Abstract
Background/Aim: Neuroblastoma (NB), the most common extracranial malignant childhood tumor accounts for about 15% of cancer-related deaths in children. Despite the intensive treatment of patients with high-risk scarification of NB, clinical outcomes indicate tumor recurrence greater than 50% and late severe adverse effects. Oxazolidinones are 5-membered heterocyclic compounds with antibacterial activity against resistant bacterial strains. Structural modifications around the oxazolidinone moiety have resulted in derivatives with anti-cancer properties against proliferation, motility, and invasion of breast cancer cells. This study aimed to examine the anti-cancer potential of novel oxazolidinones against a model of a neuroblastoma cell line. Materials and Methods: Newly synthesized and characterized triazolyl-oxazolidinone derivatives were incubated with neuroblastoma Kelly cells. The anti-proliferation and anti-progression effects of the compounds were evaluated by MTT, and adhesion with migration assays. Results: The 5-nitrofuroyl glycinyl-oxazolidinone containing 4-methyltriazolyl group demonstrated the most potent activity with an IC50=6.52 μM. Furthermore, the D-isomer of 5-nitrothiophenecarbonyl alaninyl containing derivative reduced the adhesion to fibronectin by 56.34%, while the D-isomer of 5-nitrofuroyl alaninyl derivative reduced the migration of Kelly cells by 29.14%. Conclusion: The presence of the 4-methyltriazolyl moiety seems to enhance the anti-proliferative property of triazolyl-oxazolidinone derivatives, as demonstrated by PH-145. There is little or no effect of the stereochemistry of the alanine side-chain on the antiproliferative effect, as demonstrated by the 5-nitrofuroyl D- and L-alaninyl containing derivatives with similar IC50 values. The observed differences in the inhibition of adhesion and migration by the oxazolidinones on Kelly cells provide a new therapeutic approach that needs further investigation.
- Oxazolidinones
- neuroblastoma
- Kelly cell line
- proliferation
- progression
- BSA heat denaturation
- adhesion
- fibronectin
Neuroblastoma (NB) is the most common extracranial malignant tumor in infants and children. In North America and Europe, the average annual incidence rate of NB is 10.5 per million children between 0 and 14 years old, with a slightly greater incidence in males (male:female ratio 1.2:1.0). NB accounts for approximately 10% of all malignant childhood tumors and it is the cause of up to 15% of cancer-related death in children (1). NB is an extremely heterogeneous disease and many biological, molecular, and genetic factors determine whether the disease will spontaneously regress or metastasize and become resistant to therapy (2). Most NB tumors are detected in the abdomen and are associated with the adrenal medulla or sympathetic ganglia (3). Due to the primary sites of the disease, it is commonly accepted that neuroblastoma arises from the sympathoadrenal lineage of the neural crest (4) which only present during embryonic development (5).
Of the two forms, familial NB is rare and accounts for <2% of all NB tumors (6). Commonly, familial NB is associated with a mutation in the anaplastic lymphoma receptor tyrosine kinase (ALK) gene (7). ALK is expressed in the developing sympathoadrenal lineage of the neural crest (8), and may regulate the balance between proliferation and differentiation of the lineage through multiple cellular pathways (9). In the sporadic form of NB, the most common genetic lesion is the amplification of MYCN proto-oncogene where it occurs in approximately 22% of tumors and it is associated with poor outcomes (4). MYCN is expressed in the developing neural crest and it regulates the proliferation, growth, differentiation, and survival of cells during the development of CNS through several signaling pathways (10).
There are many determinants of the clinical outcomes of NB. These include the patient's age at diagnosis, stage of disease, tumor histology, the presence of MYCN amplification, deletion of chromosomal regions, and ploidy. These determinants are now the foundations of risk-group stratification for patients with NB (2, 11). In comparison to infants, patients older than 1 to 2 years at diagnosis have a lower outcome with a predominance for patients with metastatic disease (1). Older children with NB have more indolent cancers and worse outcomes despite infrequent MYCN amplification (12). According to the International Neuroblastoma Staging System (INSS), localized tumors are staged into 1 to 3 based on the amount of resection, local invasion, and node involvement. Stage 4 defines distant metastasis, whereas stage 4S (4Special) is limited to dissemination to the liver skin and/or marrow (13). The distant metastasized tumors are also defined as M and MS by the International NB Risk Group's staging system (INRGSS) in a pattern similar to INSS 4 and 4S (14).
Pathological characteristics have been used to classify tumors as favorable and unfavorable. Shimada and colleagues provided the basic histology grading system (15) which was further developed to the recent advanced one by the International Neuroblastoma Pathology Committee (INPC). The degree of differentiation is one major factor of stratifying patients into risk groups (16). It is worth mentioning that 13-cis-retinoic acid (isotretinoin) is used as standard care for patients to drive NB differentiation (17). Another standard care of NB patients is the use of specific antibodies against GD2 as a therapy. The GD2 is a disialoganglioside antigen that is associated with neoplastic differentiation and expressed on surfaces of NB cells (18), but not on the glial lineage of neuronal stem cells (19). The most common (30-40%) chromosomal instability of NB patients with stage 4 and poor prognosis is 11q loss of heterozygosity (LOH) (1). Another genetic determinant is the hyperboloid NB tumours (DNA index >1) where there is an association with a more favorable prognosis compared to diploid ones (DNA index=1) (20, 21). However, the correlation of DNA content with survival rates is much less than in tumors with MYCN gene amplification. For example, in the current Children Oncology Group (COG), regardless of staging by the INSS, all tumors that show MYCN gene amplification are considered high-risk tumors and are treated with intensified therapies (22).
Based on the clinical and biological factors described above, NB is classified into low risk (LR), intermediate risk (IR), and high risk (HR), indicating a prediction of prognosis and risk of recurrence (14). Current treatment strategies have been successfully tailored based on patients' risk stratification where they could be limited to chemotherapeutic exposure of patients with the localized tumor to radio-therapeutic exposure of patients with more distant tumors (1). For low-risk NB, excellent survival rates resulted in patients with INSS stage 1 disease by resecting localized tumors (if not in a life-threatening organ) alone and salvage chemotherapy in rare recurrent tumors. For most low-risk patients with biologically favorable tumors, chemotherapy can be omitted. On the other hand, the localized tumors are either incompletely or completely resected in INSS stages 2B and 2A (nearby lymph nodes either do and do not contain microscopic cancer cells, respectively) and survival rate is greater than 95% of patients (23, 24). Most low-risk NB patients with stage 4S, however, without MYCN amplification undergo spontaneous regression (25, 26). IR patient includes those with INSS stage 3 and stage 4 diseases with favorable biological features and lack of MYCN amplification. Such patients are subjected to surgical resection and moderate-dose multi-agent chemotherapy using carboplatin or cisplatin, doxorubicin, etoposide, and cyclophosphamide, with a survival rate greater than 90% of patients (27, 28).
Compared to LR and IR NB patients, the HR patients require intensive treatment including chemotherapy, surgery, radiotherapy, myeloablative consolidation therapy with autologous peripheral blood stem cell rescue, combined with standard care using 13-cis-retinoic acid and anti-GD2 therapies (29) described above. Despite the recent intensive therapeutic regimen of HR, greater than 50% of patients with NB still experience tumor recurrence (30). Outcomes of HR patients (mainly stage 4 and stage 3 with MYCN amplification or with unfavorable histology tumors, all at age >18 months) remain poor despite slight improvements in survival (31, 32). For example, the event-free survival (EFS) time when a patient remains free of complications, was similarly 5-years in 30% of patients subjected to a rapid regimen of 10-day intervals (according to International Society of Pediatric Oncology Europe Neuroblastoma - SIOPEN) and in 18% of patients subjected to a standard regimen of 21-day intervals (according to COG). Both regimens include combinations of anthracyclines, alkylators, platinum compounds, and topoisomerase II inhibitors (33). Despite the minor improvement in survival, late side-effects including ototoxicity, renal dysfunction, hypothyroidism, ovarian dysfunction, and infertility (34) were reported in HR NB survivors, which are due to chemotherapy/radiation doses. Metastatic growths have been reported in 1-8% of patients enrolled in chemotherapeutic trials and in a small number of NB survivors where they have been attributed to etoposide exposure, radiation, and chemotherapy. These malignant neoplasms include myelodysplastic syndrome and acute myelogenous leukemia (35, 36).
Oxazolidinones are 5-membered heterocyclic compounds that have antibacterial activity against susceptible and resistant Gram-positive pathogenic bacteria including Mycobacterium tuberculosis. In general, oxazolidinones exemplified by linezolid (I, Figure 1) (37) exhibit their antibacterial activity by inhibiting bacterial ribosomal protein biosynthesis (38, 39). Other studies have shown that this class of compounds binds to the 50S ribosomal subunit and particularly to the A-site (40, 41). The first member of the oxazolidinone class of compounds, furazolidone (III, Figure 1), is a synthetic nitrofuran oxazolidinone derivative with the antibacterial activity known to target bacterial DNA. Furazolidone has also been reported to induce genotoxicity and carcinogenicity in certain cell types (42). However, a recent study (43) showed that furazolidone induced S phase cell-cycle arrest, suppressed cell growth, increased phosphorylated p38 (p-p38) activity and decreased the activity of phosphorylated c-Jun N-terminal protein kinase (JNK) in HepG2, a human hepatoblastoma cell line.
Chemical structures of biologically active oxazolidinone derivatives.
In addition, other studies have demonstrated anti-leukemic properties of furazolidone in acute myeloid leukemia (AML) cells in vitro, through increased stability of tumor suppressor p53 protein (44). Moreover, other oxazolidinone derivatives namely, (IV, BAY a 5830, Figure 1) (45), the 2-nitro-1H-imidazol-1-yl oxazolidinone (V, Figure 1) (46) and chloroquinoline oxazolidinone (VI, Figure 1) (47) have been shown to demonstrate anticancer properties. The natural product (-)-cytoxazone (VII, Figure 1) demonstrated cytokine-modulating activity as a selective inhibitor of the signal pathway of Th2 (48). More recently, the activity of cytoxazone-linezolid hybrid oxazolidinone derivatives exemplified by (VIII, Figure 1) against prostate (DU145) and lung (A549) cancer cell lines have been reported (49). In addition to the antibacterial and anticancer properties of oxazolidinone derivatives, other pharmacological activities including, anticoagulant, anti-thyroid, and central nervous system effects and inhibition of monoamine oxidases (44, 46, 50, 51) have been reported.
Several structural analogues of linezolid and eperezolid (I and II, Figure 1) have been evaluated in search of newer oxazolidinone derivatives with improved antibacterial activity (46, 50, 52-55). In a recent study, the anti-proliferative activity has been evaluated for selected examples of these derivatives namely, the 5-triazolylmethyl- and 5-acetamido-oxazolidinones [PH-97, IX (PH-series) and I, Figure 1]. Results showed that the 5-triazolylmethyl piperazino-oxazolidinone derivatives containing 4-N-(4-nitrobenzoyl) moiety among other compounds, showed the most potent cytostatic activity against breast cancer cells, inhibiting proliferation by up to 70%. Furthermore, the compounds also retarded motility and invasion of MDA-MB-231 cells (56).
We hereby report the in vitro anti-cancer properties of six substituted-glycinyl and -alaninyl triazolyl-oxazolidinone derivatives, coded PH-145, PH-189, PH-223D, PH-223L, PH-224D and PH-232L on a model of neuroblastoma namely, the Kelly cell line. These compounds were evaluated regarding their anti-proliferative effect on Kelly cells, where their potencies against the cells were expressed by IC50 values. By selecting a non-cytotoxic dose, two representative compounds; PH-223D and PH-2245D were subjected to investigating the adhesion and migration effects of triazolyl-oxazolidinones on Kelly cells.
Selected substituted-glycinyl and substituted-alaninyl triazolyl-oxazolidinones and the amount prepared.
Materials and Methods
All materials were purchased from Sigma (Poole, UK) unless otherwise specified. Cell culture plates and flasks were purchased from Costar (Schubert Laboratories, Germany).
Chemical synthesis of triazolyl-oxazolidinone derivatives. The N-substituted-glycinyl and -alaninyl triazolyl-oxazolidinone derivatives PH-Series (Table I) were synthesized according to previously reported methods (55-60) starting from commercially available starting materials as outlined in Figure 2. The compounds were purified by column chromatography and recrystallization to give final compounds, which were fully characterized by NMR (for proton and carbon assignments), IR, elemental analysis (%CHN) and mass spectrometric techniques.
Growth and maintenance of culture, and cell count. Human Kelly neuroblastoma cell line was purchased from the Health Protection Agency Culture Collection (HPACC, Salisbury, UK). The cells were routinely grown as monolayer cultures in T75 flask containing Dulbecco's Modified Eagle's Medium (DMEM) / Ham's Nutrient Mixture F12, supplemented with 10% fetal calf serum (FCS), 1% L-glutamine and 1% penicillin/streptomycin, and incubated at 37°C in a humidified atmosphere containing 5% CO2. As cells reached 80 to 90% confluency, cells were washed with Hank's Balanced Salt Solution (HBSS) and then dissociated by incubation in 3 ml EDTA solution for 5-10 min at 37°C. Cells were then re-suspended in 3 ml medium and centrifuged at 1000 × g for 5 min. The supernatants were discarded, and cell pellets were re-suspended in 1 ml fresh medium. Cell suspensions were transferred to a new flask containing 19 ml of fresh medium. Cells were stocked at early passages (3rd to 6th passage) and stored in liquid nitrogen where they were discarded every 10 passages thereafter. For the experimental work, 10 μl of cell suspension were placed on Improved Neubauer haematocytometer chamber and cells were counted under an optical microscope at a magnification of ×40. Cell counts were expressed as (mean of count numbers) ×104 cell/ml.
Synthesis of the triazolyl-oxazolidinone derivatives. a. MsCl, TEA, DCM; b. NaN3, DMF-H2O, 75°C; c. DME, acetylene, 100°C; d. THF-H2O, Ph3P, 75°C; e. MeOH, DIEA, 2-(1,1-dichloropropan-2-ylidene)-1-tosylhydrazine, r.t.; f. TFA, DCM, 0°C-r.t.; g. tert-boc-glycine or tert-boc-alanine, DCM-CH3CN, DCC, 1-HBT; h. nitroheteroaryl-carbonyl chloride, TEA, DCM-CH3CN; or nitroheteroaryl-carbonyl chloride, DCM-CH3CN, TEA, DC, 1-HBT.
Evaluation of the anti-proliferative effect of the triazolyl-oxazolidinones. Cells were seeded at different densities in 96-well plates and left to adhere overnight, leaving a lane in the plate with medium only to serve as a blank. Cell proliferation was evaluated using the MTT assay. Briefly, MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] reagent was added to each well at a concentration of 500 μg/ml and plates were incubated at 37°C for 4 h followed by the addition of 150 μl DMSO for dissolving the resultant blue formazan crystals. The absorbance of the colored solutions was measured at 540 nm using a plate reader. For validating the MTT assay, a standard curve was established by plotting cell densities versus the mean absorbance±SD values (subtracted from mean absorbance of the blank).
For assessing the anti-proliferative effect of the oxazolidinones, 2×104 cells were seeded in quadruplicate wells of 96-well plates and left overnight. On the next day, cells were incubated with medium only (control), medium containing DMSO (vehicle), or medium containing different concentrations of each of the six oxazolidinone derivatives dissolved in DMSO. Initially, the anti-proliferative effect of the compounds was investigated by incubating the cells for 4 days followed by the application of MTT assay described above. However, due to their high cytotoxic effect, oxazolidinones were incubated with Kelly cells overnight instead of 4 days to evaluate their effect. For calculating the anti-proliferative effect, the mean absorbance of the blank was subtracted from other mean absorbance values. The resulting values were expressed as percentages of cell viability of the vehicle-only-treated cells from three independent experiments (n=12 for each concentration of each oxazolidinone). For each oxazolidinone, a graph was established by plotting percentages of mean cell viability±standard error for the mean (SEM) versus concentrations. The half-maximal inhibitory concentrations (IC50, the concentration that produced 50% growth inhibition) of the triazolyl-oxazolidinones were calculated using non-linear regression analysis. In that analysis, the data were fitted to a dose-response-inhibition equation [log (inhibitor) vs. response (3 parameters) curve], where resultant curves were used to determine the IC50 values. These calculations and statistical analyses were conducted using the GraphPad Prism software package (version 7.04), where comparisons were also made by applying one-way analysis of variance (ANOVA) followed by Dunnett test, and p<0.05 was considered significant.
The protocol of evaluating the adhesion of Kelly cells to fibronectin.
Evaluation of the anti-progressive effect of the triazolyl-oxazolidinones Cell adhesion assay. The anti-progressive effect of the oxazolidinone derivatives on metastatic Kelly cells was further determined by assessing their potential to modulate the adhesion of the cells to a well-known extracellular matrix (ECM) glycoprotein, fibronectin. The adhesion assay was performed in 24 well plates pre-coated with fibronectin (Corning, NY, USA). The first step of the assay was to block the non-specific adsorption sites present in the fibronectin coats by incubating the wells with heat-denatured BSA. Thus, initially, the method of BSA heat denaturation was developed by incubating a 0.2-1% BSA solution prepared in PBS in a water bath set at 75°C for up to 3.5 h (61, 62). Volumes of 1 ml were withdrawn at time intervals; 0, 10, 30, 60, 90, 120, 150, 180, and 210 min, and immediately cooled on ice for 5 min.
The BSA samples were then subjected to SDS-PAGE for determining the time required to denature the BSA protein. Samples were diluted to a concentration of 25 to 50 μg in distilled water (dH2O) and incubated in one-quarter the final volume of a loading buffer composed of 0.5 M tris-HCl pH 6.8, 33.33% glycerol, 10% SDS, 0.25% bromophenol blue and 16.66% β-mercaptoethanol. Samples were then incubated in a heating block set at 95°C for 5 min. Samples were cooled to RT and then loaded onto SDS-PAGE consisting of stacking gel and resolving gel. The stacking gel was made up of 0.5 M tris-base pH 6.8, 3.9% bis-acrylamide, 0.1% SDS, 0.05% ammonium persulphate (APS) and 0.1% tetramethylethylenediamnine (TEMED), whereas the resolving gel was made up of 1.5 M tris-base pH 8.8, 6.0% bis-acrylamide, 0.1% SDS, 0.1% APS and 0.1% TEMED, both gels were prepared in dH2O. The gels were stacked in an electrophoresis tank (BioRad, UK) filled with a running buffer composed of 0.124 M tris-base, 7.2% glycine and 0.1% SDS. Alongside samples, one lane of the stacking gel was loaded with the full range rainbow molecular weight marker (Amersham Pharmacia, UK). Electrophoresis was run at 120 V for approximately 45 min. The protein bands were detected by incubating the gels for 5 min in Coomassie stain composed of 0.25% Coomassie blue G-250, 45% methanol, and 10% glacial acetic acid. The gels were then rinsed with dH2O and immersed for up to 1.5 h (or until the bands appeared clearly) in a de-stain solution composed of 50% methanol and 10% glacial acetic acid. Once bands were clearly visualized, gels were removed from the de-stain solution and kept in dH2O, and images of the gels were pictured by the ChemoDoc Imaging System (Syngene, UK).
Before evaluating the effect of selected oxazolidinone derivatives on adhesion of Kelly cells, the adhesion time of the cells to fibronectin was optimized. The non-specific adsorption sites of the fibronectin-coated wells were blocked by incubation with heat-denatured BSA for 30 min at RT (63). After this, the wells were washed with HBSS. To prepare Kelly cells for the adhesion assay, cells were harvested and volumes of 1 ml of cell suspensions at a density of 1×105 were added to 5 wells, where each well was used for a time interval. In parallel, another 5 wells were filled with 1 ml of medium only to serve as blanks. These wells were incubated at 37°C for up to 60 min, where medium and medium containing the non-adherent cells were fully withdrawn at time intervals; 0, 10, 20, 40, and 60 min. After washing the parallel wells, they were incubated with freezer-cold 100% methanol for 10-15 min at RT (this was to fix the adherent cells). After washing, wells were incubated with 0.25% w/v crystal violet solution (dissolved in 2% v/v ethanol/water) for 1 h at 37°C. After the last washing, a 2% SDS solution was added to the wells, and after 10 min the colored solutions (of blanks and cell lysates) were transferred into 96-well plates to measure absorbance using a plate reader (64). For each time point, the blank absorbance value was subtracted from the absorbance value of adherent-cells, and the obtained mean±SEM values from four independent experiments (n=4 for each time interval) were plotted against the time intervals.
For evaluating the effect of representative triazolyl-oxazolidinones on the adhesion of Kelly cells to fibronectin, 2 ml of 1×105 cell/ml were seeded in a 6-well plate and left to adhere overnight. Cells were then incubated with medium only (control), medium containing vehicle DMSO, or medium containing PH-223D or PH-224D dissolved in DMSO, for 24 h at 37°C. On the next day, cells were harvested and 1 ml of each of the four-cell suspension was added to fibronectin-blocked well to be incubated for the optimized determined time. After this, the adhesion assay was performed as described above and shown in Figure 3.
Values of the cell adhesion were expressed as mean±SEM percentages with respect to the control (untreated) cells for the vehicle-treated cells, and with respect to vehicle-treated cells for cells treated with triazolyl-oxazolidinones, from three independent experiments (n=3). The data were analyzed using the GraphPad Prism software package (version 7.04). For the percentage adhesion of vehicle-treated cells, the unpaired parametric t-test was used, and for cells treated with triazolyl-oxazolidinones, the one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test was used.
Transwell migration assay. The migration assay was performed by using cell culture inserts of which the bottom is a polyethylene terephthalate membrane with 8 μm pore size. Cells at a density of 5×103/ml were suspended in 500 μl of medium containing the triazolyl-oxazolidinones, PH-223D or PH-224D and seeded into the inserts that were situated into 24-well plate. On the other side of the membrane, 750 μl of medium containing the same concentration of the oxazolidinones was added. The plate was then incubated for 24 h at 37°C. Following the incubation period, the non-migrated cells were removed from the upper side of the membrane using cotton swabs and kimwipes, and migrated cells to the underside of the membrane were stained with crystal violet solution in the 24-well plate and counted in the insert under the microscope. A graph was established by plotting the mean number of migrated cells ±SEM versus each treatment, from three independent experiments (n=3). The data were analyzed using the GraphPad Prism software package (version 7.04) by applying the one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test.
A standard curve of Kelly cell number versus formazan absorbance. Formazan is the resultant blue product from the conversion of the yellow MTT reagent by mitochondrial dehydrogenase present in viable cells. Points represent mean±SD (n=8), r2=88.
Results
Chemical synthesis and characterization of triazolyl-oxazolidinones. The selected triazolyl-oxazolidinones were synthesized from the relevant key intermediates presented in Figure 2 to give semi scale-up quantities to afford amounts required for biological testing. The structures of the compounds were characterized by standard analytical methods as previously reported (55). The structures of the selected triazolyl-oxazolidinone derivatives are presented in Table I.
Anti-proliferative effect of triazolyl-oxazolidinones. For evaluating the effect of triazolyl-oxazolidinones on the viability of Kelly cells, the MTT assay was used. Figure 4 shows the validation of MTT assay indicated by a standard curve which was established of plotting the absorbance of the resultant formazan colored crystals versus Kelly cells at densities of 1×104-1×105, where absorbance above 10×105 cell/ml were out of range.
Cells were initially incubated with the test compounds for 4 days at concentrations ranging from 0.1 μM up to 100 μM. However, due to the high cytotoxic effect of the oxazolidinones at that concentration range (data not shown), which could prevent the possibility of comparing the levels of cytotoxicities within that concentration range or at higher concentration, treatment was performed for 1 day (24 h). To eliminate the effect that the vehicle (0.5% final concentration of the DMSO in the tissue medium) has on the viability of Kelly cells, the cells were incubated simultaneously with 0.5% DMSO in the medium for 24 h. By evaluating the cell viability using MTT assay, the effect of 0.5% DMSO was found to be significant, however, it did not affect the cell viability (95.33±10.48%) compared to the control cells (Figure 5).
The stock solutions of the triazolyl-oxazolidinones were prepared at concentrations that attain final concentrations at 0.5% DMSO in the cellular cultures. Thus, all cell viability data for cells exposed to the oxazolidinone derivatives were corrected for the effect of 0.5% DMSO (Figure 6). As shown in Figure 6, the tested triazolyl-oxazolidinones including PH-145, PH-189, PH-224D, and PH-232L showed varying significant inhibitory effects on the proliferation of Kelly cells starting from a concentration of 50 μM and up to 375 μM, where this inhibitory effect started from 10 μM for PH-223D and PH-223L. On the other hand, the oxazolidinones had varying effects on the proliferation of Kelly cells at concentrations lower than 50 μM for PH-145, PH-189, PH-224D and PH-232L, and lower than 10 μM for PH-223D and PH-223L, where these effects were persistent down to the concentration of 0.5 nM for all of the tested derivatives.
For calculating the IC50 of each oxazolidinone, the non-linear regression analysis with the [log (inhibitor) vs. response (3 parameters)] equation was applied on data of log concentrations versus mean percentages of cell viability±SD. The result dose-response curves (Figure 7) were used to determine the IC50 values which were; 6.52 μM for PH-145, 11.59 μM for PH-189, 11.10 μM for PH-223D, 11.28 μM for PH-223L, 29.83 μM for PH-224D, and 56.24 μM for PH-232L. The 5-nitrofuroyl containing glycinyl derivative, PH145 showed lowest IC50, while the 5-nitrothiophenecarbonyl L-alaninyl containing derivative, PH-232L showed the highest value. However, there is no significant difference between the 5-nitrofuroyl D- and L-alaninyl containing derivatives PH-223D and PH-223L with both having similar IC50 values. The presence of 4-methyltriazolyl moiety seems to enhance the anti-proliferative property of these derivatives as demonstrated by PH-145.
Effect of triazolyl-oxazolidinones on the adhesion of Kelly cells. The anti-progression effect of triazolyl-oxazolidinones was further evaluated by investigating their effect on the adhesion of the Kelly cells to fibronectin. Fibronectin is a glycoprotein that is present in the extracellular matrix. The effect of oxazolidinones on adhesion was studied by incubating the cells in 24-well plates pre-coated with fibronectin. To prevent the adsorption of cells to the non-specific sites present on fibronectin coats, which could give false adhesion results, these sites were blocked by using heat-denatured BSA. For this purpose, the method of denaturation was developed by incubating BSA solutions at 75°C for 3.5 h, where samples were withdrawn at different time intervals to analyze the optimum time of denaturation by performing SDS-PAGE.
A representative image of seven independent experiments of SDS-PAGE conducted for heat-treated BSA solution is shown in Figure 8. Using the rainbow ladder, it was shown that a large BSA band was clearly detected between molecular weights of 76 and 52 kDa (Figure 8), which is consistent with the BSA molecular weight of 66.430 kDa. However, as indicated in Figure 8, there were no clear denaturation bands of BSA, but there was an unknown contaminant band above 150 kDa. Additionally, a band at 225 kDa started to appear at 60 min and increased in intensity with heating time. At 120 min of heating time, aggregation of proteins was indicated by the broadening of the bands and finally tailing at 210 min (Figure 8). Thus, for blocking the fibronectin coats, BSA solutions were heated for up to 90 min, where most of the BSA band (66.4 KDa) consistently disappeared throughout the independent experiments.
The percentages of cell viability of Kelly cells treated with vehicle DMSO. Cells at density of 2×104 were incubated with 0.5% DMSO for 24 h. Cells were then subjected to MTT assay and the resulting absorbance values are expressed as percentages with respect to control-untreated cells. The bars represent mean±SEM (n=36). ***p=0.0002.
Before investigating the extent of adhesion of Kelly cells treated with tested compounds, the adhesion time of cells to fibronectin was optimized. Cells were incubated on the blocked-fibronectin coats at 37°C for 60 min. At different time intervals, the non-adherent cells were removed, and adherent cells were conducted to colorimetric measurement compared to the blanks. A graph was generated by plotting absorbances of adherent cells against the incubation time (Figure 9). As shown in Figure 9, by taking the mean absorbance±SD values of the adherent cells, it was indicated that the adhesion of Kelly cells to fibronectin was optimum at 40 min (0.178±0.04), compared to 0 time (0.029±0.168), 10 min (0.048±0.07), 20 min (0.117±0.04), and 60 min (0.113±0.08). Thus, the adhesion effect of oxazolidinones was evaluated for 40 min.
Similar to the anti-proliferative effect, the effect of DMSO vehicle on the adhesion of Kelly cells to fibronectin was also evaluated with respect to control untreated cells. As it is shown in Figure 10, cells treated with vehicle (0.5% DMSO) have higher adhesion (128.4±86.89%) compared to control untreated cells (100±22.8%). However, the increasing effect of DMSO vehicle on the adhesion of Kelly cells was not significant.
The anti-proliferative effect of triazolyl-oxazolidinone derivatives on Kelly cells. Cells at density of 2×104 were incubated with oxazolidinones for 24 h at 37°C. Cells were then subjected to MTT assay and the resulting absorbance values were expressed as percentages with respect to vehicle-treated cells. The bars represent mean±SEM (n=12). *p=0.0225, **p=0.0022-0.0001, ***p and ****p<0.0001.
The effect of the oxazolidinones on the adhesion of Kelly cells to fibronectin was evaluated with respect to vehicle-treated cells. Two analogs, the 5-nitrofuroyl alaninyl (PH-223D) and 5-nitrothiophenecarbonyl alaninyl (PH-224D) containing D-derivatives, were used as representatives of oxazolidinones in this evaluation. Cells were incubated at a non-cytotoxic concentration 1 μM (cell viability was 94.40±10.00% for PH-223D and 90.55±11.80% for PH-224D) (Figure 6) for 24 h, after which adhesion to the fibronectin wells was evaluated using the adhesion assay. As shown in Figure 11, treating the Kelly cells with PH-223D and PH-224D reduced the adhesion to fibronectin with respect to vehicle-treated cells, where the mean±SD adhesion was 66.50±20.98% for cells treated with PH-223D, 43.66±5.50% for cells treated with PH-224D, and 100±67.68% for cells treated with the vehicle alone.
The dose-response curves of Kelly cells against triazolyl-oxazolidinones. Cells at density of 2×104 were incubated with triazolyl-oxazolidinones for 24 h at 37°C. Cells were then subjected to MTT assay and the resulting absorbance values were expressed as percentages with respect to vehicle-treated cells. The curves represent mean±SD (n=12) versus log concentrations.
Effect of triazolyl-oxazolidinones on the migration of Kelly cells. The transwell migration assay was used to determine the effects of 1 μM of the same representative oxazolidinones, PH-223D and PH-224D on the motility of Kelly cells. In this assay, the number of migrated cells to the underside of the polyethylene terephthalate membrane were counted under the microscope. As shown in Figure 12, the numbers of migrated cells treated with PH-224D that were 90, 113 and 87 cells were slightly less than the number of migrated cells that were treated with DMSO vehicle that were 108, 105 and 127 cells. However, the numbers of migrated cells treated with PH-223D being 73, 88 and 86 cells, were significantly less than those treated with the vehicle. Thus, compared to cells treated with vehicle, the 5-nitrofuroyl alaninyl containing D-derivative PH-223D reduced the migration of Kelly cells by 29.14%, whereas the 5-nitrothiophenecarbonyl alaninyl containing D-derivative PH-224D reduced migration by 14.16%.
A representative pattern of SDS-PAGE for BSA solution incubated at 75°C. A solution of 1% bovine serum albumin (BSA) was prepared in PBS and heated for up to 3.5 h. Samples were withdrawn at different time intervals and placed on ice. Samples were then diluted to concentration of 50 μg to be loaded on 6% SDS-PAGE. The electrophoresis was run at 120 V for 45 min, where bands were visualized by using coomassie blue G-250 stain.
Adhesion of Kelly cells to fibronectin as a function of time. Cells at density of 2×104 were incubated in 24-well plates coated with fibronectin. At different time intervals, non-adherent cells were removed, where adherent cells were subjected to the adhesion assay. Each point represents the mean±SD from independent experiments (n=4).
Discussion
NB is the most common extracranial malignant childhood tumor resulting in 15% of cancer-related childhood deaths. With reference to “latest data by the IARC, there is a global increase of 13% in childhood cancer incidence in two decades since 1980, where 20% of cases are due to CNS tumors such as NB”. Approximately 50% of patients are diagnosed with distant metastasis where most common secondary growth sites are bone, bone marrow, and liver. More than 50% of patients with HR stratification of the disease are subjected to extensive therapeutic regimens, however, still suffer from tumor recurrence and late adverse effects. In this study, our aim was to investigate the inhibition of proliferation of an in vitro model of neuroblastoma, the Kelly cell line, by selected series of synthetic oxazolidinone derivatives coded as; PH-145, -189, -223D, -223L, -224D, and -232L.
Adhesion of Kelly cells treated with DMSO to fibronectin. Cells at density of 2×104 were incubated for 24 h with medium or medium containing 0.5% DMSO. On the next day, cells were harvested and subjected to the adhesion assay. The resulting values were expressed as percentages with respect to control-untreated cells. The bars represent mean±SEM from independent experiments (n=3).
The oxazolidinone derivatives were further utilized to examine their potential to prevent initial stages of metastasis of cancer cells. Thus, Kelly cells treated with a non-cytotoxic concentration of oxazolidinones were subjected to adhesion and migration assays, where results were compared to those of vehicle-treated cells. The proliferation analysis was carried out using the MTT assay, where this commonly used assay depends on mitochondrial dehydrogenase conversion of a water-soluble yellow MTT to a water-insoluble blue formazan product. The later can be measured spectrophotometrically after dissolution in DMSO. Compared to control-untreated cells, the incubation of cells with vehicle DMSO even though significant, did not affect the cell viability (95.33±10.48%). By incubating Kelly cells with oxazolidinones at concentrations from 0.5 nM up to 375 μM, apart from the 5-nitrofuroyl alaninyl containing D-derivative PH-223D and the 5-nitrofuroyl alaninyl containing L-derivatives PH-223L, other oxazolidinones were significantly cytotoxic from 50 μM. In contrast, PH-223D and PH-223L were significantly cytotoxic from 10 μM. All tested oxazolidinones exhibited varying effects at concentrations above the significant cytotoxic concentration. By calculating the IC50 values of the investigated oxazolidinones on Kelly cells, it was determined that the 5-nitrofuroyl glycinyl containing derivative with 4-methyltriazolyl group PH-145 had the least IC50, which was 6.52 μM, whereas PH-189, -223D and -223L had similar IC50 of 11.59, 11.1 and 11.28 μM, respectively. In contrast, IC50 for PH-224D and -232L were 29.83 and 56.24, respectively. Thus, the 5-nitrofuroyl glycinyl containing derivative with 4-methyltriazolyl group PH-145 is the most potent oxazolidinone, whereas the 5-nitrothiophenecarbonyl alaninyl containing L-derivative PH-232L is the least potent.
Adhesion of Kelly cells treated with triazolyl-oxazolidinones to fibronectin. Cells at density of 2×104 were incubated for 24 h with medium containing 0.5% DMSO, PH-223D or PH-224D. On the next day, cells were harvested and subjected to the adhesion assay. The resulting values were expressed as percentages with respect to vehicle DMSO-treated cells. The bars represent mean±SEM from independent experiments (n=3).
The adhesion of Kelly cells was further evaluated using 1 μM non-cytotoxic concentration of PH-223D and -224D, as representatives of the tested oxazolidinones. Such representation of oxazolidinones was decided due to the similar anti-proliferative results. The adhesion was carried out on 24-well pre-coated with a layer of fibronectin which is one of the ECM molecules. Such adhesion of cancer cells to ECM substrates is a measure of the metastatic progression of these cells (63). Since the fibronectin layers may have adsorption sites to which cells might adhere and give false results, these sites were blocked by incubating the wells with heat-denatured BSA. The method of heat denaturation was developed by incubating a BSA solution in a heated water bath and withdrawing samples at time intervals. The withdrawn samples were loaded on SDS-PAGE to investigate the time required to denature BSA. The BSA band was detected between molecular weights of 76 and 52 kDa, which was consistent with the BSA molecular weight of 66.43 kDa. However, no other bands with smaller molecular weight were detected to present the denaturation of BSA protein, and the intensity of the BSA band markedly decreased at 90 min of the heating time. After 90 min of heating time, contaminant unknown bands above 225 and 150 kDa started to boarder and finally tailed at 210 min, which indicates the high aggregation and unfolding of the three proteins including BSA. Thus, to block the fibronectin non-specific sites, the wells were incubated with a BSA solution heated for up to 90 min.
Migration of Kelly cells treated with oxazolidinones. Using cell culture inserts, medium containing 0.5% DMSO, PH-223D or PH-224D were added to the two sides of the inserts. Cells at density of 5×103/ml were seeded to one side of the insert. Incubation was carried out for 24 h, after which the non-migrated cells were removed, and the migrated cells to the underside of the membrane were stained using crystal violet. Migrated cells were counted under the microscope. The bars represent mean±SEM from independent experiments (n=3). *p=0.0303.
The adhesion time of Kelly cells to fibronectin was optimized by measuring the absorbance of adherent cells at time intervals from 0-60 min using a plate reader, where values were subtracted from those of blank media similarly incubated at a same time interval. The optimum adhesion time of Kelly cells using the adhesion protocol was determined to be 40 min. Consequently, Kelly cells treated with PH-223D and -224D oxazolidinones were incubated in the fibronectin wells for 40 min to evaluate their adhesion compared with the vehicle-treated cells. Compared to control untreated cells, the adhesion of vehicle-treated cells was increased by 28.4±86.89%. On the other hand, compared to the vehicle, PH-223D and -224D markedly (but not significantly) decreased the adhesion of Kelly cells by 33.5±67.68% and 56.34±5.5%, respectively. In a previous study, we indicated the presence of cell-surface polysialic acid (PSA) glycan on membranes of Kelly cells. PSA is a polymer of α-2, 8 glycosidic linked sialic acid monomers (65). It has been reported that metastatic cancer cells express highly branched glycoproteins with highly-sialylated termini on their surfaces. These glycoproteins involve in the adhesive property of cancer cells with ECM substrates and thus with the metastatic potential of these cancer cells (63). In the present study, treating Kelly cells with oxazolidinones reduced their adhesion to fibronectin (one of ECM substrates), which may provide new anti-metastatic agents that would inhibit the expression of the membrane-sialylated glycan, PSA.
Kelly cells treated with the same oxazolidinones were also subjected to a migration assay. By counting the number of the migrated cells through the polyethylene terephthalate membrane, it was indicated that the 5-nitrofuroyl D-alaninyl containing derivative PH-223D significantly inhibited the migration of Kelly cells by 29.14% compared to cells treated with DMSO vehicle. In contrast, the 5-nitrothiophenecarbonyl alaninyl containing D-derivative PH-224D inhibited the migration of Kelly cells only by 14.16% compared with cells treated with DMSO vehicle.
The approach of increasing the toxicity against cancer cells and minimizing the side-effects on normal cells was utilized in a recent study. That was done by combining the most toxic Chinese antitumor traditional medicine, arsenic trioxide (As2O3) with the natural antitumor phenol - resveratrol. This study was conducted on a human SK-N-SH neuroblastoma cell line. The viability of SK-N-SH cells treated with the mentioned combination of agents (arsenic trioxide and resveratrol) was compared to the viability of cells treated with As2O3 alone. It was shown that resveratrol at a concentration of 75 μg/ml synergistically triggered the cytotoxic effect (by suppressing cell viability) of 2 μM As2O3, the lowest ever published dose for treatment with this toxic agent. Investigation of the underlying apoptotic pathway of the adjuvant treatment at extremely low concentrations of these two agents showed an elevation of reactive oxygen species level. This may cause a significant loss of MMP, a marked reduction in the levels of the inhibitors of programmed cell death proteins; BCL2, BID, and BCL-x/L, and activation of caspase-3 and caspase-9 (66). Similarly, this approach may be used through combining the non-cytotoxic concentration of oxazolidinones that exerts a reduction effect on the adhesion and migration of Kelly cells with resveratrol or quercetin. By this combination, oxazolidinones at a concentration of 1 μM could also produce a cytotoxic effect on cancer cells. This an important finding as the use of higher concentrations of these agents would enhance cytotoxicity that may affect normal cells. Thus, treating Kelly cells with 1 μM oxazolidinones combined with resveratrol or quercetin is worthwhile to consider for future investigations. Taken together, the novel oxazolidinones may serve as cytotoxic agents against NB at IC50 of at least 6 μM, which could be otherwise affect normal cells in a tumor-surrounding tissue, while treatment with same compounds at a concertation of 1 μM alters or reduces the progression of the NB tumours and is non-cytotoxic. Thus, adjuvant chemotherapy based on combining low concentrations of oxazolidinones with natural products may enhance their cytotoxic effects and lead to an exertion of various anticancer mechanisms.
NB is the most common solid tumor in childhood after pediatric brain tumors. Likewise, the carcinogenic-expression of PSA on NCAM protein that is attached to the membrane of Kelly neuroblastoma cell line and analysis of pediatric pilocytic astrocytoma brain intracystic fluid documented post-translational modification of proteins resulting in the presence of truncated, oxidized and glycosylated proteoforms. These modifications provide important molecular characterization at the onset and development of the disease. Similar to the most common genetic amplification of MYCN proto-oncogene, the amplification of c-MYC, another member of MYC family, associates with the aggressiveness of medulloblastoma. This was shown in primary patient-derived medulloblastoma cell lines, where c-MYC amplification in these cells induced glutamine metabolism that is important for cell survival under glucose and oxygen depriving conditions present in solid tumors (67).
In conclusion, the novel triazolyl-oxazolidinones had a significant inhibitory effect on the proliferation of neuroblastoma Kelly cells, where 5-nitrofuroyl glycinyl oxazolidinone derivative containing a 4-methyltriazolyl group, PH-145 was the most potent oxazolidinone and 5-nitrothiophenecarbonyl alaninyl containing L-derivative, PH-232L was the least potent. By testing their potential activities of reducing cancer progression, it was shown that 5-nitrothiophenecarbonyl D-alaninyl containing derivative PH-224D reduced the adhesion of Kelly cells to fibronectin by 56.34%, and 5-nitrofuroyl D-alaninyl derivative PH-223D significantly inhibited cell movement by 29.14%, with respect to vehicle-treated cells. These effects of oxazolidinones on neuroblastoma cells provide a new therapeutic approach making the oxazolidinone moiety a useful structural scaffold in the discovery of potent antitumor agents. Thus, in vivo administration of the compounds for studying their pharmacokinetic properties, anti-metastatic potential and toxicities are required for such application. Also, future investigations may include the determination of the mechanism(s) of action of this novel class of anti-proliferative agents. This may lead to a discovery of more potent agents that could act at the nanomolar level with fewer side-effects.
Acknowledgements
This work was financially supported by the Research Administration, Kuwait University Research Grant PC01/17. The Authors would like to acknowledge the General Facilities Grants GS01/01, GS01/03, and GS01/05, Kuwait university for characterizing the tested compounds. The Authors would like to thank Dr. Willias Masocha, Department of Pharmacology and Therapeutics, Faculty of Pharmacy, Kuwait University for his help and support in the statistical analysis of our data.
Footnotes
Authors' Contributions
Conceptualization: Nada A. Al-Hasawi, Oludotun A. Phillips and Ladislav Novotny. Data curation: Nada A. Al-Hasawi; Methodology, Nada A. Al-Hasawi, Oludotun A. Phillips and Fatima Al-Awadhi. Synthesis of the tested substances: Oludotun A. Phillips and Leyla Sharaf. Testing of substances: Nada A. Al-Hasawi, Sanaa A. Amine and Fatima Al-Awadhi. Funding acquisition: Nada A. Al-Hasawi. Writing – original draft: Nada A. Al-Hasawi. Writing – review & editing: Nada A. Al-Hasawi, Oludotun A. Phillips and Ladislav Novotny.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received June 15, 2020.
- Revision received July 5, 2020.
- Accepted July 9, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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