Cytotoxicity of Lentinus isolates mycelial extracts on human colorectal carcinoma HCT-116 cells

Medicinal mushrooms are considered as potent natural sources for drug discovery and are of great interest in anticancer research worldwide. The cytotoxicity of rich macrofungal diversity in the Philippines remains to be largely unexplored. One of the macrofungal groups is Lentinus . Lentinus species (Polyporaceae, Basidiomycota) are wood-rotting mushrooms that naturally growing solitary or more often in groups on water-soaked logs, woods, and trunks of trees. They are important resources of functional food and bioactive metabolites. The present work evaluated the cytotoxicity of the ethanolic extracts of mycelia of the 15 Lentinus isolates against two cancer cell lines, human colorectal carcinoma (HCT-116) and hepatocellular carcinoma (HepG2), and one normal cell line, HK-2 normal kidney epithelial using the MTS proliferation assay. All Lentinus mycelial extracts showed concentration-dependent cytotoxicity against HCT-116. The IC 50 values of mushroom extracts ranged from 242.75 to 444.79 µg ml −1 for HCT-116 colon cancer cells. Among the mushroom extracts, Lentinus strigosus CL-01 extract was the most potent with the lowest IC 50 value, whereas Lentinus sajor-caju LSCBot extract registered the highest IC 50 value. On the other hand, extracts were not cytotoxic to HepG2 and HK-2 cell lines with less than 50% cytotoxicity indices. Therefore, the mycelia of 15 Lentinus isolates tested can be considered as potential sources of cytotoxic compounds for colon cancer. However, furtherance of the cytotoxicity and other bioactivity profiling of Lentinus mycelial extracts is highly recommended.


INTRODUCTION
Edible mushrooms are excellent source of nutritious and unique umami-taste food. They are rich in carbohydrates, proteins, fibers, vitamins, minerals, and low-fat content (Beelman et al., 2019;Martinez-Medina et al., 2021). They also have interesting content of minerals such as potassium, calcium, phosphorus, magnesium, iron, manganese, zinc, sodium, copper, and selenium (Murugesan, 2017;Painuli et al., 2020). Apart from the nutritional values, edible mushrooms have also been exploited for a very long time as natural alternative remedy for various diseases. The therapeutic values and medicinal properties of edible mushrooms have found to stem from numerous biologically active compounds or metabolites (Ho et al., 2020;Lu et al., 2020;Painuli et al., 2020). Mushrooms have a wide variety of bioactive compounds, which have been shown to exhibit several biological activities including antimicrobial, cytotoxic, anti-inflammatory, antioxidant antidiabetic, anti-dyslipidemia, anti-hypertension, anti-obesity, antitumor, hepatoprotective, immunomodulatory, and antiviral (Dicks and Ellinger, 2020;Kupcova et al., 2018;Lu et al., 2020;Wang, 2020).
Lentinus species, belong to Family Polyporaceae, are wild wood-rotting basidiomycetous mushrooms that grows solitary or in cluster on dead log and trunk of a tree. Their fruiting bodies have small to large, white to brown, convex at first to nearly flat, smooth to scaly, fleshy firm to tough pileus with sawtoothedge gills. The pileus is attached to centrally to eccentrically short, tough, scaly stipe. Their spore print is white to yellowish. Lentinus species are nutritious and medicinal mushroom. Lentinus tigrinus, for instance, contain carbohydrates, proteins, sugars, fiber, lipids and minerals, and show antioxidant, antibacterial and anti-hyperglycemic activities (Dulay et al., 2014(Dulay et al., , 2017. They are widely distributed in the different areas in the Philippines, mostly during the months of May to October (Arenas et al., 2015;De Leon et al., 2013;De Castro and Dulay, 2015;Dulay and Maglasang, 2017;Dulay et al., 2020). Recently, the fruiting bodies of different species of Lentinus were collected from the different areas of Luzon Island including Quezon Province, Camarines Sur, Ilocos Sur, Cagayan, Zambales, Tarlac and Nueva Ecija, and were successfully isolated through tissue culture. Mycelial biomasses of the different Lentinus isolates were mass produced using their optimal submerged culture conditions.

Mushroom culture
Pure cultures of the different isolates of Lentinus species were acquired from the culture collections of the Center for Tropical Mushroom Research and Development, and Tuklas Lunas Development Center, Department of Biological Sciences, College of Science, Central Luzon State University, Science City of Muñoz, Nueva Ecija, Philippines. The identity based on the morphological and molecular characteristics, culture code, and place of origin of the different Lentinus isolates are summarized in Table 1. Agar blocks of mycelia were sub-cultured on potato dextrose agar plates and incubated at 28°C for 7 days to allow mycelial growth. These cultures were used as source of inoculant for mass production of mycelial biomass in submerged culture.

Mass production of mycelia
Mass production of mycelia was carried out by inoculating mycelia discs into culture bottles containing 30 ml of the best medium with the optimum pH, and incubated at the required temperature, illumination, and agitation conditions. Thirty replicates of mycelial cultures for each mushroom were done. After 7 days of incubation, the mycelia were harvested, air-dried at air-conditioned room for 3 days, and prepared for extraction.

Preparation of crude extracts
The ethanol extraction protocols as described by Dulay et al. (2014) and Boukes et al. (2017) were followed with minor modifications. Five grams of each powdered mushroom mycelia were soaked in 200 ml of 95% ethanol for 2 days and then filtered using Whatman # 1 filter paper. The filtrates were concentrated using a rotary evaporator and freeze-dried. The extract yield was recorded. Extracts were dissolved to a working concentration of 2,000 µg ml −1 of the extract in a final nontoxic concentration of 0.2% dimethyl sulfoxide (DMSO) (Buhian et al., 2018;Lu et al., 2010;. These were vortexed for 30 minutes, centrifuged at 17,000 × g for 10 minutes, filter-sterilized using a 0.2 µM syringe filter and used for cytotoxicity test.

MTS proliferation assay
The cytotoxic effect of mushroom extracts was evaluated using CellTiter 96 ® Aqueous One Solution MTS Proliferation Assay Kit (Promega Co., Madison, WI). All cell lines at 90% confluence were harvested and the viable cell count was determined using trypan blue exclusion method. The cell density was adjusted to 4.5 × 10 4 viable cells ml −1 (Romero-Benavides et al.,2018). A 100 µl volume of viable cells was seeded into each well of a 96-well culture plate (Corning, NY), and subsequently incubated for 24 hours under 5% CO 2 , 95% humidity and 37°C conditions to allow attachment of cells and formation of monolayers. After incubation, 100 µl of each two-fold serial dilution of the prepared filter-sterilized working concentrations (2,000 µg ml −1 ) of mushroom extracts were added into each of the wells, resulting in final concentrations of 15.6, 31.25, 62.5, 125, 250, 500 and 1,000 µg ml −1 . The cytotoxic compound 5-flurouracil (5-FU) (Sigma-Aldrich Co., St. Louis, MO) was used as the positive control in concentrations of 15.6, 31.25, 62.5, 125, 250, 500 and 1,000 µg ml −1 , while media-DMSO dimethyl sulfoxide was used as a solvent vehicle control. Untreated wells served as the untreated negative controls. Each treatment was assayed in triplicate. Two assay trials were done in this sub-study. Assay plates were incubated under the same conditions. After 72 hours of treatment exposure, 10 µl of MTS tetrazolium solution (Promega Co., Madison, WI) were added and the set up was incubated for 30 minutes. The absorbance was measured at a wavelength of 492 nm using a microplate reader. Cytotoxicity indices were calculated using the equation, CI% = 100-{[(mean of treated absorbance values) / (mean of untreated absorbance values)] × 100}. Cytotoxicity graphs were constructed and the IC 50 values were derived via Fit Spline/LOWESS in GraphPad Prism version 9.2.0 for Windows (GraphPad Software, San Diego, CA, www. graphpad.com).

RESULTS AND DISCUSSION
Mushrooms are the store houses of new anticancer compounds (Chaitanya et al., 2019). In the Philippines, the anticancer properties of many wild mushrooms remain to be unexplored. For this reason, this current study evaluated the cytotoxic effects of ethanolic extracts of the mycelia of 15 Lentinus isolates that were mass produced in their respective optimized submerged culture conditions against HCT-116 colon cancer and HepG2 hepatocellular carcinoma cell lines, and on normal HK-2 cell line using the MTS proliferation assay. This assay detects the reduction of MTS tetrazolium compound (Owen's reagent) into a formazan product (yellow to violet) by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) or nicotinamide adenine dinucleotide hydrogen (NADH) through the activity of the mitochondrial dehydrogenase in metabolically active cells (Berridge and Tan, 1993). The cytotoxicity index values for each extract were calculated based on the absorbance readings and then plotted against the extract concentrations. The cytotoxicity index plots showing the concentration-dependent cytotoxicity of the mushroom extracts for HCT-116, wherein the cytotoxicity indices increased with increasing extract concentrations (Fig. 1). The 15 mushroom extracts were found to be not toxic to the hepatocellular carcinoma (HepG2) and normal kidney epithelial (HK-2) cells, which demonstrated less than 50% cytotoxicity indices (Figs. 2 and 3).
The IC 50 values of the mushroom extracts were determined using Fit Spline/LOWESS analysis of CI% values plotted against extract concentrations. Table 2 summarizes the IC 50 values of the mushroom extracts and 5-FU afforded by the three cell lines. For HCT-116 colon cancer cells, the IC 50 values of mushroom extracts ranged from 242.75 to 444.79 µg ml −1 . Among the mushroom extracts, CL-01 extract was the most potent with the lowest IC 50 value, whereas LSCBot extract registered the highest IC 50 value. Statistical analysis revealed that the obtained IC 50 values of mushroom extracts were significantly higher (p ˂ 0.01-0.0001) than that of the anticancer agent 5-FU (4.41 µg ml −1 ).
In the US National Cancer Institute plant screening program, a crude extract with an IC50 value ˂20 µg ml −1 after 48-72 hours exposure is generally considered to have in vitro cytotoxic activity (Boik, 2001;Kuete et al., 2013). However, for mushrooms, Boukes et al. (2017) established that crude mushroom extracts with IC 50 values of ˂100 and 100-200 µg ml −1 are considered highly and moderately cytotoxic, respectively. They also added that crude extracts with IC 50 values >100 µg ml−1 may cytotoxic compounds but at low concentrations. Accordingly, results of the present study suggest that all Lentinus extracts tested have cytotoxicity against HCT-116.
On the other hand, the IC 50 value could not be determined for all extract-treated HepG2 and HK-2 cells using the range of concentrations tested in the study. Hence, the IC 50 values were reported as >1,000 µg ml−1, which was the highest concentration tested. In contrast, 5-FU exhibited high cytotoxic activity to HepG2 and HK-2 cells with IC 50 values of 3.77 and 9.35 µg ml −1 , respectively. The vehicle control, 0.2% dimethyl sulfoxide (DMSO) in media, showed no cytotoxic activities against the three cell lines (data were not shown).
Accordingly, the degree of cytotoxicity of Lentinus species may vary depending on the type of cancer cell lines, extracting solvent used, and isolated bioactive compounds tested. Therefore, the present work cannot rule out the cytotoxicity of ethanolic extracts of the 15 Lentinus mycelia against other cancer cell lines, which were not tested in this study. The use of other extracting solvent and the isolation of bioactive components of Lentinus mycelia for cytotoxicity assessment merits further investigation.
Among the chemical components of mushroom, polysaccharides have been widely studied for several biological activities including anticancer properties (Wang, 2020). Lentinan, for instance, an isolated component of highly distilled polysaccharide from L. edodes (shiitake), has been commonly utilized as anticancer drug against lung, stomach, ovarian, liver and colon cancer in Japan (Uddin Pk et al., 2020). Additionally, lentinan has been used as adjuvant therapy for treating pancreatic, gastric, colorectal, cardiac, nasopharyngeal, lung, ovarian, and cervical cancers and for non-Hodgkin's lymphoma (Zhang et al., 2019). Aside from polysaccharides, the anticancer activity of wild medicinal mushrooms has been linked to the presence of phytochemicals, saponins, alkaloids, and phenolic compounds by interfering with particular signal transduction pathways, which play crucial role in the development and progression of cancer (De Silva et al., 2012;Lavi et al., 2012). Chemical composition analysis by silica gel chromatography and chemical structure analysis by nuclear magnetic resonance (NMR) spectroscopy of dichloromethane extract of the fruiting bodies of L. tigrinus yielded cerevisterol, stellasterol and ergosterol, which are reported to exhibit various bioactivities . Stellasterol was found to be one of the contributors of apoptosis and cell cycle arrest against the human cancer cell lines, MCF-7 and SH-SY5Y (Pereira et al., 2013).

CONCLUSION
In conclusion, the cytotocixity screening showed that ethanolic extracts of mycelia of 15 Lentinus isolates had cytotoxicity against HCT-116 human colorectal carcinoma and non-cytotoxicity to HepG2 hepatocellular carcinoma and HK-2 normal kidney epithelial cell lines. The cytotoxicity of Lentinus species and other macrofungal species in the Philippines must be further studied using different extracting solvents and cancer cell lines, including their underlying mechanisms of action.

ACKNOWLEDGMENT
The primary author would like to thank the Department of Science and Technology-Science Education Institute (DOST-SEI) in the Philippines for scholarship grant. This study was supported by the CLSU Tuklas Lunas Program funded by the Philippine Council for Health Research and Development, Department of Science and Technology. The authors would also like to express their sincerest gratitude to Ms. Dana Theresa De Leon and Mr. Rence Marrion Pineda for their technical assistance.