Materials and methods

The following section describes the general methods and routine protocols employed in performing the experiments, to test the effectiveness of the YSH on inhibiting the proliferation of cancer cells.

2.1.1 Cell Lines and tissue culture

MCF-7 (human breast adenocarcinoma, estrogen receptor positive), MDA-MB-231 (human triple-negative breast adenocarcinoma, p53-/-, mutant k-ras), and HeLa (human cervical adenocarcinoma) cells were all obtained from the American Type Culture collection, ATCC, Manassas, VA). The HBL-100 (non-malignant breast epithelial cells) were obtained from Dr K.M. Yamada, National Institutes of Health). These cell lines were maintained in Dulbecco’s Modified Essential Medium (DMEM, Hyclone Logan UT) supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone), 100 µg/ml streptomycin, and 100 U/ml penicillin (Invitrogen, Burlington, ON). The cells were cultured at 37^o C in 5% CO2. The THP-1 (peripheral blood acute monocytic leukemia) cells were obtained from ATCC and cultured in RPMI 1640 media supplemented with 10% FBS, 100 µg/ml streptomycin, and 100 U/ml penicillin.

2.1.2 Camptothecin

Camptothecin was dissolved in DMSO to obtain a stock solution of 10 mM for use as a positive control in the cytotoxicity analysis experiments. The stock solution was kept at -20° C.

2.1.3 Honey samples

Three Yemin sidr honey samples were supplied by different commercial vendors. Two of these samples were obtained from commericial markets in the Kingdom of Saudi Arabia while

the third sample was purchased from a company in the United Kingdom that sells characterized and verified product (Lote & Co., UK). The honey samples were stored at ambient temperature until required for further use. The age of the honey samples ranged from 1 to 5.5 years.

2.1.4 Preparation of honey media

Honey samples were weighed using a top-pan balance. Honey samples were diluted in serum-free culture medium (10 ml) to give a final concentration of 10% (w/v). The samples were then incubated at 37° C for 15 min to dissolve the honey into the media. Once the honey had dissolved, further dilutions were carried out in complete culture medium as described in the specific experimental procedures.

2.1.5 Treatment

The experimental design involved dividing the cell samples into test and control groups.

The YSH samples were freshly prepared for every experiment. The cells in the test group were treated with different concentration of YSH (ranging in final concentration from 0.1 - 10% (w/v) dissolved in complete culture (DMEM) media. The cells in the positive-treatment control group were treated with 10 µM camptothecin. In experiments where varying concentrations of YSH were used, cells in the negative control group were treated with a concentration of sugar (w/v) equivalent to the concentration of YSH used in that experiment. Another control group included in the experiment is a non-treated group which were grown in complete medium with no added YSH or glucose. Similar to the YSH-treated samples, the non-treated and control samples were collected at the same time points during every experiment. Moreover, the positive control groups varied with each experiment.

2.1.6 Sulphorodhamine b (SRB) assay

SRB assays are mainly performed to determine the relative cell density based on the cellular protein content. The protocol by Vichai and Kritikara (18) was adopted for SRB assays to measure the proliferation of cells treated with YSH or controls. For each experiment, 100 µl/well of each cell line was plated onto replicate 96-well plates: MCF-7, THP-1, and HLB-100 cells were suspended in media at 4000 cells/ml; and, HeLa, and MDA-MB-231 cells were suspended in media at 3000 cells/ml. The plates were then incubated for 24 h in an incubator containing 5% CO2 at 37° C and then the growth medium in each plate was replaced with a media containing different concentrations of YSH, sugar, or camptothecin, as indicated, and incubated for an additional 48 - 72 h. At the end of the experiment, the cells in each plate were fixed by incubation with 50% ice-cold tricholoracetic acid (TCA) and incubated at 4º C for 1 h, followed by repeated washing in running water and then the plates were air dried. The fixed cells were then stained by incubation in a 0.1% (w/v) SRB solution for 30 min at room temperature, and washed and then re-suspended in 200 μl of 10 mM Tris buffer, pH 10.5. The absorbance of each well was recorded at a wavelength of 530 nm using a plate reader (Molecular devices, Spectra max 340 PC) (18). The IC50 values for YSH were calculated using a sigmoidal dose-response curve (variable slope) using Graph Pad Prism V 4.02 software (Graph Pad Software, Inc.). The following formula was used to normalize the data:

(Mean OD sample - Tn) / Tp- Tn

(Where, Tp is mean O.D. of positive control and Tn is mean O.D. of negative control)

2.1.7 MTT assay (Methyl Tetrazolium Blue)

The methyl thiazol tetrazolium (MTT) assay was performed to measure cell viability and proliferation rate in cells treated with the YSH. Cancer cells were plated onto replicate 96-well plates at 2000 cells/well, with one plate per day of experiment (19), and incubated overnight in an incubator with 5% CO2 at 37° C. The cells were treated in complete culture media containing various concentrations of YSH or glucose as control. Each column of the 96 well plate (n = 8) was treated with the same conditions. The cells were incubated in the continued presence of the YSH over the course of 4 - 5 days without a media change. On each day of the experiment, one of the replica plates was treated by adding 10 μl of MTT solution in water into each well, at a final concentration of 0.25 µg/ml, and incubated for 4 h. Following the incubation, the media was removed and 100 μl/well of dimethyl sulfoxide (DMSO) was added to solubilize the converted formazan crystals. The absorbance of each well was read at 540 nm using a

(Spectramax 340PC 389) plate reader. To analyze the data, relative cell viability was determined by averaging the reading for each sample and then subtracting the culture medium background from each sample. The amount of absorbance is proportional to cell number. Statistical analysis for the absorbance corresponding to each experimental condition for each day of the experiments was performed using an ANOVA using Graph Pad Prism Software (19).

2.1.8 Cell Proliferation Using Incucyte® Proliferation Assays for Live-Cell Analysis

Cell proliferation assays are generally employed to evaluate the response of a cell population to external factors. The IncuCyte® Proliferation Assays for Live-Cell Analysis (Essen BioScience), was used in a series of cell proliferation experiments Label-free, direct cell

counting using the IncuCyteS3 cell analysis module allows cell proliferation to be quantified by counting the number of phase objects over time. By masking individual cells this enables a label-free count until the point that the cultures are densely packed and cell edges can not be

accurately determined. In addition, subsequent classification of cells into subpopulations can be performed based on properties including size and shape. For experiments, cells were collected, cell concentration determined, and then plated onto the wells of a 96 well plate and incubated at 37º C for 24 h. After incubation, these cells were treated with 1% (w/v) YSH, which is the

approximate IC50 concentration. The Incucyte was set to count the cells from 8 different areas for each sample well, for each day for 7 days, and the change in cell number was calculated and plotted for each condition.

2.1.9 Detection of Apoptosis by Acridine Orange/Ethidium Bromide

To examine changes in cellular morphology for MDA-MB-231 and MCF-7 cells treated with YSH, the cells were stained with acridine orange and ethidium bromide and visualized on a fluorescence microscope (20). The MDA-MB-231 and MCF-7 cells were grown on 6-well plates overnight at 37º C to allow for adherence of cells. Cells were then treated with different

concentrations of YSH in culture media for 24 and 48 h. For this experiment, cells were treated with 6 μg/ml of camptothecin for 24 - 48 h as a positive control group for apoptosis. Following treatment, each sample was then stained with 100 μg/ml acridine orange (Sigma-Aldrich) in culture media for 5 min and then stained with 100 μg/ml of ethidium bromide (Sigma-Aldrich) for 5 min. Finally, the cells were washed with PBS, pH 7.4, and then each sample was

visualized one at a time, as they were live and not fixed. Pictures were taken using an OLYMPYS fluorescence microscope (21).

2.1.10 Analysis of Cell Cycle Distribution

The cells were stained with the DNA stain propidium iodide and analyzed using flow cytometry to detect apoptotic cells with fractional DNA content and to identify the distribution of cells in the three major phases of the cell cycle (G1 vs S vs G2/M) (22). The following protocol was adopted in order to analyze the cell cycle distribution. MCF-7, MDA-MB-231, Hela, B16-BL6, and HBL-100 cells were exposed to YSH, glucose, or camptothecin for different time intervals, ranging from 24 h to 72 h and then harvested using trypsin. The collected cells were washed twice in PBS, pH 7.4, and fixed overnight by incubation in 75% ice-cold ethanol and stored at -20º C until analysis. On the day of analysis, the cells were collected by centrifugation and washed twice with PBS, pH 7.4. The cells were suspended in 0.5 ml PBS, pH 7.4, and 0.5 ml propidium iodide (PI) staining solution (PBS, pH 7.4, 0.3% Nonidet P-40, 100 μg/ml RNase A, and 100 μg/ml propidium iodide) was added and incubated for 1 h. The re-suspended samples were then analyzed by flow cytometry using an FC600 Flow Cytometer (Beckman Coulter) (22).

Flow profiles of ten thousand events were collected and gated to accurately measure PI intensity.

propidium iodide fluoresces at 623 nm when excited and single parameter displays were

obtained using the flow cytometric acquisition software, and the FL3-gated fluorescence signals were recorded and the proportion of cells in the sub-G1, Go/G1, S, and G2/M phases were determined.

2.1.11 Protein Extraction and Quantification

Proteins were extracted and quantified, from cultured cells treated with YSH, glucose, and control following the protocol by Santi and Lee (23). Cells were plated on 10 cm plates (Sarstedt) and incubated at 37º C for 24 h before exposing them to different concentration of

YSH. Post-treatment, exposed cells were collected at different time points (0, 24, 48, and 72 h).

Whole cell extracts were prepared by collection in RIPA buffer (PBS, pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with a protease cocktail tablet (Roche Diagnostics), 2 μM sodium orthovanadate, and 10 μM sodium fluoride. After incubating the cells in RIPA buffer on ice for 10 min, the lysates were sheared by passagethrough a 20 Ga needle, and then the cell lysates were cleared by centrifugation (Beckman Coulter, microfuge 22R) at 4º C for 15 min at 14,000 x g (23). Finally, protein quantification was carried out using a BCA Assay kit (Pierce Chemical, Fisher) and the absorbance of the samples and known concentrations of a BSA standard were read at 562 nm using a Spectramax/340 plate reader. The concentration of unknown proteins was analyzed against the BSA standard curve using the GraphPad Prism version 4.03.

2.1.12 SDS-PAGE

Protein samples, corresponding to 25 µg or 50 µg of cellular lysate protein, were subjected to electrophoresis on polyacrylamide gels containing sodium dodecylsulfate (SDS-PAGE), as described by Laemmli (24). Samples were prepared by adding 5X loading buffer (0.1 M Tris-HCl, pH 6.8, 10% [w/v] SDS, 40% [v/v] glycerol, 0.1% bromophenol blue), and heated for 5 min to denature the proteins (24). The samples were loaded per well onto a 6 – 15%

polyacrylamide gel, and the proteins were separated by electrophoresis in 1X SDS Running buffer (25 mM Trizma base, 192 mM glycine, 0.1% SDS) at 90 V for approximately 90 min. The proteins in the gel were then transferred onto a PVDF membrane (GE Health Care) which had been immersed in Transfer buffer (48 mM Tris, 39 mM glycine, 20% [v/v] methanol, 0.037%

[w/v] SDS), using a semidry transfer apparatus at 12 volts for 1 h. Transfer was verified by staining the blot with 0.1% Ponceau S in 1% acetic acid and destained in water.

2.1.13 Western Blotting

Western blot analysis was used to identify specific proteins from the complex mixture of proteins extracted from cells. The membrane was “blocked” by incubating it in Blocking buffer, 5%

Carnation non-fat skim milk in TBST (50 mM Tris-HCl, pH 7.4, 150 M NaCl, 0.1 % [v/v]

Tween 20), for 1 h. The membrane was washed with TBST before incubating it with primary antibodies against Bcl-2, Bax, Caspase 9, and PARP-1 and GAPDH and β-tublin (loading

controls) (Santa Cruz Biotech., Santa Cruz, CA) overnight (diluted in 5% Carnation non-fat skim milk, in TBST) at 4º C. After that, the blot was washed three times in TBST, and then incubated with a secondary antibody-horse radish peroxidase conjugate (Santa Cruz Biotech) (diluted in 5% non-fat skim milk in TBST) for 1 h at room temperature. This was followed by two more washes with TBST, and one wash with TBS. The horseradish peroxidase-labelled target proteins were visualized using an ECL kit (Enhanced Chemiluminescence, GE Health care) and exposed to X-Ray film.

2.1.14 Wound Healing

Cancer cells (5 × 10⁵ cells/well) were seeded in 6-well plates and grown to form a confluent monolayer for 24 h. A sterile 500 μl pipette tip was held vertically to scratch a cross in each well, Liang et al. (25),(26). The detached cells were removed by washing the well with 500 μl of fresh medium. YSH was added immediately after the wound was created and the plates incubated for 72 h. The scratch closure was monitored and imaged over the period of 24 - 72 h using the IncucyteS3 Live imaging protocol at 4 x magnification and images obtained and

time-lapse videos created for some experiments (Essen BioScience). The recovery of the scratched area can be calculated using commercially available Image J image analysis software (27),(28).

The migration of cells toward the wounds was expressed as percentage of wound closure:

% of wound closure = [(At = 0 h – At = ∆ h)/At = 0 h] × 100%,

where, At = 0 h is the area of wound measured immediately after scratching, and At = Δh is the area of wound measured 18 or 24 h after scratching.

2.1.15 Statistical Analysis

For analysis of each treatment group, data was reported as a mean and standard error of the mean (SEM). IC50 values were calculated using a sigmoidal-dose response curve which was generated by GraphPad Prism V.402. Values are means of triplicates of two or three independent experiments.