This study was undertaken to examine the growth inhibitory activity of crude extracts from the barks and fruits of G. hanburyi on human cholangiocarcinoma cell lines.
Materials and methods
Human cancer cell lines
Five human CCA cell lines used in this study including KKU-100 (poorly-differentiated adenocarcinoma), KKU-M139 (squamous carcinoma), KKU-M156 (moderately-differentiated adenocarcinoma), KKU-M213 (adenosquamous carcinoma) and KKU-M214 (moderately-differentiated adenocarcinoma) were established in the Department of Pathology, Faculty of Medicine, Khon Kaen University. All cell lines were cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS), and 100 U/ml penicillin, 100 mg/ml streptomycin. All cell lines were maintained at 37 °C in a 5% CO2 humidified incubator and were subcultured weekly.
Plant extracts
The ethyl acetate extract (VR12874), methanol extract (VR12875) and eight fractions from ethyl acetate extracts (VR12876-VR12883) from barks and an ethyl acetate extract (VR11626) and methanolic extract (VR11627) from fruits of G. hanburyi were kindly provided by Professor Vichai Reutrakul, Department of Chemistry, Faculty of Science, Mahidol University, Thailand. Stock solution of the extracts were made in DMSO and diluted in culture media to the desired final concentration.
In vitro growth inhibitory assay
The sulforhodamine B (SRB) assay was used in this study to estimate cell number indirectly by staining total cellular protein with the SRB. The protocol was based on that originally described by Skehan et al. (1990)20 with slight modification. Briefly, cells at the exponential growth phase were detached with 0.25% trypsin-EDTA (Sigma) to make single-cell suspensions. The viable cells were counted by trypan blue exclusion in a haemocytometer and diluted with medium to give final concentrations about 0.5 to 1×105 cells/ml. Each cell suspension was seeded in 96-well microtiter plates in the volume of 190 ml/well and allowed to grow overnight. The cells were then treated with plant extracts by adding 10 ml/well of each concentration in triplicate to obtain final concentration of 0.8, 4 and 20 mg/ml. The cells with same final concentration of DMSO were used as the solvent-control wells. The plates were incubated for 72 h at 37 °C in a 5% CO2 humidified incubator. At the end of drug treatment, the cells were then fixed with cold trichloroacetic acid (TCA), and washed 5 times with distilled water. The TCA-fixed cells were stained with SRB. The bound dye was solubilized with 10 mM Tris buffer (pH 10). The absorbance (OD) of each well (triplicate for each concentration) was measured by using ELISA plate reader (ELX-800; BIO-TEK INSTRUMENTS, INC.) at 510 nm. The intensity of color developed in each well was corresponded to the cell number. Percentage of cell survival will be calculated by using equation below. IC50 value was expressed as concentration of extract in microgram per milliliter that caused a 50% growth inhibition comparing with controls.
Statistical analysis
The statistical analysis was carried out by mean± SE.
Results
Effect of G. hanburyi extracts on cell growth
Of 4 crude extracts and 8 fractions from barks and fruits of G. hanburyi, the growth inhibitory activities were examined against five human CCA cell lines. After 72 h, cell viability was determined with SRB assay and the results are shown in Table1. The treatment of all cell lines with VR12874 markedly decreased cell viability with IC50 values ranging from 1.84±0.10 to 2.49±0.03 mg/ml (Table1), whereas the VR12875 showed no growth inhibitory activity on all cell lines with IC50 values >20 mg/ml. The extract VR11626 also showed strong growth inhibitory activity against all cell lines with IC50 values ranging from 1.69±0.04 to 4.41±0.10 mg/ml while the VR11627 showed no growth inhibitory activity on CCA cell lines tested (Table1).
Subsequent to these results 8 fractions from the VR12874 were also examined for their growth inhibitory activities against 5 human CCA cell lines. It was found that VR12877 and VR12881 were the two most effective extracts against these CCA cell lines with IC50 values ranging from 1.26±0.30 to 2.33±0.03 mg/ml.The VR12878, VR12882 and VR12883 also showed strong growth inhibitory activity against all cell lines with IC50 values ranging from 0.77±0.01 to 7.91±0.02 mg/ml. The low growth inhibitory activities were observed when VR12876 and VR12879 were tested against these cell lines with IC50 values ranging from 15.64±0.06 to 18.25±0.77 mg/ml (Table1).
Table1 The IC50 values of G. hanburyi crude extracts and fractions on 5 human CCA cell lines. The results are expressed as mean±SE from 3 independent experiments.
NR= None reactive
Discussion and conclusion
The search for anticancer agents from natural sources has been successful worldwide. Active constituents have been isolated and are nowadays used to treat human tumors. The ethnopharmacological knowledge is helpful in the searching for plants with potential cytotoxic activity.
In the present study we analyzed the in vitro effect of G. hanburyi extracts on five CCA cell lines. Our results demonstrate that the ethyl acetate extracts from bark (VR12874) and fruit (VR11626) of G. hanburyi had marked growth inhibitory effects on the five human CCA cell lines whereas no effect was observed in methanol extract from bark (VR12875) and fruit (VR11627). In addition, seven fractions from VR12874 also showed strong to low growth inhibitory activities against five human CCA cell lines. These results indicated that the ethyl acetate extracts might contain active constituents. Variation in the growth inhibitory activities among fractions might be depending on the type or concentration of active constituents containing in each fraction. According to the previous reports several active components have been isolated from G. hanburyi for example desoxymorellin16, isomorellin17, isomorellinol5, gambogic acid10,18, and forbesione19. In addition, some cytotoxic caged xanthones isolated from resin, fruits and whole plant of G. hanburyi have been reported4-8. Many caged xanthones have been shown to exhibit cytotoxic activities in several mammalian cancer cell lines including human gastric cancer BGC-823, human lung carcinoma SPC-A1, human gastric carcinoma MGC-803 and HeLa cells9,11,13,15, as well as anticancer and anti-tumor activities14,15. In the present study the crude extracts and fractions of G. hanburyi were used. The active components in these extracts have already been characterized. We are now in the process of studying the exact mechanism of the growth inhibitory activity and ability to induce apoptosis of these active components on human CCA cell lines. More research on the application of G. hanburyi in cancer treatment is warranted.
Acknowledgement
We gratefully acknowledge Associate Professor Banchob Sripa, Department of Pathology, Faculty of Medicine, Khon Kaen University, for providing CCA cell lines. This research was granted by the Center for Innovation in Chemistry : Postgraduate Education and Research Program in Chemistry (PERCH-CIC). We also thank Professor Vichai Reutrakul for his supply of plant extracts.
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