The Effect of Ethanol Extract of Lingzhi Mushroom (Ganoderma

lucidum) on Caspase-3 Expression in Oral Cancer Cells

Irfan Dwiandhono

, Fadli Ashar

Arsa Hadiyatama Waskito Aji

and Meta Anjay Firmansyah

Department 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

Cancer is one of the deadly diseases that provides

17.2 million cases worldwide and 8.9 million deaths

in 2016. Cancer cases increased by 28% between

2006 and 2016 (Global burden diseases, 2016).

Indonesia has a cancer prevalence that attacks all ages

of 4.1% or an estimated 347,792 people (Riskedas,

2013). The importance of oral cancer was

underscored in a recent publication on the burden of

cancer on member countries where oral cancer was

the fifth most common cancer among ASEAN

member countries contributing to 50% of all new

cancer cases (Cheong et a, 2018). .95% of cancer

cases in the oral cavity are oral squamous cell

carcinomas which appear in the form of lumps, white

or red ulcers that often attack the lips, lateral tongue,

gum, palate, and floor of the mouth (Scully & Kirby,

https://orcid.org/0000-0002-2968-9257

https://orcid.org/0000-0002-0535-1382

https://orcid.org/0000-0001-5168-9512

https://orcid.org/0000-0002-8960-362X

2014). Cancer cells can develop because molecularly

they have interference with cell death or apoptosis

program. One of the proteins that regulate the course

of apoptosis in cells, namely Caspase-3, these

proteins have become a target in cancer therapy

development. The management of oral squamous cell

cancer generally consists of surgery, radiotherapy,

chemotherapy, or a combination. However, the

actions needed to overcome this malignancy have

various shortcomings that can harm patients.

Therefore alternative cancer cell treatments are

currently being attempted through various studies.

Many studies have been carried out using natural

ingredients, all of that aim to produce medicines to

support the health care program. Also, the use of

natural ingredients used as medicine rarely causes

adverse side effects than medicine made from

synthetic materials. One of the natural materials that

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

have an anti-cancer effect is the Ganoderma Lucidum

fungus.

Research proved that the ethanol extract of

Ganoderma sp. mycelium Banyumas 1 isolate can be

an anti-cancer in HeLa cervical cancer cells.

Ganoderma sp. contains several bioactive compounds

that can be used as medicine with anti-cancer

properties (Hidayati et al, 2014). These compounds

include triterpenoids and polysaccharides (Kao et al,

2012). This study aims to determine ethanol extract

of lingzhi mushroom on apoptotic activity and

caspase-3 expression in Oral cavity cancer cells.

2 MATERIALS AND METHODS

2.1 Ethical clearance

Ensuring that the research is conducted in a

responsible and ethically accountable way leads to

beneficial outcomes. The ethics committee approved

this research's ethical clearance, Faculty of Medicine,

Universitas Jenderal Soedirman with registered

number 335/KEPK/VIII/2019.

2.2 Preparation of Ganoderma

Lucidum Ethanol Extract

Extraction of Ganoderma Lucidum fungi by

maceration with 96% ethanol. Maceration involved

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

agitation (Azwanida, 2015). The fungus is thinly

sliced and dried using an oven at 70OC for 2 hours,

then mashed using a blender to become a powder. The

powder was put in a 500 ml beaker and put into 96%

ethanol, the ratio between the powder and the solvent

was 1:5 then stirred and closed tightly with

aluminium foil. The soaked powder was left to stand

for 3 x 24 hours. The filtrate obtained was put into a

rotary evaporator at 50OC until a thick extract was

obtained and weighed.

2.3 Preparation of Test Extract and

Control Solutions

For preparing the extract, ethanol was used as a

solvent to obtain pharmacologically active

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

μl/ml was obtained. The test solution was then diluted

once to obtain a concentration of 250 μl/ml, then used

in serial dilutions for treatment group to obtain a

concentration of 500; 250; 125; 62.5; 31.25; 15,625

and 7.8 μg/ml.

2.4 Culture Activation of Oral Cavity

Cancer Cell (Kb CCL17)

Freezing is the most effective method of maintaining

a stable supply for various cell types for long term

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

hours and sprayed with 70% alcohol. Then the cells

were put into a centrifuge tube containing 10 ml of

DMEM-serum medium (DMEM was added with

10% FBS, Penicillin Streptomycin 3% and Fungizone

1%) in a laminar airflow room, then was centrifuged

for 10 Minutes at a speed of 1200 rpm, Then the

supernatant was removed, and the sediment that was

formed was added with DMEM-serum then left to

stand for 20 minutes. The cells were again centrifuged

at 1200 rpm for 10 minutes, and the supernatant was

removed, leaving 1 ml for resuspension. The cell

suspension was inserted in a tissue culture flask

(TCF) with a growth medium containing 20% FBS

and was observed under an inverted microscope. The

living cells looked round, shiny and clear. TCF was

incubated in an incubator at 37OC and 5% CO2 for

24 hours with the lid loosened.

2.5 Culture Harvesting of Oral Cavity

Cancer Cell (KB CCL-17)

Cells were taken from the CO2 incubator and

harvested after 80% confluent using Trypsin-EDTA

0.25%. The media was discarded with a sterile

Pasteur pipette, and the cells were washed twice with

PBS. Next, 50 µL of Trypsin-EDTA added as much

as 50 µL was added evenly over the cells, and then

the cells were incubated again for 2 minutes. Trypsin

inactivation was carried out by adding 2-3 ml of

DMEM-serum, trypsin is a serine protease, was

applied to cells to separate them from each other and

the underlying substratum so that they can be

transferred to a different vessel for re-plating (Sharma

et al, 2019) after then the cells were transferred into a

sterile canal. The cells were counted on a

hemocytometer.

The Effect of Ethanol Extract of Lingzhi Mushroom (Ganoderma lucidum) on Caspase-3 Expression in Oral Cancer Cells

2.6 Immunocytochemistry Assay

Immunostaining in the process of detecting specific

antigen-antibody interaction and an indirect method

using secondary antibody tagged with various labels

such as enzyme is commonly used (Kim, 2016). Cells

were distributed into the chamber slide as much as

100 μl with a density of 2 x 104 in each well and were

incubated for 24 hours in a 5% CO2 incubator to

adapted and stuck to the well. Each well then was

added with 100 μl of the test extract solution with a

concentration of 500; 250; 125; 62.5; 31.25; 15,625

and 7.8 μg/ml. then was incubated again for 24 hours.

Control used to control media in the form of a mixture

of 100 μl of culture medium with 100 μl of cell

suspension and 100 μl of DMSO. The preparation

was then soaked in a peroxidase blocking solution at

room temperature for 10 minutes. The preparations

were incubated in the prediluted blocking serum at 25

O C for 10 minutes. Then it was soaked in 25OC anti-

caspase 3 (NCL-CPP32p) monoclonal antibody for

10 minutes. The preparations were washed with

phosphate-buffered saline (PBS) for 5 minutes.

Incubation of preparations with secondary antibody

(biotin-avidin)

At 25 OC for 10 minutes. Furthermore, the

preparations were washed with PBS for 5 minutes.

Furthermore, the preparations were incubated with

peroxidase at 25°C for 10 minutes. Then the

preparations were washed with PBS for 5 minutes.

The preparations were incubated again with

chromogen Diaminobenzinidine (DAB) at 25°C for

10 minutes and with Hematoxylin Eosin for 3

minutes. The preparations were washed, cleaned, and

dripped with mounting media (Canada balsam) and

terminated by closure with a coverslip under the

running water. The preparations were observed under

a light microscope at 200x magnification. Positive

protein expression results were stained with a

brownish nucleus and cytoplasm, and cells without

protein expression were stained violet-blue. The

active p53 protein was in the cell nucleus, while the

Bax and caspase 3 proteins were in the cytoplasm

(Prokhorva et al, 2018). The count of stained cells

was expressed as a percentage.

3 RESULTS

This research began with a cytotoxic test of the

ethanol extract of the Ganoderma Lucidum fungi,

which will be used as the treatment group (TG) and

cisplatin as the positive control group (PCG). The

method used in the toxicity test of oral cavity cancer

cells in KB CCL-17 was the MTT Assay method. The

results were read on 96 well-plates using an ELISA

reader to obtain data in the form of optical density.

The absorbance results showed that the living cells

could react to the reagent to create a colour change in

MTT. The results of the MTT Assay can be seen in

Table 1.

Table 1: Inhibition percentages of KB CCl 17

No Groups

Concentrations

(µg/ml)

Cell

Inhibition

rate

1 TG1 1.95 53.94%

2 TG2 3.9 59.21%

3 TG3 7.8 63.08%

4 TG4 15.62 64.13%

5 TG5 31.35 67.42%

6 TG6 62.5 71.52%

7 TG7 125 77.49%

8 TG8 250 86.52%

9 TG9 500 92.38%

10 TG10 1000 89.45%

11 PCG1 1.56 8.89%

12 PCG2 3.125 18.26%

13 PCG3 6.25 24.09%

14 PCG4 12.5 58.41%

15 PCG5 12.5 84.4%

16 PCG6 50 94.33%

17 PCG7 100 95.11%

18 PCG8 200 70.85%

Table 1 presented the data as percentage

inhibition of cells on every Ganoderma Lucidum

fungi ethanol extract (TG) concentration and

Cisplatin (PCG). The data used to calculate the IC50

value. The IC50 value was a compound parameter

with cytostatic properties that inhibits cancer cells'

growth by 50% were obtained from the probit

analysis of the percentage of cells inhibition using the

Probit Table so that the IC50 value was obtained. The

IC50 value of the Ganoderma Lucidum fungi ethanol

extract (TG) obtained at 8.49 µg/ml and cisplatin at

IC50= 11.55 µg/ml (PCG). However, IC50 of

Ganoderma Lucidum extract on oral cancer cells from

recent studies by Syairah et al (2017) was 310 µg/ml,

on HL60, K562, and SGC-7901 cells were 440 µg/ml,

390 µg/ml and 900 µg/ml (Chen, 2016). Different

IC50 in every study has commonly occurred,

although the MTT-dependent IC50 errors analyzed in

this study were focused on the system consisting of

Ganoderma Lucidum extract, cisplatin, and oral

cancer (KB CCL-17), the obtained knowledge

concerning the reasons for the inconsistency in IC50

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 )

values is of practical importance for many other

chemotherapeutic agents and cancer systems. Indeed,

regardless of the agents or type of cancer cell lines

involved, the uneven proliferation of the control cells

at different seeding densities variations will yield

systemic errors in IC50 measurements because all of

the MTT analogue assays rely on the OD reads from

the control cells for the IC50 calculations (Haris et al,

2016; Hafner et al 2016).

Each treatment's IC50 value becomes the standard

for determining the concentration dose for the

Caspase-3 expression test. The treatment group of

ethanol extract of lingzhi mushrooms on the

expression of caspase-3 in oral cavity cancer cells

were three concentrations below the IC50, namely 8.

49, 4.24, and 2.12 µg/ml while the cisplatin group

was one concentration IC50, namely 11.55 µg/ml.

The results of the data on the expression of

caspase-3 can be seen in Figure 1

Figure.1. Caspase 3 expression on each group

The expression of caspase-3 in Figure 1 was

brown, while normal cells are blue. The caspase-3

expression was calculated by dividing the number of

positive cells by the number of all cells and

multiplying by 100 per cent with Image J software

analysis to obtain the average caspase-3 percentage.

The research data was carried out normality test

using the Shapiro-Wilk test in this study was not

generally distributed of 0.00 (p<0.05), therefore the

data was transformed with log10 so that a significant

result was obtained of 0.19 so that the data was

normally distributed (p> 0.05). The homogeneity test

was carried out. A significant value of 0.56 (p> 0.05)

was obtained. The data's variance was homogeneous

and could be continued to the parametric test—the

One Way ANOVA parametric test was carried out.

The results of the One Way ANOVA test, the

expression of caspase-3 on KB CCL-17 cells between

the ethanol extract treatment groups of lingzhi

mushroom (G. Lucidum) concentrations of 8.49, 4.24

and 2.12 μg/ml with the cisplatin group and control

had high significant differences with a p-value of

0.000 (p<0.01). Furthermore, the post hoc Least

Significance Difference (LSD) test was carried out,

aiming to determine the difference in the average

percentage of the caspase-3 expression in each group.

The Post Hoc LSD test results showed a highly

significant difference in the percentage of caspase-3

expression in each treatment group with the control

group. The difference in each group was highly

significant because of the significance value was

<0.01.

4 DISCUSSION

The apoptotic process that arises in cells is mediated

by a molecule called caspase. Caspase is a molecule

that functions to carry out apoptosis in cells. Caspase

can be divided into initiation groups and execution

groups. Caspase 3 is an example of the caspase

execution group. Caspase is activated in the extrinsic

(death ligand) and intrinsic (mitochondrial) cell

pathways (Mc Arthur & Kile, 2018). The zymogen

form caspase 3 is necessary because if it is not

regulated, caspase activity will kill all cells. In this

study, the expression of caspase-3 was strongly

expressed in the cell cytoplasm by showing a

brownish colour. Strongly expressed in the cell was

supported by the research that the apoptosis process

in KB cells is likely to be induced via extrinsic

pathways through caspase-3 activation in the cell

cytoplasm (Hutomo et al, 2014).

The apoptotic activity and expression of caspase-

3 in this study frequently increased with the addition

of the ethanol extract concentration of lingzhi

mushrooms related to the content.

Of lingzhi mushrooms which have an anti-cancer

effect. Lingzhi mushroom (G. Lucidum) contains

triterpenoids, polysaccharides, and ganoderic acids

known to cause DNA damage effects on cancer cells

to trigger apoptotic signals cells that are exposed to

lingzhi mushroom extract (Wu et al, 2013; Gurovic et

al, 2016). The content of polysaccharides in fungi

plays a role in decreasing the mitochondrial

membrane's permeability (Tian et al, 2016). This

decrease causes cytochrome c to exit the

mitochondria into the cytoplasm (Kole et al, 2011).

Cytochrome c in the cytoplasm then binds to Apaf-1

which can activate procaspase 9 to become caspase 9.

Caspase 9 will activate procaspase 3 to become

caspase-3 which acts as an effector in carrying out

apoptosis in cells (Ponde et al, 2019).

Caspase-3 can enter the nucleus through the pores

that have been made by caspase-9, removing the

substrate that causes DNA degradation. In the

nucleus, there are skeleton components in the form of

The Effect of Ethanol Extract of Lingzhi Mushroom (Ganoderma lucidum) on Caspase-3 Expression in Oral Cancer Cells

lamin A and fodrin. The breakdown of lamin by

caspase-3 will cause chromatin condensation, while

the breakdown of fodrin triggers the formation of

apoptotic bodies (Ponde et al, 2019; Zhao et al, 2020).

The chemotherapy agents' administration using

cisplatin in this study obtained the highest average

expression of caspase-3, of 9.7. Chemotherapy is

mostly via an apoptotic mechanism that involves

many proteins and genes. The most important

proteins are p53 and caspase-3 (Moningka, 2019).

5 CONCLUSIONS

The use of herbal materials as anti-cancer therapy

aimed at reducing the destructive effects of using

chemotherapy agents. Lingzhi mushrooms as herbal

plants are known to suppress cancer growth through

the apoptotic mechanism. Further research needed

regarding the effect of ethanol extract of lingzhi

mushroom in inducing various apoptotic molecules.

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The Effect of Ethanol Extract of Lingzhi Mushroom (Ganoderma lucidum) on Caspase-3 Expression in Oral Cancer Cells