SUPPLEMENTARY DATA
Suppl. Scheme 1: Chemical synthesis of Red-Br-nos
Red-Br-nos((S)-3-(R)-9-bromo-5-(4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinoline)
Experimental:
General: 1H NMR and
13
C NMR spectra were measured in CDCl3 on INOVA 400 NMR
spectrometer. All proton NMR spectra were recorded at 400 MHz. All carbon NMR spectra were
recorded at 100 MHz and were referenced with 77.27 ppm resonance of residual chloroform.
Abbreviations for signal coupling are as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet. Infrared spectra were recorded on sodium chloride discs on Mattson Genesis II FT-IR.
High resolution mass spectra were collected on Thermo Finnigan LTQ-FT Hybrid mass
spectrophotometer using 3-nitrobenzyl alcohol, in some cases with addition of LiI as a matrix.
Melting points were determined using a Thomas Hoover melting point apparatus and were
uncorrected. All reactions were conducted in oven-dried (125°C) glassware under an atmosphere
of dry argon. All common reagents and solvents were obtained from commercial suppliers and
used without further purification unless otherwise indicated. Solvents were dried by standard
methods. The reactions were monitored by thin layer chromatography (TLC) using silica gel 60
F254 (Merck) precoated glass sheets. Flash chromatography was carried out on standard grade
silica gel (230-400 mesh).
Synthesis of reduced bromonoscapine (Red-Br-nos) 2 was carried out starting from noscapine
(NOS) in two steps. The first step involved bromination of noscapine molecule using Nbromosuccinimide (NBS) in presence of triethylamine in DMF at 50°C for 4h to give 9bromonoscapine 1. In the next step, the carbonyl moiety of the lactone ring of 1 was reduced
1
efficiently by triethylsilane in presence of a moderately strong lewis acid, borontrifluoride
diethyletherate
to
produce
the
target
compound
9-bromo-5-(4,5-dimethoxy-1,3-
dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinoline 2. This new method to synthesize Red-Br-Nos is relatively better than the one described
earlier1, as it avoids the use of HBr (a strong acid) and pH adjustment of the reaction mixture
using alkali. Furthermore, it gives better yield in the second step (carbonyl reduction).
(S)-3-((R)-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (1): Noscapine (1.0 g, 2.42 mmol) and Nbromosuccinimide (1.7 g 4.11 mmol) were dissolved in 5.0 ml of anhydrous DMF followed by
the addition of triethylamine (0.67 ml, 4.84 mmol). This reaction mixture was stirred at 50°C for
8h. After TLC control the reaction mixture was concentrated and residue was taken in
chloroform (20 ml) and washed with 1M sodium bicarbonate solution followed by water and
dried over sodium sulfate. The organic layer was then concentrated under vacuum to get 2 as
yellowish solid.
MS (ESI/tandem mass spectrometry): [M+H] = 493
(S)-3-(R)-9-bromo-5-(4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinoline (2): 9-bromonoscapine 2 (0.5 g, 1.21 mmol)
was taken in 5 ml of dry THF and cooled to 0°C. To this solution was added borontrifluoride
etherate (1.0 ml) and triethylsilane (0.5 ml, 3.15 mmol). The resulting mixture was stirred
magnetically at 60°C, over an oil bath for 4h. After TLC control the reaction mixture was
concentrated under reduced pressure and the residue obtained was taken in ethyl acetate (25 ml).
The organic layer was washed with 1 M sodium bicarbonate solution, brine and water
respectively and dried over sodium sulfate. Solvent was removed in vacuo to get crude product
which was chromatographed over flash silica (CH2Cl2 and MeOH, 98:2) to get title compound 2
as yellowish solid.
MS (ESI/tandem mass spectrometry): [M+H] = 480
2
Cell ID
Group ID
#Daughters
8
S1
2
8
S2
1
8
D1
2
8
D2
1
9
M1
0
9
M2
1
9
D1
2
9
D2
1
9
D3
1
Suppl. Table 1: Sample records for representative cells 8 and 9. The first four records define
cell 8, indicating that there are four physically distinct groups of centrioles, as follows: S1 and S2
indicate that there are two groups, each containing two mothers, one sharing two daughters and
the other sharing a single daughter; there are also two distinct de novo groups, one with two
daughters and another with an isolated daughter; no isolated mother groups are present. In cell 9
there is an isolated mother centriole with no associated daughters, another mother with one
associated daughter, and three distinct de novo groups with differing numbers of daughters.
3
Suppl. Fig. 1: Attenuation of ROS upon a 4h tiron treatment prior to Red-Br-nos exposure for
12h showed a marked reduction in the proportion of cells with γ-H2AX foci as compared to drug
treatment alone. Quantitation of the percentage of cells harboring γ-H2AX foci for the indicated
treatments is shown as a bar-graphical representation.
Suppl. Fig. 2: Binding of Red-Br-nos to free tubulin and its effect on tubulin tertiary
structure. Tubulin was isolated from bovine brain by two cycles of polymerization and
depolymerization without glycerol. MAP-free tubulin was purified from MAP-rich tubulin by
phosphocellulose chromatography, was drop-frozen in liquid nitrogen, and stored at -70ºC until
used2. All fluorescence measurements were performed using a Perkin Elmer LS 50B
spectrofluorometer equipped with a constant temperature water-circulating bath. A 0.3cm pathlength quartz cuvette was used for the measurements. A. Time-dependent binding of Red-Brnos to tubulin. The binding of Red-Br-nos to soluble bovine brain tubulin was studied by
measuring the quenching of the intrinsic tryptophan fluorescence of tubulin3. Red-Br-nos (100
µM) was incubated with 1 µM tubulin in PEM buffer for 45 min at 25°C. The relative intrinsic
fluorescence intensity of tubulin was monitored at 335 nm at an excitation wavelength of 295 nm
either immediately after addition of Red-Br-nos or at 30th and 60th min after the drug addition.
The fluorescence emission intensity of Red-Br-nos at 295 nm was negligible. Fluorescence data
was corrected for the inner filter effect using the Lakowicz equation Fcorrected = Fobserved antilog
[(Aex + Aem)/2], where Aex is the absorbance at the excitation wavelength and Aem is the
absorbance at the emission wavelength. The graph represents one of the two independent
experiments. B. Effect of Red-Br-nos on 1-anilinonaphthalene-8-sulfonic acid (ANS) binding
4
to tubulin. ANS binds to hydrophobic surfaces on proteins and thus is used as a probe to study
perturbation of tertiary structure of proteins by ligands. The binding of ANS to tubulin was
measured using methods described previously4. Tubulin (2 µM) was incubated with Red-Br-nos
(0-200 µM) for 20 min in PEM buffer at 25ºC. ANS (30 µM) was added to the mixture and were
incubated for 20 min at 25ºC. The samples were excited at 380 nm and the emission readings
were recorded at 470 nm. The experiment was performed three times. Data, mean  SD.
Suppl. Fig. 3: Immunofluorescence micrographs of control untreated PC-3 cells showing
progressive cell-cycle stages. γ-tubulin is shown in green, microtubules in red and DNA (DAPI)
in blue. Interphase cells show a single centrosome and metaphase cells show the expected
bipolar spindle configuration. Scale bar = 10 m
Suppl. Fig. 4: Bar-graphical quantitation showing the percentage of PC-3 cells that display
bipolar or multipolar spindle phenotype with increasing duration of Red-Br-nos exposure (10
µM). Analysis was based on the visual determination of phenotypes from randomly selected
fields totaling to at least 200 mitotic cells under a confocal microscope using a 63X objective
(NA 1.4).
5
Suppl. Fig. 5: Red-Br-nos is much more active than the parent noscapine in inhibiting the
proliferation of various human cancer cells. The panel of 60 human tumor cell lines is
organized into subpanels representing leukemia, non-small cell lung, colon, CNS, melanoma,
ovarian renal, prostate and breast cancer lines. Cells were treated with noscapine and Red-Br-nos
at increasing gradient concentrations for 48h. The IC50 values, which represent the drug
concentration needed to prevent cell proliferation by 50% was then measured using an in vitro
Sulforhodamine B assay. Panels show bar-graphically the comparison of IC50 values of
noscapine (black bars) and Red-Br-nos (grey bars) for cancer cell lines from various tissue
origins.
6
Suppl. Fig. 6: Red-Br-nos induces ROS-mediated autophagy at 10 µM. A. Immunoblot
analysis of LC3-II expression levels in lysates from PC-3 cells treated with 10 μM Red-Br-nos
for the indicated time points. β-Actin was used as a loading control. B. Immunofluorescence
microscopy of DCF, DHE and acridine orange-stained PC-3 cells treated for 24 h with DMSO
(control) or 10 μM Red-Br-nos. Panel i shows microscopic visualization of DCF fluorescence in
control and drug-treated PC-3 cells. Panel ii shows microscopic visualization of EtBr (EB)
fluorescence in control and drug-treated PC-3 cells. Panel iii represents ncrease in number of
cells with AO accumulating acidic vesicular organelles (orange-red fluorescence) in Red-Brnos-treated cells as compared to control. Green profile depicts control cells that were stained
with AO and the red profile shows drug-treated cells with increased AO fluorescence, indicative
of numerous red AVOs.
7
Supplementary movie-1: Time-lapse recordings of MDA-MB-231 cells treated with 10 µM
Red-Br-nos for 9 hours. Cells clearly show induction of several new green dots corresponding to
numerous centrioles.
Supplementary movie-2: Time-lapse recordings of MDA-MB-231 cells stably transfected with
GFP-centrin for 12 hours. Typical centrosomes with 2 centrioles are evident and there is no
appearance of numerous or abnormal centrioles
References
1.
Zhou J, Gupta K, Aggarwal S, Aneja R, Chandra R, Panda D et al. Brominated
derivatives of noscapine are potent microtubule-interfering agents that perturb mitosis
and inhibit cell proliferation. Mol Pharmacol 2003; 63(4): 799-807.
2.
Yenjerla M, LaPointe NE, Lopus M, Cox C, Jordan MA, Feinstein SC et al. The
neuroprotective peptide NAP does not directly affect polymerization or dynamics of
reconstituted neural microtubules. J Alzheimers Dis 2010; 19(4): 1377-86.
3.
Lopus M, Oroudjev E, Wilson L, Wilhelm S, Widdison W, Chari R et al. Maytansine and
cellular metabolites of antibody-maytansinoid conjugates strongly suppress microtubule
dynamics by binding to microtubules. Mol Cancer Ther 2010; 9(10): 2689-99.
4.
Lopus M, Panda D. The benzophenanthridine alkaloid sanguinarine perturbs microtubule
assembly dynamics through tubulin binding. A possible mechanism for its
antiproliferative activity. FEBS J 2006; 273(10): 2139-50.
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