INTRODUCTION:

Cancer is a term that describes a collection of relatable diseases. Characteristic to a majority of cancers, a cell starts dividing uncontrollably and invades neighbouring tissues. Most cancers form solid tumours, except in the case of leukemia. Lifestyle and dietary measures can play a vital role in the prevention of many types of cancer¹. Cancer is a main reason for mortality involving more than one third of the global population in some way. Cancer is the cause of more than 20% of the mortality worldwide². According to the report of the International Agency for Research on Cancer of the World Health Organization published in 2014, the global incidence of cancer has been approximately 14 million new cases and is projected to register 19.3 million in 2025³. According to this report, lung cancer was the most prevalent cancer (13%) in 2012 followed by breast cancer (11.9%), colon cancer (9.7%), and prostate cancer (7.9%).

In view of the complications of the therapies currently considered for cancer, high costs of conventional therapies, and growing incidence of cancer in both developed and developing countries, it seems necessary to develop more novel approaches with higher efficiency so that the disease intensity could be declined. In this regard, there is considerable scientific and commercial interest in developing new anticancer agents from natural sources and the research aimed to develop new anticancer drugs has been turned into a significant research area. Plants produce a wide spectrum of chemical compounds with apparently no direct contribution to their growth and development, namely secondary metabolites. Terpenes, nitrogen-containing compounds, and phenolic compounds are three main classes of these compounds with various biological properties in plants that are used for a wide variety of diseases including cancer^4,5.

Bauhinia tomentosa commonly known as Yellow bell orchid tree belongs to Fabaceae family and is one of the best, versatile and most commonly used household remedy for many manifestations. Tomentosa derived from tomentose, meaning with dense, interwoven hairs. It is commonly known as ‘Kanchini’ in Tamil and ‘Phalgu’ in Sanskrit ⁶. The decoction of the plant extract is used for the treatment of liver conditions and abdominal problems. Fruit is used as a diuretic. Flowers, buds, and dried leaves are used for dysentery treatment. Root bark is used for inflammation of liver. Seeds are tonic and aphrodisiac. Infusion of stem bark is useful as an astringent gargle. Leaves have antidiabetic potential ⁷. It is used for snake bite and scorpion sting ⁸.

The anticancer activity of natural products to battle against cancer is gained through inhibition of tumor cell proliferation, inducing cytotoxicity, induction of apoptosis and suppression of metastasis and angiogenesis and eventually regulation of gene expression⁹.

The aim of the present study was to evaluate anticancer activity of ethanol leaf extract of Bauhinia tomentosa on selected A549 cancerous cell line by in vitro evaluation by 3-(4,5-dimethyl - thiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, AO/EtBr dual fluorescent staining method and DNA fragmentation method.

MATERIALS AND METHODS:

Collection and Authentication of Plant material:

The leaves of Bauhinia tomentosa Linn were collected from Villivakkam, Chennai and authenticated by Dr. S. Jayaraman, Director of Plant and Anatomy Research Centre, West Tambaram, Chennai (Authentication No. PARC /2014 /2294).

Sample preparation:

The leaves were washed with water, shade dried and powdered coarsely. Crude extract was obtained after maceration with 96% ethanol at room temperature for 72 hours, and repeated till exhaustion of the material. Thereafter, the crude ethanol extract was distilled, evaporated and dried under reduced pressure to yield ethanol extract of Bauhinia tomentosa leaves.

Chemicals:

The chemicals used in the present study, MTT, fetal bovine serum (FBS), phosphate buffered saline (PBS), and antibiotics were purchased from Sigma Aldrich and Hi media, Mumbai.

Cell lines and culture conditions:

A549 were procured from National Centre for Cell Science, Pune and was maintained in RPMI-1640 supplemented with 10% FBS, antibiotic 2% (penicillin) in a humidified atmosphere of 5% CO₂ at 37°C until confluent. The stock cultures were grown in culture flask and the experiments were carried out in 96 well plate.

Anticancer studies:

The anticancer activity of EBT was evaluated by MTT assay, AO/EtBr Assay and DNA fragmentation assay.

MTT assay:

The MTT assay was performed as described by the method of Mossman (1983)¹⁰. The MTT assay is based on the cleavage of the soluble yellow tetrazolium salt MTT into a blue colored formazan by the mitochondrial enzyme succinate dehydrogenase. This assay is extensively used for measuring cell survival and proliferation. There is a direct proportionality between the formazan produced and the number of viable cells. However, it depends on the cell type, cellular metabolism, and incubation time with MTT. This method is based on the capacity of mitochondrial enzymes of viable cells to reduce the yellow soluble salt MTT to purple blue insoluble formazan precipitate which is quantified spectrophotometrically at 570 nm after dissolving in DMSO. Cells are plated on to 96 well plates and allowed to grow in CO₂ incubator for 24 hrs (37°C, 5% CO₂). The medium is then removed and replaced by fresh medium containing different concentrations of leaf extract for 48 hrs. The cells are incubated for 24-48 hours (37°C, 5% CO₂). Then, 20μL MTT stock solution (5 mg/ml in PBS) is added to each well and incubated for 4 hrs. The medium is removed and 200 μL DMSO is added to each well to dissolve the MTT product. Then the plate is shaken at 150 rpm for 5 minutes, and the optical density is measured at 570 nm ^12,13. The cell viability is calculated using the formula, % Cell viability= ([O.D of control−O.D of test compound]/ [O.D. of control]) × 100.

Dual AO/EB fluorescent staining:

The cancer cells were seeded at a density of 0.2 X 10⁶ cells per well in 24 well plates and treated with the IC₅₀ concentration of Bauhinia tomentosa and incubated for 24 h. After incubation, the cells were washed thrice with phosphate buffered saline (PBS), stained with 10 mg/ml of dye mixture (10 mg/ml Acridine Orange and 10 mg/ml Ethidium Bromide) and cells were examined under fluorescence microscope. Acridine orange is a vital dye and will stain both live and dead cells. Ethidium bromide will stain only cells that have lost membrane integrity¹¹.

DNA Fragmentation Assay:

The inter nucleosomal cleavage of DNA was analyzed by DNA fragmentation assay as described by Alexei and James, 1994. Briefly, the cancer cells were seeded at a density of 0.2 X 10⁶cells per well in 24 well plates and treated with the IC₅₀ concentration of Bauhinia tomentosa and incubated for 24 h. After incubation, the cells were washed thrice with phosphate buffered saline (PBS) and DNA was extracted by incubating the cells with 500 µl of lysis buffer (100 mM NaCl, 1 mM EDTA, 50mM Tris–Cl (pH 8.0), 0.5 % SDS, and 1 mg/ml proteinase K) at 54°C with gentle agitation for 2h or until all solid material was digested. DNA was extracted with phenol: chloroform (24:1) followed by ethanol precipitation and the DNA pellet was air dried. The DNA was resuspended in 20 µl of TE buffer containing 50 µg/ml of RNase A, incubated for 30 min at 37°C, and analyzed on a 1.5% Agarose gel for DNA fragmentation. The gels were stained with 0.5 µg/ml of Ethidium Bromide and documented under UV transilluminator.

RESULTS AND DISCUSSION:

MTT assay of ethanol extract of B. tomentosa:

Natural products have received increasing attention over the past 30 years for their potential as novel cancer preventive and therapeutic agents^14,15. In parallel, there is increasing evidence for the potential of plant derived compounds as inhibitors of various stages of tumorigenesis and associated inflammatory processes, underlining the importance of these products in cancer prevention and therapy. Approximately 60% of drugs currently used for cancer treatment have been isolated from natural products¹⁶. At this time, more than 3000 plants worldwide have been reported to have anticancer properties. Globally, the incidence of plant derived products for cancer treatment has increased. Hence, an attempt has been made to study the cytotoxic activity of ethanol extract of B.tomentosa against the human cancerous cell line, A549.

The cytotoxic activity of the ethanol extract of B.tomentosa on A549 cell line was estimated by MTT assay. The results of the MTT assay of EBT were given in table 1 and also in figures 1 & 2 respectively. The IC₅₀ of EBT was found to be 144µg/ml whereas, methotrexate IC₅₀ was found to be 10.11µg/ml.

Dual AO/EtBr fluorescent staining:

Apoptosis is a type of genetically regulated programmed cell death that controls the development of multicellular organisms and tissues by eliminating physiologically redundant, physical damaged and abnormal cells¹⁶. The efficacy of anticancer drugs is measured by their ability to detect cancer cells and selectively promote their apoptosis. The primary mechanism by which chemotherapeutics destroy tumor cells is by inducing apoptosis. High levels of apoptosis in cancer cells are strongly associated with chemotherapeutic sensitivity¹⁷. While tumor cells undergo apoptosis in the presence of anticancer drugs, normal cells become necrotic if the drug is toxic. MTT assays cannot differentiate between these mechanisms of cell death. Therefore, the effects of drug may primarily be toxic and poisonous to normal cells. Therefore, detection of tumor cell apoptosis is more valuable than generally assessing tumor cell viability.

a. Control

b. 25μg of EBT

c. 50μg of EBT

d. 100μg of EBT

e. 250μg of EBT

f. 500μg of EBT

Figure 1. MTT cytotoxicity assay of EBT

Table 1. Percentage Cell Viability of EBT treated A549 cells

Tested concentration(µg) of EBT	% of cell viability
500	20.07
250	43.28
100	59.68
50	84.12
25	89.53
Control	100

Figure 2. MTT Cytotoxicity assay of EBT using A549 cells

Dual Acridine Orange/Ethidium Bromide (AO/EtBr) fluorescent staining, visualized under a fluorescent microscope, can be used to identify apoptosis associated changes of cell membranes during the process of apoptosis¹⁸. In addition, it allows for the distinction between normal cells, early and late apoptotic cells and necrotic cells. Therefore, AO/ EtBr staining is a qualitative and quantitative method to detect apoptosis ¹⁹. AO penetrates normal and early apoptotic cells with intact membranes, fluorescing green when bound to DNA. EtBr only enters cells with damaged membranes, such as late apoptotic and dead cells, emitting orange red fluorescence when bound to concentrated DNA fragments or apoptotic bodies²⁰. Furthermore, dual AO/ EtBr staining is able to detect mild DNA injuries¹⁸. Dual AO/EtBr fluorescent staining can detect basic morphological changes in apoptotic cells. This method can also accurately distinguish cells in different stages of apoptosis^21,22. Fluorescent staining using AO alone has been used in the past; however, detection of cell apoptosis using AO/ EtBr is a relatively new approach and few papers have reported its use²³. In comparison to AO staining, the AO/ EtBr method improves the detection of apoptosis and can distinguish between late apoptotic and dead cells. Therefore, to distinguish normal, early apoptotic, late apoptotic and dead cells, nuclear morphology by dual staining must be assessed.

Control 150µg of EBT

300µg of EBT

Figure 3. Dual AO/EtBr fluorescent staining of EBT treated A549 cells

(Uniform Green–Live cells; Green with bright green dots–Early apoptotic cells; Orange–Late apoptotic cells)

AO/EtBr fluorescent staining of EBT treated cells were depicted in figure 3. The live cells appeared uniformly green. The early apoptotic cells stained green and contain bright green dots in the nuclei as a consequence of chromatin condensation and nuclear fragmentation. The late apoptotic cells appeared orange because of the incorporation of ethidium bromide. But, in contrast to necrotic cells, the late apoptotic cells have condensed and often fragmented nuclei. Necrotic cells appeared orange, but have a nuclear morphology resembling that of viable cells, with no condensed chromatin. Thus from the above results, it was evident that EBT has induced apoptosis in lung cancer cells.

DNA fragmentation assay:

Fragmentation of DNA and chromatin is an integral process during apoptosis, and has been reported to occur in more than one distinct stage. During initial stages, high molecular weight DNA fragments of 50 kb or longer size have been observed in morphologically normal cells committed to undergo apoptosis. The low molecular weight DNA fragments are associated with late events such as formation of apoptotic bodies ²⁴, although the phenomenon of nucleosome excision (ladder formation) is also reported to initiate before any obvious apoptotic changes in cell morphology ²⁵. The extensive DNA fragmentation induced during apoptosis can be detected using techniques such as DNA ladder assay (agarose gel electrophoresis), terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labelling (TUNEL assay) and comet assay ^{26, 27}. Out of these methods, DNA fragmentation assay using agarose gel electrophoresis is the most frequent technique used for the detection of apoptosis and can easily discriminate between apoptotic and non-apoptotic (necrotic) modes of cell death, as in most cases the inter-nucleosomal cleavage of genomic DNA yielding the characteristic DNA ladder is a molecular hallmark of apoptotic cells ^28,29. In the typical DNA fragment ladder obtained, molecular weights of the genome fragments are integer multiples of 180 base pairs length associated with a nucleosome subunit. On the other hand, genomic fragments of irregular sizes are generally induced during necrotic cells, and a DNA smear is obtained during agarose gel electrophoresis.

Figure 4. Effect of EBT on DNA fragmentation

1-A549 control; 2- treated A549 (300 µg/200µl); 3- Treated A549 (150 µg/200µl); L-Gene Ruler 1 kb DNA Ladder (Fermentas)

Many of the commonly used cancer chemotherapeutic drugs exert their cytotoxic effects by inducing DNA damage, which activate cell cycle checkpoints that co-ordinate cell cycle progression with DNA repair ³⁰. DNA fragmentation occurs in apoptotic cells, caused by intrinsic activity which is induced by a variety of agents. This cleavage produces ladders of DNA fragments that are of the size of integer multiples of a nucleosome length (180– 200 bp) ³¹.

The effect of EBT on DNA fragmentation was given in figure 4. EBT has induced apoptosis in A549 cells resulting in the degradation of chromosomal DNA in to small oligo nucleosomal fragments which results in fragmentation of DNA (lane 2, 3). In case of untreated cells no fragmentation was observed and the chromosomal DNA was intact (lane 1). The result indicates that the apoptotic signal was stimulated by the treatment with EBT.

CONCLUSION:

Plants have been demonstrated as a clinical source for anticancer compounds. Hence, the development of new drugs to play an important role in cancer control is greatly desired. B.tomentosa was tested for anticancer activity based on their historical and other traditional uses. The ethanol leaf extract of B.tomentosa was prepared and tested for their potential as anticancer activity by in vitro evaluation methods, i.e., MTT assay, dual Acridine Orange/Ethidium Bromide fluorescence assay and DNA fragmentation assay. The results of the anticancer studies reveal that the EBT has appreciable cytotoxic effect against A549, lung cancer cell lines. Further studies are required to identify the mechanism of action of EBT against lung cancer.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Chitra Kalaichelvan, Vidya and S Vuruputoor. Role of nutrition in cancer. RJPT 2015; 1(6):58-63.

2. Agarwal N etal.,. Natural herbs as anticancer drugs. Int J PharmTech Res. 2012; 4:1142-1153.

3. International Agency for Research on Cancer.World Cancer Report 2014. Lyon, France: IARC Press; 2014.

4. Shirzad H et al., Correlation between antioxidant activity of garlic extracts and WEHI-164 fibrosarcoma tumor growth in BALB/c mice. J Med Food. 2011; 14: 969-974.

5. Azadmehr A et al., Evaluation of in vivo immune response activity and in vitro anti-cancer effect by Scrophularia megalantha. J Med Plants Res. 2011; 5:2365-2368.

6. Quiroga EN et al., Screening antifungal activities of selected medicinal plants. J. Ethnopharmacol. 2001; 74: 89-96.

7. Shiv Kumar Gupta. Phytopharmacognostic Investigation of Bauhinia tomentosa Linn J Adv Sci Res. 2011; 2(2):01-04.

8. R. Sumitha, V. Deepa Parvathi, N Banu. Cytotoxicity Testing of Star Fish Stellaster equestris Extracts on Pa1 celline. Research J. Pharm. and Tech. 2017; 10(9): 2851-2856.

9. Ragavan, B, Krishnakumari, S. Antidiabetic effect of T. Arjuna bark extract in alloxan induced diabetic rats. Indian J. of Clinical biochemistry, 2006; 21:123-128.

10. Mossman T. Rapid colorimetric assay for cellular growth and survival – application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-63.

11. Lalitha P et al., Anticancer potential of pyrrole (1, 2, a) pyrazine 1, 4, dione, hexahydro 3-(2-methyl propyl) (PPDHMP) extracted from a new marine bacterium, Staphylococcus sp. strain MB30. Apoptosis 2016; 21(5):566-577.

12. Alexei G et al., A rapid and sensitive assay for the detection of DNA fragmentation during early phases of apoptosis. Nucleic Acids Research, 1994; 22(13): 2714-2715.

13. Gordaliza M. Natural products as leads to anticancer drugs. Clin. Transl. Oncol. 2007; 9(12): 95-99.

14. Saklani A, Kutty SK. Plant- derived compounds in clinical trials. Drug Discov Today 2008; 13(4):161-171.

15. Gerson R et al., Complementary and alternative medicine (CAM) in Mexican patients with cancer. Clin. Transl. Oncol. 2006; 8(3):200- 207.

16. Taraphdar AK et al.,. Natural products as inducers of apoptosis: Implication for cancer therapy and prevention. CurrSci., 2001; 80: 1387–96.

17. Yamamoto M et al.,. The p53 tumor suppressor gene in anticancer agent-induced apoptosis and chemosensitivity of human gastrointestinal cancer cell lines. Cancer Chemother Pharmacol, 1999; 43: 43–49.

18. Gherghi IC et al.,. Study of interactions between DNA-ethidium bromide (EB) and DNA-acridine orange (AO), in solution, using hanging mercury drop electrode (HMDE). Talanta, 2003; 61(2): 103–12.

19. Biffl WL et al.,. Interleukin-6 delays neutrophil apoptosis via a mechanism involving platelet-activating factor. J Trauma, 1996; 40: 575–78.

20. Ribble D et al.,. A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnol, 2005; 5: 12.

21. Leite M et al.,. Critical evaluation of techniques to detect and measure cell death–study in a model of UV radiation of the leukaemic cell line HL60. Anal Cell Pathol, 1999; 19: 139–51.

22. Baskić D et al.,. Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide. Cell Biol Int, 2006; 30: 924–32.

23. Lecoeur H. Nuclear apoptosis detection by flow cytometry: influence of endogenous endonucleases. Exp Cell Res, 2002; 277: 1–14.

24. Czene S et al.,. DNA fragmentation and morphological changes in apoptotic human lymphocytes. Biochem Biophys Res Commun. 2002; 294:872–878.

25. Tanuma S et al.,. Benzylidene ascorbate induces apoptosis in L029 tumor cells. Biochem Biophys Res Commun. 1993; 15:29–35.

26. Otsuki Y. Various methods of apoptosis detection. Acta Histochem Cytochem 2000; 33:235–241.

27. Chandna S. Single-cell gel electrophoresis assay monitors precise kinetics of DNA fragmentation induced during programmed cell death. Cytometry, 2004; 61A:127–133.

28. Kerr JF et al.,. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26:239–257.

29. Pandey S et al.,. Separate pools of endonuclease activity are responsible for inter-nucleosomal and high-molecular-mass DNA fragmentation during apoptosis. Biochem cell Biol, 1994; 72:625–629.

30. Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 2001; 15: 2177.

31. Hui L et al.,. Mechanism of apoptotic effects induced selectively by ursodeoxycholic acid on human hepatoma cell lines. World Journal of Gastroenterology 2007; 3 (11): 1652-1658.

Received on 16.02.2019 Modified on 14.03.2019

Research J. Pharm. and Tech. 2019; 12(6): 2748-2752.

DOI: 10.5958/0974-360X.2019.00460.8