Open Life Sci. 2019; 14: 619–627 Research Article Peter Bober*, Michal Alexovič, Zuzana Tomková, Róbert Kilík, Ján Sabo RHOA and mDia1 promotes apoptosis of breast cancer cells via a high dose of doxorubicin treatment https://doi.org/10.1515/biol-2019-0070 Received March 8, 2019; accepted September 5, 2019 1 Introduction Abstract: Background: Transforming RhoA proteins Doxorubicin (DOX) is one of the most useful anticancer (RHOA) and their downstream Diaphanous homolog 1 agents. However, the use of higher DOX doses is limited by proteins (DIAPH1) or mDia1 participate in the regulation the cardiotoxicity [1, 2, 3]. DOX may intercalate into DNA of actin cytoskeleton which plays critical role in cells, i.e., and inhibits the progression of topoisomerase II, inducing morphologic changes and apoptosis. Methodology: To its cytotoxicity [4]. Under cytotoxic stress conditions, Rho determine the cell viability the real time cell analysis (RTCA) GTPase signalling cascade results in actin cytoskeleton and flow cytometry were used. To perform proteomic remodelling [5]. Rho family GTPases are therefore key analysis, the label-free quantitative method and post- regulators of the actin cytoskeletal dynamics [6]. It was also translation modification by the nano-HPLC and ESI-MS ion proved that DOX induces actin cytoskeleton alterations, trap mass analyser were used. Results: The results of the cell namely through Rho family RhoGTPase pathways [7]. viability showed an increase of dead cells (around 30 %) in The structural mechanisms of RHOA, RAC1 and CDC42 MCF-7/DOX-1 (i.e., 1μM of doxorubicin was added to MCF-7/ proteins are well documented, as are their regulatory WT breast cancer cell line) compared to MCF-7/WT (control) partners and post-translational modifications [8]. after 24 h doxorubicin (DOX) treatment. The signalling Moreover, it has been known that RHOA is phosphorylated pathway of the Regulation of actin cytoskeleton (p<0.0026) via multiple kinases including cAMP-dependent protein was determined, where RHOA and mDia1 proteins were kinase (PKA) and cGMP-dependent protein kinase (PKG) up-regulated. Also, post-translational modification [9, 10]. These kinases phosphorylate RHOA at serine-188 analysis of these proteins in MCF-7/DOX-1 cells revealed and deactivate RHOA by increasing its interaction with Rho dysregulation of the actin cytoskeleton, specifically the GDP-dissociation inhibitor (RhoGDI), i.e. translocation collapse of actin stress fibbers due to phosphorylation of of RHOA from membrane to cytosol [11] independent of RHOA at serine 188 and mDia1 at serine 22, resulting in their GDP±GTP cycling [12]. deactivation and cell apoptosis. Conclusion: These results mDia1 is a member of the diaphanous-related formin pointed to an assumed role of DOX to dysregulation of actin family of Rho effector proteins [13]. Unlike the deactivation cytoskeleton and cell death. mechanism of RhoA, the auto-inhibition of mDia1 through its phosphorylation at serine-22 has still not been fully Keywords: Transforming RhoA protein, Diaphanous elucidated. homolog 1 protein, actin cytoskeleton, stress fibre RHOA and mDia1 deactivated and auto-inhibited proteins in this order collapse the actin stress fibbers [11, 14]. The actin stress fibber disassembly is associated *Corresponding authors: Peter Bober, Department of Medical and with the effect of high dose of DOX, resulting in cellular Clinical Biophysics, Faculty of Medicine, University of P.J. Šafárik in apoptosis [15]. Our results confirmed that the high dose of Košice, Trieda SNP1, 04011 Košice, Slovakia, DOX (1µM) during a 24 h treatment had an impact on the cell E-mail: [email protected] viability and proteins deactivation via phosphorylation of Michal Alexovič, Zuzana Tomková, Ján Sabo, Department of Medical and Clinical Biophysics, Faculty of Medicine, University of P.J. Šafárik mDia1 and RHOA on Ser-188 and Ser-22 in this order. in Košice, Trieda SNP1, 04011 Košice, Slovakia Róbert Kilík, 1st Department of Surgery, Faculty of Medicine, Universi- ty of P.J. Šafárik in Košice, Trieda SNP1, 04011 Košice, Slovakia Open Access. © 2019 Peter Bober et al. published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 Public License. 620 P. Bober, et al. 2 Materials and methods spectrophotometer (Shimadzu, Japan) determined protein concentration. Then a 1.1 % of 0.1 M DTT (100 mMTris/HCl, pH 8) solution was added to the dissolved proteins and 2.1 Cell culture incubated at the light at 37°C for 30 min. Subsequently, the 0.1 % of 0.5 M IAA (100 mMTris/HCl, pH 8) solution Biological triplicates of MCF-7/WT and MCF-7/DOX-1 (ATCC was added and incubated in the dark at 37°C for 30 min. HTB-22, USA) were cultured during 24 h. in a 75 cm2 culture Then, the glacial acetone (-20°C) was added and left for flask (Becton, Dickinson and Company, USA). The 10 mL 60 min in a freezer at -20°C and centrifuged at 4000g for of culture medium consisted of Dulbecco’s Modified Eagle 50 min. The pellet was subsequently dried and dissolved Medium (DMEM, 4.5 g L-1 Glucose, Lonza-BioWhittaker, in 8 M urea (100 mMTris/HCl, pH=8). After that, 2 mM of Belgium) and F-12 HAM medium (Sigma, USA), prepared calcium chloride (10 mMTris/HCl, pH=8) and trypsin in in a 1:1 (v/v) ratio. This mixture was supplemented using ratio 1:100 (w/w) ratio were added and incubated at 37°C 5% FBS (GIBCO, USA) and 1% Sodium Pyruvate 100 mM for 12 h. trifluoroacetic acid (TFA) 20% was finally used (GIBCO, USA). to stop digestion of proteins. The final concentration of proteins in solution was 1 μg μL-1. Peptides were ultimately desalted by solid phase extraction using C matrix spin 2.2 xCELLigence cell proliferation assay 18 cartridges (Agilent Technologies, USA). In the first step, the 100 µL of culture medium was pipetted into a 16-well E-plate. The plate containing medium was 2.5 Off-gel fractionation inserted in the measuring device, i.e. xCELLigence system (ACEA Biosciences, USA). Subsequently, 2x104 MCF-7 The 1 mg of solution containing peptides was inserted to an cells were pipetted into the wells. Once the cells reached Agilent 3100 OFFGEL Fractionator (Agilent Technologies, one half log phase (i.e., after 21 h and 35 min), DOX was USA) for electromigration-based fractionation. Peptides added in the range 0.001-1 µM up to a total volume of 250 were resuspended in a 5% glycerol and a 1% ampholyte µL. Using the Real Time Cell Analysis (RTCA) software the (GE Healthcare Life Sciences, USA), applied to immobilized half maximal inhibitory concentration (IC ) values were 50 pH gradient (3–10) by using 12 cm strips (GE Healthcare determined during a 24 h treatment. Life Sciences, USA) and focused at default conditions with 24 hour runtimes. 2.3 Flow cytometry 2.6 LC-MS/MS analysis The viability of MCF-7/WT and MCF-7/DOX-1 cells (biological duplicates) at a density of 8x104 cells/well were measured via MCF-7/WT and MCF-7/DOX-1 samples were subjected to the Muse Cell Analyzer (Millipore, Hayward, CA, USA) using a nano-liquid HPLC system (Ultimate 3000 RSLC Nano, the Muse™ Count and Viability Kit (Millipore, Hayward, CA, Thermo Fisher Scientific, USA) on-line coupled with an USA) after 24 h. Total volume of the cells was 1 mL. amaZon speed ETD ion trap mass spectrometer (Bruker Daltonik, Germany). The CaptiveSpray (Bruker Daltonik, Germany) was used as the ion source. The procedure 2.4 Proteins extraction and in-solution was started with a 1 μL sample injection. Consequently, digestion peptides (the 1 µg solution) were loaded to an Acclaim® PepMap 100 C trap column (5 μm particles, 100 Å, 300 MCF-7/WT and MCF-7/DOX-1 cells (biological triplicates) 18 μmi.d. × 2 cm) via a mobile phase (MF, 98% water, 2% were lysed by an 8 M urea (100 mMTris/HCl, pH 8) solution. ACN containing 0.1% formic acid - FA) at 8 μL min-1 flow- Lysed cells were centrifuged for 15 min at 12000g and 4°C. rate. Then the peptides were washed out from a trapping Glacial acetone (-20°C) in a ratio of 1:5 (v/v) was added to column to an Acclaim® PepMap RSLC analytical column the supernatant. Afterwards the solution was vortexed, C (2 μm particles, 100 Å, 75 μmi.d. × 15 cm) and mass stored at -20°C (60 min) and centrifuged. The protein 18 spectrometer by MF A (98% water, 2% ACN and 0.1% pellets were ultimately dried using a Vacuum concentrator FA) and MF B (95% ACN, 5% water containing 0.1% FA). (Labconco, USA) and dissolved in 8 M urea (100 mMTris/ The gradient elution starting from 96%:4% to 65%:35% HCl, pH 8). The Bradford method and UV-Vis 3600 (MFA: MFB) was reached in a 100 min run at 0.4 μL min-1 RHOA and mDia1 promotes apoptosis of breast cancer cells via a high dose of doxorubicin treatment 621 flow-rate. For MS spectra, the acquisition parameters Software Inc.) was used. The PyTMs designed to facilitate were: positive ionization mode, enhanced resolution the introduction of post-translational modifications into mode, Ion charge control (ICC). The target was set up existing 3D-structure models was implemented as a to 400 000 compounds, maximum accumulation time plugin for the PyMOL Molecular Graphics System (Version was set up to 50 ms and scan range was 300-1300 m/z. 2.0, Schrödinger, LLC, http://www.pymol.org/). For MS/MS spectra, acquisition parameters were used: Xtreme resolution mode, ICC target was put up to 500 000 compounds, maximum accumulation time was put up to 2.10 Statistical analysis 100 ms and isolation width was 2.2 m/z.