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Antiproliferative Effect of Cassia fistula Extracts on HeLa Cells.
Background:
Cancer is a dangerous disease of mankind which continuously requires the development of better therapeutic support for the treatment/management.Cervical cancer leads to 250,000 deaths per year world wide. Medicinal plants had been studied to serve as promising treatment strategies for cancer chemotherapy.Among several traditionally well-practiced herbal medicines from India,Cassia fistula had been shown to play some role as an anti-tumor agent.Cassia fistula Linn (Hindi-Amaltas, English-Golden shower, Indian labrum)is a member of Fabacea (alt. leguminasae) family.In Ayurvedic medicinal system, C. fistula was used against various disorders such asaematemesis, pruritus, leucoderma, diabetes and other ailments.The seed and pod of C. fistula have been evaluated as an important source of secondary metabolites such as alkalanoids, flavanoids, turpenoids. Gupta et al. (1998) had reported anti-tumor activity of seed in ascited mice model. However, anti-tumor potential of C. fistula in cervical cancer is yet to be explored.
Objective:
With these background information the present study is aimed to evaluate the anti-proliferative effect of Cassia fistula seeds and pod on Human Cervical Adenocarcinoma cell line (HeLa).
Materials and Methods:
Cell Lines and Culture Conditions: HeLa cell line was obtained from National Centre for Cell Sciences (NCCS), Pune. Cells were incubated in DMEM medium supplemented with 10% foetal bovine serum, penicillin (100U/ml) and streptomycin (100U/ml) at 37oC temperature and 5% CO2 atmosphere.
Extraction of C. fistula: Fresh ripe fruits of Cassia fistula (Linn) were collected from the campus of Delhi Institute of Pharmaceutical Science and Research (DIPSAR), New Delhi, India. A voucher specimen was stored in laboratory for future reference. Seeds and pod were separated and powdered. The aqueous extract of seed and pod were obtained and lyophilized.
Cell Proliferation Assay: Proliferation of cells was assessed by MTT assay. HeLa cells (2.5 × 105/ml) were briefly cultured in a final volume of 0.2 ml in a 96-well, flat-bottom plate for 48 h and then incubated with MTT (5 mg/ml, 20 μl/well) for 4 h. The medium was replaced with DMSO (150 μl/well) and after 10 min incubation the plate was read on the iMark Microplate Reader (BioRad, USA) with a wavelength of 570 nm. In these experiments, cells were treated with various concentrations (1, 2, 4, 6, 8, 10 mg/ml) of aqueous extract of C. fistula (Seed/Pod) for 48 h, and the cytotoxicity of C. fistula extract was expressed as IC50.
Detection of Apoptosis by DNA Fragmentation Assay: After being incubated with different concentrations of extract for 48 hr, approximately 107 cells were harvested and pelleted by centrifugation (Eppendorf 5804R, Germany). Cellular DNA was extracted by a standard method and the DNA samples were analyzed by 2% agarose gel electrophoresis and visulalized as a DNA Ladder with UV.
Results:
C.fistula extracts Inhibits HeLa Cell Proliferation: C. fistula extracts concentrations ranging from 1 to 10 mg/ml were monitored after 48hrs of cultivation in the cell line and the IC50 value for both C. fistula pod and seed extracts was calculated as 4.5 mg/ml and 3.8 mg/ml respectively. On the basis of the results, cell line treated with C. fistula extracts were incubated for 48 h in the next experiments.
Validation of Apoptosis Measurement by DNA Laddering: In our results, the cells were treated with C. fistula extracts, and the DNA was directly extracted and run on agarose gel. DNA fragmentation, if presented, was seen as a stepwise ladder of DNA fragments at about 200-basepair increments below 2.3 kb. The data shows that DNA laddering is pronounced for C. fistula extracts (Pod/ Seed) in cell line.
Conclusion:
The early trends revealed that the aqueous extract of C. fistula (seed/pod) has certain level of antiproliferative effects against HeLa cell line. Their therapeutic potential is yet to be explored.
Background:
Cancer is a dangerous disease of mankind which continuously requires the development of better therapeutic support for the treatment/management.Cervical cancer leads to 250,000 deaths per year world wide. Medicinal plants had been studied to serve as promising treatment strategies for cancer chemotherapy.Among several traditionally well-practiced herbal medicines from India,Cassia fistula had been shown to play some role as an anti-tumor agent.Cassia fistula Linn (Hindi-Amaltas, English-Golden shower, Indian labrum)is a member of Fabacea (alt. leguminasae) family.In Ayurvedic medicinal system, C. fistula was used against various disorders such asaematemesis, pruritus, leucoderma, diabetes and other ailments.The seed and pod of C. fistula have been evaluated as an important source of secondary metabolites such as alkalanoids, flavanoids, turpenoids. Gupta et al. (1998) had reported anti-tumor activity of seed in ascited mice model. However, anti-tumor potential of C. fistula in cervical cancer is yet to be explored.
Objective:
With these background information the present study is aimed to evaluate the anti-proliferative effect of Cassia fistula seeds and pod on Human Cervical Adenocarcinoma cell line (HeLa).
Materials and Methods:
Cell Lines and Culture Conditions: HeLa cell line was obtained from National Centre for Cell Sciences (NCCS), Pune. Cells were incubated in DMEM medium supplemented with 10% foetal bovine serum, penicillin (100U/ml) and streptomycin (100U/ml) at 37oC temperature and 5% CO2 atmosphere.
Extraction of C. fistula: Fresh ripe fruits of Cassia fistula (Linn) were collected from the campus of Delhi Institute of Pharmaceutical Science and Research (DIPSAR), New Delhi, India. A voucher specimen was stored in laboratory for future reference. Seeds and pod were separated and powdered. The aqueous extract of seed and pod were obtained and lyophilized.
Cell Proliferation Assay: Proliferation of cells was assessed by MTT assay. HeLa cells (2.5 × 105/ml) were briefly cultured in a final volume of 0.2 ml in a 96-well, flat-bottom plate for 48 h and then incubated with MTT (5 mg/ml, 20 μl/well) for 4 h. The medium was replaced with DMSO (150 μl/well) and after 10 min incubation the plate was read on the iMark Microplate Reader (BioRad, USA) with a wavelength of 570 nm. In these experiments, cells were treated with various concentrations (1, 2, 4, 6, 8, 10 mg/ml) of aqueous extract of C. fistula (Seed/Pod) for 48 h, and the cytotoxicity of C. fistula extract was expressed as IC50.
Detection of Apoptosis by DNA Fragmentation Assay: After being incubated with different concentrations of extract for 48 hr, approximately 107 cells were harvested and pelleted by centrifugation (Eppendorf 5804R, Germany). Cellular DNA was extracted by a standard method and the DNA samples were analyzed by 2% agarose gel electrophoresis and visulalized as a DNA Ladder with UV.
Results:
C.fistula extracts Inhibits HeLa Cell Proliferation: C. fistula extracts concentrations ranging from 1 to 10 mg/ml were monitored after 48hrs of cultivation in the cell line and the IC50 value for both C. fistula pod and seed extracts was calculated as 4.5 mg/ml and 3.8 mg/ml respectively. On the basis of the results, cell line treated with C. fistula extracts were incubated for 48 h in the next experiments.
Validation of Apoptosis Measurement by DNA Laddering: In our results, the cells were treated with C. fistula extracts, and the DNA was directly extracted and run on agarose gel. DNA fragmentation, if presented, was seen as a stepwise ladder of DNA fragments at about 200-basepair increments below 2.3 kb. The data shows that DNA laddering is pronounced for C. fistula extracts (Pod/ Seed) in cell line.
Conclusion:
The early trends revealed that the aqueous extract of C. fistula (seed/pod) has certain level of antiproliferative effects against HeLa cell line. Their therapeutic potential is yet to be explored.
