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 Table of Contents  
Year : 2016  |  Volume : 2  |  Issue : 4  |  Page : 95-104

Antiproliferative and apoptotic effect of Pleurotus ostreatus on human mammary carcinoma cell line (michigan cancer foundation-7)

Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Chidambaram, Tamil Nadu, India

Date of Web Publication26-Aug-2016

Correspondence Address:
Sankaran Mirunalini
Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar 608002, Chidambaram, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2395-3977.189303

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Aim: The aim of this study was to explore the antiproliferative and apoptotic effect of Pleurotus ostreatus ethanolic extract (POEet) on human mammary carcinoma cell line (Michigan cancer foundation-7 [MCF-7]).
Methods: Gas chromatography-mass spectrometry (GC-MS) was performed to isolate and quantify the active constituent of POEet chloroform fraction. Cytotoxic property of POEet on MCF-7 and Vero cells was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. We further evaluated the apoptotic effect by measuring the mitochondrial membrane potential (ΔΨm), dual staining (acridine orange/ethidium bromide), comet assay, DNA fragmentation, and oxidant/antioxidant status in MCF-7 cells treated with and without POEet.
Results: GC-MS analysis identified 30 phytochemical constituents from the POEet chloroform fraction. MTT assay revealed a greater degree of cytotoxicity at almost all doses of POEet, with IC30 value (164.59 µg/mL) and IC50 value (1024.02 µg/mL) greatly inhibiting the cell growth. In addition, POEet could modulate the levels of oxidant/antioxidant status and induce a potent loss mitochondrial membrane potential, DNA damage, and apoptosis in MCF-7 cells.
Conclusion: The study demonstrates a potent anticancer property of P. ostreatus against human mammary carcinoma cells which might be of value in nutraceutical industry. Further investigations are essential to establish it as a treatment against breast cancer.

Keywords: Comet assay, gas chromatography-mass spectrometry, mammary cancer, Michigan cancer foundation-7 cell line, Pleurotus ostreatus

How to cite this article:
Deepalakshmi K, Mirunalini S. Antiproliferative and apoptotic effect of Pleurotus ostreatus on human mammary carcinoma cell line (michigan cancer foundation-7). Cancer Transl Med 2016;2:95-104

How to cite this URL:
Deepalakshmi K, Mirunalini S. Antiproliferative and apoptotic effect of Pleurotus ostreatus on human mammary carcinoma cell line (michigan cancer foundation-7). Cancer Transl Med [serial online] 2016 [cited 2020 Sep 23];2:95-104. Available from: http://www.cancertm.com/text.asp?2016/2/4/95/189303

  Introduction Top

Breast cancer is a major health problem worldwide and possesses an increasing risk of human affliction and economic threat, particularly in emerging countries.[1] In India, it is the second most common cancer next to cervical cancer, with an estimated 115,251 new cases every year.[2] The etiology of breast cancer is multifactorial; risk factors include environmental, hereditary, and biological factors such as age, early menarche, late menopause, and delaying first pregnancy, oral contraception, hormonal imbalance, diet, and obesity.[3]

Although majority of cancer research has been devoted to the development of antineoplastic drugs, leading to a significant improvement in the prognosis of breast cancer over the last decades, there is a high need for preventive approaches. Thus, the pursuit of cancer-preventing drugs and dietary measures has the potential to emerge as acceptable approaches to control breast cancer incidence with low toxicity.[4] In this regard, over the last decade, immense research has been carried out in plant products and the researchers have endeavored to identify some of the active components to the pharmaceutical industry. A high intake of fruits, vegetables, and mushrooms is associated with lower incidence of chronic diseases such as cancer.[5] Moreover, natural dietary agents, especially fruits, vegetables, sprouts, and mushrooms have drawn a great deal of attention both from researchers and from the public because of the role they play in the field of prevention and therapy of cancer owing to their low toxicity and biological activity.[6]

Pleurotus ostreatus, an edible fungus, is a traditional Chinese medicine which is famous for its delicious taste and high quantities of proteins, carbohydrates, minerals, and vitamins as well as low fat. Its main constituents include polysaccharides, lectin, polypeptide, amino acids, and phenol oxidase. Beneficial effects of P. ostreatus include antitumor, antioxidant, blood fat reducing, antiviral, anti-inflammatory, antibiotic, and immunoregulatory properties.[7] The polysaccharide fraction of P. ostreatus has potent antitumor activity in Ehrlich tumor and sarcoma 180, and the antitumor substance was postulated to be the β-glucan fraction.[8] Recently, in our laboratory, we already demonstrated the anticancer effect of P. ostreatus by modifying oxidant/antioxidant status in experimental rats.[9]

In spite of the global consumption of mushrooms, only two epidemiological studies demonstrated an inverse correlation between mushroom intake and the risk of cancer.[10],[11] Therefore, in the present study, we investigated the phytochemical present in the extract of P. ostreatus and further evaluated its antiproliferative activity in Michigan cancer foundation-7 (MCF-7) cell line by assessing its apoptotic property.

  Methods Top


Cell culture chemicals such as heat-inactivated fetal calf serum (FCS), glutamine, penicillin-streptomycin, Dulbecco's modified eagle's medium (DMEM), ethylenediaminetetraacetic acid (EDTA), trypsin, low-melting agarose, normal melting agarose, phosphate-buffered saline (PBS), proteinase K, acridine orange (AO), ethidium bromide (EtBr), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), rhodamine 123 (Rh-123), and tamoxifen (TAM) were purchased from Sigma Chemical Company (St. Louis, MO, USA). All other chemical and solvents used were of analytical grade.

Pleurotus ostreatus collection, extraction, and characterization

P. ostreatus mushrooms were collected in and around areas of Udhagamandalam, Nilgiris district, Tamil Nadu. The plant was taxonomically identified and authenticated. A voucher specimen (No.: 233) was deposited in the herbarium of botany, Department of Botany, Annamalai University.

Ethanolic extract

The fresh fruiting bodies of P. ostreatus were dried under the shade and pulverized in a blender to get coarse powder. Five grams of the powder was extracted with 100 mL of 95% ethanol using a Soxhlet apparatus. The solvent was evaporated on a rotary evaporator (Buchi Rotavapor, Switzerland) under reduced pressure and controlled temperature (40°C–50°C).[12] A dark semisolid material (6% yield) thus obtained was stored at 4°C until use. The dried ethanolic extract of the P. ostreatus (POEet) chloroform fraction has been used for the gas chromatography-mass spectrometry (GC-MS) analysis.

Gas chromatography-mass spectrometry analysis

GC-MS is one of the hyphenated analytical techniques which separate the components of a mixture and MS characterizes each component individually. By combining the two techniques, one can evaluate a solution (both qualitatively and quantitatively) containing a number of bioactive constituents which are used extensively in the medical, pharmacological, and law enforcement fields. GC-MS analysis was carried out on a Thermo GC-Trace ultra-version 5.0, Thermo MS DSQ II, which employed a fused silica column packed with Elite-ZB 5-MS capillary standard nonpolar column (30 m, 0.25 mm, 0.25 µm), and the components were separated using helium as carrier gas at a constant flow of 1 mL/min. The 1 µL sample extract injected into the instrument was detected by the Thermo MS DSQ II. The oven temperature was programmed at 260°C for 38.50th min (mass analyzer). The MS detection was completed in 40 min.

Cell lines and culture conditions

MCF-7, a human mammary cancer cell line, and normal Vero cell lines were purchased from National Centre for Cell Sciences, Pune, India. The cells were maintained in DMEM supplemented with 10% heat-inactivated FCS, 3% L-glutamine, 100 IU penicillin, and 100 g/mL of streptomycin. The cells were grown in 5% CO2 at 37°C and were subcultured at 80–90% confluence.

Cytotoxicity assay

The cytotoxicity of POEet on MCF-7 and Vero was determined by the MTT assay.[13] The effect of the samples on the proliferation of MCF-7 and Vero was expressed as the % cell viability, using the following formula:

% cell viability = A570 of treated cells/A570 of control cells × 100

Cells were treated with different concentrations of POEet (0, 30, 60, 180, 260, 520, 1040 μg/mL), incubated for 48 h, and the cytotoxicity was measured using MTT assay. Based on the IC30 and IC50 value, 165 µg/mL and 1024 µg/mL concentrations of POEet were chosen for further experiments. The cells were divided into four groups; Group I served as untreated control, Groups II and III were treated with 165 μg/mL and 1024 μg/mL of POEet, respectively, while Group IV treated with TAM 5 μg/mL served as positive control.

Biochemical analysis

The trypsinized cells resuspended in PBS were used for biochemical analysis. The concentration of thiobarbituric acid reactive substances (TBARS) was estimated using the method described by Niehaus and Samuelsson in 1968.[14] In this method, malondialdehyde and TBARS were measured by analyzing their reaction with TBA in an acidic condition to generate a pink-colored chromophore, which was read at 535 nm. Superoxide dismutase (SOD) was assayed using the method described by Kakkar et al. in 1984.[15] The assay is based on the inhibition of nicotinamide adenine dinucleotide (NADH), phenazine methosulfate, and nitroblue tetrazolium formazan formation. The reaction was initiated by the addition of NADH. The catalase (CAT) activity was determined using the method described by Sinha in 1972.[16] Dichromate in acetic acid was converted to perchromic acid and then to chromic acetate when heated in the presence of H2O2. The chromic acetate formed was measured at 620 nm. Glutathione peroxidase (GPx) was estimated using the method described by Rotruck et al. in 1973.[17] A known amount of enzyme was allowed to react with H2O2 in the presence of glutathione (GSH) for a specified period. Then, the GSH content remaining after the reaction was measured. The total GSH content was measured based on the development of a yellow color when 5,5'-dithiobis-2-nitrobenzoic acid was added to compound containing sulfhydryl groups.[18]

Alterations in mitochondrial membrane potential (ΔΨm)

The studies on mitochondrial and changes in the mitochondrial membrane potential have become the focus of interest in analyzing the apoptosis. The changes in ΔΨm were observed microscopically and determined fluorimetrically using fluorescent dye Rh-123 (Shimadzu RF-5301PC Spectroflurometer). After treatment with POEet for 48 h, fluorescent dye Rh-123 (10 μg/mL) was added to the cells and incubated for 30 min.[19] Then, the cells were washed with PBS and viewed under fluorescence microscope using blue filter (Nikon, Eclipse TS100, Japan).

Dual staining

AO and EtBr staining with DNA allowed visualization of the condensed chromatin of apoptotic cells.[20] The control and POEet-treated cells were seeded in a 6-well plate (3 × 10[4]/well) and incubated in CO2 incubator for 48 h. The cells were fixed in methanol: glacial acetic acid (3:1) for 30 min at room temperature washed in PBS and stained with 1:1 ratio of AO/EtBr. Stained cells were immediately washed with PBS and viewed under a fluorescence microscope (Nikon, Eclipse TS100, Japan) with a magnification of ×40. The number of cells expressing apoptotic feature was counted and expressed as a fraction of the total number of cells present in the field.

Alkaline single-cell gel electrophoresis (comet assay)

DNA damage was estimated by alkaline single-cell gel electrophoresis (comet assay) according to the method described by Singh et al. in 1988.[21] A layer of 1% normal melting point agarose was prepared on microscope slides. The untreated control and POEet-treated cells (50 μL) were mixed with 200 μL of 0.5% low melting point agarose and were pipetted onto the precoated slides. Slides were immersed in cold lysis solution at pH 10 (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris pH 10, 1% Triton X-100, 10% DMSO) and incubated at 4°C for 60 min. To allow denaturation of DNA, the slides were placed in alkaline electrophoresis buffer at pH 13 for 25 min. Subsequently, slides were transferred to an electrophoresis tank with fresh alkaline electrophoresis buffer. Electrophoresis was performed at field strength of 1.33 V/cm for 25 min at 4°C. Slides were neutralized in 0.4 M Tris (pH 7.5) for 5 min and stained with 20 μg/mL EtBr. For the visualization of DNA damage, observations were made using a ×40 objective in an epifluorescent microscope equipped with an excitation filter of 510–560 nm and a barrier filter of 590 nm. One to two hundred comets on duplicated slides were analyzed. Images were captured with a digital camera with networking capability and analyzed by image analysis software, (Version 2, Free Software Foundation, Inc., Boston, MA, USA).[22] DNA damage was quantified by tail moment (TM), tail length, and olive TM (OTM). OTM is the product of the distance (in X direction) between the center of gravity of the head and the center of gravity of the tail and the percent tail DNA.[23]

DNA fragmentation

The protocol followed for DNA fragmentation is explained elsewhere.[24] Briefly, MCF-7 cells (1 × 10[5] cells/well) treated with POEet for 48 h were collected and centrifuged at 1000× g for 5 min. The cell pellet was suspended in cell lysis buffer (Tris-HCl 10 mmol/L pH 7.4, 0.5% of Triton X-100) and kept at 4°C for 10 min. The lysate was centrifuged at 25,000× g for 20 min. The supernatant was incubated with RNase A 40 g/L at 37°C for 1 h and further incubated with proteinase K 40 g/L at 37°C for 1 h. The solution was mixed with NaCl (0.5 mol/L) and 50% 2-propanol and incubated overnight at −20°C and then centrifuged at 25,000× g for 15 min. After drying, DNA was dissolved in TE buffer and separated by 2% agarose gel electrophoresis at 100 V for 50 min.

Statistical analysis

Statistical analysis was performed using SPSS software package (version 16.0, IBM, Chicago, IL, USA). The values were analyzed by one-way analysis of variance (ANOVA), followed by Duncan's multiple range test (DMRT). All these results were expressed as mean ± standard deviation (SD) of six samples in each group. P < 0.05 was considered as statistically significant.

  Results Top

Gas chromatography-mass spectrometry analysis

GC-MS plays a key role in the analysis of unknown components of plant and mushroom origin. Based on the GC-MS results, 30 phytochemical constituents have been identified from the POEet chloroform fraction. The biological activities of phytochemical constituents were predicted by the Duke's databases. The results pertaining to GC-MS analysis lead to the identification of numerous compounds from the GC fractions of the POEet. These compounds were recognized through MS attached with GC. As shown in [Figure 1], about 30 peaks were identified in P. ostreatus by GC-MS analysis. The active principles with their retention time, molecular formula, molecular weight, and peak area (%) are presented in [Table 1]. The biological activities of some phytochemicals detected based on Dr. Jim Duke's phytochemical and ethnobotanical databases of the agricultural research service/USDA are listed in [Table 2].
Figure 1. Gas chromatography-mass spectrometry chromatogram of ethanolic extract of Pleurotus ostreatus (chloroform fraction)

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Table 1. Phytocomponents identified in the ethanolic extract of Pleurotus ostreatus by gas chromatography-mass spectrometry

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Table 2. Activity of phytocomponents identified in the ethanolic extract of Pleurotus ostreatus by gas chromatography-mass spectrometry

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Cytotoxicity assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay)

The cytotoxicity/antiproliferative activity of POEet (0–1040 µg/mL) was investigated in MCF-7 and normal Vero cell line using MTT assay. As presented in [Figure 2]a and [Figure 2]b, on incubation with POEet for 48 h, MCF-7 cells exhibited significant signs of cytotoxicity in a dose-dependent manner which was not observed in normal Vero cells. Interestingly, greater degree of cytotoxicity was observed for almost all doses of POEet. Moreover, the IC50 value of POEet was found to be 164.59 μg/mL, and IC50 value of 1024.02 μg/mL could greatly inhibit the cell growth. Hence, we have chosen the 165 µg/mL and 1024 µg/mL concentrations of POEet for further experiments.
Figure 2. Cytotoxicity of Pleurotus ostreatus ethanolic extract in (a) Vero cells and (b) Michigan cancer foundation-7 cells after 48 h. Data are expressed as mean ± standard deviation of six samples in each group (analysis of variance followed by Duncan's multiple range test). *P< 0.05 vs. control group

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Modulation of oxidant and antioxidant status

[Figure 3]a depicts the level of TBARS and antioxidants of control and treated groups in MCF-7 cell line. POEet treatment showed significantly (P < 0.05) increased levels of lipid peroxidation in MCF-7 cells. Moreover, among the two doses (165 µg/mL and 1024 µg/mL) tested, 1024 µg/mL of POEet showed maximum levels of TBARS higher than TAM (5 µg/mL). [Figure 3]b and [Figure 2]c depicts the effects of POEet on the status of enzymatic (SOD, CAT, GPx) and nonenzymatic antioxidant (GSH) status in control and treated groups in MCF-7 cells. Compared with untreated control group, POEet (at both 165 µg/mL and 1024 µg/mL) could significantly (P < 0.05) deplete the enzymatic and nonenzymatic antioxidant activities in MCF-7 cells. When compared with TAM (5 µg/mL) group, 1024 µg/mL dose of POEet could reach significance (P < 0.05) in depleting the antioxidant activity in MCF-7 cells.
Figure 3. The levels of oxidant and antioxidant enzymes in Michigan cancer foundation-7 cells of control and experimental groups. (a) The levels of lipid peroxidation marker (thiobarbituric acid reactive substances) in Michigan cancer foundation-7 cells. (b) Activities of enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase) in Michigan cancer foundation-7 cells. (c) Activities of reduced glutathione in Michigan cancer foundation-7 cells. Superoxide dismutase: U* are expressed as the amount of enzyme required to inhibit 50% of nitro blue tetrazolium reduction in 1 min. Catalase: U** μmol of hydrogen peroxide consumed per minute. Glutathione peroxidase: U*** μg of glutathione consumed per minute. The values are expressed as mean ± standard deviation of six samples in each group (analysis of variance followed Duncan's multiple range test). Bars not sharing the common superscripts differ significantly at P< 0.05

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Pleurotus ostreatus ethanolic extract modulates mitochondrial membrane potential (ΔΨm)

Mitochondrial membrane potential changes were measured after incubating untreated control and POEet-treated cells with Rh-123 fluorescence dye for 48 h. Fluorescence microscopic images [Figure 4]a show increased accumulation of Rh-123 dye in the untreated control group compared to POEet 165 µg/mL; however, an improved accumulation was observed in POEet 1024 µg/mL treated cells as revealed by a significant (P < 0.05) decrease in membrane potential. We observed similar results with positive control, TAM (5 µg/mL).
Figure 4. Morphological assessment of apoptosis in Michigan cancer foundation-7 cells by (a) Rh-123 and (b) acridine orange/ethidium bromide staining. (a [i]) A – Fluorescent microscopic images of mitochondrial membrane potential by Rh-123 staining. Arrow marks (→) represent a dye accumulation in untreated control cells, B – Arrow marks (←) represents no dye accumulation, C – Arrow marks (←) represents no dye accumulation, D – Tamoxifen 5 μg/mL. There was a little accumulation of Rh-123 in TAM 5 μg/mL group. (a [ii]) Fluorescence intensity of cells. (b [i]) A – Fluorescent microscopic images of apoptotic morphology by dual staining. Arrow mark (→) represents orange-colored cells which were late apoptotic cells. B – Pleurotus ostreatus ethanolic extract + 165 μg/mL, percentage of apoptosis. C –Pleurotus ostreatus ethanolic extract + 1024 μg/mL, D – TAM 5 μg/mL. The values are expressed as mean ± standard deviation of six samples in each group (analysis of variance followed Duncan's multiple range test). (b [ii]) Percentage of apoptosis cells. Bars not sharing the common superscripts differ significantly at P< 0.05

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Apoptotic effects

The morphological characteristics of apoptotic cells were investigated by staining with fluorescent dyes including AO and EtBr. Cells treated with POEet for 48 h showed increased percentage of cell shrinkage, condensed, and fragmented chromatin. As illustrated in [Figure 4]b, POEet at doses of 165 and 1024 µg/mL treated MCF-7 cells shows 35% and 56% significantly (P < 0.05) increased percentages of apoptotic cells, respectively, indicating formation of orange fluorescence. We also observed similar result with positive control, TAM (5 µg/mL). Whereas untreated control cells showed evenly distributed AO strain (green fluorescence), which indicates that untreated cells does not undergo apoptosis.

Pleurotus ostreatus ethanolic extract induced DNA damage

[Figure 5]a shows the photomicrographs of level of DNA damage (comet assay) in different treatment groups. The untreated control cells show largely nonfragmented DNA whereas POEet-treated cells showed fragmented DNA, which appears as a comet during single-cell gel electrophoresis. The comet lengths were analyzed by CASP software. There was no change observed in levels of DNA damage in the untreated control cells. POEet (165 and 1024 µg/mL) treatment significantly (P < 0.05) expanded tail length, percentage of DNA, OTM, and TM in MCF-7 cells. Among the two dosages tested, 1024 µg/mL of POEet demonstrated extreme tail length (56%), DNA tail (24%), OTM (30%), and TM (62%). We observed similar results with positive control, TAM (5 µg/mL).
Figure 5. Morphological assessment of apoptosis in Michigan cancer foundation-7 cells by (a) comet assay and (b) DNA fragmentation analysis. (a [i]) A – Fluorescent microscopic images of oxidative DNA damage (comet assay), B –DNA tail movement, C – Increased DNA tail movement, D – Expanded DNA tail movement. (a [ii]) Comet parameters (% tail DNA, % tail length, tail moment, and olive tail moment). (b) Lane 1 – Marker 1000 bp ladder, Lane 2 – Control, Lane 3 – Pleurotus ostreatus ethanolic extract (165 μg/mL), Lane 4 – Pleurotus ostreatus ethanolic extract (1024 μg/mL), Lane 5 – Tamoxifen (5 μg/mL). The values are expressed as mean ± standard deviation of six samples in each group (analysis of variance followed Duncan's multiple range test). Bars not sharing the common superscripts differ significantly at P< 0.05

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DNA fragmentation

To gain more insight into cell death pathways, DNA fragmentation of MCF-7 cells was detected by agarose gel electrophoresis. As illustrated in [Figure 5]b, the genomic DNA fragment appears as a series of bands which are described as DNA ladders on agarose gel, representing the formation of oligonucleosomes which are the characteristics of apoptosis. Treatment of POEet 1024 µg/mL in MCF-7 cells showed a prominent DNA fragmentation when compared to POEet 165 µg/mL. We observed similar results with positive control, TAM (5 µg/mL).

  Discussion Top

Phytoconstituents obtained from natural source have been gaining importance because of the vast chemical diversity. Therefore, there is a need to ensure the quality, safety, and efficacy of natural drugs. Out of 30 bioactive constituents, 2-methoxy-2-vinylcyclopentanol (60.43%) presents in higher quantity, pentatriacontane (0.10%) presents in less quantity, and other constituents present in considerable amount. Identified phytoconstituents such as hexadecane were reported to possess antioxidant, antibacterial, and antifungal activities.[25] The compounds named tetradecanoic acid and hexadecanoic acid ethyl ester exhibit cancer preventive, nematicide, and hypocholesterolemic activity, and triacontane possesses cytotoxic effect against MCF-7 cells.[27] Furthermore, eicosane and heptacosane possess antitumor, antioxidant, and cytotoxic effects.[28] The 9,12-octadecadienoic acid (Z, Z)-, methyl ester exhibits cancer-preventive and anti-inflammatory activity.[30] Most importantly, sterol compounds such as ergosterol possess an antitumor property which was confirmed from Dr. Duke's phytochemical and ethnobotanical database. By interpreting these compounds, it is found that P. ostreatus possesses various salutary applications. Therefore, the investigation concluded that the presence of this compound as shown in the study could contribute synergistically to the significant nutraceutical potency of P. ostreatus as well as the stronger extraction capacity of ethanol could have fashioned a number of active constituents responsible for many biological activities.

Precise evaluation of the antigrowth impacts of chemotherapeutic is massively important in cancer research about drug disclosure and toxicology safety.[33] In this context, evaluation of the cytotoxicity is a principal selection method for assessing the antitumor activities of different synthetic and natural substances. In the present study, cytotoxicity was assessed using MTT assay. As per our result, on treatment with POEet for 48 h, no cytotoxicity was observed in normal Vero cells whereas almost all doses of POEet could induce cytotoxicity in MCF-7 cells. Recent studies by Jedinak and Sliva [34] demonstrated that by the concentration of 1000 μg/mL, P. ostreatus methanolic extract inhibited the growth of MCF-7 cells and HT-29 cells by the proliferation index percentage of 70% and 17%, respectively. Consequently, the above-mentioned report also, in line with our finding, suggests that P. ostreatus suppresses proliferation of breast cancer cells without significant effect on the proliferation of normal human mammary cells.

In this study, we also performed a series of in vitro functional assays to better understand the anticancer potential of POEet against MCF-7 cell line, including an established breast cancer (in vitro) model to compare its efficacy with TAM, an established standard anticancer drug. Here, we probe the antitumor properties of POEet by investigating the effect on oxidant/antioxidant status, cell morphology, and DNA fragmentation. An altered redox status and the generation of reactive oxygen species (ROS) are playing a significant role in the common biochemical aspects in cancer cells.[35] ROS are equipped for responding with the polyunsaturated fatty acids of lipid membranes which promote lipid peroxidation. The final product of lipid peroxidation is 4-hydroxynonenal which has been thought to be a second messenger of oxidative stress. The effects of POEet on MCF-7 cells resulted in cellular oxidative stress, which apparently increased lipid peroxidation indices (TBARS), followed by decreased enzymic (SOD, CAT, GPx) and nonenzymic (GSH) antioxidant status, thus indicating their pro-oxidant role. Substantial evidence in the literature suggests that the pro-oxidant action of the polyphenolic, flavonoids, triterpenoids, and steroidal compound was mediated by copper redox cycling,[36] which may be an important mechanism of POEet for their anticancer and apoptosis-inducing properties. In addition, a recent study by Jagadeesan et al.[37] reported the pro-oxidant activity of naturally occurring steroids sapogenin and diosgenin which exhibit beneficial effect against MCF-7 and possible anticancer mechanism.[37] Thus, pro-oxidant role of POEet in MCF-7 cells proved by the data of the current study is consistent with the above-mentioned reports.

Apoptosis is an active form of cell death that occurs in response to several agents, including anticancer chemotherapeutic drugs. Mitochondria are the prime source of ROS, and mitochondrial dysfunction is considered as an early event in the apoptotic cascade. In our study, the mitochondrial membrane potential was evaluated using the fluorescent probe Rh-123, which accumulates in mitochondria of untreated control cells. Whereas POEet-treated groups show loss of Rh-123 from the mitochondria and decrease of intracellular fluorescence, which exhibit depolarization of mitochondrial membrane. This result implied that cytochrome C was released from mitochondria to cytosol in a concentration-dependent manner. Consistent with our report, Ma et al.[38] demonstrated that the chimaphilin, an extract from Passiflora incarnata Fisch-induced apoptotic cell death, was associated with loss of mitochondrial membrane potential in MCF-7 cells.

To examine whether the induction of cell death by POEet occurred by apoptosis, the analysis of nuclear morphology is highly essential. Usually, cells undergoing apoptosis display a very similar pattern of morphological membrane integrity and stain the nucleus in red.[39] In this way, live cells have a typical green nucleus; early apoptotic cells have a brilliant green nucleus with condensed or fragmented chromatin; late apoptotic cells show condensed and fragmented orange chromatin; cells died from direct necrosis have a basically ordinary orange nucleus.[40] Whereas in our study, POEet-treated groups induced apoptosis which was evidenced by formation of nuclear changes preceding the cytoplasmic blebbing and nuclear fragmentation. Moreover, it is clear that with increasing concentration of POEet, the number of apoptotic cells increased tremendously. However, recently, Sodde et al.[41] demonstrated that extracts of Macrosolen parasiticus showed apoptotic features in MCF-7 cancer cells, which coincide well with our findings.[41]

DNA is an important molecular target for tumor cell killing.[42] In the present study, we found that significant DNA damage was observed in POEet-treated MCF-7 cells by comet assay. Whereas the bioactive components such as polysaccharides, phenols, and steroids in P. ostreatus might serve as a mediator of the reactive oxygen scavenging system and have the potential to act as a pro-oxidant and an antioxidant, depending on the redox state of the biological environment. Previously, Lavi et al.[36] also reported that an aqueous polysaccharide extract from P. ostreatus induces antiproliferative and pro-apoptotic effects on HT-29 colon cancer cells. This might be the reason behind the expanded oxidative DNA damage (% tail DNA, % tail length, TM, and OTM) observed in POEet-treated malignant cells. Thus, based on the present findings along with the previous reports, it can be concluded that POEet possesses DNA damage in MCF-7 cells.

DNA fragmentation in a ladder pattern is one of the easily measured features of apoptosis, which was performed using conventional agarose gel electrophoresis that demonstrated the internucleosomal DNA cleavage.[43] The results from the present work indicated that POEet induced significant internucleosomal DNA fragmentation in MCF-7 cell line after 48 h of treatment and this activity may be attributed to the extract constituents. The fragmentation was not found to be distinct, but increased DNA damage was observed, which provide evidence for apoptotic cell death. No substantial DNA fragment was observed in untreated control cells. A recent publication demonstrated that ethanolic extract of Mimosa caesalpiniifolia leaves induced apoptosis and necrosis in the MCF-7 cell as observed from the DNA ladder.[44] Similarly, we found that the POEet induced apoptotic cell death in human MCF-7 cells. We therefore confirmed that P. ostreatus induced apoptosis, which will serve as an effective anticancer agent for the treatment against mammary cancer.

In conclusion, taken together, the results from biochemical and apoptotic assessments in our in vitro studies demonstrated that POEet has a significant anticancer effect against mammary cancer. Further studies are highly warranted to isolate the bioactive compounds from P. ostreatus in the form of nutraceuticals and to study the interaction between this active nutraceutical compounds and standard anticancer agents for various malignancies.

Financial support and sponsorship

The study was supported by University Grants Commission-Major Research project (F. No.: G7/17342/2012 (SR)), New Delhi, India.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2]

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