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The cytotoxicity assay of the ethanol extract of Rhaphidophora aurea intertwined over Lawsonia inermis and Areca catechu were tested against human breast cancer cell line by the MTT assay method. The dose-response effect and the IC50 values of the extracts were determined. Both the extracts produced significant growth inhibition against MCF-7 breast cell line with the increasing concentration. The maximum percentage inhibition was obtained at 300 Âµg/ml for both the extracts (97%). The aerial roots of Rhaphidophora intertwined over MMDOH and MBDOH displayed potent anticancer activities in vitro that can be further exploited for the development of a potential therapeutic anticancer agent.
Keywords: Rhaphidophora aurea, MCF-7 cell line, Anticancer activity,
Human cancer growth is often mainly an effect of deregulated cell cycle control and suppressed apoptosis [8-9]. Impairment of apoptosis is related to cell immortality and carcinogenesis, thus, the induction of apoptosis in neoplastic cells is therefore, important in cancer treatment . The word "apoptosis" is used to describe a common series of morphological changes involving the nucleus, cytoplasm and plasma membrane that accompanied the death of cells from a variety of tissue sources . The earliest recognized morphological changes are compaction and segregation of nuclear chromatin, chromatin margination, convolution of nuclear and cell outlines and followed by breaking of the nucleus into discrete fragments by budding or blebbing of the cell to produce a membrane-bounded apoptotic bodies . All these morphological characteristics could be observed using transmission electron microscopy.
Currently applied radiation therapy and standard chemotherapeutic drugs kill some tumor cells through induction of apoptosis. Unfortunately, however, the majority of human cancers is resistant to these therapies (3,4). It is therefore urgent to look for novel natural or synthetic apoptosis-inducing compounds as candidate antitumor agents. Along this line, plant-derived compounds have great potential to be developed into anticancer drugs because of their multiple mechanisms and low side effects (5-9).
The plant Rhaphidophora aurea recently reported to have potent microbial, wound healing, antioxidant activity and phytoconstituents like alkaloids, flavonoids, steroids, terpinoids, tannins, saponins, glycosides, anthocyanin, phenol and anthraquinin. Also the similar species Epipremnum pinnatum (L.) Engl. Exhibited cytotoxic activities against murine as well as human cell lines .
Breast cancer is the most commonly diagnosed cancer in women, representing approximately thirty percentage of all types of cancer in women (1). Based on the phytoconstituents and antioxidant properties Rhaphidophora aurea the ethanol extract of the aerial roots of Rhaphidophora aurea intertwined over Lawsonia inermis and Areca catechu were tested for anticancer activity against the MCF-7 cell line in vitro.
Materials and method
The aerial roots of Rhaphidophoraaureae intertwined over Lawsonia inermis (MM) were collected from Coimbatore District; Areca catechu (MB) were collected from Palakkad District and it was identified by the Botanical Survey of India, Coimbatrore by Joint director BSI.
The powdered MM and MB were extracted with suitable volume of solvents and refluxed for 16 hours and filtered, this process was repeated till the filtrate was colorless. The filtrate was distilled by Rotary evaporator and labeled as MMDOH and MBDOH.
The human breast cancer cell line (MCF 7) was obtained from National Centre for Cell Science (NCCS), Pune and grown in Eagles Minimum Essential Medium (EMEM) containing 10% fetal bovine serum (FBS). All cells were maintained at 370 C, 5% CO2, 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week.
Cell treatment procedure
The monolayer cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) to make single cell suspensions and viable cells were counted using a hemocytometer and diluted with medium containing 5% FBS to give final density of 1x105 cells/ml. One hundred microlitres per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 370C, 5% CO2, 95% air and 100% relative humidity. After 24 h, the cells were treated with serial concentrations of the test samples (MMDOH & MBDOH). They were initially dissolved in neat dimethylsulfoxide (DMSO) and diluted to twice the desired final maximum test concentration with serum free medium. Additional four, 2 fold serial dilutions were made to provide a total of five sample concentrations. Aliquots of 100 Âµl of these different sample dilutions were added to the appropriate wells already containing 100 Âµl of medium, resulted the required final sample concentrations. Following drug addiction the plates were incubated for an additional 48 h at 370 C, 5% CO2, 95% air and 100% relative humidity. The medium containing without samples were served as control and triplicate was maintained for all concentrations.
3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (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 48h of incubation, 15Âµl of MTT (5mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 370C for 4h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100Âµl of DMSO and then measured the absorbance at 570 NM using a micro plate reader.
The % cell inhibition was determined using the following formula.
% cell Inhibition = 100- ABS (sample) /ABS (control) x100.
Nonlinear regression graph was plotted between % Cell inhibition and Log10 concentration and IC50 was determined using GraphPad Prism software.
Cytotoxicity of MMDOH and MBDOH against MCF-7
MMDOH and MBDOH showed a dose and time dependent inhibitory effect of MCF-7. IC50 was 62.96 Î¼g/ml for MBDOH and 35.26 Î¼g/ml for MMDOH. The minimum absorbance of cell growth (0.01) was obtained at 300 Î¼g/ml. The results of cytotoxicity of both extracts against human breast cancer cells is shown in figure 1. The figure clearly shows that the control absorbance was maximum when compare to 18.75 Î¼g/ml contraction absorbance value of both the extracts. The absorbance value was decreased with increased extract concentration.
Figure 1: Absorbance of MBDOH and MMDOH against MCF-7 at 48 hours
Percentage inhibition of MBDOH and MMDOH
The maximal inhibition of cell growth 97% was obtained at 300 Î¼g/ml for both the extracts. Half the minimal inhibitory value of MBDOH is 62.96 Î¼g/ml and MMDOH is 35.26 Î¼g/ml. MMDOH percentage inhibition of 75 Î¼g/ml was equal to MBDOH 150 Î¼g/ml, when compare to MB, MM gives a maximum percentage inhibition with the minimum concentration. The results from Percentage cell inhibition and half minimal inhibitory value against human breast cancer cells is shown in Table 1 and Figure 2. The histogram of both the extracts shows a significant decrease of treated cells, it clearly shows a significant difference from the control (Image 1).
Table 1: percentage cell inhibition (48 hours) of MBDOH and MMDOH
Figure 2: Half minimal inhibitory concentration (48 hours) of MBDOH and MMDOH
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The MMDOH and MBDOH contains phytoconstinstuents like alkaloids, flavonoids, tannins, terpenoids, steroids, anthraquinon, anthocyanin, glycosides, and phenols (Arulpriya, ).
The anthracycline doxorubicin is frequently used as a chemotherapeutic agent against metastatic breast cancers . Plant alkaloids like docetaxel and paclitaxel are considered highly active chemotherapeutic agents in various cancers including those of the breast and prostate [4,5]. 1472 & 278. Plant sterols (Carmela), poly phenolics and flavonoids (Wang) are having an anticancer property.
The various solvent extracts of the aerial roots of Rhaphidophora aurea intertwined over Lawsonia inermis (MM) and Areca catechu (MB) has shown to have antioxidant properties against DPPH and Reducing power assay in vitro (Hemalatha , Arulpriya) and Minimum inhibitory concentration of both MM and MB has shown to have antimicrobial properties.
The present study confirmed that the ethanol extract of the aerial roots of Rhaphidophora aurea intertwined over Lawsonia inermis and Areca catechu has strong dose and time - dependent anticancer activity against human breast cancer cell with the IC50 and inhibited the cell growth potential of cancer cells in a dose depend manner (Figure 1 &2) and table1).
Cultured cancer cells are valuable reagents for rapid screening of potential anticancer agents as well as for the elucidation of mechanism of their activity (Singh). It was well known that human breast cancer cell line MCF-7 is Estrogen receptor (ER) positive, but approximately one third of breast cancers are ER negative, carrying a worse prognosis than the positive ones (Swami).
Conclusion the potential anticancer activity of ethanol extract of the aerial roots of Rhaphidophora aurea intertwined over Lawsonia inermis and Areca catechu against human breast cancer was investigated in this in vitro cytotoxicity study , for the first time. Both the extracts exhibited a strong inhibitory effect on MCF-7 cell lines. This experimental study suggests that Rhaphidophora area contains some constituents, which would be useful for anticancer drug discovery.