Uptake by Human Glioma Cell Lines and Biological Effects of a Peptide-Nucleic Acids Targeting

Uptake by human glioma cell lines and biological effects of a peptide-nucleic acids targeting miR-221

Eleonora Brognara1, Enrica Fabbri1, Elena Bazzoli2, Giulia Montagner1, Claudio Ghimenton3, Albino Eccher3, Cinzia Cantù5, Alex Manicardi4, Nicoletta Bianchi1, Alessia Finotti1, Giulia Breveglieri1, Monica Borgatti1, Roberto Corradini4, Valentino Bezzerri3,5, Giulio Cabrini3,5 and Roberto Gambari1

1Department of Life Sciences and Biotechnology, Ferrara, Italy; 2Department of Neuroscience/Division of Neurology/Neurooncology, AOUI Verona, Italy; 3Department of Pathology and Diagnostics, University Hospital of Verona, Italy; 4Department of Organic and Industrial Chemistry, University of Parma, Italy; 5Laboratory of Molecular Pathology, University-Hospital, Verona, Italy.

Supplementary Material

MATERIALS AND METHODS

Synthesis and characterization of PNAs

The synthesis of PNAs employed in the present paper was carried out manually using standard protocols of solid-phase synthesis of PNA and peptides. The fluorescein tag was linked to the PNA or peptide through a spacer, the products were all purified with RP-HPLC and their purity and identity was confirmed by HPLC/ESI-MS analysis as reported elsewhere (1-3). The synthesis and characterization of PNAs have been previously reported (3). Briefly, the a221 and a210 PNAs were synthesized with standard manual Boc-based chemistry using commercially available monomers (ASM, Hannover, Germany) with HBTU/DIPEA coupling. All the PNAs were synthesized in a 5 µmole scale using MBHA resin loaded with Boc-PNA-T-OH as first monomer. The R8 tail was introduced using the same coupling procedures. To obtain fluorescently labeled PNA a 2-[2-(Fmoc-amino)ethoxy)ethoxy]acetic acid [AEEA], spacer (Applied Biosystems, Foster City, CA, USA) was linked to the N-terminus of PNA and then 5(6)-carboxyfluorescein (Sigma-Aldrich) was introduced using DIC/DhBtOH coupling. PNA purification was performed by RP-HPLC as described (3). This peptide was chosen among other possible carriers as octa- and nona-arginine are the most efficient for cellular internalization (4-6). In addition, octa-arginine was efficiently used for cellular internalization of drugs (7) and PNAs (1), and can accumulate in some tumors to a higher degree than other cell-penetrating peptides (CPP) (8).

The synthesis of new R8-PNA-a221-MUT and R8-PNA-a222 was performed using a standard Fmoc-based automate peptide synthesizer (Syro II, MultiSynTech GmbH, Witten, Germany), a ChemMatrix-RinkAmide resin loaded with Fmoc-Gly-OH as first monomer and commercially available monomers (Link Technologies, Bellshill, UK) with HBTU/DIPEA coupling. After purification the PNAs were characterized by UPLC-MS on a Water ACQUITY System equipped with an ACQUITY UPLC BEH C18 (1.7μm, 2.1x50mm). Gradient: 100% A for 0.9 minutes, then from 0% to 50% B in 5.7 minutes at 0.25 ml/min flow (A: water + 0.2% formic acid; B: acetonitrile + 0.2% formic acid).

R8-PNA-a221-MUT: Rt= 2.63 minutes, calculated mw: 6188.3 g/mol; m/z found: 1238.9 [M+5H]5+, 1032.6 [M+6H]6+, 885.3 [M+7H]7+, 774.7 [M+8H]8+, 688.7 [M+9H]9+, 617.0 [M+10H]10+, 563.6 [M+11H]11+.

R8-PNA-a222: Rt= 2.94 minutes, calculated mw: 6226.3 g/mol; m/z found: 1038.7 [M+6H]6+, 890.3 [M+7H]7+, 779.0 [M+8H]8+, 692.6 [M+9H]9+, 610.7 [M+10H]10+.

Biospecific Interaction Analysis (BIA) with BIAcoreTM X100

All procedures were performed at 25°C and 5 ml/min flow rate, by using HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) (GE Healthcare) as running buffer. PNAs containing CM5 sensor chips (GE Healthcare) were obtained by amine coupling chemistry, exploiting the terminal amine group of the PNA. An Amine Coupling Kit (GE Healthcare) was used. Briefly, carboxyl groups on the sensor chip surface were first activated with a 1:1 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS) to give reactive succinimide esters. The R8-PNA-a221 was then passed over the surface at the concentration of 50 μg/ml for 10 minutes, in order to form a covalent bond. Finally, 1 M ethanolamine-HCl at pH 8.5 was injected to quench the excess of reactive groups. Hybridization to pre-miR-221 and negative control pre-miR-210 were performed in 10% HBS buffer, followed by washing with HBS and a 1 minute pulse of 50 mM NaOH for regeneration of flow cells. The BiacoreTM X100 Control Software and BiacoreTM X100 Evaluation Software, version 2.0.1 (GE Healthcare) were used for operation and data analysis, respectively.

RNA extraction

Cultured cells were trypsinized and collected by centrifugation at 1,500 rpm for 10 min at 4 °C, washed with PBS, lysed with Tri-ReagentTM (Sigma Aldrich, St.Louis, Missouri, USA), according to manufacturer’s instructions. The isolated RNA was washed once with cold 75% ethanol, dried and dissolved in nuclease free pure water before use. RNA from human tissue sections was extracted and purified with miRNeasy FFPE Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture's procedures.

Quantitative analyses of miRNAs

Reverse transcriptase (RT) reactions were performed using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA); real-time PCR was performed according to the manufacturer’s protocols. Twenty ng per sample were used for the assays. All RT reactions, including no-template controls and RT-minus controls, were performed in duplicate using the 7700 Sequence Detection System version 1.7 (Applied Biosystems, Foster City, CA, USA). The relative expression was calculated using the comparative cycle threshold method and the reference U6 snRNA was used to normalize all RNA samples, since it remains constant in the assayed samples by miR-profiling and quantitative RT-PCR analysis, as previously reported (3).

Gene expression analysis by RT-qPCR

Primers and probes used to assay p27kip1 (Assay ID: Hs00153277.m1), TIMP3 (Assay ID: Hs00165949_m1), PUMA (Assay ID: Hs00248075_m1), ICAM-1 (Assay ID: Hs00164932_m1), PTEN (Assay ID: Hs02621230_m1) were purchased from Applied Biosystems, (Applied Biosystems, Foster City, CA, USA). The relative expression was calculated using the comparative cycle threshold method and, as reference genes, the human RPL13A (Assay ID: Hs04194366_g1).

Analysis of apoptosis

Annexin V and Dead Cell assay on U251, U373 and T98G cell lines (either untreated or treated for 48 h with temozolomide and different concentrations of PNAs) were performed with “Muse”TM (Millipore Corporation, Billerica, MA, USA) method, according to the instructions supplied by the manufacturer. This procedure utilizes Annexin V to detect PS (PhosphatidylSerine) on the external membrane of apoptotic cells. A dead cell marker is also used as an indicator of cell membrane structural integrity. It is excluded from live, healthy cells, as well as early apoptotic cells. Four populations of cells can be distinguished in this assay. Cells were washed with sterile PBS 1X, trypsinized, suspended and diluted (1:2) with the one step addition of the Muse™ Annexin V & Dead Cell reagent. After incubation of 20 min at room temperature in the dark, samples were analyzed. Data from prepared samples are acquired and recorded utilizing the Annexin V and Dead Cell Software Module (Millipore).

Western blotting

Twenty mg of cellular extracts were denatured for 5 min at 98 °C in 1x SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 50 mM Dithiotreithol -DTT-, 0.01% bromophenol blue, 10% glicerol) and loaded on SDS-PAGE gel (10 cm x 8 cm) in Tris-glycine Buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). A biotinylated protein ladder (size range of 9-200 kDa) (Cell Signaling, Euroclone S.p.A., Pero, Milano, Italy) was used as standard to determine molecular weight. The electrotransfer to 20 microns nitrocellulose membrane (Pierce, Euroclone S.p.A., Pero, Milano, Italy) was performed overnight at 360 mA and 4 °C in electrotransfer buffer (25 mM Tris, 192 mM Glycine, 5% methanol). The membrane were pre stained in Ponceau S Solution (Sigma, St.Louis, MO, USA) to verify the transfer, washed with 25 ml TBS (10 mM Tris-HCl pH 7.4, 150 mM NaCl) for 10 min at room temperature and incubated in 25 ml of blocking buffer for 2 hours at room temperature. The membranes were washed three times for 5 min each with 25 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with primary rabbit monoclonal antibody (1:2000) (Cell Signaling, Euroclone S.p.A., Pero, MI, Italy) in 10 ml primary antibody dilution buffer with gentle agitation over-night at 4 °C. The day after, the membranes were washed three times for 5 min each with 20 ml of TBS/T and incubated in 10 ml of blocking buffer, in gentle agitation for 2 h at room temperature, with an appropriate HRP-conjugated secondary antibody (1:2000) and an HRP-conjugated anti-biotin antibody (1:1000) used to detect biotinylated protein marker. Finally, after three washes each with 20 ml of TBS/T for 5 min, the membranes were incubated with 10 ml LumiGLO® (0.5 ml 20X LumiGLO®, 0.5 ml 20X Peroxide and 9.0 ml Milli-Q water) (Cell Signaling, Euroclone, Pero, MI, Italy) in gentle agitation for 5 min at room temperature and exposed to x-ray film (Pierce, Euroclone, Pero, MI, Italy). As necessary, after a stripping procedure using the Restore™ Western Blot Stripping Buffer (Pierce, Euroclone, Pero, MI, Italy) membranes were reprobed with primary and secondary antibodies. X-ray films for chemiluminescent blots were analyze by Gel Doc 2000 (Bio-Rad Laboratoires, MI, Italy) using Quantity One program to elaborate the intensity data of our specific protein targets. The rabbit mAb against p27Kip1 (D37H1_3688), TIMP3 (D74B10_5673), β-actin (13E5_4970) and p70 (49D7_2708) were purchase from Cell Signaling (Euroclone, Pero, MI, Italy).

RESULTS

Specificity of R8-PNA-a221 hybridization to miR-221 sequences: a Biospecific interaction analysis

Biospecific interaction analysis showing the hybridization between different PNAs immobilized on the sensor chips and injected miR-221 or scrambled miR-221 oligonucleotides is reported in Fig S1. The following PNAs were immobilized on CM sensor chips: R8-PNA-a221, recognizing miR-221, the mutated R8-PNA-a221-MUT and the R8-PNA-a222 specific for miR-222. As expected no hybridization of the scrambled oligonucleotide to the different PNAs was detected. The miR-221 oligonucleotide hybridizes efficiently only to the R8-PNA-a221 sensor chip, generating a stable PNA/miR-221 hybrid (Fig S1A). On the contrary, the miR-221 oligonucleotide hybridizes with low efficiency to the R8-PNA-a221 sensor chip, generating an unstable PNA/miR-221 hybrid (Fig S1B). These data support the concept that the hybridization between R8-PNA-a221 and miR-221 is highly specific.

Uptake of PNA-a221 and R8-PNA-a221 by human glioma cell lines

U251, U373 and T98G glioma cell lines were cultured for 24 hours with 2 mM Fl-PNA-a221 or Fl-R8-PNA-a221 and analyzed by FACS. Fig S2 confirms that (a) as found for U251 cells, Fl-R8-PNA-a221 binds also to the other U373 and T98G glioma cell lines and (b) that no binding of Fl-PNA-a221 occurs.

R8-PNA-a221: effects on cell growth and apoptosis

A representative effect of R8-PNA-a221 and R8-PNA-a221-MUT on U251 cell line is shown in Fig S3, showing that a low antiproliferative effect was observed, but only at 4-8 mM concentrations (Fig S3A). On the contrary, R8-PNA-a221-MUT do not display any antiproliferative activity (Fig S3B). Since we planned to determine whether any effect of R8-PNA-a221 was found in association with other antiproliferative drugs we preliminarily analyzed the effects of the drug temozolomide (TMZ) on the U251 cell line and on the drug-resistant T98G cell line. As shown in Fig S4, the IC50 of U251 was found to be 18.8 ± 5.2 mM in three independent experiments. On the contrary, it was found to be higher than 400 μM in the drug-resistant T98G cell line (Fig S4).

Effects of a combination of temozolomide (TMZ) with 2 and 4 mM R8-PNA-a221

Fig S5 shows the distribution of early and late apoptotic cells in the drug-resistant T98G cells treated with TMZ (Fig S5B), with 2 and 4 mM R8-PNA-a221 (Fig S5, C and D), or with 2 and 4 mM R8-PNA-a221 in the presence of 400 mM TMZ (Fig S5, E and F). The results obtained suggest that a low increase of early and late apoptotic cells occurs in T98G cells treated with 400 TMZ alone. Treatment with 2 and 4 mM R8-PNA-a221 induces an increase of the proportion of early and late apoptotic cells. When the apoptotic cells were determined in T98G cells cultured with 2 and 4 mM R8-PNA-a221 in the presence of 400 mM TMZ, the proportion of early and late apoptotic cells was higher than expected from a simple additional inhibitory effects of TMZ and R8-PNA-a221.

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SUPPLEMENTARY FIGURES

Fig S1. Biospecific interaction analysis showing the hybridization between PNAs immobilized on the sensor chips and injected miR-221 (5’-GCT ACA TTG TCT GCT GGG TTT C-3’, solid lines) and scrambled miR-221 (5’-CTG GCT TCA TGT CAT GTA GCT G-3’, dotted line) oligonucleotides. The following PNAs has been immobilized on CMC sensor chips: R8-PNA-a221 (A), R8-PNA-a221-MUT (B) or R8-PNA-a222 (C). a, analyte injection step; b, washing step. RUin: resonance unit (RU) values before the injection; RUfin: values after injection; RUres: values after washing. Hybridization was carried out in HBS.