The Effect of Ethanol Extract of Lingzhi Mushroom (Ganoderma lucidum) on Caspase-3 Expression in Oral Cancer Cells Irfan Dwiandhono1a, Fadli Ashar1b Arsa Hadiyatama Waskito Aji1c and Meta Anjay Firmansyah1d 1Department of Dental Medicine, Jenderal Soedirman University, Jl. Dr Soeparno, Purwokerto, Indonesia Keywords: Lingzhi mushroom, Ganoderma lucidum, caspase, KB CCL-17 cells. Abstract: Oral carcinoma is cancer found in the mouth, lips, palate, gingiva, mouth floor, and cheek mucosa. Oral carcinoma is a common cause of death in Indonesia. The development of cancer cells in the oral cavity is affected by the loss of caspase-3 expression. A treatment using lingzhi mushroom is known to increase caspase-3 expression in cancer. This study aimed to know about the effect of ethanol extract of Ganoderma Lucidum on caspase-3 expression in oral carcinoma KB CCL-17. The samples were oral carcinoma KB CCL- 17 cells with five treated groups 8.49μg/ml (P1), 4.24 μg/ml (P2), 2.2 μg/ml (P3), 11.55 μg/ml (cisplatin), and one control group (K). Caspase-3 expression was analyzed using the immunocytochemistry method by counting the percentage of caspase-3 expression cells. The data were statistically analyzed using One Way ANOVA and Post Hoc LSD. The results of caspase-3 expression on each groups were 5.21 % (P1), 2 % (P2), 0.96 % (P3), 9.67 % (cisplatin) and 0.41 % (K). Ethanol extract of Ganoderma Lucidum increased caspase-3 expression in KB CCL-17 cells along with the increase of the dosage. The dosage of 8.96 μg/ml showed a higher increase of caspase-3 expression than the dosages of 4.24 μg/ml and 2.12 μg/ml. An effect of ethanol extract of lingzhi mushroom on caspase-3 expression in oral carcinoma KB-CCL17. The study using lingzhi mushroom should be more developed to determine the anti-cancer effect through various pathways. 1 INTRODUCTION 2014). Cancer cells can develop because molecularly they have interference with cell death or apoptosis Cancer is one of the deadly diseases that provides program. One of the proteins that regulate the course 17.2 million cases worldwide and 8.9 million deaths of apoptosis in cells, namely Caspase-3, these in 2016. Cancer cases increased by 28% between proteins have become a target in cancer therapy 2006 and 2016 (Global burden diseases, 2016). development. The management of oral squamous cell Indonesia has a cancer prevalence that attacks all ages cancer generally consists of surgery, radiotherapy, of 4.1% or an estimated 347,792 people (Riskedas, chemotherapy, or a combination. However, the 2013). The importance of oral cancer was actions needed to overcome this malignancy have underscored in a recent publication on the burden of various shortcomings that can harm patients. cancer on member countries where oral cancer was Therefore alternative cancer cell treatments are the fifth most common cancer among ASEAN currently being attempted through various studies. member countries contributing to 50% of all new Many studies have been carried out using natural cancer cases (Cheong et a, 2018). .95% of cancer ingredients, all of that aim to produce medicines to cases in the oral cavity are oral squamous cell support the health care program. Also, the use of carcinomas which appear in the form of lumps, white natural ingredients used as medicine rarely causes or red ulcers that often attack the lips, lateral tongue, adverse side effects than medicine made from gum, palate, and floor of the mouth (Scully & Kirby, synthetic materials. One of the natural materials that a https://orcid.org/0000-0002-2968-9257 b https://orcid.org/0000-0002-0535-1382 c https://orcid.org/0000-0001-5168-9512 d https://orcid.org/0000-0002-8960-362X 52 Dwiandhono, I., Ashar, F., Waskito Aji, A. and Firmansyah, M. The Effect of Ethanol Extract of Lingzhi Mushroom (Ganoderma lucidum) on Caspase-3 Expression in Oral Cancer Cells. DOI: 10.5220/0010487600520057 In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientific Meeting (Temilnas) Consortium of Biomedical Science Indonesia (JIMC 2020), pages 52-57 ISBN: 978-989-758-499-2 Copyright c 2021 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved The Effect of Ethanol Extract of Lingzhi Mushroom (Ganoderma lucidum) on Caspase-3 Expression in Oral Cancer Cells have an anti-cancer effect is the Ganoderma Lucidum μl/ml was obtained. The test solution was then diluted fungus. once to obtain a concentration of 250 μl/ml, then used Research proved that the ethanol extract of in serial dilutions for treatment group to obtain a Ganoderma sp. mycelium Banyumas 1 isolate can be concentration of 500; 250; 125; 62.5; 31.25; 15,625 an anti-cancer in HeLa cervical cancer cells. and 7.8 μg/ml. Ganoderma sp. contains several bioactive compounds that can be used as medicine with anti-cancer 2.4 Culture Activation of Oral Cavity properties (Hidayati et al, 2014). These compounds Cancer Cell (Kb CCL17) include triterpenoids and polysaccharides (Kao et al, 2012). This study aims to determine ethanol extract Freezing is the most effective method of maintaining of lingzhi mushroom on apoptotic activity and a stable supply for various cell types for long term caspase-3 expression in Oral cavity cancer cells. storage (Miyamoto et al, 2018). The isolated cells were taken from the liquid nitrogen tank and diluted in a water bath with a temperature of 37°C for 12 2 MATERIALS AND METHODS hours and sprayed with 70% alcohol. Then the cells were put into a centrifuge tube containing 10 ml of 2.1 Ethical clearance DMEM-serum medium (DMEM was added with 10% FBS, Penicillin Streptomycin 3% and Fungizone Ensuring that the research is conducted in a 1%) in a laminar airflow room, then was centrifuged responsible and ethically accountable way leads to for 10 Minutes at a speed of 1200 rpm, Then the beneficial outcomes. The ethics committee approved supernatant was removed, and the sediment that was this research's ethical clearance, Faculty of Medicine, formed was added with DMEM-serum then left to Universitas Jenderal Soedirman with registered stand for 20 minutes. The cells were again centrifuged number 335/KEPK/VIII/2019. at 1200 rpm for 10 minutes, and the supernatant was removed, leaving 1 ml for resuspension. The cell 2.2 Preparation of Ganoderma suspension was inserted in a tissue culture flask (TCF) with a growth medium containing 20% FBS Lucidum Ethanol Extract and was observed under an inverted microscope. The living cells looked round, shiny and clear. TCF was Extraction of Ganoderma Lucidum fungi by incubated in an incubator at 37OC and 5% CO2 for maceration with 96% ethanol. Maceration involved 24 hours with the lid loosened. soaking plant and material in a stoppered container with a solvent and allowed to stand at room temperature for a minimum of 3 days with frequent 2.5 Culture Harvesting of Oral Cavity agitation (Azwanida, 2015). The fungus is thinly Cancer Cell (KB CCL-17) sliced and dried using an oven at 70OC for 2 hours, then mashed using a blender to become a powder. The Cells were taken from the CO2 incubator and powder was put in a 500 ml beaker and put into 96% harvested after 80% confluent using Trypsin-EDTA ethanol, the ratio between the powder and the solvent 0.25%. The media was discarded with a sterile was 1:5 then stirred and closed tightly with Pasteur pipette, and the cells were washed twice with aluminium foil. The soaked powder was left to stand PBS. Next, 50 µL of Trypsin-EDTA added as much for 3 x 24 hours. The filtrate obtained was put into a as 50 µL was added evenly over the cells, and then rotary evaporator at 50OC until a thick extract was the cells were incubated again for 2 minutes. Trypsin obtained and weighed. inactivation was carried out by adding 2-3 ml of DMEM-serum, trypsin is a serine protease, was 2.3 Preparation of Test Extract and applied to cells to separate them from each other and the underlying substratum so that they can be Control Solutions transferred to a different vessel for re-plating (Sharma et al, 2019) after then the cells were transferred into a For preparing the extract, ethanol was used as a sterile canal. The cells were counted on a solvent to obtain pharmacologically active hemocytometer. compounds from the mushroom (Kumar et al, 2018). Ethanol extract of 2 mg Ganoderma Lucidum body fungi was dissolved with 1 ml of DMEM containing 10 μl DMSO. Solution with a concentration of 500 53 JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientific Meeting (Temilnas) Consortium of Biomedical Science Indonesia (KIBI ) 2.6 Immunocytochemistry Assay cells in KB CCL-17 was the MTT Assay method. The results were read on 96 well-plates using an ELISA Immunostaining in the process of detecting specific reader to obtain data in the form of optical density. antigen-antibody interaction and an indirect method The absorbance results showed that the living cells using secondary antibody tagged with various labels could react to the reagent to create a colour change in such as enzyme is commonly used (Kim, 2016). Cells MTT. The results of the MTT Assay can be seen in were distributed into the chamber slide as much as Table 1. 100 μl with a density of 2 x 104 in each well and were incubated for 24 hours in a 5% CO2 incubator to Table 1: Inhibition percentages of KB CCl 17 adapted and stuck to the well.