An in vitro based investigation of the cytotoxic effect of water extracts of the Chinese herbal remedy LD on cancer cells
© Willimott et al 2009
Received: 20 July 2009
Accepted: 28 September 2009
Published: 28 September 2009
Long Dan Xie Gan Wan (LD), a Chinese herbal remedy formulation, is traditionally used to treat a range of conditions, including gall bladder diseases, hepatitis, hyperthyroidism, migraines but it is not used for the management or treatment of cancer. However some of its herbal constituents, specifically Radix bupleuri, Radix scutellariae and Rhizoma alismatis have been shown to inhibit the growth of cancer cells. Thus, the aim of the study was to investigate the impact of LD on cancer cells in vitro.
HL60 and HT29 cancer cell lines were exposed to water extracts of LD (1:10, 1:50, 1:100 and/or 1:1000 prepared from a 3 mg/30 ml stock) and for both cell lines growth, apoptotic induction, alterations in cell cycle characteristics and genotoxicity were investigated. The specificity of the action of LD on these cancer cell lines was also investigated by determining its effect on human peripheral blood lymphocytes. Preliminary chemical analysis was carried out to identify cytotoxic constituents of LD using HPLC and LCMS.
LD was significantly cytotoxic to, and induced apoptosis in, both cell lines. Apoptotic induction appeared to be cell cycle independent at all concentrations of LD used (1:10, 1:50 and 1:100) for the HL60 cell lines and at 1:10 for the HT29 cell line. At 1:50 and 1:100 apoptotic induction by LD appeared to be cell cycle dependent. LD caused significant genotoxic damage to both cell lines compared to their respective controls. The specificity study showed that LD exerted a moderate cytotoxic action against non-proliferating and proliferating blood lymphocytes but not apoptosis. Chemical analysis showed that a number of fractions were found to exert a significant growth inhibitory effect. However, the molecular weights of compounds within these fractions did not correspond to those from the herbal constituents of LD.
It is possible that LD may have some chemotherapeutic potential. However, further studies are required to determine its cytotoxic constituents.
Long Dan Xie Gan Wan (LD) is traditionally used for the treatment of a range of conditions, including gall stones, gall bladder diseases, hepatitis, herpes, shingles, cystitis, hyperthyroidism, migraines and jaundice. The traditional ingredients typically used to make LD are Radix Scutellariae (Huang Qui), Fructus Gardeniae (Zhi zi), Radix glycyrrhizae (Gan cao), Radix rehmanniae (Di huang), Radix Gentianae (Long dan), Radix angelicae sinensis (Dang gui), Semen Plantaginis (Che qian zi), Radix Bupleuri (Cai hu) and Rhizoma alismatis (Ze xie) and also Aristolochia manshuriensis (Mu Tong). However, LD remedies containing Aristolochia manshuriensis (Mu Tong) are no longer available as Aristolochia species contain the toxic and carcinogenic aristolochic acids  and this species has been replaced in many formulations of LD by Medulla tetrapanacis (Tong cao).
Long Dan Xie Gan Wan is not traditionally prescribed in the treatment of cancer, and, to the authors' knowledge, there is no research regarding the effects of LD in any biological context. However, there are herbs within LD that are prescribed for the treatment of cancer and are reported to inhibit the growth of cancer cells in vitro. These herbal constituents are Radix bupleuri (which is traditionally derived from the dried roots of Bupleurum chinense DC and B. scorzonerifolium Willd, however other species and variants of the Bupleurum genus are also used as Radix Bupleuri ), Radix scutellariae, also known as Scutellaria baicalensis, and Rhizoma alismatis [3–9].
As LD is a commonly used Chinese herbal remedy (CHR) that contains constituents reported to possess anti-cancer activity, the aim of this study was to investigate the impact of LD on cancer cell lines in vitro to ascertain if it possesses any potential chemotherapeutic activity. The cell lines used were the HL60- (human promyelotic leukaemia) cell line  and the HT29 (human colon adenocarcinoma) cell line. These cell lines are currently being used by the authors in the characterisation of CHRs said to possess anti-cancer activity.
Results and discussion
Growth Inhibition Study
Alterations in cell cycle characteristics by LD
The effect of LD extract on unstimulated (non-proliferating) and stimulated (proliferating) primary human blood lymphocytes
Identification of bioactive fractions and chemical analysis of LD using HPLC and LC-MS
Molecular weights of compounds within active fractions
Fraction 2 DMSO:Water (50:50)
Fraction 2 MeOH:Chloroform (50:50)
Fraction 7 DMSO:Water (50:50)
Fraction 7 MeOH:Chloroform (50:50)
Molecular weights of compounds within active fractions
Fraction 2 DMSO:Water (50:50)
Fraction 2 MeOH:Chloroform (50:50)
Fraction 4 DMSO:Water (50:50)
Fraction 4 MeOH:Chloroform (50:50)
The results of this investigation show that water extracts of LD exert a growth inhibitory effect and apoptosis on both the HL60 and HT29 cancer cell lines and that there is consistency between the growth inhibitory and apoptotic effects of LD on these cell lines with growth inhibition and apoptosis being greater for HL60 than for HT29. The difference between HL60 and HT29 concerning the extent of apoptosis suffered by these two cell lines may be explained by previous studies that have shown HL60 cells to readily apoptose in response to a variety of stimuli, while HT29 cells are generally resistant to apoptosis .
There is a paucity of literature on the cytotoxic and apoptotic action of LD on cancer cells (in vivo and in vitro) however studies on the mechanism of action of some of its constituents provide some material that can be used to begin to attempt to elucidate the mechanism of action of this formulation.
Radix bupleuri, Radix scutellariae, Rhizoma alismatis and Fructus gardenia and their chemical constituents (saikosaponin D - a triterpene derivative, flavonoids including baicalin and baicalein, triterpenes and acetylated glycoside geniposide respectively) are reported to demonstrate cytotoxic and/or apoptotic activity against cancer cells in vitro, and/or in vivo [3–9, 12–18] thus, at first inspection, it is possible that the action of LD observed in this present study may be related to the actions of these herbal constituents and their chemical constituents.
The results of the cell cycle analysis reflect those of the growth inhibition and apoptosis studies as LD was generally more toxic to the HL60 cell line than to the HT29 cell line. For HL60 these results suggest that the dose-related growth inhibitory action of LD at 1:100, as seen in the growth inhibition study, was related to apoptotic induction and not cell cycle arrest. Furthermore, overlaid histograms of TUNEL data showed HL60 cells were dying irrespective of their position in the cell cycle when exposed to LD at 1:10 for 4 hours, thus suggesting the cytotoxic action of LD at 1:10 is also cell cycle-independent. For HT29 there was a clear change in cell cycle characteristics, with a dose-related decrease in cells in G1 at all time points, an increase in cells in S phase at all time points, and a small change in the number of cells in G2 compared to the control. Thus, suggesting that the growth inhibitory action of LD at 1:50 and 1:100 in this cell line may be related to transient growth arrest during S-phase of the cell cycle. However, in contrast, overlaid histograms of TUNEL data revealed that at 1:10, LD induced apoptosis in a cell cycle-independent fashion. Thus, these results may suggest that at high concentrations LD triggers apoptosis through the induction of large amounts of cellular damage and at lower concentrations, less damage is induced by LD and S phase cell cycle arrest is triggered in the HT29 cell line. As S phase arrest can be triggered by compounds that directly damage DNA or inhibit DNA synthesis  the results of the cell cycle analysis suggest that water extracts of LD may possess one or more of these types of compounds, and that these may be responsible for the ability of LD to transiently arrest S phase progression in HT29 cells at 1:50 and 1:100.
Geniposide (more specifically its acetylated derivative) a constituent of Fructus gardenia and saikosaponin D, a constituent of Radix bupleuri, have been shown to cause cell cycle arrest at the G0 and/or G1 phase(s) of the cell cycle of cancer cells in vitro via activation of pro-apoptotic genes including p53 [12, 18]. However, the results of the present study do not provide evidence of LD inducing cell cycle arrest at G1 in the HT29 cell line. This difference in results may be due to the HT29 cell line being p53 negative  and as a lack of functional p53 can prevent the prolonged arrest of cells with DNA damage at the G1 checkpoint, this could also account for the lack of G1 arrest observed in this study. However, it may be that the compounds are not present in the water extract of LD used in the present study as the preparation of both geniposide and saikosaponin D used in the studies cited differed considerably from that of the LD water extract used in the present study: the preparation of geniposide involved reflux in ether and extractions using methanol and butanol . The saikosaponin D used by Hsu et al  was a pure product dissolved in DMSO. The absence of chemical constituents in the LD water extract may also explain the differences between this extract and that of the Bupleurum scorzonerifolium Willd (from which Radix bupleuri is traditionally derived ) used by Chen et al . In the present study only slight and transient G2/M arrest was seen after 24 hours of exposure to the water extract of LD. In the study by Cheng et al  three preparations were used an acetone, a methanol and a water extract but it was only the acetone extract that induced cell cycle arrest at the G2/M checkpoint in A549 lung cancer cells.
Regarding the possible role of other chemical constituents, Lee et al  reported that baicalein, a constituent of Radix scutellariae, induced apoptosis in human lung carcinoma CH27 cells through arrest in S phase of the cell cycle. Thus, although commercially available baicalein was used by Lee et al  one could, based solely on cell culture experiments, speculate that this compound may potentially have contributed to the action of LD on the cell cycle of the HT29 cell line in the present study.
The results of the comet assay suggest that LD induced DNA damage in both cell lines through the induction of double strand breaks, single strand breaks or alkali label sites. The ability of LD or its constituents to induce DNA damage in cancer cell lines in vitro has not previously been examined. However, the ability of LD to induce DNA damage is in-keeping with the previous results of this investigation, in that LD has been found to elicit a cytotoxic action and induce apoptosis in the HL60 and HT29 cell lines, and induce cell cycle arrest in the HT29 cell line; all these actions can all occur as a result of the induction of DNA damage. Furthermore, the results of this investigation suggest that LD exerts a greater genotoxic action on the HL60 cell line than the HT29 cell line, which is also consistent with previous results obtained in this investigation.
To further elucidate the mechanism of action of LD and to characterize its chemotherapeutic potential its effect on non cancer cells was investigated. The results of this part of the study suggest that at 1:10 LD may exert a moderate cytotoxic action against non-proliferating PBLs and that LD may have induced some form of cytotoxic damage that prevented those cells from progressing through the G0/G1 checkpoint (as discussed above). However, as LD was found to significantly inhibit the growth of the HL60 and HT29 cell lines, at 1:10, these results suggest that LD may exert a more significant cytotoxic action against cancer cells than non-cancer cells.
The preliminary chemical analysis showed that for LD a number of fractions were found to exert a significant growth inhibitory effect at the 5% level (p = 0.05), however none of the fractions tested exerted a significant effect at the 1% level (p = 0.01) which may suggest that LD contains a number of moderately cytotoxic compounds that interact to elicit a significant cytotoxic effect in vitro. However, LC-MS analysis of the cytotoxic fractions revealed that the molecular weights of the compounds within these fractions do not correspond to the molecular weights of the chemical constituents discussed above. Thus, the results of this analysis suggest that previously unreported compounds are responsible for the in vitro inhibitory action of LD, on the growth of the HL60 and HT29 cancer cells lines, observed in this current study.
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 . 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 . 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 .
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 . 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 . 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.
To the authors' knowledge, this is the first investigation to suggest that LD may elicit a cytotoxic action against cancer cell lines in vitro. The results of this investigation suggest that water extracts of LD are toxic to cancer cells in vitro, triggering apoptosis possibly as a result of inducing some form of genotoxic damage. However, preliminary chemical analysis of this CHR indicates that this cytotoxic activity is due to the actions of constituents other than those discussed above. One main reason for this is linked to the differences between the preparations used in the cytotoxicity studies reported in this paper [4, 6, 8, 9, 12–18] and that used in the current study i.e. a water extract of LD. However, the possibility that interactions between the constituents in this water extract may have influenced the results observed cannot be ruled out .
In conclusion, based on the findings of this investigation, the water extract of LD appears to have some chemotherapeutic potential However, further studies are required to determine what constituents within this CHR are responsible for its cytotoxic action.
Shaun Willimot was the recipient of a Biomedical and Pharmaceutical Sciences Research Group, Kingston University, funded Ph.D. LC-MS analysis was conducted by Dr Mirza at the Institute of Cancer Research (ICR, Surrey, UK)
- Cosyns JP, Goebbels RM, Liberton V, Schmeiser HH, Bieler CA, Bernard AM: Chinese herbs nephropathy-associated slimming regimen induces tumours in the forestomach but no interstitial nephropathy in rats [abstract]. Arch toxicol. 1998, 72 (11): 738-43. 10.1007/s002040050568.View ArticleGoogle Scholar
- Yang ZY, Zhi C, Huo KK, Hui X, Tian ZP, Pan SL: ITS sequence analysis used for molecular differentiation of the Bupleurum species from northwestern China. Phyomedicine. 2007, 14: 416-422. 10.1016/j.phymed.2007.04.009.View ArticleGoogle Scholar
- Kok LD, Wong CK, Leung KN, Tsang SF, Fung KP, Choy YM: Activation of the anti-tumor effector cells by Radix bupleuri. Immunopharmacology. 1995, 30 (1): 79-87. 10.1016/0162-3109(95)00010-Q.View ArticleGoogle Scholar
- Ikemoto S, Sugimura K, Yoshida N, Yasumoto R, Wada S, Yamamoto K, Kishimoto T: Antitumour effects of Scutellariae radix and its components baicalein, baicalin, and wogonin on bladder cancer cell lines. Urology. 2000, 55 (6): 951-955. 10.1016/S0090-4295(00)00467-2.View ArticleGoogle Scholar
- Cheng YL, Chang WL, Lee SC, Liu YG, Lin HC, Chen CJ, Yen CY, Yu DS, Lin SZ, Harn HJ: Acetone extracts of Bupleurum scorzonerifolium inhibits proliferation of A549 human lung cancer cells via inducing apoptosis and suppressing telomerase activity. Life Sci. 2003, 73: 2383-2394. 10.1016/S0024-3205(03)00648-9.View ArticleGoogle Scholar
- Sonoda M, Nishiyama T, Matsukawa Y, Moriyasu M: Cytotoxic activities of flavonoids from two Scutellaria plants in Chinese medicine. J Ethnopharmacol. 2004, 91: 65-68. 10.1016/j.jep.2003.11.014.View ArticleGoogle Scholar
- Chen YL, Lee SC, Lin SZ, Chang WL, Chen YL, Tsui NM, Liu YC, Tzao C, Yu DS, Ham HJ: Antiproliferative activity of Bupleurum scrozonerifolium in A549 human lung cancer cells in vitro and in vivo. Cancer Lett. 2005, 222: 183-193. 10.1016/j.canlet.2004.10.015.View ArticleGoogle Scholar
- Huang YT, Huang DM, Chueh SC, Teng CM, Guh JH: Alisol B acetate, a triterpene from Alismatis rhizome, induces Bax nuclear translocation and apoptosis in human hormone-resistant prostate cancer PC-3 cells. Cancer Lett. 2006, 231: 270-278. 10.1016/j.canlet.2005.02.011.View ArticleGoogle Scholar
- Baumann S, Fas SC, Giaisi M, Müller WW, Merling A, Gülow K, Edler L, Krammer PH, Li-Weber M: Wogonin preferentially kills malignant lymphocytes and suppresses T-cell tumor growth by inducing PLCγ1- and Ca2+-dependent apoptosis. Blood. 2008, 111 (4): 2354-2363. 10.1182/blood-2007-06-096198.View ArticleGoogle Scholar
- Willimot S, Barker J, Jones L, Opara EI: Apoptotic effect of Oldenlandia diffusa on the leukaemic cell line HL60 and human lymphocytes. J Ethnopharmacol. 2007, 114: 290-299. 10.1016/j.jep.2007.08.030.View ArticleGoogle Scholar
- Shao RG, Shimizu T, Pommier Y: Brefeldin is a potent inducer of apoptosis in human cancer cells independently of p53. Exp Cell Res. 1996, 227: 190-196. 10.1006/excr.1996.0266.View ArticleGoogle Scholar
- Hsu Yl, Kuo PL, Lin CC: The proliferative inhibition and apoptotic mechanism of saikosaponin D in humam non-small cell lung cancer A549 cells. Life Sci. 2004, 75: 1231-1242. 10.1016/j.lfs.2004.03.008.View ArticleGoogle Scholar
- Lee HZ, Leung HW, Lai MY, Wu CH: Baicalein induced cell cycle arrest and apoptosis in human lung squamous carcinoma CH27 cells. Anticancer Res. 2005, 25 (2A): 959-964.Google Scholar
- Chan FL, Choi HL, Chen Zy, Chan PSF, Huang Y: Induction of apoptosis in prostate cancer cell lines by a flavonoid, baicalin. Cancer Lett. 2000, 160: 219-228. 10.1016/S0304-3835(00)00591-7.View ArticleGoogle Scholar
- Lee S, Kho Y, Min B, Na M, Kang S, Maeng H, Bae K: Cytotoxic triterpenoids from Alismatis Rhizoma. Arch Pharm Res. 2001, 24 (6): 524-6. 10.1007/BF02975158.View ArticleGoogle Scholar
- Chang YC, Tseng TH, Lee MJ, Hsu JD, Wang CY: Induction of apoptosis by penta-acetyl geniposide in rat C6 glioma cells. Chem-Biol Interact. 2002, 141: 243-257. 10.1016/S0009-2797(02)00073-X.View ArticleGoogle Scholar
- Peng CH, Tseng TH, Liu JY, Hsieh YH, Huang CN, Hsu SP, Wang CJ: Penta-acetyl geniposide-induced C6 glioma cell apoptosis was associated with the activation of protein kinase C-delta. Chem-Biol Interact. 2004, 147: 287-296. 10.1016/j.cbi.2004.01.003.View ArticleGoogle Scholar
- Peng CH, Tseng TH, Huang CN, Hsu SP, Wang CJ: Apoptosis induced by penta-acetyl geniposide in C6 glioma cells is associated with JNK activation and Fas ligand induction. Toxicol Appl Pharm. 2005, 202: 172-179. 10.1016/j.taap.2004.06.016.View ArticleGoogle Scholar
- American Cancer Society. [http://www.cancer.org/docroot/ETO/content/ETO_1_4X_What_Are_The_Different_Types_Of_Chemotherapy_Drugs.asp?sitearea=ETO]
- Rodrigues NR, Rowan A, Smith ME, Kerr IB, Bodmer WF, Gannon JV, Lane DP: p53 mutations in colorectal cancer. Proc Natl Acad Sci USA. 1990, 87 (19): 7555-7559. 10.1073/pnas.87.19.7555.View ArticleGoogle Scholar
- Wang CJ, Tseng TH, Lin JK: Penta-acetyl geniposide: isolation, identification and primary effect on C6 glioma cells in vitro. Anticancer Res. 1992, 12: 911-916.Google Scholar
- Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C: A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescin labelled annexin V. Journal Immunol Methods. 1995, 184: 39-51. 10.1016/0022-1759(95)00072-I.View ArticleGoogle Scholar
- Lukas J, Lukas C, Bartek J: Mammalian cell cycle checkpoints: signalling pathways and their organization in space and time. DNA Repair. 2004, 3: 997-1007. 10.1016/j.dnarep.2004.03.006.View ArticleGoogle Scholar
- Vanhuyse M, Kluza J, Tardy C, Otero G, Cuevas C, Bailly C, Lansiaux A: Lamellarin D: a novel pro-apoptotic agent from marine origin insensitive to P-glycoprotein-mediated drug efflux. Cancer Lett. 2005, 221: 165-175. 10.1016/j.canlet.2004.09.022.View ArticleGoogle Scholar
- Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu J-C, Sasaki YF: Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000, 35: 206-221. 10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J.View ArticleGoogle Scholar
- Serke S, Serke M, Brudler O: Lymphocyte activation by phytohaemagglutinin and pokeweed mitogen. Journal Immunol Methods. 1987, 99: 167-172. 10.1016/0022-1759(87)90122-0.View ArticleGoogle Scholar
- Yuan R, Lin Y: Traditional Chinese medicine: an approach to scientific proof and clinical validation. Pharmacol Therapeut. 2000, 86: 191-198. 10.1016/S0163-7258(00)00039-5.View ArticleGoogle Scholar