Extended Abstract

©2015 Dustri-Verlag Dr. K. Feistle ISSN 0946-1965

DOI 10.5414/CPXCES14EA07e-pub: September 23, 2015

Correspondence to Prof. Dr. Christoph A. Ritter Ernst-Moritz-Arndt-Universität Greifswald, Institut für Pharmazie, Klinische Pharmazie, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifs-wald, Gemany ritter@uni-greifswald.de

Key wordsbreast cancer – Brk – small molecule inhibitors – loss of adherence – cell death

Novel 4-anilino-α-carboline derivatives induce cell death in nonadhesive breast cancer cells through inhibition of Brk activityMarkus Oelze1, Kazem Ahmed Mahmoud2, Wolfgang Sippl2, Tom Wersig2, Andreas Hilgeroth2, and Christoph A. Ritter1

1Institute of Pharmacy, Clinical Pharmacy, Ernst-Moritz-Arndt-University of Greifswald, Greifswald, and 2Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry, Martin-Luther University Halle-Wittenberg, Halle, Germany

Introduction

The cytoplasmatic nonreceptor protein ty-rosine kinase Brk (breast tumor kinase, PTK6) is overexpressed in several epithelial tumors, particularly in breast cancer [1]. In vitro ex-periments in nontumor and breast cancer cell lines revealed that overexpression promotes cell proliferation and cellular transformation by sensitizing cells for stimulation of the epi-dermal growth factor receptor (ErbB) family [2, 3]. Moreover, Brk appeared to promote a migratory and invasive phenotype in breast cancer cell lines [4]. In some of the investi-gated cell models, effects of Brk, such as ac-tivation of Signal transducer and activator of transcription 3 (STAT3), which is associated with stimulated cellular proliferation [5], rely on the kinase activity [6]. On the other hand, scaffolding characteristics of the protein seem to be responsible in other contexts, for instance in promoting hepatocyte growth factor (HGF)-induced migration of breast cancer cells due to kinase-activity independent formation of Brk/ERK5 complexes [7]. In order to further in-vestigate the role of Brk as a therapeutic target and dissect functional cell model- and context-specific characteristics, specific inhibitors of this kinase are necessary. Here, we present ef-fects of the first, newly synthesized, 4-anilino-α-carboline derivatives on Brk signaling and growth of mammary carcinoma cells.

Material and methods

Cell culture conditions

T-47D cells were obtained from DSMZ (Braunschweig, Germany) and cultivated

in RPMI-Medium (PAA, Paching, Austria) with a 10% FCS supplement (Invitrogen, Carlsbad, USA). For suspension growth, cultivation plates were coated with a 5 mg/mL solution of poly(2-hydroxy-ethyl-meth-acrylic acid) (polyHEMA) in 96% ethanol by evaporation in a 37 °C air-tight environ-ment. Cells were plated in the appropriate cellular density and cultured in the medium described above. For incubation experiments with 4-anilino-α-carboline derivative inhibi-tors, stock solutions of MK138 and MK150 [8] were prepared in DMSO in a concentra-tion of 10 mM, and working solutions were diluted using culture medium.

Expression of Brk and related signaling proteins

After cultivation under respective culture conditions, cells were lysed using standard lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich, Taufkirchen, Ger-many), and protein content was assessed by modified Bradford assay (Roti(R)-Nanoquant, Carl Roth GmbH, Karlsruhe, Germany). Proteins were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Specific pro-tein bands were visualized by incubating membranes with primary antibodies against STAT3, pSTAT3 (Cell Signaling Technolo-gies, Danvers, MA, USA, 1 : 500 dilution), GAPDH (Meridian Life Sciences, Memphis, TN, USA, 1 : 3,000 dilution) and a secondary horseradish peroxidase-conjugated antirab-bit IgG antibody (Cell Signaling Technolo-gies, Danvers, MA, USA, 1 : 3,000 dilution).

International Journal of Clinical Pharmacology and Therapeutics, Vol. 53 – No. 12/2015 (1052-1055)

4-anilino-α-carboline derivatives induce cell death in nonadhesive breast cancer cells by inhibition of Brk 1053

Figure 1. Top: 4-Anilino-α-carboline derivatives reduce STAT3 phosphorylation. A: T-47D cells were plat-ed in 6-well plates at 105 cells/well and treated with depicted concentrations of MK138 (upper panel) or MK150 (lower panel). 50 µg of total protein was separated on 10% SDS-polyacrylamide gel, protein was transferred to nitrocellulose membrane, specific bands were visualized by immunostaining with polyclonal specific antibody followed by HRP-conjugated antibody, and chemoluminescence was documented by ChemoCam imaging system. Phosphorylation of STAT3 (pSTAT3) was detected by immunoblotting using total STAT3 loading control. Representative blots are shown. B: T-47D cells were transfected with siRNA targeting Brk (Brk siRNA) or nontargeting (nt) siRNA for negative control. Western blot analysis was per-formed as described in (A) using polyclonal rabbit antibodies against phosphorylated (pSTAT3) and total STAT3. C: Prior to western blot analysis, T-47D cells were treated with 100 nM of the Brk inhibitor MK150 or 0.02% DMSO as solvent control for 24 h. Western blot analysis was performed as described in (A) and (B). Representative blots are shown. In each western blot analysis, GAPDH was detected as loading con-trol. Bottom: Effect of 4-anilino-α-carboline derivatives (MK 138 & MK 150) on adherent and nonadherent T-47D cells. 105 cells were grown in each well of 6-well plates with regular surface (no surface coating) and surface coating with polyHEMA for 24 hours after seeding and were subsequently treated with 100 nM of either MK138 or MK150 or DMSO as solvent control. Cell proliferation and proportion of dead cells in T-47D cell populations were assessed by flow cytometry using the dead cell discrimination assay with prop-idium iodide staining measuring either total cell count (D) or cells gated for propidium iodide (PI) positive nuclei as marker for cell death (E). Bars in the bar graphs shown represent mean+standard error of at least n = 3 replicates.

Oelze, Mahmoud, Sippl, et al. 1054

Chemoluminescence was generated using Immun-StarTM WesternCTM Kit (Bio Rad Laboratories, München, Germany) and de-tected by ChemoCam Imaging System (In-tas, Göttingen, Germany). For Brk knock-down, cells were plated in 6-well plates, left to adhere overnight, and were then transfected with siRNA targeting Brk (ON-Target plus siRNA 9, Human PTK6) or non-targeting siRNA using transfection reagent (DharmaFect(R)TR) (Thermo Scientific, Rockford, IL, USA). Cells were incubated for 24 hours and, after exchange of medium, subsequently cultivated for another 48 hours prior to protein expression analysis.

Determination of cell viability

Cell count and dead cell discrimination analysis was performed using flow cytom-etry. Cells were incubated in trypsin/EDTA solution to yield single-cell suspensions, incubated with propidium iodide solution (Miltenyi, Bergisch Gladbach, Germany) and single-cell fluorescence was assessed us-ing the MACSQuant flow cytometer (Milt-enyi).

Results

Brk phosphorylates STAT3 in T-47D breast cancer cells

To evaluate consequences of Brk inhibi-tion by 4-anilino-α-carboline, phosphoryla-tion of STAT3 was assessed in the presence of increasing concentrations of MK138 and MK150 (Figure 1A). It was observed that 100 nM and 1,000 nM of the inhibitors were able to reduce STAT3 phosphorylation in T-47D cells. In order to confirm that STAT3 is a direct substrate of Brk in T-47D cells, Brk was knocked down using specific siRNA, and phosphorylation of STAT3 was assessed by western blotting using a pSTAT3-specific antibody. siRNA transfection resulted in a reduction of Brk in T-47D cells and a sub-sequent reduction of STAT3 phosphorylation (Figure 1B). A similar effect was observed when T-47D cells were incubated with the 4-anilino-α-carboline derivative MK150 (Figure 1C). These data suggest that STAT3

is phosphorylated in the presence of active Brk in T-47D cells.

Inhibition of Brk induces cell death under loss of adherence conditions

Furthermore, we investigated effects of 4-anilino-α-carboline derivatives on T-47D cells in cell suspension assays compared to adhesive monolayer growth. In general, cellular growth was reduced when cells grew in suspension compared to adherent cell growth (153 ± 37 vs. 287 ± 19 counts/µL cell suspension) (Figure 1D), howev-er, this was not due to increased cell death (Figure 1E). While cell counts were not af-fected by incubating adherently growing T-47D cells in the presence of 4-anilino-α-carboline derivatives, addition of 100 nM MK138 and 100 nM MK150 to the culture medium reduced cell counts in suspension cultures significantly (113 ± 10 and 103 ± 28 counts/µL cell suspension, respectively, vs. 153 ± 37 counts/µL cell suspension in con-trol cultures, mean ± SD, p < 0.05) (Figure 1D). In addition, Brk inhibition by MK138 or MK150 in the state of loss of adherence significantly increased the fraction of cells staining positive with propidium iodide from 6.4 ± 1.8% in DMSO-treated cultures to 10.0 ± 1.1% and 9.7 ± 1.1% in cultures incu-bated with MK136 or MK150, respectively (mean ± SD, p < 0.05) (Figure 1E). These re-sults suggest that Brk plays a role in the abil-ity of cells to grow under loss of adherence conditions, which is partly blocked by selec-tive inhibitors of this protein tyrosine kinase.

Conclusions

We found a concentration-dependent reduction of STAT3 phosphorylation by 4-anilino-α-carboline derivatives. Further-more, when forced to grow in a nonadher-ent environment due to polyHEMA-coating of the growth surface, addition of inhibitors to culture medium resulting in a concentra-tion of 100 nM led to a significant increase in cells staining positive for propidium io-dide. Moreover, the tested inhibitors reduced cell count significantly under loss of adher-ence condition compared to control cultures, whereas no effect was detected for adherent

4-anilino-α-carboline derivatives induce cell death in nonadhesive breast cancer cells by inhibition of Brk 1055

growth of T-47D cells under the described conditions. Furthermore, we portrayed the effect of pharmacological Brk inhibition and its constraining effect on loss-of-adherence growth ability in T-47D breast cancer cells for the first time.

Acknowledgment

This work was supported by the German Research Foundation (Deutsche Forschun-gsgemeinschaft DFG, Grant No. HI 687/10-1, SI 868/10-1, RI 1196/4-1).

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