Designed Tetra-peptide Interacts with Tubulin and Microtubule

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Designed Tetra-peptide Interacts with Tubulin and Microtubule Batakrishna Jana, Prasenjit Mondal, Abhijit Saha, Anindyasundar Adak, Gaurav Das, Saswat Mohapatra, Prashant Kurkute, and Surajit Ghosh Langmuir, Just Accepted Manuscript • Publication Date (Web): 30 May 2017 Downloaded from http://pubs.acs.org on June 1, 2017

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Designed Tetra-peptide Interacts with Tubulin and Microtubule Batakrishna Jana,‡1 Prasenjit Mondal,‡1,2 Abhijit Saha,1 Anindyasundar Adak,1 Gaurav Das,1 Saswat Mohapatra,1,2 Prashant Kurkute1 and Surajit Ghosh*1,2 1. Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. Fax: +91-33-24735197/0284; Tel: +91-33-2499-5872 2. Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Biology Campus, 4 Raja S. C. Mullick Road, Kolkata 700 032, India. CORRESPONDING AUTHOR INFORMATION: Fax: +91-33-2473-5197/0284; Tel: +91-33-2499-5872; E-mail: [email protected] KEYWORDS: SLRP, tubulin/microtubule, GTP, cancer cell, antimitotic activity ABSTRACT: Microtubule regulates eukaryotic cell functions, which have tremendous implication in tumor progression. Thus, design of novel approaches for controlling microtubule function is extremely important. In this manuscript, a novel tetra-peptide Ser-Leu-Arg-Pro (SLRP) has been designed and synthesized from small peptide library consisting of 14 tetrapeptides, which perturbs microtubule function through interaction at “anchor region”. We have

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studied the role of peptide on microtubule function on a chemically functionalized 2D platform. Interestingly, we have found that SLRP binds with tubulin and inhibits the kinesin driven microtubule motility on kinesin immobilized chemically functionalized 2D platform. Further, this peptide modulator interacts with intracellular tubulin/microtubule and depolymerizes the microtubule networks. These interesting findings of perturbation of microtubule function both on engineered platforms and inside the cell by this small peptide modulator inspired us to study the effect of this tetra-peptide on cancer cell proliferation. We found that the novel tetra-peptide modulator causes moderate cytotoxicity to the human breast cancer cell (MCF-7 cell), induces the apoptotic death of MCF-7 cell and activates the tumor suppressor proteins p53 and cyclindependent kinase inhibitor 1 (p21). To the best of our knowledge, this is the shortest peptide discovered, which perturbs microtubule function both on engineered 2D platform and inside the cell. 1. INTRODUCTION Microtubule dynamics play important role in maintaining cell structure, function and division.1-4 Many anticancer drugs are developed targeting microtubule dynamics.5-10 However, clinical successes of most of the drugs are unsatisfactory due to their poor bioavailability.11-12 Thus, development of highly bioavailable drugs is the key prerequisite for the treatment of cancer. Although, in this direction, recently attempts have been made through liposomal formulation of existing drugs,13 nanoparticle-based delivery of drugs14 and antibody-conjugated delivery15 but, success rate is still unsatisfactory in clinical stage. Thus, further development of biocompatible anticancer molecules is necessary to develop novel anticancer molecules. Over the last decade, development of peptide-based drugs has drawn the tremendous attention due to its excellent biocompatibility and easy to modulate.16-17 Anticancer peptides (< 50 amino acids and are often


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cationic in nature) are found to be a potential candidate for cancer therapy due to high specificity, high tumor penetration, low production cost and ease of synthesis and modification over the existing small anticancer molecules and drugs.18-19 We are currently working on development of anticancer molecules through design of various peptides targeting to tubulin/microtubules surface. Our design principle is mainly focused on the development of peptides from various drugs and nucleotide binding pockets of tubulin using computational technique and previously described interacting amino acids partners.20 In this manuscript, we have designed and developed a novel short tetra-peptide Ser-Leu-Arg-Pro (SLRP) from small library of 14 tetra-peptides, originating from exchangeable GTP/GDP binding pocket of tubulin. We have shown computationally and experimentally that this novel short tetra-peptide interacts at "anchor region", which is close to the exchangeable GTP/GDP binding pocket of tubulin. We found that the tetra-peptide interacts with tubulin on 2D micropatterned surface, interacts with microtubule lattice and inhibits the kinesin driven microtubule motility, inhibits tubulin polymerization, shows moderate cytotoxicity to human breast cancer cell (MCF-7 cell), depolymerizes the intracellular microtubules, induces the apoptotic death of MCF-7 cell and activates the tumor suppressor proteins p53 and cyclin-dependent kinase inhibitor 1 (p21). In brief, our design concept was emanated from exchangeable GTP/GDP binding site of β-tubulin. GTP plays a crucial role in the polymerisation of microtubule from α, β-tubulin heterodimer.21 Thus, designing of small molecules/peptides from exchangeable GTP/GDP binding pocket is essential as they can perturb the GTP binding efficiency with β-tubulin. As a result, tubulin polymerization process is perturbed and ultimately the cell division is affected. PDB structure of exchangeable GTP/GDP site of β-tubulin (1JFF)2 is consisted of specific polar [Threonine (Asparagine (N206, N228), Aspartic acid (D179) and Tyrosine (Y224)] and non-


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polar [Valine (V171) and Cystine (C12)] amino acids (Fig. 1a). Recently, specific interaction partners of polar and non-polar amino acids, shown by Faure et al. provide important concept of designing the peptide sequences in a protein surface.20 These two interesting fact motivated us to design a novel peptide RLPS (Fig. 1a). 2. EXPERIMENTAL 2.1 Chemicals Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH and Fmoc-Rink Amide AM resin were purchased from Novabiochem. D-Biotin was purchased from Thermo Fisher. E, Z-Link Biotin-NHS was purchased from Thermo Scientific. Diamino-polyethylene glycol with MW 3000 Da (NH2-PEG3000-NH2) and Mono Boc Diamino-polyethylene glycol with MW 3000 Da

(N2H-PEG3000-NHBOC)

were

purchased

from

Rapp

Polymer.

(3-

glycidoxypropyl)trimethoxysilane (GOPTS) and Diisopropylcarbodiimide were purchased from Fluka. OtBu protected Tris-(nitrilo Tris-acetic acid) [(OtBu)Tris-NTA], Catalase and Glucose oxidase were received from Dr. Thomas Surrey's laboratory in EMBL, Heidelberg Germany (Currently at London Cancer Research Institute, UK). Sodium Chloride, Ninhydrin GR, Sodium hydrogen carbonate, Sodium hydroxide, di-Sodium hydrogen phosphate dihydrate, Potassium hydroxide, Phenol, Ethanedithiol (EDT), Trifluoroaceticacid, Ethanol, Hydrogen peroxide (30% solution), Acetone, Dichloromethane, N, N-Dimethylformamide (DMF), Magnesium chloride hexahydrate and Potassium dihydrogen phosphate were purchased from Merck. Potassium Chloride, Pyridine, Sulphuric acid and Ether were purchased from Fisher Scientific. Glucose was purchased from Qualigens. Pipyridine, Dimethyl sulphoxide, O-(1H-Benzotriazol-1 yl) N, N, N′, N′-Tetramethyluronium hexa fluorophosphate (HBTU) and Diisopropylethylamine (DIPEA)


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were purchased from Spectrochem. Acetonitrile was purchased from J. T. Baker. Methanol was purchased from Finar. Triton-X-100 was purchased from SRL. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Kanamycin sulfate, 5(6)-Carboxy Fluorescein (FITC), Dulbecco’s Modified Eagle’s Medium (DMEM) medium, Trypsin-EDTA solution, Adenosine 5′-triphosphate disodium salt hydrate (ATP), Guanosine 5′-triphosphate sodium salt hydrate (GTP), PIPES, β-casein, Ethylene glyol-bi(2-aminoethylether)-N, N, N′, N′-tetraacetic acid (EGTA), cell cultured DMSO, DAPI, Neutravidin and formaldehyde were purchased from Sigma Aldrich. 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid (HEPES) was purchased from Himedia. Penicillin-Streptomycin and fetal bovine serum were purchased from Invitrogen. Rabbit monoclonal anti-alpha Tubulin (EP 1332Y) and Goat pab to Rb IgG (Cy3.5 ®) ab 6941 polyclonal rabit were purchased from Abcam. Bisbenzimide H 33258 (Hoechst) was purchased from Calbiochem. p53 (F-8) mouse monoclonal IgG, p21 (F-5) mouse monoclonal IgG, Goat anti mouse IgG (H+L) RPE human adsorbed and annexin V apoptosis detection kit were purchased from Santa Cruz Biotechnology. All compounds were used without further purification. 2.2 Docking study We have performed docking study using Autodock-Vina version 1.1.2.22 98X60X64 affinity grids were centered on the receptor tubulin [PDB ID: 1JFF]2 for docking with SLRP and its’ thirteen analogues. Also, we have performed another docking study taking SLRP peptide at the anchor region of tubulin. The affinity grid having dimension 112X80X70 was centered on the receptor tubulin [PDB ID: 1Z2B]23. All the images were seen and produced in PyMOL (The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC).


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2.3 Synthesis, purification and characterization of the fourteen peptides We have synthesized all the fourteen peptides following solid phase peptide synthesis method in Liberty 1 CEM microwave peptide synthesizer using Rink Amide AM resin. 20% piperidine in DMF was used for Fmoc-deprotection at the time of synthesis. Finally, the crude peptide was cleaved from the resin by using standard cleavage cocktail solution (TFA 91%, Phenol 3%, EDT 3%, Milli Q 3%), purified by HPLC (Shimadzu) and characterized by MALDI-TOF Mass Spectroscopy 2.4 Cell culture MCF-7 (human breast cancer cell line) cell line and WI38 (human lungs fibroblast normal cell line) were purchased from NCCS, pune (India) and cultured in Dulbecco Modified Eagle medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum at 37 oC and 5% CO2 atmosphere in our lab. 2.5 MTT assay Anticancer property of the short tetra-peptide SLRP and its analogues in MCF-7 cell line was assessed by % cell viability assay, which is well-known as MTT assay. The principle of MTT assay is the reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) to its insoluble purple coloured formazan by cellular oxidoreductase enzymes, present in the viable cells. Thus, intensity of the purple colour of the formazan corresponds to the number of viable cells present, which is quantified by measuring the absorbance of the control and treated cells after MTT reduction at 550 nm wavelength. The procedure is as follows. The MCF-7 cells were plated at a density of 10000 cells per well in a 96-well plate one day prior to the treatment. Then the cells were incubated with different concentrations of SLRP and its analogues


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(maximum concentration used for the experiment is 50 µM in all the case) differently in serum free DMEM medium for 4 h. There was no treatment in the control cells. Then the medium was exchanged with complete medium and kept for 20 h. Then each well was treated with MTT solution (concentration-5 mg/mL) for 4 h keeping the background well aside. 1:1 mixture of DMSO and methanol was added to each well after discarding the medium and the absorbance of each well was recorded at 550 nm wavelength in ELISA plate reader. Results were expressed as percent viability = [(A550 (treated cells)-background)/(A550 (untreated cells)-background)] x 100. The MTT assay of SLRP in WI38 cell line and MCF-7 cell line (for lower concentration of SLRP) was performed following the above procedure. 2.6 Protein biochemistry Tubulin was isolated from goat brain and purified following previously described procedure.24 Labeling of tubulin with Alexa-Fluor-568 carboxylic acid succinimidyl ester was performed to obtain the Alexa-568-labeled tubulin. Kinesin612-His10 proteins were expressed in E. coli and purified through Ni-NTA column in our laboratory. 2.7 Tubulin polymerization assay/Tubulin turbidity assay Effect of SLRP on the tubulin polymerization was studied by measuring the tubulin turbidity with time in presence of GTP. The assay is known as tubulin turbidity assay. 20 µM tubulin, 4 mM GTP, 10% dimethyl sulfoxide and SLRP with different concentrations (200, 100 and 50 µM) were mixed in Brinkley Reassembly Buffer 80 (BRB 80) in ice and loaded into 37 °C heated quartz cuvettes of path length 10 mm. To the control, there was no SLRP. The absorbance of the solution was measured at 350 nm for 40 minutes in the UV-Vis Spectrophotometer


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(G6860A Cary 60 UV-Vis Spectrophotometer, Agilent Technologies). DMSO was used to initialize the polymerization and the data was analyzed in origin software. 2.8 Microtubule assembly assay Microtubule assembly assay was performed to know the role of SLRP on the tubulin polymerization, which was monitored by measuring the DAPI fluorescence of the solution during tubulin polymerization.25-26 Fluorescence intensity of DAPI solution increases as it binds with microtubule. Therefore, the amount of tubulin polymerization i.e. the microtubule formation was quantified in presence and absence of SLRP (by measuring the fluorescence intensity of DAPI solution with time. A mixture of 10 µM DAPI in BRB80 buffer (BRB 80; 80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, pH 6.9) containing 100 µM tubulin, 10 mM GTP and SLRP having different concentrations was prepared. We used 200 and 100 µM SLRP for the experiment. The solution was excited at 355 nm wavelength at 37 oC and the emission spectra of the solution were recorded in region from 400 nm to 600 nm wavelengths for 60 minutes in five minutes time interval in Quanta Master Spectrofluorometer (QM-40), which is equipped with Peltier for controlling the temperature during experiment. Control experiment was carried out under same condition in absence of SLRP. The data was calculated in origin Pro 8.5 software. 2.9 Determination of binding affinity of SLRP by fluorescence intensity quenching study of intrinsic Tryptophan residue of tubulin Intrinsic Tryptophan fluorescence intensity of tubulin is quenched in presence of a drug, which binds with the tubulin. So, intrinsic Tryptophan fluorescence intensity of tubulin was measured in presence of different concentrations of SLRP. From that, binding constant was determined using a modified Stern-Volmer equation.21 10 µM of tubulin was mixed with different concentrations


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of SLRP in BRB80 buffer in ice and the fluorescence emission spectra were recorded from 310 to 450 nm, upon excitation of sample at 295 nm at 4 °C using Quanta Master Spectrofluorometer (QM-40) equipped with Peltier for controlling the temperature. 2.10 Determination of stoichiometric binding of SLRP tetra-peptide with tubulin by UVabsorbance The stoichiometric binding of SLRP peptide with tubulin was determined by recording absorbance of the tubulin-peptide complexes at 280 nm. The absorbance of the complexes were varied with different mole fraction of the peptide. The mole fraction of the SLRP peptide was varried between (0.05-0.95). The molar ratio of the binding was calculated using Job Plot.

2.11 Binding of SLRP with tubulin on biotin micropatterned surface Preparation of biotin micropatterned glass surfaces and flow chamber were performed as described previously.27 The flow chamber was equilibrated with β-casein (1 mg/mL) and 0.3 µM neutravidin in HEPES buffer successively followed by washing with HEPES buffer in ice. Then the flow chamber was exchanged with BRB80 buffer. 50 µM solution of biotin-SLRP was loaded to the flow chamber and kept for 10 minutes in ice followed by washing with BRB80 to remove excess peptide. 20 µL solution of BRB80 buffer containing 20 µM tubulin, 3.75 µM Alexa-fluor-568 labeled tubulin, 4 mM GTP, 25 mM MgCl2, 1 mg/mL glucose oxidase, 0.5 mg/mL catalase and 1 mM glucose was loaded to flow chamber. After that, the flow chamber was sealed and imaged under TIRF microscope using 561 nm laser through 60X objective (Olympus) and an Andor iXon3 897 EMCCD camera. Control experiment was carried out following the same procedure without incubating the flow chamber with biotin-SLRP.


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2.12 FRET study Tubulin bound colchicine and FITC-SLRP FRET distance measurement

+

IA

γ

IA ID

Efficiency of FRET, εFRET = In our FRET studies,

As obtained from fig. 2d, εFRET = 0.44 From the spectral overlap graph the Forster distance (R0) between Tub-col complex and FITCSLRP was calculated to be ~ 29.5 ±1 Å. Now the distance (RDA) between Tub-col complex (donor) and FITC-SLRP (acceptor) was calculated by the following equation.

FRET FRET

DA

 −ε = Ro   ε

  

1111 6666

1111

RRRR

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RDA was calculated to be ~ 30.35 ±1 Å 2.13 Preparation of Alexa-fluor-568 labeled GMP-CPP microtubules Alexa-Fluor 568 labeled GMP-CPP microtubules preparation: 1. (A) Tubulin mixture on ice: 0.75 µL Alexa-Fluor-568 labeled tubulin (15 mg/mL, 65% labeling ratio), 4.25 µL tubulin (20 mg/mL) and 44.5 µL BRB80 (80 mM PIPES, 1 mM MgCl2, pH adjusted to 6.8 by using KOH solution) were mixed on ice; (B) Final mixture on ice: 5 µL GMP-CPP (10 mM), 1 µL MgCl2 (100 mM), 10 µL tubulin mixture from 1.(A) and 34 µL BRB80 were mixed in ice and incubated


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for 2 h at 37 °C. 2. The final mixture was centrifuged for 7 minutes at 12000 rpm. 3. The coloured pellet was resuspended in 20 µL warm BRB80 at 37 °C and stored. 2.14 Effect of SLRP on microtubule motility Tris-NTA and biotin dual functionalized glass surface and the flow chamber with this were prepared following previously described procedure. First, the flow chamber was equilibrated with BRB80 followed by incubation with β-casein for 5 minutes. Then, it was washed with BRB80 to remove excess β-casein. Next, 0.3 µM neutravidin was loaded to the flow chamber and kept for 10 minutes. Excess neutravidin was removed by washing the flow chamber with BRB80. After that 1 mM biotin-SLRP was flowed into the flow chamber and incubated for 10 minutes followed by washing with BRB80 to remove the excess unbound peptide. Next, 50 nM kinesin612-His10 in BRB80 was loaded into the chamber and incubated for another 10 minutes followed by washing the flow chamber with BRB80. All the steps up-to this was performed in ice condition. After that the motility buffer, containing 3 mM Mg-ATP and an oxygen scavenger system (50 mM glucose, 1 mg/mL glucose oxidase and 0.5 mg/mL catalase) in BRB80 was flowed to the flow chamber after warming it to room temperature. Five chamber volumes of preformed GMP-CPP stabilized Alexa-Fluor-568 labeled microtubules in motility buffer were flowed into the chamber and microtubules gliding were observed by time-lapse TIRF microscopy at 37 °C. Control experiment was performed following the same procedure without addition of biotin-SLRP to the flow chamber. 2.15 Cellular uptake studies Cellular uptake of FITC-SLRP was carried out in MCF-7 cell line as follows. The cells were plated at a density of 5000 cells per cover glass bottom dish one day before the treatment. Then


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the cells were incubated with FITC-SLRP in serum free DMEM medium for 4 h followed by the treatment of Hoechst (1 µg/mL) for another 1 h. Then the cells were exchanged with complete medium and kept overnight. Finally, the cells were washed with colourless serum free medium and viewed under an NIKON inverted microscope (Model Ti-U). 2.16 Cellular uptake studies by flow cytometry Cellular uptake of FITC-SLRP was further carried out in MCF-7 cell line by flow cytometry. The cells were plated at a density of ~2X105 cells per well in a 6 well plate one day prior to the treatment. Then the cells were incubated with FITC-SLRP in serum free DMEM medium for 4 h. Then, the cells were trypsinized and washed with colourless serum free medium and analyzed by BD LSRFORTESA flow cytometer using emission filters at 530 nm. Data was analyzed using FACS DIVA software. 2.17 Interaction with microtubule network inside the MCF-7 cells The interaction of SLRP with the microtubule network inside the MCF-7 cells was studied as follows. The cells were plated at a density of 5000 cells per cover glass bottom dish one day prior to the treatment. After that, the cells were incubated with FITC-SLRP in serum free DMEM medium for 4 h followed by the exchange with the fresh complete DMEM medium containing 10% FBS for 20 h. There was no treatment in the control. Then the cells were fixed with 4% paraformaldehyde solution and permeabilized with 0.1% Triton-X-100. Then, the cells were treated with primary antibody Anti-alpha Tubulin, clone EP 1332Y Rabit monoclonal antibody (1:300)) for 2 h followed by the incubation with secondary antibody Goat pab to Rb IgG (Cy3.5 ®) ab 6941 polyclonal rabit (1:500)) for another 2 h. Then, Bisbenzimide H 33258 (Hoechst) (1 µg/mL) was treated for 30 minutes to stain the nucleous. Following the termination


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of the experiment, the cells were washed with 1X PBS and observed in an Andor spinning disc confocal microscope with a 60X objective (Olympus) and an Andor iXon3 897 EMCCD camera in bright field, 405, 488 and 561 nm wavelength laser lights. 2.18 Measurement of subcellular co-localisation by Image J software Cellular image data was analysed with the help of JACop (Just Another Co-localisation Plugin) using Image J software in order to confirm subcellular co-localisation as previously described. Co-localisation of green (FITC-SLRP) and red (microtubule network) signals was analysed via mask of the co-localisation pixels in white (co-localisation map). From the Costes’ statistical analysis the p-value was found to be 100%, which indicates co-localisation in the regions masked in white (colocalisation map) is highly probable. The Pearson's Coefficient of FITCSLRP for co-localisation with tubulin/microtubule was found to be 0.718. Cytofluorogram between FITC-SLRP and tubulin/microtubule network exhibits good co-localization. Also, from cross-correlation function (CCF), high CCF value for FITC-SLRP with tubulin/microtubule both at minimum and maximum pixel shift (dx) was found. Thus, we have observed from above analysis using Costes’ approach, Pearson’s coefficient and Van Steensel’s CCF approach that FITC-SLRP is nicely co-localized with tubulin/microtubule network. 2.19 Effect of SLRP with the microtubules network of MCF-7 cells Effect of SLRP on microtubule network of the MCF-7 cells was studied following previously described procedure as in the ‘Interaction with microtubule network inside the MCF-7 cells’ section. Only the change is that, here the MCF-7 cells were treated with 50 µM SLRP instead of FITC-SLRP. Following the completion of the experiment, the cells were washed with 1X PBS


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and observed in the inverted microscope (Model Nikon Eclipse Ti-U) with a 40X objective in DIC mode, 405 and 561 nm fluorescence channels. 2.20 Annexin V and PI assay by fluorescence microscopy MCF-7 cells were seeded at a density of 5000 cells per cover glass bottom dish one day before the treatment. Then the cells were treated with 50 µM SLRP in serum free DMEM medium for 4 h keeping one cover glass bottom dish as a control. Then the SLRP treated cells were exchanged with complete medium containing 10% fetal bovine serum (FBS) and kept for another 20 h. There was no SLRP treatment in the control. Then the cells were washed with PBS and assay buffer successively. 200 µL of assay buffer containing 5 µg/mL annexin V and 1.25 µg/mL Propidium Iodide (PI) was added to each well and kept for 15 minutes followed by washing with assay buffer. Then the cells (both treated and untreated) were imaged under an NIKON inverted microscope (Model Ti-U) using 40X objectives in 488 and 561 nm channel. 2.21 Apoptosis study by Flow Cytometry MCF-7 cell was plated at a density of ~5 X 105 cells per well in a 6-well plate one day before the treatment. The cells were incubated with 50 µM SLRP in serum free DMEM medium for 4 h followed by exchange of the fresh DMEM containing 10% FBS and kept for another 20 h. To the control, no SLRP was treated. After that, the cells were trypsinized and pelleted by centrifugation at 3000 rpm for 3 minutes. Then the pellet was incubated with 100 µL solution of assay buffer containing 1.25 µg/mL of Propidium Iodide (PI) and 5 µg/mL of annexin V for 15 minutes at 37 oC after washing with 1X PBS. After that, another 400 µL of assay buffer was added to each pellet and analyzed by BD LSRFORTESA flow cytometer using emission filters at 530 and 610 nm. Data was analyzed using FACS DIVA software.


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2.22 Immunoblotting MCF-7 cells (~5-6×106) were seeded overnight followed by treatment with 50 µM of SLRP for 24 h. Cells were lysed and total protein concentration was determined using Bradford assay. Protein was separated using SDS-PAGE and transferred to PVDF membrane. PVDF (Immobilon-P Polyvinylidene fluoride membranes; Millipore Corporation, Bedford, MA, USA) membrane was blocked for nonspecific interaction using skimmed milk solution followed by overnight incubation with primary monoclonal antibodies such as anti-p53 (Sigma), anti-p21 (Santa Cruz Biotechnology, Heidelberg, Germany) and anti-α-tubulin (Merck Millipore, Darmstadt, Germany). Then membrane was incubated with secondary antibody conjugated with peroxidase. Protein expression was analysed using HRP substrate (Luminata Forte Western HRP substrate, Merck Millipore, Darmstadt, Germany) and chemiluminescence detection system. Protein band intensity has been normalized with loading control to evaluate relative expression of p53 or p21 to that of control. 2.23 Immunofluorescence microscopy of p53 and p21 Expression level of tumor suppressor protein p53 and cyclin-dependent kinase inhibitor 1 (p21) were checked in MCF-7 cell after SLRP treatment. MCF-7 cells were seeded at a density of 5000 cells per cover glass bottom dish 18-24 h before the treatment. After that, the cells were treated with 50 µM SLRP in serum free DMEM medium for 4 h followed by the exchange with the fresh complete DMEM medium containing 10% FBS for 20 h, keeping one cover bottom dish for control. Then the cells were fixed and permealized with 4% paraformaldehyde solution and 0.1% Triton-X-100 respectively. Then mouse monoclonal IgG p53 (F-8) antibody with dilution 1:300 and mouse monoclonal IgG p21 (F-5) antibody with dilution 1:300 was added for p53 and


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p21 assay respectively after washing with PBS and kept for 2 h. Afterthat secondary antibody Goat anti mouse IgG (H+L) RPE human adsorbed with dilution 1:600 was treated for another 2 h. Then, Bisbenzimide H 33258 (Hoechst) (1 µg/mL) was treated for 30 minutes to stain the nucleous. Following the completion of the experiment, the cells were washed with 1X PBS and observed in the inverted microscope (Model Nikon Eclipse Ti-U) with a 40X objective in DIC mode, 405 and 561 nm fluorescence channels. 2.24 Data Analysis Microscopic images were analysed using Image J software and the spectroscopic and statistical data were analysed in Origin 8.5 software. 3. Results and Discussion 3.1 Molecular docking study for calculation of binding energy of tetra-peptides and binding of SLRP in “Anchor region” of tubulin We performed the molecular docking study to understand how RLPS tetra-peptide binds with tubulin and found that binding energy is -7.2 Kcal/mol (Fig. S1). As this tetra-peptide binds with tubulin, the sequence of this peptide was scrambled and another thirteen scrambled peptides was constructed by sequence suppling in order to develop the best antimitotic peptide (Fig. 1). It was found that all the thirteen tetra peptides are binding close to the exchangeable GTP site of the tubulin and the binding energy is minimum in case of SLRP (-7.4 Kcal/mol) (Fig. S1). So, the binding affinity of SLRP to the tubulin is strongest among all the fourteen peptides. Fig. 2a represents the binding of SLRP close to the GDP/exchangeable GTP site of the tubulin. The binding is being stabilized by the hydrogen bonding interaction between Asn-228, GLn-15, Ser140, Val-171 and Thr-145 of tubulin with SLRP (Fig. S2). It was earlier described that


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molecules, bind closed to the GDP/exchangeable GTP, which is "anchor region" causes inhibition of tubulin polymerization.28 It was also described that this "anchor region" was exposed at the outer site of microtubule and near to the exchangeable GTP binding site.28 Docking data indicates that our SLRP tetra-peptide may bind at "anchor region". However, this needs to be confirmed using another assay. From the molecular docking of SLRP peptide at the "anchor region" close to the exchangeable GTP/GDP binding site of tubulin, we found that this peptide has significant binding at the "anchor region". The binding energy is very high (-8.0 kcal/mol) in this site. We have shown the "anchor region" by red square in the tubulin heterodimer (Fig. S3a) and the binding of SLRP peptide at the anchor region (Fig. S3b) which is approx. 33 Å apart from tubulin bound colchicine. 3.2 Synthesis, purification and characterization of the fourteen peptides For further screening, we have synthesized all the fourteen peptides following solid phase peptide synthesis method in Liberty 1 CEM microwave peptide synthesizer using Rink Amide AM resin. Crude peptides were purified by reverse phase HPLC with C18 column using water and acetonitrile mixture as binary solvent and characterized by ESI and MALDI Mass Spectroscopy (Fig. S4-S17). 3.3 MTT assay for screening best peptide We have studied the cell viability of all the fourteen peptides by MTT assay. From the MTT assay, it was found that all the analogues of RLPS are less-toxic to the MCF-7 cells compared to SLRP peptide at 50 µM concentration (Fig. S18). Tetra-peptide SLRP is killing ~40% MCF-7 cancer cells at 50 µM concentration, which suggests that SLRP has moderate cytotoxicity in MCF-7 cancer cell. This result reveals that among fourteen peptides SLRP shows best anticancer activity, thus all the future studies we proceed with this peptide. Next, we checked the


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cytotoxicity of SLRP in lower concentration in the MCF-7 cell line and found that SLRP is not toxic at the lower concentration (Fig. S19). We have calculated the IC50 value of SLRP, which is 165±7.4 µM (better than recently discovered antimitotic dodecapeptide 184.3±12.3 µM).29 Next, we checked that whether SLRP is toxic to the normal cell or not. For that we performed the MTT assay of SLRP in WI38 (human lungs fibroblast normal cell line) normal cell line and found that SLRP is not toxic to the normal cell (Fig. S20). Next we checked the stability of SLRP in human blood serum (HBS) for 24 h and found that the peptide is almost 90% degraded in 24 h (Fig. 21).30 3.4 Turbidity assay reveals SLRP tetrapeptide inhibits tubulin polymerization As SLRP shows best anticancer activity and it is designed from exchangeable GTP/GDP binding pocket of tubulin surface, we planed to study whether this peptide has any role in tubulin polymerization or not. Generally the effect of small molecules on tubulin polymerization is studied by tubulin turbidity assay.21 So, we studied the effect of SLRP on tubulin polymerization by tubulin turbidity. It was observed that the turbidity of solution in presence of SLRP decreases in a concentration dependent manner compare to the control i. e. the rate of tubulin polymerization decreased compared to the control in presence of SLRP (Fig. 2b). So, it can be stated that, SLRP interacts with tubulin and inhibits the tubulin polymerization in vitro. 3.5 Microtubule assembly assay reveals SLRP tetrapeptide inhibits tubulin polymerization In order to confirm the inhibition of tubulin polymerization by SLRP, we further performed microtubule assembly assay in presence and absence of SLRP following previously described method.25,26 It was observed that rate of increase of DAPI fluorescence in presence of SLRP is less compare to control (Fig. 2c), which further supports the inhibition of tubulin polymerization by SLRP.


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3.6 Determination of binding constant of SLRP tetra-peptide by Tryptophan fluorescence quenching After that, the binding affinity of SLRP with tubulin was determined by measuring the rate of Tryptophan (Trp) fluorescence quenching of tubulin due to interaction with different concentration of SLRP (ESI) and the binding constant was calculated using a modified SternVolmer equation.21,31 The binding constant of SLRP to the tubulin was found to be 0.0181X104 M-1 (Fig. S22b). Fig. S22a represents the fluorescence emission spectra of SLRP-tubulin complex. 3.7 Determination of stoichiometric binding of SLRP tetra-peptide in tubulin by UV-absorbance The stoichiometric binding of SLRP peptide in tubulin is determined by taking absorbance value of the tubulin-peptide complex system at 280 nm. The absorbance of the complex is varied with different mole fraction of the peptide. The values were analysed by plotting a JOB Plot of the experiment. It was found that at the maximum point peptide‘s mole fraction is ~50% which indicate 1:1 binding of SLRP peptide with tubulin ( S22c). 3.8 Synthesis, purification and characterization of Biotin-SLRP and FITC-SLRP After knowing that SLRP is the best candidate, we have synthesized biotin-SLRP and FITCSLRP following solid phase peptide synthesis method in Liberty 1 CEM microwave peptide synthesizer using Rink Amide AM resin. Crude peptides were purified by reverse phase HPLC with C18 column using water and acetonitrile mixture as binary solvent and characterized by ESI and MALDI Mass Spectroscopy (Fig. S23-S24). 3.9 SLRP tetra-peptide immobilized biotin micropatterned surface selectively capture tubulin indicates interaction of tetra-peptide and tubulin on 2D engineered micropatterned surface


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The interaction of SLRP with tubulin was further performed in an engineered chemically functionalized 2D platform (ESI).27 Interestingly, red coloured square-shaped micropatterns were observed (Fig. 3a). Fig. 3b represents the cartoon diagram of how SLRP interacts with tubulin on engineered 2D micropatterned surface. Control experiment was carried out following the same procedure without immobilization of biotin-SLRP and no red coloured micropatterns were observed (Fig. S25), which clearly indicates the binding of Alexa-Fluor-568 labeled tubulin only on biotin-SLRP immobilized micropatterns. It supports the interaction of SLRP with tubulin on engineered chemically functionalized 2D platform. 3.10 FRET study for confirmation of binding site of SLRP tetrapeptide into the tubulin surface As we have described before that our design concept of tetrapeptide library originating from GTP/GDP exchangeable binding site, where screened SLRP tetra-peptide shows good binding energy. However, it is difficult to confirm the exact binding site of SLRP tetrapeptide at tubulin surface. Thus, we have performed Förster Resonance Energy Transfer (FRET) experiment32-33 to determine the probable binding site of SLRP in tubulin using FITC-SLRP as a donor and tubulin bound colchicine (Tub-col complex) as an acceptor. We have seen that the binding site of peptides or small molecules, those bind to tubulin can be determined if they have a significance spectral overlap region with colchicine fluorescence. Colchicine’s weak fluorescence becomes much higher upon binding to tubulin (λex = 350 nm, λem = 427 nm).21 So, we performed the FRET experiment between Tub-col complex (donor) and FITC-SLRP (acceptor). Interestingly, it was found that the FRET is occurring between Tub-col complex and FITC-SLRP (Fig. 2d). The calculated R0 value between the aforesaid donor-acceptor pair is 29.5±1 Å. So, the measured average RDA distance between them is ̴ 30.3±1 Å. It indicates that SLRP binds to tubulin in a site, which is ̴ 30.3 Å apart from colchicine binding site in tubulin, which mostly matches with the


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distance between the colchicine site and GDP/exchangeable GTP. Above results clearly indicate that SLRP is binding close to the GDP/exchangeable GTP site to the tubulin not the exact GDP/exchangeable GTP site. So, it matches with the earlier report that molecules bind closed to the GDP/exchangeable GTP, which is "anchor region" causes inhibition of tubulin polymerization.28 3.11 Interaction of SLRP tetra-peptide with microtubule lattice: Kinesin and SLRP immobilized surface creates strong friction on microtubule lattice resulted inhibition of microtubule gliding speed FRET and molecular docking study reveals that our SLRP peptide is probably interacting and binding close to the exchangeble GTP binding site of tubulin, which is "anchor region". As we know that "anchor region" is exposed at the outer site of microtubule and near to the exchangeble GTP binding site,28 we have designed an assay to confirm whether our tetra-peptide can interact with microtubule lattice or not through "anchor region". For this assay, we have prepared trisNTA and biotin dual functionalized glass surface following previously described procedure.34 Then biotin-SLRP and kinesin (Kinesin612-His10) were immobilized on it followed by loading of very short freshly prepared microtubules using GMP-CPP and observed the motility of the microtubules immediately under TIRF microscope at 37 oC. Control experiment was carried out in the absence of biotin-SLRP following the same procedure. We calculated the gliding speed from the kymographs, acquired from time lapse images. The average gliding speed of the microtubules was found to be 37.117 µm/min (Fig. 3d and supplementary movie S1) and 70.52 µm/min (Fig. 3c and supplementary movie S2) in presence and absence of biotin-SLRP, respectively. It was found that, the gliding speed of the microtubules decreases 1.9 times (Fig. 3e) in presence of biotin-SLRP. Above result clearly states that, biotin-SLRP strongly binds with


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the microtubules lattice, which creates strong friction and resulted slow down of the gliding speed of the microtubule. Fig. 3f represents the cartoon diagram of how SLRP interacts with microtubule lattice on kinesin and peptide immobilized engineered 2D surface. This experimental result supports that SLRP tetra-peptide binds at "anchor region" of tubulin. 3.12 Visualization of FITC-SLRP tetra-peptide and its interaction with intracellular microtubule network inside the MCF-7 cell. As SLRP is showing binding with tubulin in vitro, we became interested to study the effect of SLRP on intracellular tubulin/microtubule. For that, first we have checked whether FITC tagged peptide (FITC-SLRP) is entering into MCF-7 cell or not (ESI). From microscopic image, it was found that, there are green signals inside the MCF-7 cells in 488 nm channel (Fig. S26), which indicates the uptake of FITC-SLRP into the MCF-7 cells. The cellular uptake was further confirmed by flow cytometry (Fig. 4a and S27). Next, we investigate whether SLRP tetrapeptide interacts with intracellular microtubule or not. For that, we treated the MCF-7 cells with FITC-SLRP for 4 h in serum free DMEM medium. Then the cells were incubated with complete medium for overnight followed by antibody and Hoechst treatment (ESI) and imaging through an Andor spinning disc confocal microscope. Interestingly, it was found that there are yellow coloured small aggregates inside the MCF-7 cells obtained from the merged image (Fig. 4b and S28e). Fig. S28a, S28b, S28c and S28d represent the DIC image, nucleus, green aggregates of FITC-SLRP inside the MCF-7 cells and microtubules network of MCF-7 cells respectively. The merged image clearly indicates the binding of FITC-SLRP with microtubules inside the MCF-7 cells. Cellular image data was analysed with the help of JACop (Just Another Co-localisation Plugin) using Image J software as previously described to further confirm subcellular colocalisation.35,36 Above analysis using Costes’ approach, Pearson’s coefficient and Van


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Steensel’s

CCF

approach

confirms

that

FITC-SLRP

is

nicely

co-localized

with

tubulin/microtubule network (Fig. S29 and S30). 3.13 SLRP tetra-peptide disrupts the intracellular microtubule networks of MCF-7 cells As SLRP interacts with tubulin and perturb tubulin polymerization in vitro, we became interested to study the effect of SLRP tetra-peptide on microtubule networks of MCF-7 cells (ESI). Immunocytochemistry images were captured under fluorescence microscope, which reveals that intracellular microtubule network of MCF-7 cells were completely disrupted and shrinked upon treatment with SLRP tetra-peptide (Fig. 4d and S32). Fig. 4c and S31 represent the nice microtubule network of the control MCF-7 cells. We quantify the shrinking of MCF-7 cell sizes due to disruption of microtubule networks using Image J software through measuring the circumference of the MCF-7 cells upon treatment with SLRP tetra-peptide (Fig. S33). 3.14 Nature of cell death by SLRP tetra-peptide reveals apoptotic death: Fluorescence microscopy Now, we tried to investigate the pathway of killing of MCF-7 cell by SLRP tetra-peptide. So, first we studied whether the killing is following the apoptotic pathway or not by fluorescence microscopy using annexin V and propidium iodide (PI) (ESI). It was observed that, there is red and green signal inside the SLRP treated MCF-7 cells (Fig. S35) whereas there is no such red and green signal in the control cells (Fig. S34). It indicates that both annexin V and PI enter into the SLRP treated MCF-7 cells, which gives the preliminary idea of apoptotic pathway of killing by SLRP. 3.15 Nature of cell death by SLRP tetra-peptide reveals apoptotic death: Flow cytometry


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In order to confirm the apoptotic pathway, we analyzed the % of apoptotic and necrotic cells compare to the healthy cells after 50 µM SLRP treatment by flow cytometry. From the flow cytometry analysis, it was found that % of apoptotic MCF-7 cells treated with SLRP is ~40 (Fig. 5b), whereas it is ~10 in SLRP untreated cells (Fig. 5a). % of necrotic cells is negligible in both the case. Fig. 5c represents the comparative histogram of % of apoptotic, necrotic and healthy cells in both SLRP treated and untreated MCF-7 cells. So, it was confirmed from the above data that, the killing of MCF-7 cells by SLRP follows the apoptotic pathway. 3.16 Evaluation of p53 and p21 proteins expression by immunoblotting experiment Generally, tumor suppressor protein p53 and cyclin-dependent kinase inhibitor 1 (p21) are activated upon treatment with an anticancer compound inducing apoptotic cell death. So, we studied whether p53 and p21 are activated or not upon treatment with SLRP by immunoblotting experiment. Interestingly, it was found from immunoblotting experiment that both p53 and p21 were activated upon treatment with SLRP (Fig. 5d). Further, protein expression was quantified and normalized against loading control (α-Tubulin). A 1.5 fold rise in p53 protein expression and 5.9 fold rises in p21 protein expression were observed in SLRP treated MCF-7 cells compared to control A549 cells (Fig. 5e). 3.17 Evaluation of p53 and p21 proteins expression by immunofluorescence microscopy Further, we have evaluated its cellular localization using immunocytochemistry experiment. Both the proteins p53 and p21 is activated in SLRP treated MCF-7 cells as we observed strong red signals inside the SLRP treated MCF-7 cells after the treatment of p53 (Fig. S37) and p21 antibody (Fig. S39), whereas there was insignificant signals inside the untreated MCF-7 cells after the treatment of p53 (Fig. S36) and p21 antibody (Fig. S38). Fig. 6a and 6b represent the


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histograms of the fluorescence intensity of p53 and p21 proteins in MCF-7 cell respectively, after SLRP treatment along with control. 4. Conclusion In summary, we have discovered a cell penetrating, antimitotic tetra-peptide SLRP, which perturbs microtubule function. We have shown that it strongly interacts with tubulin at "anchor region", which is close to the exchangeable GTP/GDP binding pocket and inhibits tubulin polymerization. We have studied the role of peptide on microtubule function on a chemically functionalized 2D platform and found that it strongly binds with tubulin. Further, this short tetrapeptide modulator interacts with intracellular tubulin/microtubule and disrupts the microtubule networks. Finally, we have shown that it causes moderate anticancer activities to MCF-7 cells through apoptotic cell death via activating the tumor suppressor proteins p53 and cyclindependent kinase inhibitor 1 (p21). ASSOCIATED CONTENT Supporting Information This materials are available free of charge via the internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Dr. Surajit Ghosh E-mail: [email protected].

Author Contributions


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BJ and PM synthesized and purified all the peptides and performed various in vitro microtubule assays (microtubule assembly assay, turbidity assay, tryptophan quenching experiment), cellular uptake studies, microtubule gliding assay, binding of SLRP with tubulin on biotin micropatterned surface, interaction and effect of SLRP on microtubule network, apoptosis studies and helped SG in writing manuscript. PM and AA performed FRET experiment and docking studies. PK, AS, GD and SM performed immunoblotting assay and helped BJ and PM in performing other experiments. SG conceived the idea, supervised the project and wrote the manuscript. ‡These authors contributed equally ACKNOWLEDGMENT Authors wish to thank Dr. Partha Chakrabarti for accessing his laboratory and NCCS-Pune for cell line. BJ, PM, AS and PK thank CSIR, AA and SM thank UGC for their fellowships. GD thanks ICMR fellowship. SG kindly acknowledges DST-grant (EMR/2015/002230) for financial assistance. ABBREVIATIONS Ser-Leu-Arg-Pro, SLRP; Human breast cancer cell, MCF-7 cell; Förster Resonance Energy Transfer, FRET; Hour, h. REFERENCES 1) Nogales, E.; Whittaker, M.; Milligan, R. A.; Downing, K. H. High-resolution Model of the Microtubule. Cell 1999, 96 (1), 79-88. 2) Lowe, J.; Li, H.; Downing, K. H.; Nogales, E. Refined Structure of Alpha Beta-tubulin at 3.5 A Resolution. J. Mol. Biol. 2001, 313 (5), 1045-1057.


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36) Jana, B.; Mohapatra, S.; Mondal, P.; Barman, S.; Pradhan, K.; Saha, A.; Ghosh, S. α‑ Cyclodextrin Interacts Close to Vinblastine Site of Tubulin and Delivers Curcumin Preferentially to the Tubulin Surface of Cancer Cell. ACS Appl. Mater. Interfaces 2016, 8 (22), 13793-13803.

Fig. 1. (a) Docking image showing various amino acids at exchangeable GTP/GDP binding cavity of β-tubulin and designing of RLPS from relative frequency of amino acids interaction. (b) Peptide sequence of RLPS and its analogues.


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Fig. 2. (a) Docking image of SLRP and tubulin shows that SLRP is binding close to the GDP/exchangeable GTP site of the tubulin. It is also showing the amino acid residue of tubulin involved in hydrogen bonding interaction with SLRP. (b) Tubulin turbidity assay in presence SLRP indicating that tubulin turbidity decreases in presence of SLRP in a concentration dependent manner compare to control, which indicates inhibition of tubulin polymerization in presence of SLRP. (c) Microtubule assembly assay in presence of SLRP indicates that there is decrease in the rate of enhancement of DAPI fluorescence in tubulin polymerization in presence of SLRP, compare to control, which supports the inhibition of polymerization in presence of SLRP. FRET study: (d) Energy transfer graph between donor (Tub-col complex) emission and acceptor (FITC-SLRP) absorption indicating that FRET is occurring between Tub-col complex and FITC-SLRP.


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Fig. 3. (a) Binding of tubulin on Biotin-SLRP immobilized micropatterned surfaces: Red coloured micropatterns, obtained from the microscopic image reveal that the tubulin specifically binds with Biotin-SLRP, immobilized on neutravidin loaded micropatterned surfaces, which further supports the interaction of SLRP with tubulin. Scale bars correspond to 20 µm. (b) Cartoon represents how SLRP interacts with tubulin on engineered 2D micropattern surface. Gliding assay of microtubule: (c) Histogram of gliding speed of microtubules in absence of SLRP. (d) Histogram of gliding speed of microtubules in presence of SLRP. (e) Comparative diagram of average gliding speed of microtubule in presence and absence of SLRP (error bar indicates standard deviation of the value, *p< 0.05). (f) Cartoon represents how SLRP interacts with microtubule on kinesin and peptide immobilized engineered 2D surface.


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Fig. 4. (a) Comparative diagram indicating the relative cellular uptake of 100 µM FITC-SLRP (SLRP_100) and 200 µM FITC-SLRP (SLRP_200) in MCF-7 cell by flow cytometry (error bar indicates standard deviation of the value, *p< 0.05). (b) Binding of FITC-SLRP with microtubule in MCF-7 cells. Scale bar corresponds to 20 µm. (c) Microtubules network of control MCF-7 cell. (d) Microtubules network of SLRP treated MCF-7 cell. Scale bars correspond to 30 µm.


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Fig. 5. Apoptotic study by FACS analysis indicates that the amount of apoptotic MCF-7 cell population increases after treatment with (b) SLRP compared to (a) control. (c) Comparative bar diagram represents the % of apoptotic (Q2), necrotic (Q1) and healthy (Q4) MCF-7 cells after SLRP treatment along with control (Error bar indicates standard deviation of the value, *p< 0.05). (d) Immunoblotting experiment showing more activation of p53 and p21 proteins in MCF-7 cells after SLRP treatment compared to control. (e) Bar diagram shows higher expression of p53 and p21 protein in MCF-7 cells after SLRP treatment compared to control. Error bar indicates standard deviation of the value, *p< 0.05.


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Fig. 6. (a) Quantitative analysis reveals fluorescence intensity inside the MCF-7 cells in 561 nm channel due to p53 activation after SLRP treatment along with control. (b) Quantitative analysis reveals fluorescence intensity inside the MCF-7 cells in 561 nm channel due to p21 activation after SLRP treatment along with control (Error bar indicates standard deviation of the value, *p< 0.05).


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Designed Tetra-peptide Interacts with Tubulin and Microtubule

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