IAETSD JOURNAL FOR ADVANCED RESEARCH IN APPLIED SCIENCES ISSN NO: 2394-8442

In-vitro anticancer activity of on - Jurkat cell line and Human Breast cancer - MCF-7.

Seethalaxmi Radhakrishna*1, Sarvanakumari. B.S 2 1 Research scholar, Research and Development Center Bharathiar University, Coimbatore. India 2 Professor, PG and Research Department of Microbiology, Shree Narayana Guru College, Coimbatore, India. Corresponding author - ¹[email protected] ²[email protected]

Abstract:- Breast cancer and T cell leukemia are the most leading cause of death all over the world. Breast cancer is the most frequent malignancy in women and T cell leukemia is a cancer of immune system’s own T cells. Baliospermum montanum is a red listed with medicinal properties. In our present study we have tried to evaluate the anticancer properties of methonolic leaf extract of Baliospermum montanum against these 2 cancers using cell lines. We investigated the antiproliferative activity using MTT assay on 2 cancer cell lines MCF-7 [breast cancer] and Jurkat [T cell leukemia]. Results showed dose dependent cytotoxic effect on Jurkat and MCF-7. MCF-7 exhibited very less cytotoxicity compared to Jurkat with an IC₅₀ value of 298 µg/ml. Further analysis was carried out with Jurkat cell line. Phase contrast microscopy showed cytotoxic and apoptotic activity. DNA fragmentation analysis of Jurkat showed DNA ladder pattern commonly associated with . Cell cycle analysis were conducted by FACS to find out the phase of growth arrest. Cell cycle arrest was 12.79% at G2/M phase whereas untreated cells 6.62%. Our study clearly demonstrates the anticancer activity of Baliospermum montanum on T cell leukemia [Jurkat cells] and can be a good source for anticancer drug. No significant proliferation inhibition was observed in breast cancer [MCF-7].

Key words: - MTT, cytotoxic, FACS, antiproliferative, apoptosis.

I. INTRODUCTION

Medicinal have played an important role in development of modern medicine and their therapeutical uses are well documented. Side effects of several allopathic drugs and development of resistance to currently used drugs have led to increased emphasis on the use of plant materials as a source of medicines for a wide variety of human ailments [1]. Plant based compounds are inherently better tolerated in the body than synthetic chemicals and have higher chances to be approved as drugs [2],[3]. Exploring new plants and plant derived natural compounds are the subject of extensive research worldwide. Approximately 60% of currently used anticancer chemotherapeutic drugs (vinblastin, vincristine) are derived from plant resource [4],[5], With cancer being a widespread threat to humanity, plants play an important role in cancer prevention as well as therapy[6].

Despite significant progress in the treatment of certain forms of cancer it remains a major cause of death throughout the world [7]-[9]. Breast cancer is the most frequent malignancy in women and the second leading cause of cancer-related deaths worldwide. Due to its major impact on population, this disease represents a critical public health problem that requires further research at the molecular level in order to define its prognosis and specific treatment. Basic research is required to accomplish this task and this involves cell lines as they can be widely used in many aspects of laboratory research and, particularly, as in vitro models in cancer research [10].

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Lymphoma is the most common blood cancer, Lymphoma occurs when cells of the immune system called lymphocytes, a type of white blood cell, grow and multiply uncontrollably. Adult T-cell leukemia/lymphoma (ATL or ATLL) is a rare cancer of the immune system's own T-cells. T-cell leukemia describes several different types of lymphoid leukemia which affect T cells. Human T cell leukemia/lymphotropic virus type 1 (HTLV-1) is believed to be the cause of it, in addition to several other diseases [11]-] 14].

Baliospermum montanum (Muell – Arg) is an important aromatic medicinal plant belonging to the family includes 280 genera with 730 species with the largest Euphorbia [15]. Root, leaf and seeds of B. montanum are used medicinally and are documented from Asian countries including Nepal, Burma, Malaya and India. The plant is commonly referred as Naga Danti a threatened medicinal plant [16]. B. montanum is known for its ethanobotanical and traditional use [17]. Plant is well known for its antioxidant [18], anti-inflammatory activity [19] and anti-microbial activity.

The plant is also well known for its anticancer avtivity. Alcoholic extract showed Cytotoxicity activity on HT– 29 human cancer cells[20]. The nano particles exhibited a significant reduction in the colony forming ability and wound healing capacity of the prostate cancer cells [21]. The cytotoxixicity effect of silver nano particals was studied by MTT assay against Vero cell lines and Hep2 cell lines [22].

Thus, in our present study we have tried to evaluate the anticancer and phytotherapeutic potential of this traditionally important red listed medicinal plant Baliospermum montanum against 2 major Cancer viz breast and T cell leukemia . Alcoholic leaf extract of the plant was used in the investigation. We have investigated the antiproliferative activity of this plant using 2 cell lines MCF-7 [breast cancer] and Jurkat [T cell leukemia]. MCF-7 is a breast cancer cell line isolated in 1970 from a 69-year-old Caucasian woman [23]. MCF-7 human breast cancer cells are used widely for studies of tumor biology and hormone mechanism of action [24]. Jurkat cells are an immortalized line of human T lymphocyte cells that are used to study acute T cell leukemia, T , and the expression of various chemokine receptors susceptible to viral entry, particularly HIV. Jurkat cells are useful in science because of their ability to produce . Their primary use, however, is to determine the mechanism of differential susceptibility of cancers to drugs and radiation [25].

II. MATERIALS

A. Plant material and Preparation of extract

The plant material was collected from Gandhi Krishi Vignan Kendra (GKVK) Bangalore. Soon after collection leaves were removed, washed, shade dried and made to a fine powder (particle-0.25mm size). 100 grams of plant powder was taken in a soxhlet extractor and extracted in 500 ml of methanol (48 h). The solvent was recovered by distillation. The extract was concentrated to a residue which was stored for subsequent experiments.

B. Cell cultures Cancer cell lines were obtained from American type culture collection center, United States. The stock cultures of Jurkat [T cell Leukemia] and MCF -7 [breast cancer] cell lines were cultured in DMEM [Dulbecco’s Modified Eagle Medium] and RPMI [Rosewell Park Memorial Institute] media with 10% FBS and 1% streptomycin and incubated at 37°C with 5% CO₂ overnight in a humified atmosphere. Cell viability was determined by the tryphan blue dye exclusion method.

III. METHODS

In the study, human breast cancer cell lines MCF-7 and T- cell leukemia jurkat cell lines were treated with alcoholic extract of Baliospermum montanum. Demonstration of viability of the cells, cytotoxicity, apoptosis and growth or cell cycle arrest were carried out by the MTT assay, Microscopic examination, DNA fragmentation analysis and Flow cytometry- FACS.

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A. Cytotoxicity Studies

Cytotoxicity of the cell lines were evaluated using MTT (3-(4,5-Dimethyl hiazole-2-yl)-2,5-diphenyltetrazolium bromide ) assay [26]with some modification. 70 -80 % of confluent cell lines of Jurkat and MCF-7 were collected and centrifuged. 1 x 10⁵ cells / well and 5 x 10⁵ cells / well of Jurkat and MCF -7 were seeded respectively in a 96 well plate and incubated for 24 hrs at 37°C and 5% CO₂ . Plant extract to be tested were added ranging from 0 – 320 µg/ml. [Conc. 0, 10, 20, 40, 80, 160 and 320 mg/ml] and were incubated for 24 hrs. After incubation plates were centrifuged at 2,000 rpm for 5 min. 100 µl/well (50 µg/well) of the MTT (5mg/10ml of MTT in 1X PBS) working solution were added to the respective wells and incubated for 3-4 hrs. 100 µl of DMSO was added to rapidly solubilise the formazan. Absorbance were measured at 570 nm. The cytotoxicity of the plant extract was expressed as IC ₅₀ value concentration of the extract inhibiting cell growth by 50%.

B. Microscopic analysis

Phase contrast microscope were used to investigate the morphological changes occurring in the cell leading to apoptosis after extract treatment. Jurkat cells (1 x 10⁵) were seeded in 6- well plate and were stained with ethidium bromide {5µg/ml]. Cells were observed under inverted microscope.

C. DNA ladder assaying

Jurkat cells treated with the extracts which induced toxicity were incubated at 37°C and 5% CO₂ for 24hrs to establish the process of DNA fragmentation and apoptosis. After incubation media with cells were centrifuged. Pellet was collected and resuspended in PBS. Cells were harvested and lysed with 500µl of lysis buffer. DNA was treated with RNase at 50°C for 30 minutes. Sample was treated with proteinase K and incubated at 50°C for 30 minutes. Samples were loaded onto 2% agarose gel. One kb & 50 kb ladder and non-treated Jurkat cells were also loaded alongside [27].

D. Cell cycle analysis

Cell cycle analysis of the treated cells were conducted by Flow Cytometry to screen the phase of cell cycle arrest [28]. 1 x 10⁵ cells ( Jurkat ) were cultured in a 6- well plate containisng 1 ml of serum free media. After 4-6 hrs of incubation, cells were treated with and without 100 µg/ ml of sample and 1% DMSO as control and incubated for 24 hrs. After 24hrs of treatment, cells were collected and centrifuged at 1,500 rpm for 5 minutes at room temperature and the supernatant was discarded .The pellet was washed twice gently with 1XPBS and fixed overnight at 4 °C in 500 µl of fixing solution (containing 15% FBS and 15 % PBS in 70 % ethanol ). The cells were then centrifuged at 1,500rpm for 5 minutes at room temperature and the supernatant was discarded. Cell pellet was washed two times with cold 1XPBS. Cells were incubated for 1 hr at room temperature in 500 µl of propidium iodide solution containing 0.05 mg/ml propidium iodide, 0.1 mM EDTA and 0.05mg/ml RNase A in PBS. The percentage of cells in various stages of cell cycle were determined using FACS Canto II BD Biosciences, San Jose, CA ) and analyzed by flow Jo 7.5.5 ( Tree Star Ashland OR ).

IV. RESULTS

The anticancer activity of the plant Baliospermum montanum was evaluated against MCF-7 and Jurkat cell lines. Our preliminary studies showed that the major mechanisms of inhibition included cell cytotoxic effect and apoptosis. Jurkat cells showed significant amount of cytotoxicity.

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A. MTT Assay

Cell viability was measured by MTT assay. Effects demonstrated a dose dependent cytotoxic effect on both Jurkat and MCF-7 cells. The number of viable cells reduced significantly as the dose increased [Fig1]. Cytotoxicity was expressed as the concentration of the extract inhibiting cell growth by 50% [ IC ₅₀]. IC ₅₀ value of Jurkat cells was 298 µg/ml. However MCF-7 exhibited very less cytotoxicity at higher concentration. Jurkat cells showed significant inhibitory action when compared to MCF-7. Cell Cytotoxicity may be due to the presence of high content of flavonoids in the plant [18].

Figure I. MTT assay showing percentage of inhibition versus extract concentration in Jurkat cell line. Decrease in cell viability were observed in jurkat cells incubated with various concentration (10, 20, 40, 80, 160, 320µl) of Baliospermum montanum extract. Percentage of inhibition were observed in a dose dependent manner.

B. Microscopic Observation

In order to evaluate the cause of growth inhibition of Jurkat and MCF-7 cells by Baliospermum montanum, morphological changes in the cell and characteristic features of apoptosis were studied under an inverted microscope. Treated Jurkat cells showed prominent morphological changes like cell shrinking, rounding of cells, formation of membrane blebs and appearance of apoptotic bodies’ characteristic features of cytotoxicity and apoptosis [1]. Plant extract showed very less effect on MCF-7 cells. Hence, only jurkat cells were taken up for further analysis.

C. DNA Fragmentation Analysis

Jurkat cell lines treated with extract were subjected to DNA fragmentation analysis. Agarose gel electrophoresis showed that treatment with extract resulted in the nuclear condensation and the formation of DNA fragments. This characteristic feature is commonly associated with the apoptosis process, in which DNA is cleaved into fragments by the endogenous endonuclease. The pattern of laddering was clearly observed in the gel [29].

D. Cell Cycle Analysis

Jurkat cell lines were further analysed to evaluate the phase of cell cycle arrest with FACS, which revealed that treated sample cell cycle arrest was 12.79% at G2/M phase whereas untreated cells 6.62%. The result suggests that sample arrest occurred at G2/M phase and at the same time it induced apoptosis [Figure 2].

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Fig-II. Flow cytometer analysis. Jurkat cells were treated with 360µg/ml of Baliospermum montanum extract and analysed by flow cytometer (FACS) . The result suggested that the treated sample cell cycle arrest was 12.79% at G2/M phase and untreated cells 6.62%.

V. DISCUSSION

In our present study the anti-cancer potential of B. montanum were evaluated and the results were discussed in detail. Previous studies have reported that this plant has been used as folk medicine for centuries because of its rich medicinal phytoconstituents. Antitumor activity of the plant was carried out investor against human MCF-7 and Jurkat cell lines. In our study Jurkat cells treated with extract exhibited reasonable antiproliferative activity indicating the presence of potent cytotoxic and biologically active components in the plant. The antiproliferative effect of the plant may be because of the rich naturally occurring antioxidants and flavonoids present in the plant. The cell viability and percentage of inhibition was assessed by MTT assay which is based on the reduction of MTT by the mitochondrial dehydrogenase of intact cells to form a purple Formosan product.

The results exhibited decline in cell density in a dose dependent manner [Figure1]. IC₅₀ values were calculated. Microscopic observation of the treated cells showed the morphological changes such as cell shrinking and rounding of cells confirming the toxicity caused by the plant extract. DNA fragmentation analysis by agarose gel electrophoresis also assessed the inhibitory activity of the plant. Clear laddering pattern was observed on the gel confirming nuclear condensation and DNA fragmentation which are the characteristic feature of apoptosis. Further to confirm the apoptosis induction activities, cells were stained with propidium iodide and analysed by flow cytometer [FACS]. The result suggest that the sample arrest occurred at G2/M phase and at the same time it induced the apoptosis. The percentage of cells in each phase of the cell cycle were screened by FACS which indicated that the treated cells were 12.79% at G2/M phase and untreated cells 6.62%.

Above results justifies that Baliospermum montanum has the potential to scavenge the Jurkat cells and maybe a promising therapeutic tool for treatment of Adult T cell leukemia or ATL. Human T-cell leukemia virus type 1[HTLV-1] is the causative agent of aggressive form adult T cell leukemia. ATL continues to have poor prognosis, mainly because of its resistance to conventional as well as high dose chemotherapy. Therefore the establishment of new therapeutic strategies for ATL is important [30]. This data may serve to utilize the thereupetic properties of the plant and could be effective in preventing and curing patients with ATL. Isolation of individual compound from the plant may act as a good and natural source of drug against T-cell leukemia. Earlier studies have also confirmed the anticancer, antibacterial and antioxidant potential of this plant.

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However, MCF-7 showed very less inhibitory activity when treated with Baliospermum montanum extract. Different extraction methods may also influence the cell cytotoxicity in jurkat as well as MCF-7 cell lines. In future the plant may serve as a good pharmaceutical agent in preventing or slowing down cancers of the immune system.

VI. CONCLUSION

Our current work clearly confirms that the Baliospermum montanum extract induced cytotoxicity against Jurkat cells and can be a potent anticancer candidate for the drug against T- cell leukemia caused by Human lymph tropic virus type 1 (HTLV-1) infection. Further studies are recommended to assess their potential components, their relevant molecular mechanism of action and effective natural therapeutic remedies. The plant is in the stage of extinction and needs conservation.

ACKNOWLEDGEMENT

Authors are thankful to Dr Shubharani, Surana college, Bangalore, Dr V Sivaram , Department of botany, Bangalore university, Bangalore. We are also thankful to Dr Somashekar and Dr Ravikumar, and Manohar from scandal life sciences for their valuable support and guidance.

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