An in vitro evaluation of the cytotoxic potential of medicinal mushrooms against human breast cancer cell lines

The aim of the present study was to demonstrate the effect of AB, CS, and IA in the treatment of human breast cancer cells (MCF- 7 and MDA-MB-231) and bone marrow stromal cells (HS-5) in vitro. MCF-7 cells have the tumor suppressor gene p53 as wild type, while MDA-MB-231 cells have p53 mutations (19). In addition, MCF-7 cells are estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptor positive and are classified as luminal type (low grade). MDA-MB-231 cells, on the other hand, show negative expression of ER, PR and HER2 [triple negative breast cancer (TNBC)] and are classified as basal type (high grade) (20, 21). Although TNBC accounts for only 15–20 % of all breast cancers, there are limited treatment options for this type of cancer as these cells do not respond to hormones or targeted therapies (22). Given the lack of validated molecular targets and the poor treatment outcomes in patients with TNBC, there is a clear need to develop better therapies. For this reason, it is of great importance to find agents that can act on these highly metastatic cells.

The results obtained in this study show that all three extracts reduce the viability of MDA-MB-231 cells. Figures 1, 2, and 3 show the effects of the concentration ranges (25, 50, 100, 200, 400, and 800 μg/mL) of AB, CS, and IA on the proliferation/viability of the MCF-7, MDA-MB-231, and HS-5 cell lines.

IA had the best effect on MDA-MB-231 cells with an IC₅₀=343.3 μg/mL (Figure 1). This corresponds to a 7 % higher efficacy than AB (IC₅₀=368.4 μg/mL) (Figure 2) and 78 % higher than CS (IC₅₀=613 μg/mL) (Figure 3). Treatment of the second breast cancer cell line (MCF-7) with IA for 72 hours resulted in some degree of cytotoxic effect, but the IC₅₀ was not reached at any of the concentrations tested. In addition, the cytotoxic effect of AB was demonstrated both in MDA-MB-231 and MCF-7 cells (IC₅₀ of 96.7 μg/mL for MCF-7). Moreover, CS reached its 50 % inhibitory concentration only in MDA-MB-231 cells. The most potent of the three compounds was AB, reaching the IC₅₀ in both cancer cell lines, with the treatment having the strongest overall effect on MCF-7 cells. Toxicity to healthy cells (HS-5) was significantly lower for all treatments and did not reach the IC₅₀ at any of the concentrations tested. These data are noteworthy as they indicate that the extracts are more toxic to cancer cells than to healthy cells.

Figure 1

Evaluation of the cytotoxic effects of Immune Assist on HS-5, MCF-7, and MDA-MB-231 cell lines using MTT assay measured after 72 hours treatment. Results are shown as mean±SEM. Statistical significance was assessed using Student’s t-test. *p<0.05,**p<0.005 vs. control

Figure 2

Evaluation of the cytotoxic effects of Agaricus blazei on HS-5, MCF-7, and MDA- MB-231 cells using MTT assay measured after 72 hours treatment. Results are shown as mean±SEM. Statistical significance was assessed using Student’s t-test. *p<0.05,**p<0.005 vs. control

Figure 3

Evaluation of the cytotoxic effects of Cordyceps sinensis on HS-5, MCF-7, and MDA- MB-231 cell lines using MTT assay measured after 72 hours treatment. Results are shown as mean±SEM. Statistical significance was assessed using Student’s t-test. *p<0.05,**p<0.005 vs. control

It has been documented that exposure of the MCF-7 and MDA-MB-231 cell lines to cordycepin (3-deoxyadenosine) from CS resulted in a dose-dependent inhibition of cell growth and reduced cell viability (23). Research into the pharmacological effects of mushroom extracts that are components of IA (A. blazei, C. sinensis, G. frondosa, G. lucidum, L. edodes, and T. versicolor) and their bioactive compounds in cancer treatment and prevention has already shown that they have suppressive activity against MCF-7 and MDA-MB-231 cells through various forms of cytotoxic effects. An aqueous extract from the fruiting body of Lentinula edodes showed significant dosedependent inhibitory effects on the proliferation of MCF-7 cells (24). The viability and cell migration of MDA-MB-231 cells were impaired by an extract from Ganoderma lucidum (25), while oral administration of the extract for one month decreased the expression of genes involved in the invasive behavior of MDA-MB-231 cells and prevented their migration (26). In addition, the ethanol extract of Trametes versicolor suppressed the proliferation of the MCF-7 and MDA-MB-231 breast tumor cell lines in a dosedependent manner (27). In turn, MycoPhyto^® Complex, a mixture of fungal mycelia from Cordyceps sinensis, Coriolus versicolor, Ganoderma lucidum, Grifola frondosa, Agaricus blazei and Polyporus umbellatus as well as β-1,3-glucan from yeast, suppressed the proliferation of MDA-MB-231 in a dose- (0–0.5 mg/mL) and time-dependent (24, 48, 72 h) manner and arrested the cells in the G2/M phase of the cell cycle (28). Moreover, treatment with polysaccharides from Grifola frondosa increased the accumulation of reactive oxygen species and induced mitochondrial dysfunction in MCF-7 and MDA-MB-231 breast cancer cells (29). Ganodermanontriol from G. lucidum inhibited the proliferation and colony formation of MDA-MB-231 cells (30).

A mushroom extract that is successful against cancer should be able to eliminate cancer cells while protecting healthy cells, such as HS-5, from excessive damage. Previously, latcripin-7A extracted from Lentinula edodes was shown to induce cell cycle arrest and promote autophagy, leading to apoptotic cell death in MCF-7 and MDA-MB-231 cells without affecting the survival of healthy breast MCF-10A cells (31). Our results are consistent with this and show that 72-hour incubation with different concentrations of AB, CS and IA (25–800 μg/mL) induces some degree of cytotoxicity in HS-5 cells, but does not reach the half-maximal inhibitory concentration. The MTT assay as a simplified 2D standard model for cytotoxicity provides a basis and insight into possible future investigations of the effects of the tested substances. Due to the method used, we cannot speculate on the mechanisms and reasons for the effects obtained based on our results. However, based on the above-mentioned in vitro and in vivo studies of medicinal mushrooms and their constituents, it is plausible to assume that the effect of some active ingredients in AB, CS and IA from groups of polysaccharides, polyphenols, sterols or catechols may influence the overall outcome. Studies have shown to some extent that the anticancer effects may be due to their antioxidant and free radical scavenging activity, inhibition of metabolic activation and enhancement of detoxification of carcinogens, direct cytotoxicity, antiproliferation and modulation of signal transduction molecules, and induction of cell cycle arrest and apoptosis (11, 12, 23, 28–31). This preliminary study provides evidence for further analysis of the effects of AB, CS and IA, their underlying mechanisms and the possibility of evaluating combined therapies with conventional treatments.

Our study has shown that AB, CS and IA can suppress the growth of highly metastatic MDA-MB-231 cells. AB proved to be the most potent and cytotoxic extract for MCF-7 and MDA-MB-231 cell lines. To our knowledge, this is the first study to show that traditional medicinal mushrooms can suppress the growth of human breast cancer cell lines in vitro, while affecting the survival of healthy cells to a much lesser extent. This study provides an initial structure for future research to determine the exact molecular mechanism responsible for the anticancer effect of AB, CS, and IA. In addition, AB, CS, and IA should be further investigated in vivo for use in combination with chemotherapeutic agents as adjuvants in breast cancer therapy.