Siddhartha Sankar Ghosh, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, for the award of the degree of Doctor of Philosophy. Cell-based studies establish the anti-proliferative and anti-invasive role of recombinant membrane-permeable PTEN-Long protein in primary glioblastoma cell line.
OH Ammonium hydroxide
8 Time-dependent uptake study of PTEN-Long in U-87 MG cells based on the determination of cell viability by MTT assay.
LIST OF TABLES
Introduction
Loss of function of the cell signaling regulators is the cardinal reason for disease initiation and. Uncontrolled manifestation of the AKT protein causes physiological disturbances in the cell leading to cancer initiation and progression.
Literature Review
- Cellular Role of the Akt Signaling Pathway
One such commonly deregulated signaling pathway associated with pathogenesis is the signaling pathway Akt (Robertson 2005, Tokunaga et al., 2008). Akt positively affects Skp2 activity with phosphorylation at Serine72 (Gao et al., 2009).
Illustration of substrates phosphorylated by Akt to regulate cellular environment 2.4 Regulation of Akt Pathway by PTEN
- Current Status of the Akt Pathway Inhibition and Limitations
- PTEN Based Therapy
- Delivery Strategies for Intracellular Recombinant Protein Therapy
- Key Features and Scope of the Work
- Objectives
One of the main functions of the protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) is to antagonize the function of PI3Kinase (Stambolic et al., 1998). PTEN has been reported to be inactivated by mutation in prostate cancer (Cairns et al., 1997).
Materials and Methods
Materials and Methods
Materials
Maintenance of Cell Lines
Cloning of Human PTEN-Long and PTEN
The lagged product was transformed into competent Escherichia coli DH5α by standard heat shock procedure. Transformed cells were spread on ampicillin-LB agar plates coated with X-gal/IPTG to aid in blue-white screening. After overnight growth in an incubator at 37 °C, selected white and blue colonies were screened for positive clones by restriction digestion with BamHI and XhoI restriction enzymes.
The PCR amplification conditions for cloning the PTEN gene consisted of 30 cycles of 95°C for 30 seconds, 50°C for 1 minute, and 72°C for 1 minute 20 seconds using gene-specific forward primer 5'-CGGGATCCGACATGACAGCCATCATCAAAG -3' with a BamHI overhang and reverse primer 5'-CCCTCGAGGACTTTTGTAATTTGTGTATGC-3' with a XhoI overhang. After growth overnight in a 37°C incubator, selected white and blue colonies were screened for positive clones by restriction digestion with BamHI and XhoI enzymes.
Cloning into Bacterial Expression Vector
Bacterial Expression of the Recombinant PTEN-Long and PTEN Proteins
Purification of the Recombinant PTEN-Long and PTEN Proteins
The bacteria were lysed by sonication with probes on ice at an amplitude of 30% for 30 cycles of 2 seconds ON and 25 seconds OFF. Supernatant collected after centrifugation at 12,000 rpm for 20 min was diluted with an equal amount of HBS (50 mM Hepes and 150 mM NaCl, pH 7.4) and filtered through a 0.45 μm syringe filter. The lysate was allowed to bind to glutathione-agarose beads under slow shaking conditions on ice for 1 hour, followed by 8 washes of 5 minutes each with HBS.
Eluted fractions were subjected to buffer exchange by dialysis against 25 mM Hepes pH 7.4 at 4 °C for 4 h. The dialyzed protein was concentrated by centrifugation at 5000 rpm (30kDa MWCO) Spin-X UF concentrator and verified by 12% SDS-PAGE analysis.
Thrombin Cleavage of the Recombinant Proteins
Characterization of the Recombinant Proteins .1 Western Blot
- MALDI TOF-TOF Analysis
- Secondary Structure Analysis by Circular Dichroism Spectroscopy
- Protein Phosphatase Assay
Purified GST-PTEN-Long and GST-PTEN proteins separated on a 12% SDS gel were stained with colloidal coomassie G-250. After staining, the desired band was cut into small pieces from the SDS-PAGE gel. The gel pieces were destained with a destaining solution from the IGD kit (Sigma).
The purified proteins GST-PTEN-Long and GST-PTEN were dialyzed against 10 mM Tris pH 7.4, and further concentrated by centrifugation at 5000 rpm for the desired time using (30 kDa MWCO) Spin-X UF concentrator. Samples in 10 mM Tris pH 7.4, taken in a cuvette with a path length of 5 mm, were analyzed using a JASCO-815 spectrometer. Assessment of the in vitro phosphatase activity of GST-labeled and unlabeled PTEN-Long and PTEN was performed using para-nitrophenyl phosphate (PNPP) as substrate.
The reaction buffer was composed of 25 mM Hepes pH 7.4, 10 mM DTT and 1 μg of purified GST-tagged and untagged enzymes. Stop solution consisting of 1N NaOH added after the specified incubation period served a dual purpose; terminate the reaction and intensify the color of the product measured by TH. Under alkaline conditions, the product para-nitrophenol (PNP) is converted to para-nitrophenolate which was measured by absorbance at 405 nm (Martin et al., 2014) using Perkin Elmer Victor X3 multiplexed reader.
Cellular Internalization Study of PTEN-Long
Cell Viability Assay
The product was measured by absorbance at 570 nm along with a background measurement at 690 nm using a multiplate reader (Tecan, Infinite M200PRO). For all PTEN-Long experiments, U-87 MG cells were seeded at a cell density of 3500 cells per well in a 96-well plate. For all PTEN experiments, U-87 MG cells and MCF7 cells were seeded at a cell density of 4000 and 6000 cells per well, respectively, in a 96-well plate.
Scratch Assay
The medium was then discarded and DMSO was added to dissolve the formazan crystals formed by enzymatic conversion in living cells. Immediately after wounding, images of the wound were taken with a Nikon ECLIPSE Ti microscope. Cells were then treated with recombinant proteins and incubated at 37 °C, under humid conditions of 5% CO2 for the desired period of time.
Then, at the end of the experiment, the control and treated dishes with tissue culture were examined under a microscope.
Determination of Cell Cycle Pattern
The schematic representation of the scratch assay
- Modulation of Cellular Signalling
- Synthesis of Silica Nanoparticles
- Characterization of Silica Nanoparticles .1 Surface Morphology Study
- Determination of Immobilization of GST-PTEN onto Silica NPs
- Release Studies of GST-PTEN from Silica NPs
- Evaluating GST-PTEN-Silica Nanoparticles Interaction .1 Evaluation of Structural Integrity
- Protease Protection Assay
For PTEN-Long experiments, U-87 MG cells were treated with 100 n m protein for 2 h and 4 h in DMEM serum medium. A FESEM and TEM study was performed to determine the morphology of the synthesized silica nanoparticles. FTIR analyzes of lyophilized samples were performed by mixing the samples with KBr to form.
The peaks obtained were analyzed to study binding of the recombinant protein to the silica nanoparticles. The supernatant was collected and intrinsic fluorescence of the protein was examined at 280 nm to determine the amount of protein released using Fluoromax‐4, Horiba JobinYvon, Edison, NY, USA. To study the structural changes induced upon nanoparticle interaction, the secondary structure of the GST-PTEN released from the nanoparticle surface was analyzed by means of circular dichroism spectroscopy.
Assessment of the in vitro phosphatase activity of GST-PTEN bound to silica nanoparticles was performed using both protein and lipid substrates. DTT is an important part of the reaction buffer because it is necessary to maintain the cysteine residue in the active site of the enzyme in the reduced state (Spinelli and Leslie 2015). After incubation, a stop solution consisting of 1 N NaOH was added to terminate the reaction and intensify the color of the product, para-nitrophenol (PNP).
Schematic representation of the protease protection assay design
- Characterization of Silver Nanoclusters
- Binding Study of GST tagged PTEN and Silver Nanoclusters
- Polymer Encapsulation and Characterization of PTEN Bound Nanoclusters
- Cellular Internalization of the PTEN-Nanocomposites
- RNA Isolation and Expression Study
- Generation of Spheroids
- Calcein-AM/ PI Dual Staining of U-87 MG and MCF7 Spheroids
- Alamar Blue Assay for Spheroid Viability Study
- Cell Cycle Analysis of the Spheroids
- Statistical analysis
Lysozyme-stabilized silver nanoclusters were synthesized by a minor modification of the protocol developed by Zhou et al. (Zhou et al., 2012). Change in the color of the solution from colorless to brown indicated the reduction of silver. The fluorescence spectrum of the synthesized nanoclusters was recorded from 500 nm to 800 nm after excitation at 480 nm using Fluoromax-4, Horiba JobinYvon, Edison, NY, USA.
The interaction between protein and nanoclusters was determined by probing the fluorescence of the nanoclusters. Where, Fluorescence IntensityNCS is the fluorescence of the nanoclusters, and Fluorescence IntensityBound NCS is the fluorescence of the nanoclusters after binding to GST-PTEN. The concentration of the nanocomposites is expressed as nM protein (PTEN) and μg/ml silver in the cluster.
The luminescent property of silver nanoclusters has been exploited to study the intracellular delivery of protein cargo. U-87 MG and MCF7 cells were seeded in 6-well plates at a cell density of 1 x 105 cells per well in DMEM medium supplemented with 10% FBS and incubated at 37 °C under humidified 5% CO2 conditions for cell adhesion. After treatment of U-87 MG and MCF7 spheroids with PTEN-nanocomposites and nanocomposites, the alamar blue assay was performed.
Results and Discussion
- Bacterial Expression, Purification and Physical Characterization of the Recombinant Proteins
- Functional Characterization of the Recombinant Proteins
- Evaluation of Therapeutic Potential of PTEN‐Long
- Assessment of Co‐therapy Module with PTEN‐Long
- Synthesis and characterization of Silica Nanoparticles for Recombinant PTEN Immobilization
- Evaluation of Structural and Functional parameters of Recombinant PTEN immobilized onto Silica Nanoparticles
- Synthesis and Characterization of PTEN‐Nanocomposites
- Cellular Internalization Study of PTEN‐Nanocomposites
- Effect of PTEN‐Nanocomposites on Cell Viability and Combination Therapy To determine the effect of PTEN-nanocomposite on the proliferation of MCF7 and U-87 MG
- Generation of Spheroid Model
- Internalization and Effect of PTEN‐Nanocomposites
- Cell cycle analysis of Spheroids
- Gene Expression Profile of Spheroids
73 Figure 4.6 Kinetic profile of the recombinant proteins against para-Nitrophenyl phosphate (A) GST tagged and untagged PTEN (B) GST tagged and untagged PTEN-Long. Before studying the effect of PTEN-Long on the U-87 MG, the cellular internalization of the protein was assessed. Inhibition of cyclin B1 led to accumulation of the PTEN-Long-treated cells in the S phase.
TEM images of GST-PTEN immobilized silica nanoparticles revealed no significant change in the average size and morphology of the nanoparticles upon binding of the protein (Figure 4.18A). The loading rate of the PTEN nanocomposites was determined by a protocol developed by Sah et al. Confocal microscope images of MCF7 and U-87 MG cells showed red fluorescence of the nanocomposites after incubation with PTEN nanocomposites for 4 h.
The study thus confirms endocytosis-dependent uptake of the nanocomposites in MCF7 and U-87 MG cells. Inhibition of cyclin B1 resulted in the accumulation of the PTEN nanocomposites treated MCF7 cells in the S phase. The results revealed a dose-dependent reduction in cell viability after treatment with increasing concentrations of PTEN nanocomposites for MCF7 (Figure 4.41A) and U-87 MG cells (Figure 4.41B).
IC50 was obtained for MCF7 cells by concentration of PTEN nanocomposites of PTEN (36 nM)-Cluster (30 μg/ml) Composite. U-87 MG were treated with varying concentrations of erlotinib (2.5 to 10 μM) and fixed concentration of PTEN nanocomposites [PTEN (24 nM) cluster (20 μg/ml) composite].
Conclusion and Future
- Cloning expression and Purification
- Functional Characterizations
- Intracellular Delivery of PTEN
- Intracellular Tracking
- Modulation of Cellular Signaling
- Effect on Spheroids
- Future Prospects
- Neha Arora and Siddhartha Sankar Ghosh (2016), Functional Characterizations of Interactive Recombinant PTEN Silica Nanoparticles for Potential Biomedical
- Neha Arora, Lalitha Gavya S, Siddhartha Sankar Ghosh (2018), Multi-facet Implications of PEGylated Lysozyme Stabilized-Silver Nanoclusters Loaded
- Neha Arora, Rajib Shome, Siddhartha Sankar Ghosh (2018), Effect of PEGylated Lysozyme Stabilized-Silver Nanoclusters Loaded Recombinant PTEN Cargo on 3D
- Poster Presentation, Neha Arora and Siddhartha Sankar Ghosh, Functional Stabilization of Recombinant PTEN onto Silica Nanoparticles for Potential
- Poster Presentation, Neha Arora and Siddhartha Sankar Ghosh, PEGylated Silver Nanoclusters Mediated Cytosolic Delivery of Tumor Suppressor Protein PTEN to
- Poster Presentation, Neha Arora and Siddhartha Sankar Ghosh, Understanding Therapeutic Potential of PEGylated Silver Nanoclusters Loaded Recombinant PTEN,
Intracellular delivery of PTEN was mediated by binding of the recombinant protein to silica nanoparticles. The structural integrity was further confirmed by analyzing the functional integrity of the recombinant enzyme. As a result, each formulation step requires a careful examination of protein function.
The functional integrity of the recombinant protein was analyzed after binding to nanoclusters and also after PEG encapsulation. Reproduced by permission of The Royal Society of Chemistry (RSC) on behalf of the Center National de la Recherche Scientifique (CNRS) and the RSC. RSC) on behalf of the European Society for Photobiology, the European Society for Photochemistry and the RSC.
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