Background
Around one quarter of deaths in the US are accounted for by Cancer. According to the American Cancer Society, on an average, 559,312 people die of the disease each year
There are a number of alternative medicine systems based on traditional theories and philosophy that have originated in specific geographical areas and evolved over the years. The most widely practiced of these include the
Apoptosis an active physiological process resulting in cellular self-destruction of unwanted cells is absent in cancer cells. Apoptosis is characterised by distinct morphologic changes, including cell shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation, and the formation of apoptotic bodies
In present study we chose to investigate the extracts of
The bioactivity of the NS seeds has been investigated both in vitro as well as in vivo for a number of therapeutic properties like antioxidant, antihistaminic, anti inflammatory, immune-modulatory, anti viral, anti helimenthic, anti bacterial
Cervical cancer forms an important part of cancer sub-types among women and is more common in Hispanic and African-American women than in Caucasians. It is the fifth most common type of cancer among women with 471,000 new cases each year with 11,070 cases in US in 2008 and 3,870 deaths according to the data released by the National Cancer Institute. Considering the significance of this cancer sub-type accounting to a death every 2 minutes, we used HeLa cell lines to analyze the anticancer activity of the NS seed extract and sought to investigate the ability of NS to induce apoptosis and to determine the biochemical mechanisms underlying apoptosis in HeLa cells. To determine the species' antiproliferative activity, we examined the effects of various solvent fractions on cell viability by clonogenic inhibition assay. Induction of apoptosis was assessed by DNA fragmentation, TUNEL assay and western blot for the expression of the anti-apoptotic protein Bcl-2 & bcl-XL and of the pro-apoptotic proteins caspase-9, caspase-8 and caspase-3 and p53. The results led us to hypothesize that certain bioactive compounds in NS seeds might be capable of effectively causing death of cancer cells by up-regulating the expression of certain pro-apoptotic gene and simultaneously down-regulating the expression of anti-apoptotic genes.
Results and Discussion
NS extract-induced clonogenic inhibition of HeLa cells
The methanolic extract (IC50 2.28 μg/mL) (Fig
A NS-Methanolic Extracts induced Clonogenic inhibition in HeLa cells
A NS-Methanolic Extracts induced Clonogenic inhibition in HeLa cells. 1B. NS n-haxane Extracts induced Clonogenic inhibition in HeLa cells. 1C: NS-Chloroform Extract induced Clonogenic inhibition in HeLa cells
Quantification of Apoptosis by DNA fragmentation and TUNEL assay
Apoptosis is a form of programmed cell death characterized by cytoplasmic condensation, plasma membrane blebbing and nuclear pycnosis, leading to nuclear DNA breakdown into multiples of ~200 bp oligonucleosomal size fragments. The detection of apoptosis in cultured cells relies heavily on techniques involving the extraction of nuclear DNA and characterization of such oligonucleosomal ladders by gel electrophoresis and detection of DNA damage by end labeling using terminal transferase-mediated dUTP-digoxigenin-end labeling (TUNEL). HeLa cells were treated with respective IC50 concentration of NS seed extracts as follows: chloroform extract at 0.41 ng/ml, the n-hexane extract at 2.20 μg/ml and the methanolic extract at 2.28 μg/ml to determine whether the bioactive compounds of NS seeds induced HeLa cell death via apoptosis or by necrosis. HeLa cells treated with these extracts showed active apoptosis after 24 hours of treatment; the increase in apoptosis over the untreated population was 21.1% in methanolic extract treated cells, 30% n-hexane extract treated cells and 42% chloroform extract treated cells (Fig
NS extracts induced apoptosis in HeLa cells
NS extracts induced apoptosis in HeLa cells. The bar graphs show the percentage of TUNEL-positive cells. Apoptosis in the treated cells was normalized to the baseline apoptosis in the untreated cells. The data shown are the means from two independent experiments.
DNA laddering
DNA laddering: DNA fragmentation on 1% Agarose Gel Electrophoresis shows; Lane 1 100 bp marker, Lane 2 untreated cells, Lane 3 Chloroform extract treated cells, Lane 4 n-hexane extract treated cells and Lane 5 methanolic extract treated cells.
Altered Apoptotic Gene Activity and Caspase activation by NS Seed extract treatment in HeLa Cells
To determine the mechanism of NS-induced apoptosis, the expression of anti- and pro-apoptotic proteins following NS treatment was examined by western blotting. It was clear from western blot results that the expression of
Expression of anti-apoptotic genes; bcl-2 and bcl-XL in HeLa cells treated with methanolic, choloroform and n-haxane extract
Expression of anti-apoptotic genes; bcl-2 and bcl-XL in HeLa cells treated with methanolic, choloroform and n-haxane extract. The graph shows the intensity of bands (ImageJ sofware).
Expression of caspase -3, -8 and -9 in HeLa cells treated with methanolic, choloroform
Expression of caspase -3, -8 and -9 in HeLa cells treated with methanolic, choloroform. and n-haxane extract. The graph shows the intensity of bands (ImageJ sofware).
Expression of pro-apoptotic gene, p53 in HeLa cells treated with methanolic, choloroform
Expression of pro-apoptotic gene, p53 in HeLa cells treated with methanolic, choloroform. and n-haxane extract. The graph shows the intensity of bands (ImageJ sofware).
Conclusion
The drug discovery process is becoming more and more complex and capital-intensive, and it calls for systematic and critical review of methods and approach towards the entire process, a need to rediscover the discovery process afresh.
The Indian Systems of Medicine are in the list approved by the National Council for Alternative Medicine (NCAM), USA, although most of these drugs that claim to cure various ailments are yet to be validated scientifically. According to Parasnis
Screening of medicinal plants for potential anticancer properties has increased greatly over the past few decades. For instance, the US National Cancer Institute has implemented a large-scale project of acquisition and screening of compounds isolated from medicinal plants originating in various parts of the world. These medicinal plants are identified based on ethnopharmacological, chemosystemic and ecological information.
Therefore, there is a need for more effective anticancer agents since the most common tumors in adults are resistant to almost all presently available anticancer drugs and the majority of the available drugs have limited anti-solid tumor activity
Apoptosis is an active physiological process resulting in cellular self-destruction that involves specific morphological and biochemical changes in the nucleus and cytoplasm
The mechanism of action of many anticancer drugs is based on their ability to induce apoptosis
The NS seed powder was subjected to three different organic extractions with increasing polarity
Efforts towards finding a practical solution in combating this dreadful disease could prove to be of paramount importance. There is no paucity on the availability of umpteen numbers of medicinal plants known to regional communities practicing indigenous healing techniques, which can on scientific evaluation provide an alternative to the presently available options that do not go without severe and painful side-effects. Many herbs have been evaluated in clinical studies and are currently being investigated phytochemically to understand their tumouricidal action against the insidious nature cancer.
Ayurvedic therapy has been found to cure these chronic diseases like caner better, which are previously not amenable to treatment by western medical practices
Herbal decoctions consisting of multiple herbs each possessing tremendous potential for a cancer cure are commonly used in Ayurveda. These formulations are reported to work on multiple biochemical pathways and are capable of influencing several organ systems simultaneously. The benefit of a herbal decoction is that it can nourish the body as a whole by supporting various organ systems. More importantly as it has no synthetic part to it, there is very less likelihood of an unexpected adverse effect. Many of the herbs have been scientifically-proven for anti-cancerous properties and are used for the treatment of various cancers
However, the potent extracts should be tested on several other cancer cell lines, biochemical and molecular studies carried out using the extracts in animal models to establish their therapeutic efficacy as well as toxicity, and the most potent extracts subjected to HPLC and GC&MS analyses to identify and characterize the efficacious phytotherapeutic and/or bioactive compound(s) in NS.
Materials and methods
Preparation of organic extract
Air dried NS seeds were pulverized using a milling machine and extracted with Methanol,
Extraction and partitioning of bioactive compounds of
Extraction and partitioning of bioactive compounds of
Maintenance of HeLa cell line
HeLa cells were procured from the National Centre of Cell Sciences (NCCS), Pune, India. Cell lines were maintained and propagated in 90% Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were cultured as adherent monolayer and maintained at 37°C in a humidified atmosphere of 5% CO2. Cells were harvested after subjecting them to brief trypsinization. All Chemicals were research grade.
Cell Viability Assay
Cell viability was assayed where and as required by trypan blue exclusion test
Colonogenic Inhibition Assay
The Colonogenic inhibition assay was performed as described previously by us
(Where PE is the plating efficiency).
Determination of IC50 Concentration
The IC50 value (the concentration of the drug which is capable of bringing about 50% inhibition of colony formation) of the drug used for treatment was determined by plotting a graph with Survival fraction (Y-axis) against the concentrations of the extracts (X-axis) [Fig
Analysis of DNA Fragmentation
HeLa cells were treated with respective concentration (IC50) of each extract for 24 hours and then harvested. Genomic DNA was extracted by standard salting out method with slight modification
TUNEL Assay
To quantify apoptosis, the ApopTag™ in situ apoptosis detection kit (Oncor, Gaithersburg, MD) was used as per the instructions of the manufacturer. The ApopTag kit detects DNA strand breaks in single cells by terminal transferase-mediated dUTP-digoxigenin-end labeling (TUNEL). Briefly, cells were seeded in chamber slides and the next day they were exposed to each organic extracts. After 24 hrs the DNA was tailed with digozigenin-dUTP and cojugated with an anti-digoxigenin fluorescein. The cells were then counterstained with propidium iodide and antifade. The stained cells were observed using Carl-Zeiss epifluorescence microscope using triple band-pass filter. To determine the percentage of cells indicating apoptosis, a total of 1000 cells were counted in each experiment.
Western Blot Analysis
After treatment, the cells were collected and washed twice with cold PBS. The cells were then lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO3, 10 mM NaF, 1 mM DTT, 1 mM PMSF, 25 lg/ml aprotinin, and 25 lg/ml leupeptin) and kept on ice for 30 min. The lysates were then centrifuged at 12,000 g at 4°C for 20 min; the supernatants were stored at -70°C until use. The protein concentration was determined by the Bradford method. Aliquots of the lysates (30 μg of protein) were separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane using transfer buffer (192 mM glycine, 25 mM Tris-HCl, pH 8.8, and 20% methanol [v/v]). After blocking with 5% non-fat dried milk, the membrane was incubated for 2 h with primary antibodies, followed by 30 min with secondary antibodies in milk containing Tris-buffered saline (TBS) and 0.5% Tween. Anti-human p53, caspase-3, caspase-8, -caspase-9, -Bcl-2, and bcl-XL antibodies were used at a 1:1000 dilution as the primary antibodies, while horseradish peroxidase-conjugated horse anti-rabbit IgG (Sigma Chemicals, USA) was used at a 1:5000 dilution as the secondary antibody. The membrane was then exposed and protein bands were detected using Enhanced Chemiluminescence.
Statistical analysis
The results of each series of experiments (performed in triplicates) are expressed as the mean values ± standard deviation of the mean (SD).
Abbreviations
TUNEL: terminal transferase-mediated; dUTP: digoxigenin-end labeling; CAM: Complementary and alternative medicine; NS:
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
GS is a researcher working in cancer biology and carried the overall study. GS and TNH undertook the Statistical analysis. AM along with JA and DKYL designed the work and interpreted the results. AM, GS and AAA contributed to the writing of the manuscript, which was edited, revised critically and co-ordinated by DKYL. All the authors read and approved the final manuscript.