2.1 Plant source
C. nutans obtained from HERBagus Sdn Bhd, Malaysia was recognised by the Herbarium Department of the School of Biological Sciences, Universiti Sains Malaysia with voucher number assigned was 11536 (Fig. S1). Additionally, C. nutans was authenticated using deoxyribonucleic acid (DNA) barcoding markers with accession numbers of KX014785.1 to KX014787.1 (matK) and KU985403.1 to KU985412.1 (rbcL and psbA-trnH) (Ismail et al. 2018).
2.2 Subfractionation of CN-Dcm extract
The CN-Dcm extract was prepared based on the method described by Ismail et al. (2020). In this study, CN-Dcm extract (10 g) was chromatograped using 0.040 to 0.063 mm silica gel (Merck, Darmstadt, Germany) packed into the column chromatography (40 mm) (Fig. 1). The slurry silica gel was prepared by dissolving the silica gel with n-hexane solvent. The CN-Dcm extract was then added to the packed column at the top. The column was then subjected to solvent systems containing varying ratios of n-hexane to ethyl acetate (H:EA). Following that, a column was filled with another solvent mixture, chloroform to methanol (CHL:MEOH). Finally, 100% methanol was flushed into the column. A small eluent from subfraction extracts was placed on a thin-layer chromatography (TLC) plate (Merck, Darmstadt, Germany) and those that had similar retention factor (Rf) values were pooled together. Then, the TLC plate was sprayed with vanillin with sulphuric acid (Qrec (Asia), Malaysia). The vanillin (15 g) was dissolved in 250 mL of ethanol. The dissolved vanillin was diluted with 2.5 mL of sulphuric acid. The TLC plate was dried for 15 to 20 mins in a 100°C hot air oven. The subfraction extracts were lyophilised and stored at -80°C until needed.
2.3 Cell culture
Both cells were acquired from the American Type Culture Collection (Manassas, Virginia, USA). The normal breast, MCF 10A cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Paisley, UK) complemented with 0.5 mg/mL hydrocortisone (Gibco, Paisley, UK), 20 mg/mL epidermal growth factor (Gibco, Paisley, UK), 5% horse serum (Gibco, Paisley, UK), 1% PenStrep and 10 µg/mL insulin (Gibco, Paisley, UK). The MCF7 cancer cells were cultured in the Roswell Park Memorial Institute Medium 1640 (RPMI-1640) supplemented with 10% (v/v) fetal bovine serum (FBS) (Nacalai Tesque, Kyoto, Japan) and 1% (v/v) PenStrep (Gibco, Paisley, UK). Both cells were incubated with 5% carbon dioxide (CO2) in a 37°C incubator. When the cells reached confluence, the old medium was decanted from the flask. An amount of 3 mL phosphate-buffered saline (PBS) (Nacalai Tesque, Kyoto, Japan) was transferred to the flask in order to remove the old media and dead cells. The flask was then filled with 200 µL of 0.25% Trypsin-EDTA (Gibco, Paisley, UK) and incubated for 5 mins in the incubator to detach the cells. The trypsinization process was significantly enhanced by delicately tapping the flask. In order to inactivate the trypsin, 3 mL of medium was subsequently added to the flask and centrifuged for 5 mins at 300 x g. After removing the supernatant, the cell pellet was resuspended with a new medium.
2.4 MTS assay
Both cells with density of 1 × 104 cells/mL were added to a 96-well plate and incubated for 24 h. The extracts were resuspended in 100% dimethyl sulfoxide (DMSO). The MCF7 cells were treated with serial diluted crude and fraction extracts (0 - 300 µg/mL) and subfraction extracts (0 - 200 µg/mL). In addition, 0.1% DMSO was used as a negative control while tamoxifen (Nacalai Tesque, Kyoto, Japan) was served a positive control. To ensure that the maximum concentration of DMSO in the treatment of cells was within the range of 0.1–0.5% (Scambia et al. 1994), the 0.1% of DMSO working solution was used. Additionally, MCF 10A cells have been treated to measure the selectivity index (SI) values. The MCF 10A cells were treated with serial dilution ranging from 0 to 1200 µg/mL for crude and fraction extracts and 0 to 1000 µg/mL for subfraction extracts.
The 20 µL of MTS solution (Promega, USA) was added to each of the 96 well-plates after 72 h. Then, the cells were incubated in the incubator for 4 h. The antiproliferative activities were obtained by measuring the cells viability at an absorbance of 490 nm using a microplate reader (OMEGA BMG Labtech, Malaysia). The treatment was repeated in triplicate and the cell viability was calculated using the following formula: Cell viability (%) = Absorbance (Treated cells – Untreated cells)/Absorbance (Untreated cells – Untreated cells) × 100%. The values are expressed as the mean ± standard deviation (SD). The half-maximal inhibitory concentration (IC50) of both cells were determined.
2.5 Selectivity Index (SI)
As described by Robertson et al. (2003), the SI value was calculated using the subsequent equation: SI = IC50 of MCF 10A / IC50 of MCF7. The extract with the highest SI value was selected for further analysis. The most effective extract, with the greatest antiproliferative effect on MCF7 cancer cells, was used in an additional cell viability assay with incubation periods that were varied (24, 48, and 72 h).
2.6 Cell morphology of MCF7
The MCF7 cells (7.5 × 104 cells/mL) were cultured in a 6-well plate and allowed to grow in a complete medium for 24 h. The dose concentrations of IC50 from the selected extract and the positive control, tamoxifen, were subsequently transferred to MCF7 cells and incubated for 72 h. Then, the cells were stained with acridine orange/propidium iodide (AO/PI) dual-fluorescence dye (Sigma Aldrich, USA) (10 µg/mL) and incubated for 15 mins. Afterwards, 1 mL of PBS was added to each well twice to wash the stained cells. Finally, an inverted fluorescence microscope (Olympus, USA) was used to capture the cell morphology of the stained MCF7 cells at 40× magnification.
2.7 Apoptosis analysis
Briefly, the MCF7 cells (2 × 105 cells/mL) were cultured in a 25 cm2 culture flask at 24 h. The dose concentration of the IC50 of the selected extract and tamoxifen were added to the flask. The MCF7 cells were treated for 72 h. The apoptotic effect was carried out at 1x105 cells/mL cell density. Annexin V-FITC kit (Miltenyi Biotec, Germany) were used to evaluated the apoptotic effect of treated cells. The apoptosis effect was analysed using a flow cytometer (Moflo XDP Cell Sorter, Beckman Coulter, USA).
2.8 Western blot
The MCF7 cells with a density of 7.5 × 105 cells/mL were grown in the 75 cm2 culture flasks for 24 h. The IC50 of the selected extract and tamoxifen were used to treat the cells for 72 h. The old media was decanted after treatment, and the attached cells were washed with cold PBS. The radioimmunoprecipitation assay (RIPA) buffer with 1% protease inhibitor cocktail (Cell Signaling Technology, USA) were used to extract protein from MCF7 cells. The 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the protein according to its molecular weight. Then, the protein bands were transferred to a polyvinylidene fluoride (PVDF) membrane (Axon Scientific, Malaysia). The 5% blocking buffer (skimmed milk) (SunLac, Malaysia) was used to block the PVDF membrane for 1 h. Then, the PVDF membrane was washed with Tris-buffered saline with 0.1% Tween 20 detergent (TBST). The membrane was washed for 10 mins with gentle agitation four times. The membrane was blotted with diluted primary antibodies overnight at 4˚C. The primary antibodies were β-actin (PA1-183), BID (PA5-29159) and BCL-2 (PA5-20068) were obtained from Invitrogen, USA and P53 (ab1431), BAX (ab7977), caspase-9 (ab25758), caspase-3 (ab13847) and caspase-8 (ab25901) were obtained from Abcam, UK. The next day, the PVDF membrane was rinsed four times with TBST. Anti-rabbit IgG, horseradish peroxidase-linked antibody (7074) (Cell Signaling Technology, USA) was used as a secondary antibody to blot the membrane for 1 h. In order to determine the targeted proteins, the enhanced chemiluminescence (ECL) clarity reagent (Bio-Rad, USA) was added to the blotted membrane and eventually the protein bands were captured by the Versa Doc Imaging System (Bio-rad, USA). The ImageJ was used to analyse the band intensities and fold changes (Rueden et al. 2017). The following calculation was made using the equations: Normalized band intensity = band intensity of target protein ÷ band intensity of β-actin
The MCF7 cells with a concentration of 2 × 105 cells/mL were cultured in the 25 cm2 culture flasks for 24 h. The cells were treated for 72 h with the IC50 of the selected extract and tamoxifen. After treatment, the ribonucleic acid (RNA) was isolated using the RNeasy Mini Kit (Promega, USA). The purity of RNA was measured using a Nanodrop® ND100 spectrophotometer (Thermo Fisher Scientific, USA). The complementary DNA (cDNA) was synthesised according to the Tetro™ cDNA Synthesis kit (Meridian Bioscience, USA). The SensiFAST™ SYBR® Hi-ROX kit (Meridian Bioscience, USA) was used to determine the expression of P53, BID, BCL-2, BAX, caspase-9, caspase-3 and caspase-8 genes in treated MCF7 cells. The β-actin gene was used as an internal control. The gene sequences obtained from De et al. (2019), Devarajan et al. (2002), Ferreira and Cronjé (2012), Quispe-Soto et al. (2016) and Baharara et al. (2015) were presented in Table S1. The final volume of each reaction mixture was 10 µL, comprised of 5µL of 2x SensiFAST SYBR® Hi-ROX Mix, 1 µL of synthesised cDNA (10 ng/µL), 1 µL of reverse and forward primers of each gene (10 µM) and 3.2 µL of sterile distilled water. The reaction for each gene was biologically repeated three times. The StepOne Plus™ Real-Time PCR System instrument (Applied BioSystems, USA) was set up to run under the following program: an initial denaturation step at 95°C for 2 mins followed by 40 cycles at 95°C for 5 s, 60°C for 5 s, and 72°C for 20 s. The gene expression was measured using comparative CT (ΔΔCT) to obtain the fold change of targeted genes between the treated and untreated MCF7 cells.
The 5mg/mL of selected extract was mixed in 1 mL of methanol and filtered using a 0.45 µM polytetrafluoroethylene filter. Then, the extract was conducted using an Agilent 1290 LC-QTOF-MS system consisting of an Agilent 1290 Infinity liquid chromatography system with the 6520 Accurate Mass Quadrupole Time of Flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The LC separations were conducted using an Agilent Zorbax Eclipse XDB-C18 column (2.1 × 150 mm, 3.5 µm) at 25°C. The mobile phase consisted of 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B) was used. The parameters included: fragmentor voltage, 125 V, N2 gas flow rate, nebulizer, 45 psig and 10 L/min; N2 temperature, 300°C. The mass range in positive modes was recorded between m/z 100 and 3200. Additionally, the mass spectra for Agilent MassHunter Qualitative Analysis have been sorted out with an add-in to the METLIN database. The correct elemental composition structure has been produced with an accurate m/z for compound identification (Agilent Technologies, Santa Clara, CA, USA) (Sana et al. 2008).
2.11 Statistical analysis
For statistical computing, the statistical analysis was carried out using PRISM Graph Pad version 8.0. Analysis of variance (ANOVA) was used to calculate the significance of differences through comparison of the mean values. The p-value of less than 0.05 was statistically significant.