Anti-cancer property of Lenzites betulina (L) Fr. on cervical cancer cell lines and its anti-tumor effect on HeLa-implanted mice

In global scenario cervical cancer is increasing. New drugs from natural compounds are in search. Mushrooms are now recognized as miniature pharmaceutical factories producing hundreds of novel constituents. We have taken ethanolic extract Lenzities betulina (LBE) wild mushroom for evaluation of its as anti-cancer property against cervical cancer cell lines e.g. HeLa, CaSki and SiHa and anti tumor activity against HeLa implanted tumor on mice. The extraction was done by dip and stirring method in 90% ethanol for 72 h. For evaluation of anti-cervical cancer, several assays were performed such as MTT assay, cell morphology by phase contrast microscope and F-action polymerization by Laser scanning confocal microscope and nuclear morphology DAPI staining under inverted fluorescence microscope, MMP, ROS, cell cycle, autophagy and stem cell population by flow cytometry and DNA laddering were done. Western blotting was done for protein expression. To evaluate anti-metastatic activity, anti-cologenic assay and wound healing assay were adopted. For chemo-analysis of the LBE, GC-MS was done. The results from Cytotoxicity assay showed that at highest dose of LBE (1000 µg/ml) after 24 h, percentage of cell inhibitions were 85.13 %, 77.13 % and 47.70 % against HeLa, CaSki and SiHa respectively and the calculated IC50 values were 492.52 ± 2.6 µg/ml, 612.22 ± 4.2 µg/ml, and 1210.30 ± 6.4 µg/ml respectively. Depending upon the cytotoxicity screening, HeLa cell line was considered for the further studies. Cell morphology study exhibited that LBE treated HeLa cells became round from normal spindle shape. DAPI staining showed that LBE treated nucleus became condensed and fragmented. DNA fragmentation at 230 and 300 base pair zone from agarose gel assay was observed. LBE induced ROS generation and reduced MMP. It up regulated the expression of apoptotic genes and p53 while down regulated Bcl2, pro-caspase 3 and pro caspase-9 gene. Cell cycle was arrested at G2/M checkpoint. Autophagic induction was exhibited by vacuole formation in treated cells. CSC population of treated cells was reduced and F-actin polymerization was observed in treated cells. In addition, LBE suppressed metastatic nature by inhibition of cell migration and colonization. The inhibition of growth of the tumors in HeLa cell-implanted mice showed that treatment with 50 mg LBE/kg of body weight of mice led to a marked reduction in the volume (93.22 ± 9.2 %) and weight (90.42 ±9.55 %) of the tumors. The GC-MS profile of LBE shows that out of 69 compounds, 9, 12-Octadecadienoic acid (Z, Z) and Ergosta-5, 8, 22-trien-3-ol, (3.beta22E) are in a significantly higher proportion with the percentage peak area 22.13 and 19.72 respectively. Library search for bioactivity showed that these compounds are anti-cancerous and interestingly 4’-Hydroxy-6-methoxyaurone binding with P-glycoprotein inhibits the cancer cells to become drug resistant. In conclusion, LBE is very prominent anti-cervical cancer having a lot of anti-cancerous compounds which are probably acting synergistically. This report of anti-cervical cancer property of L. betulina is probably first time in oncology. Its therapeutic use in human model is urgent for new drug development.


INTRODUCTION
Mushrooms or macrofungi offer high sources of new isolable bioactive compounds with diversified chemical structures, which are considered potent sources for drug discovery. In the stone era, human went for hunting and the same time collect food including mushrooms for organoleptic properties such as flavor and texture [1][2][3] and then realized that some of mushrooms have properties for curing certain ailments. Local consumption of wild mushrooms are also increasing because of their medicinal properties 4,5 due to the presence of secondary metabolites having pharmaceutical importance. 3,4 There are more than 14,000 mushrooms out of 5.1 million estimated fungi, among which near about 700 exhibit medicinal properties 6 . Lenzitis betulina (L) Fr. belongs to the family Polyporaceae, order Polyporales and phylum to Basidiomycota, and grows on logs and fallen woods. Various species of Lenzites are well distributed around the world. Lenzitis spp have no value as food since it has an extremely tough exterior 7 . There have been different studies on the various medicinal uses of Lenzites spp. antimicrobials 8,9 , anti-viral 10 , and immunosuppresser 11 . Phytochemical analysis of compounds presents in Lenzites spp. using three different solvents (ethanol, water and petroleum ether) showed that phenolic and steroids were present in the ethanol extract; flavonoids, tannins and steroids were present in the petroleum ether and only saponins were present in the aqueous extract 9 . Since many of the compounds have been shown to act synergistically, it is worth testing the anti-proliferative effects of the whole mushroom extract rather than its individual components. This principle (synergy) is compatible with similar natural biological products like the essential oils, which are more effective when used as whole products, while quenching or nullifying potential unwanted side-effects by the presence of individual components 12 . Among mushroom extracts, an ethanol extract probably finds the most extensive application 13 . Cancer is a major public health problem and one of the leading causes of death in the world today 14 . Cervical cancer is the second most common cancer among women worldwide 15,16 . One third of newly diagnosed cancers among women in the India are cervical cancers and that is for the infection with some types of HPV (Human Papilloma Virus). Every year in India, 122,844 women are diagnosed with cervical cancer and 67,477 die from the disease 17 18 . The current anti-cancer drugs i.e. chemo-drugs available in market are not target specific and pose several sideeffects and complications in clinical management of various forms of cancer 19 , which highlights the urgent need for novel effective and nontoxic natural compounds 20,21 . Epidemiological studies showed that among the Chinese women who regularly intake powder of Agaricus bisporus with tea have less breast and gastrointestinal (GI) cancers 22 . Consumption of mushroom has inverse effect in breast cancers occurrence among premenopausal women 23 . Lucas et al. 24 got the first credit to show that mushroom extract has antitumor properties using extracts of Boletus edulis and other basidiomycetes against Sarcoma-180 and also on HeLa cell lines presenting significant growth inhibition on those types of cancer. Ikekawa et al 25 published one of the first scientific reports on antitumor activity of extracts of mushrooms against implanted Sarcoma 180 in animals. Soon after, three major anticancer drugs, Krestin from cultured mycelium of Trametes (Coriolus versicolor), Lentinan from fruiting bodies of Lentinus edodus and Schizophyllan from Schizophyllum commune, were developed 26-28 . We came to know from the review work of Dai Yu-C et al 29 , that 200 to 331 of mushroom species have anticancer activity. Near about 650 species of higher basidiomycota have been found anti-cancer activity 26. Although Lenzites betulina is worldwide distributed, there has been minimal or no extensive research work on it for cancer remedy. Therefore, the main objectives of this work are to screen

Preparation of ethanol extract (LBE)
The collected and identified fruit bodies of L. betulina (LB) after oven dried at 50 º C temperature for 48 h were ground by mixture grinder (Beckman food mixer, India). This smashed biomass (100 g) was suspended in 500 ml of 90% ethanol and incubated for 72 h at 200 rpm and 37˚C. The suspension was filtered on what man No. 4 paper to remove the biomass. This procedure was repeated thrice. The supernatant was concentrated and ethanol was subsequently removed from the extract using a rotary vacuum evaporator at 40ºC, and the remaining solvent was removed with a freeze-drier. The resulting dried powder stored at 4˚C. The stock solution of LBE(LBE) was prepared by dissolving in dimethylsulphoxide (0.2% DMSO) at a concentration 10 mg/ml and stored at 4˚C.

Cell lines, culture medium and subculture of cell line
Human Cervical Cell lines (HeLa, CaSki and SiHa) were grown and maintained in DMEM (Dulbecco's Modified Eagle's) Medium supplemented with L Glutamine, 10% FBS, sodium bicarbonate in T25 cell culture flasks to the 80-90% confluence. The media also supplemented with 1000 μg/ml streptomycin (Himedia) and 1000 IU/ml penicillin (Himedia) at 37°C in a humidified atmosphere of 5% CO 2 . Maintenance cultures were passaged weekly, and the culture medium was changed twice a week 33 .

Cytotoxicity / cell proliferation assay
MTT colorimetric assay method was employed to evaluate cell viability in this cytotoxic assay. Cells of HeLa, SiHa and CaSki were seeded in a 96 well plate (1x10 4 cells/well in 100 μl of medium) separately and treated for 24, 48 and 72 h with 100, 250, 500, 750 and 1000 µg/ml LBE. After incubation the media was discarded and 100 μl/well MTT (Himedia Laboratory) reagents were added into each well and the cells incubated for another 2-4 h until purple precipitates were clearly visible under a microscope. Flowingly, the medium together with MTT (100 µl) were aspirated off the wells, DMSO (100 µL) was added and the plates shaken for 5 min. The absorbance for each well was measured at 540 nm in a micro-titre plate reader (Bio-Rad i-mark). The IC 50 values of LBE were calculated The concentration which led to a 50% killing (IC 50 ) was calculated by plotting a dose response graph of the cytotoxicity values obtained using the formula given below: % X= , Where x= % of cell Cytotoxicity, y= Control, Z= test. Data points represent the mean ± SD in one experiment repeated at least thrice.

Morphological examination of HeLa cancer cells
HeLa cancer cells were grown in 6 well -culture plate and treated with LBE (0, 500 μg /ml and1000 μg /ml). Cells were examined under phase contrast inverted microscope (Olympus,Japan) and photographs were taken using a digital camera (Q Imaging) for analysis.

4, 6-Diamidino-2-phenylindole (DAPI) nuclear staining
HeLa cells were cultured in cover slip in 6 well plates and treated with 500 and 1000 µg/ml of LBE and incubated at 37°C for 24 h. After incubation cells were washed with PBS in three times and the cells were fixed in 3.7% formaldehyde for 10 min then the cells were rinsed into PBS+ for three times, 0.2% Triton X-100 was added and incubated for 5 min. After incubation the cells were washed with PBS and DAPI was added, and incubated for 5 min, the solution was discarded and again the cells were washed with PBS in three times. After washing cells were observed under the fluorescence microscope (Olympus inverted fluorescence microscope CKX 53)

Effect of LBE on the mitochondrial membrane potential (MMP) of HeLa cells
Mitochondrial outer membrane permeability was measured by flow cytometry using the MitoProbe™ JC-1 Assay Kit, Thermo Fisher Scientific. HeLa cells were cultured in 6 well plates and treated with 250 and 500 µg/ml of LBE and incubated at 37°C for 6 h and 12 h. After incubation cells were washed twice with PBS and incubated with 10 µl of 200 µM JC1 at 37 0 C, 5% CO 2 for 30 min, cells were washed once by adding 2 ml of warmed phosphate-buffered saline (PBS) to each tube of cells. The cells were pelleted by centrifugation and re-suspended by gently flicking the tubes. 500 μl PBS was added to each tube and analyzed on a flow cytometer with 488 nm excitation using emission filters appropriate for Alexa Fluor 488 dye and R-phycoerythrin. A gate on the cells, excluding debris. Using the CCCP-treated sample, standard compensation intensity was determined by flow cytometry. Cells with reduced fluorescence were counted as having lost some of their mitochondrial membrane potentials.

Detection of Intracellular ROS(Reactive oxygen species) by H 2 DCFDA
ROS were detected by the modified method of Deem TL, Cook-Mills JM. 6 1x10 5 cells were cultured in 35 mm plates and treated with 500, 750 µg/ml of LBE and incubated at 37°C at 2 h. After incubation cells were trypsinized and suspended in 500µl PBS (1x), 5µl H 2 DCFDA (Thermo Fisher Scientific) was added and incubated for 30 min at 37ºC.then analyze the sample in Flow Cytometer (FACS Calibur, BD Bioscience). Where Ascorbic acid acts as a positive control and H 2 O 2 acts as a negative control. Also we observed ROS by inverted fluorescence microscopy.

Cell cycle analysis
For the determination of cell cycle phase distribution of nuclear DNA. Cells were treated with 100, 250, 500 µg/ml LBE and incubate for 6 and 12 h. After incubation cells were fixed with 3% pformaldehyde, permeabilized with 0.5% Triton X-100, and nuclear DNA was labeled with propidium iodide (PI, 125 mg/ml, Santacruze Biotechnology, USA) after RNase treatment. Cell cycle phase distribution of nuclear DNA was determined on FACS Calibur using Cell Quest software (Becton Dickinson). Histogram of DNA content (x-axis, PI fluorescence) versus counts (y-axis) has been displayed. Cell Quest statistics was employed to quantities the data at different phases of the cell cycle.

Annexin V-FITC apoptosis assay
In general phosphatidyl-serine (PS) exists in internal face of plasma membrane but in early apoptosis it comes to external face of the plasma membrane, the high affinity PS binding protein Annexin V (conjugated to fluoresce in isothiocyanate FITC) which has high affinity to PS binding is used to identify the early event of HeLa cell killing caused by the LBE. On the other hand, simultaneously counterstaining propidium iodide (PI) is applied which allowed the differentiation of necrotic and apoptotic cells. An Annexin v-fluoresce in isothiocyanate (FITC)/ propidium iodide apoptosis detection kit (Santa cruze Biotechnology, USA), according to the manufacturer's protocol, was applied to calculate cell apoptosis. In this study, percent of apoptotic and necrotic cells were determined after treatment with the LBE (250, 500, 750 and 1000 µg/ml) by flow cytometry using the AnnexinV-FITC apoptosis detection kit (Santacruze Biotechnology). Briefly, PI and Annexin-V were added to the cell line .The mixture was incubated for 15 min at 37 0 C, and then analyzed on FACS Verse. Electronic compensation of the instrument was done to exclude overlapping of the emission spectra. Total 10,000 events were acquired, the cells were properly gated and dual parameter dot plot of FL1-H (x-axis; FLUOS-fluorescence) versus FL2-H (y-axis; PI-fluorescence) was performed and shown in logarithmic fluorescence intensity.

DNA fragmentation assay/ DNA Laddering assay
The DNA ladder assay was sensitive, cost-effective and most useful method for estimating apoptosis. 2x10 5 cells were cultured into 6 well plates and treated with 500, 750 and 1000 µg/ml of LBE and incubate at 37°C for 48h. After treatment, DNA was isolated from HeLa cell line and visualized after the separation by gel electrophoresis (1.8% agarose gel containing Etbr in 40Mm Trisacetate with electrophoresis at 75V at 4 h.) Autophagy assay by acridine orange 2x10 5 cells were cultured in 6 well plates and treated with 250 and 500 µg/ml of LBE and incubated at 37°C at 24 h. After incubation cells were trypsinized and suspended in 500 µl PBS (1x), add 1µg/ml acridine orange and incubate for 30 min at 37 0 C. The sample was then detected using Flow cytometry (FACS Calibur, BD Bioscience) and data were analyzed F-actin staining 2x10 5 cells (HeLa) were cultured in 6 well plates and incubated at 37°C at 24 h. After incubation cells were treated with LBE (750 µg/ml) and incubate at 37°C at 24 h. After incubation cells were washed with 1x PBS and fixed with 3.7% formaldehyde. After fixation 0.2% Triton X was added and incubated for 2 min and then Alexa flour 488 Phalloidin (1:500) was added for 1h in room temperature. Finally cells were stained by DAPI and the sample was then detected using confocal microscopy (Olympus, Japan) and data were analyzed.
Hoechst efflux assay for cancer stem cell population study 2x10 5 cells (HeLa) were cultured in 6 well plates and treated with 250 and 500µg/ml of LBE and incubated at 37°C at 24 h. After incubation cells were trypsinized and suspended in 500 µl PBS (1x), verapamil 50 µg/ml was added and incubated for 30 min at 37℃. After verapamil treatment, cells were washed with PBS (1x) for 3 times, Hoechst (1µg/ml) was added and incubated for 30 min in 37°C.
The sample was then detected using Flow cytometry (FACS Calibur, BD Bioscience) and data were analyzed.

Western blot analysis
HeLa (2x10 5 ) cells were treated with 100, 200, 500 and 750 µg/ml of LBE for 24 h. After treatment, cells were lysed with RIPA buffer (Abcam). The effect of treatment on the expression of certain cell cycle proteins such as p53, and on apoptotic proteins such as Bcl-2, pro-caspase-3, pro-caspase-9, and PRAP (Santacruze Biotechnology, USA) was determined. Proteins were detected by incubation with the corresponding primary antibodies, and antibodies followed by blotting with the HRP-conjugated secondary antibody. The blots were then detected using Luminol (Bio-Rad).

Wound healing/ scratch assay
HeLa (50x10 3 ) cells were seed in a 24 well plate and incubate at 37°C for 24 h. After 80-90% confluences, 200 μl micro tips used to press firmly against the top of the cell culture plate and a vertical wound down through the cell monolayer was swiftly made and then discarded the media carefully and each wall was washed with 1x PBS and finally treated with 500, 1000 µg/ml of LBE and a snap (zero h) through inverted fluorescence microscope (Olympus inverted fluorescence microscope, Japan) were taken and the culture plate was placed into CO 2 incubator (37°C, 5% CO 2 ). After 24 h incubation plates were removed and placed it in an inverted fluorescence microscope to take a snap (Olympus inverted fluorescence microscope, Japan) and sample was analyzed by Q Capture pro-7.

Cologenic assay
HeLa (500) cells per well were seeded in a 6 well cell culture plate and incubated at 37 °C. Immediately after attachment the cells were treated with (250, 500,750, 1000 and 1250 µg/ml) LBE for 4 days of interval. Cells were treated with same concentration of extract then colonies were fixed with 3.7% formaldehyde and stained with (0.5% w/v) crystal violet. Viable colonies were counted using Olympus inverted fluorescence microscope (CKX 53).Colonies consisting of more than 50 cells were counted by using a colony counter and the results were reported as a percentage of colonies formed using the following equation: % Colonies formed = Colonies formed in treated sample /Colonies formed in untreated sample X100.

Antitumor effects of LBE in HeLa cell-implanted mice
Male Swiss Albino mice (20-30 g) were used for the present investigation. The animals were kept in well ventilated cages (one group per cage) and provided with standard laboratory rodent diet ad libitum along with free access of water .Prior to commencement of experiments, all the animals were kept for four weeks under controlled temperature (25 ± 2 0 C) and humidity (50-70%) with 12 h day/night cycle for acclimatization. All mice were housed under hygienic /pathogen-free air conditions in a room maintained at 24°C with 50% relative humidity and a 12 h/12 h light-dark cycle. All animal experiments were approved and performed according to the regulations of the Institutional Animal Ethics Committee, Government of India (approval no: 12/P/S/ IAEC/2018). The mice were divided into 6 groups:) control mice supplemented with pellet (control + vehicle; =6);ii) controls supplemented with LBE (500 mg/kg of body weight) (control + LBE; =6); iii) HeLa cell-implanted mice supplemented with pellet (HeLa + vehicle; =6); iv) HeLa cell-implanted mice supplemented with LBE (10 mg/kg of body weight) (HeLa + LBE 10; =6);v) HeLa cell-implanted mice supplemented with LBE (25 mg/kg of body weight) (HeLa + LBE 25, =6); and vi) HeLa cellimplanted mice supplemented with LBE (50 mg/kg of body weight ) (HeLa + LBE 50, =6).
For tumor generation, a suspension of 2x10 6 HeLa cells in 0.2 ml DMEM was subcutaneously injected on the dorsal surface of the right hind legs of the Swiss albino mice, where as the control groups were injected with DMEM. The tumors were measured with Vernier calipers every 3-4 days by using the formula 2 x x 0.52 (where is the shortest diameter and b is the longest diameter). When the tumor volume was measured to be 0.50-0.90 cm 3 , the mice were randomized. Subsequently, the mice were supplemented daily with vehicle or LBE at the doses of 10, 25, and 50 mg/kg of body weight for 28 days. After 28 days, all the mice in groups 3-6 were sacrificed, and the tumors were excised from their legs and then weighed. Visceral organs (i.e., the kidney, liver, spleen and lungs) were morphologically observed for possible side effects of LBE in the mice.

Statistical analysis
All data were analyzed using Graph Pad Prism 5 software (Graph Pad Software, Inc., La Jolla, CA, USA), and one-way analysis of variance was performed to compare differences between the groups. All results are presented as the mean ± standard error of mean from three independent experiments performed in a parallel manner, unless otherwise indicated. All figures shown in the present study were obtained from at least three independent experiments. P<0.05 was considered to indicate a statistically significant.

GC/MS analysis was done by Agilent Technologies, GC-6860N Network System with inert Mass
Selective Detector and the search library used by C:\Database\NBS75K.L. Applying HP-5MS (19091S-602) Column the length and thickness of column was respectively30m and 0.25mm.The carrier gas was Helium gas (99.99%) used at a flow rate of 1ml/min and an injection volume of 2µl. Injector temperature was 280˚C, 70ev ion source temperature 280˚C. The oven temperature was 50˚C isothermal for 2.0 min, with an increase of 10˚C/min to 280˚C, then10˚C/min to 300˚C, and ending with 10 min isothermal at 280˚C.

Cytotoxicity / anti proliferative of LBE against HeLa cell line by MTT assay
It was investigated that whether LBE affected HeLa, CaSki and SiHa cell proliferation or not. The cell proliferation graph of the HeLa cell line (Fig1A, B, C) exhibited that untreated (negative control) cells proliferated at the expose time periods (24, 48 and 72 h). In contrast, cells were treated with LBE at all tested concentrations, showed reduced proliferation in concentration and time dependent manners in all cell lines (HeLa, CaSki and SiHa). Cells were treated with higher concentration (1000 µg/ml) of LBE proliferated least (14.87%, 22.87%, 52.30%) respectively. It indicated the percentage of cell inhibition were 85.13 %, 77.13 % and 47.70 % against HeLa , CaSki and SiHa (Fig 2A, B) at 24 h and 1000 µg/ml of LBE .In case of HeLa percentage of cell inhibition was increase as dose and time dependent manner. After calculating the data presented in graph 1, the IC 50 value i.e. the concentration of LBE that resulted in a 50% reduction in absorbance compared with the control (negative) after 24 h of treatment, were 492.52 ± 2.6 µg/ml, 612.22 ± 4.2 µg/ml, and 1210.30 ± 6.4 µg/ml (HeLa, CaSki and SiHa) respectively.

Study of morphological changes of HeLa cells by LBE under Phase contrast microscopy and Fluorescence Microscopy
The cells, treated with various concentrations of LBE, and after 24 h cells, were examined under a phase contrast microscope for monitoring their morphology. Untreated cells (negative control) exhibited normal spindle shape and reached 90% confluency after 24 h culture. Cells, treated with 500 µg/ml and 1000 µg/ml of LBE were round and shrunken and cell membrane blebbing were observed in cells treated with 1000 µg /ml of LBE (Fig.3A, B and C). DAPI staining under fluorescence microscopy showed that increase of concentration of LBE, the intensity of fluorescence increased and nuclear shrinkage, fragmentation, chromosome condensation and apoptotic bodies, and the number of apoptotic cells increased in compare to the negative control (

Mitochondrial membrane potential assay
Mitochondrial membrane potential, in situ, is an important indicator of mitochondrial function and dysfunction and the loss of mitochondrial transmembrane potential (ΔΨm) leading to damage of mitochondrial membrane is the main step for mitochondrion-dependent apoptotic pathway. HeLa cells were treated with the two optimum concentrations of LBE such as 250 µg/ml in 6 h (Fig.4D, E ,F) and in 12 h (Fig.5D, E, F) and 500 µg/ml in 6 h (Fig.4G, H, I) and in 12h (Fig.5G, H ,I) in vitro with compare to the control (Fig.4A, B, C;) Fig. 5A, B, C) and we examined the effect of LBE on inverted fluorescence microscopy (Olympus, Japan) by JC1 retention as compared with negative control counterparts and it showed that LBE treatment significantly reduced JC1 fluorescence in HeLa cells , suggesting that the mitochondrial pathway is involved in LBE-induced apoptosis. In addition to that we also studied it by flow cytometry (FACS Calibri BD), and the ( Fig.6A, B, C, D, E, F, G) showed that LBE disrupted and did dysfunction of mitochondrial transmembrane potential.

Intra cellular ROS generation assay
To investigate the intracellular levels of ROS, the cell-permeable probe H 2 DCFDA was utilized. Nonfluorescent H 2 DCFDA, which is hydrolyzed to DCFH inside the cells, yields highly fluorescent DCFDA in the presence of intracellular H 2 O 2 and associated peroxides. Whether or not LBE increased or decreased the generation of ROS was subsequently examined. As shown in (Fig.7), LBE treatment (500, 750 µg/ml) increased the generation of ROS in HeLa cells, as determined by monitoring DCF fluorescence, when the dose of LBE was gradually increased. Also we studied ROS by FACS (Fig. 8

Cell cycle distribution
We study the cell cycle distribution of HeLa cells in FACS using PI staining. With the treatment of LBE proportion of the cell in the G 2 /M region increased as a dosage dependent and time dependent manner. The G 2 /M population of HeLa cell in vitro increase from 16.11 % (control), to 19.55 % (100 µg/ml), 20.21% (250 µg/ml), and 20.62 % (500 µg/ml) respectively in 6 h time duration (Fig.9A, B, C, D, E) In addition to 12 h time duration the HeLa cell was arrested in G 2 /M phase 17.62% (100 µg/ml), 17.64% (250 µg/ml), and 20.04% (500 µg/ml) with compared to the control 16.87% (Fig.10A, B, C, D, E).

Annexin-V assay
In annexin-V assay four different phenotypes were observed.  Figure12. DNA ladder assay. Genomic DNA from HeLa cells was isolated and subjected to electrophoresis in agarose gel (1.8%) and Control (C) lane showed no fragmented DNA but 1000 μg/ml showed fragmented DNA in the form of ladder after 24 h DNA fragments were visualized under a UV trans-illuminator and compared with a standard marker.

Induction of Autophagy by LBE
Recently autophagy has gained much attention as the potent alternative mechanism to fight cancer. In fact autophagy is a self-digestive process of cells within auto phagosomes, and is delivered to the lysosome for subsequent degradation and recycling. In our autophagy experiment analyzed by FACS, autophagic cell distribution in negative control was 11.57% (Fig. 13D). It was increased to13.02% (Fig.13F) and 26.76% (Fig.13E) in 250 µg/ml and 500 µg/ml respectively. Our result was also validated by fluorescent microscopic analysis. In negative control, acidic vacuole was not present (Fig.13A), while it was increased with increasing concentrations (Fig.13B, C).

Cancer stem cell population study by Hoechst efflux assay
The Hoechst efflux assay was done by flow cytometer The Side Population(SP) tail contains cells that are able to efflux the Hoechst 33342 dye, The SP of HeLa cells in negative controls was 68.04% (15A) , while in LBE treatment, SP of HeLa cells was 46.34% in 250 µg/ml (15B,), 20.81% 500 µg/ml (15C), respectively. Verapamil (positive control) treated HeLa cells showed 2.10% of SP in 50 µg/ml (15D) .Percentage of side population was also presented in bar diagram (1E).

Study of induction of apoptosis by protein expression in Western blotting assay
In order to understand how LBE induced apoptosis of cancer cells and inhibited cell proliferation, the gene expressions of pro-and anti-apoptotic genes such as, pro-caspase 3, pro-Caspase 9, P53, PARP and Bcl-2, were detected. We found that treatment with LBE decreased the expression of gene, procaspase 3, pro-caspase 9, Bcl-2 and PARP (Fig.16) in HeLa cells in vitro. More over p53 gene, one of important cancer guard genes showed high expression i.e. up regulated by LBE.

Anti-migration by LBE against HeLa cells by wound healing /scratch assay
Cell migration was an important property of cancer cells. The effect of LBE on cell migration was examined by a wound healing or scratch assay in vitro (Fig 17). Cells were cultured in agarose plates 24 h to come 80% confluency. Cells in the plates were scratched by a 200 µl pipette tip in the center of the well and exposed with various concentrations of LBE. Briefly, 50x10 3 HeLa cell per well were cultured in 24 well plate. After reaching 90% confluency, the center of the culture dishes was scratched with 200 µl pipette tip. Then the cells were washed frequently by using PBS and incubated with LBE at concentration of 0, 500 and 1000 µg/ml. After 24 h image of the cells was taken under 10X magnification.

Cologenic assay
We tried to examine the capacity of the LBE to inhibit the colony formation of HeLa cells on agarose plate. The results showed maximum colony formation of HeLa cells in control (negative) (18A) where as in treatment, LBE decreased the colony formation of this cancer cells gradually when we increased doses from 250 µg/ml to 1250 µg/ml (18 B, C, D, E & F) and in maximum dose 1250 µg/ml (18 F) no cells were found in 24 h. Treatment. The property of anchorage-independent growth of cancer cells, in vitro is one of the important properties, and colony formation is correlated with the in vivo oncogenic potential of cancer cells.

Antitumor effects of LBE treatment in HeLa-implanted mice
Growth of the tumors in HeLa cell-implanted mice is shown in (Fig19), and tumor growth (volume) in 50 mg/kg LBE treatment groups were plotted in line plot (Fig.20). Tumors in the HeLa + vehicle group increased in volume after 7 days (0.65±0.10 cm 3 ), reaching a maximum of 4.10± 1.59 cm 3 after 28 days. The tumor volume for the 50 mg/kg LBE treatment groups on days 11, 15, 21, and 28 were 0.49± 0.07, 0.23 ±0.05, 0.14±0.03, and 0.04 ± 0.01cm 3 , respectively (Table 1). When the weight of the tumors in this group was measured on day 28, following the sacrifice of the mice, the tumors were of the minimum weight (214 ±12.34 mg), whereas, in the HeLa + vehicle control group (negative), the tumors attained the maximum volume (4.10±1.59 cm 3 ). Treatment with 50 mg LBE/kg of body weight of mice led to a marked reduction in the volume (93.22±9.2% cm 3 ) and weight (90.42 ±9.55 %) of the tumors. However, the reduction of tumor volume in the HeLa + LBE 10 treatment group (3.95 ± 2.00 cm3) did not reach a significant level compared with the HeLa+ vehicle group (4.10± 1.59 cm 3 ).The visceral organs (kidney, liver, spleen, and lungs) were morphologically observed, and they were revealed to be normal and intact: No disorganization, no lesions and no abnormal growth were identified.

Gas chromatography-mass spectrometry (GC-MS) analysis
GC-MS analysis is powerful tool for qualitative and quantitative analysis of various compounds present in natural products. And widely being applied in medical, biological and food research 34-37 . In present study the GC-MS analysis of the LBE of L. betulina from the NIST inbuilt, Library Data Bank showed the presence of 69 compounds (Fig 21). Of the 69 compounds obtained, 32 compounds had the higher matching probability above 50% with the standard compounds of the NIST Library data Bank ( Table 1). Out of which, 14 compounds found to be in higher proportion with the percentage peak area >1.

DISCUSSION
The current findings demonstrated that LBE of L. betulina is very potent killer of HeLa, CaSki and SiHa as validated by MTT assay, and present elaborate experimental works on various parameters such as inhibition of proliferation, induction of apoptosis, reduction of mitochondrial membrane potential, generation of intracellular ROS, induction of autophagy of HeLa cell line, de-polymerization of Factin of cytoplasm, cancer stem cell population study and anti-metastatic assay (migration and cologenic assay) were not conducted by any other worker but previously extracts of different mushrooms on other cell lines were reported by other scientists. As per the report of Liu et al. 36   Apoptosis, a genetically regulated form of cell death, plays an important role in homeostasis condition in our body 44 . Its defects can contribute to diseases like cancer, so interest surrounding the apoptosis has grown highly among oncologists. Apoptosis is suggested to be one  [61][62][63] .Whereas arrest at G2/M phase was induced in prostate 62 , hepatoma 64 and bladder cancer cells 65 . On the other hand, Dudhgaonkar et al. 66 isolated triterpenes from G. lucidum and observed that they triggered cell cycle arrest at G0/G1 and G2/M phase in macrophages; therefore it suggested that cell cycle arrest depends on the specific biologically active compounds as well as particular cells. .
We also examined activity of the LBE as anti-metastasis by performing cologenic assay and antimigration assay against HeLa cell line. Ganoderic acids (GA-A and GA-H) from Ganoderma lucidum demonstrated inhibition on colony formation of invasive breast cancer cells 67 . Pleurotus ostreatus (aqueous extract) exhibited a reduction in the number of colonies of COLO-205 (oral cancer) cells with 43.8 ± 3.5% in contrast with 100% proliferation on untreated cells 68 . Our Cologenic assay also showed anti colonization property as indicated by other workers in other cell line. The migration property of cancer cells is another hall mark of metastasis. In our migration scratch assay, LBE showed very good anti migration agent against HeLa cell line. It was also supported by F actin filament polymerization caused by LBE in our Laser Scanning Confocal microscopy (LSCM) study. Actin polymerization status as measured by an increase in F/G-actin ratio (increased F-actin or decreased G-actin), is a marker of cancer 69 . Our current experiment showed that cytoplasmic F-G actin was depolymerized by LBE in HeLa cells as seen under LSC Microscopy. Lu et al. 65 observed that Ganoderma extracts (ethanolic) triggered anti proliferation, arrested cell cycle at G2/M, and induced actin polymerization in bladder cancer cells and mentioned the impact of actin polymerization in cell migration. Since actin polymerization is a cell cycle-related event 70 , we may infer that the observed inhibition of cell proliferation and arrest in G2/M check point and inhibition of cell migration by LBE in HeLa cells may be linked with actin remodeling. However, to draw the exact relationships between actin remodeling, G 2/M arrest, and growth inhibition need more elaborated research. As we know, cytoplasmic actin filament forms podia of HeLa cells by which cells migrate and their polymerization/ shrinkage invariably inhibits podia extension and migration.
Recently autophagy has gained much attention as the potent alternative mechanism to fight cancer. Although the impact of autophagy on cancer cell death is a topic of intense debate 71,72 , one school of oncologists with their data supports the alternate mechanism of autophagy in the death of cancer cells [73][74][75] . There are emerging evidences that autophagy, a caspase-independent cell death process, plays critical roles in the generation of antineoplastic responses and mediates caspase-independent malignant cell death 76 . Other scientists studied that the deletion of Atgs and Bif-1(autophagy-regulating genes) also validated that autophagy suppressed tumor functions 77,78 . We examined first that LBE also induced autophagy on the HeLa cell that is caspase independent cell death process. Curcumin triggered the occurrence of autophagy in SUP-B15 cells at exact 4 h and 8 h after treatment of it 79 . Similarly, we infer that in addition to apoptosis, autophagy is another mechanism of LBE to inhibit the proliferation of HeLa cell line and tumerogenesis in mice as supported from finding of Santi et al 74 who recorded that metformin prevents cell tumorigenesis through autophagy-related cell death.
Currently analysis of cancer stem cell has gained impetus in therapeutic study, as whether therapeutic strategy is able to kill all cancer stem cells (CTC) in tumor or not. Otherwise cancer/tumor will relapse after time being. So population of cancer stem cell analysis is new addition in cancer biology. We also estimated the population of stem cell of HeLa after treatment by LBE and our results showed that treatment of LBE significantly reduced CTC population after the time. A study of mice in vivo with implanted BGC-823 cancer cells revealed that the weight and volume of the tumors were reduced after 15 days' treatment with P. ostreatus mycelium polysaccharide 2 (POMP2) 80 . In another study, polysaccharides were extracted from Lepista sordida, and when applied against laryngo carcinoma, these inhibited cell growth in vitro and caused a reduction in tumor size in vivo 81 The compound ergosterol was isolated from Agaricus brasiliensis, and led to a retardation in tumor growth in sarcoma 180-bearing mice. In studies in vivo, ergosterol was shown to inhibit neovascularization, as well as being potentially an inhibitor of angiogenesis 82 A marked reduction in tumor fluid volume, and changes in various other properties, were identified in Dalton's lymphoma ascites-bearing mice following Calocybe indica (milky mushroom) treatment, in comparison with negative control group mice 83 In the present study, 50 mg LBE /kg body weight of mice also led to a marked reduction in tumor volume in HeLa cell-implanted mice. None of the previous study found mechanistic clue of compounds exist in EE of mushrooms. GC-MS profile of LBE exhibited that out of 69, 14 compounds were in higher proportion with the percentage peak area > 1. Two of the 14 compounds, 9, 12-Octadecadienoic acid (Z, Z) and Ergosta-5, 8, 22-trien-3-ol, (3.beta22E) were in a significantly higher proportion with the percentage peak area 22.13 and 19.72 respectively. We started to search data base (Swiss target prediction, www. Swisstarget pridiction. ch ) to find out the binding affinity cites of these two compounds. We found that Ergosta-5, 8, 22-trien-3-ol, (3. beta22E) has maximum binding affinity to Cytochrome P 450 of mitochondria, so it can attach with later and induces it for further regulation of caspase 9 apoptotic gene. Similarly, 9, 12-Octadecadienoic acid (Z, Z) has affinity to bind with fatty acid binding protein of membrane, peroxisome proliferator activated receptor, and tyrosyl DNA phosphodiesterase. The ergo sterol of Amauroderma rude, when injected in murine cancer infected mice, enhanced survival time of the mices, indicating that ergosterol might be the leading anticancer substance 84 .The inhibitors of COX-1 and COX-2 enzymes are the compounds -ergosterol, ergosta-4,6,8 85 , 22-tetraen-3-one and 1-oleoyl-2-linoleoyl-3-palmitoylglicerol were isolated from Grifola frondosa 86,22 . In our LBE has much % of peak area of Ergosterol, so it conferred anti HeLa. In LBE another important fatty acid compound N-hexadecanoic acid is present and it showed significant cytotoxicity against human colorectal carcinoma cells (HCT-116) 87 .Harada et al. 87 , 88 reported that Nhexadecanoicacid blocked proliferation of human fibroblast cells by inhibiting DNA topoisomerase-I. Lai et al. 89 reported that it binds to microtubule associated protein 'tau', and inhibits cell division. Other workers demonstrated that both oleic and α -linolenic acid showed a proliferation inhibition effect on prostate carcinoma cells 90,91 . More over phenolic compounds such as Phenol, 2,4-bis(1,1dimethylethyl) (1.50 ) and Phenol, 2,4,6-tris (21icycle[2.2.1]h ept-2-yl) are also found in LBE. Role of phenolic compounds as anticancer is now well established. A recent study showed that 2, 4-bis (1,1dimethylethyl)-(1) is cytotoxicity against Hela cell 92 . Mechanistic studies have shown that phenolic compounds can produce reactive oxygen species (ROS) under cell culture conditions and induce apoptosis in cancer cells [93][94][95][96][97][98] . It has been reported that polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in humans, when up to 1.0 gm is ingested daily from a diet rich in fruits and vegetables 99 .Only four phenolic acids, however, were analyzed in Agaricus (white &brown) and shiitake mushrooms 100 . In our experiment LBE showed potent anti proliferative property with IC 50 at 492.52±2.6 µg/ml, 612.22 ± 4.2 µg/ml, and 1210.30 ± 6.4 µg/ml for 24 h against HeLa, CaSki and SiHa cell line respectively. Liu et al. 101 isolated eight compounds including 5 sterols from L. betulina, while Fakoya & Oloketuyi 9 , reported phenolic and steroids compounds are present in EE of this mushroom. Lupeol, one terpenoid, is also present in LBE, library search exhibited that it facilitated significant inhibition of growth of breast cancer cell line 102 , prostate 103 , colorectal 104 , and gastric cancer 105 . GCMS profile of LBE also showed the presence of 4'-Hydroxy-6-methoxyaurone and this compound inhibits cancer cells to acquire drug resistant and also cytotoxicity against mouse RAW264.7 cell 106 . Recently multidrug resistance (MDR) of tumor cells to cytotoxic drugs is a major problem in cancer chemotherapy 106 . It has been well established that the mechanism by which tumor cells acquire multidrug resistance (MDR) is the over expression of aPgp (P-glycoprotein: ATP dependent membrane glycoprotein) that binds a drug and drags out it from the cell [107][108][109] . The driving force of drug efflux is Pgp-mediated ATP hydrolysis, the liberated energy of which is used for the drug transport [110][111][112] . 4-Hydroxy-6-methoxyaurone has high binding affinity to the nucleotide-binding domain of Pgp 113 and by binding with Pgp it may inhibit the cancer cells to become drug resistant. 112 In conclusion, LBE (LBE of L.betulina) is a mixture of potential compounds for inhibition of proliferation of HeLa, CaSki and SiHa cell lines. By apoptosis as shown by ROS generation, MMP reduction, regulating pro-caspase 3 & 9, and cancer guard gene p53, it was found that LBE is potent anti-HeLa cell line. Moreover it induced caspase independent autophagy of HeLa cells and prevents tumerogenesis. It blocked the cell division by arresting G2/M checkpoint. Anti-metastasis property of it was also revealed from anti-migration and anti-cologenic capacity. Its anti-migration property was also validated from F-G actin polymerization capacity in HeLa cells. Efficacy to reduce cancer stem cell population is one another important anti-HeLa property of this mushroom. Antitumor effects of LBE treatment in HeLa-implanted mice model, in our experiment also validated that LBE is one important anti cervical cancer activity. GC-MS profile also exhibited a lot of anti-cancerous fatty acids, phenol and terpenoid. One interesting compound i.e. 4'-Hydroxy-6-methoxyaurone which inhibits the cancer cells to become drug resistant. Taken together, the anticancer activity of ethanol extract of L. betulina may be the result of the synergistic effects of various compounds, which suggests that this mushroom is excellent source of anticancer agents in the treatment of cervical cancer.