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Abstract As part of a permanent screening programme, which considers the search for plants and natural products with anticancer properties, the plants are subjected to bioscreening assay testing for cytotoxity. Another crucial component of pre-clinical oncology drug development is the study and monitoring of cell death in tumour and normal tissues. Therefore, methanolic extract stem bark of cassia fistula L was tested for in vitro cytotoxicity and apoptogenic potential by MTT assay, DAPI assay, mitosensor assay and caspase assay.
Keywords: Casssia fistula, MTT, DAPI, Mitosensor, Caspases, human cell lines.
with different organic solvents of increasing polarity in a Soxhlet apparatus. The methanol extract (MEC) was found to possess maximum activity in pilot studies. The extract was dried with a rotary evaporator under reduced pressure at 40-450c. The yield of the extract was 18%. The extract was dissolved in 0.1% DMSO. Four different concentrations of the extract namely 37.5, 75, 150, 300 (ug/ml/) were used for the experiments. MTT assay: MTT assay was performed as per Black and Speer 4. A panel of human cell lines consisting of MCF7, SW480, HeLa, HCT116 and IMR32, were seeded in micro titre plates @ 5000 cells/well and allowed to grow until 85% confluence was reached. Then the medium (DMEM) was removed and MEC dissolved in 0.1%DMSO was mixed with medium in 4 different concentrations namely, 37.5,75,150 and 300ug/ml. For control cells medium without drug was added. The cells were seeded in duplicates and one plate was assayed after 24 hrs of incubation and the other plate assayed after 48 hrs of incubation by MTT assay. Apoptogenic potential of MEC: In this part of the study, apoptogenic potential of MEC was estimated by observing nuclear condensation or pyknosis of MEC treated cell lines, change in the mitochondrial membrane potential and Cytochrome C release of MEC treated cell lines. The induction of caspases in the MEC treated cell lines was also estimated to find out the induction of apoptosis. a) DAPI staining of cells: Chromatin condensation is a late apoptotic event. DAPI, a bisbenzimide dye, is a cell permanent, minor group binding DNA stain that fluoresce bright blue upon binding to DNA. Cells are scored as apoptotic if they have fragmented nuclei. The celllines were grown in 96 well plates in presence of different concentrations of MEC. About 60 l of the medium was removed from the wells and the same amount of diluted dye was added. The cells were incubated at 37 oC in a 5% CO2 incubator for 15 minutes. 60 l of the medium was removed from the wells and observed under fluorescence microscope with UV filter. b) Mitosensor assay: The loss of mitochondrial membrane potential is a hallmark for apoptosis. The JC 1 is a cationic dye used to signal the loss of the mitochondrial membrane potential. In healthy cells, the dye stains the mitochondria bright red. The negative charge established by the intact mitochondrial membrane potential allows the lipophilic dye, bearing a delocalized positive charge, to enter the mitochondrial matrix where it accumulates. When the critical concentration is exceeded, JC-1 aggregates
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Introduction
Cassia fistula L. belonging to the family of Caesalpinaceae is widely distributed throughout tropics. The English common name of the plant is Indian Laburnum. This is a tree of 6-9m height with straight trunk, smooth bark which is pale grey when young and dark brown when old. Plant is widely distributed in India, Sri Lanka, Malaysia and China. All the parts of the tree are medicinally important in traditional systems of medicine. In Cambodia the bark and wood is used to treat dysentery. Every part of the plant is prescribed in combination with other drugs for the treatment of snakebite and scorpion sting (Charaka, Sushruta, Yoga Ratnakara). Besides this, the plant has been shown to possess several medicinal values such as hypoglycemic, hepatoprotective3, antibacterial27 hypocholesterolaemic9 and antidiabetic10 in experimental animals. The methanolic extract of pods of the plant has shown remarkable antitumour activity against EAC cell lines in mice.14 The bark of the plant has remarkable antioxidant properties also. In addition the chemical constituents of different parts of the plant have also been reported by various authors24.
Conclusion
The long term cytotoxicity and antiproliferative activity of the MEC was assayed by a tetrazolium assay namely MTT, using
Fig.1: Percentage cytotoxicity for different cell lines with varying concentrations MEC
Fig.2: % Cells showing apoptosis in MCF 7 cells treated with different concentrations of MEC (DAPI staining)
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Fig.3: Percentage increase in total caspase activity when treated with varying concentrations of MEC
Plate 2: b) MCF7 cells treated with 75g/ml MEC and showing MMP
Plate 2: c) MCF7 cells treated with 150g/ml MEC and showing MMP
References
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