Preparation of LD
LD was obtained from a local TCM practitioner (London, UK). This formulation of LD, which was in capsule form, contains: Radix gentianiae (Long Dan), Radix bupleuri (Cai hu), Rhizoma alismatis (Ze xie), Semen plantaginis (Che qian zi), Radix rehmanniae (Di huang) (the approximate relative quantity of each of these herbal constituents in this formulation of LD is 14.29%) Fructus gardeniae (Zhi zi), Radix scutellariae (Huang qin) Radix angelicae sinensis (Dang gui) Radix glycyrrhizae (Gan cao), Medulla tetrapanacis (Tong cao) (the approximate relative quantity of each of these herbal constituents in this formulation of LD is 7.1%). So as to adhere as closely as possible to the traditional method of preparation and ingestion of this CHR, aqueous extracts were prepared as follows: LD was obtained in tablet form and was therefore not boiled, instead 3 g of the contents of each pill were crushed then incubated in water (30 ml) at 37°C for 1 hour, after which time the water extract was centrifuged and sterile filtered using a 0.45 μm sterile filter (Nalgene, Hereford, UK). This water extract of 3 g in 30 ml was the stock solution used to prepare the concentrations (1:10, 1:50, 1:100 and 1:1000) of the LD water extract used in the experiments described below.
HL60 (human promyelotic leukaemia) and HT29 (human colon adenocarcinoma) cancer cell lines were obtained from The European Collection of Cell Cultures (ECACC, Sailsbury, UK). The HL60 cell line was grown in RPMI 1640 medium (Gibco, Paisley, UK) containing 10% foetal bovine serum (FBS) (Gibco, Paisley, UK). The HT29 cell line was grown in minimum essential medium (MEM) (Gibco, Paisley, UK) containing 10% FBS, 2 mM L-glutamine and 2 mM NEAAs. Cells were incubated at 37°C in an atmosphere containing 5% CO2.
Growth Inhibition Study
For the growth inhibition study, the growth inhibitory actions of water extracts of LD were investigated at final concentrations of 1:10, 1:100 and 1:1000, prepared using the stock solution described above, on each of the cell lines over a period of 72 hours. Growth curves were carried out in 24-multiwell plates; the seeding concentration was 1 × 105cells/ml. Three identical plates were made up for each of the experiments at 0 hrs, and then one of these was counted and discarded after 24, 48 and 72 hours. Cell viability was measured by determining whole cell numbers using a haemocytometer, and viable cells were excluded using trypan blue (Gibco, Paisley, UK).
Apoptosis studies were carried out to ascertain the mode of cell death. The Annexin V assay, propidium iodide staining and the TUNEL assay were all used to investigate not only the impact of LD on apoptosis but to determine at what stage this process is induced by this CHR.
The Annexin V assay
The Annexin V assay provides information about one of the early morphological characteristics unique to apoptotic cell death, which is the loss of membrane asymmetry before loss of membrane integrity. In the Annexin V assay, the translocation of phosphatidylserine (PS) residues from the internal to the outer face of the plasma membrane before loss of membrane integrity is used as a marker of apoptotic induction [22
]. Annexin V conjugated with the fluorochrome fluorescin isothiocyanate (FITC) and the vital dye propidium iodide (PI) were used to distinguish between viable, early apoptotic and late stage apoptotic/dead cells.
The Annexin V assay was performed using the BD Pharmingen™ Annexin V-FITC Apoptosis Detection Kit (Becton Dickenson, Oxford, UK) and 5 μl of 50 μg/ml of propidium iodide (Sigma, Poole, UK) were used.
HL60 and HT29 cell lines (1 × 105cell/ml) were exposed to LD (1:10) for 6,12 16 and 24 hours (for HT29 only) then analysed using a BD FacsCalibur Flow Cytometer running CellQuest Pro (Becton Dickenson, Oxford, UK). The fluorochrome FITC was analyzed using the FL1 detector, while PI was detected using FL2.
PI staining for sub-G1 peaks
Although propidium iodide (PI) staining was used as part of the Annexin V assay to distinguish cell populations, separate PI staining provides evidence of the presence of one of the later characteristics of apoptosis which is fragmentation of the genome by identifying reductions in DNA content.
HL60 and HT29 cell lines at a starting concentration of 1 × 105cell/ml were made up and exposed to water extracts of LD (1:10 - prepared as described above) for 4, 8 and 24 hours (for HT29 cells only). Following exposure, cells were permeabilised using ice-cold 70% ethanol. Cells were then washed twice in ice cold PBS and re-suspended in PBS (1 ml). To remove double stranded RNA, 1 unit of DNAse free RNAse A (Promega, Hertfordshire, UK) was added to the cell suspension, and incubated for 30 minutes at 37°C. Finally, PI (100 μl of 50 μg/ml) was added to the cell suspension, and cells were stored on ice and protected from light until analysis.
Flow cytometry was used to analyse samples. For apoptotic analysis 10,000 of all events detected by the flow cytometer were counted and saved. In order to generate accurate histograms gating was performed to separate single cells that had passed through the flow cell from two (doublets) or more cells that had passed through the flow cell at the same time (which are scored as a single event by the flow cytometer).
The TUNEL assay was performed using the Promega DeadEnd™ Fluoremetric TUNEL system (Promega, Southampton, UK). This assay is similar to PI staining in that it looks for evidence of genomic fragmentation however as a marker it is more specific as it provides evidence of 3' hydroxyl-termini DNA strand breaks within cells.
Flasks were seeded with HL60 and HT29 cells at a starting concentration of 1 × 105 cells/ml. After exposure to water extracts of LD (1:10 - prepared as described above)for 4, 8 and 24 hours (for HT29 cell only)
Cell cycle analysis using PI staining and TUNEL
To address the issue of whether any inhibitory effects of LD on HL60 and HT29 cells occurred as a result of cell cycle arrest, the effect of LD on cell cycle characteristics was investigated by determining how it affected cell cycle checkpoints that, via a series of sensors, can detect cellular damage and ultimately inhibit cell cycle progression, to allow for repair, or induce apoptosis if the damage is irreparable [23
]. These cell cycle check points are G1 (during which cells prepare for replication), S (during which DNA is replicated and a complete copy of each chromosome is made) and G2/M (M is the last phase of the cell cycle during which new chromosomes are equally segregated between two daughter cells before division).
To examine the role of cell cycle arrest as a consequence of any growth inhibitory actions due to LD HL60 and HT29 cell lines were seeded at a starting concentration of 105
cells/ml and water extracts of LD added at final concentrations of 1:50 and 1:100 (prepared using the stock solution described above) for 24, 48 and 72 hours. Following incubation, cells were permeabilized using ice-cold 70% ethanol, then washed twice in ice cold PBS and re-suspended in PBS (1 ml). To remove double stranded RNA, 1 unit of DNAse free RNAse A was added to the cell suspension, and incubated for 30 minutes at 37°C. Finally, PI (100 μl) was added to the cell suspension, and cells were stored on ice and protected from light until analysis. For cell cycle analysis 15,000 of all events were saved and the percentage of cells in each stage of the cell cycle calculated using the freeware Cyclred [24
TUNEL data for HL60 and HT29 cells exposed to water extracts of LD (1:10 - prepared as described above)for 4 hrs and 24 hrs respectively were collected (as described above) to analyse the cell cycle stage in which apoptosis was induced for each of these cell lines. Analysis of the cell cycle stage in which apoptosis was being induced are expressed as overlaid histogram plots showing all cells, viable cells and apoptotic cells, with data gated from TUNEL density plots and FL2-Area versus FL2-Width plots.
Determination of genotoxicity using the Comet assay
To look for evidence of DNA damage (genotoxicity) which can trigger apoptosis, the comet assay was used. The pH>13 version of the experiment was used, which looks for evidence of single strand breaks, alkali labile sites, DNA-DNA/DNA-protein cross linking and single strand breaks (SSB) associated with incomplete excision repair sites [25
]. Media (1.5 ml) containing 1 × 105
cells/ml was placed in 24 well multi-well plates. HL60 and HT29 cell lines were exposed to water extracts of LD at 1:10, 1:50 and 1:100 (prepared as described above); a control was also set up. Cells were incubated at 37°C in an atmosphere containing 5% CO2
for 4 hours. After the incubation, the cells were centrifuged at 800 rpm for 5 minutes and re-suspended in low melting point (LMP) agarose (Promega, Southampton, UK) (0.5%) heated to 37-42°C and pipetted on to slides. The slides were placed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris and 1%v/v Triton®
X-100 (added immediately before use)) for 1 hour at 4°C. The slides were then removed, washed in PBS and placed in electrophoresis solution (0.3 M NaOH, 1 mM EDTA, pH>13) for 30 minutes at 4°C, then subject to electrophoresis for 30 minutes at 4°C at 25 V/300 mA. After electrophoresis, slides were washed in neutralisation buffer (0.4 M Tris, pH 7.5 with 10 M HCl) 3 times for 5 minutes each. Slides were then dried and ethidium bromide (20 μg/ml) (Sigma, Dorset, UK) was pipetted on to the gel. Slides were analyzed using a Zeiss Axioskop microscope (Carl Zeiss LTD, Hertfordshire, UK) connected to a Nikon DN100 digital camera (Nikon, Kingston upon Thames, UK). Tail length was measured live at 1000× magnification using the Eclipse Net image analysis package (Nikon, Kingston upon Thames, UK) The comet assay was carried out in duplicate and for each run 50 tails scored, yielding a total of 100 counts per experiment.
The effect of LD extract on unstimulated (non-proliferating) and stimulated (proliferating) primary human blood lymphocytes
To determine if the cytotoxic effect of LD was cancer cell specific its effect on non cancer cells peripheral human blood lymphocytes, PBLs (both dividing (stimulated) and non-dividing (non-stimulated) was investigated.
Isolation of PBLs
Primary human blood lymphocytes (PBLs) were isolated from 20 ml heparinized venous whole blood using Ficoll-Paque Plus. Whole blood (15 ml) was diluted 1:1 with PBS then layered on top of 20 ml Ficoll in a 50 ml falcon tube and centrifuged for 30 minutes at 800 rpm. Lymphocytes were removed from the interface between the plasma and Ficoll layers.
Culture and stimulation of PBLs
Isolated PBLs were cultured in RPMI 1640 media containing 10% FBS supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C. Cells were incubated at 37°C in an atmosphere containing 5% CO2
. The effects of LD on non-proliferating and proliferating PBLs were examined. In order to stimulate isolated PBLs to proliferate in vitro
, phytohaemagglutinin (PHA) was used. Phytohaemagglutinin is a plant lectin isolated from the red kidney bean (Phaseolus vulgaris
), and is a potent mitogen [26
]. To stimulate PBLs, 10 mM PHA (Sigma, Dorset, UK) was added to appropriate flasks. Flasks were seeded at 1 × 105
cells/ml and incubated with water extracts of LD at 1:10 (prepared as described above) for 24, 48 and 72 hours in the presence and absence of PHA. These exposure times were used so as to allow the PHA-stimulated PBLs to progress through the cell cycle in the presence of LD. Controls, for each time point, were also set up. Experiments were carried out in duplicate.
Evidence of apoptotic induction and alterations in cell cycle characteristics were investigated using PI staining and FACS analysis, as described above. In order to analyse the cell cycle-related and apoptosis-inducing effects of LD on proliferating and non-proliferating PBLs using PI staining, singlets had to be gated and used for analysis. Data are represented as histograms showing cell number (x-axis) versus relative PI fluorescence intensity (y-axis).
Identification of bioactive fractions and chemical analysis of LD using HPLC and LC-MS
LD was split into fractions using a LichroCART® 250-10, LIChrospher® 100 RP-18e (10 μm) HPLC column (Merck, Darmstadt, Germany). LD 500 ul was injected into the column and the flow rate set at 3 ml/minute. Fractions (3 ml) were collected at 1 minute intervals. Water was used as the mobile phase. The HPLC prep column was attached to a Perkin-Elmer series 410 LC pump and a Perkin-Elmer LC-235 Diode array detector. Absorbance was measured at 253 nm. Individual fractions were sterile filtered using a 0.45 μm sterile filter. HL60 cells at a starting concentration of 1 × 105/ml were exposed to LD fractions for 48 hours then cell numbers compared to control cell growth using the trypan blue exclusion assay.
LC-MS analysis was carried out on those fractions that exerted the strongest statistically significant (p = 0.01) growth inhibitory effects using atmospheric pressure chemical ionization (APCI) and sample detection using a Finnigan LCQ MS detector. Samples (1.5 ml) were initially found to be too dilute, and along with a distilled water control were evaporated under vacuum at 55°C. These were then taken up in DMSO: Water 50:50 (200 μl) and sonicated before analysis. Any remaining residue was taken up in MeOH: Chloroform 50:50 (200 μl) and also sonicated before analysis.
Data presentation and statistical analysis
Data for the growth inhibition study are expressed as mean ± standard deviation from the mean. The mean values of each concentration at each time point were compared to control values using ANOVA. Annexin V data were collected in the form of dot plots and then represented as density plots. Cells in the bottom left quadrant of the density plots are viable (Annexin V-FITC and PI negative), cells in the lower right quadrant are apoptotic (Annexin V-FITC positive and PI negative), and cells in the upper right quadrant are late stage apoptotic/necrotic (Annexin V-FITC and PI positive). PI staining data are presented as histograms with the x axis showing relative DNA content and the y axis showing cell number. TUNEL data are represented as density plots showing DNA content on the x axis (using FL2) and the relative number of DNA strand breaks on the y-axis (using FL2). Cell cycle analysis data are represented as histograms showing cell number (x-axis) versus relative PI fluorescence intensity (y-axis). Data from the comet assay are expressed as mean ± SD. The tail lengths of DNA from the exposed cells were compared to those of the controls using the t-test. HPLC data are represented as graphs showing relative growth inhibition compared to control cell growth (± standard deviation). The mean growth inhibitory effect of each fraction was compared to control cell growth using ANOVA.