Nigrospora Sphaerica Products from the Flowering Dogwood Exhibit Antitumorigenic Effects Via the Translational Regulator, Ps6 Ribosomal Protein

Asha Maheshwari^1,2, Margaret Mmbaga¹ & Quincy Quick, A.^2*

¹Department of Agricultural and Environmental Sciences, Tennessee State University, Nashville, TN, USA
²Department of Biological Sciences, Tennessee State University, Nashville, TN, USA

*Correspondence to: Dr. Quincy Quick, A., Department of Biological Sciences, Tennessee State University, 3500 John A. Merritt Blvd, Nashville, TN, 37209 USA.

Copyright © 2018 Dr. Dr. Quincy Quick, A., et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background
Endophytic fungi are organisms that colonize the internal tissues of plants without damaging the host [1], and have proven to be a robust source for the discovery of novel secondary metabolites that exhibit a wide range of biological and biomedical applications [2-4]. A candidate source and potential host of endophytic fungi with biomedical applications is the flowering dogwood, Cornus florida L., which has been shown to produce antiparasitic compounds for the treatment of malaria [5]. Germane to the present study C. florida has also been shown by Vareed et al., [6] to produce the phenolic pigment anthocyanin, which displayed antitumor activity in colon, breast, lung, stomach, and central nervous system cancers. To this end the current study examined the antitumor activity of crude extracts prepared from the endophytic fungus Nigrospora sphaerica isolated from C. florida, which has previously been described as an endophyte resource for several biomedical applicable compounds such as antibiotics [7-8], antiviral anthraquinones and azaphilones [9], and the antifungal griseofulvin [10]. Of specific significance the alkaloid chemotherapeutic drug, vinblastine, is also a product of the endophytic fungus N. sphaerica albeit isolated from Catharanthus roseus (Madagascar periwinkle) [11]. However, the clinical caveat with the utility of the microtubule poison vinblastine for the treatment of human cancers has been its associated toxicities, making it imperative to identify sources for chemotherapeutic agents with improved toxicity profiles. Here we examined the antiproliferative effect of crude extracts from N. sphaerica on lung and glioblastoma cancer cell lines, as well as the ability of N. sphaerica extracts to inhibit cancer cell migration. The results of this study provide experimental evidence that the endophytic fungus N. sphaerica in flowering dogwoods is a promising source of bioactive metabolites with potential antitumor and antimetastasis activities.

Methods
Sample Collection and Fungal Isolation
Healthy stem samples were collected from mature C. florida plants at Otis L. Floyd Nursery Research Centre (Tennessee State University, McMinnville, TN). The samples were placed in sterile bags, stored at 4^°C and processed within 24 hours of sampling. Endophytic fungi were isolated using methods as previously described by Schulz et al., [12] with some modification. Stem samples were cleaned thoroughly under running tap water and cut into 1.0-1.5cm pieces. Samples were next surface sterilized with 70% ethanol for 1 minute and 10% sodium hypochlorite for 3-5 minutes. Samples were subsequently rinsed three times with sterile distilled water, blotted on paper towel and allow to air dry. Next five segments of vascular tissue (3-4mm) were transferred onto petri dishes containing acidified potato dextrose agar, incubated at room temperature and observed for appearance of mycelial growth. Pure fungus was obtained by at least two or three successive sub-culturing on potato dextrose agar (PDA; Sigma-Aldrich, St. Louis, MO).

Molecular Identification and Phylogenetic Analysis
Fungal genomic DNA was extracted using the FastDNA kit (MP Biomedicals, Santa Ana, CA) per manufacturers’ instructions. Next PCR of the Internal Transcribed Spacer (ITS) region was performed using the universal primers ITS1 and ITS4 [13]. Subsequently, the PCR product was purified using Exosap (USB-Affimetrix, Santa Clara, CA), and sequenced by Eurofin Genomics (Louisville, KY). Next the ITS sequence was analyzed using BLASTN (GenBank National Centre of Biotechnology Information-NCBI; https://www.ncbi.nlm.nih.gov) and the identity of the organism was determined based on the closest match in the GenBank database. DNA sequence similarities of ≥ 99-100% homology were used to identify the fungus N. sphaerica studied here. Subsequently, the N. sphaerica ITS sequence and reference sequences retrieved from GenBank were aligned using ClustalW prior to phylogenetic tree construction in MEGA7 software [14]. The neighbor-joining (NJ) method was used to infer evolutionary history [15]; the p-distance method was used to compute evolutionary distances [16]; and bootstrap analysis with 1000 replication was used to assess the robustness of the phylogenetic tree [17].

Crude Extract Preparation
For crude extract preparation 5-7, 6mm² agar plugs of fungal culture were inoculated in 1000ml erlenmeyer flasks containing 300ml of potato dextrose broth (Sigma Aldrich, St Louis, MO; USA). Flask were then incubated at 28^°C on a rotary shaker at 160rpm for 10 days. After incubation, the fermented medium was centrifuged at 4000rpm to separate mycelia from the supernatant. Subsequently, supernatant was filter sterilized (0.22μm) and extracted three times with equal volumes of ethyl acetate (Fisher Scientific, Fair Lawn, NJ; USA) using separatory funnel. Next the organic phase was removed using rotary evaporator under vacuum and resultant crude extract was lyophilized. The stock solution of 2.0mg/ml crude extract was prepared in dimethyl sulfoxide (DMSO; Amresco Solon, OH; USA) and stored at -20^°C for further analysis.

Cells Culture Conditions and Reagents
U251 human glioblastoma cells were purchased from Sigma-Aldrich (St. Louis, MO; USA) and A549 human lung carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA; USA). Both cell lines were maintained in Dulbecco’s Modified Eagles Medium-DMEM (Invitrogen, Carlsbad, CA; USA) supplemented with 10% Fetal Bovine Serum (Invitrogen, Carlsbad, CA; USA), 2mM L-glutamine (Invitrogen, Carlsbad, CA; USA), 100nM MEM non-essential amino acids (Invitrogen, Carlsbad, CA; USA), and penicillin-streptomycin (Invitrogen, Carlsbad, CA; USA) at 37^oC and 5% CO₂.

Crystal Violet Cell Proliferation Assay
U251 and A549 cells were plated in 24 well plates and treated with 2μg/ml, 4μg/ml, 8μg/ml, and 10μg/ ml of fungal crude extract, while vehicle treated control cells were treated with DMSO. For dose response experiments cells were treated with N. sphaerica (2μg/ml, 4μg/ml, 8μg/ml, and 10μg/ml) fungal extract or vehicle and allowed to incubate for 48h, while for time course experiment cells were plated, treated as explained above and incubated for 2, 4 and 6 days. At the end of each time point the tissue culture medium was removed, the cell monolayer was fixed with 100% metha¬nol for 5 minutes and stained with 0.5% crystal violet in 25% methanol for 10 minutes. Cells were then washed three times five minutes each with distilled water to remove excess dye and allowed to dry overnight at room temperature. The incorporated dye was then solubilized in 0.1M sodium citrate (Sigma-Aldrich St. Louis, MO; USA) in 50% ethanol. Next, 100μl of experimental samples were transferred to 96 well plates and optical densities were recorded at 595 nm using an X-mark microplate absorbance spectrophotometer (BioRad, Hercules, CA; USA).

Cell Migration
Motility assays were conducted according to manufacturer’s instructions (CellBiolabs Inc, San Diego, CA; USA). Cell suspensions containing 0.5-1.0 x 10⁶ cells/ml were treated with DMSO (vehicle control) or 2μg/ml of crude extract in serum free media, while 500μl of media containing 10% fetal bovine serum was added to the lower chamber of the migration plate. Next 300μl of cell suspension containing vehicle control or 2μg/ml of crude extract were then added to the inside of each insert and allowed to incubate for 24 hours at 37^°C and 5% CO₂. Subsequently non-migratory cells were removed from plate inserts (per manufacturer’s instructions), while migratory cells were stained with crystal violet and the dye solubilized (per manufacturer’s instructions). Optical densities were read at 595nm using an X-mark microplate absorbance spectrophotometer (BioRad, Hercules, CA-USA).

Caspase 3 Activity
To assess caspase 3 activity cells were plated in serum-free DMEM for 24h, treated with 2μg/ml, 4μg/ml, 8μg/ml crude extract, or vehicle (DMSO) and allowed to incubate for 24h. After incubation, cells were rinsed with cold phosphate buffer saline (PBS; Bio-Rad Laboratories, Hercules, CA, USA) and lysed in CelLytic M cell lysis reagent (Sigma-Aldrich, St Louis, MO, USA). Protein concentrations were then determined using the Bradford method. Next, caspase 3 activity was determined using the CaspACE Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Western Blotting
For western blot experiments cells wells were treated with 2μg/ml, 4μg/ml, 8μg/ml crude extract or vehicle (DMSO) for three hours, rinsed with PBS, and lysed with CelLytic M cell lysis reagent (Sigma-Aldrich, St Louis, MO; USA). Protein concentrations were subsequently determined using the Bradford method. Proteins were next run in 4-15% SDS- polyacrylamide gels (BioRad, Hercules, CA; USA) and transferred to nitrocellulose membranes. Nitrocellulose membranes were then incubated overnight at 4^°C with primary antibodies to detect the protein expression of pAkt, pS6 ribosomal protein, p90 RSK, 4EBP1, GAPDH, and β actin (CellSignaling Technology, Danvers, MA; USA). Nitrocellulose membranes were next washed three times 5 minutes each in PBS containing 0.05% Tween 20, incubated with an HRP-conjugated secondary antibody for 3h at room temperature and washed. Proteins were visualized and analyzed by enhanced chemiluminescence (Thermo Scientific, Nashville, TN; USA) and a UVP BioSpectrum imaging system (UVP, Upland, CA; USA).

Statistical Analysis
Cell proliferation, migration, and time-course experiments were each performed at least three times in duplicate. Means were determined by averaging duplicate samples within each independent experiment. Students t-tests and ANOVA with Sidak’s posthoc statistical analysis were used to evaluate significance.

Result
The endophyte N. sphaerica has been previously reported in a number of plant species [11, 18 - 20]. To our knowledge this study is the first report to show the association of N. sphaerica with C. florida as confirmed using ITS-rDNA sequence and phylogenetic analysis (Figure 1).

Additionally, few studies have evaluated the anticancer properties of secondary metabolites from N. sphaerica. Initial experimental studies on the effect of the endophytic fungus N. sphaerica on human cancer cells was determined via dose response analysis on lung cancer and glioblastoma cell lines, which displayed a concentration dependent effect (Figures 2 and 3). Glioblastoma cells treated with 2μg/ml of N. sphaerica crude extract showed a 25% decrease in cell viability and an 84% decrease in cell viability when treated with 10μg/ml, while lung cancer cells treated with these concentrations displayed a 17% and 62% decrease, respectively, as compared to vehicle treated control cells (Figure 2). The quantitative effect of this Nigrospora fungus on lung and glioblastoma cancer cell viability was further supported by ANOVA analysis which showed a statistically significant difference (p < 0.05) across all concentrations examined. Subsequently, time course analysis were performed on lung and glioblastoma cancer cells exposed to crude extracts of N. sphaerica.

Consistent with dose response experiments cells treated with N. sphaerica crude extracts ranging in concentration between 2μg/ml and 10μg/ml also displayed concentration dependent effects of crude extracts on glioblastoma and lung cancer cells, with the highest doses having the most pronounced effect on decreasing cell viability (Figure 4). This is supported by a 93% and 96% decrease in viable lung cancer and glioblastoma cells, respectively, observed 6 days’ post-exposure with 10μg/ml of N. sphaerica crude extract, as compared to vehicle treated control cells examined at this same time point (Figure 4). Additionally, treatment with 10μg/ml of N. sphaerica induced a cytostatic cellular response in lung cancer cells and a cytotoxic cellular response in glioblastoma cells over the six-day time period (Figure 4). These data are consistent with the antiproliferative effects of N. sphaerica products observed in leukemia and breast cancer cells [11, 21].

To gain insight into the role that apoptotic cell death played in the reductions in cancer cell viability as a consequence of exposure to N. sphaerica crude extracts, caspase 3, a known executioner of apoptosis, was evaluated in lung cancer and glioblastoma cells (Figure 5) post-exposure to crude extracts. Our data showed a diminutive increase in caspase 3 activity in both cell lines treated with N. sphaerica crude extracts (Figure 5). Additionally, we assessed the alternative cell death mechanism autophagy, as a means that underlie reduced cell viability of lung and glioblastoma cells treated with N. sphaerica crude extract. Immunoblotting data showed no change in the autophagy marker LC3 in response to N. sphaerica crude extract exposure (data not shown). We further analyzed the expression of proteins that regulate translational control (pAkt, pS6 ribosomal protein, 4EBP1) and ERK (p90RSK) signaling pathways as mechanistic contributors of the anti-proliferative effect of N. sphaerica crude extract on solid tumor cell lines studied here. N. sphaerica crude extract exposure caused a demonstrative decrease in the expression of the translational regulator pS6 ribosomal protein in lung cancer cells (Figure 6) but no measurable change in the ERK signaling protein p90RSK. In contrast N. sphaerica crude extract had no effect on translational control and ERK signaling proteins evaluated here in glioblastoma cells (data not shown).

Because tumor recurrence is a manifestation of the resistance of many cancers to clinically used chemotherapeutic agents, we next examined the efficacy of N. sphaerica crude extracts to inhibit lung cancer and glioblastoma cell migration. Boyden chamber assays revealed that cells treated with 2μg/ml of N. sphaerica crude extracts decreased (p < 0.05) tumor cell migration as compared to vehicle treated control cells (Figure 7), paralleling recent findings by Mady et al., [22] that showed the endophytic fungus Penicillium chrysogenum impaired breast cancer cell migration.

Discussion
We have shown here that metabolic products of the endophytic fungus N. sphaerica has antitumorigenic properties as exhibited by the antiproliferative effects of N. sphaerica crude extracts from C. florida on lung cancer and glioblastoma cells. Our findings are consistent with previous experimental studies that have also shown secondary metabolites produced by N. sphaerica isolated from C. roseus and Asteraceae inhibited the proliferation of breast cancer and leukemia cells, respectively. More specifically Ayob et al., [11] demonstrated that the vinca alkaloid, vinblastine produced by N. sphaerica from C. roseus inhibited breast cancer cell growth. It should be mentioned that the concentration however of vinblastine used to inhibit breast cancer cells by Ayob et al., [11] was three times that of the highest concentration of N. sphaerica crude extract used in this study to inhibit the growth of solid cancer cell lines. This suggest that cancer cells may be more sensitive to N. sphaerica products extracted from C. florida and that endophytic fungi products from this plant are more effective at inhibiting cancer cell proliferation. Additionally, Gallo et al., [21] showed that diterpene aphidicolin, a N. sphaerica product of Asteraceae had an antiproliferative effect on leukemia cells that was attributed in part to G2/M cell cycle arrest, apoptosis, and autophagy. In contrast N. sphaerica crude extracts in our study did not induce apoptotic cell death or an autophagic response, but instead promoted a decrease in the translational regulator, pS6 ribosomal protein an effector molecule in the PI3K/Akt/mTOR signaling pathway regulated by mTORC1 (Raptor-mTOR) a known regulator of cell growth, glucose and lipid metabolism, autophagy, and protein synthesis [23]. Although the change in pS6 ribosomal protein expression provides a mechanistic basis for the antiproliferative effects of lung cancer and glioblastoma cells seen in response to N. sphaerica crude extracts, the downregulation of this translational regulatory protein has also been associated with impeding cancer cell migration. In a study by Kim et al., [24] it was demonstrated that genetic downregulation of pS6 ribosomal protein inhibited esophageal cancer cell migration and invasion. This was further supported in studies by Chen et al., [25] who observed this same phenomenon in non-small cell lung cancer. Taken together these investigations support the notion that impairment of lung cancer and glioblastoma cell migration to N. sphaerica crude extracts isolated from C. florida is also a consequence of pS6 ribosomal protein reduction.

Conclusion
The anti-migratory effects in conjunction with the antiproliferative properties of N. sphaerica crude extracts from C. florida on solid tumor cell lines observed in our study provide evidence that endophytic fungi isolated from this plant is able to impede the continued development and recurrence of human cancers. Furthermore, findings here expand the pool of botanical resources that can be used to evaluate secondary metabolites from endophytic fungi for their anticancer properties to include the flowering dogwood, C. florida.

Acknowledgements
This work was partly supported by United States Department of Agriculture, National Institute of Food and Agriculture: Award Number 2010-38821-21477. The authors declare that they have no competing interests.

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