Human Breast Adenocarcinoma Cells

Published Date: 02 Nov 2017

Human breast adenocarcinoma cells (MFC-7) were obtained from National Centre for Cell Science (NCCS), Pune and preserved in Eagles Minimum Essential Medium (EMEM) containing 10% fetal bovine serum (FBS). All cells were maintained at 370 C, provided with 5% CO2, 95% air and 100% relative humidity in a CO2 incubator. Maintained cultures were weekly passaged, and the culture medium was changed twice a week.

Preparation of cell suspension

The monolayer cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) to make single cell suspensions. The viable cells were counted using a hemocytometer and diluted with medium containing 5% FBS to give final density of 1x105 cells/ml. About one hundred microlitres of cell suspensions were seeded into 96-well plates at plating density of 10,000 cells/well. The plates were incubated at 370C provided with 5% CO2, 95% air and 100% relative humidity to allow for cell attachment. After 24 hours the cells were treated with serial concentrations of the test samples.

Preparation of test plant extracts

About 6 mg of plant extract was dissolved in 2 mL of sterile DMSO (serum free medium) to obtain a stock concentration of 6000 µg/2 mL. One mL of the stock plant extract was kept undispensed and another 1 mL was serial two fold diluted in sterile tubes containing 1mL of DMSO, so as to obtain a drug concentration ranging from 3000, 1500, 750, 375 and 187.5 µg/ mL. Aliquots of 100 µl of different sample dilutions were added to the appropriate wells already containing 100 µl of the medium. Following drug addition the plates were incubated for an additional 48 h at 370 C, 5% CO2, 95% air and 100% relative humidity. The medium amended without plant samples were served as control and triplicate was maintained for all concentrations.

MTT assay

The 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) assay is a colorimetric assay for measuring the activity of cellular enzymes that reduce the tetrazolium dye, MTT, to its insoluble formazan, giving a purple color. MTT is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Therefore, the amount of formazan produced is directly proportional to the number of viable cells.

After 48 hours of incubation, 15µl of MTT (5mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 370C for 4 hours until purple precipitates were clearly visible under a microscope. Flowingly, the medium together with MTT was then flicked off and the formed formazan crystals were solubilized in 100µl of DMSO and measured the absorbance at 570 NM using a micro plate reader. The percentage cell inhibition was determined using the following formula:

:

Percentage cell viability (CV)

100- Average ABS of duplicate drug wells

=

x100

Average ABS of control wells

Statistical analysis

Data were presented as mean ±SEM. Nonlinear regression graph was plotted between % cell inhibition and Log10 concentration. A dose-response curve were plotted to enable the calculation of the concentrations that kill 50% of the MCF-7 cells (IC50) using Graph Pad Prism software.

In the present study, the cytotoxic effect of Couroupita guianensis crude aqueous and organic solvent leaf extracts on MCF-7 cells were evaluated by MTT assay. MTT assay is a well-established in vitro method for cytotoxicity against cancer cell lines and non-cancer cell lines (Abu-Dahab et al., 2007). Among the four extracts used, the ethanolic extract was found to be highly antiproliferative on MCF-7 cell lines with an IC50 value 71.39 µg/ml, while the chloroform leaf extracts were moderately antiproliferative with an IC50 value of 150 µg/ml. It is of great interest that the ethanolic extracts of C. guianensis had shown an encouraging activity against MCF-7 proliferative cells when compared to other extracts. On the other hand, we found the methanolic leaf extract was less active on MCF-7 cell lines and its IC50 value was > 300 µg/ml. Early reports on cytotoxicity assays using plant extracts suggested cytotoxicity screening models provide important preliminary datas to identify plant extracts with potential antineoplastic properties for future work (Cardellina et al., 199; Baskar et al., 2010).

In vitro cytotoxicity assays may sometimes pave a way to identify certain anticancer drugs from the herbal sources and its application towards in vivo treatment. The neoplastic activity of the plant extract may be due to the presence of phytoconstituent specifically flavonoids and plant herbs rich in flavonoids have been reported with therapeutic properties. Since the flavonoids are the ingredients of vegetables, fruits and other plant parts, the consumption of flavonoid rich plant products have been reported to reduce the risk of cancer (Ramos, 2007; Kanadaswami et al., 2005; Ren et al., 2003). In the present study the preliminary phytochemical screening of C. guianensis leaf extracts were found to posses flavonoids in the organic solvent fractions and its presence may elicit the antiproliferative property on MCF-7 cell lines. Further investigations are essential to isolate and identify the types of flavonoids in the leaf extracts of C. guianensis. Flavonoids are widely distributed in plants fulfilling floral pigmentation producing yellow or red or blue pigmentation, UV filtration, symbiotic nitrogen fixation, chemical messenger or physiological regulator, cell cycle inhibitors, inhibitory activity against organisms that cause plant diseases. Flavonoids might induce mechanisms that affect cancer cells and inhibit tumor invasion.

Flavonoids or bioflavonoids are a class of plant secondary metabolites referred to as vitamin P with a permeability of vascular capillaries.

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