong
antimigration activities with moderate inhibition of cell growth at
the same time. It was noted that chlorine substitution at meta position
(14e) is far superior to ortho position (14b). Adding a methyl group on the thiazole nitrogen to some of these
analogues appears to endow more potent cytotoxicity while increasing
the overall inhibitory effect on cell motility. Thus, 15b. 15c, 15d, 15e, 15f, and 15g all displayed potent antimigration activities
with IC50 values ranging from 0.096 μM (15f) to 1.07 μM (15g).Attempts to modify both
thiazole phenyl and amide phenyl ring yielded
eight analogues (14k through 14r). These
compounds showed almost no antimigration activities except 14o with an IC50 value of 1.03 μM. However, if these
analogues are modified further by adding a thiazole N-methyl substitution (15k through 15m),
some modest increases in the activity are obtained. For example, 15l exhibited an IC50 value of 0.758 μM.
On the other hand, if the methyl substitution is on the amidenitrogen
(16g through 16m), the results are mixed.
For instance, compared to 14k, IC50 value
of 16g improved from >25 to 0.312 μM, an increase
of potency by nearly 100-fold. Also, from 15o to 16j, the position change of methyl substitution from thiazolenitrogen to amidenitrogen resulted in an increase of antimigration
activity by 4 times.
Antiinvasion
To determine the antiinvasion activities
of the thiazole compounds, the most potent antimigration analogues
were selected for a Matrigel invasion assay. As shown in Figure 2, at 10 μM, all tested compounds demonstrate
50% or greater inhibition of cell invasion through the Matrigel. Notably,
compound 5o exhibited the greatest potency in blocking
the cancer cell invasion. It is also noted that the antiinvasion activities
of the selected thiazole derivatives do not always correlate with
their antimigration activities.
Figure 2
Antiinvasion activities of selected thiazole
compounds.
Antiinvasion activities of selected thiazole
compounds.
Antiangiogenesis
Given the association of cell migration
with angiogenesis[11−14] and the possible involvement of fascin in angiogenesis,[15] we next investigated if the thiazole compounds
might have any antiangiogenic properties using the chick chorioallantoic
membrane (CAM) assay. In this test the vascular system of a fertilized
chicken embryo is used as a model. Figure 3 demonstrates the effects of 5o and 5p on
the development of embryonal blood vessels compared to a negative
control (PBS) and a positive control (10 ng/plug basic fibroblast
growth factor (bFGF) and 30 ng/plug vascular endothelial growth factor
(VEGF)) without the thiazole compounds. The antiangiogenesis activity
of the three thiazole compounds was determined by the suppression
of angiogenic action of BV (bFGF + VEGF) when each compound was added
to a collagen containing BV and placed on the chorioallantoic membrane
of 10-day old chick embryos for 3 days. As shown in Figure 3, the analogues 5o and 5p were seen to potently block the angiogenic action of BV.
Figure 3
Effect of 5o and 5p on angiogenesis in
chick embryo ex ovo chorioallantoic membrane (CAM) assay. Angiogenesis
was scored by two scorers in a blinded fashion on a scale from 0 to
3 where 0 contained no new vessels and 3 exhibited extreme angiogenesis
determined by the presence of new vessels radiating out from the plug
in addition to the regular repeating pattern of the normal CAM vasculature.
Dashed lines in representative images (below) indicate the top edges
of the collagen and drug-containing plugs from which angiogenesis
was determined. In these experiments, the thiazole analogues 5o and 5p significantly inhibited the induction
of angiogenesis by bFGF and VEGF (BV) after 3 days.
Effect of 5o and 5p on angiogenesis in
chick embryo ex ovo chorioallantoic membrane (CAM) assay. Angiogenesis
was scored by two scorers in a blinded fashion on a scale from 0 to
3 where 0 contained no new vessels and 3 exhibited extreme angiogenesis
determined by the presence of new vessels radiating out from the plug
in addition to the regular repeating pattern of the normal CAM vasculature.
Dashed lines in representative images (below) indicate the top edges
of the collagen and drug-containing plugs from which angiogenesis
was determined. In these experiments, the thiazole analogues 5o and 5p significantly inhibited the induction
of angiogenesis by bFGF and VEGF (BV) after 3 days.
Mechanism of Action
Our previous
study[10] found that the thiazole derivatives
blocked migration and
invasion of cancer cells by impairing the cytoskeleton dynamics accompanied
by reduced colocalization of the actin-bundling protein fascin. To
provide further evidence that fascin might be involved in the antimigratory
action of the newly synthesized thiazole analogues, we overexpressed
fascin in MDA-MB-231 cells to investigate the resulting cell motility
and the effect of the thiazole compounds on the cells overexpressing
fascin. Figure 4 shows the higher level of
fascin expression in the transfected cells than the native level of
fascin in the control cells (Figure 4A). Consistent
with previous findings,[3,16−23] the fascin+ cells exhibited significantly higher (2.4-fold increase)
migration than the control cells (vector). Treatment of the fascin+
cells with our most potent thiazole analogues, 5o and 5p, nearly completely abrogated the enhanced migration as
illustrated in Figure 5. At 10 μM 5o and 5p, the control MDA-MB-231 cells lost
about 80% migratory capacity. The thiazole compounds also inhibited
the fascin+ MDA-MB-231 cells by about the same percentage (Figure 5). These results provided additional evidence that
the thiazole derivatives likely acted through interaction with fascin.
Figure 4
Overexpressing
fascin in MDA-MB-231 leads to enhanced migration.
Figure 5
Enhanced cell migration induced by fascin-overexpression is abrogated
by thiazole analogues 5l, 5o, and 5p.
Overexpressing
fascin in MDA-MB-231 leads to enhanced migration.Enhanced cell migration induced by fascin-overexpression is abrogated
by thiazole analogues 5l, 5o, and 5p.
Effect on F-Actin Structure
To investigate if the thiazole
analogues inhibit cell migration and invasion by impairing the F-actin
network, actin filaments of HeLa cells treated with selected thiazole
analogues were stained with phalloidin tagged with fluorescent agent
(Acti-stain 488 fluorescent phalloidin). As shown in Figure 6, the control cells showed a distinct presence of
F-actin filaments along the cells and had more evenly distributed
F-actin structures in the cytoplasm. In contrast, after treatment
with the three thiazole analogues 5o, 5p, and 8b each at 10–6 M, the cells
exhibited various degrees of disorganization of actin filaments and
a decrease of F-actin staining intensity near the cell membranes.
These results suggest that the antimigration and antiinvasion activities
of the thiazole analogues may be mediated by interfering with the
actin filament network.
Figure 6
Effects of thiazole analogues 5o, 5p,
and 8b on the F-actin structure of HeLa cells. Cells
were plated on glass coverslips and incubated with vehicle (DMSO), 5o, 5p, or 8b for 24 h and were
fixed and stained with Acti-stain 488 phalloidin. Cells were observed
under a fluorescent microscope, a digital CCD camera, and 40×
objective. Data are representative of three independent experiments.
Effects of thiazole analogues 5o, 5p,
and 8b on the F-actin structure of HeLa cells. Cells
were plated on glass coverslips and incubated with vehicle (DMSO), 5o, 5p, or 8b for 24 h and were
fixed and stained with Acti-stain 488 phalloidin. Cells were observed
under a fluorescent microscope, a digital CCD camera, and 40×
objective. Data are representative of three independent experiments.
Conclusion
In
summary, the 63 new thiazole derivatives designed and prepared
by further structural modifications of the previously discovered lead
compounds 1 and 2 produced more potent fascin
inhibitors. Among the active compounds, the 5 series
of analogues with longer alkyl chain substitutions on the thiazolenitrogen exhibited greater antimigration activities than those with
other structural motifs. The most potent analogue, 5p, inhibited 50% of cell migration at a concentration of 24 nM. As
expected, all thiazole derivatives that were selected for Matrigel
invasion assays were found to have potent antiinvasion activities.
In addition to antimigration and antiinvasion properties, two representative
thiazole derivatives, 5o and 5p, also showed
strong antiangiogenesis activity, blocking new blood vessel formation
in a chicken embryo membrane assay. A functional study in which enhanced
cell motility due to fascin overexpression was nearly completely abrogated
by the most potent analogues 5o and 5p provided
additional evidence that the action of the thiazole derivatives involved
targeting the F-actin bundling protein fascin. This proposed mechanism
of action is further supported by the observation that cellular F-actin
structures were significantly impaired by the treatment of the thiazole
analogues.
Experimental Section
All
reagents and solvents were purchased
from AK Scientific, Sigma-Aldrich Chemical Co., Fisher Scientific,
ACROS, and Pharmco-AAPER and were used as received. All organic solvents
(Pharmco-AAPER) used were of reagent grade quality and were used without
further purification. NMR spectra were recorded on a Bruker Fourier-300
spectrometer (Bruker Inc., Billerica, MA) in ppm. Crude synthetic
products were purified by the following methods: chromatography on
silica gel (60–100 mesh, Fisher Scientific) column. Analytical
thin layer chromatography (TLC) was performed on 250 μm fluorescent
plates (Agela Technologies, DE, USA) and visualized by using UV light.
For all products, the purity was ascertained to be greater than 95%
by the HPLC method using a Shimadzu (Columbia, MD) 2010 HPLC–UV/MS
system with a C-18 reverse phase column and by GC–MS analyses
using an Agilent Technologies 5975C inert MSD mass spectrometer.
General Procedure for N-Alkylation of 3 and 4
To a cooled mixture of NaH (0.26 g, 60% in oil,
6.5 mmol) in THF (20 mL) was added a solution of compound 3 or 4 (5 mmol) in THF (10 mL) dropwise. The mixture
was warmed to room temperature and stirred for 20 min. After that,
the mixture was cooled to 0 °C again and MeI or RBr (6.5 mmol)
was added dropwise. The mixture was then warmed to room temperature
and stirred for 2 h. Water (5 mL) was added to quench the reaction,
and the mixture was further diluted with water (50 mL). The mixture
was extracted with CH2Cl2 (3 × 50 mL).
The combined organic phase was dried with anhydrous MgSO4. After removal of all the solvent, the residue was purified by silica
gel chromatography (hexane/EtOAc = 4:1) to afford products 5 (more polar) and 6 or 7 (more polar) and 8 as solids.
Synthesis of N-(4-(2,4-Dimethylphenyl)thiazol-2-yl)benzenesulfonamide
(10)
To a solution of 9 (0.61g,
3.0 mmol) and pyridine (0.96 mL, 12 mmol) in anhydrous dichloromethane
was added benzenesulfonic chloride (0.76 mL, 6 mmol) in dichloromethane
dropwise at 0 °C. After being stirred at room temperature for
2 h under nitrogen atmosphere, the reaction mixture was concentrated
in vacuo. The saturated Na2CO3 solution was
added to quench the reaction, and the solution was extracted with
ethyl acetate, dried over MgSO4, and concentrated in vacuo.
The residue was purified by flash chromatography to give product 10 (0.57 g, 55% yield) as a solid. 1H NMR (300
MHz, CDCl3): 9.85 (bs, 1H), 7.93 (d, J = 7.8 Hz, 2H), 7.53–7.43 (m, 3H), 7.16 (d, J = 7.8 Hz, 1H), 7.08–7.04 (m, 3H), 6.26 (s, 1H), 2.34 and
2.32 (ds, 6H). 13C NMR (75 MHz, CDCl3): 168.7,
141.9, 140.1, 136.3, 136.1, 132.2, 132.0, 128.9, 128.7, 127.2, 126.5,
125.7, 103.6, 21.2, 20.4. GC–MS: 344 (M+). HRMS
(ESI(+)) calcd for C17H17N2O2S2 (M + H): 345.0731. Found: 345.0727.
Methylation
of 10 To Afford N-(4-(2,4-Dimethylphenyl)-3-methylthiazol-2(3H)-ylidene)benzenesulfonamide (11) and N-(4-(2,4-Dimethylphenyl)thiazol-2-yl)-N-methylbenzenesulfonamide (12)
To a cooled
mixture of NaH (0.07 g, 60% in oil, 1.6 mmol) in THF (5 mL) was added
a solution of compound 10 (0.34g, 1 mmol) in THF (5 mL)
dropwise. The mixture was warmed to room temperature and stirred for
20 min. After that, the mixture was cooled to 0 °C again and
MeI (0.25 mL, 4.0 mmol) was added dropwise. The mixture was then warmed
to room temperature and stirred for 2 h. Water (3 mL) was added to
quench the reaction, and the mixture was further diluted with water
(10 mL). The mixture was extracted with CH2Cl2 (3 × 10 mL). The combined organic phase was dried with anhydrous
MgSO4. After removal of all the solvent, the residue was
purified by silica gel chromatography (hexane/EtOAc = 4:1) to afford
products 11 (more polar) (0.27 g, 75% yield) and 12 (0.034 g, 9% yield) as solids.11: 1H NMR (300 MHz, CDCl3): 8.05–8.02 (m, 2H),
7.58–7.43 (m, 3H), 7.12–7.01 (m, 3H), 6.27 (s, 1H),
3.19 (s, 3H), 2.36 (s, 3H), 2.11 (s, 3H). 13C NMR (75 MHz,
CDCl3): 166.8, 142.3, 140.6, 139.1, 137.6, 131.9, 131.4,
130.5, 128.6, 127.1, 126.6, 126.4, 103.5, 33.6, 21.3, 19.5. GC–MS:
358 (M+). HRMS (ESI(+)) calcd for C18H19N2O2S2 (M + H): 359.0888. Found:
359.0879.12: 1H NMR (300 MHz, CDCl3):
7.83 (d, J = 7.2 Hz, 2H), 7.58–7.38 (m, 4H),
7.02–6.98 (m, 2H), 6.88 (s, 1H), 3.45(s, 3H), 2.32 and 2.30
(ds, 6H). 13C NMR (75 MHz, CDCl3): 160.0, 151.2,
137.9, 136.8, 135.9, 133.8, 131.8, 131.4, 129.4, 129.3, 127.4, 126.6,
112.0, 36.7, 21.2, 21.1. GC–MS: 358 (M+). HRMS (ESI(+))
calcd for C18H19N2O2S2 (M + H): 359.0888. Found: 359.0880.
General Procedure for the
Acylation of 13 To Afford 14
To
a solution of 13 (0.01 mol) and
DMAP (1.24 g, 0.01 mol) in anhydrous dichloromethane was added the
acyl chloride in dichloromethane dropwise at 0 °C. After being
stirred at room temperature for 2 h under nitrogen atmosphere, the
reaction mixture was concentrated in vacuo. The saturated Na2CO3 solution was added to quench the reaction, and the
solution was extracted with ethyl acetate, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash chromatography
to give product 14 as a solid.
To a cooled mixture of NaH (0.26 g, 60% in oil,
6.5 mmol) in THF
(20 mL) was added a solution of compound 14 (5 mmol)
in THF (10 mL) dropwise. The mixture was warmed to room temperature
and stirred for 20 min. After that, the mixture was cooled to 0 °C
again and MeI (6.5 mmol) was added dropwise. The mixture was then
warmed to room temperature and stirred for 2 h. Water (5 mL) was added
to quench the reaction, and the mixture was further diluted with water
(50 mL). The mixture was extracted with CH2Cl2 (3 × 50 mL). The combined organic phase was dried with anhydrous
MgSO4. After removal of all the solvent, the residue was
purified by silica gel chromatography (hexane/EtOAc = 4:1) to afford
products 15 (more polar) and 16 as solids.
MDA-MB-231 cell line was a gift from Dr.
KiTani Johnson, and HeLa cell line was a gift from Dr. Thomas Wiese,
both of Xavier University of Louisiana College of Pharmacy. A549 cells
were generously provided by Dr. Mandip Sachdeva of Florida A&M
University College of Pharmacy. These cancer cell lines were routinely
cultured in DMEM medium supplemented with 10% FBS, 4 mM glutamine,
1 mM sodium pyruvate, 100 IU/mL penicillin, 100 μg/mL streptomycin,
and 0.25 μg/mL amphotericin. Cultures were maintained in 5%
carbon dioxide at a temperature of 37 °C.
In Vitro Migration Assays
Migration assays were performed
following the manufacturer’s instructions (BD Falcon). Briefly,
MDA-MB-231, A549, or HeLa cells were seeded at a density of 2.5 ×
104 in 500 μL of serum-free and phenol red-free media
in the upper chamber of a 24-well Transwell system. DMEM supplemented
with FBS (5%) was used as a chemoattractant in the lower wells. After
24 h, membranes were scrubbed, fixed with 10% phospho-buffered formalin,
permeabilized with 100% ice-cold methanol, and stained with 0.1% crystal
violet in 20% methanol. Membranes were removed and mounted on glass
slides for visualization by light microscopy. For dose dependent migration
assays, MDA-MB-231 cells were seeded in the Transwell migration chambers
as described above and treated with 10–8, 10–7, 10–6, and 10–5 M concentrations of each thiazole compound for measurement of resulting
cell migration; 0.1% DMSO was used as vehicle control. Data are represented
as % of migrated cells treated with vehicle per 100× field of
view (100×).
Cell Survival/Growth Assay
For growth
assay in the
presence of 10–5 M individual thiazole compound,
MDA-MB-231 cells were plated in six-well plates at a density of 50 000
per well in DMEM medium supplemented with 10% FBS. The cells were
then cultured for 5 days, while equal treatment volumes of DMSO were
used as vehicle control. Cell numbers were counted with a Coulter
instrument (Beckman-Coulter). The ratio of thiazole compound treated
cell numbers to vehicle treated cell numbers was defined as survival
ratio. Experiments were conducted in triplicate and data represented
as the mean ± SD.
Invasion Assays
Matrigel-coated
invasion chambers (BD
Biosciences, NJ) were used to determine the inhibitory effect of the
thiazole derivatives on MDA-MB-231 cells. Briefly, cells in exponential
phase of growth were serum-starved for 24 h prior to seeding, detached
by brief trypsinization, and resuspended in medium containing DMSO
or thiazole derivatives. The Matrigel invasion inserts were prepared
following the manufacturer’s instructions. MDA-MB-231 cells
(2.5 × 104 cells/well) were seeded in the upper chamber,
and medium supplemented with 10% FBS as chemoattractant containing
DMSO or 10 μM thiazole derivatives was added to the bottom well.
After incubation for 24 h, the noninvasive cells were removed from
the upper surface of the membrane by “scrubbing”, and
the invasive cells on the under surface of the membrane were fixed
with 4% formaldehyde for 10 min at room temperature and stained with
0.04% crystal violet, counted microscopically at 100× magnification.
Five fields per membrane were randomly selected and counted in each
group. The relative invasion of cells was calculated as the percentage
invasion through the Matrigel membrane relative to that of DMSO treated
cells.
Overexpression of Human Fascin 1. Construction
of Fascin Vector
The fascin total RNA samples were extracted
from HeLa cells using a PureLink total RNA purification system (Invitrogen)
and quantitatively analyzed using a Nanodrop spectrophotometer (Thermo
Scientific). The fascin cDNA was generated using a Superscript III
one-step RT-PCR system (Invitrogen) with the following primers: fascin1-F
(sense) 5′-GAA TTC ATG ACC GCC AAC GGC ACA GC-3′ and
fascin1-R (antisense) 5′-AAG CTT CTA GTA CTC CCA GAG CGA GGC-3′.
The RT-PCR reaction was carried out as follows: step 1, 45 °C
for 30 min and 94 °C for 2 min; step 2, for 35 cycles 94 °C
for 15 s, 51 °C for 30 s, and 72 °C for 1 min and 30 s;
step 3, 72 °C for 5 min and hold at 4 °C. Humanfascin cDNA
(1.5 kilobases [kb]) samples were then cloned into a pcDNA 3.1 vector
(Invitrogen).
Overexpression of Fascin in MDA-MB-231 Cell
For fascin
overepression, the MDA-MB-231 cells were seeded at a density of 5
× 105 cells par well in six-well plates. At 70–80%
confluence, the cells were transiently tranfected with 4 μg
of the fascin-expressing plasmid pcDNA-fascin. The transfection was
achieved by incubating the cells overnight at 37 °C in a CO2 incubator with Lipofectamine 2000 reagent (Invitrogen). The
next day, cell medium was replaced with 1000 μg/m G418 followed
by medium replacement every 2–3 days for a total 10–12
days. Stable clones were isolated after selection with 500 μg/mL
G418 for 2–3 weeks, and fascin expression levels were determined
by Western blot.
Chick Embryo Chorioallantoic Membrane (CAM)
Assay
Fertilized
embryos (Charles River Laboratories, Charleston, SC) were incubated
at 37.5 °C for 3 days, removed from their shell using a Dremel
tool, and placed into a covered weighing boat for 7 further days of
incubation. Solidified 30 μL onplants containing 2.1 mg/mL rat
tail collagen (BD Biosciences, Bedford, MA) and 10 ng of bFGF and
30 ng of VEGF in the presence or absence of thiazole analogues 5o and 5p were placed on the CAM over two pieces
of nylon mesh approximately 0.5 cm2. Four collagen onplants
were added per egg on at least three separate eggs. After 3 additional
days of incubation, images were taken of each plug on surviving embryos
using a mini-Vid camera (LW Scientific; Lawrenceville, GA) and quantified
in a masked fashion on a scale from 0 to 3 with 0 representing no
angiogenesis and 3 representing extreme angiogenesis. Data from one
scorer (confirmed by a second masked scorer) are presented as the
mean ± standard error of the mean. Statistical significance was
determined by one-way analysis of variance (ANOVA) followed by Dunnett’s
multiple comparison test (GraphPad Prism 6, La Jolla, CA).
Cell Phalloidin
Staining
HeLa cells were grown on glass
coverslips until about 50% confluent. Vehicle or thiazole analogues
treated HeLa cells were fixed for 15 min with 3.7% paraformaldehyde
in PBS. Cells were washed with PBS and permeabilized in pemeabilization
buffer (0.5% Triton X-100 (w/v) in PBS) for 10 min and washed again
with PBS. The cells were then treated for 30 min with 200 μL
of 100 nM Acti-stain 488 phalloidin in the dark. The coverslips were
rinsed with PBS and inverted on a drop of antifade mounting media
on a glass slide. Cells were observed under a fluorescence microscope
equipped with a 480Ex/535Em filter set, a digital CCD camera, and
40× objective.
Authors: Shilong Zheng; Qiu Zhong; Quan Jiang; Madhusoodanan Mottamal; Qiang Zhang; Naijue Zhu; Matthew E Burow; Rebecca A Worthylake; Guangdi Wang Journal: ACS Med Chem Lett Date: 2013-01-15 Impact factor: 4.345
Authors: Giuseppe Pelosi; Felice Pasini; Filippo Fraggetta; Ugo Pastorino; Antonio Iannucci; Patrick Maisonneuve; Gianluigi Arrigoni; Giovanni De Manzoni; Enrica Bresaola; Giuseppe Viale Journal: Lung Cancer Date: 2003-11 Impact factor: 5.705
Authors: Yafeng Ma; Louise E Reynolds; Ang Li; Richard P Stevenson; Kairbaan M Hodivala-Dilke; Shigeko Yamashiro; Laura M Machesky Journal: Biol Open Date: 2013-09-19 Impact factor: 2.422
Authors: Nuria Díaz-Argelich; Ignacio Encío; Daniel Plano; Aristi P Fernandes; Juan Antonio Palop; Carmen Sanmartín Journal: Molecules Date: 2017-08-02 Impact factor: 4.411
Figure 1
Scheme 1
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Figure 6