Evaluation of Anticancer Property of Claviceps Purpurea Secondary Metabolites on Different Cancer Cell Lines

The present study deals with the secondary metabolites of Claviceps purpurea extract, which has been conrmed as an empirically potential cure for cancer. The purpose of this research was to prove scientically the anticancer potential of ethyl acetate extract as its effects on the cell cycle, membrane apoptosis, DNA fragmentation and nuclear staining of lung cancer (A549) cell lines. The MTT method was used to measure cytotoxic effects, ethyl acetate extract of Claviceps purpurea showed IC 50 value of 56.38 µg/ml compare to other cancer cell lines like breast cancer (MCF-7) cell lines (90.04 µg/ml) and cervical cancer (HeLa) cell lines (84.97 µg/ml) and normal cell lines broblast (3T3L1) does not show IC 50 values due to lesser percentage of inhibition. The effect of inhibition of cell cycle and induction of cell apoptosis was measured by the ow cytometry method. DNA fragmentation studied by the gel electrophoresis method. The results showed, the ethyl acetate extract of Claviceps purpurea gives an induction of apoptosis effect on A549 cancer cell through inhibition of cell cycle in the G0-G1, Synthesis, and G2/M phases. In necrotic cell regions apoptosis was found. In DNA fragmentation 80 μg/ml concentration of extract showed signicant results against A549 cell lines. In the nuclear staining study, membrane blebbing will be found. It indicates the extract was very active against cancer cell lines and it occurs in late apoptotic phase. In this study of all parameters, Colchicine was used as standard for cell cycle studies, for membrane apoptosis studies vinblastine and DNA fragmentation studies hydrogen peroxide was used.


Introduction
The Claviceps purpurea (Fr.) Tul. (Hypocreaceae) is probably the best-known species of the Claviceps genus due to its pharmacological activities. The fungus produces different ergot alkaloids (ergoline and clavine type) with a wide range of biological activities. The broad spectrum of ergot alkaloids effects is mostly based on their interactions with adrenergic, serotoninergic or dopaminergic receptors, as well as on their interference with some cellular and molecular processes. The ergoline alkaloids are used in the treatment of uterine atonia, postpartum bleeding, migraine, orthostatic hypotension, cerebral insu ciency, hyperprolactinemia and Parkinson disease. The antitumoral effect observed in the case of some clavinetype alkaloids reinforced the interest for Clavicepspurpurea as a possible source of new onchotherapeutical agents (Craita et al., 2011). The therapeutic application of microbial metabolites provided the opportunity for the discovery of antibiotics (e.g., Penicillin, Erythromycin, Streptomycin, Amphotericin etc.) and anticancer agent (e.g., Daunorubicin, Doxorubicin, Bleomycin and Pentostatin).
Microorganism produce secondary metabolites such as enzyme, growth hormone, antimicrobial and anticancer substances. The secondary metabolites were potential drugs for treatment of newly developing diseases in humans, plants and animals. Lack in advance knowledge of fungi and their medicinal activity has encouraged researchers to investigate the importance of fungal microorganisms particularly the secondary metabolites produced by these microorganisms as potential anticancer agents (Kumala et al., 2007). The antibiotics have been useful in our battles against infectious bacteria and fungi for over 50 years. However, many antibiotics are used commercially, or are potentially useful, in medicine for activities other than their antibiotic action. They are used as antitumor agents, immunosuppressive agents, hypocholesterolemic agents, enzyme inhibitors, antimigraine agents, and antiparasitic agents (Demain, 1999).
Traditionally, the in vitro determinations of toxic effects of unknown compounds have been performed by counting viable cells after staining with a vital dye. Alternative methods used are measurement of radioisotope incorporation as a measure of DNA synthesis, counting by automated counters and others which rely on dyes and cellular activity. The MTT system is a means of measuring the activity of living cells via mitochondrial dehydrogenases. The MTT method is simple, accurate and yields reproducible results. The key component is (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) or MTT, is a water-soluble tetrazolium salt yielding a yellowish solution when prepared in media or salt solutions lacking phenol red.
The two most obvious features of the cell cycle are the synthesis and duplication of nuclear DNA before division, and the process of division itself -mitosis. These two components of the cell cycle are usually indicated in shorthand as the synthesis phase (S phase) and mitosis (M). When the S phase and M phase of the cell cycle were originally described, it was observed that there was a temporal delay or gap between mitosis and the onset of DNA synthesis, and another gap between the completion of DNA synthesis and the onset of mitosis. These gaps were termed G1 and G2 respectively (Joany et al., 1998). The use of fungus in the regression of certain forms of cancer has been recognized for more than a century. Important advances have been made to study and develop live fungal products such as proteins, enzymes, immunotoxins and secondary metabolites which speci cally target cancer cells and cause tumor regression through growth inhibition, cell cycle arrests or apoptosis induction (Vijaya et al., 2014).
Apoptosis and necrosis are two distinct forms of cell death in mammals. Unlike necrosis, which can invade large populations of cells, apoptosis normally triggers the death process only in a single cell.
Furthermore, necrosis is an accidental cell death that occurs as a result of severe physical or chemical changes. while apoptosis not only occurs during natural fetal development but can be also induced by various stimulators, including drug treatment and other stress conditions. Since mis regulation of apoptosis, including both excessive or reduced cell death, normally leads to various human diseases such as cancer, apoptosis-based therapies are considered new biological approaches for the treatment of such abnormalities (Azadeh et al., 2014).
Acridine orange/ethidium bromide (AO/EB) staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a uorescence microscope and counted to quantify apoptosis. The aim of the study was to screen metabolites from Claviceps purpurea for study of in vitro anticancer properties against human cancer cell lines by nuclear staining studies, DNA fragmentation analysis, cell cycle analysis and membrane apoptosis studies.

Materials And Methods
Cell lines and culture medium Human lung carcinoma (A549), MCF-7 (breast adenocarcinoma cells), Hela (cervical adenocarcinoma cells) and nonmalignant broblast 3T3L1 were used for anticancer activity. The stock cells were cultured in DMEM/RPMI supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100 µg/ml) in a humidi ed atmosphere of 5% CO 2 at 37 o C until con uent. The cell was dissociated with cell dissociating solution (0.2 % trypsin, 0.02 % EDTA, 0.05 % glucose in PBS). The viability of the cells is checked and centrifuged. Further, 50,000 cells /well was seeded in a 96 well plate and incubated for 24 hrs at 37 o C, 5 % CO 2 incubator.
Cytotoxicity studies by MTT assay The monolayer cell culture was trypsinized and the cell count was adjusted to 5.0 x 10 5 cells/ml using respective media containing 10% FBS. To each well of the 96 well microtiter plate, 100 µl of the diluted cell suspension (50,000 cells/well) was added. After 24 hrs, when a partial monolayer was formed, the supernatant was icked off, washed the monolayer once with medium and 100 µl of different test concentrations of drugs were added on to the partial monolayer in microtiter plates. The plates were then incubated at 37 o C for 24 hrs in 5% CO 2 atmosphere. After incubation the test solutions in the wells were discarded and 100 µl of MTT (5 mg/10 ml of MTT in PBS) was added to each well. The plates were incubated for 4 hrs at 37 o C in 5% CO 2 atmosphere. The supernatant was removed and 100 µl of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 590 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (IC 50 ) values is generated from the dose-response curves for each cell line. The IC 50 value is determined by using the formula i.e. % Inhibition = (OD of Control -OD of sample)/OD of Control) x 100 (Vijay et al., 2014).

Cell cycle Analysis
The number of cells (1x10 6 ) were seeded and cultured for 24 hrs in a 6 well plate containing 2 ml of media. Cells were then treated with desired concentrations of given samples were prepared in media and incubated for another 24 hrs cells were then harvested and centrifuged at 2000 rpm for 5 minutes at room temperature and supernatant was discarded carefully retaining the cell pellet. Cell pellet was washed by resuspending in 2 ml of 1XPBS. The washing was repeated another time with the same conditions. Supernatant was discarded retaining the pellet. Cells were xed by resuspending in 300 µl of Sheath uid followed by addition of 1 ml of chilled 70% EtOH drop by drop with continuous gentle shaking and another 1 ml of chilled 70% EtOH added at once. The cells were then stored at 4°C for or overnight. Post xing, the cells were centrifuged at 2000 rpm for 5 minutes. The cell pellet was washed twice with 2 ml of cold 1XPBS. Cell pellet was then resuspended in 450 µl of sheath uid containing 0.05mg/ml PI and 0.05 mg/ml RNaseA and incubated for 15 minutes in dark. The percentage of cells in various stages of cell cycle in fungal extracts treated, untreated and control (colchicine) populations were determined using FACS Caliber (BD Biosciences, San Jose, CA) (Ramesh et al., 2011).

Membrane Apoptosis Studies
The day before induction of apoptosis, plated 1X10 6 cells per well for a 6 well plate using DMEM cell culture medium. After 18 hrs, the wells for oating (dead) cells and removed by pipette. Replaced with new culture medium to the original volume. Treated cells to induce apoptosis with samples at different concentrations, and incubate for 24 hrs Later, collected cell culture medium into 15 ml tubes. Using policeman, the cells were detached from the dish and added 1 ml of medium to each well and transferred the contents to the 15 ml tubes. Centrifuged and discarded the supernatant. Washed the cells twice with cold PBS and then resuspend cells in 1 ml 1X binding buffer at a concentration of 1x10 6 cells/ml. 500 µl of cell suspension is aliquoted and 10 µl of Propidium Iodide and 5 μl Annexin V is added. The suspension is incubated for 15 minutes at RT in the dark. Post incubation, the cells were analyzed by ow cytometer as soon as possible (within 1 hour) (Seamus et al., 1995).

DNA Fragmentation Studies
Cells were seeded at a concentration of 1x10 6 per 35 mm dish incubated at 37°C/5% CO 2 incubator for 24 hrs. The con uent cells grown after 24 hrs of incubation were treated with sample concentration. After treatment, cells were trypsinized, and both adherent and oating cells were collected by centrifugation at

Statistical analysis
The differences observed between the control and treated groups for cell proliferation. Comparison between two groups were made using one-way ANOVA to assess the IC 50 value. The results were expressed as the Mean ± SEM (standard error of the mean) from three different replicates and a value of p < 0.05 was considered statistically signi cant.

Results And Discussion
Cytotoxicity studies by MTT Assay In the present study, the in vitro cytotoxic effects of ethyl acetate extract of Claviceps purpurea on several cell lines was evaluated by micro-culture tetrazolium assay (MTT). MCF-7, HeLa, A549 cell lines and normal cell lines (3T3L1) were evaluated. The cytotoxicity of the fungal extracts was also investigated against the normal Fibroblast cell line. The ethyl acetate extract of Claviceps purpurea showed an exponential increase in cytotoxic activity against both the cells are MCF-7, A549, and HeLa. In A549 cell lines the ethyl acetate extract showed IC 50 value is 56.38 µg/mL (Table 1) The increasing number of cancer cases around the world is demanding for more improved strategy for prevention as well as treatment of this dreaded disease. Approaches, which are more than just a treatment mode rather which may be included as a nutritional supplement in our regular diet, can be a better strategy to prevent carcinogenesis (Arunchand et al., 2018).

Cell cycle studies
Ethyl acetate extract in cell cycle studies, the results were found to be as followed in order to determine the cell cycle pattern of A549 cells, vinblastine and ethyl acetate extracts were treated to cells were analyzed in ow cytometric method. The cell cycle phase distribution was quantitated from 3 independent sets of measurements that showed relatively similar pattern to ethyl acetate extract which was different than standard Colchicine. The results were showed in gure 1-5 and table 2. The ability of anticancer agents to suppress the growth of cancer cells can also be associated with blocking cell-cycle progression. Based on the signi cant inhibition of A549 cell proliferation by ethyl acetate extract, it was postulated that the extract might modulate the cell-cycle progression of these cancer cells. Cell-cycle distribution analysis showed this extract caused a reduction in S phase population, which was associated with the accumulation of cells in the G0/G1 phase at a concentration of 40 µg/ml. A secondary metabolite of Epanorin (EP) exhibited a G1 cell cycle arrest which was similar in magnitude to that shown by TXM, a clinically used anti proliferative drug for estrogen positive breast cancer cells. Moreover, the effects on cell cycle progression were comparable to the reported for coumetarol on the MCF-7 Cells (Juan et al., 2019).

Apoptosis detection using PI Annexin V-FITC staining of A549 cells by ow cytometry
Apoptotic and necrotic cell populations in both control and treated cancer cells were studied by FACS using Annexin V-FITC and Propidium Iodide. The concentration of ethyl acetate extract 40 µg/ml and 80 µg/ml has induced necrosis in A549 cells with 27.54% and 35.54% cells respectively; and Standard Vinblastine showed 11.06% and 10.22% early and late apoptotic cells when compared to control cells with 56.11% also has shown necrosis of 22.61%. The results were showed in gure 6-10 and table 3.
Apoptosis is a programmed cell death characterized by cleavage of chromosomal DNA into oligonucleosomal fragments. Apoptosis is an active form of cell death that occurs in response to several agents, including chemotherapeutic drugs. Apoptosis was initially described by its morphological characteristics, including cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. Analysis of nuclear morphology is highly essential to examine the induction of cell death (Ramya et al., 2017).
DNA fragmentation studies on A549 cell line DNA fragmentation is an important feature in cells undergoing apoptosis. To con rm that ethyl acetate of Claviceps purpurea makes cell death in A549 cells via apoptosis and not necrosis, DNA fragmentation patterns in treated cells were analyzed. The activation of endogenous endonucleases results in the cleavage of chromatin into inter-nucleosomal fragments of approximately 180-200 bp; it is believed that DNA fragmentation is carried out by Caspase-activated DNase (CAD), leading to the cleavage of nuclear DNA into multiple fragments with low molecular weights. A549 cells treated with two different concentrations of ethyl acetate extract for approximately 2hrs showed inter-nucleosomal DNA fragmentation in the form of a DNA ladder, typical of apoptotic DNA fragmentation (Fig 11). The exact DNA fragmentation and the absence of a DNA smear due to non-speci c DNA degradation show the apoptosis-inducing ability of ethyl acetate extract. In this study, A549 cells were treated with Sample 2 (Ethyl acetate extract) and it was found that Sample has induced comparatively more DNA damage at 80 μg/ml when compared to Standard H 2 O 2 with 200 µM concentrations.
The fragmentation of genomic DNA was con rmed by DNA laddering assay to support the apoptosis induction by the extract in the cancer cells. It was observed that the treated cells showed DNA laddering pattern and a single genomic DNA band was in untreated cells. The cells treated with control positive had also DNA laddering pattern.
In ethyl acetate extract of Claviceps purpurea showed signi cant result of DNA fragmentation studies in A549 cellines. The concentrations of 40µg/ml and 80µg/ml of treated cells were showed signi cant DNA fragmentation compare to control cells these treated cells showed better results. The standard H 2 O 2 also showed more DNA damaged in cells and this activity may be contributed by the extract constituents. The fragmentation was not found in distinct, but increased DNA damage was observed, which provide evidence for apoptotic cell death.
Cancer chemotherapy drugs usually exert cytotoxic effects on malignant cells by inducing apoptosis.
Major apoptotic events include cell cycle arrest and the formation of reactive oxygen species, leading to loss of mitochondrial membrane potential and DNA fragmentation. It is believed that caspase 3, a major downstream effector of apoptosis, mediates proteolytic cleavage of poly (ADP ribose) polymerase (PARP), leading to DNA fragmentation. These apoptotic events, accompanied by the potent growth inhibition of HeLa cells, were detected when the cells were treated with partially puri ed VCR from T. radicus-CrP20 (Padmini et al., 2015).

AO/EtBr Staining
Dual staining was examined under a uorescent microscope. Normal cells are seen with circular nucleus uniformly distributed in the center of the cell which is seen in the control gure 12.
In treated sample 40 μg/ml show in the early stage apoptotic cells, where cell's nucleus is showing yellow-green uorescence by Acridine orange (AO) staining and concentrated into a crescent or granular that located in one side of cells. Staining was localized asymmetrically within the cells and membrane blebbing was seen in gure 13.
In treated sample 80 μg/ml show in the nucleus of cells were orange uorescence by EtBr staining and gathered in concentration and located in bias, this is late apoptotic phase. Cells that have taken up complete EtBr are the necrotic cells as in Figure 14. In standard Vinblastine at 25 µM/ml showed on A549 cellines in gure 15.
Ethyl acetate extract of Claviceps purpurea showed at 40 µg/ml concentration yellow-green uorescence by Acridine orange (AO) staining and concentrated into a crescent or granular that located in one side of cells. Staining was localized asymmetrically within the cells. At 80 µg/ml concentration orange uorescence by EtBr staining and gathered in concentration and located in bias. This is late apoptotic phase in A549 cell lines. Membrane blebbing will be found. It indicates ethyl acetate extract of Claviceps purpurea is very active against cancer cell lines.
According to Tayyaba et al., 2018 state that the results of acridine orange (AO) and ethidium bromide (EB) staining of cells treated with AHC. The results of acridine orange (AO) and ethidium bromide (EB) staining of cells treated with AHC. AO is a cell permeable uorescent dye and stains nuclear DNA in both live and dead cells, whereas EB is a uorescent dye that only stains nuclear DNA in cells that have lost their membrane integrity. We observed that after AO/EB staining viable cells were uniformly stained green, early apoptotic cells were stained greenish yellow or displayed green yellow fragments, late apoptotic cells were stained orange or displayed orange fragments, and necrotic cells showed orange to red uorescing nuclei with no indication of chromatin fragmentation.

Conclusion
In cytotoxicity studies of fungal extract of Claviceps purpurea showed signi cant anticancer activity during preliminary study by MTT Assay. In MTT assay there are 3 cancer cell lines were used against ethyl acetate extract of Claviceps purpurea. This extract showed signi cant IC 50 values on A549 cell lines (IC 50 56.38 µg/ml). This is used for further assays of anticancer activity. In cell cycle study, ethyl acetate extract is increased and showed signi cant results in synthesis and G2M phases these studies were evaluated by ow cytometric method also studied membrane apoptosis studies. In membrane apoptosis study, ethyl acetate extract acts as a cell arrest in necrotic cells region. DNA fragmentation study, ethyl acetate treated extract showed signi cant damaged DNA bands on A549 cell lines. In Nuclear staining method ethyl acetate extract showed early stage apoptotic cells to found in DNA damage and membrane blebbing will be occur on A549 cell lines. Ethyl acetate extract of Claviceps purpurea, some of the secondary metabolites acts as a signi cant anticancerous activity against different cancer cell lines. Tables   Table:1

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.