Dulbecco’s Modified Eagle’s Medium (DMEM) Medium from Gibco Invitrogen, USA; penicillin/streptomycin from Nacalai Tesque Inc., Kyoto, Japan; fetal bovine serum (FBS) from Tico Europe, Amstelveen, Netherlands; phosphate buffer saline (PBS), (3-(4,5-dimetiltiazol-2-il)-2,5-difenil tetrazolium bromide) (MTT) from Sigma-Aldrich, UK; dimethylsulphuxide (DMSO) and hydrochloric acid (HCl) form Fisher Scientific, UK; sodium hydrogen carbonate (NaHCO3) and potassium hydrogen phosphate (KH2PO4) from Systerm, Malaysia; sodium chloride (NaCl) and sodium dihydroxide (NaOH) from EMSURE, Jerman; disodium hydrogen phosphate (Na2HPO4) from HmbG Chemicals, German; potassium chloride (KCl) from J.T Baker, USA; dihidroethidium (DHE) stain and dichlorofluorescin-diacetate (DCFH-DA) stain from Eugene; Disodium ethylenediaminetetraacetate dihydrate (Na2EDTA), low-melting point agarose (LMA) and normal melting agarose (NMA) from Sigma-Aldrich, St. Louis, MO, USA; Tris from Bio-Rad Laboratories, Hercules, CA, USA; 70% alcohol and distilled water from the FSK laboratory.
Compounds 4-Peperidinone,3,5-bis[(4-hydroxy-3-methoxyphenyl) methylene]-,(3E,5E) (FLDP-5) and 4-Peperidinone, 3,5-bis[(4-hydroxy-3-methoxyphenyl) methylene]-1-Methyl(3E,5E) (FLDP-8) were synthesized and contributed by Dr. Lam Kok Wai from Centre for Drug and Herbal Development, Faculty of Pharmacy, Universiti Kebangsaan Malaysia (Kuala Lumpur, Malaysia). Curcumin and hydroquinone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of FLDP-5 and hydroquinone were prepared at 50 mM, while stock solutions for FLDP-8 and curcumin were prepared at 25 mM. All compounds were dissolved in solvent dimethyl sulfoxide (DMSO). Treatment for all compounds on LN-18 cells was performed in dark condition due to the compounds’ photosensitive characteristics.
LN-18 human GBM cells were established in 1976 from cells taken from a patient with a right temporal lobe glioma whereas HBEC-5i cells were established in 1994 which were derived from small fragments of human cerebral cortex obtained from patients who had died of various causes and were devoid of any pathologic abnormalities. Both cell lines were obtained from American Type Culture Collection (ATCC). The culture medium used throughout these experiments was Dulbecco’s Modified Eagle’s Medium (DMEM) medium (GIBCO) for both cell lines supplemented with fetal bovine serum (FBS) (5% for LN-18 cells and 10% for HBEC-5i cells) and 1% penicillin/streptomycin. Both LN-18 and HBEC-5i cells were used between passages 3-12 for the experiments and maintained at 37°C
and 5% CO2.
MTT cytotoxicity assay
The cytotoxic effects of curcumin and the curcuminoid analogues (FLDP-5 and FLDP-8) were assessed as previously described (Mosmann 1983). Briefly, LN-18 cells were seeded in a 96-well plate at a density of 5 × 104 cells per well in a volume of 200 µL and were treated with curcuminoid analogues (FLDP-5 and FLDP-8), curcumin and hydroquinone (positive control) with respective concentrations. The treated cells were incubated for 24-h under 5% CO2 at 37°C After 24-h incubation, 20 µL of 5 mg/mL MTT was added to each treated cells and further incubated for 4-h at 37°C . MTT salt was reduced forming a purple coloured crystal formazan by the active viable cells with dehydrogenase enzyme. Then, 180 µL of the medium was discarded from the treated cells before adding 180 µL of dimethyl sulfoxide (DMSO) to solubilize the crystal formazan, respectively. The plate was further incubated for 15 min to completely dissolve the crystal formazan, followed by gentle resuspension for each well. The cytotoxic effects of each compound were detected by measuring the absorbance of each well at 570 nm using iMarkTM microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The inhibitory concentration that kills 50% of the cell population (IC50) was obtained from the plotted of each compounds concentrations versus the percentage of the cell viability.
The selective index (SI) of the compounds were calculated according to the equation as previously established (Popiołkiewicz et al. 2005). The calculation is done in order to determine the degree of selectivity of the compound tested against cancerous cells, in which values larger than “100” indicates the compound is selective toward cancerous cells and confers minimal toxicity in normal non-malignant cells. In this study, the SI values were determined by IC50 of normal human cerebral microvascular endothelial cell (HBEC-5i) divided by IC50 of LN-18 cells for each compound. It was simplified as follows:
Blood-brain barrier (BBB) and ADMET prediction
PubChem database was applied to get the smiles structures of FLDP-5 and FLDP-8 curcumioid analogues, and was further used for the BBB and ADMET prediction using two different online platforms, AlzPlatform (www.cbligand.org/AD/) and ADMETlab 2.0 (https://admetmesh.scbdd.com/) (Hu et al. 2020; Liu et al. 2014; Xiong et al. 2021; Zhao et al. 2007). AlzPlatform was built by using the support vector machine (SVM) and LiCABEDS algorithms on PubChem fingerprint from 1593 reported compounds. By entering the smiles structures of the compounds, the online predictor calculated the BBB score, which showed whether a compound could pass the blood-brain barrier (BBB+) or not (BBB-). ADMETlab 2.0 which is an integrated online platform, was used to computationally predict the pharmacokinetic properties of compounds such as absorption, distribution, metabolism, excretion, and toxicity (ADMET).
Reactive oxygen species assessment
The level of reactive oxygen species (ROS), specifically for superoxide anion and hydrogen peroxide were assessed as previously described (Chan et al. 2010; Ooi et al. 2021). Briefly, the treated LN-18 cells were administered at different time-point intervals before being harvested. The treated LN-18 cells were then collected by centrifugation at 220 × g for 5 mins. After the supernatant was discarded, the pellet was resuspended with 1 mL of fresh pre-warmed FBS-free DMEM media and with the addition of 1 µL of 10 mM DHE and 10 mM DCFH-DA stains. The cells suspension with DHE and DCFH-DA staining were incubated at 37°C
for 30 min. After the incubation period, the cells were centrifuged at 220 × g for 5 min. Then, the cells were washed with 1 mL chilled PBS, and the supernatant was discarded, followed by resuspension of the pellet by 500 µL of ice-cold PBS. The stained cells were transferred to flow cytometric analysis tubes and analyzed using FACSCanto II flow cytometer (BD Bioscience, USA) on 10,000 cells.
Alkaline comet assay
As previously described, the alkaline comet assay was performed to access DNA damage induced by curcumin and the curcuminoid analogues (FLDP-5 and FLDP-8) (Chan et al. 2006; Tan et al. 2020). Treated LN-18 cells were harvested and washed twice with Ca2+, Mg2+-free PBS. Cell pellets were then mixed thoroughly with 80 µL of 0.6% low melting point agarose (LMA) and laid on hardened 0.6% normal melting agarose (NMA). The agarose was allowed to solidify and subsequently placed in a chilled lysis buffer (2.5 M NaCl, 100 Mm EDTA, 10 mM Tris, and 1% Triton-X) for lysis to occur. Slides were then incubated in an electrophoresis buffer (0.3N NaOH, 1 Mm EDTA) for 20 minutes to facilitate DNA unwinding. Electrophoresis was performed under 25 V, 300 Ma for 20 minutes. Subsequently, slides were rinsed with neutralizing buffer (400 Mm Tris) thrice prior to staining with 50 µg/mL ethidium bromide solution. The slides were observed under Olympus BX51 fluorescence microscope (Olympus, Japan) equipped with 590 nm filter. The tail moment, the product of tail length and fraction of total DNA in the tail, was assessed using Comet ScoreTM software (TriTek Corp, Sumerduck, VA, USA) on 50 cells per slide.
Scratch/ wound-healing assay
A monolayer wound healing assay was performed following the protocol from previous studies with slight modifications (Nordin et al. 2021; Senft et al. 2010). LN-18 cells were seeded at a density of 2 × 105 cells per well in a 12-well plate (Nest Biotechnology, Jiangsu, China). After reaching 90% confluency, the cells monolayer were then scraped in a straight line creating a “scratch” using a sterile 200 uL pipette tip. The cells were then washed with PBS before taking photographs of the scratched area using a camera attached to an inverted phase contrast microscope (Olympus, Japan). The scratch area were photographed, and the location on the plate was noted. The cells were then treated with FLDP-5 curcuminoid analogue (1.25 and 2.5 µM), FLDP-8 curcuminoid analogue (2.5 and 5 µM) and curcumin (12.5 and 25 µM). Untreated cells were used as a control for the experiment. Cells were further incubated for 24-h and 48 h before the same area were photographed, and the cells migration area was measured using Image J software before the percentage of wound closure was calculated.
Boyden chamber invasion assay
The principle of this assay is based on two medium containing chambers separated by a porous membrane through which cells transmigrate (BOYDEN 1962). This assay was conducted following the protocol provided by QCMTM Collagen Cell Invasion Assay, 24-well (8 μm), Colorimetric kit purchased from Merck, Germany. Generally, LN-18 cells were starved for 24-h prior to assay in serum-free DMEM medium. Then, 250 μL of harvested cell suspension with concentration of 7 × 105 cells/mL in chemo-attractant free media was added to the insert/upper chamber of the well containing collagen-coated membrane. After that, 500 μL of DMEM containing respective compounds treatment will be added to the bottom chamber of the well. After 24-h incubation, the insert coated with the membrane will be fixed and stained with 400 μL Cell Stain. A cotton-tipped swab was used to remove the non-invading cells/collagen layer from the interior of the insert. The stained insert was transferred to a new well containing 200 μL of Extraction Buffer, and 100 μL of the dye mixture was transferred into a 96-well plate. The Optical Density (OD) of invaded cells were measured using iMarkTM microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at 560nm.
Cell cycle analysis
Cell cycle distribution was determined following protocol as previously described (Leong et al. 2016). Cells were seeded at 2 × 105 cells per well in a 6-well plate before being treated with curcuminoid analogues (FLDP-5 and FLDP-8) and curcumin for 24-h. The treated cells will be harvested and washed with chilled PBS before being fixed with 70 % alcohol for at least overnight before staining. After fixing, cells will be washed with PBS and later stained with PI/RNase staining buffer (500 μL) (BD Bioscience) for 15 minutes at room temperature. Stained cells will then be analysed by using FACSCanto II flow cytometer (BD Bioscience, USA) on 20,000 cells, and the content of DNA will be determined by using ModFit LT™ software (Verity Software House).
The data are expressed as the mean ± standard error of mean (S.E.M.) from at least three independent experiments. The statistical significance was evaluated using one-way ANOVA with the Dunnet post hoc test to assess significance difference with negative control (NEG) or Tukey post hoc test to determine the significance of differences between multiple treatment groups. Differences were considered statistically significant with a probability level of p<0.05.