Impact of ethanolic extract of Mikania glomerata on human breast cancer (MCF 7) cell line

The ethanol extract of Mikania glomerata has anti-proliferative effect on the human breast cancer cell lines. The object of the present work is to investigate the anti-cancer effect of Mikania glomerata ethanolic extract on breast cancer. Soxlet fractions using crude ethanolic extract of Mikania glomerata was prepared by standard extraction protocols. To check the antiproliferative effect of this extract, the extract chosen was tested for cell viability on the breast cancer cells MCF 7 in different concentrations. Cell viability was evaluated by MTT assay for 24 hour and 48 hours. The LD50 value was calculated and different morphometric assays were performed with the effective dose of the extract. The effect of the extract on the normal cell was evaluated as well. Cell proliferation, cell cycle, Clonogenic survival, Apoptosis and MTT assays were performed. The ethanolic extract showed a dose-dependent and time dependent inhibition on cell proliferation in the breast cancer cell lines. It showed low cytotoxicity in the normal cells and inhibited cellular adhesion and wound healing in treated cancer cells. The present study suggests that the leaf extract from Mikania glomerata induces anticancer effect on the breast cancer cells. Further study might help to confirm it as an anti-cancer drug.


Introduction
Herbal medicine is the oldest form of healthcare known to the human race with a history of documentation for almost 4000 years. It has survived for ages and is still a vibrantly alive branch of medicine that is being used actively in many cultures throughout the world today. The World Health Organization estimates that 80% of the world's population, mainly in the developing countries relies on herbs for their primary health care needs. This is principally due to the popular belief that herbal drugs are without any side effects besides being inexpensive and obtainable in the neighborhood.
Cancer is one of the major threats of modern life, which is considered as the second cause of death after myocardial infarction. Millions of people die every year in different types of cancer despite tremendous efforts to find methods of control and cure. In the last century, great advances were made in modern medical science to control disease. But many diseases like cancers are not yet curable fully. To find out new and authentic therapies, scientists are working with traditional or folk medicines in parallel of modern medicine. Mikania glomerata has been used for medicinal purposes for centuries.
The active ingredients of M. glomerata have beneficial effects against many diseases, including cancers. There are not so many research works done in this field and very few review articles exist in this area and hence this study.
www.ssjournals.com on a water bath to a syrupy mass. The dried substance was dissolved in ethanol and stored in cold room for future use.

Cell lines and growth conditions
The cell line used for the proliferation study was MCF7 Human breast cancer cell lines. MCF-7 cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in monolayer in Nunc culture dishes (six wells of 9.6 cm 2 /plate). Cells were maintained in Dulbecco's minimum essential medium (Gibco) supplemented with 5% PCS (Gibco Bio-Cult, Paisley, Scotland), glutamine (2 mmol/litre), streptomycin (100 ug/ml) and penicillin (100 lU/mL), and phenol red as pH indicator (standard medium). During growth experiments cells were grown in the absence of the pH indicator phenol red (11), and CSFCS (Charcoal stripped foetal calf serum) replaced the normal PCS (experimental medium). The charcoal stripping was performed by shaking 200 mL of FCS with 1.0 g of Norit A charcoal and 0.1 g of dextran (Sigma Chemical Co., St. Louis, MO) at 37Â°C for 3 h.
The serum was then centrifuged at 9000 x g for 15 min. Growth factors and inhibitors added to the medium were: insulin, EGP, and estradiol-17/3 (obtained from Sigma), interleukin-1Â« and IGF-1 (Insulin like growth factor 1-gene) and TGF/3 (Transforming Growth Factor-3). The cell layer was briefly rinsed with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contain trypsin inhibitor. 2.0 to 3.0 mL of Trypsin-EDTA solution was added to flask and cells were observed under an inverted microscope until cell layer was dispersed (usually within 5 to 15 minutes). 6.0 to 8.0 mL of complete growth medium was added and cells were aspirated by gently pipetting. Appropriate aliquots of the cell suspension were added to new culture vessels. The cultures were incubated at 37°C.

Cell proliferation assay
All cells were grown as described earlier. For cell proliferation effects, four different concentrated samples (10,20,30, 40 µg/mL) were seeded at 20,000 cells per flask. All cells were grown for 24 hours and cell counts were taken. the cell lines study was used to screen the activity of ethanolic extract of Mikania glomerata leaves against for MCF7 Human breast cancer cell line.

Cells counting using haemocytometer
Assessment of cell viability and distinction of cell types was carried using Neubauer counting chamber. Cover-slip was moistened and affixed to the haemocytometer. Care was taken to ensure that the cover-slip and haemocytometer were clean and greasefree. Alcohol was used for cleaning purpose. A small amount of trypan blue-cell suspension was transferred to one of the chambers of the haemocytometer by carefully touching the cover slip at its edge with the pipette tip and allowing each chamber to fill by capillary action. Overfilling or under filling of the chamber was avoided.

Determination of the number of cells (total and viable)
The cells were viewed under a microscope at 100x magnification. Focusing was done to see a grid of 9 squares. Then the microscope was focused on one of the 4 outer squares in the grid. Adjustments were made so that the square contained 16 smaller squares. Cell counting was carried in the four 1 mm corner squares. Procedure was repeated based on either concentration or dilution. Trypan Blue is the "vital stain"; excluded from live cells. Live cells appear colourless and bright (refractile) under phase contrast. Dead cells stain blue and are non-refractile. The cells were counted as per the procedure illustrated. 4 corner squares were counted and the average was calculated. Each large square of the haemocytometer, with coverslip in place, represents a total volume of 0.1 mm 3 (1.0 mm X 1.0 mm X 0.1 mm) or 10 -4 cm 3 . Since 1 cm 3 is equivalent to approximately 1 ml, the total number of cells per ml was determined using the following calculations: %Cell

Assessment of cell density and evaluation of viable cells
The samples were diluted to appropriate concentrations and the absorbance of the sample was measured by means of spectrophotometer at 550 nm. Consistent wavelength must be used while measuring the cell density of a particular cell line. An independent calibration curve was generated for each cell line [1]. After spectrophotometer analysis, actual counting of cells was executed using counting chamber.

Fluorescence assay for cell cycle distribution
At the end of 24 hours after treatment, the cells were smeared on to glass microscope slides, fixed with methanol and air dried. The cells were then stained with propidium iodide (0.1µg/mL) and scored under fluorescence microscope fitted with PI filter. A total of 100 cells were scored for intensity of DNA staining in the nucleus. Initially the slides were scored for determining the 1x fluorescence intensity using intensity tool from Adobe Photoshop. This was determined by counting the cells and the majority of www.ssjournals.com cells will fall into this category. This is the G1 cells fluorescence intensity. Based on this intensity other phases of cell cycle were determined 2.6 Clonogenic survival assay The clonogenic cell survival assay determines the ability of a cell to proliferate indefinitely, thereby retaining its reproductive ability to form a large colony or a clone. This cell is then said to be clonogenic. For cell proliferation effects, cells were seeded at 20,000 cells per flask.
Three flasks were used per concentration. All cells were grown for 24 hours. The various treatment conditions on MCF7 breast cancer cell line with different concentration of ethanolic extract of Mikania glomerata. At the end of 24 hours' treatment, the cells were removed and plated on to separate flasks. Trypsinize the stock flask of cells containing the cells that have to be tested. Make sure that the cells are in single-cell suspension and obtain an accurate cell count. The cells were counted using a hemocytometer. Using a Pipette, add 20,000 cells (the cell number can vary depending on the cell type) to the 5 mL of medium in each T-25 flask. Shake gently to distribute the cells evenly. The flasks were then incubated for 12-14 days at 37ºC. The flasks were then stained with Gentian violet and scored for number of colonies.
Clonogenic assay serves as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells which can often only be determined microscopically. Clonogenic assay is the method to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. The following protocol has been modified from a published version [2].

Apoptosis
Apoptosis induction was determined by measuring Annexin V activity using Annexin V apoptosis kit. The kit was sourced from Promega corporation, Southapton, UK and consisted of Annexin V buffer and lyophilized Annexin V substrate. Cells were seeded at the concentration of 104 cells per well in 96 well microtitre plate and incubated for 48 hours. Aliquot of 100 µL of fresh media containing appropriate concentration of extracts were transferred to the assigned respective wells. Stauroporine 0.1 µg/mL was used as positive control and untreated wells were treated as negative control. Total of six wells were assigned for each treatment. The plate was allowed to equilibrate to room temperature after 24 hours of incubation prior to performing the assay. 100 µL of Annexin V reagent was added to each well and mixed for 60 seconds and incubated for further 1 hour at room temperature. An aliquot of 100 µL of contents from each well was transferred to white-walled 96 well plate. The light emitted was measured by Packard lumicount microplate luminometer and measurement was recorded using THERMOmax TM plate reader linked to a computer using SoftMax Pro software (Muñoz-Alonso et al., 2008). The results are depicted in the tabular form.

MTT assay
MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide, a yellow tetrazole), is sliced by mitochondrial dehydrogenase of viable cells, resulting in a purple formazan product that could be measured spectrophotomerically. The formazan product formed is in proportion to the number of viable cells and inversely proportional to the amount of cytotoxicity. The absorbance of formazan product can be measured at a wavelength between 510 and 600 nm using a spectrophotometer [3] [4]. The positive control treatment consisted of 1 % phenol. Positive control reagents are chemicals known to bring a standard positive response in a test system. The positive control chemical is used to show that the test system is responding satisfactorily and that the test is valid. The negative control used consisted of the sterile distilled water. Qualitative and quantitative evaluations of viability of cells were determined using MTT assay. Cells were seeded at the density of approximately 1x10 5 cells per well in 96 well plates in 100 µL medium. After an incubation of 48 hours' culture medium was removed and replaced with aliquots of 100 µL of medium containing appropriate concentration of extracts particles. 1 mg/mL of phenol was treated as positive control and untreated cells served as negative control. Total of six wells were assigned for each treatment. The cells were treated with the test material and positive control for 24 hours. Following the incubation period the cells were examined microscopically to assess the morphological alterations indicative of qualitative evaluation of toxicity. A 100 µL of MTT reagent was added to each well and mixed at least for 60 seconds and the plates were incubated for approximately 4 hours. 100 µL of Dimethyl Sulfoxide (DMSO) was transferred to each well to lyse the cells. Absorbance of the lysate was measured at 560 nm to determine the quantitative evaluation of cytotoxicity using the THERMOmax TM plate reader linked to a computer using SoftMax Pro software.
The culture medium was removed from the plate and the wells were washed with phosphate buffered saline. Aliquots (100 μL) of the culture medium containing appropriate concentrations of the test article, positive controls, and negative control were applied to the plate wells. Plate was incubated at

Results and Discussion
Different classes of compounds were previously isolated from various Mikania parts, which can be associated to this plant's pharmacological activities. The main groups are: coumarins and derivatives, sesquiterpenes, sesquiterpenes lactones, diterpenes, phytosterols/terpenoids and flavonoids. Caffeoylquinic acid derivatives beyond other chemical compounds are found in smaller amount. Diterpenes such as kaurenoic acid and benzoylgrandifloric acid (class of kauranes), have also attracted interest for their pharmacological action. Moreover, detailed screenings revealed the presence of other substances in species of Mikania as alcohols, acids, esters, aldehydes and organic esters [5] [6]. These ingredients and their derivatives are found to have analgesic activity [7][8], antibacterial [9][10] and anticancer properties [11]. One of the main species of genus Mikania is M. glomerata popularly known as 'guaco' with a long standing record application and has a reasonable reputation to have many therapeutic properties was taken for screening of any anti-cancer activities. Hence, the effects of alcoholic of M. glomerata were investigated on cell proliferation, cell cycle analysis, clonogenic survival assay, apoptosis and cytotoxicity using MCF7 (human breast cancer cell lines) cell lines.

Cell proliferation assay
In the cell proliferation assay on MCF7 cells using ethanolic extract of Mikania glomerata, initial cell counts were done in the triplicate culture vessels and 24hrs counts were made to ensure cell proliferation in the taken test cell line. The initial count had a mean value of 20,777 cells and the 24hr count had a mean value of 50,914 cells. Control cell lines with a mean value of 50,444 cells were set up without the test material to assess the inhibitory effect. The results indicated that the ethanolic extract of Mikania glomerata on the MCF7 human breast cancer cell line did not produce cell growth inhibitory effect in the cell proliferation assay. Significant result was not obtained, even in higher test dose (Table: 1; Figure: 1).
Development and optimization of in vitro assays provides for a simple, reliable, sensitive, reproducible, inexpensive and high-throughput method with which to assess the efficacy of novel chemotherapeutic pharmaceuticals on cell survival or proliferation early in the discovery process of drug development [12]. Anti-proliferative effect of the extract of the root of Polygonum multiflorum on MCF-7 human breast cancer cells and the possible mechanisms was demonstrated by Chen et al [13]. Methanol extracts of Scrophularia species, a medicinal plant widely used in folk medicine since ancient times exhibited strong anti-proliferative properties on HL-60 leukaemia cells leading to strong growth inhibition and high cell death rates [14] [15]. Reagan-Shaw et al [16] in 2010 reported antiproliferative effects of apple peel extract against cancer cells. Similarly, hexane extract of Raphanus sativus root exerts potential chemopreventive efficacy and induces apoptosis in cancer cell lines throµgh modulation of genes involved in apoptotic signaling pathway [17]. Inhibitory effect of bitter melon (Momordica charantia) on cell proliferation of adrenocortical cancer cells was illustrated by Brennan et al [18] in 2012. Aqueous extract of Trametes robiniophila induced inhibitory effect on both MCF-7 and MDA-MB-231 cells. In line with these authors cell proliferation assay was conducted on three human cancer cell lines using extracts of M. glomerata.

IJASR|VOL 02|ISSUE 04|2016
www.ssjournals.com The cell cycle governs the transition from quiescence (G0) to proliferation while ensuring the fidelity of the genetic transcript. The phases associated with DNA synthesis (S phase) and mitosis (M phase) are separated by the gaps G1 and G2. Cell cycle checkpoints are control mechanisms that ensure the fidelity of cell division in eukaryotic cells. These checkpoints verify whether the processes at each phase of the cell cycle have been accurately completed before progression into the next phase. Multiple checkpoints have been identified in the cell cycle process [19]. The key parts of the cell cycle machinery are the cyclin-dependent kinases (cdks) and the regulatory proteins called cyclins. The cdks are rational targets for cancer therapy because their expression in cancer cells is often aberrant and their inhibition can induce cell death. Inhibitors of cdks can also block transcription [20]. Momordica charantia extract inhibits breast cancer cell proliferation by modulating cell cycle regulatory genes [21].

Clonogenic survival assays
Apoptosis, or programmed cell death is a cascade of normal physiologic process for removal of unwanted cells. The occurrence of this mechanism was detected on MCF7 human breast cancer cell line using fluorochrome-labeled Annexin V after 24 hrs exposure of ethanolic extracts in graded concentrations. The Phosphatidyserine (PS) levels (RFUs) in cells from the untreated controls served as a baseline indicator for normal Phosphatidyserine levels. The cultures treated with the positive control, stauroporine, 0.1 µg/mL produced a significant increase in Relative Fluorescence Unit (RFU), indicating release of Phosphatidyserine to the cell surface and thereby demonstrating the initiation of apoptosis. The results showed no significant apoptosis activity following treatment with 10, 20, 30 and 40 µg/ml (24 hrs.) of ethanolic extract of Mikania glomerata on MCF7 cells (Table: 3; Figure: 3).
The clonogenic assay estimates the regenerative potential of the extract or drug treated cells. Any subtle metabolic effect rendered by drug may get reflected by reduced ability of the cells to proliferate. It can be detected by number and size of the colonies formed by cells after exposure to drµg [22]. It is a routinely used assay to look at cell survival following radiation exposure. This is gold standard method for looking at long term cell growth and differentiation. Hudson et al [23] in 2000 reported that in the clonogenic assay, the brown rice bran extract decreased colony formation in SW 480 and MDA MB 468 cells (human-derived tumor cell lines) and further recorded that the effect was particularly potent against MDA MB 468 cell line. Combinatorial cytotoxic effects of Curcuma longa and Zingiber officinale on the PC-3M prostate cancer cell line using clonogenic assay was investigated and the potent chemopreventive activity of the test substances against human cancer cell line was concluded by Kurapati et al [24] in 2012.

Apoptosis using Annexin V
Apoptosis, or programmed cell death is a cascade of normal physiologic process for removal of unwanted cells. The occurrence of this mechanism was detected on MCF7 human breast cancer cell line using fluorochrome-labeled Annexin V after 24 hr exposure of alcoholic extracts in graded concentrations. The Phosphatidyserine (PS) levels (RFUs) in cells from the untreated controls served as a baseline indicator for normal Phosphatidyserine levels. The cultures treated with the positive control, stauroporine, 0.1 µg/mL produced a significant increase in Relative Fluorescence Unit (RFU), indicating release of Phosphatidyserine to the cell surface and thereby demonstrating the initiation of apoptosis. The results showed no significant apoptosis activity following treatment with 10, 20, 30 and 40 µg/ml (24 hr) of alcoholic extract of Mikania glomerata on MCF7 cells (Table: 4; Figure: 4).
The binding of Annexin V to phosphatidylserine is a common assay used as a measure of drug toxicity as it reflects translocation from the inner to the outer leaflet of the plasma membrane, which is one of the earliest features of cellular death via apoptosis [25] [26]. Different researchers have documented the effect of plant extracts on cancer cell lines by using Annexin V for the reading of apoptosis. According to Mukherjee et al [27], ethanolic leaf extract of Thuja occidentalis blocks proliferation of A549 cells (lung carcinoma cells) and induces apoptosis in vitro. Anti-cancer potential study of plant extracts adopting Annexin V on Ziziphus mauritiana in vitro against HL-60, Molt-4, and HeLa cancer cell lines [28], algal extract on the AML cell lines HL-60 and MV-4-11 [29], Sutherlandia frutescens (cancer bush) extract on SNO oesophageal cancer cell line [30], Chaetoceros calcitrans extract on human breast cell line (MCF-7) [31], Mikania hirsutissima leaf extract fraction on U87 human glioblastoma cell line [32] and Melilotus officinalis (sweet clover) extract fraction on human TK6 lymphoblastoid cell line [33] were studied and their apoptotic property has been recorded.

Cytotoxicity using MTT assay
The optical density (OD) mean value calculated for untreated cells was 0.82 ± 0.08. The positive control (phenol 1%) computed mean OD value was 0.192 ± 0.07 indicating the validity of the experiment. Introduction of cultures to the extract for 24hrs in the graded concentrations of 10 µg/ml, 20 www.ssjournals.com µg/ml, 30 µg/ml and 40 µg/ml was done to assess the cytotoxic effect.

Conclusion
The anti-cancer activities of Mikania glomerata components were recognized thousands of years ago but proper scientific research with this important traditional medicine is a very recent story. More research works should be emphasized behind this because it is a safe and promising anticancer agent. Specially, researchers should investigate the active ingredients more broadly, because, there is very few authentic reports about the chemical composition of seeds or oil of M. glomerata exist. Also, the exact molecular mechanisms of components on different cancers should be investigated with more emphasis because current understandings are mostly unclear. For example, it is reported that M. glomerata oil can protect cells from radiation, but the molecular mechanisms behind this is not properly understood. Currently, in some parts of the world, there is a renaissance of interest in traditional remedies. Many investigators now believe that traditional medicine is a promising source of new therapeutics against cancer. Extensive research with M. glomerata may contribute to the discovery of new anticancer strategies.