|Year : 2018 | Volume
| Issue : 2 | Page : 321-327
Anticancer potential of leaf and leaf-derived callus extracts of Aerva javanica against MCF-7 breast cancer cell line
Desingu Kamalanathan, Devarajan Natarajan
Department of Biotechnology, Natural Drug Research Laboratory, Periyar University, Salem, Tamil Nadu, India
|Date of Web Publication||8-Mar-2018|
Dr. Devarajan Natarajan
Department of Biotechnology, Natural Drug Research Laboratory, Periyar University, Salem - 636 011, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Aerva javanica is an exotic and medicinal plant in India.
Aim of the Study: The main goal of this study was to evaluate the antiproliferative properties of leaf and leaf-derived callus extracts against human breast cancer cell line MCF-7.
Methods: The plant parts were sequentially extracted with hexane, chloroform, ethyl acetate, acetone, and methanol. The extract was concentrated to yield the crude extract, which was tested for anticancer potentials. The anticancer potential of cytotoxic extracts was determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and DNA fragmentation assays in human breast cancer cell lines (MCF-7).
Results: All the tested extracts showed significant antiproliferative activities in a concentration- and time-dependent manner. The inhibitory concentration of extract was tested against target cell line, and the results show in vitro leaf of A. javanica has higher inhibitory effect against the tested cancer cells at lower concentration (about 11.89 and 22.45 μg/ml) followed by other samples extracts.
Conclusion: The results of the present study conclude in vitro plant sample having more potent anticancer property and support the need of further studies to isolate potential anticancer drug with cancer cell-specific cytotoxicity.
Keywords: 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide and DNA fragmentation assay, Aerva javanica, human breast cancer cell line
|How to cite this article:|
Kamalanathan D, Natarajan D. Anticancer potential of leaf and leaf-derived callus extracts of Aerva javanica against MCF-7 breast cancer cell line. J Can Res Ther 2018;14:321-7
|How to cite this URL:|
Kamalanathan D, Natarajan D. Anticancer potential of leaf and leaf-derived callus extracts of Aerva javanica against MCF-7 breast cancer cell line. J Can Res Ther [serial online] 2018 [cited 2019 Nov 22];14:321-7. Available from: http://www.cancerjournal.net/text.asp?2018/14/2/321/171210
| > Introduction|| |
Cancer is an abnormal growth and proliferation of cells and considered as one of the most life-threatening diseases, which possess many health hazards around the world with 6 million mortalities every year.,, More than 30% of cancers are caused by modifiable behavioral and environmental risk factors, including tobacco and alcohol use, dietary factors, insufficient regular consumption of fruit and vegetable, overweight and obesity, physical inactivity, chronic infections from Helicobacter pylori, hepatitis B virus, hepatitis C virus and some types of human papillomavirus, environmental and occupational risks including exposure to ionizing and nonionizing radiation. Cancer may be uncontrollable and incurable, and may occur at any age, in any part of the body., The most common types of cancers prevailed between men and women are lung, prostate, colorectal, stomach, breast, and cervical cancer. Among them, breast cancer is one of the leading cancers globally with the highest rate of cancer deaths in women. Tissue invasiveness and metastatic spread of breast cancer cells are liable for most of the morbidity and mortality allied with the disease. One among eight women are facing lifetime risk (at any time and age) of developing invasive breast cancer in the world., Hence, there is an urgent need to diagnose at an early stage, though early detection of breast cancer has an advantage of hormone therapy, radiotherapy, and surgery as remedy, there are huge risk of reoccurrences of malignancy or metastatic cancers. MCF-7 is a breast cancer cell line with characteristics of differentiated mammary epithelium, having the ability to process estradiol via cytoplasmic estrogen receptors and capability of forming domes, is selected for present study. The search for novel anticancer drugs from plant sources started in early 1950s, the development of potential anticancer drugs such as vinca alkaloids, vincristine, and vinblastine laid the strong foundation for development of several plant-based drugs., Previously, several medicinal plants were screened for the isolation of anticancer compounds (podophyllotoxin, vinorelbine, vindesine, etc.) and they were found successful in treatment of several cancer including Kaposi sarcoma, breast, and lung cancers. Earlier, paclitaxel analogs have provided a major renewable natural source and were used mostly in breast cancer and Kaposi sarcoma, it was later found to be unproductive in several cases. An imperative need existing among the treatments for breast cancer without damaging healthy cells and hence the identification of naturally occurring phytocompounds from medicinal plants to combat cancers has become highly sought after in recent decades due to their very low side effects. Keeping this view in mind, the present attempt was focused on the anticancer activity of leaf and leaf-derived callus extracts from Aerva javanica against breast cancer cell lines (MCF-7) under in vitro.
| > Methods|| |
The auxiliary buds of A. javanica were collected from wayside thickets, Thuraiyur Town, Tiruchirappalli District, Tamil Nadu, India, during the month of October 2012. The nomenclature of the plant was authenticated by Botanical Survey of India (Government of India), Coimbatore, Tamil Nadu, India (BSI/SRC/5/23/2013-2014/Tech/2083). The herbarium specimen was deposited in the department (PU-Biotech-NDRL-04) for further reference.
Micropropagation of explants
Fresh and healthy explants (nodal segments) were processed aseptically and inoculated in the Murashige and Skoog (MS) medium supplemented with different concentrations of cytokinins and auxins (such as 6-benzyl adenine, kinetin, spermidine (SPM), and 2,4-dichlorophenoxy acetic acid for shoot and callus induction. All the cultures were maintained under cool-white fluorescent light at 24°C with a 16 h photoperiod. The regenerated shoot buds and callus explants were isolated and repeatedly subcultured at regular time intervals (4 weeks) on same or improved medium for the development of multiple shoots and higher callus mass. The well-developed shoots (about 5 cm average) were transferred to MS medium supplemented with Indole-3-butyric acid (IBA) alone and combination with α-naphthalene acetic acid and indole-3-acetic acid (IAA) and incubated as same conditions for roots formation. In vitro, matured plantlets were acclimatized and transferred to the greenhouse in the polycups containing sterile soil and sand (1:1 ratio).
Extraction of plant materials
Matured callus,in vitro and in vivo leaves (about 1.5 months) were harvested and washed with distilled water. Then it was dried under laboratory hot air oven for not exceeding 24 h at minimal temperature. Dried plant samples were ground to make a fine powder. The plant powder (2 g) was weighed and extracted sequentially using organic solvents (such as hexane, chloroform, ethyl acetate, acetone, and methanol) were kept in orbital shaker for 48 h. The extracts were filtered through Whatmann No. 1 filter paper and then extracts were reduced to 10% of its original volume.
Cell lines source
The human breast cancer cell lines (MCF-7) was procured from National Centre for Cell Sciences Pune. Cell line was grown as monolayer cultures maintained in Dulbecco's modified Eagle's medium supplemented with heat-inactivated fetal bovine serum (10%) (GIBCO BRL), 2 mM L-glutamine (Sigma Chemical Co) and mixed with antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin). The cultures were sustained at 37°C in an atmosphere of 5% CO2 incubator with 95% humidified the air. The stock solution was prepared in dimethyl sulfoxide (DMSO) and stored at −20°C until use. The aliquots used in this study were freshly prepared for each experiment with a final DMSO concentration of 0.1%. All the experiments were performed as three replicates.
Assessment of cell viability
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay
Cell viability was measured by 3-(4,5-dimethylthiazole-2 -yl)-2,5-diphenyl tetrazolium bromide (MTT) assay described by Mossman with few modifications. Approximately, 5 × 103 MCF-7 cells were plated in each well (of 96 well plates) and incubated for 24 h for the cell attachment with the medium. After incubation, supernatant of the media was replaced with an equal amount of fresh medium containing different concentrations of plant samples (methanolic extracts of in vivo,in vitro leaf and callus extracts) and 5-fluorouracil (5-FU) (positive control) dissolved in DMSO. After the appropriate incubation period, MTT solution was added to the plate (at a final concentration of 5 mg/mL). The cells were incubated for 4 h in dark at 37°C. The resultant MTT-products were dissolved in DMSO. Viability was calculated by measuring optical density (at 570 nm) using reference wavelength of 650 nm in ELISA reader. From the optical densities, the percentage growth was calculated using the following formula:
Percentage growth = 100 × ([T − T0]/[C − T0])
If “T” is greater than or equal to: “T0,” and if “T” is less than “T0”
Percentage growth = 100 × ([T − T0]/T0)
where “T” = optical density of test
“C” = the optical density of control
“T0” = the optical density at time 0.
A dose response curve was generated by using the growth percentage and IC50 values were interpolated from the growth curves.
After 48 h of incubation, the cells were observed under high-resolution microscopes for clear cell viability, cell morphology analysis and images of each concentration was captured and recorded.
DNA fragmentation assay
MCF-7 cells (1 × 106) were placed in culture plates (30 mm) and the cells reached approximately 70% confluence, add increasing concentrations of samples (methanol extracts of in vivo,in vitro leaf and callus extracts) (IC50 concentration) and incubated for 48 h. The cells were harvested and pelleted by centrifugation at 8000 rpm for 2 min. The harvested cells were washed twice with ice-cold phosphate buffer saline. The cell pellet was lysed in a buffer containing 10 mM tris HCl, 10 mM ethylenediaminetetraacetic acid (EDTA) and 0.2% triton X-100 (pH 7.5). After 10 min on ice, the lysate was centrifuged (13,000 rpm) for 10 min at 4°C. Then, the supernatant (containing RNA and fragmented DNA, but not intact chromatin) was extracted first with phenol followed by phenol:chloroform:isoamyl alcohol (24:25:1) as per the method of Sambrook and Russell. The aqueous phase was brought to 300 mM NaCl, and the nucleic acids were precipitated with 2 volume of ethanol. The pellet was washed with 70% ethanol, air-dried, and dissolved in 20 μL of TE buffer (10 mM tris HCl, 1 mM EDTA, pH 7.5). Then, the sample was digested with RNAse A (0.6 mg/ml, at 37°C for 30 min). The obtained DNA from samples was analyzed by 2% agarose gel electrophoresis. The gels were stained with ethidium bromide and visualized as a DNA ladder under ultraviolet transillumination.
Each experiment was performed in triplicates for statistical analysis (mean ± standard deviation). Data were also subjected to analysis of variance and mean values were compared by Dunnett T3 post-hoc multicomparison tests. Differences at P < 0.05 and 0.01, 0.0001 were considered to be significant.
| > Results|| |
The micropropagation results show cytokinins (benzyl aminopurine [BAP]) alone at the concentration of 1.0, and 2.0 mg/L has produced a considerable amount of multiple shoots in Aerva species. SPM hormone at 0.5 mg/L (A. javanica) induced high rates of shoot regeneration and auxiliary bud proliferation. On the other hand,in vitro flowering was induced by the effect of polyamines (SPM) at the concentration of 1.0 + 0.5 mg/L BAP + SPM. Supplement of auxins alone or in combinations IBA + IAA (1.0 + 1.0 mg/L) have produced maximum root length and mass roots in A. javanica [Figure 1]. The different forms of callus mass were achieved during this investigation [Figure 2].
|Figure 1: Regeneration of A. javanica using shoot-tip and nodal explants (a) Regenerated shoot tip explant, (b) Nodal explant, (c) Callus regeneration, (d) Multiple shooting, (e) In vitro flowering, (f) Shoot elongation, (g) In vitro roots, (h) Matured plants (hardened)|
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|Figure 2: Nature of different forms of callus in A. javanica. (a) Pale yellow green callus (Better callus mass), (b) Brown callus (Best callus mass), (c) Yellow callus (Good callus mass), (d) Greenish callus (Good callus mass) (Non embryo forming cells)|
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Anticancer potential of Aerva javanica extract by 3-(4,5- dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay
Based on the results of MTT assay, methanolic extracts of in vivo,in vitro leaf and callus materials showed the potential inhibition of cancer cell and reduced the risk of further proliferation. MCF-7 cell lines were treated with different concentrations of plant extracts (ranged from 5 to 500 μg/mL). The viability of the cell lines were recorded after optimum incubation period (4 h in dark at 37°C). Highest inhibition of cancer cell growth with minimal viability was observed in the in vitro leaf extracts (at the concentration of 10 μg/mL), followed by the callus extracts (25 μg/mL). The inhibition of MCF-7 cell lines by in vivo leaf extracts was obtained at 100 μg/mL concentration [Figure 3], [Figure 4], [Figure 5].
|Figure 3: Different concentrations of A. javanica in vivo leaf extract in MTT assay|
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The cytotoxic potential of different concentrations of A. javanica extracts showed 100% cell death (MCF-7 cell lines) with an increased concentration of the plant extracts tested. Further, the IC50 values of test extracts were calculated, and the results are tabulated [Table 1]. The IC50 of in vitro leaf material was recorded as 11.89; callus was 27.18 and in vivo leaf was 114.43 μg/mL, respectively. Based on the results,in vitro leaf and callus materials were found to have higher anticancer potential than the in vivo materials, and it was compared with the commercial standard (5-FU) which showed IC50 of 22 μg/mL. Further, these concentrations were tested by DNA fragmentation assay for the confirmation of the cell apoptosis. Henceforth, the lower concentrations of in vitro leaf and callus materials could be used in the anticancer activity.
|Table 1: Antiproliferative effect of A. javanica samples on human Breast cancer cell line (MCF-7)|
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DNA fragmentation assay
The arrest of cell cycle and control of its proliferation was done by calculating the fragmentation analysis of the DNA. The optimum IC50 values were used for the determination of the DNA fragmentation, and formation of the typical ladder fragments. The fragmented DNA bands at the base pair range of 1500–100 bp as noticed in the agarose gel. Clear fragmentation was observed in all the tested samples (in vivo, in vitro leaf and callus). Nonfragmented control DNA showed single band in the agarose gel. Fragmentation of DNA by in vivo, in vitro leaf and callus materials was similar to each other [Figure 6].
|Figure 6: DNA fragmentation of MCF – 7 cell line. M – Marker, L1 – Control, L2 – In vivo sample, L3 – In vitro sample and L4 – Callus sample|
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| > Discussion|| |
Breast cancer is the second main deadly disease for women worldwide. Phytochemicals isolated from medicinal plants are considered as promising agents for the clinical treatment of breast cancer. The present study reported anticancer potentials of in vivo, in vitro leaf and callus extract of methanol solvent of A. javanica was tested against human breast cancer (MCF-7) cell line by MTT assay and DNA fragmentation analysis. The different concentrations of aliquots with optimum IC50 values recorded potential anticancer property.In vitro leaf of A. javanica has higher inhibitory effect against the tested cancer cells at a lower concentration (about 11.89 and 22.45 μg/ml) followed by other samples extracts. The induction of cytotoxicity (alteration in morphologic characteristics) and DNA fragmentation was recorded in a concentration dependent manner, the increased concentration of extracts induced a higher rate of cellular damages (MTT and DNA fragmentation assay). Similar kind of observation was recorded by Monga et al., in MCF-7 cell line. DNA fragmentation was noticed (as a ladder) in the agarose gel electrophoresis with the multimers of 100 bp. Formation of DNA fragmentation is one of the characteristic features observed during apoptosis of the cells and is considered as a biochemical hallmark of apoptosis. It directly correlate with the early morphological signs of apoptosis and has been widely used as a distinctive marker of the apoptosis process.,, The methanolic extracts of A. lanata was effective against ehrlich ascites carcinoma (EAC) at a low LC50 which was <50 μg/mL. Similarly, cytotoxic properties and solid tumor effects of Aerva lananta was studied by Nevin and Vijayammal. Siveen and Girija who reported that the ethanol extract of A. lanata was successful in elimination of Dalton's lymphoma ascites (DLA) and EAC cells at a concentration of 500 μg/mL. Rajesh et al., reported potential anticancer activity on DLA by the aerial part extracts of A. lanata which strongly support the results of the present investigation. Callus extracts proved promising anticancer activity than the wild leaf extracts of Moringa oleifera against Hela cells.
Few other reports witnessed the potential use of various medicinal plants for control of the proliferation of human breast cancer. The induction of apoptosis in human breast cancer cell by betulinic acid of jujube fruits was reported by Sun et al., Cytotoxic activity of extracts from pikutbenjakul preparations from Thai traditional medicine preparation, composed of five plants (Piper chaba, Piper sarmentosum, Piper interruptum, Plumbago indica, and Zingiber officinale) against breast cancer cell (MCF-7) was documented by Sakpakdeejaroen and Arun. Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells using the extracts of Antrodia camphorata was reported by Ling et al., Anticancer effect of Cassia auriculata leaf extract in vitro through cell cycle arrest and induction of apoptosis in human breast (MCF-7 cell lines) and larynx cancer cell lines was reported by Prasanna et al., Recently, Nazeema et al., and Gitanjali and Debasish reported the potential in vitro anti-breast cancer activity of Datural metel and Limonia acidissima against MCF-7 cell line. Similarly, cytotoxic activity of Broussonetia papyrifera on MCF-7, HELA and HepG2 cell lines were reported by Naveen Kumar et al. Ali et al. and Pavan Kumar et al. reported the promising antiproliferative and apoptotic activity by Lavandula dentata and Silybum marianum extracts against human breast cancer adenocarcinoma cells. Henceforth, our result also ensures the anticancer potential of in vitro leaf and callus methanolic extracts from A. javanica on human breast cancer. This is the 1st time report on anticancer properties of wild and micropropagated Aerva species.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]