Sialic Acid Metabolic Engineering: A Potential Strategy for the Neuroblastoma Therapy

<div><p>Background</p><p>Sialic acids (Sia) represent negative-charged terminal sugars on most glycoproteins and glycolipids on the cell surface of vertebrates. Aberrant expression of tumor associated sialylated carbohydrate epitopes significantly increases during onset of cancer. Since Sia contribute towards cell migration ( =  metastasis) and to chemo- and radiation resistance. Modulation of cellular Sia concentration and composition poses a challenge especially for neuroblastoma therapy, due to the high heterogeneity and therapeutic resistance of these cells. Here we propose that Metabolic Sia Engineering (MSE) is an effective strategy to reduce neuroblastoma progression and metastasis.</p><p>Methods</p><p>Human neuroblastoma SH-SY5Y cells were treated with synthetic Sia precursors N-propanoyl mannosamine (ManNProp) or N-pentanoyl mannosamine (ManNPent). Total and Polysialic acids (PolySia) were investigated by high performance liquid chromatography. Cell surface polySia were examined by flow-cytometry. Sia precursors treated cells were examined for the migration, invasion and sensitivity towards anticancer drugs and radiation treatment.</p><p>Results</p><p>Treatment of SH-SY5Y cells with ManNProp or ManNPent (referred as MSE) reduced their cell surface sialylation significantly. We found complete absence of polysialylation after treatment of SH-SY5Y cells with ManNPent. Loss of polysialylation results in a reduction of migration and invasion ability of these cells. Furthermore, radiation of Sia-engineered cells completely abolished their migration. In addition, MSE increases the cytotoxicity of anti-cancer drugs, such as 5-fluorouracil or cisplatin.</p><p>Conclusions</p><p>Metabolic Sia Engineering (MSE) of neuroblastoma cells using modified Sia precursors reduces their sialylation, metastatic potential and increases their sensitivity towards radiation or chemotherapeutics. Therefore, MSE may serve as an effective method to treat neuroblastoma.</p></div

Invasion assay.

<p>CIM plate upper chamber was coated with ECM protein for 4 h and the SHSY5Y cells were cultured in the presence or absence of 10 mM ManNAc, ManNProp or ManNPent followed by (A) monitoring the impedance for 24 h. (B) Non-natural Sia precursor treated cells significantly decreased the invasion ability (** p<0.0001) compared with untreated and ManNAc treated cells (**P<0.0002), ns: not significant.</p

Migration assay.

<p>SHSY5Y cells were cultured in the presence of 10 mM ManNAc, ManNProp or ManNPent. Cells were seeded in the CIM plate, using complete medium as a chemo -attractant. A. Cell migration was measured by impedance and read out as cell index. B. Changes in the 1 h of measurement are represented as bars along with standard deviation. ManNProp and ManNPent inhibit the migration with higher significance (P<0.0001, P<0.0001), ns: not significant.</p

Cytotoxicity assay with 5-Fluorouracil.

<p>0.05×10⁶ SHSY5Y cells were seeded in the RTCA E plate and cultured in the presence or absence of 10 mM ManNProp or ManNPent followed by treatment with 5-FU. A. Cytotoxicity induced by 5-FU towards the engineered cells was measured. B. ManNProp and ManNPent treated cells displayed significant increase in cytotoxicity at 10-fold reduced 5-Fluorouracil concentration compared to untreated cells. ** P<0.0001 for ManNProp and ManNPent treatment until 25 µM 5-Flurouracil.</p

Radiation treatment impact on migration.

<p>SHSY5Y cells were cultured in the presence or absence of 10 mM ManNAc, ManNProp or ManNPent. Cells were radiated at a dose rate of 2 Gray/min for the total of 6 Gray. Radiated and control cells were subjected to migration assay (RTCA). (B) ManNProp and ManNPent pretreated cells displayed diminished migration ability at a significant rate (**P<0.0004, ***P<0.0001). Radiation treated control cells display reduced migration but not as significant (p<0.05) as compared to Sia engineered cells, ns: not significant.</p

MTT Assay for Cisplatin Treatment on Sia Precursor Pretreated SHSY5Y Cells.

<p>SHSY5Y cells were cultured in the presence of 10 mM ManNProp or ManNPent followed by cisplatin treatment for 48 h. Complete medium was supplemented with MTT reagent and the absorbance at 560 nm was measured. ManNPent pretreatment displayed significant cytotoxicity (P<0.0001 to P<0.0056) for 0.1 to 5 µM cisplatin concentration. ManNProp treatment does not yield a significant difference in comparison to control cells.</p

Chromatographic polySia and total Sia analysis of SHSY5-cells cultured with Sia precursors.

<p>A. PolySia chains were directly released from cell homogenates and labeled with DMB in a one-pot reaction. Resulting fluorescently tagged sialic acid polymers were separated on an anion-exchange column according to the polySia chain length. Respective chain length of polySia residues is given for selected peaks. B. To analyze possible alterations in the relative proportions of individual polySia chains, Peak areas corresponding to polySia chains ≥DP 8 were calculated to obtain the individual chain length distribution of polySia in lysates from SHSY5Y cells treated with ManNAc, ManNProp and ManNPent as well as untreated cells. Data are means ±S.D. of four independent experiments. Values obtained for untreated cells were set to 100%. The statistical evaluation was performed by Student's t test (unequal variances, two-tailed). Significance levels are indicated by *, p<0.05, **, p<0.005, ns: not significant. C. SHSY5Y cells were cultured in the presence or absence of 10 mM ManNAc, ManNProp or ManNPent. Sia were released with 2 M formic acid and purified by anion-exchange chromatography. Purified sialic acids were tagged with DMB and analyzed by reversed phase HPLC. Quantification of Sia exhibited that ManNProp treatment led to significant reduction of Sia up to 84% and similarly ManNPent treatment reduced Sia to 60% (*, P<0.01, P<0.03). ManNAc treatment increases the sialic acid concentration 5% more than the untreated control but not significantly (ns: not significant) (A, B).</p