Tyrosine kinases are important mediators of cellular functions such as proliferation, differentiation, metabolism, and survival. As tyrosine kinases are frequently deregulated in e.g. inflammatory diseases and cancer they are among the most attractive drug targets [1
]. Spleen tyrosine kinase (SYK) is a 72 kDa non-receptor tyrosine kinase consisting of two SRC homology 2 (SH2) domains and a kinase domain joined by two linker regions, interdomains A and B [4
]. A shorter SYK splice variant lacking 23 amino acids in the linker region B (SYK(S) or SYK B) has also been described in various cell types [5
SYK is a multifunctional protein. It mediates inflammatory responses by linking immune cell receptors to various intracellular signaling networks and exhibits, for example, a pivotal role in B-cell development [4
]. Among its various functions, SYK promotes, in concert with PKCδ, the expression of anti-apoptotic Mcl-1 in B-cell chronic lymphocytic leukemia (CLL) [10
] and regulates actin filament assembly and dynamics through phosphorylation of cortactin and cofilin in ovarian cancer, thereby promoting migration and invasion [11
]. Furthermore, phosphorylated SYK has been observed in specific cell types and areas of the developing nervous system and diverse functions have been described [12
Widely expressed in hematopoietic cells [15
], SYK is a promising therapeutic target in inflammatory diseases (including rheumatoid arthritis, allergies, systemic lupus erythematosus, and chronic immune thrombocytopenia) [17
] as well as in different hematological malignancies such as CLL [10
], non-Hodgkin lymphoma [22
], and acute myeloid leukemia (AML) [23
However, SYK expression is not restricted to hematopoietic cells and its presence has been described, among others, in epithelial, endothelial, and neuronal cells as well as in different solid tumors [24
]. The role of SYK in the tumorigenesis of both hematological and solid malignancies is highly complex, tumor-specific and cell type-dependent, as both tumor promoting and suppressing functions have been described [25
A tumor-suppressing role for SYK has been demonstrated in e.g. breast cancer [26
], pancreatic cancer [27
], melanoma [28
], hepatocellular carcinoma [29
] as well as childhood pro-B-ALL [30
]. In contrast, SYK has been shown to be pro-tumorigenic in prostate cancer [31
], small-cell lung cancer [32
], ovarian cancer [11
], glioma [34
], pediatric retinoblastoma [35
], and Ewing sarcoma [36
Of note, distinct functions of the two SYK splice variants have also been demonstrated. While the longer SYK variant suppressed breast cancer invasiveness, the shorter variant SYK B did not [37
Neuroblastoma is a malignancy of early childhood with 90% of patients diagnosed below the age of 10 [38
]. Worldwide, neuroblastoma accounts for 12.5% of cancer cases in children of age 0 to 4 years [39
]. Arising from the developing sympathetic nervous system, the majority of primary tumors occur in the adrenal gland [38
]. About 50% of neuroblastomas are classified as low- and very-low-risk with a very good prognosis and receive minimum therapy [38
]. In contrast, the treatment of high-risk neuroblastomas remains a challenge. Despite intensive therapy, about 50% of patients are refractory to first-line treatment or relapse within two years [40
With the increasing understanding of neuroblastoma biology and the identification of druggable protein kinase targets such as ALK and Aurora A kinase as well as the MAPK and PI3K/mTOR/Akt signaling pathways targeted therapies, both alone and in combination with conventional drugs, provide new promising treatment options [41
]. The aim of the present study was to investigate the expression of SYK in neuroblastoma tumor tissues as well as neuroblastoma cell lines and to evaluate its use as a potential therapeutic target.
There is a need for further advancements in the treatment of neuroblastoma to improve the survival of high-risk patients and reduce acute and long-term toxic effects in neuroblastoma survivors. Targeted therapies exhibit great potential used either alone or more particularly in combination with conventional drugs [41
In two other pediatric cancers, retinoblastoma and Ewing sarcoma, SYK promotes tumor cell survival and SYK inhibition, using small molecule inhibitors, was identified as a promising treatment option for these diseases [35
In the present study, we observed that SYK expression was higher in four different neuroblastoma cohorts compared to neural crest cells and benign neurofibroma. Moreover, we demonstrate that SYK is present in neuroblastoma tissues and to a lesser extent in neuroblastoma cell lines. Inhibition of SYK using small molecule inhibitors alone or in combination with chemotherapeutic drugs as well as knockdown of SYK by siRNA impaired the cell viability of SYK expressing neuroblastoma cells. Additionally, SYK inhibition decreased phosphorylation of Akt and ERK1/2 indicating Akt and MAPK signaling as potential downstream targets of SYK in neuroblastoma. Furthermore, constitutive active SYK increased neuroblastoma cell viability independent of endogenous SYK expression. Taken together, our findings indicate a tumorigenic involvement of SYK in neuroblastoma.
We observed the presence of SYK protein in the majority of neuroblastoma tissues analyzed in this study. A positive staining of SYK and p-SYK was observed in both the cytoplasm and the nucleus with a more pronounced nuclear staining for p-SYK. The presence of SYK in different cellular compartments (cytoplasm, nucleus, membrane) has previously been described [37
Furthermore, we compared the presence of SYK protein in MYCN
-amplified and non-MYCN
-amplified tumor tissue and did not observe differences between the two groups. Of note, by examining publicly available gene expression datasets, we detected a negative correlation between MYCN
in neuroblastoma. In contrast, a positive correlation between SYK
was discerned. MYCN
-amplification occurs in about 20% of neuroblastomas and is associated with aggressive tumors and poor survival [47
]. Furthermore, MYC
has also been identified as an independent prognostic marker for poor survival in neuroblastoma [50
] and is predominantly expressed by non-MYCN
-amplified tumors [51
]. A link between SYK and MYC has been previously demonstrated in Ewing sarcoma and hematopoietic cells [36
]. Therefore, a potential connection between SYK and MYC in neuroblastoma is highly interesting and warrants further investigation.
In contrast to neuroblastoma tumor tissue, SYK protein was only detected in two out of ten neuroblastoma cell lines by western blot. This is in accordance with previous findings by Alaminos et al. reporting more frequent methylation of the SYK
promoter in neuroblastoma cell lines (60%) compared to tumor tissue (11%) [53
]. In subsequent work by Margetts et al. and Grau et al. no aberrant hypermethylation of the SYK
promoter was observed in tumor tissue or highly infiltrated bone marrow [54
]. Yu et al. recently demonstrated that EGF stimulates SYK-mediated migration and invasion in ovarian cancer cells. The authors suggested that SYK function might be regulated by environmental stimuli [11
]. Therefore, one could speculate that the absence of a specific stimulus, which is present in the tumor microenvironment, could lead to the downregulation of SYK
expression in neuroblastoma cells lines. However, additional studies are necessary to determine if known factors, such as specific cytokines and chemokines, present in the neuroblastoma tumor microenvironment may affect SYK expression and function.
Complex phosphorylation events on tyrosine residues are required for the regulation of SYK functions by mediating conformational changes and creating docking stations for other proteins [8
]. Phosphorylation of Tyr352 and/or Tyr348 (Tyr346 and Tyr342 in mouse SYK) provides binding sites for various proteins such as phospholipase Cγ, Vav-1 and 2 as well as Akt, and ERK, linking SYK to different signaling pathways and cellular functions [8
]. Furthermore, Tyr352 has been linked to constitutive SYK activation [63
]. The Tyr525/526 residues are located in the SYK activation loop. These phosphorylation sites likely provide important docking sites for other proteins thereby mediating intracellular signaling. However, mutations in these sites affect the in vitro catalytic activity only marginally [8
]. Phosphorylation of Tyr323 in SYK promotes binding of Cbl protein family members causing ubiquitination and possibly degradation of SYK. However, Tyr323 is also an important binding site for PI3K indicating multiple functions of this phosphorylation site (reviewed in [8
We determined the status of these three well-established SYK phosphorylation sites (Tyr352, Tyr525/526, and Tyr323) and found that all were phosphorylated under normal growth conditions in the SYK expressing SH-SY5Y neuroblastoma cells, suggesting the presence of catalytically active SYK.
We observed a weaker nuclear staining of both total SYK and p-SYK in neuroblastoma cell lines as compared to neuroblastoma tissue. It has previously been demonstrated that SYK splice variants display differences in cellular localization and function [37
] and that EGF can modulate SYK splicing pattern [66
]. These findings indicate that the cellular localization of SYK may be affected by environmental stimuli and changes in splicing pattern. Therefore, the presence of specific stimuli in the neuroblastoma tumor microenvironment as well potential differences/changes in SYK splicing pattern compared to neuroblastoma cell lines may contribute to the observed differences in staining pattern. In our study, we observed that siRNA-mediated SYK downregulation reduced neuroblastoma cell growth. However, we did not achieve a complete SYK knockdown. Since SYK is a protein kinase, residual SYK protein could provide an explanation for the significant, but modest effect on the cell viability.
Therefore, we investigated the effects of commercially available SYK inhibitors BAY 61-3606 [68
], R406 [69
], PRT062607 (P505-15) [70
] and GS-9973 [71
] on neuroblastoma survival. Using increasing inhibitor concentrations, we compared the effect on cell viability in neuroblastoma cells with and without detectable SYK protein levels (SH-SY5Y and SK-N-BE(2), respectively). We determined at least one concentration for each inhibitor at which a significant reduction in cell viability was observed in SH-SY5Y but not SK-N-BE(2) cells, indicating an effect on cell viability that can likely be attributed to specific SYK inhibition.
PRT062607 and GS-9973 significantly reduced the viability of SH-SY5Y but not SK-N-BE(2) cells at a concentration of 1 μM. This is in line with recent work by Sun et al. where 1 μM PRT062607 and GS-9973 significantly impaired clonogenicity and cell viability of Ewing sarcoma cell lines [36
]. A dose-dependent impairment of cell viability as well as increased caspase-3 activity has been previously reported in retinoblastoma cells after treatment with BAY 61-3606 and R406 [35
]. We observed that these inhibitors significantly decreased SH-SY5Y cell viability as compared to SK-N-BE(2) cells at concentrations of 0.1–0.6 μM and 0.8–1 μM, respectively.
Of note, inhibitor concentrations >1 μM also significantly reduced the cell viability of SK-N-BE(2) cells that exhibit no apparent SYK expression suggesting off-target effects for all four inhibitors when used at higher concentrations. Kinase inhibitors commonly display off-target effects that can be beneficial but need to be carefully evaluated at the mechanistic level. For example, the SYK inhibitor BAY 61-3606 has been reported to inhibit JNK [72
]. JNK inhibition has previously been demonstrated to reduce the apoptotic effect of chemotherapeutic drugs such as doxorubicin in SH-SY5Y cells [73
]. Therefore, off-target effects of BAY 61-3606 on JNK could potentially impair doxorubicin function and not display a potentiating effect as seen in our studies. Furthermore, work by Colado et al. demonstrated that GS-9973 and R406 can impair T-cell function via off-target effects on the SYK homolog ZAP-70 [74
]. We screened the neuroblastoma cell lines used in this study for ZAP-70 presence and found it expressed at protein level only in SK-N-DZ cells (Supplementary Figure S6
). Since this cell line was not used in the inhibitor studies, potential off-target effects on ZAP-70 can be excluded. However, these are just two of many potential proteins that may be the target of non-selective inhibition.
Various downstream targets for SYK have been described in health and disease [4
]. ERK- and Akt-mediated signaling is known to be affected by SYK inhibition in CLL and Ewing sarcoma [20
]. We observed that Akt and ERK1/2 phosphorylation was decreased by the SYK inhibitors BAY 61-3606, R406 and PRT062607, but not GS-9973. A possible explanation could be differences in the kinetics of GS-9973 mediated SYK inhibition. Since we only examined two time points, (4 h and 24 h) rapid and transient effects might not have been detected. PI3K/Akt- and MAPK-mediated signaling was previously shown to promote neuroblastoma tumorigenesis [75
]. Therefore, a decrease in Akt and ERK1/2 phosphorylation and activity could contribute to the impaired cell viability.
Yu et al. demonstrated an increased expression of SYK in paclitaxel-resistant ovarian cancer cells and that paclitaxel in combination with the SYK inhibitor R406 increased apoptosis in vitro and impaired tumor growth in vivo [33
]. Paclitaxel is a chemotherapeutic drug that is rarely used for the treatment of neuroblastoma. It was, however, included in this study to determine if the additive effect seen in ovarian cancer cells [33
] could also be observed in neuroblastoma cell lines. We furthermore analyzed the effect of three drugs used in first-line treatment and/or refractory and relapsed neuroblastoma: cisplatin, doxorubicin, and temozolomide.
We compared the cell viability of neuroblastoma cells treated with cytostatic drugs alone or in combination with the pharmacological SYK inhibitor BAY 61-3606. Synergetic and additive effects were observed in SYK expressing SH-SY5Y cells for paclitaxel- BAY 61-3606 and cisplatin- BAY 61-3606 combination, respectively. Furthermore, 0.4 μM BAY 61-3606 potentiated the effects of doxorubicin and temozolomide in SH-SY5Y cells. Interestingly, a synergetic effect of BAY 61-3606 in combination with paclitaxel was also observed in SK-N-BE(2), a cell line without significant expression of SYK protein. Although BAY 61-3606 concentrations applied in this study are rather low, off-target effects are likely the cause for the observed effect.
Furthermore, we observed increased amounts of cleaved PARP in the SYK-positive SH-SY5Y cells after treatment with BAY 61-3606 (0.4 and 0.8 μM) in combination with the tested drugs after 24 h, particularly when 0.8 μM BAY 61-3606 was used. We propose that the decrease in cell viability in SH-SY5Y cells may be attributed to an increase in apoptosis, whereas the decrease in cell viability in SYK-negative SK-N-BE(2) cells might rather be due to reduced proliferation than increased apoptosis. However, further experiments are required to determine the detailed mechanisms.
In addition, we also demonstrate that transient transfection with a constitutively active SYK variant increased the cell viability of neuroblastoma cell lines independent of endogenous SYK expression. This suggests that SYK has tumor-promoting functions in neuroblastoma. Previous work reported a tumor-suppressing role for SYK in breast cancer, among others. Transfection of breast cancer cells with a wild-type SYK encoding vector suppressed invasive outgrowth in Matrigel and impaired tumor growth and metastasis in mice [26
]. We did, however, not observe any inhibitory effect on the cell viability of transfected neuroblastoma cell lines expressing exogenous SYK. Taken together, this suggests that SYK functions as a tumor-promoting molecule in neuroblastoma rather than having a tumor-suppressing effect.
To further evaluate the potential therapeutic use of SYK inhibitors in neuroblastoma both as a single agent and in combination with existing chemotherapeutic drugs, in vivo studies are necessary. Since SYK is widely expressed by hematopoietic cells, potential negative effects on the immune cells of the tumor microenvironment have to be carefully evaluated using immunocompetent neuroblastoma animal models. Recent work in glioma demonstrated that SYK inhibition impaired the mobility and infiltration of B cells and CD11b+ leukocytes in addition to reducing proliferation and migration of tumor cells [34
4. Materials and Methods
4.1. Microarray Gene Expression
Gene expression analysis was performed using the MegaSampler feature of the publicly available R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl
4.2. Reagents and Antibodies
The selective SYK inhibitors GS-9973 (Entospletinib) and R406 were purchased from Selleck Chemicals Europe (Munich, Germany). BAY 61-3606 and PRT062607 were obtained from Calbiochem/Merck (Merck Life Science AS, Oslo, Norway) and ApexBio (Houston, TX, USA), respectively. Paclitaxel, cisplatin, doxorubicin, and temozolomide were bought from Sigma-Aldrich Norway AS (Oslo, Norway). The antibodies used in this study are listed in Table 3
4.3. Human Tissue Samples and Cell Lines
Human tissue samples were obtained, with informed consent (written or verbal) provided by the parents or guardians for the use of tumor samples in research, in accordance with the ethical approval from the Stockholm Regional Ethical Review Board and the Karolinska University Hospital Research Ethics Committee (approval ID 2009/1369-31/1 and 03-736). Neuroblastoma tumor tissue was collected at the Karolinska University Hospital, snap-frozen in liquid nitrogen and stored at −80 °C until further use.
The human cell lines SK-N-AS, SK-N-SH, SK-N-DZ, SK-N-FI, SH-EP1, Kelly, SH-SY5Y, and IMR-32 as well as THP-1, Jurkat E6.1, and U937 cells were obtained from the ATCC (American Type Culture Collection, LGC Standards GmbH, Wesel, Germany). SK-N-BE(2) cells were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). The cell lines were cultivated in RPMI-1640 medium containing L-glutamine and sodium bicarbonate (Sigma-Aldrich Norway AS, Oslo, Norway) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA, USA). LAN-6 cells were a kind gift from Deborah Tweddle, Newcastle University and were grown in Iscove’s Modified Dulbecco’s Medium (Sigma-Aldrich Norway AS, Oslo, Norway) supplemented with GlutaMAX™ and 10% heat-inactivated FBS. All cell lines were cultivated at 37 °C in humidified air with 5% CO2 and mycoplasma tests were performed regularly using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland). The identity of the human neuroblastoma cell lines was confirmed by STR-profiling performed at the Centre of Forensic Genetics, University of Tromsø, Norway.
4.4. Immunohistochemistry (IHC)
Formalin-fixed, paraffin-embedded tissue sections were deparaffinized using xylene (VWR International, Oslo, Norway) and a series of graded alcohols (Sigma-Aldrich Norway AS, Oslo, Norway) followed by rehydration and washing in phosphate buffered saline (PBS, Biochrom GmbH, Berlin, Germany). Antigen retrieval was performed in sodium citrate buffer (pH 6) in a microwave oven. After blocking of endogenous peroxidase with 0.3% H2O2 for 15 min, unspecific antibody binding sites were blocked with 5% BSA in PBS (AppliChem, Darmstadt, Germany) for 45 min. The sections were incubated with the primary antibody overnight at 4 °C. The following day sections were thoroughly washed in PBS and incubated with SignalStain® Boost IHC Detection Reagent, HRP, Rabbit (Cell Signaling Technology, Leiden, Netherlands) and kept for 1 h at room temperature. Following washes in PBS, the sections were incubated with Liquid DAB+ Substrate solution (Dako, Agilent Technologies, Inc., Santa Clara, CA, USA). A matched isotype control was used as a control for nonspecific staining. The sections were examined with a BX43 microscope (Olympus, Tokyo, Japan) and images were acquired with an Olympus DP26 camera.
4.5. RNA Isolation and RT-PCR
Total RNA was isolated using the NucleoSpin® TriPrep Kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) and RNA quantity and quality was determined with a NanoDrop™ 2000 spectrometer (Thermo Fisher Scientific Inc.). One μg RNA was used as input for cDNA synthesis with the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories AB, Oslo, Norway).
PCR was set up as a 25 μl reaction mix containing 2 μL cDNA, 12.5 μL AccuStart™ II GelTrack PCR SuperMix (Quanta Biosciences, Gaithersburg, MD, USA), 400 nM of each primer (Sigma-Aldrich Norway AS, Oslo, Norway) and 10.1 μL of ultra-pure H2
O (Biochrom GmbH, Berlin, Germany). The PCR run was performed in a T100™ Thermal Cycler (Bio-Rad Laboratories AB) as follows: 2 min at 94 °C and 35 cycles of 94 °C for 20 s, 61 °C for 30 s and 72 °C for 90 s. The following intron spanning primer sets were used: APRT (housekeeping) 5′ CCCGAGGCTTCCTCTTTGGC 3′ (sense) and 5′ CTCCCTGCCCTTAAGCGAGG-3′ (antisense) [80
], SYK 5′ CATGTCAAGGATAAGAACATCAT AGA 3′ (sense) and 5′ AGTTCACCACGTCATAGTAGTAATT 3′ (antisense) [26
], SYK L/S 5′ TTTTGGAGGCCGTCCACAAC ‘3 (sense) and 5′ ATGGGTAGGGCTTCTCTCTG 3′ (antisense) [37
All primer sets used in this study were intron-spanning to avoid false positive signals caused by amplification of residual traces of genomic DNA. PCR products were analyzed by gel electrophoresis. The 2% SeaKem® LE Agarose gel (Lonza) was stained with GelRed™ (Biotium, Inc., Hayward, CA, USA) and visualized under UV light in the BioDoc-It™ Imaging System (UVP, LLC, Upland, CA, USA).
4.6. Immunocytochemistry (ICC) and Western Blot
Cells were grown in 8-well µ-Slide dishes (iBidi GmbH, Munich, Germany) until they reached approximately 70% confluence. Following a brief rinse with PBS the cells were fixed with 4% formaldehyde (Alfa Aesar, VWR International, Oslo, Norway) for 20 min. After three washes with PBS, unspecific binding sites were blocked with 5% goat serum (Sigma-Aldrich Norway AS) in PBS containing 0.3% Triton-X-100 (Sigma-Aldrich Norway AS) for 1 h. The cells were incubated with primary antibodies diluted in 1% bovine serum albumin (BSA; AppliChem, Darmstadt, Germany) in PBS containing 0.3% Triton-X-100 overnight at 4 °C. After three washes with PBS, the cells were incubated with the secondary antibodies diluted in 1% bovine serum albumin in PBS containing 0.3% Triton-X-100 for 1 h at room temperature, protected from light. Following three washes with PBS, the nuclei were stained with Hoechst 33342 (ImmunoChemistry Technologies, LLC, Bloomington, IN, USA) for 10 min. The cells were washed 3x with PBS and covered with Mounting Medium for fluorescence microscopy (iBidi GmbH). Subsequently, the cells were examined using a Zeiss LSM780 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). Images were taken with the same microscope settings (laser intensity, gain etc.) and identical image processing parameters were applied to allow comparison between the cell lines. Western blots were performed as previously described [81
4.7. Immunoprecipitation (IP)
Cells were washed twice with cold PBS and lysed by addition of lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Triton X-100, 1 mM DTT and 1 mM EDTA (Sigma-Aldrich Norway AS, Oslo, Norway) as well Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific Inc.). Following sonication and centrifugation, the protein concentration was determined using a Protein Quantification Assay (MACHEREY-NAGEL GmbH & Co. KG). A sample of the supernatant was taken as “input control”, supplemented with NuPAGE® LDS Sample Buffer (4X) (Thermo Fisher Scientific Inc.) as well as 100 mM DTT (AppliChem, Darmstadt, Germany) and incubated for 10 min at 70 °C. Cell lysate containing approximately 800 μg protein was pre-cleared with an irrelevant IgG2a antibody (same class as the SYK antibody used for IP) to reduce unspecific binding. Afterwards, the samples were incubated with the anti-SYK antibody (1 μg) rotating at 4 °C overnight followed by incubation with 80 μl of a 50% Sepharose G beads/lysis buffer solution (GE Healthcare, Oslo, Norway) for 1 h rotating at 4 °C. The beads were washed three times with lysis buffer and two times with 50 mM Tris-HCl, pH 8.0. Afterwards, the beads were incubated in sample buffer containing NuPAGE® LDS Sample Buffer (4×), ultrapure H2O as well as 100 mM DTT for 10 min at 70 °C. The samples were subsequently used for western blot analysis.
4.8. siRNA-Mediated SYK Silencing
Two pre-designed SYK siRNAs were used in this study: ID-s13679 (siRNA 1) and ID-s13681 (siRNA 2) as well as a scramble control #1 siRNA (cat# 4392420 and 4390843, Ambion, Thermo Fisher Scientific Inc.). Cells were seeded into 24- and 6-well plates for cell viability assay and western blot analysis, respectively. After 24 h, the cells were transfected with the different siRNAs using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific Inc.) according to the manufacturer’s specifications. Briefly, cells were incubated with 5 pmol (24-well plate) or 30 pmol siRNA (6-well plate) per well in Opti-MEM (Thermo Fisher Scientific Inc.) for 4 h followed by the removal of the transfection mix and addition of fresh Opti-MEM. After 72 h the cell viability was assessed and cells were harvested for western blot analysis. Densitometry was performed using Fiji software [82
4.9. Cell Viability Assay
To assess the effect of the commercially available SYK inhibitors BAY 61-3606, R406, GS-9973 (Entospletinib) and PRT062607 alone as well as BAY61-3606 in combination with paclitaxel, cisplatin, doxorubicin and temozolomide on the cell viability of SH-SY5Y and SK-N-BE(2) neuroblastoma cells the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazodium bromide)-assay was used [83
]. The cells were seeded in 96-well plates in full growth media. As an exception, cells treated with GS-9973 and PRT062607 were seeded in Opti-MEM to reduce cell viability variations attributed to residual serum. After 24 h, the cells were washed once with Opti-MEM before incubation with SYK inhibitors alone for 24 and 48 h or a combination of chemotherapeutic drugs with BAY61-3606 for 48 and 72 h. Control cells received the corresponding drug vehicle at the highest concentration present in the drug-treated cells. BAY 61-3606 and doxorubicin were dissolved in water, R406, GS-9973, PRT062607, and temozolomide in DMSO, paclitaxel in ethanol, and cisplatin in 0.9% saline. After 24, 48 or 72 h the MTT solution (10 μL of 5 mg MTT, Sigma-Aldrich Norway AS, per ml phosphate buffered saline) was added to each well and incubated for additional 3 h. To facilitate formazan crystal solubilizing, 70 μL of the solution were carefully removed from each well, 100 μL isopropanol containing 0.04 M HCl were added and mixed thoroughly. In addition, the plates were placed on an orbital shaker for 1 h at room temperature. The absorbance was measured with a CLARIOstar plate reader (BMG LABTECH, Ortenberg, Germany) at 590 nm. The experiment was performed at least three times with at least three parallels per treatment. The cell viability was calculated as the ratio of the mean OD of treated cells over vehicle treated control cells (100% living cells). The cell viability assay for the siRNA and SYK plasmid studies were performed in 24-well plates. The amounts of MTT solution and acidic isopropanol were adjusted correspondingly.
4.10. Cell Signaling Study
To investigate the effect of commercially available SYK inhibitors on MAPK- and Akt- mediated signaling SH-SY5Y cells were seeded in 6-well plates in full growth medium. The next day, the cells were washed in Opti-MEM and treated with BAY 61-3606, R406, GS-9973 (entospletinib), PRT062607 or corresponding vehicle controls (water for BAY 61-3606 and DMSO for R406, GS-9973, and PRT062607) for 4 or 24 h. Following incubation, the cells were washed with PBS and harvested in RIPA Lysis and Extraction Buffer containing Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific Inc.) and analyzed by western blot. Densitometry was performed using Fiji software. Phosphorylated and total protein were normalized to their respective GAPDH loading control (pERK/GAPDH, ERK/GAPDH, pAkt/GAPDH, Akt/GAPDH). Ratios of pERK/ERK and pAkt/Akt were calculated using the normalized values. The respective vehicle control was set as 1 and the ratios were calculated.
4.11. Transfection with SYK Plasmids
The following, previously described FLAG-tagged plasmids were used in this study: pCAF1 empty expression vector, SYK wt = SYK wild type, SYK B = short SYK splice variant B, SYK Y130E = constitutive active SYK, SYK K402R = kinase dead SYK, SYK RR42/195KK = SYK with inactive SH2 domains [26
]. Cells were seeded in 24- (cell viability assay) or 6-well plates (western blot) in full growth media. The following day the cells were transfected using jetPRIME® transfection reagent (Polyplus-transfection®, Illkirch, France) according to the provided manual (1 μg DNA per well in a 6-well plate and 0.25 μg DNA per well in a 24-well plate). After 5 h, the media containing the transfection mix was removed and fresh media was added. After 48 h, cell viability was determined and cells were harvested for western blot.
4.12. Drug Combination Analysis
To determine whether the chemotherapeutic drug-SYK inhibitor combinations displayed a synergistic or additive effect, highest single agent, response additivity and Bliss independence calculations were performed on the cell viability data as described in [84
]. Highest single agent method proves the superiority of the drug combination compared to its single agents and was assessed with statistical testing (two-way ANOVA). Response additivity and the Bliss Independence model both compares the observed drug combination effect to the expected additive effect and thereby calculates a combination index. For Response additivity the expected additive effect is calculated as following: E(A) + E(B) and for the Bliss Independence: E(A) + E(B) − E(A)*E(B) where E is the effect produced by drug A and B. A combination index <0.9 was defined as synergistic, 0.9–1.1 as additive and >1.1 as antagonistic.
4.13. Statistical Analysis
GraphPad Prism software (versions 7 and 8, GraphPad Inc., San Diego, CA, USA) was used for statistical analysis and graph design. Fisher’s exact test was used to test the statistical significance of the association between two categories. Two-way ANOVAs and Dunnett or Bonferroni post-tests were applied to assess two independent variables (differences between cell lines and the effect of treatment). Two-tailed one sample t-tests were used for the statistical analysis of the cell signaling studies.