This article is part of the outcome of work commissioned and paid for by PIP charity and completed at UCLan in 2015.

We thank Professor Kalaminder Singh for this summary and refer readers to the article on Acyl homoserine lactone on this page.

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The anti-cancer activity of palmitoyl ascorbate (PA) was investigated against the human glioblastoma cell line (U87 MG) and the normal human foetal glial cells (SVG-P12). As PA has poor aqueous solubility, sodium taurocholate (NaTC) a natural surface-active agent was used to solubilise PA for cytotoxicity testing as determined by the PrestoBlue cell viability assay. Initial experiment was carried out to determine the concentration of NaTC which was not toxic to the cells so that it can be used for solubilisation of PA

Materials

The cell lines used in the study were U87 MG and SVG P12.  The palmitoyl ascorbate (PA) (BioXtra, Purity 99.0%), Sodium taurocholate hydrate (powder form, 86339, Purity 97.0%), cis-Diammineplatinum(II) dichloride (Crystalline form, P4394), Diphenyleneiodonium chloride (D2926, Purity 98.0%) and 0.1mM H₂O₂ were purchased from Sigma-Aldrich, UK. The ROS assay was performed using a ROS Kit (Orange, MAK144) purchased from Sigma-Aldrich, UK. The PrestoBlue cell viability reagent used in the study was purchased from life technologies, UK. All cell culture essentials including Eagle’s minimal essential medium (EMEM), sodium pyruvate, L-glutamine, foetal calf serum (FCS), non-essential amino acids, trypsin and PBS were purchased from Lonza, UK. Other cell culture consumables such as 75cm² and 25cm² filter cap angled neck tissue culture flasks, pipettes, tips and 96-well plates were purchased from Fisher, UK.

Methods

Solubility Assay

In procedure, 5mg of PA was taken in a clear glass test tube and 5mL of diluted ethanol was gradually added (10µL each time) to the test tube while shaking gently and then vortexed. Alternatively, 5mg of PA was dissolved in absolute ethanol (same as described procedure) by shaking gently and then the solution was diluted to make it up to 0.1% and 0.5% ethanol both in water and media. The solubility of PA was determined in NaTC solution. Firstly, 200mM NaTC stock solution was prepared by dissolving 107.6mg of NaTC hydrate powder (MW = 538) in 1mL of both distilled water and EMEM. The same previous procedure was followed to dissolve PA (by adding 10µL each time into 5mg of PA) in the NaTC solution shaking gently.

Cell Culture

The cells (both SVG-P12 and U87 MG) were cultured in 75 cm² filter cap angled neck tissue culture flask using Eagle’s minimal essential medium (EMEM) supplemented by  L-glutamine (2mM), foetal calf serum (10%), non-essential amino acids 1% and 1mM sodium pyruvate and the flasks were incubated at 37⁰C and 5% CO₂ conditions. Further, cells were split every time when flasks became 70% confluent approximately.

Cell viability assays and time response results

Both U87 MG and SVG-P12 cells were seeded at 5 x 10³ cells per well per 100µL in sterile 96-well microtiter plates and incubated for 24h at 37⁰C and 5% CO₂ conditions. Then the media was removed gently and 90µL of appropriate test solution was added. For NaTC cytotoxicity, range of solutions of NaTC (0.2mM to 200mM) were prepared with EMEM by serial dilution method and added to the wells. Similarly, for PA cytotoxicity assay, series of dilutions of PA (50µM to 500µM) were prepared by dissolving it in respective molar ratio of NaTC solution ( PA: NaTC = 1:4) and added to the 96-well plate. The plates were then incubated for 48h (both NaTC and PA cytotoxicity) before addition of 10µL of PrestoBlue reagent to each well. For the time course, cells were treated with 400µM PA dissolved in 1.6mM of NaTC solution (Molar ratio 1:4) for 30 min, 2h, 6h, 24h and 48h before addition the PrestoBlue reagent. The plates were further incubated for 4h and then the bottom-read fluorescence was measured using a microtiter ELISA plate reader at an excitation and emission wave length of 535 nm (25 nm band width) and 615 nm (10nm band width) respectively. Cell viability was expressed as a percentage of the viability of untreated control cells. As a positive control, cells were treated with 50µM cisplatin for all viability assays.

Furthermore, 2.5 x 10⁵ cells (5mL, 5 x 10⁴ cells per mL) were seeded in sterile T25 filter cap angled neck tissue culture flask and incubated for 24h at 37⁰C and 5% CO₂ conditions. Then the media was removed and 5mL of 400µM PA (dissolved in 1.6mM NaTC solution in media) was added. The flasks were incubated for 6h and 24h, then seen and imaged (10X lens) under an inverted microscope.

 

Fig – 1 Cell viability results of the NaTC against SVG-P12 and U87 MG cell lines following 48h incubation. Each set of data is the mean (±SEM) from 3 independent samples (N=3)

 

NaTC showed no apparent cytotoxicity at low concentrations (0.2mM – 2 mM against both SVG-P12 and U87 MG cell lines. However, at 10 mM and higher concentrations, NaTC was found to cause significant cytotoxicity to both the cell lines tested. Based on these results NaTC at 2 mM or lower concentrations were chosen as a solubilising agent for PA for subsequent studies.

Fig 2: Cell viability studies of PA (50µM to 500µM) on SVG-P12 cell lines and U87 MG cell lines following 48h incubation. At a molar ratio of 1:4, concentrations of PA (50µM; 100µM; 200µM; 300µM; 400µM and 500µM) dissolved in respective concentrations of NaTC (0.2mM; 0.4mM; 0.8mM; 1.2mM; 1.6mM and 2mM) were tested. Each set of data is the mean (±SEM) from 3 independent samples (N=3)

(I)	(II)	(III)
(IV)	(V)	(VI)

Fig 3:The images taken by an inverted microscope of the cells cultured in a T25 flask and treated with 400µM PA dissolved in 1.6mM NaTC (Ratio 1:4). (I) Untreated SVG-P12 cells, (II) SVG-P12 cells treated for 6h, (III) SVG-P12 cells treated for 24 h. (IV) Untreated U87-MG cells, (V) U87-MG treated for 6h, (VI) U87-MG treated for 24h.

Conclusion

This study shows that PA exhibits potential cytotoxicity at concentration of 200 µM and above when tested in vitro on U87 MG cells. In addition, PA exhibited higher cytotoxicity towards U87 MG than SVG-P12 at higher concentrations. The study shows some promising results of PA reducing cell viability of U87 MG concentrations above 200 µM and warrants further investigations.

Recommendation(s)

Lipid microparticles could probably be further investigated as carrier for the food grade chemical mixture supplied by PIP. Suitability of various other lipids and/or surfactants need to be explored to arrive at the appropriate formulation with better particle size and flow properties. A suitable analytical method needs to be developed for quantitation of ascorbyl palmitate in the food grade chemical mixture to determine its content and carry out dissolution studies. Palmitoyl ascorbate has shown cytotoxicity towards human glioblastoma cell line (U87 MG). In addition, it would be of interest to determine its mechanism of action. Further work on determination of cytoxicity on other cancer cell lines may also be of interest.