Cellular, genomic and transcriptomic effects of secondary metabolites of the Hybrid Butterbur on the HeLa cell line

Elena Yu. Zlatnik; Yaroslav S. Enin; Oleg N. Burov; Elena S. Bondarenko; Alexander B. Sagakyants; Denis S. Kutilin; Yulia V. Dzigunova; Inna A. Novikova; Yury V. Przhedetskiy

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South Russian

Journal of Cancer..

Vol. 5

No. 3, 2024

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South Russian

Journal of Cancer..

Vol. 5

No. 3, 2024

South Russian Journal of Cancer. 2024. Vol. 5, No. 3. P. 50-63

https://doi.org/10.37748/2686-9039-2024-5-3-5

https://elibrary.ru/koukit

ORIGINAL ARTICLE

Cellular, genomic and transcriptomic effects of secondary metabolites..

of the Hybrid Butterbur on the HeLa cell line..

E. Yu. Zlatnik1, Ya. S. Enin1 , O. N. Burov2, E. S. Bondarenko1, A. B. Sagakyants1, D. S. Kutilin1,

Yu. V. Dzigunova2, I. A. Novikova1, Yu. V. Przhedetskiy1

1 National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

2 Southern Federal University, Rostov-on-Don, Russian Federation

Dendro51@yandex.ru

ABSTRACT

Purpose of the study. To evaluate the cellular, genomic (gene copy number) and transcriptomic (gene expression) effects of

P.hybridus (L.) secondary metabolites when they affect the HeLa cell line.

Materials and methods. The isolation

of secondary metabolites

from plant

material

and its

identification

were carried out

by

preparative chromatography. The composition was determined using mass spectrometric analysis, and the final verification

of structural formulas was carried out by nuclear magnetic resonance at the Department of Natural Compounds, the Faculty

of Chemistry of the Southern Federal University. The subsequent phase of the study was conducted using both cultural and

molecular methods. HeLa cells

were cultivated under standard conditions in a MEM medium. Once the confluence level was

reached 75�80

%, the nutrient medium was replaced with the introduction of the studied compounds (at a concentration of 4

micrograms/ml) and cultivated for 72 hours. Cell mortality was determined using a NanoEnTek JuliFl counter (Korea) in the

presence of 0.4 % trypan blue. The assessment of apoptosis following secondary metabolite exposure was conducted on

a BD

FACSCanto II flow cytometer using the

FITC

Annexin

V Apoptosis

Detection

Kit

I.

The

level

of replication

and expression

of the genes responsible for apoptosis was assessed by digital droplet PCR (ddPCR).

Results. The following compounds were isolated and verified, and were assigned the following sequence numbers to facilitate

their use in the experiment: No. 2 � 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one, No. 3 � 5-(hydroxymethyl) furan-2-carbaldehyde,

No. 5.3 � 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde, P. hybridus (L.) At the stage of cell death assessment, it was

found that

the greatest

effect

was

achieved in the compound under ordinal

No. 2. However, the evaluation of the copy number

and expression of the CASP8, CASP9, CASP3, BAX, BCL2, TP53, MDM2, CDKN1B, CDK1, CCND1, CCND3, and RB1 genes by

DD-PCR revealed the presence of apoptosis initiation in tumor cells at the molecular level under the action of compounds No.

2 and No. 5.3 obtained from P. hybridus (L.).

Conclusion. The outcomes were multifeatured. Only compound 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one exhibited a pronounced

cytostatic effect out of all compounds utilized in the experiment. Concurrently, the compound 2,2,8-trimethyldecahydroazulene-

5,6-dicarbaldehyde was found to induce an increase in the expression of the CASP3, CASP8, TP53, and BAX genes.

Keywords:

secondary plant metabolites, apoptosis, gene expression, copy number variation, HeLa cell line, digital droplet PCR

For citation: Zlatnik E. Yu., Enin Ya. S., Burov O. N., Bondarenko E. S., Sagakyants A. B., Kutilin D. S., Dzigunova Yu. V., Novikova I. A., Przhedetskiy Yu. V.

Cellular, genomic and transcriptomic effects of secondary metabolites of the Hybrid Butterbur on the HeLa cell line. South Russian Journal of Cancer. 2024;

5(3): 50-63. https://doi.org/10.37748/2686-9039-2024-5-3-5, https://elibrary.ru/koukit

For correspondence: Yaroslav S. Enin � Junior Researcher, Laboratory of Molecular Oncology, National Medical Research Centre for Oncology, Rostov-on-Don,

Russian Federation

Address: 63 14 line str., Rostov-on-Don 344037, Russian Federation

E-mail: Dendro51@yandex.ru

ORCID: https://orcid.org/0000-0002-4572-1579

SPIN: 7683-2286, AuthorID: 840050

Scopus Author ID: 57196464479

Funding: this work was not funded. The work was performed with scientific equipment provided by the Central Research Institute of the National Medical

Research Center for Oncology: https://ckp-rf.ru/catalog/ckp/3554742/

Conflict of interest: the authors declare that there are no obvious and potential conflicts of interest associated with the publication of this article

The article was submitted 19.06.2024; approved after reviewing 07.08.2024; accepted for publication 25.08.2024

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ORCID: https://orcid.org/0000-0002-4572-1579

SPIN: 7683-2286, AuthorID: 840050

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South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 50-63

Zlatnik E. Yu., Enin Ya. S. , Burov O. N., Bondarenko E. S., Sagakyants A. B., Kutilin D. S., Dzigunova Yu. V., Novikova I. A., Przhedetskiy Yu. V. Cellular, genomic and

transcriptomic effects of secondary metabolites of the Hybrid Butterbur on the HeLa cell line

INTRODUCTION

Cervical cancer is one of the main causes of female

mortality. Every year, more than 528,000 new

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cases of breast cancer and more than 266,000

deaths

from this

disease

are

detected

[1, 2]. The

HeLa cell line is a very convenient and simple object

for conducting model experiments in vitro. This cell

line was obtained on February 8, 1951 from a cervical

tumor

of a patient named Henrietta Lacks at the

Baltimore

hospital

[3]. In

our study, we

used this

cell

line to evaluate the cytotoxic effect of the organic

compounds of plant origin that we obtained.

Plants

synthesize a huge number of secondary

metabolites, and in fact it is these metabolites that

form the basis of many commercial pharmaceuticals,

as well as herbal medicines. Many secondary

metabolites, such as alkaloids, terpenoids and phenylpropanoids,

are being considered for drug development

[4].

Secondary metabolites of plants are structurally

diverse compounds that do not directly participate in

the growth, development and reproduction of plants,

but

more often perform a protective function. These

compounds with different chemical structures can

act as potential multi-purpose anticancer

agents [5].

For the first time in history, the term secondary metabolite

was proposed by the German biologist Albrecht

Kessel in 1891. when he gave a lecture "On the

chemical composition of cells" for the Berlin Society

of Physiologists, in which he said: "I propose to call

compounds that are important for each cell primary,

and compounds that are not present in any plant cell

secondary" [6]. Currently, the secondary metabolites

of plants are divided into several large groups. Terpenoids

(isoprenoids) cover more than 40,000 structures

and form the largest class of all known plant

metabolites. They represent

a class of hydrocarbons,

i. e. products

of biosynthesis

of the general formula

(C5H8) n, with a carbon skeleton that is a derivative

of isoprene CH2=C(CH3)�CH=CH2. Alkaloids are

characterized as heterocyclic compounds containing

a nitrogen molecule in a heterocycle and count

about 21,000 compounds. Phenolic compounds are

aromatic compounds

with

a

benzene

ring containing

at least one hydroxyl group [7].

The species selected in our work for the isolation

of secondary metabolites is the hybrid Petasites hibridus

(L.) Gaertn., B.

Mey. & Scherb is a herbaceous

perennial plant of the Asteraceae family, found in the

European territory of Russia, and, in particular, in the

Krasnodar Territory and the Republic of Adygea. The

reasons for the interest in this species are that various

representatives of the genus Petasites, including

P. hibridus (L.)

itself, contain compounds

with

cytotoxic effects on tumor cells of various nosolo

gies

[8]. So in

the

Japanese

White-collar Petasites

japonicus (Siebold & Zucc.) Maxim. sesquiterpene

I and sesquiterpene II were detected, which showed

cytotoxic effect against both human astrocytoma

U-251MG tumor cells, as well as against the MDA

MB-231 breast cancer cell line [9].

Various methodological approaches are used to

study the effect of secondary plant metabolites on

tumor

cells,

including cytometry

and flow

cytofluorometry,

model experiments on cell cultures and

molecular genetic studies. The latter include the

assessment of the level of replication and gene

expression. CNV (copy number

variation) is a type

of genetic polymorphism that leads to a change in

the number of certain genetic loci and, as a result,

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a change in the expression of these genes

and their

products � proteins and non-coding RNAs [10].

Studies

of the effect of secondary plant metabolites on

the expression and replication of genetic loci regulating

apoptosis and the cell cycle in cervical cancer

are currently few, so this aspect requires additional

study. This is what this work is dedicated to.

The study purpose was to evaluate the cellular,

genomic (gene replication) and transcriptomic (gene

expression) effects of secondary metabolites of

P. hibridus (L.) when they are exposed to the HeLa

cell line.

MATERIALS AND METHODS

Extraction of metabolites. The primary plant material

was collected and determined with the participation

of the staff of the Department of Botany of

the Academy of Biology and Biotechnology of the

D. I. Ivanovsky Southern Federal University. Isolation

and verification of secondary metabolites of

P. hibridus (L.) were carried out by employees of

the

Department of Natural and High Molecular Weight

Compounds of the Faculty of Chemistry of the Southern

Federal University. Tetrachloroethylene was used

as

a solvent

for primary extraction, which

was

poured

into mechanically purified and crushed rhizomes.

��-�� 2024. �. 5, � 3. �. 50-63

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The primary extraction process lasted for four

months. To extract tetrachloroethylene from vegetable

raw materials, the decantation method was

used, followed by concentration of the solution by

distillation of the solvent in a distillation unit. Tetrachloroethylene

was

used

as

a

solvent

to reduce

the amount of polar compounds (mono- and disaccharides,

amino sugars, etc.). The next step was the

separation of the resulting concentrated solution using

column chromatography using silica gel as

a sorbent

on column 20*2. Various

eluents were used:

first, tetrachloroethylene, which allowed to obtain

10 fractions of various colors, from colorless to light

yellow. Then methylene chloride was used, which

gave 10 more fractions. After that, the eluent was

changed,

and the column was filled with a mixture

of methylene

chloride

and alcohol

in

a ratio of 10/1,

which led to the production of two more fractions.

All fractions were concentrated by evaporation on

a rotary evaporator.

The method of high-performance liquid chromatography

with mass detection was used to identify

the isolated compounds. The mass spectra were

analyzed using NIST 2011 biotechnology, which confirms

the results of studies with alkaloids and other

biologically active compounds.

Fractions containing higher fatty acids, nitrogenous

bases of nucleic acids and their glycosides

were excluded from further work. In addition, the

previously isolated fractions

were further purified

using column

chromatography, and

the

purified

compounds

were identified using nuclear magnetic resonance

(1H NMR). The identification of purified frac

tions using NMR made it possible to determine the

purity and confirm the structure of compounds previously

assumed using mass detector chromatogra

phy. The following main names have been identified

for experimental

use: No. 2

� 2,4-dihydroxy-2,5-dimethylfuran-

3(2H)-oh, No. 3

� 5-(hydroxymethyl)furan-

2-carbaldehyde, No. 5.3

� 2,2,8-trimethyldecahydroazulene-

5,6-dicarbaldehyde (Fig. 1).

Assessment of biological effects

The biological effect of the isolated compounds

was evaluated on the HeLa CCL2 cell line. The cell

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line was obtained from the biobank of the National

Medical Research Centre for Oncology, which

works in accordance with the recommendations on

the organization of the structure of biorepositories

and the ethical requirements of the latest edition

of the ISBER Best Practices and based on the ISO

9001 standard [11]. The cells were cultured at 37 �C

and 5 %CO2 in a nutrient medium

Igla MEM (BioloT)

containing 10 % fetal serum from cows (HyClone,

USA), up to a

number of 1

. 106 cells. When 80 %

confluence was achieved, the nutrient medium was

replaced with a similar

one with the addition of 4

micrograms / ml of furfural and azulene derivatives

to the test samples, and without the addition of the

studied substances in the negative control. The

exposure time was 72 hours. After that, the cells

were removed from culture vials with 0.1 % trypsin

solution. The number of living and dead cells was

determined using an automatic NanoEnTek JuliFl

counter (Korea) with 0.4 % trypan blue staining. Cells

removed from culture vials were preserved in an RNA

2,4-dihydroxy-2,5-dimethylfuran-

-3(2H)-one (2)

Chemical Formula: C6H804

Molecular Weight: 144,13

5-(hydroxymethyl)furan- -2-carbaldehyde

(3)

Chemical Formula: C6H603

Molecular Weight: 126,11

2,2,8-trimethy|decahydroazulene-5,6-

dicarbaldehyde (5.3)

Chemical Formula: C15 02

H24

Molecular Weight: 236,3499

Fig. 1. Structural formulas of three compounds isolated from the hybrid P. hibridus (L.) protein

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 50-63

Zlatnik E. Yu., Enin Ya. S. , Burov O. N., Bondarenko E. S., Sagakyants A. B., Kutilin D. S., Dzigunova Yu. V., Novikova I. A., Przhedetskiy Yu. V. Cellular, genomic and

transcriptomic effects of secondary metabolites of the Hybrid Butterbur on the HeLa cell line

medium (IntactRNA Eurogene). Cellular apoptosis

was

assessed

on

a

BD

FACSCanto II

flow cytofluorometer

using the

FITC

Annexin

V Apoptosis

Detection

Kit I. Cells stored in an RNA medium were divided

into two equal aliquots, from which total DNA/RNA

preparations were extracted using the commercial

DNA-sorb-B and Trizol kit, respectively.

Molecular methods

The evaluation of copy number variations and

gene expression was performed by digital drip PCR

using the QX200� ddPCR� EvaGreen Supermix

kit (Bio-Rad, USA). The Droplet Digital polymerase

chain reaction system (ddPCR�) was developed

for high-precision absolute quantitative analysis of

target sequences of nucleic acids encapsulated in

discrete droplets of water-oil emulsion determined by

volumetric method. Using a droplet generator, each

sample of the studied locus was divided into 20,000

droplets in three repeats. Amplification was carried

out to the end point (40 cycles) on the C1000 Touch

Thermal Cycler Bio-Rad.

After the amplification was completed, a QX200

Bio-Rad reader was placed on the sample plate,

which counted droplets giving fluorescent positive

and negative signals to calculate the concentration

of target DNA and ctDNA. The principle of measuring

the level of copyness and expression indicators

using digital drip PCR technology was to directly

count events via the FAM channel. In positive droplets

containing at least one copy of the target DNA,

the droplet reader

shows fluorescence,

unlike negative

droplets in which amplification did not occur.

QuantaSoft v1 software.7.4 measures the number

Fig. 2. Screenshot of the QuantaSoft v1 software.7.4 during the

result processing

of positive and negative droplets in each sample,

and then applies an algorithm for calculating the

Poisson distribution function to determine the initial

concentration of target DNA molecules in units of

"copies/.l" (Fig. 2).

The level of CNV and gene expression was calculated

as follows. According to the formula, the

concentration of each studied locus / concentration

of the reference locus . the number of copies of the

reference loci in the genome (as a rule 2).

Statistical data processing

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Statistical data processing was carried out using

the Statistica 19.0 program (StatSoft Inc., USA). To

assess the significance of the differences, a singlefactor

analysis of variance was used (the critical

level of statistical significance was p < 0.05).

STUDY RESULTS

At

the first

stage of the study, the purification

and verification of compounds that can exhibit cytotoxic

effects on tumor cells of various nosologies

was carried out. The identification of the isolated

compounds was performed by mass spectometry

and nuclear

magnetic

resonance (NMR);

during it,

2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one was verified,

which was assigned the serial number 2,5-(hydroxymethyl)

furan-2-carbaldehyde with the serial

number 3, as well as 2,2,8-trimethyldecahydroazulene-

5,6-dicarbaldehyde with the serial number 5.3.

All three compounds are isolated from the rhizomes

of P. hibridus (L.). The data obtained by evaluating

the cytotoxic effect of the compounds involved in the

experiment on the NanoEnTek JuliFl cell counter are

presented in Table 1.

As can be seen from the data presented in Table

1, all compounds according to the results of the

trepan blue test had an approximately equivalent

effect

on

HeLa tumor cells; in

experimental

samples,

the number of dead cells exceeded the control by

1.97�2.44 times. The images below, obtained using

an inverted microscope (Leica DM IL LED), show

a comparison of a control sample of the HeLa cell

line

with

a

sample

treated

with

compound

No. 2.

After exposure, a

violation

of the

monolayer in

the

experimental sample is seen associated with weaker

cell attachment or lysis, and a large number of cells

are "scalded" (Fig. 3).

��-�� 2024. �. 5, � 3. �. 50-63

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The following images show the effect achieved

when exposed to compound No. 3 in comparison

with the control,

a dense monolayer

of cells is observed,

at

the

same

time

a large

number of scalding

cells (Fig. 4).

Figure 5 shows a comparison of the control of

Hela cells with cells that were exposed to compound

5.3, in the experimental sample there is a dense

monolayer of cells and an increase in the number

of scalding cells that exceeds that observed in the

control.

The results of the evaluation of the antitumor effect

of the metabolites used by us are also

confirmed

by the data of flow

cytofluorometry presented below.

The most pronounced cytotoxic effect was shown

by (2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one) at

No. 2

at a concentration of 4

micrograms /ml at

exposure for 72 hours. The remaining compounds

used did not have such an effect according to flow

cytofluorometry (Fig. 6�8, table 2).

As can be seen from Figure 6, 72-hour incubation

with (2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one)

had a cytostatic effect on HeLa cells, expressed in

an increase in the number of cells in the state of

early apoptosis from 7.2 to 13.3 %, and late apoptosis

from 5.8 to 8.3 %. The total number of cells

in the state of apoptosis after exposure (2,4-dihydroxy-

2,5-dimethylfuran-3(2H)-one) increases 1.6

times.

Based on

the

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data in

Fig.7, it

follows

that

a 72-hour

incubation with 5-(hydroxymethyl)furan-2-carbalde

hyde did not have a cytotoxic effect on HeLa cells,

the difference with the control in early apoptosis

changed from 7.2 to 7.3 %, and late apoptosis � from

5.8 to 6.2 %. The total number of cells in the state

of apoptosis after exposure to 5-(hydroxymethyl)

furan-2-carbaldehyde increases by 0.5 times.

Figure 8 demonstrates that 72-hour incubation

with 5-(hydroxymethyl)furan-2-carbaldehyde also did

not have a cytotoxic effect on HeLa cells, the difference

with the control in early apoptosis changed

from 7.2 to 7.5 %, and late apoptosis � from 5.8 to

6.8 %. The total number of culture cells in the state

of apoptosis after exposure to 2,2,8-trimethyldecahydroazulene-

5,6-dicarbaldehyde increased 1.3 times.

Digital drip PCR was used to evaluate changes

in CNV and expression (CNV/EXP) indices under

the

influence of secondary metabolites

from

P. hibridus (L.) isolated by us. When exposed to

2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde

at a concentration of 4 micrograms/ml exposure

for 72 hours, there were increases in the level of

expression of CASP3 in relation to the control by

28.28times (p < 0.05), and

CASP8 by 46.71 times

(p < 0.05). At the same time, the expression of the

CASP9 locus increased by 3.43 times (p

<

0.05). Exposure

to 5-(hydroxymethyl) furan-2-carbaldehyde at

a concentration of 4 micrograms/l and an exposure

of 72

hours

had

the

following effect: the

expression

level of CASP3 increased by 4.57 times relative to

the control (p < 0.05), it also increased the expression

level of CASP8 by 10.48 times (p < 0.05). When

using 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one at

a concentration of 4

micrograms/ml

exposure for 72

hours, the expression of the CASP3 locus relative to

the control increased by 3.95 times (p < 0.05), and

CASP8 by 3.38 times (p < 0.05). At

the

same time,

the indicators of the copy level (CNV) of the CASP8,

CASP9, and CASP3 loci did not undergo major changes

(Fig. 9).

At the same time, the assessment of changes in

the levels of copy number variability (CNV) and expression

(EXP) at the TP53 and MDM2 loci showed

the following results. The compound 2,2,8-trimeth-

Table 1. The number of

living and

dead

HeLa cells after exposure to

isolated

secondary metabolites after staining with trypan blue

Compounds, concentrations 72 hours, living cells 72 hours, dead cells

Control 93.52 % 6.48 %

� 2, 4 .g /ml

87.23 % 12.77 %

� 3, 4 .g /ml

86.66 % 13.34 %

� 5.3, 4 .g /ml

84.16 % 15.84 %

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 50-63

Zlatnik E. Yu., Enin Ya. S. , Burov O. N., Bondarenko E. S., Sagakyants A. B., Kutilin D. S., Dzigunova Yu. V., Novikova I. A., Przhedetskiy Yu. V. Cellular, genomic and

transcriptomic effects of secondary metabolites of the Hybrid Butterbur on the HeLa cell line

�B

Fig. 3. HeLa cells after exposure to 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one. A � control specimen; B � experimental specimen

�B

Fig. 4. HeLa cells after exposure to terpenoid 5-(hydroxymethyl)furan-2-carbaldehyde. A � control specimen; B � experimental specimen

�B

Fig. 5. HeLa cells

after incubation

with the terpenoid 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde. A � control

specimen;

B � experimental specimen

��-�� 2024. �. 5, � 3. �. 50-63

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�� HeLa

yldecahydroazulene-5,6-dicarbaldehyde at an exposure

of 72

hours

and a concentration of 4

micrograms/

ml increased the TP53 copy level by 1.05

times (p < 0.05), and MDM2 decreased by 0.26

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.

times (p < 0.05)

relative to the control. The difference

between them was 4 times. In addition, 2,4-dihydroxy-

2,5-dimethylfuran-3(2H)-one increased TP53

expression level by 1.46 times (p < 0.05)

at

exposure

of 72 hours and concentration of 4 micrograms/ml,

30_05_19-Kontr

105

104

while MDM2 decreased by 0.88 times (p < 0.05). The

difference was 1.66 times (Fig. 10).

The following data were obtained when evaluating

changes in the level of replication and expression of

the BAX and BCL2 loci. The terpenoid 2,2,8-trimethyldecahydroazulene-

5,6-dicarbaldehyde increased

the level of CNV of the I locus by 0.9 times relative to

the control (p < 0.05), the

level

of BCL2 decreased by

0.13times (p < 0.05). The difference between them

30_05_19-2_4 mkg

105

104

PIPE-A

-7,508

Q1-1 Q2-1

Q3-1 Q4-1

PIPE-A

-2.190 -103 0 103 104 105

Ann V FITC-A

-7,399

Q1-1 Q2-1

Q3-1 Q4-1

-2.223 -103 0 103 104 105

Ann V FITC-A

103

0

-103

�

B

Fig. 6. Effect of (2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one) on necrosis/apoptosis of the HeLa cell line: A � control specimen;

B � experimental specimen, (Q3-1 � living cells, Q4-1 � early apoptosis, Q2-1 � late apoptosis/necrosis, Q1-1 � dead cells)

30_05_19-Kontr 30 05_19-5 3 4 mkg

105

104

PIPE-A

-7,508

Q1-1 Q2-1

Q3-1 Q4-1

PIPE-A

-2.190 -103 0 103 104 105

Ann V FITC-A

-7,326

Q1-1 Q2-1

Q3-1 Q4-1

-657 0 103 104 105

Ann V FITC-A

103

0

-103

�

B

Fig. 7. Exposure to 5-(hydroxymethyl)furan-2-carbaldehyde on necrosis/apoptosis of the HeLa cell line: A � control specimen;

B � experimental specimen, (Q3-1 � living cells, Q4-1 � early apoptosis, Q2-1 � late apoptosis/necrosis, Q1-1 � dead cells)

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 50-63

Zlatnik E. Yu., Enin Ya. S. , Burov O. N., Bondarenko E. S., Sagakyants A. B., Kutilin D. S., Dzigunova Yu. V., Novikova I. A., Przhedetskiy Yu. V. Cellular, genomic and

transcriptomic effects of secondary metabolites of the Hybrid Butterbur on the HeLa cell line

was 6.92 times. Also, when exposed to the same

compound, the expression level of BAX increased by

1.73 times (p < 0.05), and BCL2 decreased by 1.19

times (p < 0.05). The difference between them was

1.45 times in favor of an increase in BAX (Fig. 11).

The furan and azulene derivatives of P. hibridus

(L.) metabolites used in our study changed

the level of replication and expression of CDKN1B,

CDK1, CCND1, CCND3 and RB1 loci as follows. Thus,

2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde

increased CCND3 expression by 20.66 times relative

to the control (p < 0.05), RB1 expression increased by

7.35 times (p < 0.05) at an exposure of 72

hours and

a concentration

of 4

micrograms/ml. In turn

5-(hydroxymethyl)

furan-2-carbaldehyde with an exposure

of 72

hours

and a concentration of 4

micrograms/ml

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.

also increased the expression level of CCND3 by

5.23 times (p

<

0.05).

At the same time,

2,4-dihy30_

05_19-Kontr

105

104

droxy-2,5-dimethylfuran-3(2H)-one at similar concentrations

and exposures increased the CCND3 copy

level by 3.48 times (p < 0.05), and

the

expression

level

increased by 2.42 times relative to the control (p <

0.05). At the same time, 2,4-dihydroxy-2,5-dimethylfuran-

3(2H)-one at the point of 4 micrograms/ml with

an exposure of 72 hours increased the expression

level of the RB1 locus relative to the control by 4.51

times (p < 0.05) (Fig. 12).

DISCUSSION

Since the early 2000s, many works have been published

worldwide on the search for new compounds

of natural origin, including plant origin ones, with cytostatic

or cytotoxic effects on tumor cells of various

diseases

[12]. In

our study, we

conducted not

only

a model experiment to assess the level of cytotoxic

30 05_19-5 3 4 mkg

105

104

PIPE-A

-7,508

Q1-1 Q2-1

Q3-1 Q4-1

PIPE-A

-2.190 -103 0 103 104 105

Ann V FITC-A

-7,326

Q1-1 Q2-1

Q3-1 Q4-1

-657 0 103 104 105

Ann V FITC-A

103

0

-103

�

B

Fig. 8. The effect of 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde on necrosis/apoptosis of the HeLa cell line: A � control specimen;

B � experimental specimen, (Q3-1 � living cells, Q4-1 � early apoptosis, Q2-1 � late apoptosis/necrosis, Q1-1 � dead cells)

Table 2. The number of HeLa cells in a state of apoptosis after exposure to isolated secondary metabolites

(72 hours exposure)

Compound Concentration,

.g /ml

Alive cells

Q3-1

Early-stage

apoptosis

Q4-1

Late-stage

apoptosis / necrosis

Q2-1

Dead cells

Q1-1

Control 87.0 % 7.2 % 5.8 % 0 %

No. 2 4 78.3 % 13.3 % 8.3 % 0 %

No. 3 4 91.1 % 2.5 % 6.3 % 0 %

No. 5.3 4 94.2 % 2.6 % 3.2 % 0 %

��-�� 2024. �. 5, � 3. �. 50-63

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�� HeLa

effect of the P. hibridus (L.) secon dary metabolites

that we obtained, but also used the digital drip PCR

method to register molecular genetic changes in

loci responsible for suppressing tumor growth and

apoptosis in HeLa tumor cells.

The data obtained in the study show mixed

results. The most pronounced change in the expression

level of the CASP8 and CASP3 loci was

revealed when exposed to 2,2,8-trimethyldecahydroazulene-

5,6-dicarbaldehyde. Cytosolic caspases

are cysteine-asparagine proteases, which are the

main family of proteins involved in the transmission

of cell death signals. Caspases are divided into three

groups:

initiatory,

inflammatory and effector.

They

are directly involved in the initiation of apoptosis.

As is known, the CASP8 protein, which is an initia

50

46,713,4328,2845

40

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35

30

25

20

15

10,480,834,5710

5

3,380,693,950,890,941,550,930,980,810,02 0,750,180

Compound No. 2,

4 .g/ml,

72 hours EXPCompound No. 2,

4 .g/ml,

72 hours CNVCompound No. 3,4 .g/ml,

72 hours EXPCompound No. 3,4 .g/ml,

72 hours CNVCompound No. 5.3,4 .g/ml,

72 hours EXPCompound No. 5.3,

4 .g/ml,

72 hours CNV

CASP8 CASP9 CASP3

Fig. 9. Changes in the level of replication and expression of the CASP8, CASP9, and CASP3 loci under the action of 2,4-dihydroxy-2,5-dimeth-

ylfuran-3(2H)-one (No. 2), 5-(hydroxymethyl)furan-2-carbaldehyde (No. 3) and 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde (No. 5.3)

1,6

1,460,881,4

1,201,381,2

1,040,771,031,141,191,081,050,261,0

0,8

0,6

0,4

0,2

0

Compound No. 2, Compound No. 2, Compound No. 3,Compound No. 3,Compound No. 5.3,Compound No. 5.3,

4 .g/ml,

72 hours EXP

72 hours CNV

72 hours EXP

72 hours CNV

72 hours EXP

72 hours CNV

TP53 MDM2

Fig. 10. Changes in the level of replication and expression of TP53, MDM2 loci when exposed to 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one

(No. 2), 5-(hydroxymethyl) furan-2-carbaldehyde (No. 3) and 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde (No. 5.3)

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 50-63

Zlatnik E. Yu., Enin Ya. S. , Burov O. N., Bondarenko E. S., Sagakyants A. B., Kutilin D. S., Dzigunova Yu. V., Novikova I. A., Przhedetskiy Yu. V. Cellular, genomic and

transcriptomic effects of secondary metabolites of the Hybrid Butterbur on the HeLa cell line

tor, is associated with tumor necrosis factor (TNF)

located on the cell surface, as well as the FAS ligand

(FasL), and induces apoptosis (CD95). Activation of

the CASP8 protein via the external apoptosis pathway

triggers BID-mediated activation of BAX and

BAK proteins on the outer membrane of mitochondria,

which leads to the release of cytochrome C

and subsequent activation of CASP9, which, in turn,

activates CASP3 and CASP7, thereby performing

the process of apoptosis along the mitochondrial

pathway [13]. It should be noted that when exposed

to 5-(hydroxymethyl)furan-2-carbaldehyde showed

a change

in

the

expression

level

of CASP8 and

CASP3 of a similar

profile.

1,081,691,731,191,6

1,4

1,2

0,870,950,911,131,001,170,900,131,0

0,8

0,6

0,4

0,2

0

Compound No. 2, Compound No. 2, Compound No. 3,Compound No. 3,Compound No. 5.3,Compound No. 5.3,

4 .g/ml,

72 hours EXP

72 hours CNV

72 hours EXP

72 hours CNV

72 hours EXP

72 hours CNV

BAX BCL2

Fig. 11. Changes in the level of replication and expression of BAX, BCL2 loci when exposed to 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one

(No. 2), 5-(hydroxymethyl) furan-2-carbaldehyde (No. 3) and 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde (No. 5.3)

25

20

15

10

5

0

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1,601,691.2820.667,351,361,181,342,424,510,840,921,063,481,151,301,151,365,232,561,181,260,610,900,800,751,120,920,471,06

Compound No. 2, Compound No. 2, Compound No. 3,

Compound No. 3,

Compound No. 5.3,

4 .g/ml,

72 hours EXP

72 hours CNV

72 hours EXP

72 hours CNV

72 hours EXP

72 hours CNV

CDKN1B CDK1 CCND1 CCND3 RB1

Fig. 12. Changes in the level of replication and expression of CDKN1B, CDK1, CCND1, CCND3, RB1 loci under the influence of 2,4-dihydroxy-

2,5-dimethylfuran-3(2H)-one (No. 2), 5-(hydroxymethyl)furan-2-carbaldehyde (No. 3) and 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde

(No. 5.3)

��-�� 2024. �. 5, � 3. �. 50-63

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�� HeLa

At the same time, 2,2,8-trimethyldecahydroazulene-

5,6-dicarbaldehyde increased the expression level

of TP53 and significantly reduced the expression

of MDM2, which may indicate a specific targeting

of the action of this compound. It should be borne

in mind that TP53 has tumor suppressive activity,

which largely explains its ability to induce cell death,

including apoptosis, through transcription-dependent

and transcription-independent mechanisms [14]. In

addition, the nuclear protein p53 transcriptionally activates

the expression of many pro-apoptotic genes

of the BCL-2 family, such as NOXA, PUMA, BID, BAD,

BIK, BAX, etc., whereas it inactivates the expression

of anti-apoptotic BCL-2, BCL�Xl and MCL1, leading

to mitochondrial apoptosis

[15]. The relationship

between changes in the expression level of TP53 and

BAX loci was also reflected in the results obtained.

As in the case of the TP53/MDM2 locus bundle, exposure

to the terpenoid 2,2,8-trimethyldecahydroazulene-

5,6-dicarbaldehyde affected the expression

level of BAX/BCL2 loci.

The changes in the expression level of CCND3 and

RB1

loci under the influence of 2,4-dihydroxy-2,5-dimethylfuran-

3(2H)-one were also revealed. This

compound is the only one used in our study, that led

to a decrease in the expression level of the CCND3

locus relative to RB1 (the expression level of RB1

was almost 2 times higher than the expression

level of CCND3). As is known, d-type cyclins (d1,

d2 and d3) are cell cycle regulators that activate

cyclin-dependent kinases cdk4 and cdk6, which are

often overexpressed in malignant neoplasms. The

CCND3 gene product interacts with the Rb tumor

suppressor protein and participates in its phosphorylation.

CDK4 activity is associated with this CCND3,

which is necessary for the transition of the cell cycle

to the G2 phase. Inhibition of CCND3 and cyclin-d

cdk4/6

kinase in tumor cells with a high content of

retinoblastoma rb1 protein causes cell cycle arrest.

However, reducing only the level of rb1 in tumor cells

does not lead to

a stop in proliferation [16]. The data

on the change in the expression of CCND3 and RB1

are consistent with the data of objective control from

photographs obtained using an inverted microscope

and data from flow cytofluorometry.

CONCLUSION

The study made it possible to establish the multidirectional

effect of secondary metabolites of P. hibridus

(L.)

on

the

death

and apoptosis

of HeLa cells.

The data obtained by

digital drip PCR revealed a maximum

increase in the expression of genes responsible

for regulating apoptosis (CASP3, CASP8, TP53, BAX)

under the action of 2,2,8-trimethyldecahydroazulene-

5,6-dicarbaldehyde, as well as a change in the

expression of CCND3 and RB1 genes under

the influence

of 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one. At

the same time, according to

cytometry and flow

cytofluorometry,

a more pronounced proapoptogenic

(cytotoxic)

effect was detected in 2,4-dihydroxy-2,5-dimethylfuran-

3(2H)-one. It should be noted that in our

work, the expression index reacted most actively to

the studied substances, which, in some cases, was

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dissonant with both gene replication and the level

of mortality and apoptosis of tumor cells. Perhaps

chemical

modifications

of the

compounds

used by

us will have a more pronounced effect both at the

molecular genetic level and at the cellular level.

References

1.

Hegde YM, Theivendren P, Srinivas G, Palanivel M, Shanmugam N, Kunjiappan S, et al. A Recent Advancement in Nanotechnology

Approaches for the Treatment of Cervical Cancer. Anticancer Agents Med Chem. 2023;23(1):37�59.

https://doi.org/10.2174/1871520622666220513160706

2. Kutilin DS, Mogushkova HA. Effect of anthracycline antitumor antibiotics upon transcription activity of cancer-testis antigens

in model experiments with hela cells. Medical Immunology. 2019;21(3):539�546. (In Russ.).

https://doi.org/10.15789/1563-0625-2019-3-539-546,

EDN:

SIWZEN

3.

Cox E. Performing HeLa: theatrical bodies and living remains. Med Humanit. 2023 Sep;49(3):447�456.

https://doi.org/10.1136/medhum-2022-012524

4.

Li Y, Kong D, Fu Y, Sussman MR, Wu H. The effect of developmental and environmental factors on secondary metabolites

in medicinal plants. Plant Physiol Biochem. 2020 Mar;148:80�89. https://doi.org/10.1016/j.plaphy.2020.01.006

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 50-63

Zlatnik E. Yu., Enin Ya. S. , Burov O. N., Bondarenko E. S., Sagakyants A. B., Kutilin D. S., Dzigunova Yu. V., Novikova I. A., Przhedetskiy Yu. V. Cellular, genomic and

transcriptomic effects of secondary metabolites of the Hybrid Butterbur on the HeLa cell line

5.

Fakhri S, Moradi SZ, Farzaei MH, Bishayee A. Modulation of dysregulated cancer metabolism by plant secondary metabolites:

A mechanistic review. Semin Cancer Biol. 2022 May;80:276�305. https://doi.org/10.1016/j.semcancer.2020.02.007

6.

Zagoskina NV, Nazarenko LV. Secondary Plant Metabolites: Distribution, History of Study, Practical Application Bulletin of

the Moscow City Pedagogical University. Series "Pedagogy and Psychology". 2019;(2(34)):8�19.

https://doi.org/10.25688/2076-9091.2019.34.2.1

7.

Mohiuddin AK. Chemistry of Secondary Metabolites. Annals of Clinical Toxicology. 2019 Jan 29;2.

https://doi.org/10.25107/2641-905X-v2-id1014

8. Apostolova S, Oreshkova T, Uzunova V, Georgieva I, Maslenkova L, Tzoneva R. A Standardized Extract of Petasites hybridus

L., Containing the Active Ingredients Petasins, Acts as a Pro-Oxidant and Triggers Apoptosis through Elevating of NF-.B

in a Highly Invasive Human Breast Cancer Cell Line. Front Biosci (Landmark Ed). 2023

Jun 12;28(6):111.

https://doi.org/10.31083/j.fbl2806111

9.

Matsumoto T, Imahori D, Saito Y, Zhang W, Ohta T, Yoshida T, et al. Cytotoxic activities of sesquiterpenoids from the aerial

parts of Petasites japonicus against cancer stem cells. J Nat Med. 2020 Sep;74(4):689�701.

https://doi.org/10.1007/s11418-020-01420-x

10. Kutilin DS, Kecheryukova MM. From bioinformatic screening of genetic markers to minimally invasive diagnostics of metastases

in the lymph nodes of patients with cervical cancer. Kazan Medical Journal. 2022;103(5):725�736. (In Russ.).

https://doi.org/10.17816/KMJ2022-725, EDN: OTTJXB

11.

Timofeeva SV, Filippova SYu, Sitkovskaya AO, Gnennaya NV, Mezhevova IV, Shamova TV, et al. Bioresource collection of

cell lines and primary tumors of the National Medical Research Center of Oncology of the Ministry of Health of Russia.

Cardiovascular Therapy and Prevention. 2022;21(11):44�50. (In Russ.). https://doi.org/10.15829/1728-8800-2022-3397,

EDN: MWVYOR

12.

Bouabdallah S, Al-Maktoum A, Amin A. Steroidal Saponins: Naturally Occurring Compounds as Inhibitors of the Hallmarks

of Cancer. Cancers (Basel). 2023

Jul 31;15(15):3900. https://doi.org/10.3390/cancers15153900

13.

Pang J, Vince JE. The role of caspase-8

in inflammatory signalling and pyroptotic cell death. Semin Immunol. 2023

Nov;70:101832.

https://doi.org/10.1016/j.smim.2023.101832

14.

Voskarides K, Giannopoulou N. The Role of TP53 in Adaptation and Evolution. Cells. 2023 Feb 3;12(3):512.

https://doi.org/10.3390/cells12030512

15.

Hao Q, Chen J, Lu H, Zhou X. The ARTS of p53-dependent mitochondrial apoptosis. J Mol Cell Biol. 2023

Mar

29;14(10):mjac074.

https://doi.org/10.1093/jmcb/mjac074

16.

Wang H, Nicolay BN, Chick JM, Gao X, Geng Y, Ren H, et al. The metabolic function of cyclin D3-CDK6

kinase in cancer cell

survival. Nature. 2017 Jun 15;546(7658):426�430. https://doi.org/10.1038/nature22797

Information about authors:

Elena Yu. Zlatnik � Dr. Sci. (Med.), MD, Professor, Chief Researcher, Laboratory

of Immunophenotyping of Tumors, National Medical Research Centre

for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-1410-122X, SPIN: 4137-7410, AuthorID: 327457, ResearcherID: AAI-1311-2020, Scopus

Author ID: 6603160432

Yaroslav S. Enin � Junior Researcher, Laboratory

of Molecular Oncology, National Medical Research Centre for Oncology, Rostov-on-Don, Russian

Federation

ORCID: https://orcid.org/0000-0002-4572-1579, SPIN: 7683-2286, AuthorID: 840050, Scopus Author ID: 57196464479

Oleg N. Burov � Cand. Sci. (Chem.), Associate Professor, Department of Natural and High Molecular Compounds, Faculty

of Chemistry, Southern

Federal University, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-7704-033X, SPIN: 5269-7656, AuthorID: 642948, ResearcherID: A-8428-2014, Scopus Author ID: 23033004000

Elena S. Bondarenko � Junior Researcher, Laboratory of Immunophenotyping of Tumors, National Medical Research Centre for Oncology,

Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-8522-1026, SPIN: 3117-4040, AuthorID: 865798, Scopus Author ID: 57200132337

Alexander B. Sagakyants � Cand. Sci. (Biol.), Head of the Laboratory

of Immunophenotyping of Tumors, National Medical Research Centre for

Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0003-0874-5261, SPIN: 7272-1408, AuthorID: 426904, ResearcherID: M-8378-2019, Scopus Author ID: 24329773900

��-�� 2024. �. 5, � 3. �. 50-63

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�� HeLa

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.

Denis S. Kutilin � Cand. Sci. (Biol.), Leading Researcher, Laboratory

of Molecular Oncology, National Medical Research Centre for Oncology,

Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-8942-3733, SPIN: 8382-4460, AuthorID: 794680, Scopus Author ID: 55328886800

Yulia V. Dzigunova � Senior Lecturer, Department of Botany, Academy

of Biology

and Biotechnology. DI. Ivanovoskogo Southern Federal University,

Rostov-on-Don, Russian Federation

SPIN: 2204-2967, AuthorID: 1062681

Inna A. Novikova � Dr. Sci. (Med.), MD, deputy director for science, National Medical Research Centre for Oncology, Rostov-on-Don,

Russian Federation

ORCID: https://orcid.org/0000-0002-6496-9641, SPIN: 4810-2424, AuthorID: 726229, ResearcherID: E-7710-2018, Scopus Author ID: 7005153343

Yury

V. Przhedetskiy

� Dr. Sci. (Med.), MD, Professor, Head of the Department of Reconstructive Plastic Surgery

and Oncology, National Medical

Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0003-3976-0210, SPIN: 3888-6265, ResearcherID: ATT-7598-2020, Scopus Author ID: 57188731912

Contribution of the authors:

Zlatnik E. Yu. � manuscript editing;

Enin Ya. S. � concept and design of the study, experiment coduction, writing the manuscript;

Burov O. N. � isolation and verification of compounds from plant material;

Bondarenko E. S. � cytofluorimetric analysis;

Sagakyants A. B. � analysis of the cytofluorometry results;

Kutilin D. S. � manuscript editing;

Dzigunova Yu.V. � collection and determination of plant material;

Novikova I. A. � design of the bibliography, manuscript editing;

Przhedetskiy Yu. V. � statistical data processing.

Cellular, genomic and transcriptomic effects of secondary metabolites of the Hybrid Butterbur on the HeLa cell line Текст научной статьи по специальности «Клиническая медицина»

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