Berberine Induces Toxicity in HeLa Cells through ... - ACS Publications

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A - ANS B - Berberine

Biochemistry C --Colchicine TRP 21B α-Tub C β-Tub

B ACS Paragon Plus Environment A

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Berberine induces toxicity in HeLa cells through perturbation of microtubule polymerization by binding to tubulin at a unique site

Darpan Raghav, Shabeeba M Ashraf, Lakshmi Mohan and Krishnan Rathinasamy* School of Biotechnology, National Institute of Technology Calicut, Calicut, Kerala, India.

Corresponding Author *Dr. K. Rathinasamy (), Asst. Professor, School of Biotechnology, National Institute of Technology Calicut, Calicut-673601, India E-mail: [email protected] Phone: +91-4952285455, Fax: +91-4952287250

ACS Paragon Plus Environment

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Biochemistry

Abstract Berberine has been used traditionally for its diverse pharmacological actions. It exhibits remarkable anticancer activities and is currently under clinical trials. In this study, we report that the anticancer activity of berberine could be partly due to its inhibitory actions on tubulin and microtubule assembly. Berberine inhibited the proliferation of HeLa cells with an IC 50 of 18 µM and induced significant depolymerization of interphase and mitotic microtubules. At its IC50, berberine exerted a moderate G2/M arrest and mitotic block as detected by FACS analysis and fluorescence microscopy respectively. In a wound closure assay, berberine inhibited the migration of HeLa cells at concentrations lower than its IC50 indicating its excellent potential as an anticancer agent. In-vitro studies with tubulin isolated from goat brain indicated that berberine binds to tubulin at a single site with a Kd of 11 µM. Berberine inhibited the assembly of tubulin into microtubules and also disrupted the preformed microtubules polymerized in the presence of glutamate and taxol. Competition experiments indicated that berberine could partially displace colchicine from its binding site. Results from FRET, computational docking and molecular dynamic simulations suggest that berberine forms a stable complex with tubulin and binds at a novel site 24 Å away from the colchicine site on the β-tubulin. Data obtained from synchronous fluorescence analysis of the tryptophan residues of tubulin and from the FTIR spectroscopy studies, revealed that binding of berberine alters the conformation of tubulin heterodimer which could be the molecular mechanism behind the depolymerizing effects on tubulin assembly. Keywords: Berberine, cancer, computational docking, molecular dynamic simulations, FRET and tubulin.


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INTRODUCTION

Berberine (5,6-dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium) is an isoquinoline alkaloid which is present in a wide variety of medicinal plants such as Berberis aristata (tree turmeric), B. vulgaris (Barberry), B. aquifolium (Oregon grape), Hydrastis canadensis (Goldenseal), Coptis chinensis (Coptis or Goldenthread) and several other species (1). Berberine has been found to be effective against diseases such as diarrhea, diabetes, metabolic syndrome, polycystic ovary syndrome, coronary artery disease, hyperlipidemia, obesity and fatty liver disease (2). Berberine is reported to exhibit antifungal, antiprotozoal and antimicrobial activity against a wide range of bacteria like Klebsiella pneumonia, Proteus vulgaris, Mycobacterium smegmatis, Mycobacterium tuberculosis, Helicobacter pylori, methicillin-resistant Staphylococcus aureus and Escherichia coli (3-7). The broad antimicrobial spectrum of berberine could be due to its DNA damaging effects and inhibition of cell division (2, 3). Berberine has been reported to inhibit the functions of FtsZ, the prokaryotic homolog of mammalian cell division protein tubulin (8). Recently, it has also been shown that berberine and its synthetic analogs bind to ftsZ of several other bacteria and exhibit growth inhibitory effects (9 - 11). In addition to its antimicrobial activity berberine also exerts significant anticancer activity (2). Since the 1900’s, intensive research has been carried out to investigate the antineoplastic properties of berberine. Berberine induced apoptosis in a large number of murine and mammalian cell lines such as HL-60, WEHI-3, HCC, HeLa, PC-3, MCF-7, L1210, EAC, U937, HepG2 and HCT-8 (3, 12). Several mechanisms have been proposed for its cytotoxicity like loss of mitochondrial membrane potential, activation of caspases, upregulation of p53, inhibition of DNA topoisomerase I and inhibition of DNA synthesis (1215). There are several contradicting reports regarding its mechanism of action. Berberine has shown to inhibit the proliferation of prostate cancer cells by inducing G1 arrest followed by apoptosis in one study (16), while it was shown to induce G2/M arrest in several cancer cells (17-20). It is apparent from the literature that the cytotoxic effects of berberine might involve multiple targets and mechanisms. Since berberine inhibited the proliferation of several cancer cell lines through multiple mechanisms including the G2/M block, we tried to decipher the mechanism behind the G2/M block. Tubulin, the major component of microtubules play a crucial role in cell division, and any interference in the functioning of tubulin leads to mitotic block and cell cycle arrest


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Biochemistry

which makes it an effective target for antimitotic drugs (21, 22) and cancer chemotherapy. Microtubule-targeted agents remain the most classical and reliable antimitotic agents till date (21). The microtubule-targeted antimitotic drugs are generally classified into two major groups, the microtubule-destabilizing agents, and the microtubule-stabilizing agents. The socalled ‘stabilizing agents’ enhance microtubule polymerization whereas the ‘destabilizing agents’ inhibit microtubule polymerization when present at very high concentrations; however, both the agents suppress microtubule dynamics without significantly altering the microtubule polymer mass at their half-maximal inhibitory concentration (21, 22). Most of the depolymerizing agents prefer binding at either of the two well-characterized domains on tubulin heterodimer, i.e., the “Vinca” domain or the “colchicine” domain. Vinca site binding agents include the Vinca alkaloids, the cryptophycins, the dolastatins, eribulin, spongistatin, rhizoxin and maytansinoids. Colchicine-site binding agents include colchicine and its analogs, podophyllotoxin, combretastatins, steganacins and curacins (21, 22). In this study, we show that berberine binds to tubulin and depolymerizes the interphase and mitotic microtubules in HeLa cells. Our experimental evidence indicates that berberine binds to tubulin at a novel site that is located between the colchicine binding site and ANS/bis-ANS binding site in the β subunit. Binding of berberine resulted in the inhibition of microtubule polymerization and rapid disruption of preformed microtubules. The FTIR studies indicated that berberine altered the secondary structure of tubulin upon binding. Our data collectively indicate that the antiproliferative activity of berberine could be partly through perturbation of microtubule assembly. Berberine also suppressed HeLa cell migration at concentrations lower than its observed IC50, indicating towards its clinical relevance. Our findings suggest that berberine can be used as an anticancer drug either alone or in combination with other established anticancer drugs for better therapeutic outcomes. 2. EXPERIMENTAL PROCEDURES 2.1. Chemicals Berberine chloride hydrate was purchased from Alfa Aesar (Haverhill, Massachusetts, United States). Paclitaxel, vinblastine sulfate, 1-anilinonaphthalene-8-sulfonic acid (ANS), podophyllotoxin, colchicine, 5,5´-dithiobis-2-nitrobenzoic acid (DTNB), sulforhodamine B (SRB), Hoechst 33342, guanosine 5´-triphosphate (GTP), propidium iodide, EGTA, MgCl2, piperazine-N,N′-bis (2-ethanesulfonic acid) (PIPES), mouse monoclonal anti-α-tubulin IgG and FITC conjugated anti-mouse IgG were purchased from Sigma-Aldrich (St. Louis, MO,


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USA). Fetal bovine serum (FBS) and Alexa Fluor 568 conjugated anti-mouse IgG were purchased from Invitrogen (Thermo Scientific). Acridine orange (AO), L-tryptophan, Minimal essential medium (MEM), cell culture tested antibiotic solution, and phosphatebuffered saline (PBS), were purchased from HiMedia (Mumbai, India). All the other reagents used in this study were of analytical grade. 2.2. Cell culture and inhibition of cell proliferation HeLa cells were obtained from NCCS, Pune, India and were grown in 25 cm2 tissue culture flasks in a humidified atmosphere containing 5% CO2 and 95% air at 37 oC. Cells were maintained in minimal essential medium (MEM) supplemented with 10 % (v/v) fetal bovine serum, sodium bicarbonate and antibiotic solution containing 100 units of penicillin, 100 µg of streptomycin, and 0.25 µg of amphotericin B per mL. Berberine stock solution was prepared in 100% dimethylsulfoxide (DMSO). The effect of berberine on HeLa cells was determined by incubating the cells with different concentrations of berberine (0 -100 µM) for 24 h in 96-well tissue culture plates. In all the experiments the final DMSO concentration was kept ≤ 0.1% (v/v) and 0.1% DMSO alone was used as a vehicle control. The inhibition of proliferation was studied using the standard sulforhodamine B (SRB) assay (23). 2.3. Determination of cell cycle distribution by flow cytometric analysis HeLa cells (0.5×105 cells/mL) were grown in 25 cm2 tissue culture flasks and treated with increasing concentrations of berberine for 24 h. At the end of the treatment the cells were trypsinized and harvested by centrifugation at 5000 RPM for 5 min and were fixed in 70 % ethanol. The cells were then washed twice with PBS solution and were resuspended in 0.5 mL of PBS solution containing 10 µg/mL PI and 50 µg/mL RNase A. After incubation in the dark for 1 h at 37 ○C, cells were passed through the cell strainer (pore size 40 µm) and the cell cycle distribution was analyzed by using BD FACSAria flow cytometer (BD Biosciences, San Jose, CA). 2.4. Immunofluorescence microscopy To determine the mitotic index and to visualize the interphase and mitotic microtubules, HeLa cells (0.5×105 cells/mL) were grown on poly-l-lysine coated glass coverslips (12 mm) in 24-well tissue culture plates and were subsequently treated with different concentrations of berberine or colchicine (7 and 20 nM) for 24 h. The tissue culture plates were gently centrifuged in a cytospin for 10 mins and the cells were then fixed with


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Biochemistry

3.7% (v/v) formaldehyde solution in PBS for 30 min at 37 oC. The cells were permeabilized with cold methanol at -20 oC for 15 min. Immunostaining was performed using mouse monoclonal alpha-tubulin antibody and goat anti-mouse IgG conjugated to Alexa Fluor 568 (24, 25). The DNA was stained using Hoechst 33342. The coverslips were mounted on clean glass slides with the mounting medium containing 1, 4-diazabicyclo [2.2.2] octane (DABCO) as an anti-quenching agent. The number of cells in the mitotic phase was determined by observing the organization of the chromosome and the organization of the microtubules under the Nikon ECLIPSE Ti-E inverted fluorescent microscope (Tokyo, Japan). The mitotic index was calculated as the percentage of cells blocked at mitosis (23, 24), at least 1,000 cells were scored for each concentration of berberine and colchicine. Immunofluorescence images were acquired using the CoolSNAP digital camera and were processed by using ImageJ (NIH, USA). 2.5. Acridine orange (AO) staining for detection of early and late apoptosis HeLa cells (0.5×105 cells/mL) were incubated with either a vehicle (0.1% DMSO) or with different concentrations of berberine (10, 20, and 50 µM) for 24 h. The live cells were immediately viewed under an inverted fluorescent microscope after adding AO (2 µg/mL) and the images were captured using the CoolSNAP digital camera. 2.6. Wound closure assay HeLa cells (1x106 cells/mL) were grown in Minimum Essential Medium supplemented with 10% FBS in 35 mm cell culture dishes. At 90% confluence, a wound was made using a sterile micropipette tip. The floating cells were removed immediately after wounding, and the media was changed with fresh one containing different concentrations of berberine (0, 5, and 10µM). Cells were observed at 24, 48 and 72 h intervals and the bright field images of the wound closure were recorded using the Nikon ECLIPSE Ti inverted microscope. Percentage wound healing was calculated by using the formula: % Cell migration = [1-(width of scratch at specific time point ‘t’/ width of the scratch at zero time)] × 100

2.7. Purification of goat brain tubulin Tubulin was isolated from goat brains by two cycles of polymerization using glutamate and depolymerization in cold as described earlier (26, 27). Tubulin plus microtubule-associated proteins (MAPs) was isolated using 4M glycerol as described earlier (26-28). The purity level of isolated tubulin was determined by Coomassie Brilliant Blue-


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stained 10% SDS-PAGE. The protein concentration was determined by using a molar extinction coefficient of 115,000 M-1cm-1 for tubulin at 280 nm. The protein was stored in aliquots at -80 oC until further use. All the experiments with tubulin were performed in PEM Buffer (25 mM PIPES, 1 mM EGTA, 3 mM MgCl2, pH 6.8). 2.8. Spectral measurements All the fluorescence measurements were done using a JASCO FP-8300 (Tokyo, Japan) spectrofluorometer equipped with a thermostatted cell holder directly connected to a circulating water bath for maintaining constant temperature. In order to minimize the inner filter effects of berberine, a 0.3 cm path-length cuvette was used for all the fluorescence spectral measurements. The absorbance data was recorded using a Perkin-Elmer UV-visible spectrophotometer (USA) in a 1 cm path-length cuvette. The FTIR spectra was recorded using Perkin-Elmer FTIR Spectrometer (USA). The data were analyzed and processed using SigmaPlot 12.5. 2.9. Determination of berberine binding to tubulin using fluorometric analysis A primary stock solution of berberine was prepared by dissolving berberine chloride crystals in 100 % DMSO. The final concentration of DMSO was kept ≤ 2% in all the experimental sets.

The concentration of berberine was determined by using a molar

extinction coefficient (Ɛ) of 22,500 Mˉ1 cm-1 at 344 nm (Figure 1). The binding of berberine to tubulin was determined by incubating tubulin (1 µM) with increasing concentrations of berberine (0-100 µM) for 30 min at 37 oC. The samples were excited at 295 nm to excite the intrinsic tryptophan residues of tubulin, and the emission spectrum was recorded. The binding of berberine to tubulin was also determined by using the hydrophobic fluorescent probe ANS (27). Tubulin (2 µM) was incubated with 40 µM ANS for 15 min to form a stable tubulinANS complex at room temperature followed by addition of increasing concentrations of berberine (0-100 µM). The reaction mixtures were then incubated at 37 oC for an additional 30 min. The mixtures were excited at 400 nm, and the emission spectrum was recorded. The background fluorescence contributed by the PEM buffer and free berberine were routinely subtracted from all the spectral measurements. Inner filter correction was done according to the equation F = Fobs antilog [(Aex + Aem)/2], where Aex is the absorbance of berberine at the excitation wavelength and Aem is the absorbance of berberine at the emission wavelength (24, 27). The fraction of binding sites (X) occupied by berberine was calculated by using the equation X = (Fo

–

Fc)/ΔFmax, where Fo and Fc represent the fluorescence intensity of


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tubulin/tubulin-ANS complex in the absence and presence of different concentrations of berberine respectively. ΔFmax, which is the maximum change in the fluorescence intensity of tubulin/tubulin-ANS complex after all the binding sites were occupied with the ligand, was determined from the Y-intercept of the graph 1/ ΔFmax vs. 1/ [berberine]. The dissociation constant (Kd) was calculated assuming a single binding site of berberine per tubulin dimer using the relationship, 1/X = 1 + (Kd/Lf), (where Lf is the concentration of free berberine (24, 27). The experiment was repeated three times.

2.0

Optical Density (OD)

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Biochemistry

1.5

1.0 Berberine

0.5

0.0 250

300

350

400

450

500

550

600

Wavelength (nm) Figure. 1. Absorbance spectrum of 40 µM berberine. The inset shows the chemical structure of

berberine

(5,

6-dihydro-9,

10-dimethoxybenzo[g]-1,

3-benzodioxolo

[5,

6-a]

quinolizinium). 2.10. Job’s plot Job’s method of continuous variation was used to determine the stoichiometry of berberine binding to tubulin (27-29). Several mixtures of tubulin and berberine were prepared by continuously varying the concentrations of tubulin and berberine in the mixtures keeping the total concentration of berberine plus tubulin constant at 2 µM. The samples were then incubated at 37 oC for 30 min and were excited at 295 nm and the emission spectrum was recorded. The experiment was repeated three times. 2.11. Time dependent quenching of tubulin fluorescence by berberine The kinetics of berberine binding to tubulin was monitored at 37 oC for 55 minutes. Berberine (10 µM) was added to tubulin (1 µM) and the time-dependent quenching of


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tryptophan fluorescence by berberine was monitored. The samples were exciting at 295 nm at every 2 min interval. Emission at 336 nm was recorded and plotted after solvent correction and correction for the inner filter effect (29). 2.12. Investigation of tubulin conformation upon berberine binding by synchronous fluorescence spectroscopy technique The synchronous fluorescence spectroscopic technique introduced by Lloyd is a useful method to detect the micro-environmental changes occurring in the close proximity of the chromophores (30). In this study, the synchronous fluorescence spectrum was recorded by using the excitation and emission monochromators of the spectrofluorometer simultaneously by maintaining a fixed wavelength difference (Δλ) between them. To check the effect of berberine on tryptophan and tyrosine residues, tubulin (1 µM) was incubated with increasing concentrations of berberine (0-40 µM) at 37 oC for 30 min and synchronous fluorescence spectrum was recorded by fixing Δλ at 60 and 15 nm respectively (31, 32). The background fluorescence of the buffer at the same Δλ value was routinely subtracted for all the samples. Inner filter correction was done as described earlier. 2.13. Inhibition of tubulin assembly by berberine To check the effect of berberine on tubulin polymerization, the standard sedimentation assay was performed as described earlier (28). Briefly, tubulin (12 µM) was incubated with different concentrations of berberine (0, 20, 40, 60, and 100 µM) in the polymerization buffer containing 25 mM PIPES, 1 mM EGTA, 3 mM MgCl2, 0.8 M glutamate, and 1 mM GTP, pH 6.8 at 37 oC for 45 min. The polymers were sedimented by centrifugation at 50,000 x g for 45 min at 30 oC. The pellet and supernatant fractions were separated and the protein concentration present in the supernatant was determined using Bradford assay (33). A similar experiment was performed to analyze the effect of berberine on polymerization of MAP rich tubulin (15 µM). MAPs can induce the polymerization of tubulin in the presence of GTP without any inducer. All the experiments were performed three times. The inhibition of microtubule assembly by berberine was also studied by a light scattering assay (27-29). Tubulin (12 µM) was mixed with different concentrations of berberine (0, 20, 40, 60, 80, 100 µM) in the polymerization buffer. Colchicine (20 µM) was used as a positive control for the experiment. The polymerization reaction was initiated by incubating the sample in a thermostatted cuvette holder directly connected to a circulating


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Biochemistry

water bath maintained at 37 oC. The assembly kinetics was monitored by light scattering at 350 nm for 15 min using JASCO FP-8300 spectrofluorometer. 2.14. Effect of berberine on preformed microtubules To analyze the disruptive effects of berberine on preformed microtubules two different approaches were employed. In both the approaches, we used the change in relative light scattering at 350 nm as a measure of the polymer mass (27). In the first approach, tubulin (12 µM) was allowed to polymerize in the polymerization buffer containing 0.8 M glutamate for 10 min at 37 oC, and then 100 µM berberine was added to the preformed microtubules. Relative light scattering at 350 nm was monitored for the next 15 min. In the second approach, tubulin (12 µM) was allowed to polymerize in the presence of paclitaxel (20 µM) for 10 min at 37 oC, and then berberine (100 µM) was added, and the light scattering was monitored for the next 15 min. 2.15. Effect of berberine on the binding of Vinblastine to tubulin Vinblastine is a non-fluorescent compound whose binding site on tubulin is well characterized (21, 22). Berberine exhibits a weak fluorescence in aqueous buffers when excited at 430 nm (34). Taking advantage of berberine fluorescence a competition assay involving berberine and vinblastine was performed. Tubulin (2 µM) was incubated with 20 µM berberine for 30 minutes at 37 oC to form a stable tubulin-berberine complex. Increasing concentrations of vinblastine (0, 2, 5, 10 and 20 µM) were then added to the complex, and the mixtures were further incubated for 20 minutes at room temperature. The samples were then excited at 430 nm and the emission spectrum was recorded. The background fluorescence from the buffer was routinely subtracted from all the samples. 2.16. Effect of berberine on the binding of colchicine and podophyllotoxin to tubulin Colchicine is one of the well-characterized tubulin binding agents. One remarkable property of this compound is that in aqueous solutions the fluorescence of colchicine is negligible; however, upon binding to tubulin its fluorescence increases by several folds (35). Tubulin (2 µM) was incubated with colchicine (10 µM) for 1 h at 37 oC to form a stable tubulin-colchicine (T-C) complex. Different concentrations of berberine (0, 40, 80, and 100 µM) were then added and the reaction mixtures were incubated for an additional 30 min at 37 o

C. All the samples were excited at 360 nm and the emission spectra was recorded. The


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background fluorescence from the buffer and the free ligands were routinely subtracted from all the samples and the inner filter correction was done as described earlier. To study the effect of berberine on the binding of podophyllotoxin, a non-fluorescent colchicine site agent, tubulin (1 µM) was incubated with berberine (40 µM) for 30 min at 37 o

C to form a stable berberine-tubulin complex. Different concentrations of podophyllotoxin

(0, 40, 60, 80, and 100 µM) were then added which was followed by incubation for further 30 min at 37 oC. Samples were then exited at 430 nm, and the emission was recorded from 450 to 600 nm. 2.17. Probing the binding site of berberine on tubulin using computational docking The X–ray crystal structure of tubulin-colchicine: stathmin-like domain complex (PDB ID: 1SA0, resolution: 3.58 Å) was selected from the protein data bank (36). This structure is a tetramer containing two α-tubulin (A and C) subunits and 2 β-tubulin subunits (B and D) arranged in an alternate manner. The protein was prepared by detaching subunits C and D along with stathmin-like domain complex. Heteroatoms like colchicine, GTP and GDP were also removed. An energy minimization step was performed for this modified 1SA0 structure (now onwards 1SA0*) using SPDBV 4.10 software in order to repair the distorted geometries (37). Molecular docking was performed by using Autodock 4.0 running on an Ubuntu Operating System (38). Chemsketch was used to create clean and 3D structures of berberine and colchicine in MOL file format (39). OpenBabel was used to convert the MOL files into PDB files (40). Autodock module was used for processing and preparing the molecules. Briefly, polar hydrogens, Kollman charges and AD4 type atoms were added to the protein. Docking runs were performed by keeping the receptor as a rigid and ligand as a flexible molecule. Grid parameters and Dock parameters were maintained constant for all the runs. Lamarckian Genetic algorithm of 100 iterations was repeated 15 independent times and the results were clustered with a root mean square distance (RMSD) of 2.00 Å. The results were analyzed from the clustering graph. The clusters were compared on the basis of cluster size and binding energy. The cluster with the highest number of confirmations and least binding energy was selected as the most favorable cluster. Binding conformation and binding energy were analyzed with the help of AutoDock Tools 1.5.6 and Chimera 1.9 (41). To validate our docking analysis, the molecule 1SA0* was subjected to blind docking with colchicine. For this, the entire molecule 1SA0* was enclosed in a grid box of 126 x 126 x 126 grid points


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Biochemistry

with a grid spacing of 0.70 Å (42). After successful validations, the molecule 1SA0* was subjected to docking analysis with berberine using similar grid, dock and Lamarckian Genetic algorithm parameters. 2.18. Molecular dynamics simulations The molecular dynamics simulations of tubulin-berberine complex was carried out using GROMACSv5.1.3 with Gromos53a6 force field (43, 44). The PRODRG server was used to generate topology and parameter files of berberine (45). The molecular system was solvated with 44,486 Simple point charge water molecules (SPC/E) in a dodecahedron box and neutralized by adding 32 Na+ ions. Energy minimization of the system was done with 50,000 steps of steepest descent method during which bad contacts in the structure were removed. This was followed by applying positional restraints to the energy minimized system, which was then equilibrated for 100ps with NVT and NPT ensemble equilibration protocol for about 50,000 steps. Linear constraint (LINCS) algorithm was applied to fix all the hydrogen-related bond lengths and Particle Mesh Ewald (PME) was employed to treat long-range electrostatic interactions. Finally, MD simulations were performed for 10 ns under constant number of particles at constant temperature and pressure. The RMSD and distance analyses between berberine and tubulin were carried out using the GROMACS tools. The tubulin-berberine structure obtained after MDS was subjected to redocking using Autodock 4.0 and the results were clustered with a root mean square distance (RMSD) of 2.00 Å. The final docked structure of berberine on tubulin was analyzed using LigPlot (46). 2.19. Fluorescence Resonance Energy Transfer (FRET) and distance measurement of berberine from ANS binding site, colchicine binding site and TRP 21B. FRET is a nondestructive spectroscopic method that is routinely used for monitoring the proximity and the relative angular orientation of the donor and acceptor fluorophores separately or attached within the same macromolecule (47, 48). A stable tubulin-colchicine complex (Tubulin 5 µM + colchicine 5 µM) was prepared in the absence (Fo) and presence (F) of berberine (5 µM) to calculate the distance between the colchicine and the berberine binding site on the tubulin heterodimer. Similarly, to calculate the distance between ANS and berberine, a tubulin tubulin-ANS complex (Tubulin 2 µM and ANS 2 µM) was prepared in the absence (Fo), and in the presence of berberine (2 µM) (F). The distance between Trp 21B and berberine was calculated by incubating tubulin (5 µM) in the absence (Fo) and presence (F) of berberine (5 µM). The samples containing ANS and those containing colchicine were


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

excited at 400 and 360 nm respectively. The intrinsic tryptophan residues of tubulin were excited at 295 nm. Efficiency (E) of FRET was calculated by using the formula E = 1 – F/Fo, where Fo is the fluorescence intensity in the absence of the acceptor and F is the corrected fluorescence intensity in the presence of the acceptor (47-49). The emission intensity at 470, 430, 336 nm were used for the calculations involving ANS, colchicine and intrinsic tryptophan residues of tubulin respectively. The quantum yields (QD) of the tubulin-ANS complex, tubulin-colchicine complex and tubulin (intrinsic tryptophan residues) were considered as 0.47 (50), 0.03 (35) and 0.06 (51) respectively. The distance (r) between the donor and acceptor was estimated from the equation E = (Ro6) / (Ro6 + r6) where Ro is the critical distance between the donor and the acceptor when the efficiency of transfer is 50 % (47, 48). The spectral overlap integral, J was calculated by using a|e - UV-Vis-IR Spectral Software 1.2, FluorTools, www.fluortools.com. J was obtained in units of M-1 cm-1 (nm) 4 when the wavelength was plotted in nm. As per the equations defined by Lakowicz (52), Ro in Å was calculated using R06 = 8.79 × 10-5(ĸ2 n-4 QD J (λ)) where ĸ2 = 2/3 (the orientation factor for random orientation in fluid solution), n = 1.33 i.e. the average refractive index of the medium, QD is the fluorescence quantum yield of the donor in the absence of acceptor, and J is the effect of the spectral overlap between the fluorescence emission spectrum of the donor and the absorbance spectrum of the acceptor.

2.20. Monitoring the reaction kinetics for cysteine residue modification with DTNB 5, 5´-dithiobis-2-nitrobenzoic acid (DTNB) is a sulfhydryl-specific reagent that forms a complex with thiol groups present in tubulin. This unique property makes DTNB a useful probe for monitoring the conformational changes in tubulin due to ligand binding (27, 53, 54). Tubulin (3 µM) was incubated with berberine (0, 40, and 80 µM) at room temperature for 30 min. DTNB (200 µM) was then added to the reaction mixtures and the rate of sulfhydryl group modification was monitored by measuring the change in absorbance at 412 nm at different time points. The absorbance contributed by the buffer, and free berberine at the same wavelength was subtracted from all the samples. The number of sulfhydryl groups modified after 40 min was determined by using a molar extinction coefficient of 12,000 M1

cm-1 for TNB- at 412 nm.

2.21. Analysis of tubulin conformation upon berberine binding


Page 14 of 48

Page 15 of 48

The effect of berberine on the secondary structure of tubulin was examined by performing an FTIR analysis. Tubulin (10 µM) was incubated with berberine (40 µM and 80 µM) for 30 minutes at 37 oC. The samples were subjected to FTIR analysis by using attenuated total reflectance (ATR) method (55). For analysis, the obtained FTIR spectrum was converted into absorbance spectrum using formula Abs = 2- log [%T]. To decipher the narrow changes occurring in the amide I region of the tubulin spectrum upon berberine binding, a second derivative of the FTIR spectrum was generated from the absorbance spectrum using OriginLab 2016.

3. RESULTS 3.1. Inhibition of HeLa cell proliferation by berberine We investigated the antiproliferative effect of berberine on HeLa cells using the standard SRB assay. After 24 hours of treatment, berberine inhibited the proliferation of HeLa cells with a half-maximal inhibition (IC50) at 18 ± 0.1µM (Figure 2). The inhibition of proliferation was concentration dependent for example 10, 20, 40 and 60 µM berberine inhibited the cell proliferation by 35, 52, 78 and 88% respectively (Figure 2).

% Inhibition of cell proliferation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

120 100 80 60 40 20 0 0

20

40

60

80

100

120

Berberine (µM) Figure 2. Berberine inhibited the proliferation of HeLa cells with an IC50 of 18 ± 0.1µM. Berberine (0-100 µM) was incubated with HeLa cells for 24 h and a standard SRB assay was performed to determine the half maximal inhibitory concentration. 3.2. Berberine induced moderate mitotic block in HeLa cells


Biochemistry

Cells treated with different concentrations of berberine for 24 h were analyzed in FACS to determine the effect on cell cycle progression. As evident from Figure 3, berberine treatment increased the population of cells in G2/M phase in a concentration dependent manner. For example, 6.9, 13.6, 15.4 and 18.0 % cells were found to be in G2/M phase of the cell cycle in the absence and presence of 10, 20 and 40 µM berberine respectively (Table 1). Since berberine blocked the cell cycle progression at G2/M, we counted the number of cells blocked at the mitotic phase using fluorescence microscope and found that 3.5, 8.3, 10.2 and 12.4 % cells were blocked at mitosis when treated with 0, 10, 20 and 40 µM berberine respectively (Table 1). At higher concentrations, berberine did not increase the percentage of mitotic cells, instead induced cell death. As reported earlier, Colchicine at its IC50 induced a mitotic block of 7 % in HeLa cells (56). At 20 nM concentration, the mitotic block was found to be 26 %.

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 48

Control

Ber 10 µM

Ber 20 µM

Ber 40 µM

DNA Content ACS Paragon Plus Environment

Page 17 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 3. Berberine arrested the cell cycle progression of HeLa cells in G2/M phase of the cell cycle. HeLa cells (0.5×105 cells/mL) were grown and treated with increasing concentrations of berberine. After 24 h, the cells were collected and fixed with 70 % ethanol. The cellular DNA was stained with propidium iodide as detailed in Materials and Methods.

Table 1: Effect of berberine on cell cycle progression and mitotic index of HeLa cells Sample

% of cells in different phases of cell cycle (% Parent population) determined using FACS

Mitotic Index calculated using Immunofluorescence Microscopy

G1

S

G2/M

Control

78.7

9.7

6.9

3.5

Berberine 10 µM

71.3

12.1

13.6

8.3

Berberine 20 µM

65.8

12.3

15.4

10.2

Berberine 40 µM

62.4

13.4

18.0

12.4

3.3. Effects of berberine on the interphase and spindle microtubules of HeLa cells The

organization

of

the

microtubules

and

chromosomes

was

analyzed

by

immunofluorescence microscopy using antibodies against alpha-tubulin and Hoechst 33342 staining of the chromosomes. As shown in Figure 4, the control cells exhibited a typical interphase microtubule organization. At 10 µM concentration, berberine exhibited moderate depolymerization of the interphase microtubules. Interestingly, at 20 µM concentration, berberine strongly disrupted the interphase microtubular network. At concentrations twice and five times higher than its IC50, berberine treated cells exhibited complete loss of interphase microtubule network. Colchicine, a well-known microtubule depolymerizing agent, did not significantly alter the interphase microtubular network of HeLa cells at its reported IC50 concentration (7 nM); however, at concentration three times its IC50 (20 nM), exhibited a moderate depolymerization of the microtubule network.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Microtubule

DNA

Page 18 of 48

Merged

A Control

B BER 10 µM

C BER 20 µM

D BER 50 µM

E

BER 100 µM

F Col 7 nM

G

Col 20 nM

Figure 4. Berberine depolymerized the interphase microtubules in HeLa cells. HeLa cells grown on cover slips were incubated with the indicated concentrations of berberine/colchicine for 24 h. Microtubules (red) and DNA (blue) were visualized as described in section 2. Arrows indicate abnormalities in interphase microtubule network. Scale bar represents 15 µm.


Page 19 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Microtubule

DNA

Merged

Control

Ber 10µM

Ber 20µM

Col 7 nM

Col 20 nM

Figure 5. Effect of berberine on the mitotic spindles of HeLa cells. At its IC50, berberine disrupted the mitotic spindle apparatus and caused misalignment of chromosomes at the metaphase plate (indicated by the arrow). Colchicine (7 and 20 nM) was used as a positive control for the experiment. Spindle microtubules (red) and DNA (blue) were visualized as described in section 2. Scale bar represents 30 µm. When observed under the fluorescent microscope, control mitotic cells showed normal bipolar spindle morphology with chromosomes aligned compactly at the metaphase plate (Figure 5). Cells treated with 10 µM berberine exhibited nearly normal spindles;


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

however, the spindle length was shorter compared to the control cells. Cells treated with 20 µM berberine exhibited significant depolymerization of the spindle microtubules, and the chromosomes were not aligned compactly at the metaphase plate. As reported earlier, treatment with 7 nM colchicine did not significantly affect the organization of the mitotic spindle apparatus but significantly disturbed the alignment of chromosomes at the metaphase plate (56). Higher concentration of colchicine like 20 nM induced moderate depolymerization of mitotic spindles and caused condensation of the chromosomes in HeLa cells. It is understandable from the data obtained from the immunofluorescent images that berberine at its IC50 exhibits remarkable depolymerization of microtubules unlike other known depolymerizing agents like colchicine which do not depolymerize the microtubules at its IC 50 concentrations (Figures 4 and 5). Agents like colchicine and vinblastine induce mitotic block at their IC50 by suppressing the dynamics of microtubules and induce depolymerization of microtubules only at concentrations several folds higher than their IC50 (21, 22). 3.4. Berberine induced apoptosis in HeLa cells in a concentration-dependent manner Berberine at higher concentration induced cell death, hence we used AO staining to quantify and characterize the cell death since it can accurately differentiate the different stages of apoptosis (57). The onset of apoptosis is marked with the condensation of chromatin. Late events occurring in apoptosis include further condensation of chromatin, nuclear disassembly and cell shrinkage (58). It is evident from Figure 6A that berberine treatment increased the number of apoptotic cells in a concentration-dependent manner. For example, 10, 20, and 50 µM berberine induced 9.5, 16.7 and 41.5% apoptosis after 24 hours of incubation (Figure 6B). The control cells showed a normal morphology with less number of apoptotic cells (5%) as compared to the berberine treated samples. Interestingly, cells treated with 10 µM berberine displayed a condensed chromatin indicating the onset of apoptosis. These cells showed abnormal cell morphology with deformities in the cell membrane as observed from the DIC images. Cells treated with 20 and 50 µM berberine were found to be in the late apoptotic stage. These cells displayed cell shrinkage along with membrane blebbing which are hallmarks of late apoptosis.


Page 20 of 48

Page 21 of 48

AO

A

DIC

Merged

Control

10µM

20µM

50µM

60 50

% Apoptosis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

B

40 30 20 10 0

0

10

20

50

Berberine (µM) Figure 6. Berberine induced apoptosis in HeLa cells in a concentration dependent manner. (A) HeLa cells (0.5×105 cells/mL) were incubated with different concentrations of berberine. After 24 h of incubation, AO was added and the live cells were viewed as described in the section 2. Shown are the photomicrographs of HeLa cells. Scale bar represent 30 µm. (B) Graph represents percentage of apoptotic cells observed after 24 h of treatment with different concentrations of berberine. Atleast, 600 cells were counted for each concentration. The experiment was repeated thrice, data represent mean ± SD.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3.5. Berberine suppressed the migration of HeLa cells at concentrations lower than the IC50 Cell migration is a critical event in cancer metastasis (59). Scratch wound healing assay has been accepted as a reliable method to show cell migration (20). In the present study, we checked the migration of HeLa cells upon treatment with lower concentrations of berberine (5 and 10 µM). Berberine effectively inhibited the migration of HeLa cells in a dose and time dependent manner (Figure 7A). After 24 hours, 5 and 10 µM berberine treated cells showed 21% and 6% migration respectively; at the same time control cells exhibited 55% migration. After 48 hours 5 and 10 µM berberine treated cells showed 31% and 11% migration respectively compared to 80% migration observed in the control cells. After 72 hours, the wound was completely closed in the control cells while in 5 and 10 µM berberine treated cells the migration was only 41% and 14% respectively (Figure 7B). The results indicate that berberine could effectively prevent the migration of cancer cells even at concentrations lower than its IC50.

A


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Page 23 of 48

B

120 100

% Migration

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Control 5 µM BER 10 µM BER

80 60 40 20 0

24

48

72

Time (h) Figure 7. Berberine inhibited the migration of HeLa cells at low concentrations. (A) Migration of HeLa cells in the absence and presence of berberine at different intervals of time (0, 24, 48, 72 hours) was monitored as discussed in section 2. Arrows indicate the width of the wound at different intervals of time. Scale bar is 200 µm. (B) Berberine (0, 5 and 10 µM) significantly inhibited the cell migration of HeLa cells in a time dependent manner. % Cell migration at specific time intervals, was calculated by using the formula mentioned in section 2.6. The experiment was repeated three times, data represents mean ± SD. 3.6. Binding of berberine to tubulin using fluorimetric analysis The fluorimetric approach is a practical tool to study the key mechanisms responsible for molecular interactions between ligands and macromolecules. Details such as binding affinity, the number of binding sites, and intermolecular distances between the interacting molecules can be deciphered using fluorimetry (27-29, 31, 32). Since berberine induced depolymerization of interphase and spindle microtubules, we decided to study its effect on goat brain tubulin isolated in-vitro using fluorescence spectroscopy. It was observed that there was a progressive decrease in the intrinsic tryptophan fluorescence of tubulin upon binding of berberine in a concentration-dependent manner (Figure 8A). The Figure 8B shows the change in the fluorescence intensity of tubulin incubated with various concentrations of berberine. A dissociation constant (Kd) of 27 ± 2 µM was obtained from the double reciprocal plot (Figure 8B Inset). A binding stoichiometry of 1:1 was obtained for tubulin and berberine by Job’s method of continuous variation (Figure 8C).


Biochemistry

Berberine at higher concentrations (20-100 µM) has huge inner filter effects due to its significant absorbance at the excitation (295 nm) and emission (336 nm) wavelengths (Figure 1). Hence to calculate Kd of berberine with minimal inner filter effects the environment sensitive fluorescent probe ANS which has an excitation and emission maxima at 400 and 470 nm respectively was used (27, 39). As shown in Figure 8E, berberine quenched the fluorescence of the tubulin-ANS complex in a concentration dependent manner. The Figure 8F shows the change in the fluorescence intensity of tubulin-ANS complex incubated with various concentrations of berberine. A dissociation constant (Kd) of 11 ± 1 µM was obtained from the double reciprocal plot (Figure 8F inset). The scatchard analysis of the data obtained from the same experiment indicated that berberine has a single binding site on tubulin (Figure

500

A 400 300 200 100 0 320

340

360

300

B

250 200 150

1/X

Change in Fluorescence Intensity (336 nm)

Relative Fluorescence Intensity

8G).

100 50 0 0

380

50

C

40 30 20 10 0 0.0

0.2

0.4

0.6

0.8

Kd = 27 µM

20

40

60

80

100 120 140 160

Berberine µM

1.0

Relative Fluorescence Intensity (336 nm)

60

35 30 25 20 15 10 5 0

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1/Lf

Wavelength (nm)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 48

500 480 460 440 420 400 380 360 340 320 300

D

0

Mole Fraction [Ber] / [Ber + Tubulin]


10

20

30

40

Time (mins)

50

60

Biochemistry

E 600

400

200

440

460

480

500

520

350

F

300 250

2.6 2.2 2.0

150

1.8 1.6

100

1.4 1.2 1.0 0.00

50

0.05

0.10

0.15

0.20

0.25

1/Lf

0 0

20

40

60

80

100

Berberine (µM)

Wavelength (nm)

0.07

Kd = 11 µM

2.4

200 1/X

Change in Fluorescence Intensity ( 470 nm)

800

0 420

G

0.06 0.05

X/Lf

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 25 of 48

0.04 0.03 0.02 0.01 0.00 0.4

0.5

0.6

0.7

0.8

0.9

1.0

X

Figure 8. Berberine bound to tubulin in a time and concentration dependent manner at a single site. (A) Berberine decreased the intrinsic tryptophan fluorescence of tubulin in a concentration dependent manner. Tubulin (1 µM) was incubated with berberine 0 (●), 5 (○), 10 (▼), 20 (∆), 40 (■), 60 (□), 80 (♦), 100 (◊) and 150 (▲) µM for 30 min at 37 ºC. The samples were excited at 295 nm and the emission spectrum was recorded. (B) The change in the tryptophan fluorescence intensity was plotted against different concentrations of Berberine. Inset shows the double reciprocal plot which yielded a Kd of 27 ± 2 µM. (C) Job’s plot of continuous variation for berberine-tubulin interaction. Several mixtures of tubulin and berberine were prepared as discussed in section 2.10. The Job’s plot indicated that tubulin and berberine interact with a stoichiometry of 1:1 confirming a single binding site on tubulin. (D) Binding kinetics of berberine on tubulin. Tubulin (1 µM) was incubated with berberine (10 µM) for 55 minutes and the emission at 336 nm was recorded by exciting the samples at 295 nm at regular time intervals. (E) Berberine decreased the fluorescence of tubulin-ANS


120

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

complex in a concentration dependent manner. Tubulin-ANS complex was incubated with berberine 0 (●), 5 (○), 10 (▼), 20 (∆), 40 (■), 60 (□), 80 (♦), 100 (◊) µM for 30 min at 37 ºC. The samples were excited at 400 nm and the emission spectrum was recorded. (F) The change in the fluorescence intensity of tubulin-ANS complex was plotted against different concentrations of Berberine. Inset shows the double reciprocal plot which yielded a Kd of 11 ± 1 µM. (G) Scatchard analysis of the data indicated that berberine has a single binding site on tubulin heterodimer. The bound/free fraction (X/Lf) was plotted against the bound (X) fraction of berberine and the number of binding sites were determined from the x-intercept. 3.7. Berberine quenched the tryptophan fluorescence in a time-dependent manner Berberine was able to quench the tryptophan fluorescence in a time-dependent manner (Figure 8D). For example, 10 µM berberine reduced the tubulin tryptophan fluorescence by 14, 30, and 33 % after 20, 40, and 55 min respectively. Our observation suggests that the kinetics of berberine binding on tubulin is slow, as it took almost 55 min for the protein to get saturated with the berberine. 3.8. Berberine alters the hydrophobicity of the aromatic amino acid residues upon binding to tubulin Earlier studies have shown the usefulness of synchronous fluorescence spectroscopy in understanding the changes occurring in the protein microenvironment upon ligand binding (31, 32). Each tubulin heterodimer contains 8 tryptophan residues and 14 tyrosine residues. When Δλ between excitation and the emission wavelength was fixed at 60 nm, the synchronous fluorescence spectra displays the spectral characteristics of tryptophan residues exclusively. When the Δλ was fixed at 15 nm, the synchronous spectra displays the spectral characteristics of tyrosine residues (31, 32). The synchronous fluorescence spectra of tryptophan and tyrosine residues of tubulin upon addition of different concentrations of berberine are shown in Figure. 9A and 9B. A red shift of 2 nm was observed in the emission maximum of tryptophan residues suggesting that the polarity around these residues was increased upon berberine binding. No such shift was observed in the emission maximum of tyrosine residues. It is apparent from the data that berberine induced a conformational change in the tubulin structure upon binding.


Page 26 of 48

Biochemistry

400

A 300

200

100

0 280

285

290

295

300

305


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Relative fluorescence intensity

Page 27 of 48

500

B 400 300 200 100 0

290

Wavelength (nm)

300

310

Wavelength (nm)

Figure 9. Berberine induced a red shift in the synchronous fluorescence spectrum of tryptophan residues without affecting the tyrosine residues. Tubulin (1 µM) was incubated with different concentrations of berberine 0 (●), 5 (○), 10 (▼), 20 (∆), 40 (■), 60 (□) µM at 37 ºC for 30 min and synchronous fluorescence spectrum was recorded by fixing Δλ at 60 which is specific for tryptophan (A) and at and 15 nm which is specific for tyrosine residues (B). The arrows indicate the shift in the emission maximum in both cases. 3.9. Berberine inhibited the assembly of goat brain tubulin in-vitro As we confirmed the binding of berberine on tubulin, we wanted to check the effects of berberine on tubulin assembly. The effects of berberine on polymerization of purified goat brain tubulin and MAP-rich tubulin into microtubules was analyzed using two complementing assays, the sedimentation assay, and the light scattering assay. As shown in Figure 10A, berberine inhibited the glutamate-induced polymerization of tubulin into microtubules in a concentration dependent manner. As compared to the control, berberine 20, 40, 60, and 100 µM reduced the polymer mass by 5 ± 2.5%, 8 ± 5.3%, 11± 4.2%, and 31 ± 4.2% respectively. However, in the case of MAP rich tubulin we observed a better depolymerizing effect with 20, 40, 60, and 100 µM berberine reducing the polymer mass by 29 ± 2.5%, 44 ± 4.3%, 57 ± 3.2%, and 72 ± 3.1% respectively. In the light scattering assay, tubulin (12 µM) was allowed to polymerize into microtubules in the presence of different concentrations of berberine. The polymerization reaction was induced by addition of glutamate and GTP. As shown in Figure 10B, berberine


320

Biochemistry

inhibited the polymerization of tubulin in a concentration dependent manner. For example, in the presence of 20, 40, 60, 80 and 100 µM berberine, tubulin polymerization was inhibited by around 43, 59, 62, 78 and 87 % respectively as compared to the control. Colchicine (20 µM) inhibited the tubulin polymerization by 87 %. These observations strongly suggest that berberine binds to tubulin and inhibits its polymerization into microtubules similar to other microtubule depolymerizing agents.

3500

120 Relative light scattering (350 nm)

% Polymer mass

A 100 80 60 40 20

B

3000 2500 2000 1500 1000 500 0

0

20

40

60

80

100

120

0

Berberine (µM)

200

400

600

800

Wavelength (nm)

1000

Relative Light Scattering (350 nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 48

C 800 600 400 200

200

400

600

800

1000 1200 1400 1600

Time (s)

Figure 10. Effect of berberine on the assembly of pure tubulin, MAP rich tubulin and preformed microtubules. (A) Berberine inhibited the assembly of pure tubulin and MAP rich tubulin. Different concentrations of berberine (0-100 µM) were incubated with pure tubulin (12 µM) (●) and MAP-rich tubulin (15 µM) (○) and a sedimentations assay was carried out to determine the percentage of polymer mass. The experiment was done three times, data represent mean ± SD. (B) Berberine inhibited the glutamate induced assembly of tubulin. Pure tubulin was allowed to polymerize at 37 ºC in the absence and presence of


1000

Page 29 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

berberine 0 (○), 20 (□), 40 (●), 60 (♦), 80 (▼) and 100 µM (∆) in the polymerization buffer containing 0.8 M glutamate, and 1 mM GTP respectively. Colchicine 20 µM (■) was added as a positive control for the experiment.

The polymerization was monitored by light

scattering at 350 nm for 15 min. (C) Berberine disrupted the preformed microtubules. Pure tubulin (12 µM) was polymerized in the presence of 0.8 M glutamate (●) and 20 µM Paclitaxel (○) for 10 min followed by addition of berberine (100 µM) (indicated with an arrow). The change in relative light scattering was monitored at 350 nm for the next 15 min. 3.10. Berberine exhibited a rapid disruption of the preformed microtubules As berberine inhibited the polymerization of microtubules in the presence of glutamate, we wanted to find out whether berberine could depolymerize the preformed microtubules. Effect of berberine on preformed microtubules was determined by two different approaches. In the first approach, where tubulin (12 µM) was polymerized in the presence of 0.8M glutamate for 10 min, we observed a steady decrease in the light scattering upon addition of berberine (100 µM) (Figure 10C). In another approach, where tubulin (12 µM) polymerization was induced in the presence of 20 µM taxol for 10 min, we observed a rapid depolymerization of the preformed microtubules upon addition of berberine (100 µM) (Figure 10C). These observations collectively suggest that berberine could effectively depolymerize the preformed microtubules. 3.11. Berberine does not share the binding site with vinblastine or colchicine Since berberine depolymerized microtubules in a manner similar to that of the classical “depolymerizing agents”, we wanted to determine whether berberine shares the binding site with vinblastine or colchicine on tubulin. As evident from Figure 11A and 11B, increasing concentrations of vinblastine (0, 2, 5, 10 and 20 µM) had no significant effect on the fluorescence of the berberine-tubulin complex indicating that vinblastine does not compete with berberine and both the ligands can bind to the tubulin simultaneously. Since vinblastine had no effect on the binding of berberine on tubulin, a competitive assay involving colchicine and berberine was performed. It was found that berberine was able to decrease the T-C fluorescence at 430 nm in a concentration dependent manner (Figure 11C and 11D). The results indicate that both the ligands influence the binding of each other suggesting that they may have overlapping binding site on tubulin. It was also observed that the quenching of T-C fluorescence by berberine at 430 nm was associated with a concomitant increase in the fluorescence emission of berberine at 545 nm (Figure 11C). Based on the


Biochemistry

above findings it is reasonable to suggest that the binding site of berberine on tubulin may not exactly overlap with that of colchicine and the quenching of T-C complex fluorescence by berberine could be due to intermolecular FRET. The binding pocket of berberine was further analyzed by using podophyllotoxin a non-fluorescent colchicine site binding agent (21, 22). We found that podophyllotoxin weakly reduced the fluorescence of berberine-tubulin complex (Figure 11E), indicating that they may have distinct binding sites on the tubulin



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Biochemistry

Figure 11. Competition experiments with vinblastine, colchicine and podophyllotoxin for localizing the binding site of berberine on tubulin. (A) Berberine (20 µM) was allowed to form

a stable complex with tubulin (2 µM) for 30 min at 37 ºC. This was followed by addition of 0 (●), 2 (○), 5 (▼), 10 (∆) and 20 (■) µM vinblastine. All the samples were excited at 430 nm and the emission spectrum was recoded. Berberine alone (♦) and Tubulin alone (□) at the same excitation wavelength had very weak fluorescence. (B) Graph showing the change in the fluorescence intensity of tubulin-berberine complex at 545 nm upon addition of different concentrations of vinblastine. (C) Colchicine (10 µM) was allowed to form complex with tubulin (2 µM) for 1 hour at 37 oC and berberine 0 (●), 40 (○), 80(▼), and 100 (∆) µM, was added to the complex. The samples were excited at 360 nm and the emission spectrum was recoded. (D) Graph showing the change in the fluorescence intensity of tubulin-colchicine complex upon addition of increasing concentrations of berberine. (E) A stable berberinetubulin complex was allowed to form for 30 min at 37 ºC and different concentrations of podophyllotoxin (0-100 µM) were added. The change in the fluorescence intensity of berberine-tubulin complex at 545 nm was plotted against increasing concentrations of podophyllotoxin. 3.13. Probing the binding site of berberine using computational docking analysis and MD simulations To identify the binding site of berberine on tubulin, amino acids involved in berberine-tubulin interactions and the types of interactive forces involved, a systematic molecular docking and simulation approach was employed. Validation of the docking analysis was done by blind docking of colchicine with tubulin (1SA0*). Our results are in excellent agreement with the previously reported data, where colchicine is found to interact closely with amino acids like THR 179A, ASN 255B, VAL 318B, and ILE 378B (42). After successful validation, berberine was docked with the molecule 1SA0*. To determine the stability of berberine tubulin complex, the best docked pose of berberine on tubulin heterodimer was taken and a MD simulation was performed. As evident from Figure 12A, the RMSD of berberine tubulin complex was found to be around 3.9 nm, indicating that a stable berberine-tubulin complex was formed and the fluctuations were less during the course of simulation. The distance between berberine and tubulin was found to be 0.25 nm at the end of the dynamic simulation, suggesting that berberine and tubulin were present in close proximity to each other throughout the simulation process (Figure 12B). Furthermore, upon re-docking


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the energy minimized berberine-tubulin complex obtained after MDS, a binding energy of 6.98 kcal/mol was obtained from Autodock 4.0. As shown in Figure 12C, berberine was docked at a novel site on β tubulin. Upon superimposing the best docked pose of berberine obtained from docking analysis with the crystallographically determined binding conformation of colchicine on tubulin dimer, it was observed that the binding site of berberine is closer to the colchicine site (Figure 12C). Analysis by LigPlot suggested that berberine formed hydrogen bonds with LEU 132B and ARG 164B. Berberine also showed close interactions with other amino acids like CYS 131B, GLU 159B, GLU 160B, TYR 161B, PRO 162B and ASP 163B (Figure 12D).

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Figure 12. Berberine forms a stable complex with tubulin and binds at a distinct site in the heterodimer. (A) RMSD between berberine and tubulin after 10 ns of MDS. (B)


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Biochemistry

Distance between berberine and tubulin after MDS of 10 ns. (C) Superimposition of final docked conformation of berberine (yellow) with the crystallographically determined binding conformation of colchicine (green) and TRP 21B (cyan) on tubulin heterodimer. (D) Interacting residues involved in berberine and tubulin interactions. 3.15. Localization of the berberine binding site on the tubulin heterodimer using FRET analysis Since podophyllotoxin did not significantly reduce the fluorescence of berberinetubulin complex and also our docking analysis predicted a distinct binding site of berberine on the tubulin heterodimer, we tried to locate the binding site of berberine on tubulin by using FRET analysis. The conditions required for the energy transfer to happen include: (i) the donor should have significant fluorescence; (ii) the fluorescence spectrum of the donor and the absorbance spectrum of the acceptor should overlap; and (iii) the average distances between a donor fluorophore and the acceptor fluorophore should be between 2 – 8 nm (31, 32, 63, 64). As shown in Figure 13A, the fluorescence spectrum of T-C complex shows good overlap with the absorbance of berberine. The spectral overlap between the emission spectrum of the tubulin-colchicine complex and the absorbance spectrum of berberine was found to be J = 3.4 ×1013 M-1 cm-1 (nm) 4. The efficiency of the transfer was calculated to be E = 0.05. Under these conditions, Ro, the critical distance when the efficiency of transfer is 50 % was 16 Å and the value of r = 24 Å between the colchicine and berberine binding site may be accepted with a high degree of confidence. Further, the distance between the binding site of berberine and the ANS binding site on tubulin heterodimer was also calculated. Figure 13B shows the spectral overlap between the fluorescence spectrum of tubulin-ANS complex and the absorbance spectrum of berberine. Here, we found that J = 5.33×1013 M-1 cm-1 (nm) 4, E = 0.144 and Ro = 27 Å. A value of r = 37 Å between the ANS and berberine binding site may be accepted with a high degree of confidence. Our docking analysis indicated that berberine was bound to a site that was closer to the microenvironment of TRP 21B (Figure 12C). Since TRP 21B is one of the tryptophan residue that contributes to the fluorescence emission of tubulin, it is possible that the emission by TRP 21B is directly quenched by berberine and the probability of energy transfer between the two molecules is likely to happen. The spectral overlap between the fluorescence emission spectra of tubulin and the absorbance spectra of berberine is shown in Figure 13C. Here, we estimated that J = 1.5 ×1014 M-1 cm-1 (nm) 4, E = 0.1, Ro = 23 Å and r = 33 Å. In all


Biochemistry

the three distance calculations, the distance between the donor and the acceptor fluorophores are on a scale of 2-8 nm and 0.5Ro < r < 1.5Ro which indicates that the energy transfer from the donor to acceptor occurs with high probability (31, 32, 63, 64).

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Figure 13. Spatial relationship between berberine binding site and other well characterized sites in the tubulin heterodimer. (A) The spectral overlap between the fluorescence emission spectrum (solid line) of tubulin-colchicine complex (donor) and the absorbance spectrum (dashed line) of berberine (acceptor). (B) The spectral overlap between the fluorescence emission spectrum (solid line) of tubulin-ANS complex (donor) and the absorbance spectrum (dashed line) of berberine (acceptor). (C) The spectral overlap between


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the fluorescence emission spectrum (solid line) of intrinsic tryptophan residue of tubulin (donor) and the absorbance spectrum (dashed line) of berberine (acceptor).

(D) Two-

dimensional model representing the relative positions of various ligand binding sites on the tubulin heterodimer. Ruthenium red (RR) and 4-(dicyanovinyl)julolidine (DCVJ) binding sites are well characterized in the α tubulin. Based on the computational analysis and distance measurements by FRET, we could locate that berberine (B) binds to a novel site that is 24 Å away from the colchicine (COL) binding site towards the β subunit and 37 Å away from the ANS site. The distance between berberine and the nearest Tryptophan residue, TRP 21B was calculated to be 33 Å. 3.16. Binding of berberine induced a structural change in tubulin thereby reducing the number of cysteine residues accessible to DTNB It is know that the modification of few sulfhydryl groups of cysteine residues in tubulin could inhibit its polymerization into microtubules (27, 53). DTNB, a sulfhydrylspecific reagent forms complexes with the thiol groups in tubulin and induces chemical modifications (27, 53, 54). We reasoned that the amount of chemical modifications induced by DTNB in the absence and presence of berberine could be used to analyze the effect of berberine on the conformation of tubulin. Figure 14 shows that berberine significantly reduced the number of cysteine residues accessible to DTNB in a concentration dependent manner. For example, in the absence of berberine there were 12.70 sulfhydryl residues accessible to DTNB per tubulin dimer, however in the presence of 40 and 80 µM berberine, the accessibility was to DTNB was reduced to 11.45 and 9.89 respectively. This indicates that the binding of berberine induces a conformational change in tubulin structure. 0.4

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Biochemistry

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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 14. Berberine protected cysteine residues against chemical modifications by DTNB. Tubulin (3 µM) was incubated with berberine 0 (●), 40 (○), and 80 (▼) µM for 30 minutes at room temperature. DTNB (200 µM) was then added to the reaction mixtures and the absorbance was monitored at 412 nm for the next 50 min. 3.17. Binding of berberine alters the secondary structure of tubulin Tools like FTIR spectroscopy are of extreme importance when it comes to monitoring changes in the secondary structures of proteins (60). In this study, the effect of berberine binding on the secondary structure of tubulin was examined using FTIR spectroscopy. Figure 15A shows the comparison between FTIR spectra of tubulin in the absence (black line) and presence of 40 (red line) and 80 µM (blue line) berberine. Tubulin in the absence of berberine exhibited the characteristic bands designated as amide I (1600-1700 cm-1), amide II (14801580 cm-1), and amide III (1229-1301 cm-1) (55, 60, 61). It is evident that, upon binding to berberine (40 and 80 µM) there is a substantial decrease in the intensity of bands for the secondary structures. Upon comparison, we observed that in the absence of berberine, amide I gave a signature at 1650 cm-1 but in the presence of berberine 40 and 80 µM, the peak was shifted to 1654 and 1656 cm-1 respectively. Amide II band gave a signature at 1550 cm-1 in the absence of berberine but upon binding to 40 and 80 µM berberine, the peak was shifted to 1543 cm-1. Also, in the presence berberine we observed the appearance of peak at 1437 cm -1. A red shift was observed for the peak located at 1400 cm-1, which shifted to 1406 cm-1 upon berberine binding. Similarly, we observed a shift in the peak located at 1465 cm-1. Upon binding of 80 µM berberine, this peak was shifted to 1468 cm-1. For comparing the minute changes happening at the amide I region of tubulin which is most sensitive to the protein secondary structures in the absence and presence of berberine, a second derivative of absorbance spectra was generated (60, 61) (Figure 15B). In the control, the bands appearing between 1688 and 1662 cm-1 are assigned to β-turn, the band appearing at 1654 cm-1 is assigned to α-helix. Bands appearing between 1645 and 1637 cm-1 are assigned to unordered structures and the band appearing at 1648 cm-1 is assigned to the random coil structures (55, 60, 61). In the presence of berberine (40 µM) we observed the appearance of several new peaks. For example, peak appearing at 1671 cm-1 is assigned as β-turns, the peaks at 1648 and 1640 cm-1 are assigned as random coils, and the peak at 1627 cm-1 is assigned as β-sheet. All these changes in the FTIR spectra of tubulin upon berberine binding suggest that berberine induced a change in the secondary structure of tubulin upon binding.


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Biochemistry

Wavenumber (cm-1)

Figure 15. Berberine altered the secondary structure of tubulin. Tubulin (10µM) was incubated with berberine (40 µM and 80 µM) for 30 minutes at 37 ºC and FTIR analysis was performed as mentioned in section 2. (A) The absorbance spectrum of tubulin alone (black line), tubulin in the presence of 40 µM (red line) and 80 µM (blue line) berberine. (B) Second


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

derivative of absorbance spectra of tubulin in the absence (black line) and presence (red line) of 40 µM berberine. 4. Discussion Berberine is reported to exert a variety of pharmacological actions (1-15). It has shown to induce G2/M arrest in cancer cells of different origins (17-20). In this study we show that the potential mechanism behind the G2/M arrest and the anti-cancer activity of berberine could be through its inhibitory activity on tubulin polymerization. Berberine inhibited the proliferation of HeLa cells and blocked the cell cycle progression at G2/M phase of the cell cycle. It induced a moderate mitotic arrest and strong microtubule depolymerizing activity at its IC50 concentration. Recently, Saha et al. have also reported the disruption of tubulin network by berberine at 250 – 300 µM which is closer to their observed IC50 (62). As a whole, the data suggests that berberine induces depolymerization of microtubules at its IC 50 which is in contrast to the other known microtubule depolymerizing agents like colchicine, vinblastine, griseofulvin and benomyl. These depolymerizing agents induce significant depolymerization of microtubules only at concentrations 2-4 folds higher than their IC50 (2124). Since berberine inhibited the proliferation of HeLa cells and induced apoptotic cell death, we wanted to study its effect on the migration of HeLa cells using the wound healing assay. Results of wound healing assay indicate that berberine strongly inhibited the migration of HeLa cells at 5 and 10 µM (Figure 7). At these concentrations the inhibition of HeLa cell proliferation was found to be approximately 23 and 35 % respectively indicating that berberine could inhibit the HeLa cell migration at concentrations that were less toxic to the cells. This result is consistent with a previous report where berberine at lower concentrations inhibited the migration of HONE1 cells (20). Since microtubules play an important role in cell migration, it is possible that the inhibitory action of berberine against HeLa cell migration could be due to its inhibitory effects on the functioning of the microtubules. The depolymerization effect of berberine observed on interphase and mitotic spindles of HeLa cells encouraged us to study its effect on purified tubulin isolated from goat brains using fluorescence spectroscopy. Berberine quenched the intrinsic tryptophan fluorescence of tubulin and the fluorescence of tubulin-ANS complex in a concentration dependent manner. The Kd for berberine binding on tubulin was found to be 26 ± 2 and 11 ± 1 µM by using the tryptophan fluorescence of tubulin and the fluorescence of tubulin-ANS complex respectively


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Biochemistry

(Figure 8). Since the absorbance of berberine at 400 and 470 nm is very minimal (Figure 1), the Kd calculated using tubulin-ANS fluorescence could better represent the binding affinity of berberine on tubulin. Berberine took around 55 minutes to get completely complexed with tubulin, indicating a slow binding. This binding kinetics of berberine on tubulin was found to be similar to that of colchicine which also exhibited a slow and steady binding (35). The synchronous fluorescence spectrum analysis suggested that berberine increased the polarity specifically around tryptophan residues as we observed a red shift of 2 nm in the emission maximum of tryptophan residues without affecting the tyrosine residues. It was also observed that upon addition of berberine, the quenching of the fluorescence intensity of tryptophan residues was stronger than that of the tyrosine residues suggesting the predominant involvement of tryptophan residues behind the quenching of tubulin fluorescence by berberine. Similar results were observed when the binding of ligands such as triphenyltin and brucine were analyzed using synchronous fluorescence spectroscopy with major macromolecules like BSA, hemoglobin and HSA respectively (31, 32). The binding of berberine to tubulin resulted in a blue shift in the tubulin emission maximum when excited at 295 nm (Figure 8A). We also observed a change in the pattern of the tubulin emission spectrum in the presence of higher concentrations of berberine (20-100 µM) as a new peak appeared at 365 nm. A similar blue shift was also observed in a previous study where berberine was shown to bind with human serum albumin (HSA) and bovine serum albumin (BSA) (47, 48). A blue shift upon ligand binding in general refers to an increased hydrophobicity of the region surrounding the tryptophan residues. We feel that berberine may not increase the hydrophobicity of the region surrounding the tryptophan residues; since we found that berberine induced a red shift of 2 nm in the synchronous fluorescence spectrum analysis. To confirm whether berberine really increased the hydrophobicity around the tryptophan residues we conducted the fluorometric analysis with pure L-tryptophan and different concentrations of berberine (0-100 µM) in PEM buffer. The fluorescence emission spectrum of the complex was monitored by exciting the samples at 295 nm. As expected, we observed a decrease in the emission maximum of tryptophan (blue shift) with a simultaneous appearance of new peak at 365 nm in samples containing higher concentrations of berberine (40-100 µM) (data not shown). This observation clearly suggests that berberine at higher concentrations might modify the emission maximum of tryptophan without increasing the hydrophobicity.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The results from the sedimentation assay and the light scattering assay are in excellent agreement with the results obtained from the cell culture, where berberine completely disrupted the microtubule network of HeLa cells. Berberine was also able to rapidly disrupt the preformed microtubules polymerized in the presence of 0.8 M glutamate and taxol (20 µM) (Figure 10C). This depolymerization effect of berberine was similar to that of quercetin, which has shown to inhibit tubulin polymerization and also induces depolymerization of preformed microtubules (27). Based on these observations it is tempting to suggest that berberine upon binding to soluble tubulin induces a conformational change in such a manner that the berberine bound tubulin dimers are unable to polymerize into microtubules even in the presence of inducers. To the preformed microtubules, berberine might get incorporated in the ends of the growing polymers and induce conformational changes that are unfavorable for further dimer addition. Such conformational changes might cause thermodynamic instability in the microtubule lattice resulting in its rapid disruption. To locate the binding site of berberine on tubulin we carried out competitive binding assays with vinblastine and colchicine which are well characterized microtubule depolymerizing agents. Our observations indicated that berberine did not share the binding site with colchicine and vinblastine. To gain insights into the binding site of berberine and to predict its spatial location on the tubulin heterodimer, FRET experiments were performed. Results from FRET analysis support the data obtained from the docking studies that berberine binds to a novel site on β tubulin that is 24 Å away from the colchicine binding site and 37 Å away from the ANS binding site. Previous studies have reported that the distance between ANS binding site and the colchicine binding site is around 50 Å (63, 64). From our study, the sum of the distances between ANS – berberine and colchicine – berberine sites is 37 + 24 = 61 Å, which suggests that berberine binding site may lie between the ANS and the colchicine site on the N-terminal domain of the β tubulin (Figure 13D). Furthermore, our docking analysis indicated the presence of a tryptophan i.e. TRP 21B closer to the binding site of berberine (Figure 12C). Out of 8 tryptophan residues in the tubulin heterodimer, TRP 346A, TRP 407A, TRP 21B, and TRP 407B are the likely candidates for contributing to the emission of the free tubulin (51). Using the similar approach the distance between berberine and TRP 21B, a tryptophan residue closer to the berberine binding site, was calculated to be 32 Å. Our observations establish the spatial distances between the known ligand binding sites and the putative binding site of berberine on tubulin heterodimer. The presence of TRP 21B closer to the berberine binding site also justifies the data obtained from the synchronous fluorescence spectroscopy where a red shift was observed at Δλ value of 60 nm which is


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Biochemistry

specific for tryptophan residues (Figure 9A). It is also possible that berberine specifically influenced the polarity of this particular residue indicating towards a possible structural modification in the tubulin structure. Another line of evidence for the structural changes induced by berberine comes from DTNB assay in which berberine protected the modification of cysteine residues by DTNB (Figure 14). Docking analysis confirmed the presence of two cysteine residues i.e. CYS 129B and CYS 131B, closer to the berberine binding site on the β tubulin. Similarly, colchicine was also reported to protect the cysteine residues from modifications induced by alkylating agents (27) indicating that both the ligands could exert similar kinds of conformational changes on the tubulin structure upon binding. The conformational change induced by berberine upon binding to tubulin was further confirmed by FTIR analysis. It is evident from the FTIR spectrum that berberine upon binding to tubulin decreased the intensity of bands for the secondary structures indicating a conformational change. The appearance of new peaks in the second derivative of absorbance spectra of tubulin also confirms the change in secondary structure of tubulin. Similar results were obtained when the secondary structure was monitored using FTIR spectroscopy for several other proteins and ligands (60, 61). 5. Conclusions and future perspectives Berberine has shown to exhibit anticancer activity either alone or in combination with several other known anticancer agents with synergism (1-10). It has also shown to exert preferential cytotoxic activity against cancer cells; hence its toxicity and side effects in normal cells are expected to be much lower (65, 66). Berberine is currently under clinical trials for the prevention of colorectal adenoma recurrence [Study ID Numbers: NCT02226185 and NCT02365480]. In this study, we report that berberine inhibited the proliferation of HeLa cells and the antiproliferative activity of berberine correlated well with its ability to depolymerize the interphase and mitotic spindles. Hence, the anticancer efficacy of berberine can be explored in combination with other clinically established anticancer drugs having diverse cellular targets. 6. Conflicts of Interest The authors declare that there are no conflicts of interest

7. Funding sources and acknowledgment


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This work is supported by the grant from the DST/SERB, Government of India (SR/SO/BB0013/2010) to Dr. Rathinasamy K. The authors D.R, S.M.A and L.M are thankful to NITC and MHRD, Government of India for their fellowship. The authors gratefully acknowledge the support provided by Dr. Aji A. Anappara, Department of Physics, NITC for FTIR facility. We also thank Mr. Jomon Sebastian for the molecular dynamics simulations and Mr. Susobhan Mahanty for critical reading of the manuscript.

References

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