Chemicals
Erbium (III) oxide nanoparticles (Er2O3-NPs) were purchased from Sigma-Aldrich Chemical Company (Saint Louis, USA) with pink appearance and product number (203,238). Powders of Er2O3-NPs with 99.9 trace metals basis were suspended in deionized distilled water to prepare the required concentrations and ultra-sonicated prior use.
Cell line
Human hepatocellular carcinoma (Hep-G2) cells were obtained from Nawah Scientific Inc., (Mokatam, Cairo Egypt). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) media supplemented with streptomycin (100 mg/mL), penicillin (100 units/mL) and heat-inactivated fetal bovine serum (10) in humidified, 5% (v/v) CO2 atmosphere at 37 °C.
Characterization of Er2O3-NPs
The purchased powders of Er2O3-NPs were characterized using a charge coupled device diffractometer (XPERT-PRO, PANalytical, Netherlands) to determine its X-ray diffraction (XRD) pattern. Zeta potential and particles’ size distribution of Er2O3-NPs were also detected using Malvern Instrument Zeta sizer Nano Series (Malvern Instruments, Westborough, MA) equipped with a He–Ne laser (λ = 633 nm, max 5mW). Moreover, transmission electron microscopy (TEM) imaging was done to detect the shape and average particles’ size of Er2O3-NPs suspension.
Sulforhodamine B (SRB) cytotoxicity assay
Sulforhodamine B (SRB) assay was conducted to assess the influence of Er2O3-NPs on the proliferation of cancerous Hep-G2 cells12. Aliquots of 100 µl of Hep-G2 cells suspension containing 5 × 103 cells were separately cultured in 96-well plates and incubated for 24 h in complete media. Hep-G2 Cells were then treated with five different concentrations of Er2O3-NPs (0.01, 0.1, 1, 10 and 100 µg/ml) incubated for 24 h or (0.1, 1, 10, 100 and 1000 µg/ml) incubated for 72 h. After 24 or 72 h of Er2O3-NPs exposure, cultured cells were fixed by replacing media with 10% trichloroacetic acid (TCA) and incubated for one hour at 4 °C. Cells were then washed five times with distilled water, SRB solution (0.4% w/v) was added and incubated cells in a dark place at room temperature for 10 min. All plates were washed three times with 1% acetic acid and allowed to air-dry overnight. Then, protein-bound SRB stain was dissolved by adding TRIS (10 mM) and the absorbance was measured at 540 nm using a BMG LABTECH-FLUO star Omega microplate reader (Ortenberg, Germany).
Cells treatment
Cancerous Hep-G2 cells were cultured at the appropriate conditions and dived into control and treated cells. The control cells were treated with an equal volume of the vehicle (DMSO; final concentration, ≤ 0.1%), while the treated cells were treated with the IC50 of Er2O3-NPs. All cells were left for 72 h after nanoparticles treatment and were harvested by brief trypsinization and centrifugation. Each treatment was conducted in triplicate. Cells were washed twice with ice-cold PBS and used for different molecular assays.
Estimation of genomic DNA integrity
The impact of Er2O3-NPs exposure on the integrity of genomic DNA in cancerous Hep-G2 cells was estimated using alkaline Comet assay13,14. Treated and control cells were mixed with low melting agarose and spread on clean slides pre-coated with normal melting agarose. After drying, slides were incubated in cold lysis buffer for 24 h in dark and then electrophoresed in alkaline electrophoresis buffer. Electrophoresed DNA was neutralized in Tris buffer and fixed in cold absolute ethanol. For analysis slides were stained with ethidium bromide, examined using epi-fluorescent microscope at magnification 200× and fifty comet nuclei were analyzed per sample using Comet Score software.
Estimation of intracellular ROS generation
The effect of Er2O3-NPs exposure on intracellular ROS production in cancer Hep-G2 cells was studied using 2,7-dichlorofluorescein diacetate dye15. Cultured cells were washed with phosphate buffered saline (PBS) and then 2,7-dichlorofluorescein diacetate dye was added. Mixed cells and dye were left for 30 min in dark and spread on clean slides. The resultant fluorescent dichlorofluorescein complex from interaction of intracellular ROS with dichlorofluorescein diacetate dye was examined under epi-fluorescent at 20× magnification.
Measuring the expression levels of apoptotic and anti-apoptotic genes
Quantitative real time Polymerase chain reaction (RT-PCR) was conducted to measure the mRNA expression levels of apoptotic (p53 and Bax) and anti-apoptotic (Bcl2) genes in control and treated Hep-G2 cells. Whole cellular RNA was extracted according to the instructions listed by the GeneJET RNA Purification Kit (Thermo scientific, USA) (Thermo scientific, USA) and using Nanodrop device purity and concentration of the extracted RNAs were determined. These RNAs were then reverse transcribed into complementary DNA (cDNA) using the instructions of the Revert Aid First Strand cDNA Synthesis Kit (Thermo scientific, USA). For amplification, RT-PCR was performed using the previously designed primers shown in Table 116,17 by the 7500 Fast system (Applied Biosystem 7500, Clinilab, Egypt). A comparative Ct (DDCt) method was conducted to measure the expression levels of amplified genes and GAPDH gene was used as a housekeeping gene. Results were expressed as mean ± S.D.
Analysis of cell cycle distribution
Distribution of cell cycle was analyzed using flow cytometry. Control and treated cancer Hep-G2 cells with IC50 of Er2O3-NPs for 72 h were harvested, washed with PBS and re-suspended in 1 mL of PBS containing RNAase A (50 µg/mL) and propidium iodide (10 µg/mL) (PI). Cells were incubated for 20 min in dark at 37 C and analyzed for DNA contents using FL2 (λex/em 535/617 nm) signal detector (ACEA Novocyte flow cytometer, ACEA Biosciences Inc., San Diego, CA, USA). For each sample, 12,000 events are acquired and cell cycle distribution is calculated using ACEA NovoExpress software (ACEA Biosciences Inc., San Diego, CA, USA).
Estimation of apoptosis induction
Apoptotic and necrotic cell populations were determined using Annexin V- Fluorescein isothiocyanate (FITC) apoptosis detection kit (Abcam Inc., Cambridge Science Park Cambridge, UK) coupled with two fluorescent channels flow cytometry. After treatment with Er2O3-NPs for 72 h and doxorubicin as a positive control, Hep-G2 cells were collected by trypsinization and washed twice with ice-cold PBS (pH 7.4). Harvested cells are incubated in dark with Annexin V-FITC/ propidium iodide (PI) solution for 30 min at room temperature, then injected via ACEA Novocyte flowcytometer (ACEA Biosciences Inc., San Diego, CA, USA) and analyzed for FITC and PI fluorescent signals using FL1 and FL2 signal detector, respectively (λex/em 488/530 nm for FITC and λex/em 535/617 nm for PI). For each sample, 12,000 events were acquired and positive FITC and/or PI cells are quantified by quadrant analysis and calculated using ACEA NovoExpress software (ACEA Biosciences Inc., San Diego, CA, USA).
Statistical analysis
Results of the current study are expressed as mean ± Standard Deviation (S.D) and were analyzed using the Statistical Package for the Social Sciences (SPSS) (version 20) at the significance level p < 0.05. The student t-test was used to compare between the untreated and treated cancer Hep-G2 cells.
Source: Ecology - nature.com
