Gold nanoparticles-induced cytotoxicity in triple negative breast cancer involves different epigenetic alterations depending upon the surface charge

Gold nanoparticles (AuNPs) are used enormously in different cancers but very little is known regarding their molecular mechanism and surface charge role in the process of cell death. Here, we elucidate the molecular mechanism by which differentially charged AuNPs induce cytotoxicity in triple negative breast cancer (TNBC) cells. Cytotoxicity assay revealed that both negatively charged (citrate-capped) and positively charged (cysteamine-capped) AuNPs induced cell-death in a dose-dependent manner. We provide first evidence that AuNPs-induced oxidative stress alters Wnt signalling pathway in MDA-MB-231 and MDA-MB-468 cells. Although both differentially charged AuNPs induced cell death, the rate and mechanism involved in the process of cell death were different. Negatively charged AuNPs increased the expression of MKP-1, dephosphorylated and deacetylated histone H3 at Ser10 and K9/K14 residues respectively whereas, positively charged AuNPs decreased the expression of MKP-1, phosphorylated and acetylated histone H3 at Ser 10 and K9/K14 residues respectively. High-resolution transmission electron microscopy (HRTEM) studies revealed that AuNPs were localised in cytoplasm and mitochondria of MDA-MB-231 cells. Interestingly, AuNPs treatment makes MDA-MB-231 cells sensitive to 5-fluorouracil (5-FU) by decreasing the expression of thymidylate synthetase enzyme. This study highlights the role of surface charge (independent of size) in the mechanisms of toxicity and cell death.


1) Experimental procedures a) Synthesis of citrate-capped AuNPs
Citrate-capped AuNPs were prepared by the reduction of Gold (III) chloride trihydrate (HAuCl4.3H2O) with trisodium citrate according to the Turkevich and Frens method 1,2 .
Briefly, the reaction was carried out in a 250 mL round bottomed flask with the centre neck attached to a reflux condenser. First, 250 mL of 0.25 mM HAuCl4.3H2O solution was heated to boiling. Then, 4 mL of aqueous solution of 1% trisodium citrate was added to it under vigorous stirring and the boiling was continued for another 15 min until the solution turns to a deep red colour. During the reaction, the colour of the solution changed initially from yellow to colourless and finally to wine red. It was then stirred until it reached room temperature to control the particle size and thus achieving a narrow particle size distribution.

b) Synthesis of cysteamine-capped AuNPs
Cysteamine-capped gold nanoparticles were prepared by the reduction of gold (III) chloride trihydrate with sodium borohydride in the presence of cysteamine 3 . Briefly, 400 μL of 213 mM cysteamine hydrochloride was added to 40 mL of 1.42 mM gold (III) chloride trihydrate in a conical flask and then subjected to stirring for 20 min at room temperature. Sodium borohydride was dissolved in cold distilled water immediately before use. After 20 min, 10 μL of 10 mM NaBH4 was added quickly into the mixture solution. Vigorous stirring was maintained from the addition of NaBH4 to another 30 min. The colour of mixture solution during the reaction process was changed from yellow to brownish. After further mild stirring, the gold nanoparticle solution was stored in the dark condition at 4 C.

c) Zeta size, Zeta potential and TEM
Dynamic light scattering for characterization of hydrodynamic size of AuNPs dispersed in water was performed on Nano-ZS, Malvern Instruments, Malvern, UK, taking the average of 5 measurements. Zeta potential was also measured to determine the amount of aggregation of

d) Transmission electron microscopy (TEM) of AuNPs treated cells
Ultra-thin sections of cells were analyzed using TEM to see the distribution of AuNPs according to the modified method as described 5 . Briefly, cells were treated with AuNPs (100 μg/mL, 250 μg/mL and 500 μg/mL) for 24 h. At the end of incubation, the cells were washed with PBS to remove any unbound AuNPs. Cells were then fixed in 2.5% glutaraldehyde for 30 min at 4 C. Fixed cells were scraped and washed 5 times with PBS. Before dehydration with increasing concentrations of alcohol, cells were further treated with 1 % osmium tetroxide for 2 h at 4 C. Alcohol and spurr's low viscosity resin were used in the ratio 2:1, 1:1, 1:2 and 1:3.
Finally 100 % spurr resin was added and the beam capsule was incubated for 18 h at 70 C.
RMC ultra-microtome is used for cutting ultra-thin sections of 60 nm thickness. Sections were stained with 0.5% uranyl acetate and analyzed under FEI TF-20 TEM at 120 kV.

e) Isolation of total proteins and western blotting
After AuNPs treatment for 24 h, isolation of total proteins and histones, using 0.25 M HCl were done by the methods as described 6 . Protein estimation was performed by Lowry's method and then reduced by using 1X laemmli's sample buffer. Equal amounts were run on SDS PAGE and were electrophoretically transferred onto PVDF membrane using semi-dry transfer apparatus (Bio-Rad). Immunoblot analysis was performed using anti-phospho-p38 the membranes were stripped in stripping buffer and re-probed with another antibody. The immunoblots were quantified by densitometry scanning using NIH Image J software.

f) Total RNA isolation
Briefly, total RNA was extracted from cells using TRIzol reagent (Invitrogen, CA, USA) and was purified according to the manufacturer's protocol using an RNeasy kit (AuPrep RNeasy Mini Kit; Life Technologies Pvt. Ltd., India) as described 7 . The RNA quality and integrity of each sample was assured using NanoDrop spectrophotometer (ND-1000) by A260/280 absorbance ratio and agarose gel electrophoresis respectively.

g) Reverse transcription and RT-PCR
For checking the mRNA levels of genes, cDNA synthesis from RNA was carried out by using verso cDNA synthesis kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions as described 7 and then Quantitative real time-PCR was carried out by using Light Cycler 2.0 (Roche Diagnostics) according to the method as described 6