Abstract
Objectives
This study aims to determine the effects of Hericium erinaceus extracts on cell viability and the effects of H. erinaceus water extract on the telomerase activity of MCF-7 cells. H. erinaceus is an edible mushroom widely used in traditional Chinese medicine. Although its various therapeutic properties, the literature has not yet submitted evidence about H. erinaceus for its effects on the telomerase activity of MCF-7 cells.
Methods
MCF-7 cells were treated with ethanol, ethanol-water, ether, ethyl acetate, methanol-water, and water extracts to determine the effects on cell viability using the WST8 method. The TeloTAGGG Telomerase PCR ELISA kit was used to assess telomerase activity.
Results
The water extract was determined to be the most efficient extract to decrease cell viability. The water extract’s half-maximal inhibitory concentration was 250 μg/mL at 72 h. It is found that H. erinaceus has no statistically significant effect compared to positive control on reducing telomerase activity. We found a statistically significant difference in telomerase activity % between H. erinaceus water extracts and negative control (p<0.05).
Conclusions
Consequently, these differences in telomerase activity are a significant association rather than inferring action. It is considered that water extract shows its cell viability inhibition effects through different mechanisms.
Introduction
Breast cancer incidence and mortality rates are increasing every day and have overtaken lung cancer worldwide by 2020 cancer statistics [1]. It is known that different treatment methods, such as chemotherapy, hormone therapy, surgery, and radiotherapy have various side effects on breast cancer patients [2]. Therefore, developing new treatment strategies with lower side effects is essential to treat breast cancer. It is known that several natural products are used in the treatment of diseases such as cancer. Previous studies have reported that different natural products have an in vitro cell viability inhibition effect on MCF-7 cells [3], [4], [5].
The medicinal mushroom Hericium erinaceus, also named Yamabushitake or Lion’s Mane mushroom, grows over old or dead broad-leaved trees. This fungus belongs to the class basidiomycetes and has been used for many years in alternative medicine treatments in Far East countries, especially Japan and China [6, 7]. H. erinaceus has antioxidant, hypolipidemic, antimicrobial, anti-aging, anti-inflammatory, antiangiogenic, immunomodulating, anticancer, and antimetastatic properties [8], [9], [10], [11], [12]. The mycelium and fruit organs of H. erinaceus contain many bioactive compounds which serve as medicines [9]. Studies have shown that aromatic components, including hericenones, erinacines, hericerin, and hericenes, found in H. erinaceus, exhibit broad in vitro and in vivo bioactivities [13]. It is reported that Y-A-2, cytotoxic phenols, hericone A and hericone B, erinapyrone A, and erinapyrone B, isolated from the fruit organ of H. erinaceus using ethanol or acetone, showed inhibitory activity against the proliferation of HeLa cells [14].
There is a lack of studies about developing new therapies that can increase the lifespan of metastatic breast cancer patients. It has become an essential topic of cancer research in recent years. Interest in medical fungi has been raised to develop alternative therapies, and scientific studies are being conducted to investigate the effects of fungi and fungus extracts on various cancer types. Many basidiomycetes contain biologically active polysaccharides in fungi, fruit organs, mycelial cultures, and culture waters. These polysaccharides have diverse chemical structures, many belonging to the β-glucan groups. It has been reported that their basic glucan chains have β- (1–3) branches and, additionally, β-(1–6) side branches for antitumor effects. However, fungal polysaccharides do not directly attack cancer cells; they activate antimetabolites by activating different immunological responses [15].
H. erinaceus is known to be rich in some physiological components. In particular, β-glucan polysaccharides are responsible for fungal anticancer, immunomodulatory, hypolipidemic, antioxidant, and neuroprotective activities [6]. Although the mechanism of action has not been fully explored, it is thought that the anticancer properties of H. erinaceus are caused by various chemical components which are primarily contained.
Previous studies have reported that telomerase activity is at significant levels in a thumping majority of human cancer cells [16]. Also, cancer cells have been shown to provide replicative immortality via telomerase activity [17, 18]. Overexpression of telomerase activity in cells has been associated with resistance to both apoptosis and senescence induction. Instead, suppression or decrease in telomerase activity can cause telomeric shortening. In this study, we investigated the effects of H. erinaceus extracts on cell viability and the telomerase activity of MCF-7 breast cancer cells.
Materials and methods
Mushroom materials
H. erinaceus samples were supplied by the Ege University Department of Bioengineering. The infrastructure of the mushroom production laboratory in the Ege University Department of Bioengineering was utilized. MCC61, MCC63, MCC64, MCC66, and MCC67 strains were used for the micelle form of the organism.
Growing environment
The mixture of 60 % oak shavings, 30 % wheat straw, and 10 % wheat bran was soaked to 60–65 % moisture level to obtain the Basidiocarp form. The mixture is filled in polypropylene bags with a weight of 1 kg. Bags were autoclaved for 45 min at 121 °C under 1.5 atmospheric pressure. After reaching room temperature, the substrate was inoculated with a micelle and was incubated in a 23–24 °C incubation chamber. The room was illuminated after the mycelium was wrapped entirely, and the bags were drilled from various locations. It was observed that the hat form was formed from the punctured points. After the ripening was completed, the harvest was done.
Liquid culture medium
100 mL of 2 % malt medium was added to the conical flask and sterilized. After cooling, from each culture of H. erinaceus, inoculation was performed with 6 mm diameter syringes. Conical flasks were incubated at 24–25 °C and shaken daily to prevent fungal felting. The liquid culture was filtered on the 20th day of incubation, and the micelle was harvested.
Preparation of extracts
Six different H. erinaceus ethanol-water, ethanol, methanol-water, water, ethyl acetate, and ether extracts were prepared at the end of the harvest. Maceration was obtained from H. erinaceus ethanol-water (1:1), ether, methanol, ethanol, and ethyl acetate extracts; however, water extract was obtained by infusion (2 %). During the process, samples were stirred under the solvent/macrofungi sample ratio of 15/1. Filtration of solvents was done with a syringe filter. Shortly after, the filtration samples were evaporated to generate gel-like mushroom extract. Lyophilization was done to the dried residues, and then was weighed to obtain the yield. The following formula was used to calculate the percentage of yield extracts: Yield (%)=(Extract weight after solvent evaporation/H. erinaceus powder weight) × 100.
Lyophilized extracts were used before they were dissolved in dimethyl sulfoxide (DMSO). Extracts were stored at −20 °C at a 25 mg/mL final concentration.
Cell culture
MCF-7, an estrogen receptor-positive [ER(+)] breast cancer cell line, was obtained from American Type Culture Collection. Roswell Park Memorial Institute 1640 (RPMI-1640), containing 1 % penicillin/streptomycin and 10 % Fetal bovine serum (FBS), was used as a culture medium. Cells were plated in 25 and 75 cm2 flasks; in a 37 °C incubator supplying 5 % partial CO2 pressure.
Cell viability assay
According to the instruction manual, the Cell Counting Kit-8 (CCK-8, Sigma-Aldrich, Darmstadt, Germany) was used to perform the cell viability assay. 104 cells were seeded in 96 well plates. Subsequently, the cells were treated with six different extracts of H. erinaceus with various concentrations (0.01, 0.1, 1, 10, 100 μg/mL) for 24, 48, and 72 h. Absorbance was measured at 450 nm using a Thermo Multiskan EX multiple reader (Thermo Fisher Scientific, Waltham, USA).
Telomerase activity
According to the cell viability assay results, water extract was selected for further analysis regarding telomerase activity assay. The telomerase activity assay kit TeloTAGGG Telomerase PCR ELISA (Roche Applied Science, Darmstadt, Germany) was used to determine the telomerase activity following the manufacturer’s instructions [19].
Statistical analysis
The experiments were performed in triplicate. Results were analyzed using one-way analysis of variance (ANOVA) with Post-Test using GraphPad Prism v.5.0b (GraphPad Software, San Diego, USA) for statistical comparisons. A value of p<0.05 was considered statistically significant.
Results
Effects of H. erinaceus extracts on MCF-7 cell viability and telomerase activity
MCF-7 cells were treated with ethanol-water, ethanol, methanol-water, water, ethyl acetate, and ether extracts of H. erinaceus for 24, 48, and 72 h. As shown in Figure 1A, treatment with ethanol-water extract for 72 h caused a reduction in MCF-7 cell viability by 18 % (p<0.05) at a concentration of 1 μg/mL and 29 % (p<0.001) at 10 μg/mL concentration. The ethanol extract inhibited cell viability by about 8 and 9 % at 1 and 10 μg/mL concentrations at the end of 72 h of incubation, respectively (Figure 1B). However, these inhibitions were not significant (p>0.05). Besides, no significant decrease was found in cell viability with methanol: water extract (Figure 1C). As shown in Figure 1D, treatment with 1, 10, and 100 μg/mL water extract for 24 h showed significant inhibition on MCF-7 cells since cell viability was reduced by 36 , 29, and 39 %, respectively (p<0.019). The half-maximal inhibitory concentration (IC50) value of Hericium erinaceous water extract was determined as 250 μg/mL for MCF-7 cells at 72 h (Figure 2). The effects of ethyl acetate extract on MCF-7 cell viability are shown in Figure 1E. No significant cell viability inhibition was determined at 24, 48, and 72 h (p>0.05). On the contrary, cell viability induction was 46 % on 100 μg/mL concentration at the end of 48-h incubation (p<0.01). Figure 1F illustrates that ether extract induced suppression of the viability of MCF-7 cells in a dose-dependent manner at the end of 72 h of incubation. It was determined that cell viability was suppressed by 19 , 22, 30, and 39 % (p<0.01) at 0.1, 1, 10, and 100 μg/mL, respectively. However, a physiologically significant IC50 value could not be calculated for ether extract.
Effects of Hericium erinaceus extracts on the cell viability of MCF-7 cells. Percentage of viable cells after 24, 48, and 72 h of incubation with Hericium erinaceus (A) ethanol-water, (B) ethanol, (C) methanol-water, (D) water, (E) ethylacetate, and (F) ether extracts with six different concentrations (0, 10–100 ng/mL, 1–10–100 μg/mL) are shown. Values are mean ± Standard Error (SE) of three replications. *Indicates p-value <0.05, and **indicates p-value <0.001.
A half-maximal inhibitory concentration (IC50) curve for MCF-7 cells treated with Hericium erinaceus water extract. Percentage cell viability is plotted against the logarithm of treatment concentrations (0.0025, 0.25, 2.5, 25, 250 μg/mL). The IC50 value of H. erinaceous water extract was determined as 250 μg/mL for MCF-7 cells at 72 h. Values are mean ± Standard Error (SE) of three replications. *Indicates p-value <0.05 and **indicates p value <0.001.
Effects of H. erinaceus water extract on telomerase activity
We found that H. erinaceus water extract was the most effective extract among others in inhibiting cell viability. The MCF-7 cells were treated with different concentrations of water extract (62.5, 125, 250, 500, and 1,000 μg/mL) for 72 h to assess the effects on the telomerase activity. As shown in Figure 3, the telomerase activity % was determined with different concentrations of H. erinaceus water extracts compared to the control. We found that H. erinaceus water extracts at different concentrations showed statistically similar telomerase activity % by using telomeric repeat amplification protocol compared to the positive control of the kit. We found a statistically significant difference in telomerase activity % between H. erinaceus water extracts and negative control (p<0.05).
Telomerase activity % of Hericium erinaceus water extract-treated MCF-7 cells using the telomeric repeat amplification protocol. The MCF-7 cells were treated with different concentrations of water extract (62.5, 125, 250, 500, and 1,000 μg/mL) for 72 h, and telomerase activity % was determined compared to the control. Values are mean ± standard error (SE) of three replications. **Indicates p value <0.001.
Discussion
In Asian countries, fungi have been collected and cultivated from nature to treat diseases for centuries [20]. One of the edible mushrooms, H. erinaceus, has been reported to have therapeutic properties such as anticancer, antioxidant, immunomodulating, neuroprotective, and hypoipidemic effects [21]. In the scope of this study, we aimed to understand the effects of different H. erinaceus extracts on MCF-7 estrogen receptor-positive breast cancer cell viability and telomerase activity. Previous studies have shown the anticancer and antimetastatic effects of H. erinaceus in other cell lines [10, 11]. In the present study, ethanol-water, ethanol, methanol-water, water, ethyl acetate, and ether extracts were used, and it was determined that other extracts except the water extract caused low or no cell viability inhibition on MCF-7 cells. The H. erinaceus water extract is more effective than other extracts can be explained by the high amounts of water-soluble polysaccharides in the fungus [22]. A different study shows that tumor growth in the CT-26 colon cancer mouse model can be inhibited by H. erinaceus hot water and ethanol, acidic and alkaline extracts. The study has also shown that hot water and ethanol extracts contain a higher level of β-glucan than others. It is emphasized that hot water and ethanol extracts are more effective than acidic and alkaline extracts [23]. It has also been determined that H. erinaceous hot water and ethanol extracts induce apoptotic cell death in CT-26 cells [24]. Besides, the ethanol extract has been reported to have anticancer potential on hepatocellular carcinoma cells HepG2 and Huh-7, colon cancer HT-29, and stomach cancer NCI-87 cell lines [25]. In the results of our literature review, we summarized that water and ethanol extracts of H. erinaceus have more impact on cancer cells than other extracts.
Similarly to this finding, our results support the anticancer activity of water extract. This data demonstrates the importance of investigating the mechanism under water extract that affects the MCF-7 cell viability. Telomerase activity is one of the main hallmarks of cancer cells [26]. In most cancer types, telomerase reactivation causes immortality and malignant transformation of cancer cells [27]; therefore, it becomes a promising therapeutic target for novel anticancer drugs. Previous studies reported that Cordyceps militaris, a medicinal mushroom, caused a decrease in telomerase activity on A549 non–small cell lung cancer (NSCLC) cells with its water extract. Ganoderma lucidum is another mushroom that reduces the telomerase activity in MCF-7 cells [28]. Based on all the data, we performed a telomerase activity assay to determine the effects of H. erinaceus water extract on the telomerase activity of MCF-7 cells. However, we could not detect a significant inhibition of the telomerase activity of MCF-7 cells.
Together, these results have shown us that the cell viability inhibition effect of H. erinaceus water extract on MCF-7 cells cannot be explained through a decreased telomerase activity. To explain the mechanism underlying MCF-7 cell viability inhibition, the relationship with different metabolic pathways should be investigated.
Our results indicate that H. erinaceus water extract exhibited cell viability inhibition activity on the MCF-7 breast cancer cell line. However, it is understood that water extract inhibits the viability of MCF-7 cells through another molecular mechanism except for decreasing telomerase activity. Further in vitro and in vivo studies are needed to explain the mechanism underlying the effects of H. erinaceus on the regulation of telomerase activities in malign and non-malignant cells.
Funding source: Ege Ãœniversitesi
Award Identifier / Grant number: Project ID 1527
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Research funding: This study was funded by the Ege University under the Scientific Research Projects Coordination Unit (BAP) scientific support program with the project ID: 1527.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: Authors state no conflict of interest.
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Informed consent: Not applicable.
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Ethical approval: Not applicable.
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