Effects of Grifola frondosa Secretions on Tumorigenic and Non-tumorigenic Mammary Epithelial Cells
MacKenzie Thomas
Oral Robert University
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MacKenzie Thomas is currently a student at Oral Roberts University, from which he will complete his Bachelor of Science in May 2023. He is a Medical Molecular Biology major and is a Fellows Honors Scholar. He looks forward to starting medical school in the Fall of 2023.
Panpsychism: A Beautiful Circumvention
Abstract
With cancer being the second deadliest killer in the world, trailing closely behind ischemic heart disease, it is no mystery why novel treatment options are sought after. It is estimated that by the year 2060, cancer will become the leading cause of death, further increasing the urgency to find new cures. While there are many different types of cancer, breast cancer is the second most prevalent and the deadliest malignancy among women. Common therapeutic strategies to combat this include surgery, radiotherapy, and chemotherapy.
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Unfortunately, these methods lack substantial specificity to target malignant growths and preserve healthy tissues. Mushrooms have long been used for medicinal purposes, and more recently, they have been studied for cancer suppression. Grifola frondosa (G. frondosa), also known as maitake, is a Basidiomycetes fungus that provides many benefits, including antidiabetic, antioxidant, anti-inflammation, hepatoprotective, antiviral, and antitumor properties. Several studies have shown that G. frondosa’s bioactive molecules, such as polysaccharides and polypeptides, have the significant immunomodulatory effects of activating and upregulating many immune-related cells such as macrophages, natural killer cells, and cytotoxic T-cells. Also, G. frondosa biomolecules increase the release of cytokines which promote apoptosis and have antiproliferative activity. Thus, the compounds that maitake produces are a potential avenue for cancer research. Full spectrum G. frondosa secretions, as well as separated fractions, were tested on both MDA-MB-468 triple-negative mammary epithelia and MCF10A human breast epithelial cell lines. Cell viability assays were performed, yielding results that suggest G. frondosa secretions contain compounds that significantly impair breast cancer cells.
Brief Look at Breast Cancer
Unfortunately, cancer has impacted most households. The disease has become the second most prevalent cause of death worldwide, trailing closely behind the top killer, ischemic heart disease. In 2016 alone, 8.97 million cancer-related deaths were recorded worldwide (Mattiuzzi and Lippi 2019). Lung, breast, and prostate cancers are the most common, and since breast cancer mostly affects women, its prevalence is quite high in the female population. It is estimated that 13% of females 15-49 years of age have breast cancer, and this neoplasm is the second leading cause of death among women (Mattiuzzi and Lippi 2019). While this malignancy is broadly categorized, varying forms of the disease have different implications.
Four main subtypes of breast cancer are differentiated based on hormone receptor (HR) status and immunohistochemical properties. There are HR+/HER2+, HR-/HER2+, HR+/HER2-, and HR-/HER2- tumors. HR-/HER2- cancer is the most aggressive and has high metastatic potential and is referred to as triple-negative breast cancer (TNBC). TNBC can pose the most significant threat to patients since these cancers lack hormone receptors and HER2 expression. Thus, no targeted treatment to halt the progression of TNBC is available (Saha et al. 2021).
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Human epidermal growth factor receptors (HER) are transmembrane receptors that respond to signals from the extracellular environment (Moasser 2007). HER2 receptors are common in breast cells and control cell growth, division, and repair (BreastCancer.org 2022). Given its role in integral steps of the cell cycle, it is clear why HER2 is an oncogene. In order to be activated, the HER2 must associate with another HER family protein since no ligand binds to it (Gutierrez and Schiff 2011). The intracellular domain of HER2 has tyrosine kinase activity which, when activated by dimerization, initiates several secondary messenger pathways to influence many genes’ expression. The HER2 protein can be dysregulated either by mutation, transcriptional deregulation, or gene amplification, which can lead to constitutive activation of the pathways associated with this protein (Moasser 2007). While there is variation between breast cancers based on cell surface receptors, there are sustained features of how they develop.
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Researchers have outlined ten hallmarks of all breast cancer, regardless of subtype. When these features are combined, they pose a deadly threat to the body. The first hallmark of breast cancer is the ability to sustain proliferative signaling outside of the normal parameters. Usually, this involves the deregulation of growth signals that control the cell cycle, causing the cell to grow at an increased pace. For example, HER2 gene amplification or overexpression is found in 20-30% of all breast cancers, which confers cell growth and proliferation, increasing the odds of a poor clinical outcome (Saha et al. 2021). Underlying factors of signaling dysregulation are mutation and genomic instability, which comprise another hallmark. For there to be abnormalities in the cellular pathways, the cancer cell must have a high mutation rate, which usually occurs by disrupting genomic maintenance and repair machinery. Some of the most common breast cancer mutations occur within the BRCA1 and BRCA2 genes (Saha et al. 2021). BRCA, meaning “breast cancer susceptibility” genes, are involved with double-stranded DNA break repair (Saha et al. 2021). Thus, if there happened to be a mutation in either of the two, there would be a lack of DNA damage repair, enabling the cell’s genome to undergo further damage.
Cancer cells also develop the ability to resist cell death. Usually, when there are damages to the cellular processes that are beyond repair, pro-apoptotic proteins recognize this and initiate a self-destruct. However, these rogue cells lack this fail-safe, often having dysregulation in the proteins involved (Saha et al. 2021). Thus, the cell is able to persevere and multiply. Neoplastic cells also enable replicative immortality by manipulating telomeres. Telomeres are ends of chromosomes that generally shorten with each cell division, acting as a biological clock and contributing to aging. All adenocarcinomas express telomerase, which is an enzyme that prevents telomere shortening (Holysz et al. 2013). With the active telomerase, cancer cells evade the natural aging process, which immortalizes them and enables continual division and metastasis.
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For a tumor to gain mass, the individual cells must grow at an abnormally rapid pace while evading growth suppressors. Dysregulation of the cell cycle leads to uncontrolled growth, which is commonly seen with TP53 mutations. TP53 is a gene that encodes the p53 protein, which regulates functions such as gene expression, metabolism, and apoptosis (Saha et al. 2021). These qualities make p53 a key regulator of certain checkpoints that determine if the cell is healthy and able to proceed in proliferation. Thus, TP53 knockout mutations allow the cell to continually divide despite massive damage to the cellular machinery. In order to fuel this swift growth and division, energy metabolism must be reprogrammed. Commonly, cancerous cells shift from aerobic mitochondrial metabolism to glycolytic energy production (Saha et al. 2021). Increase in glycolysis enables the rogue cell to produce energy even while lacking an oxygen supply. However, as the tumor expands and metastasizes, it will require more nutrients and gases. Angiogenesis is another hallmark of breast cancer, which is the formation of blood vessels within the tumor (Saha et al. 2021). By generating a vascular system, the tumor can utilize oxygen from the blood in aerobic metabolism as well as expel waste back into the bloodstream. Vascular endothelial growth factor (VEGF) typically drives angiogenesis and is seen to be active within breast tumors (Saha et al. 2021).
Interestingly, researchers have discovered that inflammation is a hallmark of breast cancer. It was originally thought that this immune response was the body’s attempt to eradicate the growth. However, recent discoveries show that inflammation encourages tumorigenesis by contributing growth factors and reactive oxygen species that can damage DNA (Saha et al. 2021). While the immune system can aid the development of cancer by encouraging genetic mutations, the tumor must evade destruction. There are several ways neoblasts circumvent eradication by the immune response, one being the release of immunosuppressive factors (Saha et al. 2021). The tumor either must mask its presence or limit the immunological response to survive, which aids the final hallmark of breast cancer, the ability to invade and metastasize. The leading cause of death in breast cancer patients is the initial tumor’s ability to spread into other tissues in the body (Saha et al. 2021). Once the primary tumor metastasizes into the secondary tumor, there are greater difficulties in combating the disease. Metastasis is a common stage of breast cancer, and hopefully, future treatments will target this stage to prevent unfavorable outcomes.
Current Treatments for Breast Cancer
Treatment options vary depending on what subtype of breast cancer a patient has and how far the malignancy has progressed. Stage I breast cancer is the earliest stage when the tumor is small and has not spread anywhere else, whereas Stage IV is metastatic, meaning that it has spread to other sites distant from the primary tumor (Moo et al. 2018). While each patient presents a unique scenario, breast-conserving therapy (BCT) and mastectomy are common procedures to help remove the disease. However, aggressive breast cancers, like TNBC, generally are not treated with these procedures since the local recurrence rates are high (Moo et al. 2021). BCT involves the surgical removal of the tumor, followed by radiation therapy on the whole breast (Moo et al. 2021). By excising solely the growth, the majority of the breast is conserved, and the radiation kills any remaining neoplastic cells missed during the surgery. On the other hand, mastectomy removes the entire mammary gland. The skin-sparing method allows for a flap that can be used in reconstructive surgery if the patient requests and it is deemed safe (Moo et al. 2018). For advanced breast cancers, mastectomy will be paired with radiation therapy to help prevent the survival of malignancies. Certain breast cancers, such as the HER2+ subtypes, can be biologically specific.
Therapies targeting HER2 receptors are generally coupled with chemotherapy. Stage I HER2+ cancer is treated with paclitaxel, a chemotherapy agent, and trastuzumab, a HER2 targeting drug (Moo et al. 2018). Combining broad and narrow spectrum treatments can produce favorable outcomes for the patient. More developed tumors, such as stage II and III HER2+, are treated with trastuzumab and a combination of docetaxel and carboplatin, which are chemotherapy agents (Moo et al. 2018). While trastuzumab offers selectivity, recent advances have improved its specificity. Pertuzumab, a monoclonal antibody that inhibits the dimerization of HER2, has been added to trastuzumab to increase its specificity for HER2+ cancer cells and prevent their proliferation (Moo et al. 2018).
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Endocrine therapy is useful for breast cancers with hormone receptors (HR+) but can present a more rigorous process. Patients needing this type of treatment are administered endocrine therapy for 5-10 years (Moo et al. 2018). Given that hormones are manipulated, many areas of the body may be affected during such treatments. Tamoxifen is one type of drug used in this process, which is coupled with aromatase inhibitors in post-menopausal women. Aromatase inhibitors diminish estrogen production, which, when used in conjunction with Tamoxifen, can lead to unpleasant side effects, such as arthralgia, vaginal dryness, hot flashes, and myalgia (Moo et al. 2018). Tamoxifen also can lead to musculoskeletal problems and increases the likelihood of uterine cancers and veinous blood clots (Moo et al. 2018). Unfortunately, not all breast cancers offer the opportunity for selective targeting with current treatment options. TNBC lacks the HER2 and hormone receptors, leaving these high-risk patients with the option of chemotherapy (Moo et al. 2018).
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A recent discovery in plant biology has led to the development of Taxol, which is a widely used anticancer drug for pancreatic, ovarian, breast, and other cancers. Taxol is synthesized in the phloem tissue of the bark in Pacific Yew trees (Talbot 2015). The drug is used by the tree as an immune defense against pathogens. Given the tree’s long lifespan of up to 3000 years, Taxol and other defense mechanisms must be very effective. Interestingly, endocytic fungi that colonize within the Yew tree are also able to produce the anticancer agent (Talbot 2015).
Taxol inhibits microtubule breakdown, which proves exceedingly effective since a cell cannot properly replicate without microtubule function (Talbot 2015). Microtubules are necessary for the separation of sister chromatids during mitosis, as well as cell division in cytokinesis. By disrupting these processes, Taxol prevents the continual proliferation of cancerous cells. The discovery of this drug shows that plants, and the fungi that inhabit them, have incredible potential in the treatment of cancer.
Grifola frondosa Overview
Mushrooms have long been treasured by various cultures for their great taste and medicinal qualities. More than 2000 species of fungi are found in nature, yet only about 25 are used commercially (Valverde et al. 2015). There are many types of poisonous fungi, and consumption of these will lead to detrimental health problems. However, those that are non-toxic offer excellent nutritional value. Mushrooms generally have all the essential amino acids, a high protein content, a healthy fatty acid profile, and vitamins while also being low calorie (Valverde et al. 2015). Due to their chemical profile, mushrooms are a great fix for the metabolic needs of a vegetarian diet.
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Grifola frondosa, also known as “hen of the woods” and “maitake,” is a Basidiomycetes fungus that offers a wide range of benefits and tastes delicious. Throughout Japan’s history, naturally occurring locations of maitake were sacredly guarded, referred to as “treasure islands,” which were only revealed to an heir upon the owner’s death (Rogers 2011). Hen of the woods, which can weigh up to 22 pounds and resembles a small hen covered with leaves, is found mainly in Eastern North America and Asia under trees (Rogers 2011). Since they can be relatively difficult to locate, several methods of cultivation have been developed.
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Several alternative mediums can facilitate G. frondosa growth, but fungi have shown partiality to coarse materials. Sawdust, wheat straw, millet, and rye are excellent substrates for growing via three general methods: bottle culture, bag culture, or outdoor bed culture (Wu et al. 2021). Bottle and bag methods allow for controlled humidity for the growing fungus, while the outdoor bed culture simulates a natural growing environment. Alternatively, mycelia, the vegetative part of the fungus resembling thin filaments, can be grown on agar plates for a more controlled setting (García-García et al. 2014). As the mycelia grow and expand, it digests the substrate and releases byproducts into the environment, which can be collected using the proper methods. In one method, a layer of sterile autoclaved deionized Millipore water is added on top of the fungus, which allows the compounds secreted from the mycelia to be suspended in the liquid. Thus, by collecting the suspension, the secretions are also gathered.
For centuries, Grifola frondosa has been used in traditional medicine across Asia, but it was not until recently that the benefits were examined. Lately, research has shown that G. frondosa confers antiviral, antidiabetic, anti-inflammatory, and anti-tumor effects while also having an immunomodulatory role (Wu et al. 2021). In 1983, a paper was published that outlined a maitake extract, now known as the D-fraction, that exhibits anti-tumor effects (Ohno et al. 1984). This extract is a β-glucan complex that is 30% protein by weight and has garnered more attention for its interleukin-1-stimulating effect (Rogers 2011). Recently, this D-fraction was used in addition to chemotherapy agents, which yielded results that indicate the treatment was effective against breast, liver, and lung cancers (Wu et al. 2021).
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Several polysaccharide fractions that have been elucidated from maitake show anti- cancer and immune-enhancing effects. Compounds from G. frondosa have exerted anti-tumor effects by enhancing the immune system, which protects healthy cells, prevents metastasis, and inhibits tumor growth (Wu et al. 2021). Maitake extractions have been shown to inhibit 86% of cancer growth, which is likely due to it increasing the activity of macrophages, cytotoxic T-cells, N-killer cells, and macrophages (Rogers 2011). Currently, most studies focus on the polysaccharides that the mushroom produces, but several bioactive proteins and peptides have been isolated and further research is required.
Given that Grifola frondosa has been used medicinally for centuries, it is an excellent avenue for clinical research. The mushroom has specifically been shown to contain bioactive molecules that improve the outcomes of breast cancer, such as the D-fraction. Since both proteins and sugars from maitake possess anti-tumor capabilities, it is reasonable to test G. frondosa secretions on TNBC, which is the most aggressive form of the disease and lacks a direct treatment method. The purpose of the experiments outlined in this report is to see the potential of maitake as a selective breast cancer treatment. It is hypothesized that G. frondosa secretions will not reduce TNBC viability but have significant differences in effect when compared to non-tumorigenic breast epithelia.
Methods
Two experiments were conducted to assess TNBC viability after exposure to G. frondosa secretions. In the first experiment, a 96-well plate was seeded with MDA-MB-468 cells (Table 1). Cancer cells were administered varying amounts of G. frondosa secretion, as well as water to serve as a control. After incubation with the Abcam colorimetric indicator solution, the plate was read in the Bio-Rad iMark Microplate Reader, which measured the absorbance at 570 nm and 605 nm. The absorbance ratios of treatment and control groups were compared to determine cell viability and secretion efficacy (Figure 1). Experiment 2 followed a similar approach as experiment 1. However, both MDA-MB-468 and MCF10A cells were treated to compare the secretion’s cytotoxicity on the cancerous cell line and healthy breast epithelial cells.
Results: Experiment Set 1
In experiment set 1, a 96-well plate was prepared (Table 1). Every well labeled as “veh” contained only autoclaved deionized Millipore water as the additive instead of mushroom secretions. Each well labeled with “G. Frons” was administered the specified volume of drug (G. frondosa secretion). Adding water instead of drug to the MDA-MB-468 cells offers a control to compare the cell viability between treatment and control groups. Also, no cell controls were used, labeled as “ncc.” These wells provided a cell-free background reading to compare absorbance values to when calculating average percent cell viability.
Rows C and D provide no cell controls, or background readings, which lack cells but contain water or the drug. Each well labeled “468” was seeded with 20,000 MDA-MB-468 cells in 100 μL. Rows with “1:100” were administered a diluted drug sample, being one hundredth of the original concentration of drug. The same was done for wells labeled as “1:10,” except the drug was diluted to a tenth of the original G. frondosa secretion sample. The cells were treated with either the drug or water in varying volumes in a series of five technical replicates. Rows A and B were administered 10 μL of the respective diluted drug, whereas the other rows were given 2 μL, 4 μL, 6 μL, or 8 μL of drug or water, as specified by Table 1. The concentration of the drug in the stock G. frondosa secretion sample was determined to be 0.724 μg/μL via the Bio-Rad NGC Chromatography System. Thus, the “1:100” wells were treated with 0.00724 μg of drug, the “1:10” wells with 0.0724 μg of drug, the 2 μL wells having 1.448 μg of drug, the 4 μL wells having 2.896 μg of drug, the 6 μL wells having 4.344 μg of drug, and the 8 μL wells were administered 5.792 μg of drug.
After treatment, the absorbance of each well was measured at 570 nm and 605 nm.
Measuring the absorbance of the cells at 570 nm and 605 nm gives a reading of the metabolic output of the cells. Thus, it is inferred that cells less metabolically active are less viable. These data were then used to find the ratio of the optical density at 570 nm (OD570) to the optical density at 605 nm (OD605). These ratios (Table 2) are used to calculate cell viability via the following formula:
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For each experimental group, these calculations are recorded in Table 3.
The results in Table 3 indicate that TNBC cells exhibit less viability with increasing amounts of G. frondosa secretion. It is interesting to note how in small quantities, like the 1:100 and 1:10 diluted secretions, the breast cancer cells experienced growth, having viabilities above
100. However, when the amount of drug increased, viability decreased. Standard deviation was calculated by comparing replicate viabilities. These findings are represented by Figure 1, constructed using the values in Table 3.
Figure 1: MDA-MB-468 cells were treated with varying amounts of G. frondosa secretions or a water control. After incubating with either the drug or control treatment, the cell viabilities were calculated by comparing the control samples’ absorbances to the drug samples’ absorbances at 570 nm and 605 nm. The error bars represent the standard deviation of viability between replicates.\
Increasing the volume of G. frondosa secretion, and thus increasing the micrograms of drug, correlates with a decrease in cell survivability (Figure 1). While the viability between the replicates treated with the drug was compared, the differences between the control group and the drug group were also analyzed. A two-tailed, two sample T-test was performed between the drug treatment and control treatment (Table 3). Significance was established if the p-value is less than 0.05. Drug and control groups showed significant differences (Figure 2).
igure 2: The comparison of 570 nm/605 nm absorbance between the control treatment group and the drug treatment group is depicted above. The 96 well plate (Table 1) was treated with either water or the drug. After incubation, the Bio-Rad iMark Microplate Reader was used to measure the absorbance change at 570 nm and 605 nm. The Control bars reflect the cell population’s absorbance change of Replicate Groups that were treated with water instead of the drug. The bar lines represent standard deviation of the samples, while the asterisk indicates significance (p < 0.05), determined by a two-tailed T-test between the Control and Drug groups.
There is a statistically significant difference between the effects of water and the effects of the drug on MDA-MB-468 cell populations when the amount of G. frondosa drug is 1.448 μg (2 μL) or greater (Figure 2). The secretions can reduce the cancer cells’ ability to survive. The data from Experiment 1 depict that maitake has potential as a cancer-killer, leading to another experiment that compared the drug’s effects on TNBC cells and non-tumorigenic breast epithelial cells.
Experiment 2
In the second set of experiments, two 96-well cytotoxicity assays were performed, one using MCF10A cells and the other using MDA-MB-468 cells. However, before the assays were performed, the drug was characterized via fast protein column chromatography (FPLC) to examine the protein and peptide composition of the G. frondosa secretion (Figure 3).
Figure 3: Characterization of the proteins and peptides within G. frondosa secretion via FPLC. The maitake secretion was run through the Bio-Rad NGC Chromatography System to generate its protein profile. The secretion was fractioned and separated into 26 individual test tubes. The columns labeled A/1 – A/26 represent which test tube the fraction was collected. The graph lines represent the level of absorption the sample had at 215 nm (top), 280 nm (middle), and 490 nm (bottom).
Fractions A/13 – A/16 appear to contain the majority of peptides and proteins (Figure 3). Thus, the fractions were divided into three separate groups. The tubes A/3 – A/12 were grouped together to form the “3-12” drug, samples A/13 – A/16 were combined to form the “13-16” drug, and A/17 – A/26 were combined to yield the “17-26” drug sample.
The 96-well plate seeded with MCF10A cells was prepared following the layout of Table 4. The type of drug varied between replicates, since the wells were treated with the either the “3- 12,” “13-16,” or “17-26” drug fraction (Table 4). The complete secretion was tested as well, referred to as “full.” Each treatment group had two replicate non-cell control wells which provided background readings used in calculating cell viability, labeled as “ncc” (Table 4). 10 μL of either drug or water was added to each well, as outlined by Table 4.
After incubating the treated plate and the addition of Abcam assay solution, the absorbance of each well at 570 nm and 605 nm was recorded. Then, these values were used to create the ratio of OD570/OD605 which indicates cell viability (Table 5).
Values were generated (Table 5) by dividing the read absorbance at 570 nm by the absorbance at 605 nm. From these data, cell viability for each treatment group was calculated by the following formula:
Thus, the ability of each fraction, as well as the full secretion, to kill MCF10A cells was calculated. For each drug type (3-12, 13-16, 17-26, Full), calculations were made (Table 6).
The 3-12 G. frondosa fractions (Fraction 1) showed to have an insignificant effect on MCF10A viability, having a p-value of 0.55 when comparing the control group to the experimental group. MCF10A cells treated with Fraction 1 experienced little change compared to the group treated with the 13-16 fractions (Fraction 2). MCF10A cells showed 56.22% viability when treated with the secretion’s main peptides and proteins. The cells treated with the full secretion experienced the greatest death, having a viability of 0.46%. Dissimilarly, the cells treated with the 17-26 fractions (Fraction 3) showed a viability above 100%, likely meaning that the cell population grew slightly. However, this change in cell viability is insignificant with a p- value of 0.2. Significance between control and drug groups were calculated using a two-tailed T- test, whose p-values are listed (Table 6). Calculations from Table 6 have been visualized (Figure 4), and a direct comparison between each sample’s control group and drug treatment group was made (Figure 5).
Figure 4: MCF10A cells were treated with differing fractions of G. frondosa secretions. After incubation with the drug, the 96 well plate (Table 4) was read by the Bio-Rad iMark Microplate Reader. The absorbance values for drug and control treatments were averaged and compared. Average viability was determined by comparing the drug treatment’s absorbance to its respective control treatment absorbances. Averages of MCF10A viability for each treatment group – cells treated with Fractions 1, 2, 3, and the full secretion – is compared. The error bars represent standard deviation between the replicate group’s averaged viabilities
Figure 5: The comparison of 570 nm/605 nm absorbance (OD570/OD605) between the control treatment group and the drug treatment group is depicted above. The 96 well plate (Table 4) was treated with either water or the drug. After incubation, the Bio-Rad iMark Microplate Reader was used to measure the absorbance change at 570 nm and 605 nm for each replicate. The Control bars reflect the cell population’s average absorbance change of replicate wells that were treated with water instead of the drug. The bar lines represent standard deviation of the samples, while the asterisk indicates significance (p < 0.05), determined by a two-tailed T-test between the Control and Drug groups.
The same methods used in determining the cytotoxicity of G. frondosa fractions were used on MDA-MB-468 cells. The 96 well plate for the TNBC was prepared the same as the MCF10A plate with a similar layout (Table 7).
Similar to the MCF10A 96-well plate, each treatment group was done with five replicates to normalize data. After the plate was treated and incubated, the absorbances of each well at 570 nm and 605 nm were determined via the Bio-Rad iMark Microplate Reader and the ratio of OD570/OD605 was calculated to determine cell viability (Table 8).
Table 8 shows the 96-well plate (Table 7) with the ratio of OD570/OD605 in each well. Using these values, the viability of MDA-MB-468 cells can be calculated for each treatment group using the following formula:
The result of these calculations is seen in the “Viability” column (Table 9).
The MDA-MB-468 cell population experienced insignificant growth when treated with the 3-12 fractions of G. frondosa, having a viability of 103.18%. However, Fraction 2, which contains the main peptides and proteins, had a significant effect on the cells, yielding a 4.78% viability. Likewise, the full secretion also significantly killed the cells, which had a 1.68% viability. Fraction 3 had a significant effect on the cells when compared to the control group, having a 97.07% viability with a p-value of 0.042. Thus, while it was not as drastic as the change seen in the “Full G. frond” group, the difference between control and drug treatment still significantly changed the cell population’s health. A graphical representation of MDA-MB-468 sample viabilities were made (Figure 6).
Figure 6: MDA-MB-468 cells were treated with differing fractions of G. frondosa secretions. After incubation with the drug, the 96 well plate (Table 7) was read by the Bio- Rad iMark Microplate Reader. The absorbance values for drug and control treatments were averaged and compared. Average viability was determined by comparing the drug treatment’s absorbance to its respective control treatment absorbances. Averages of MDA- MB-468 viability for each treatment group – cells treated with Fractions 1, 2, 3, and the full secretion – is compared. The error bars represent standard deviation between the replicate group’s averaged viabilities.
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​Distinct difference in potency between the different types of drug administered is apparent (Figure 6). The cells treated with Fraction 2 or the full G. frondosa secretion are significantly less viable than those treated with Fractions 1 and 3. A comparison between replicates’ Drug and Control groups was made (Figure 7).
Figure 7: The comparison of 570 nm/605 nm absorbance (OD570/OD605) between the control treatment group and the drug treatment group is depicted above. The 96 well plate (Table 7) was treated with either water or the drug. After incubation, the Bio-Rad iMark Microplate Reader was used to measure the absorbance change at 570 nm and 605 nm for each replicate. The Control bars reflect the cell population’s average absorbance change of replicate wells that were treated with water instead of the drug. The bar lines represent standard deviation of the samples, while the asterisk indicates significance (p < 0.05), determined by a two-tailed T-test between the Control and Drug groups
Figure 8: The effects of G. frondosa were compared between MCF10A and MDA-MB-468 cell lines. Both cultures were exposed to Fractions 1 – 3 and the Full Secretion. After drug treatment and incubation, the absorbance changes of each cell type were recorded. Average viability was determined by comparing the drug treatment’s absorbance to its respective control treatment absorbances. The error bars represent standard deviation between replicates, while the asterisks indicate significance with a p-value < 0.05. Two-tailed T-tests were performed between the cell type for each treatment group.
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In order to elucidate the differences between MCF10A and MDA-MB-468 cell types that have been treated with G. frondosa, the two datasets were compared (Figure 8). A two-tailed T- test was calculated for each treatment group to compare viability between cell types. Treating both MCF10A and MDA-MB-468 cells with Fraction 1 led to insignificant differences in cell viability with a p-value of 0.956. However, it is evident that the two cell lines reacted differently to Fraction 2, having significantly different average percent cell viability with a p-value of 1.64E-08. When treated with Fraction 2, TNBC exhibited less viability than the non-tumorigenic cell line. Also, the MCF10A cells had significantly higher viability than the MDA-MB-468 cells when treated with the Fraction 3, with a p-value of 3.92E-05. Finally, both cell types reacted similarly when treated with the full G. frondosa secretion, having a p-value of 0.274.
Discussion
Experiment sets 1 and 2 have interesting implications, as the mushroom secretion significantly lowers cancer cell viability. Experiment set 1 revealed that G. frondosa secretion has the potential to kill tumorigenic mammary epithelia. Increasing the amount of drug given to the cells led to greater MDA-MB-468 population death (Figure 1). Unfortunately, the dataset had some shortcomings. When looking at the ratio of 570 nm and 605 nm absorbances between replicate wells, the 4 μL treatment group had a large standard deviation of 12.91 (Table 2, Table 3). Well F10 had an uncharacteristically low viability compared to the other replicates in row F, thus skewing the standard deviation. The lower OD570/OD605 ratio could have been due to a number of reasons, the most likely being pipetting error or accidental agitation of the cells.
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Regardless, the difference in viability between the control and drug groups still was significant with a p-value of 1.04E-04. The 2 μL treatment group was the first to show a considerable lowering of cell viability (Figure 2). Only 1.448 μg of the drug was needed to decrease the cell population, which shows the potency of this particular mushroom secretion. While G. frondosa showed potential for killing TNBC, its effect on healthy breast cells was yet to be determined. Ultimately, a drug that kills both healthy and cancerous cells would not be advantageous to the patient. Thus, experiment set 2 helped elucidate the drug’s effect on MCF10A cells, which represent healthy breast epithelial cells.
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Experiment set 2 had two main goals: to compare the effects of G. frondosa on two different cell lines and to investigate the cytotoxicity of the secretion’s components. Figure 3 shows the FPLC of the G. frondosa secretion. Based on the protein and peptide profile, the tubes containing A/13 – A/16 were collected as Fraction 2. This separation allowed for a comparison of the secretion constituents’ effects.
Looking at the MCF10A data (Figures 4 and 8), the full G. frondosa secretion dramatically decreased cell viability. The MDA-MB-468 cells responded similarly, and the difference between cell type populations was not significant. Thus, the effect of these secretions was equivalent between cell populations. However, by fractioning the secretions, we were able to achieve differential cell viability. Fraction 2 treatment showed substantial differences in cell viability, leaving only 4.78% of the MDA-MB-468 population viable and 56.22% of the MCF10A cells viable. Treatment of the MCF10A cells with Fractions 1 and 3 yielded insignificant changes (Figure 5), showing that the proteins and peptides of the secretion are the key contributors to its cytotoxicity.
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Since the full secretion lowered viability more than Fraction 2, it is likely that a combination of compounds within the drug, which were separated by the chromatography machine, causes its potency against mammary breast epithelia. When considering G. frondosa’s effects on normal breast cells, more experiments will need to be done to elucidate the toxic compounds from the harmless or beneficial compounds. It would be interesting to see how partitioning Fraction 2 into smaller fractions would affect MCF10A and MDA-MB-468 cells.
Also, varying the concentration of Fraction 2 could yield a higher average percent viability of MCF10A cells. In the experiment, MCF10A cells were administered 10 μL of drug. According to the FPLC data, Fraction 2 had a concentration of 6.424 mg/mL. Thus, 64.24 μg of Fraction 2’s constituents resulted in 56.22% MCF10A cell viability. Future experiments should focus on diluting the concentration to achieve greater non-tumorigenic cell viability, which would also increase MDA-MB-468 cell viability. Thus, an optimal concentration should be established which maintains the difference in cell type viability while minimizing the damage to healthy cells.
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In these experiments, G. frondosa secretions appear to cause significant differences between TNBC and non-tumorigenic cell viability, even in small concentrations. While these results are exciting, there should be more experiments to reproduce and validate these findings. Also, further analysis of the secretion’s fractions should be done to elucidate the proteins, peptides, sugars, and other compounds that affect cell viability. Based on experiment sets 1 and 2, maitake looks to be a promising avenue for future treatment options for TNBC.
Literature Cited
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