





































Novel wild edible mushroom Astraeus hygrometricus (Pers.) Morgan induces robust apoptosis on human acute lymphoblastic leukemia cells through a RONS-subsisted mitochondria-dependent pathway


 

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    Abstract— The purpose of this study is to investigate the potential medicinal benefits of the Astraeus 

hygrometricus (Pers.) Morgan, a newly discovered wild edible fungus, on cells that have developed acute 

lymphoblastic leukaemia in humans.Methods: Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 

bromide (MTT) assay, a battery of tests was conducted to determine the antiproliferative and chemopreventive 

potential of various extracts from five wild mushrooms: A. hygrometricus, Phallus sp., Lentinus sp., Tricholoma 

sp., and Serpula sp. The results were evaluated against a panel of six cancer cell lines and normal cells. The 

following were examined using flow cytometry: cell cycle profiling, apoptosis determination, intracellular 

reactive oxygen and nitrogen species (ROS and RNS), and mitochondrial membrane potential. We analysed the 

expression pattern of mitochondrial proteins and used colorimetric methods to quantify caspase activity. A. 

hygrometricus methanol extract showed the most antiproliferative effect, whereas MOLT-4 cells were the most 

sensitive, according to the results. Mushroom extract effectively halted cell cycle development at the G0/G1 

stage and produced strong selective apoptosis in MOLT-4 cells. In MOLT-4 cells, the extract increased ROS 

generation and altered the potential of the mitochondrial membrane. By activating the caspase cascade, 

downregulating Bcl-2 expression, and boosting Bax expression, the methanol extract triggered apoptosis. In 

conclusion, the new edible wild mushroom may have biomolecules that may be used in the creation of 

antileukemic medications. 

 

 

 
 
 
 
 

Novel wild edible mushroom Astraeus hygrometricus (Pers.) Morgan 

induces robust apoptosis on human acute lymphoblastic leukemia 

cells through a RONS-subsisted mitochondria-dependent pathway 

Pal a, Ribhu a, Chouni a, Subhadwip Hajra b, Santanuna Paul a, * 

a Department of Botany, University of Calcutta, Kolkata 700019, India 
b Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute, Kolkata 700026, India 

 
 

 

 

1. Introduction 
 

Among all human diseases, cancer is now the 

leading killer.American Cancer Society estimates 

put the number of new cancer cases at 1,918,030 

in 2022, with 609,360 deaths attributed to the 

disease.3 In 91 out of 172 nations, cancer ranks as 

the leading or second-leading cause of death, 

indicating a high mortality risk.1  

Radiotherapy and chemotherapeutic medications 

are the principal cancer therapies now available, 

however they have the potential to damage 

healthy cells and slow patients' recovery after 

treatment.5 There is an urgent need to produce a 

safe anticancer medication from natural sources 

with decreased side effects in order to eradicate 

the issues related to the administration of a 

nonspecific chemotherapeutic agent.  



 

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Numerous bioactive substances, such as phenols, 

flavonoids, volatile oils, organic acids, 

polysaccharides, glycosides, carotenoids, and 

alkaloids, have been discovered in mushrooms, 

which has led to their inclusion in the quest for 

alternative natural anticancer medications. Novel 

anticancer, antibacterial, antioxidant, 

immunomodulatory, anti-inflammatory, 

cardiovascular, and antidiabetic medicines may be 

derived from these chemicals or from alternative 

natural sources.6e11 – Traditional, all-natural 

medicine derived from mushrooms has a long 

history of use in some  

 

 

 

 

 

Southeast Asian nations. For many centuries, 

various components originating from mushrooms 

have been used as nutritional supplements and 

herbal remedies in traditional Chinese medicine 

(TCM).12 Unfortunately, only a small fraction of 

the world's mushroom species have undergone 

any kind of medicinal screening, and even less 

have been studied in this area.13 Numerous 

mushroom species have shown promise as 

anticancer agents; they include Agaricus, 

Antrodia, Albatrellus, Cordyceps, Calvatia, 

Clitocybe, Inocybe, Inonotus, Russula, 

Schizophyllum, and Trametes.13 This trustworthy 

alternate supply of anticancer chemicals, however, 

seems to be understudied.  

Previous research indicated that methanol extract 

of Astraeus hygrometricus (Pers.) Morgan (A. 

hygrometricus) had an antiproliferative action on 

Jurkat cells with only a small impact on normal 

peripheral blood mononuclear cells (PBMCs). The 

five species of mushrooms tested in this study 

were Phallus sp., A. hygrometricus, A. 

hygrometricus, Lentinus sp., Tricho-loma sp., and 

Serpula sp.14 Here, we included six more cancer 

cell lines in the screening and apoptotic efficacy 

study: MOLT-4, Reh, and NALM-6, which are 

leukemic; Hep G2, which is hepatocellular 

carcinoma; A549., which is lung carcinoma; 

MCF-7, which is breast cancer; and BEAS-2B, 

which is normal. Investigating the role of reactive 

nitrogen species (RNS) and reactive oxygen 

species (ROS) and their potential effect on 

mitochondrial membrane potentiality was another 

extension of our research into the potential 

mechanism of cancer inhibition. Additionally, we 

tested the most sensitive cell line for the 

apoptogenic potential of the strongest extract. To 

conclude, we sought to determine the molecular 

mechanism of apoptotic induction by examining 

how the mushroom extract affected cell cycle 

progression and the expression levels of apoptotic 

mediators, caspases, and many important 

mitochondrial proteins.  

 
2. Materials and methods 

 
2.1. Sample collection and identification 

 
Wild mushroom fruit bodies were collected between 2015 

and 2019 from diverse habitats between 22.1203◦N, 

88.3943◦E and 27.0206◦N, 88.5650◦E, spanning an area from 
the hinterland of the 

Bay of Bengal and lateritic region of the western part of West 

Bengal state   to   the   great   Himalayan   foothills.   The   fruit   

bodies   of 
A. hygrometricus (Jamboni, 22.4502◦N, 86.8998◦E) and 
Lentinus sp. 

(Sigram, 22.4479◦N, 86.8967◦E) were collected from the 
laterite region of Jhargram District; those of Tricholoma sp. 

and Serpula sp. (Mathurapur, 22.1203◦N,  88.3943◦E) were 
collected from areas 

with alluvial soil in the South 24-Parganas District; while 
those of 

Phallus sp. were collected from wood logs associated with 

moist humus soil (Lolegaon, 27.0206◦N, 88.5650◦E) in the 
Kalimpong 

District of West Bengal, India. Mushroom species were 

identified using various published standard indentation keys 

and molecular validation, and voucher specimens were 

deposited at the Calcutta University Herbarium.5,14 

 
2.2. Chemicals 

 



 

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¼ 

× 

All chemicals used were of molecular grade. Dulbecco's 

modi- fied eagle medium (DMEM, Gibco, New York, NY), 

Roswell Park Memorial Institute Medium (RPMI 1640, Gibco, 

New York, NY), fetal bovine serum (FBS, Gibco, New York, NY), 

penicillin-streptomycin (Gibco, New York, NY), amphotericin b 
(Himedia, Maharashtra, India), 2 mM L-glutamine, phosphate 

buffer saline (PBS, Gibco, New York,   NY),   3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 

bromide  (MTT,  Sigma  Aldrich,  Burlington,  MA),  40,6-
diamidino-2-phenylindole (DAPI, Sigma Aldrich, Burlington, 
MA), methanol, hexane, ethyl acetate, DMSO (Sigma Aldrich, 
Burlington, MA), annexin-V/propidium iodide (PI) apoptosis 
detection kit (BD- Pharmingen, Bergen, NJ), CM-H2DCFDA 
(Invitrogen, Carlsbad, CA), JC-1 kit (Invitrogen, Carlsbad, CA), 
propidium iodide (PI, Himedia, Maharashtra, India), caspase 
detection kit (BioVision, Waltham, MA), and primary and 
secondary antibodies against poly (ADP- ribose) polymerase 
(PARP) and b-actin (Abcam, Cambridge, UK) were purchased 
from the respective manufacturers. 

 
2.3. Preparation of mushroom extracts 

 
The samples were dried under a shed and then ground 

using a mixer grinder. Each mushroom powder sample was 

extracted by selectively applying a solvent system based on 

solvent polarity, and 50 g of starting material was primarily 

percolated in hexane for 3 d to remove the fatty substances. 

The residual material was then sequentially extracted with 

ethyl acetate followed by methanol solvent.  Each  extract  

was  evaporated  in  a  rotary  evaporator, 

lyophilized at reduced pressure, and finally stored at 4◦C for 
further 

experiments. 

 
2.4. Cell culture and maintenance 

 
The MOLT-4 (T-cell acute lymphoblastic leukemic), Reh 

(B-cell acute lymphoblastic leukemic), and NALM6 (B-cell 

acute lympho- blastic leukemic) cell lines were kindly gifted 

by Dr. Santu Ban- dyopadhyaya and Dr. Chitra Mandal (IICB, 

Kolkata, India). Hep G2 (human hepatocellular carcinoma) 

cells were gifted by Prof. Sanjit Dey from the Department of 

Physiology, University of Calcutta (Kolkata, India). The A549 

(human lung carcinoma), MCF-7 (human breast cancer), and 

BEAS-2B (human nontumorigenic lung epithelial) cell lines 

were procured from the National Centre for Cell Sciences 

(NCCS, Pune, India). All cell lines were maintained in either 

DMEM or RPMI 1640 supplemented with 2 mM L-glutamine, 

10% (v/ 
v) FBS (heat-inactivated), and 10 U/mL 

penicillin/streptomycin and incubated at 37◦C in a humidified 
atmosphere of 5% CO2 incubator 

(HF90, Heal Force, Hong Kong, China). 

 
2.5. Cell viability assay 

 
The antiproliferative effect of the crude extracts of the 5 

mushroom species was assessed against 6 different cancer 

cell lines, that is, three leukemic cell lines (MOLT-4, Reh, and 

NALM-6), one hepatocellular carcinomic cell line (Hep G2), 

one lung carci- nomic cell line (A549), and one breast cancer 

cell line (MCF-7) as 

well as against one normal cell line (BEAS-2B) and PBMCs from a 

healthy donor by the MTT assay.15,16 In brief, 5 × 104 cells were 
incubated with various concentrations (10e100 mg/mL) of 

mush- room extract in a 96-well plate for 24 h at 37◦C. 
Subsequently, 3 h before completion, 25 mL MTT solution (5 
mg/mL in PBS) was added 

to each well, and finally, formazone complex was dissolved in 

DMSO, and the optical density (OD) was recorded at 560 nm. 

The absorbance of fully lysed (100%) cells was measured by 

treating them with 5% sodium dodecyl-sulfate (SDS) lysis 

buffer before measurement at 560 nm. The percentage of cell 

viability was calculated using the following equation: 

 

Cell viability ð%Þ¼
 

ODsample — OD100% lysis

 
 

.  
OD0% lysis  — OD100% lysis

   

×100 

Regarding the isolation of PBMCs, the percoll density 

gradient centrifugation (d 1.082 g/L) method was applied to 

heparinized venous blood from a healthy donor and desired 

PBMCs were 

 
recovered from the interface layers. The recovered cells were 

washed in PBS and resuspended in RPMI 1640 supplemented 

with 2 mM L-glutamine, gentamicin, and 10% heat-inactivated 

FBS, for further study.17 

 
2.6. Effect of methanol extract on cellular morphology-

DAPI staining 

 
To    investigate    the    effect    of    the    methanol    extract    
of 

A. hygrometricus on the nuclei of MOLT-4 cells, DAPI staining 

was performed.18,19 The cells were incubated with increasing 

concen- 

trations (0e25 mg/mL) of mushroom extract for 24 h at 37◦C. 
Cells 

were fixed with 4% paraformaldehyde, followed by washing 

with PBS. The cells were then stained with 2 mg/mL DAPI for 

15 min in the dark and viewed under a fluorescent microscope 

at 400 1 magnification (Dewinter Optical, Delhi, India). Based 

on the status of the nucleus, cells were categorized as 

apoptotic (condensed or fragmented chromatin) or normal 

(smooth nuclear structure). 

 
2.7. Cell cycle profiling assay by propidium iodide staining 

 
For this experiment, MOLT-4 cells and PBMCs were treated 

with increasing concentrations of mushroom extract (0e25 

mg/mL) for 24 h. Subsequently, cells were harvested into a 

single-cell suspen- 

sion and incubated overnight at —20◦C with 70% ethanol. Cells 



 

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× 

× 

× 

were resuspended in PBS and incubated for 2 h with RNaseA 
(20 mm) at 

37◦C. Eventually, PI was added, and after incubation for 20 

min at 25◦C, cells were subjected to flow cytometry 
analysis.4,19 

 
2.8. Measurement of cellular apoptosis by annexin V-PI staining 

 
Induction of apoptosis by the mushroom extract was 

measured by  flow  cytometry  after  annexin  V-FITC/PI  

staining.16,20  Briefly, 

1.5 × 105 MOLT-4 cells and PBMCs were incubated with an 
increasing concentration (0e25 mg/mL) of methanol extract 
for 
24 h. After incubation, cells were washed with  PBS  and  resus- 

pended in 100 mL of binding buffer for 30 min. Finally, 5 mL 

annexin V-FITC and 5 mL PI were added to cells and 

incubated for 15 min, and fluorescent signals were recorded. The 

cell population negative for both annexin V and PI was recorded 

as viable cells. Based on the annexin V/PI ratio, stained cells were 

categorized as early apoptotic (positive for annexin V/negative 

for PI)  or late  apoptotic  (positive for annexin V/positive for PI). 

 
2.9. Measurement of mushroom-induced cellular ROS using 

DCFDA 

 
The levels of intracellular ROS were measured using the 

DCFDA method  according  to  the  manufacturer's  

instructions.  In  brief, 

1.5 × 105 MOLT-4 cells were incubated with different 
concentra- tions of mushroom extract (0, 5, 10, 25, and 50 
mg/mL) for 6 h. After 

incubation, cells were washed with PBS and reincubated in 
serum- free media containing 5 mM CM-H2DCF-DA for another 

30 min at 37◦C. Finally, fluorescent signals of the intracellular 
production of 

ROS were recorded using a flow cytometer. (Attune NxT, 

Boston, MA).17 

 
2.10. Measurement of mushroom-induced intracellular 

RNS by DAF-2 

 
The levels of intracellular RNS induced by the mushroom 

extract were measured using the DAF-2 method according to 

the manu- facturer's instructions. For the measurement of the 

concentration- dependent formation of RNS, 1.5 105 MOLT-4 

cells were incubated with different concentrations of mushroom 

extract (0, 5, 10, 25, and 50 mg/mL) for 6 h. To evaluate the 

time-dependent formation of 

RNS, 1.5 105 MOLT-4 cells were induced with 5 mg/mL of 

mush- room extract every 60 min for up to 300 min. After 

incubation, the cells were washed with PBS and reincubated 

in serum-free media containing diaminofluorescein-FM (DAF-

FM) for another 30 min. The cells were then subjected to flow 

cytometry and fluorescent signals of intracellular production 

of RNS were recorded. 

 

2.11. Measurement of mitochondrial permeability 

 
To explore the involvement of mitochondria in the 

apoptosis process, we measured the mitochondrial 

membrane potential (DJm), using JC-1. In this procedure, 1.5 

105 MOLT-4 cells were incubated with increasing 

concentrations of mushroom extract (0e25 mg/mL) for 6 h. 

The cells were then stained with JC-1 (10 mM in PBS), 

incubated for 15 min, and subjected to flow cytometry. The 

recorded data was further analyzed by applying a quadrant 

plot to distinguish monomers from J-aggregates and by using 

the FlowJo software (FlowJo, Ashland, OR). Results were 

presented as the ratio of J aggregates/monomers, effectively 

indicating the cellular mito- chondrial transmembrane 

potential.17 

 
2.12. Determination of caspase activity 

 
The activity of caspase-3, -8, and -9 was measured in cell 

lysates (100 mg protein in 50 mL lysis buffer) using a 
colorimetric assay according to the manufacturer's instructions. 

In brief, 5 ×  105 MOLT-4 cells were incubated with 5 mg/mL of 

methanol extract for 0e180  min  at  37◦C.  Cell  lysates  were 
then prepared,  according  to 

the manufacturer's instructions, and the protein 

concentration was measured, spectrophotometrically. Lysates 

were supplemented with 50 mL reaction buffer (10 mM DTT) 

and DEVD-para- nitroanilide (pNA, 4 mM, 5 mL; caspase-3 

substrate)  or  LEHD- pNA  (4  mM,  5  mL;  caspase-9  

substrate)  or  IETD-pNA  (4  mM, 

5 mL; caspase-8 substrate) and incubated at 37◦C for 0e3 h. 
The 

activity of each caspase was evaluated by quantitatively 

measuring the levels of the  resulting  chromophore  pNA  at  

405  nm  every 30 min for 3 h. For validation of the 

mushroom extract-induced activation of each caspase, MOLT-

4 cells were further coincubated with methanol extract and a 

pancaspase inhibitor, Z-VAD-FMK, for 24 h, and cell viability 

was measured by the MTT assay.18,21 

 
2.13. Immunoblotting 

 
Cell lysates were prepared from MOLT-4 cells treated with 5 

mg/ mL of mushroom extract for 12 and 24 h using the RIPA 

buffer method.4 For immunoblotting, 20e35 mg proteins were 

resolved on 8%e15% SDS-PAGE gels and transferred onto 

polyvinylidene difluoride membranes (Sigma Aldrich, 

Burlington, MA). Mem- branes were blocked with 5% BSA in 

1 TBST and probed with respective primary monoclonal 

antibodies, followed by incubation with the corresponding 

secondary antibodies. Finally, the blots were visualized using 

the ECL method on the Chemidoc MP system (Bio-Rad, 

Hercules, CA). 

 
2.14. Data analysis 

 
All experiments were performed independently in 



 

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duplicates or triplicates. All numerical data are expressed as 

mean (standard deviation). One-way ANOVA followed by 

Dunnett's multiple com- parison test or Turkey's test 

(wherever applicable) was used to compare differences 

among the experimental groups. In a specific case, the 

Student's t-test was also performed. Statistical significance was 

based on the following P value thresholds. All analyses were 

performed using GraphPad Prism 7.00 (GraphPad Software, 

San Diego, CA). 
 



 

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3. Results 

 
3.1. Differential antiproliferative activity induced by ethyl 

acetate and methanol extract of 5 different mushrooms against a 

panel of cancer cell lines 

 
We aimed to study the antiproliferative efficacy of 

methanol and ethyl acetate extracts of 5 different mushrooms 

against 6 cancer cell lines and normal cells by performing MTT 

assays. We cultured the 6 cancer cell lines, that is, NALM6, Reh,  

MOLT-4, A549, Hep G2, and MCF-7, one normal cell line BEAS-

2B, and PBMCs isolated from a healthy donor with different 

concentra- tions (0, 10, 25, 50, and 100 mg/mL) of mushroom 

extracts for 24 h. The results showed that the majority of 

extracts exhibited a dif- ferential pattern of dose-dependent 

inhibition of cancer cell pro- liferation (Fig. 1A). All tested 

extracts exhibited minimal cytotoxicity against normal cells, 

that is, PBMCs, and  BEAS-2B cells. The antiproliferative 

efficacies of 2 representative mush- room extracts, in terms of 

“half-maximal inhibitory concentration” (IC50), are presented 

in Fig. 1B and C. We categorized the resulting IC50 values as 

promisingly good (IC50 less than 25 mg/mL), moderately good 

(IC50 between 25 mg/mL and 250 mg/mL), or 

nonconsiderable (IC50 more than 250 mg/mL). The Selectivity 

Index (SI), which implies the effectivity and safeness of any 

drug, was calculated by dividing the “half-maximal inhibitory 

cytotoxic concentration” (CC50) value of this drug in normal 

cells with the IC50 value of this drug in each cancer cell line. 

The respective SI values are presented in Supplemental 

Tables 1 and 2. 

Among  all  the  5  tested  ethyl  acetate  mushroom  

extracts (Fig. 1B), A. hygrometricus showed the highest 

effectivity against the MOLT-4 cell line with an IC50 of 16.10 

(1.22) mg/mL. The ethyl acetate extract of A. hygrometricus 

also exhibited a high selectivity index against the MOLT-4 cell 

line, in contrast to other cell lines, with SI values of 43.63 

(1.30) against BEAS-2B, and 34.52 (1.77) against PBMCs. The 

ethyl acetate extracts of all 5 mushrooms were more effective 

against leukemic than other cancerous cell lines. The same 

trend was observed in the methanol extracts (Fig. 1C). The 

meth- anol extract of A. hygrometricus was the most effective 

against the MOLT-4 cell line with an IC50 value of 7.25 (0.69) 

mg/mL, exhibiting SI values of 64.20 (0.59) and 127.67 (1.57) 

against BEAS-2B and PBMCs, respectively. 

Both ethyl acetate and methanol extracts of A. 

hygrometricus produced promising to moderately good 

efficacy against all cancerous cell lines, except for the A549 

cell line, which appeared to be the most resistant when 

treated with ethyl acetate extracts. Compared with other 

mushrooms, both extracts of Lentinus sp. showed the least 

efficacy against all cell lines. The methanol ex- tracts of 

Phallus sp. and Tricholoma sp. were the most effective 



 

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Fig. 1. In vitro antiproliferative efficacy of both ethyl acetate and methanol 

extracts of 5 mushrooms were measured by MTT assay in the presence of an 

increasing concentration of the extract (0e100 mg/mL) in various cell lines, 

including NALM6, Reh, MOLT-4, A549, Hep G2, and MCF-7; one normal cell line 

BEAS-2B and PBMC isolated from a healthy donor (A); the IC50 value of different 

cancer cell lines, treated with increasing ethyl acetate extract of 5 different 

mushrooms (B); the IC50 value of  different  cancer  cell  lines  treated with 

increasing methanol extract of five different mushrooms (C). 

Note: PBMC: peripheral blood mononuclear cell. 
 

against the breast cancer cell line MCF-7 with IC50 values less 
than 

10 mg/mL. Methanol extract of all the 5 mushrooms 

produced moderately good efficacy against Reh and NALM6 

cell line. Both extracts of all 5 mushrooms showed mediocre 

efficacy against the NALM6  cell  line.  We  concluded  that  

the  methanol  extract  of 

A. hygrometricus was the most effective and safe anticancer 

extract, and MOLT-4 was the most sensitive cell line; hence, we 

performed further experiments on MOLT-4 cells using the 

methanol extract of 

A. hygrometricus. 

 
3.2. Mushroom extract induced nuclear condensation on 

MOLT- 4 cells in a concentration-dependent manner 

 
Following DAPI staining, MOLT-4 cells treated with an 

increasing concentration of the methanol extract (0e25 

mg/mL) for 24 h exhibited characteristic features of increased 

apoptosis in a concentration-dependent manner (Fig. 2AeC) 

with respect to control. Treated cells displayed bright, 

fragmented, condensed, and unorganized nuclei, whereas the 

nuclei of control cells appeared dimly blue and organized (Fig. 

2A and B). The apoptotic effect of the extract was initiated at 

the concentration of 5 mg/mL, resulting in 26.33% 

morphologically altered MOLT-4 cells and increased 

concentration-dependently. The percentage of apoptotic 

MOLT- 

4 cells at the highest treatment concentration reached  74.66% 

(Fig. 2C). 

 
3.3. Methanol extract interfered with cell cycle 

progression by targeting cells at sub-G0/G1 phase 

 
To verify the effect of the methanol extract of A. 

hygrometricus on the cell cycle distribution profile, we treated 

both MOLT-4 cells and PBMCs with increasing concentrations 

of the extract (0e25 mg/ mL) for 24 h, and acquired the cell 

cycle histogram by flow cytometry. The results showed that 

the methanol extract specif- ically targeted cells residing at the 

sub-G0/G1 phase, whereas no significant effect was noticed in 

normal cells (Fig. 3AeC). A signif- icant decrease occurred in 

the numbers of the cell population at the G0/G1 phase in cells 

treated with 5 mg/mL extract, which further increased 

analogously  in  a  concentration-dependent  manner (Fig. 3A). 

There was a drastic shift in the number of cells from the G0/G1 

phase to the sub-G0/G1 zone, which indicated that the extract 

specifically targeted the G0/G1 population, as evidenced by cell 

cycle analysis. The sharp shift observed in the number of cells in 

the sub-G0/G1 phase indicated nuclear fragmentation 

resulting from apoptosis-like cell death. This finding suggested 

that these wild edible mushrooms may promote apoptosis-like 

cell death, specifically in MOLT-4 cells but not in normal cells. 

Overall, our findings suggested that the methanol extract of A. 

hygrometricus demonstrated antileukemic activity against 

MOLT-4 cells by tar- geting the sub-G0/G1 population. 

 
3.4. Methanol extract of A. hygrometricus augmented 

robust apoptosis, specifically in MOLT-4 cells, had 

negligible impact on normal cells 

 
Using annexin V/PI dual staining, we further corroborated 

the A. hygrometricus-induced apoptosis in cancer cells. We 

treated both MOLT-4 cells and PBMCs with or without 

increasing concentrations of the methanol extract (0e25 

mg/mL) for 24 h, and subjected them to flow cytometry after 

staining with annexin V/PI. Our analysis revealed a dose-

dependent increase in the number of apoptotic MOLT-4 cells; 

whereas, the methanol extract exhibited minimal activity 

against PBMCs isolated from healthy donors (Fig. 3D and E). At 

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24 h after treatment, the percentage of apoptotic cells was 

increased from 52.22% at the lowest treatment 

concentration of 5 mg/mL to 94.93% at the highest treatment 

concentration of 25 mg/ mL. The apoptotic-related IC50 value 

of the A. hygrometricus extract was 6.91 (0.91) mg/mL, which 

was very close to the IC50 concen- tration obtained by the 

MTT assay. At the same range of treatment concentrations, 

PBMCs showed very modest apoptosis. With these results, we 

inferred that the methanol extract of A. hygrometricus 

specifically induced robust apoptosis in MOLT-4 cells but not 

in normal PBMCs. 

 
3.5. Methanol extract of A. hygrometricus induced the 

production of high levels of ROS in leukemic cells 

 
ROS may trigger the intrinsic apoptotic cascade via in- 

teractions with proteins of the mitochondrial permeability 

transition complex. Therefore, we  aimed  to test the 
involvement of intracellular ROS in the A. hygrometricus 
methanol extract- induced apoptosis in MOLT-4  cells.  We  

treated  MOLT-4  cells with increasing concentrations (0e50 

mg/mL)  of  methanol extract for 6 h, followed by labeling 

with CM-H2DCFDA (5 mM) and  measurement  of  the  

number  of  DCFþ cells  by  flow cytom- 
etry.  The  methanol  extract  triggered  the  generation  of  a  
high 

 
 

Fig. 2.  Impact of methanol extract of A. hygrometricus on cellular and nuclear morphology in MOLT-4 cells: dose-dependent change in the condensed nuclear structure of 

MOLT- 4 cells treated with 5,10, and 25 mg/mL extract for 24 h and studied by DAPI (A); changes in the cellular shape and membrane blebbing were clearly observed in MOLT-

4 cells in a dose-dependent manner (B); the bar graph indicates a concentration-dependent increase in mushroom-induced altered morphology containing MOLT-4 cells (C). 

Notes: Data are expressed as mean (standard deviation). Magnification: 400 × 1. Scale bar ¼ 10 mm. ****P < .0001 vs. control group. 

 



 

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Fig. 3. MOLT-4 and PBMC, isolated from healthy donor cells, were treated with an increasing concentration of mushroom extract (0, 5, 10, and 25 mg/mL) for 24 h: The bar 

graph represent a dose-dependent augmentation of the cells at the G0/G1 phase in MOLT-4 cells (A); in contrast, no noticeable change was observed in PBMC (B); 

posttreatment, cells were harvested and fixed with ethanol and stained with PI. the representive histogram showing the distribution profile of the cell cycle analyzed by flow 

cytometry (C); A concentration-dependent increase in the annexin V-FITC/PI-positive population of MOLT-4 cells was observed in response to increasing concentration of 

methanol extract treated for 24 h, whereas a very light effect was observed in PBMC cells (D); The contour plot depicts that with increasing concentration of the extract, the 

percentage of apoptosis significantly increased in MOLT-4 cells in contrast to PBMC (E). 

Notes: PBMC: peripheral blood mononuclear cell. t-test was performed to compare multiple groups means (MOLT-4) vs. PBMC control. **P < .01 vs. control group. 

 

yield of ROS in a concentration-dependent manner as the fluo- 

rescence intensity of DCF was increased with the increase in 

the concentration of the extract (Fig. 4A and B). To confirm 

that the induction of apoptosis was a downstream event of the 

increased production of intracellular ROS, we preincubated a 

set of MOLT- 

4 cells with the antioxidant, L-acetyl cysteine (L-NAC). The 

percentage of apoptotic cells decreased from 90.56% to 

22.83% after L-NAC treatment, indicating that L-NAC rescued 

cells from apoptosis by scavenging ROS. In another set of 

MOLT-4  cells, media was supplemented with GSH,  hence  

inhibiting  the depletion of intracellular GSH. We found that 

the percentage of apoptotic cells decreased from 90.56% to 

28.56% following GSH supplementation in the medium (Fig. 

4C). This further confirmed the involvement of intracellular 

ROS in inducing apoptosis following treatment of  MOLT-4  

cells  with  the  methanol  extract of A. hygrometricus. 

 
3.6. Methanol extract of A. hygrometricus induced the 

production of high levels of RNS in leukemic cells 

 
As reactive nitrogen species (RNS) are essential signaling 

and effector molecules that play a role in apoptosis along with 

ROS, we examined the effect of the methanol extract of A. 

hygrometricus on the  production  of  RNS  using   DAF-2DA.  We  

observed  that  MOLT- 4 cells treated with an IC50 concentration, 

that is 7.25 (0.69)  mg/mL of methanol extract of A. 

hygrometricus showed a time-dependent increase in the 

generation of RNS  from  the  basal  level,  which peaked at 180 

min, and subsequently plateaued (Fig. 5C). The production of RNS 

reached a maximum after treatment with 25 mg/ mL methanol 

extract, and gradually decreased with increasing concentrations 



 

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(Fig. 5A and B). There was a gradual increase in the fluorescence   

of   DAFþ  cells   along   with   concentration   gradient 
treatment, suggesting that the methanol extract of A. 
hygrometricus 

also induced the generation of RNS along with ROS in MOLT4 
cells. 

 

3.7. Depolarization of mitochondrial membrane potentials in MOLT-4 cells treated with methanol extract of A. hygrometricus 

 

Loss of mitochondrial membrane potential (DJm) is one of the significant consequences of ROS-induced apoptosis. The mito- 

chondrial disruption includes an imbalance in the mitochondrial membrane potential and associated changes in the oxidation- reduction 

state inside the mitochondria. Depolarization of the mitochondrial membrane potential prevents the accumulation of the JC-1 dye in 

the mitochondria and results in the dispersion of the dye throughout the cell leading to a shift from red (J- 

 

 
 

Fig. 4. Flow cytometric data indicated a concentration-dependent increase in intracellular ROS in MOLT-4 cells in response to the increasing concentration of the mushroom 

extract: the fluorescent signals from the cells were acquired by flow cytometer (A, B); The bar graph depicts a significant decrease in apoptosis, after treating with ROS 

scavenger NAC and GSH (C). 

Notes: Posttreatment, DCFDA (5 mM final) in serum-free media was added and incubated for 30 min; cells were washed in 1 × PBS. Finally, the fluorescent signals from the 

cells 
were acquired by flow cytometer. One-way ANOVA, followed by Dunnett's multiple comparisons tests, were performed to compare multiple groups means (treated sets) vs. 
control. 

****P < .0001, vs. control group. 

 



 

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Fig. 5. Flow cytometric data indicated both concentration-dependent (A, B) and 
time-dependent (C) increases in intracellular RNS production in MOLT-4 cells. 

Notes: One-way ANOVA, followed by Dunnett's multiple comparisons tests, was 

performed to compare multiple groups' means (treated sets) vs. control. ****P < 

.0001, vs. control group. 

A. hygrometricus methanol extract-treated cells. The results 

revealed a shift in the fluorescence emission from red to green, 

indicating a massive loss of DJm in A. hygrometricus methanol 

extract-treated MOLT-4 cells (Fig. 6A and B). Consequently, 

this decrease in the red/green fluorescence intensity ratio 

indicated mitochondrial depolarization (Fig. 6C). We detected 

that the per- centage of green monomers increased from 

18.7% to 89.0% in a concentration-dependent manner. We 

thus concluded that the methanol extract induced the 

depolarization of mitochondrial membranes, leading to 

apoptosis. 

 
3.8. Methanol extract of A. hygrometricus induced cleavage of 

PARP 

 
The DNA repair enzyme, poly (ADP) ribose polymerase 

(PARP) plays a vital role in DNA repair. However, when cells 

receive an apoptotic stimulus and activate their caspase 

cascades, PARP serves as a substrate for active caspase-3, thus 

preventing the DNA repair process. Hence, the cleavage of PARP 

is considered a prerequisite for apoptosis.17 Therefore, we 

evaluated the effect of the methanol extract on PARP cleavage. 

The amount of cleaved-PARP was significantly increased at 12 

and 24 h in MOLT-4 cells treated with 5 mg/mL of methanol 

extract compared with that in the untreated control MOLT-4 

cells (Fig. 7B). 

 
3.9. Methanol extract of A. hygrometricus induced 

caspase- dependent apoptosis 

 
Caspases, a group of proteases, are effector molecules of 

the apoptotic pathway and play a significant role in 

programmed cell death. Therefore, we measured the 

activities of caspase-3, -8, and 

-9 after treatment with the methanol extract of A. 

hygrometricus (5 mg/mL) every 30 min for 3 h. There was an 

exponential increase in the activities of two caspases 

(caspase-3 and -9) up to 90 min of treatment. In contrast, 

there was no significant change in the level of activity of 

caspase-8 (Fig. 7A). To validate the methanol extract- induced 

activation of caspases, we coincubated MOLT-4 cells with a 

nontoxic concentration of Z-VAD-FMK (20 mM), a pancaspase 

in- hibitor, and measured cell viability using the MTT assay. 

We found that Z-VAD-FMK attenuated the extract-induced 

cytotoxicity in MOLT-4 cells by increasing the IC50 of the 

methanol extract of A. hygrometricus. Hence, we concluded 

that the methanol extract of 

A. hygrometricus induced apoptosis in a caspase-dependent 

manner. 



 

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3.10. Methanol extract of A. hygrometricus triggered 

mitochondria- dependent apoptosis in MOLT-4 cells 

 
The increased production of intracellular reactive oxygen 

and nitrogen species (RONS), followed by the disruption of 

mitochon- drial membrane potentiality and elevated levels 

of caspase-3 and 

-9, is correlated with the induction of the mitochondria-

involved intrinsic apoptotic pathway. To confirm our 

hypothesis, we checked 3 other proteins related to 

mitochondria-dependent apoptosis. One of the related 

consequences of intrinsic apoptosis is the downregulation of 

the antiapoptotic proteins and upregula- tion of proapoptotic 

proteins.17,22 We found that the levels of the anti-apoptotic 

Bcl-2 protein were downregulated in a time- dependent 

manner in methanol extract-treated MOLT-4 cells; in contrast, 

the levels of the proapoptotic protein (Bax) were upre- 

gulated (Fig. 7B). Cytochrome c is a significant mediator 

molecule of 

 
 

 
 

Fig. 6. Flow cytometric data indicated a concentration-dependent increase in green monomer and a decrease in Red J-aggerates in MOLT-4 cells (A, B), a sharp decrease in Red 

to green ratio was observed in a concentration-dependent manner (C). 

 



 

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Fig. 7. Lysates of MOLT-4 treated with mushroom extract in a time-dependent 

manner were subjected to study the caspase profile (caspase 8, caspase 9, and 

caspase 3): both Caspase 9 and 3 upregulated in a time dependant manner (A); 

western blot images showing the expression profile of pro-apoptotic (Bax), and 

anti-apoptotic Bcl-2 protein involved in time-dependent extract induced 

apoptosis in MOLT-4 cells, the expression profile of cytochrome-c and PARP 

were also measured (B); the mushroom extract selectively induced the RONS in 

MOLT-4 cells and positively induced pro-apoptotic Bax protein, followed by 

the release of cytochrome-c, which ultimately exhibited cellular apoptosis 

through the intrinsic pathway (C). 

Note: Data are expressed as mean (standard deviation). 

the intrinsic apoptotic pathway. In mammalian cells, various 

apoptotic stimuli cause the release of cytochrome c from mito- 

chondria, inducing a series of biochemical reactions, and 

finally cellular death through the induced activation of 

caspases.23 Hence, an increased amount of cytosolic 

cytochrome c confirms the occurrence of intrinsic apoptosis. 

The extract-treated MOLT-4 cells showed an elevated amount 

of cytosolic cytochrome c in a time- dependent manner (Fig. 

7B). 

 
4. Discussion 

 

Fatalities caused by cancer, particularly during the 

recovery phase, are mostly attributable to the 

ineffectiveness and adverse effects of currently 

available cancer treatments.24, 25 Poor 

individuals, particularly in less developed nations, 

may be unable to afford cancer treatments due to 

their high cost.26 Consequently, there is a need 

for new anticancer medications that are more 

selective for cancer cells and have fewer adverse 

effects.  

The anticancer properties of many biomolecules 

originating from mushrooms have been shown. 

These include b-glucans, b-proteoglycans, 

ergosterol, lectins, triterpenes, ergothioneine, and 

selenium.27 Few studies have examined 

mushrooms' medicinal potential, especially in the 

context of drug development. We set out to assess 

the anticancer potential of mushrooms—an 

upcoming arsenal for possible anticancer 

medications—in order to circumvent one of the 

major roadblocks to cancer chemoprevention: the 

unwanted toxicity of pharmaceuticals to normal 

cells, which causes unpleasant side effects.  

 

We screened ethyl acetate and methanol extracts 

of five wild mushrooms for antiproliferative 

potential: A. hygrometricus, Phallus sp., Lentinus 

sp., Tricho-loma sp., and Serpula sp. This was 

motivated by the extensive ethnobotanical history 



 

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of mushroom usage as alternative medicine. Out 

of the five species we examined, our research 

found that the new edible fungus A. 

hygrometricus had the least amount of 

carcinogenic chemicals. No studies have shown 

the impact of astrakurkurone and astrakurkurol on 

leukemic cells, while prior research has 

investigated their effects on hepatocarcinoma 

(Hep G2, Thle 2, and Hep 3B) and renal cell 

carcinoma (RCC) cell lines.28e30 Prior to this, we 

documented that an A. hygrometricus methanol 

extract was effective in killing a moderately 

leukemic cell line.  

In 2022, leukaemia accounted for 7% of all cancer 

cases and 7% of all cancer deaths worldwide, 

according to cancer statistics.3 The incidence of 

acute lymphoblastic leukaemia (ALL) is 1.6 per 

100,000 in the US, making it a very common 

malignancy. There is an estimated 80% incidence 

rate of ALL, making it the most prevalent 

childhood cancer.3,31 The antileukemic effects of 

wild edible mushrooms have been the subject of 

few investigations so far. The antileukemic 

potential of A. hygrometricus, a wild edible 

fungus, was investigated in the current research. 

More specifically, we detail the molecular 

mechanism by which A. hygrometricus induces 

cell death in drug-resistant robust T-cell acute 

lymphoblastic leukaemia cells (MOLT-4).  

Our research showed that A. hygrometricus 

methanol extract had strong apoptogenic potential, 

and that after 24 hours of treatment with different 

quantities of mushroom extract, MOLT-4 cells 

showed a dramatic increase in cell population in 

the sub G0-G1 stage. Nonetheless, normal cells 

did not show any discernible alterations. Nandi et 

al. found that the A. hygrometricus compound 

astrakurkurol showed the same  

 

Halting in the G0/G1 phase in Hep G2 cells.29 

This discovery provides further evidence that A. 

hygrometricus may interrupt the cell cycle and 

cause cells to die in cancer cells, but it has no 

effect on normal cells.  

There are two well-established routes for 

signalling cell death: intrinsic and extrinsic.32 An 

important component of the extrinsic apoptotic 

pathway is the recognition and propagation of 

extracellular stimuli by specific membrane 

receptors. On the other hand, RONS successfully 

triggers mitochondria-centered apoptosis via the 

intrinsic route, which is primarily a caspase-

dependent process involving a regulatory system 

centred on mitochondria. After caspase-8 

mediates the extrinsic route and caspase-9 initiates 

the intrinsic pathway, caspase-3 cleaves the 

downstream executor protein to activate 

apoptosis.33 Mushroom extract increased caspase-

9 and -3 levels but had no discernible effect on 

caspase-8 levels. Based on our findings, it seems 

that the intrinsic apoptotic pathway may have 

been responsible for the strong apoptosis in 

MOLT-4 cells that was triggered by mushrooms. 

An integral part of the intrinsic apoptotic route 

includes the release of cytochrome c, collapse of 

mitochondrial membrane polarity, and 

mitochondrial malfunction mediated by reactive 

oxygen species (ROS).34 Nevertheless, proteins 

belonging to the Bcl-2 family regulate the last 

effector molecules of apoptosis, caspase-3 and 

caspase-9. Notably, the ratio of proapoptotic 

(Bax) to antiapoptotic (Bcl-2) proteins determines 

the extent of apoptosis.16 Our results show that 

the mushroom extract confirmed a time-dependent 

cleavage of the apoptosis controller Bcl-2 

proteins, leading to cell death. The Bcl-2 protein 

and caspase-9, the last effectors of apoptosis, are 

connected via cytochrome c. Inferring from the 

increased ROS production by the A. 

hygrometricus methanol extract, we found that it 

exerted its antileukemic effects against MOLT-4 

cells via the mitochondria-dependent apoptotic 

pathway (Fig. 7C). Additionally, it was shown 



 

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that the levels of RNS were elevated after extract 

administration.  

 
5. Conclusion 

 

A. hygrometricus methanol extract significantly 

inhibited the proliferation of MOLT-4 cells 

relative to BEAS-2B and PBMCs derived from a 

healthy donor, out of five wild mushrooms 

evaluated against six human cancer cell lines. The 

aggressive, resilient, and resistant T-cell acute 

lymphoblastic leukaemia known as MOLT4 is 

characterised by PI3K-AKT deregulation. By 

controlling Bcl-2 and Bax, our results revealed 

that the intrinsic apoptotic route, which is 

mediated by cytochrome c and caspases, is the 

principal mechanism by which A. hygrometricus 

induces apoptotic-like cell death in MOLT-4 cells. 

In MOLT-4 cells, the mushroom extract increased 

ROS and RNS production and produced strong 

apoptosis via the mitochondria-dependent route. 

Since A. hygrometricus is a native mushroom, we 

infer that it contains active myco-compounds that 

may be useful in the fight against leukaemia. 

 
 

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