Full length research paperIn vitro cytotoxic activity of methanolic extract of stem bark of Cassia fistula L.
Mathew Linu a,*, Shashidhar Shankar b
a. School of Biosciences, M.G. University, Kottayam, Kerala, India
b. Azeezia Medical College, Kollam, Kerala, India
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 were tested for their in vitro
cytotoxicity and apoptogenic potential by MTT assay, DAPI assay, mitosensor assay, and
caspase assay
Key words: Casssia fistula, MTT, DAPI, Mitosensor, Caspases, human cell lines
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 (Charaka, Sushruta,
Yoga Ratnakara) 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
corresponding author 91-481-2591790, 9447505690, [email protected]
plant has show 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. However, information on the anticancer properties of the stem
bark of the plant is not t available. Hence a preliminary screening for the cytotoxic activity using a panel of
human celllines of the methanolic extract of the stem bark of the plant was done in the present
investigation.
Materials and Methods
Plant material
Stem bark of C. fistula was collected from the premises of School of Biosciences, M G University,
Kerala, India and authenticated by Dr. V. T. Antony, Taxonomist, St. Berchman’s College,
Changanacherry, Kerala, India and a Voucher specimen was deposited in the Regional herbarium Kerala
(Specimen. No. 4589) in St. Berchman’s College, Changanacherry, Kerala, India. The bark was dried in
shade and 100g of powdered bark was extracted with different organic solvents of increasing polarity in a
soxlet 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 24hrs of incubation
and the other plate assayed after 48hrs 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
were 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 different
concentrations of MEC. About 60 μl 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 μl 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 become fluorescent red. In
apoptotic cells the mitochondrial membrane potential collapses and the JC-1 cannot accumulate within the
mitochondria. In these cells JC-1 remains in the cytoplasm in a green fluorescent monmeric form.
Apoptotic cells, showing primarily green fluorescence, are easily differentiated from healthy cells that
show red and green fluorescence. The aggregate red form has absorption/emission maxima of 585/590 nm.
The green monomeric form has absorption/ emission maxima of 510/527 nm. For mitosensor assay the
cells were grown in 96- well plate and using MEC at two concentrations namely 150ug/ml and 300ug/ml
for inducing apoptosis. The lyophilized JC-1 reagent was reconstituted with 500μl DMSO to obtain
100X stock solution.JC-1 reagent was diluted to 1X immediately prior to use (2μl /ml of DMEM
medium.The cell culture media was removed and replaced with enough diluted 1X JC-1 reagent
sufficient to cover the cells (50μl/well).The cells were incubated at 37 oC in a 5% CO2 incubator for 15
minutes. The dye was removed and washed with serum free medium by adding 50μl of serum free
medium and observed under fluorescence microscope.
c) Assay of total caspases
A synthetic peptide substrate is labeled with AFC (7-amino – 4 trifluromethyl coumarin), a
fluorescent molecule, to form a fluorogenic compound that can be used for measuring caspase activity.
When AFC is attached to the substrate, it produces a blue fluorescence upon exposure to light
(excitation max ~ 400nm). Caspase enzymatically cleaves the AFC substrate and releases free AFC.
Free AFC produces a yellow fluorescence (emission max: ~ 505nm). Fluorometer is first calibrated
with known amounts of free AFC. The release of AFC in the reaction mixture is monitored with
fluorometer. Caspase activity in the sample is proportional to the amount of free AFC produced. A
unit is defined as the amount of / caspase required for producing 1 pmol of AFC / min at 25 o C at
saturating substrate concentrations. Cells were counted and harvested by centrifugation followed by
washing with PBS. cells were again resuspended to the desired concentration using ice cold lysis
buffer and Incubated for 5 minutes in ice, (Cell lysis buffer: 50 mM HEPES, 100mM NaCl, 0.1%
CHAPS, ImM DTT, 100mM EDTA pH 7.4) followed by centrifugation at 10,000xg, 10 minutes at
4oC. The supernatant was saved and held on ice until use (Extracts can be flash frozen in an
acetone /ethanol bath and stored at – 70oC for later use.).Total protein of the sample was estimated and
adjusted the protein concentration to 50 mg per reaction500 μM stock solution of caspase substrate
was prepared in in DMSO. 500 uM stock solution of caspase inhibitor was prepared in DMSO.
(Assay Buffer: 100 mM HEPES, 10% sucrose, 10mM DTT, 500 uM EDTA. Adjust pH to 7.5 using
0.1 N NaOH or HCl.). The reaction was set up by adding 30ul of substrate of substrate, 440ul of assay
buffer, and 30ul of sample (containing 50 ug proteins) and incubated the reaction mixture at 37 oC for
1 hour. The reading was taken at 400nm. The relative increase in caspase (RIC) activity was
expressed with respect to control in percentage.
RIC =
Results and discussion
A panel of cultured human cancer cell lines was used in order to find out the cytotoxic potential of
MEC of bark of C. fistula in a time and dose dependent manner for an extended period of time. The 4
doses of MEC selected were 37.5, 75, 150 and 300ug/ml, which was, tested for 2 time periods namely
24hrs and 48hrs (Fig.1). The cell lines used were MCF7, HCT116, SW480, IMR32, and HeLa. The
IC50 values of MEC for the cell lines MCF7 (breast carcinoma), and IMR 32 (brain cancer) were around
150mg/ml and 75mg/ml for 24 and 48hrs of incubation respectively. But for the cell lines HeLa
(cervical carcinoma), HCT116 (colon cancer) and SW480 (colon carcinoma), IC 50 values were around
300mg/ml and 150mg/ml for 24hrs and 48hrs respectively. It is reported that many plant derivatives have
antiproliferative and cytotoxicity effect against cultured human cancer cell lines 31, 12, 32, 5, 19, 30, 6. In a
continuing search for naturally occurring antineoplastic agents from higher plants, cytotoxic activities
against human cancer cell lines could be considered as a reliable source of information. Due to the
limitations of common methods for determining cell viability namely trypan blue exclusion,
incorporation of radioactive nucleotides and clonogenic assays, there was a need for the development of
a colorimetric assay, which would have an upper hand over the earlier assay methods. In one of the
earliest efforts to develop a practical in vitro drug sensitivity assay, Black and Speer4 utilized a
tetrazoluim/ formazan method to assess inhibition of dehydrogenase activity by cancer chemotherapeutic
drugs of excised tissue. As an in situ vital staining process, this phenomenon has been used for
identifying viable colonies of mammalian cells in soft agar culture 28 and for facilitating in vitro drug
sensitivity assays with human tumour cell populations in primary culture1. Since then tetrazoluim
reagents have become popular as convenient non-radioactive alternatives for determining the number of
viable cells in proliferation and cytotoxic assays. Moreover tetrazoluim assays can be semi automated
with the use of 96 microtitre plates to provide easy and rapid analysis of large number of samples 11.
Based on such a tetrazolium assay the MEC was found to possess appreciable cytotoxicity against
different cell lines. Since it is well known that different cell lines might exhibit different sensitivities to
a cytotoxic compound, the use of more than one cell line, is therefore considered necessary in the
detection of cytotoxic compounds18 . Here a panel of 5 human cancer cell lines with different origins;
morphology and tumourigenicity were selected and used for MTT assay. The results were summarised
in Fig.1 which shows that MEC exhibited a dose and time dependent inhibitory effect on all the human
cancer cells examined with varying degree of effect on the different cell lines i.e. the methanolic extract
showed maximum cytotoxicity against MCF7 and IMR 32. IC50 values of these cell lines were around
75ug/ml for 48hrs of incubation. But for the cell lines HeLa, HCT 116 and SW 480 the IC 50 value was
around 150ug/ml for 48 hrs of incubation. It is clear from this that MEC shows varying activity towards
different cell lines.
Next an attempt was made to find out, whether the antiproliferative action was due to apoptogenic
effect of the MEC and for this three different experiments were conducted using the MCF7 cellline.
Chromatin condensation and apoptotic body formation, which was confirmed by morphological evaluation
of the MEC treated cells by DAPI staining. In MCF 7 cells treated with MEC, there was an increase in the
no. of cells showing apoptotic morphology in a dose dependent manner (Fig2, Plate1). The cells treated
with 150 ug/ml of MEC for 24 hours showed Fifty percent cell death with respect to control.
Apoptosis usually affects the cells that are aged, dysfunctional, or damaged by external
stimuli. It is an active, energy requiring process leading to a well regulated degradation of the cell.
Early pathomorphological features are chromatin condensation and marginalization in the nucleus, DNA
fragmentation into mono and oligo nucleosomal units, cellular shrinkage, packing or organelles and
dilatation of the endoplasmic reticulam16. Qualifying cell death and cellular proliferation can provide
information about the process of carcinogenesis and the response to antitumour treatment. Since the
MEC could induce apoptosis in cancer cell lines as evident from DAPI staining and morphological
observation of condensation, it can be said that the MEC has promising anticancer potential .It is also
reported that depending on the type and dosage of the chemotherapeutic drugs, the modality of
radiotherapy and the sensitivity of tissue, cellular damage might bring about the cell cycle arrest
followed by insufficient repair; induction of active apoptotic cell death might result 15.
In the second experiment with MCF 7 cell line the loss of mitochondrial membrane potential
was assayed by JC-1 dye. JC-1 is a cationic fluorescent dye, staining the mitochondria bright red in
healthy cells. 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 29. When the critical concentration of the dye is exceeded, aggregates of JC-1 form which
become fluorescent red. In apoptotic cells, the mitochondrial membrane potential collapses, thereby JC-
1 fails to accumulate within the mitochondria. In these cells JC-1 remains in the cytoplasm in a green
fluorescent monomeric form. Apoptotic cells showing primarily green fluorescence are easily
differentiated from healthy cells, which showed red and green fluorescence.
The mitochondrial permeability transition is an important step in the induction of cellular
apoptosis. During apoptosis the electrochemical gradient referred to as A across the mitochondrial
membrane collapses. The collapse is thought to occur through the formation of pores in mitochondria by
dimerized Bax or activated Bid, Bak or Bad proteins. Activation of these pro apoptotic proteins is
accompanied by the release of Cytochrome C into the cytoplasm25, 22, 2, 8.
It is clear from the Plates 2, that MEC could induce Cytochrome C release by damaging the
mitochondrial membrane in MCF 7 celllines. When the cell line wasc treated with MEC, there was
remarkable release of Cytochrome C into the cytosol, in a dose dependent manner, as compared to the
controls. This supports the claim that MEC could induce apoptosis. The pores created by proapoptotic
proteins cause the release of Cytochrome C into cytosol 8. Therefore it is evident that proapoptotic
proteins of intrinsic pathway of apoptosis are induced by the treatment with MEC.
Mitochondria play a key role in the apoptotic machinery of the cell by releasing caspase
activators such as Cytochrome C, releasing caspase independent death effectors and causing the loss of
essential mitochondrial functions 33, 13. During apoptosis, the outer mitochondrial membrane becomes
permeabilized, allowing inner membrane proteins to be released and activate the downstream apoptotic
machinery including Cytochrome 26. Mitochondrial membrane permeabilisation alone can trigger
apoptosis or necrosis, even in cancer cells23. If the MEC is able to overcome the cancer cells’ resistance
to MMP, then the cells will have no choice but to undergo apoptosis.
Induction of apoptosis is an effective mechanism used to eradicate, transformed or deleterious
cells. Many chemotherapeutic or chemopreventive agents act through triggering of apoptotic pathways in
tumour cells 21. The cellular apoptotic machinery is formed by protein interactions and protein
modification. Protein kinases as well as various cysteinyl-specific aspatate proteases or caspases have been
proposed to mediate apoptosis induced by cytokines, chemotherapeutics and cellular stress through a highly
organized network at different signaling levels7, 20, 17. In the third experiment the cell death associated
caspases (caspase 2, 3, 7, 8, 9, and 10) were collectively assayed by using the common caspase substrate, in
MCF7 breast cancer cell line in a dose dependent manner. As shown in Fig 3 the activities of one or more
of caspases 2, 3, 7, 8, 9 and 10 were increased significantly on MEC treated cells as compared to the
activities in control cells. The MEC dose of 150 ug/ml showed a 50% increase in caspase activity on
treated cells as compared to control cells. The increased caspase activity in MEC treated cells as compared
to control cells, supported that the treated cell were undergoing apoptotic cell death.
Conclusion
The long term cytotoxicity and antiproliferative activity of the MEC was assayed by a tetrazolium
assay namely MTT, using a panel of 5 human cancer cell lines namely MCF7, SW 480, HCT 116, HeLa and
IMR32. This was done in a dose and time dependent manner. The MEC showed maximum activity towards
MCF7, HCT116 and IMR 32.Again the apoptogenic effect of MEC was estimated in cultured human breast
cancer cell line MCF7 , by DAPI assay, Mitosensor assay and caspase assay. DAPI assay was performed to
visualize the morphological changes in cells treated with MEC. The MEC treated cells showed nuclear
condensation or pyknosis indicating apoptosis. Similarly mitosensor assay proved MMP and Cytochrome C
release in MEC treated cell lines, which is an indication of apoptosis. The MEC did increase the total cellular
caspase activity in treated cell lines, which also showed that MEC could induce apoptosis. It is clear from the
above mentioned studies that DNA damage, MMP and caspase activation is evident and that MEC is
able to induce apoptosis in cancer cell lines. Hence the mechanism by which MEC impart its antitumour
activity might be by the induction of apoptosis in cancer cells.
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