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, linubinoi@gmail.com

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.

References

1 Alley M. C. and Leibor M. M., Improved optical detection of colony enlargement and drug cytotoxicity in

primary soft agar cultures of human solid tumour cells. Br. J. Cancer., 49, 225 (1984.).

2 Basanez G., Nechushtan A., Drozhinin O., Chantruiya A., Choe E., Tutt S., Wood K. A., Hsu Y. T.,

Zimmerberg J.and Youle; R. J., Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer

membranes at subnanomolar concentrations. Proc. Natl. Acad. Sci. USA., 96, 5492 (1999).

3 Bhakta T., Mukherjee P. K., Mukherjee K., Banerjee S., Mandal S.C., Marty T. K. and Pal M. Saha. ,

Evaluation of hepatoprotective activity of Cassia fistula leaf extract.J Ethnopharmacol., 66, 277 (1999).

4 Black M. M. and Speer E. D., Effects of cancer chemotherapeutic agents on dehydrogenase activity of

human cancer tissue in vitro. Am J Clin Path..,23, 218 (1953).

5 Chaya N., Jerauchi K., Yamagala Y. and Kinjo Okabe H., Antiproliferative constituents in plants 14.

Coumarins and acridone alkaloids from Boenninghausenia japonica. Biol. Pharm. Bull.,27(8),1312 (2004).

6 Chia-Nan Ehen, Hsin – Haiku Huang, Chia –Li Wu, Coney;P.C., Kubm Higb, T. A. Hsu, Hsing –

Pang Hsieh, Shung-En Chuang and Gi-Ming Lai., Isocostunolide, a sesquiterpene lactone, induces

mitochondrial membrane depolarization and caspase-dependent apoptosis in human melanoma cell Cancer

Lett., 246,237 (2007).

7 Daniel P. T., Dissecting the pathways to death. Leukemia., 14, 2035 (2000).

8 Desagher S., Osen – Sand A., Nichols A., Eskes R., Montessuit S., Lauper S., Maundrell K., Antonsson

B and Martinou J.C., Bid-induced conformational change of Bax is responsible for mitochondrial

cytochrome c release during apoptosis. J. CellBiol., 144 (5), 891(1999).

9 El-Saadany; S. S., El-Massry R. A., Labib S. M. and Sitohy M. Z,. The biochemical role and

hypocholesterolaemic potential of the legume Cassia fistula in hypercholesterolaemic rats. Nahrung., 35,

807(1991).

10 Esposit Avella M., Diaz A., De Gracia I., De Tello R. and Gupta M.P., Evaluation of traditional

medicine: effects of Cajanus cajan L. and of Cassia fistula L. on carbohydrate metabolism in mice.Rev.

Medical Panama., 16, 39 (1991).

11 Finlay G. J., Baugloy B. and Landwilson W. R. A , Semiautomated microculture method for investigating

growth inhibitory effects of cytotoxic compounds on exponentially growing carcinoma cells. An. Biochem.,

139,272 (1984).

12 Girija Kuttan, Vasudevan D. M. and Ramadasan Kuttan., Effect of a preparation from Viscum album on

tumor development in vitro and in mice.J Ethnopharmacol., 29,35 (1990).

13 Green D.and Kroemer G., The pathophysiology of mitochondrial cell death. Science.,305,626 (2004).

14 Gupta; M., Mazumder; U. K., Rath; N., Mukhopadhyay; D. K., Antitumor activity of methanolic

extract of Cassia fistula L. seed against Ehrlich ascites carcinoma.J Ethnopharmacol.,72,151(2000).

15 Hengartner M. O., Apoptosis: corralling the corpses. Cell., 104,325( 2001).

16 Holdenrieder S. and Stieber P., Apoptotic markers in cancer. Clin. Biochem., 37,605 (2004).

17 Joza N., Kroemer G. and Penninger J. M.,Genetic analysis of the mammalian cell death

machinery.Trends Genet., 18,142 (2002).

18 Kamaljit Kaur, Husheem Michael, Saroj Arora, Pirkkao Harkonen, and Subodh Kumar., In vitro bioactivity-

guided fractionation and characterization of polyphenolic inhibitory fractions from Acacia nilotica (L.)

Willd. ex Del.. J Ethnopharmacol., 99, 353( 2005).

19 Kang S A., Park H J., Kim M J., Lee S Y., Han S W. and  Leem K H., Citri Reticulatae Viride

Pericarpium extract induced apoptosis in SNU-C4, human colon cancer cells. J Ethnopharmacol., 97,

231(2005).

20 Kaufmann S. H. and Earnshaw W. C. ,Induction of apoptosis by cancer chemotherapy. Exp Cell Res.,

256,42 (2000).

21 Kutuk O, Pedrech A, Harrison P., and Basaga, H,. Pramanicin induces apoptosis in Jurkat leukemia

cells: a role for JNK, p38 and caspase activation. Apoptosis., 10, 597 (2005).

22 Luo X., Budihardio I., Zou H., Slaughler C. and Wang S., Bid, a Bcl2 interacting protein, mediates

cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell., 94,

481(1998).

23 McLachlan A., Kekre N., McNulty J.and Pandey S., Pancratistatin: a natural anti-cancer compound that

targets mitochondria specifically in cancer cells to induce apoptosis. Apoptosis.,10, 619 (2005).

24 MurtyV. N., Rao T. V. P. and Venketeswarlu V,. Chemical constituents of Cassia fistula L.

Tetrahedron., 23, 515 (1966).

25 Narita M., Shimizu S., Ito T., Chittenden T., Lutz R.J., Matsuda H., and Tsujimoto Y., Bax interacts with

the permeability transition pore to induce permeability transition and cytochrome c release in isolated

mitochondria. Proc. Natl. Acad. Sci. USA ., 95,14681 (1998).

26 Orrenius S., Mitochondrial regulation of apoptotic cell death. Toxicol. Lett.,149,19 ( 2004).

27 Perumal Samy R., Ignacimuthu S., Sen A., Screening of 34 Indian medicinal plants for antibacterial

properties. J Ethnopharmacol.,62, 173 (1998).

28 Schaeffer W. I. and Friend K., Efficient detection of soft agar grown colonies using a tetrazolium salt.

Cancer Lett.,1, 275 (1976).

29 Smiley S. T., Reers M., Mottola - Hart shorn C., Lin M., Chen A., Smith T. W., Steele G. D. and Chen

L. B., ntracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming

lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA.,88, 3671 ( 1991).

30 Tan M. L., Sulaiman S. F., Najimuddin N., Samian M. R. and Teng Ku Muhammad T. S., Methanolic

extract of Pereskia bleo (Kunth) DC. (Cactaceae) induces apoptosis in breast carcinoma, T47-D cell line. J

Ethnopharmacol.,96, 287 (2005).

31 William E. C ., Jerald J. N., David W. G ., Jauma B , Carles C , Frances V , Peter J. S .and Carl F.A.,

Cytotoxic and antimalarial alkaloids from Brunsvigia littoralis. Planta Med., 64, 91(1998).

32 Young-Kyoon Kim, Seok Keun Yoon and Shi Yong Ryu.,Cytotoxic triterpenes from stem bark of

Physocarpus intermedius. Planta Med., 66, 485 (2000).

33 Yuan H., Mutaomba M., Prinz I. and Gohlieb R., Differential processing of cytosolic and mitochondrial

caspases. Mitochondrion., 1, 61 (2001).