5-Fluorouracil and Rumex obtusifolius extract combination trigger A549 cancer cell apoptosis: Uncovering PI3K/Akt inhibition by in vitro and in silico approaches

Abstract

In this study, the objective was to explore novel strategies for improving the efficacy of anticancer therapy. The focus was on investigating the antiproliferative effects of combining Rumex obtusifolius extract (RO) with the chemotherapeutic agent 5-Fluorouracil (5-FU) in non-small A549 lung cancer cells (NSCLC). Key factors such as the PI3K/Akt cell signaling system, cytokines, growth factors (TNFa, VEGFa), and enzymes (Arginase, NOS, COX-2, MMP-2) were analyzed to assess the impact of the combination treatment. Results revealed that the combined treatment of 5-FU and RO demonstrated a significant reduction in TNFa levels, comparable to the effect observed with RO alone. RO was found to modulate the PI3K/Akt pathway, influencing the phosphorylated and total amounts of these proteins during the combined treatment. Notably, COX-2, a key player in inflammatory processes, substantially decreased with the combination treatment. Caspase-3 activity, indicative of apoptosis, increased by 1.8 times in the combined treatment compared to separate treatments. In addition, in silico analyses explored the binding affinities and interactions of RO's major phytochemicals with intracellular targets, revealing a high affinity for PI3K and Akt. These findings suggest that the combined treatment induces apoptosis in A549 cells by regulating the PI3K/Akt pathway.

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5-Fluorouracil and Rumex obtusifolius extract
combination trigger A549 cancer cell apoptosis:
Uncovering PI3K/Akt inhibition by in vitro and in
silico approaches
Mikayel Ginovyan
Yerevan State University
Hayarpi Javrushyan
Yerevan State University
Svetlana Hovhannisyan
Yerevan State University
Edita Nadiryan
Yerevan State University
Gohar Sevoyan
L.A. Orbeli Institute of Physiology NAS RA
Tigran Harutyunyan
Yerevan State University
Smbat Gevorgyan
Denovo Sciences Inc
Zaruhi Karabekian
L.A. Orbeli Institute of Physiology NAS RA
Alina Maloyan
Knight Cardiovascular Institute, Oregon Health & Science University
Nikolay Avtandilyan
Yerevan State University
Article
Keywords: Rumex obtusifolius, 5-Fluorouracil, lung adenocarcinoma, phytochemicals, PI3K/Akt pathway,
apoptosis
Posted Date: April 25th, 2024

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DOI: https://doi.org/10.21203/rs.3.rs-4254380/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License
Additional Declarations: No competing interests reported.

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Abstract
In this study, the objective was to explore novel strategies for improving the ecacy of anticancer
therapy. The focus was on investigating the antiproliferative effects of combining
Rumex obtusifolius
extract (RO) with the chemotherapeutic agent 5-Fluorouracil (5-FU) in non-small A549 lung cancer cells
(NSCLC). Key factors such as the PI3K/Akt cell signaling system, cytokines, growth factors (TNFa,
VEGFa), and enzymes (Arginase, NOS, COX-2, MMP-2) were analyzed to assess the impact of the
combination treatment. Results revealed that the combined treatment of 5-FU and RO demonstrated a
signicant reduction in TNFa levels, comparable to the effect observed with RO alone. RO was found to
modulate the PI3K/Akt pathway, inuencing the phosphorylated and total amounts of these proteins
during the combined treatment. Notably, COX-2, a key player in inammatory processes, substantially
decreased with the combination treatment. Caspase-3 activity, indicative of apoptosis, increased by 1.8
times in the combined treatment compared to separate treatments. In addition,
in silico
analyses explored
the binding anities and interactions of RO's major phytochemicals with intracellular targets, revealing a
high anity for PI3K and Akt. These ndings suggest that the combined treatment induces apoptosis in
A549 cells by regulating the PI3K/Akt pathway.
1. Introduction
The continuous increase in cancer rates, failure of conventional chemotherapies to control the disease,
and excessive toxicity of chemotherapies (in some cases including immunotherapy) clearly demand
alternative approaches.Natural products contain many constituents that can act on various targets in the
body to induce pharmacodynamic responses1–3. Modulating biochemical and immune functions using
medicinal plants and their products combined with chemotherapeutic agents has recently become an
accepted therapeutic approach4.Lung cancer is one of the most common causesof cancer-related
deaths worldwide. According to the American Cancer Society, approximately85% of all lung cancer
deaths were theresult of non-small-cell lung cancer (NSCLC).
There are several reasons for targeting the PI3K/Akt pathwaysignaling pathway and related extracellular
and intracellular components. Correctly regulating the activity and quantity of these components can
have a strong anticancer effect on cancer cells.Several pro-tumorigenic processes converge on
hyperactive PI3K/Akt signaling, and it is becoming increasingly evident that reactive oxygen species
(ROS) metabolism is no exception to this5.Metabolic reconguration and the resulting generation of
ROS are vital for facilitating the development of tumors. Dysregulated PI3K/Akt signaling is crucial in
regulating numerous molecular processes that elevate ROS levels. This occurs either by directly
inuencing mitochondrial energy production and activating NADPH oxidases (NOXs), or indirectly by
generating ROS as a metabolic byproduct6.Comprehending the intricate relationship between ROS and
PI3K/Akt signaling is important for devising effective therapeutic approaches to combat tumors reliant
on this pathway. This signicance is underscored by recent clinical trials showing limited ecacy of
PI3K/Akt pathway inhibitors and the emergence of resistance. It could lead to the discovery of new

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biomarkers and metabolic vulnerabilities, as well as the development of more potent therapeutic
combinations that disrupt redox balance and specically target PI3K-driven tumors6,7.Chemotherapy
and radiotherapy stand as primary treatments for cancer patients, and they induce apoptosis in cancer
cells partly by increasing ROS levels. Agents like 5-FU, cisplatin, and other chemotherapeutic drugs trigger
ROS production by inuencing the electron transport chain. The heightened ROS levels then trigger
cascades such as caspase activation, release of cytochrome C, and DNA damage, ultimately leading to
apoptosis 6. Given the potential pro-oxidative properties of herbs, we incorporated a chemotherapeutic
compound alongside 5-FU in our model. This approach allows for the concurrent inhibition of the
PI3K/Akt pathway while maintaining or increasing signicant levels of ROS and reactive nitrogen species
(RNS). Such elevation can effectively promote the induction of apoptosis.
PI3K pathway promotes metastasis by promoting tumor neovascularization, which is required for the
metastatic spread of tumors. PI3K forms a complex with E-cadherin, β-catenin, and VEGFR-2 and is
involved in endothelial signaling mediated by VEGF through the activation of the PI3K/Akt pathway 8.
The PI3K/Akt signaling pathway also promotes TNF-induced endothelial cell migration and regulates
tumor angiogenesis.TNF-α plays a signicant role in promoting the survival and metastasis of lung
cancer. The levels of TNF-α in tumor tissues and serum collected from patients with NSCLC substantially
increase with the clinical stage of the tumor. Additionally, matrix metalloproteinases (MMPs) and
cyclooxygenase-2 (COX-2) contribute to tumor angiogenesis. COX-2 stimulates endothelial angiogenesis
primarily through the upregulation of the antiapoptotic protein Bcl-2 and activation of the PI3K/Akt
signaling pathway 5.A recent study indicates thatCOX-2inhibitionprotects
againsthypoxia/reoxygenation-inducedcardiomyocyteapoptosis
via
AKT-dependent enhancement of
iNOS expression 9. Preconditioning the cells with the COX-2 inhibitor NS398 resulted in decreased
expressions of TNFα, prostaglandin E2 (PGE2), and interleukin-6 (IL-6) pro-inammatory factors. It was
observed that inhibiting COX-2 could mitigate the increased release of nitric oxide (NO) and expression of
inducible nitric oxide synthase (iNOS) induced by Giardia. Studies unveiled a crucial role of COX-2 in
modulating the pro-inammatory response and defense-related NO production in interactions between
Giardia and macrophages10. MMP-2 transcriptional suppressiondecreased VEGF, PI3K protein levels,
and AKT phosphorylation in lung cancer cells. MMP-2 suppression disrupted phosphatidylinositol 3-
kinase (PI3K) dependent VEGF expression; ectopicexpression of myr-AKT restored VEGF inhibition.
Studieswith either function blocking integrin-αVβ3 antibody or MMP-2 specic inhibitor (ARP-100)
indicate that suppression of MMP-2 decreased integrin-αVβ3-mediated induction of PI3K/AKT leading to
decreased VEGF expression. A549 xenograft tissue sections from mice thatweretreated with MMP-2
siRNA showed reduced expression of VEGF11.Numerous studies provide evidence of the inuence of
natural compounds on the components within this pathway. For instance, the natural ubiquinone
Coenzyme Q0 (CoQ0) has shown ecacy against the proliferation of various cancer cell lines such as
HepG2, A549, and SW480, promoting apoptosis by elevating ROS levels. Treatment with LY249002, which
blocks the PI3K/AKT pathway, signicantly reduced NFκB activation and MMP-9 levels .

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The analysis of the literature reveals that TNF-α, VEGF-α, COX-2, MMP-2, Caspase-3, and NOS are
interconnected in various cancer processes. Their interrelations are mediated through the PI3K/Akt
signaling pathway. This study focused on employing a combination of natural compounds and a
chemotherapeutic agent to target these specic components to attain potent anticancer effects.
We hypothesized that phytoextracts, either single or in combination with chemotherapy compounds, may
effectively modulate the immune system(TNFa/COX-2/Arginase),inhibit angiogenesis andprogression
ofmetastasis(VEGFa/NOS/NO/MMP-2)via regulation of PI3K/Aktsignaling pathway. In our previous
researchworkswe showedthepromisinganticancer effect of
Rumex obtusifolius
(RO) inan
in vivo
experimental breast cancer rat model, in parallel, its cytotoxic effect was elucidated against two cell
cultures: MCF-7, and HT2912.The combined anticancer effects of inhibitors targeting the metabolic
pathway of L-arginine have also been investigated. The ndings demonstrate signicant anti-tumor
properties, including reductions in tumor size, number, and mortality. Changes in various biochemical
parameters in the blood, associated with participants in the metabolic pathway of L-arginine, were
observed. However, alterations in key factors that would provide a detailed understanding of the exact
molecular mechanisms underlying the anticancer effects were not noted13. This research elucidated the
mechanisms of the anticancer effects of
R. obtusifolius
extract, both independently and in combination
with the conventional chemotherapeutic compound 5-FU. Specically, the impact of the herbal extract of
R. obtusifolius (0.25mg/mL) on the TNFα-VEGFα/PI3K/Akt/NOS/COX-2-MMP-2 pathway was assessed
separately and in combination with 5-FU (40µM) in non-small lung adenocarcinoma A549 cells.In
addition, the possible interaction of compounds identied by HPLC/MS/MS of RO on PI3K/Akt in the
active site pocket was also elucidated by
in silico
study in comparison with its ligand care. By uncovering
the molecular mechanisms of anticancer effects, identifying the most active phytochemicals, and
clarifying the specic targets of these compounds, there is potential to inhibit, prevent, or delay cancer
development with minimal side effects.
2. RESULTS
2.1. Modulation of growth-inhibiting properties of 5-
Fluorouracil with
R. obtusifolius
extract tested by MTT
assay
Growth inhibiting properties of RO seed ethanol extract on A549 cancer cells were tested with
in vitro
MTT assay. Based on the obtained data the RO extract even at the highest tested concentration (0.5 mg
DW/mL) and at any of the tested exposure times (4, 24, and 72 h) did not show any statistically
signicant impact on the growth of A549 cells (Fig.1A).
Further modulating activity of none-inhibitory concentrations of RO extract (0.25 mg DW/mL) toward
uorouracil (5-FU) on A549 cells (Fig.1B, C, D) was investigated using
in vitro
MTT assay. Based on the
obtained data, statistically signicant strong modulation of 5-FU was observed at 24 h exposure time on

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all tested 5-FU concentrations (Fig.1C). Considerable modulation was detected on 4 h exposure time as
well (Fig.1B). However, during 72 h exposure time, no modulation was observed (Fig.1D).
2.2. R. obtusifolius extract alone and in combination with 5-FU downregulate quantitative changes of
TNFa, VEGFa, COX-2, and MMP-2.
Quantitative changes in TNFα-related COX2 and VEGF-related MMP2 were assessed in the next step
using ELISA. RO extract decreased the quantities of TNFα (Fig.2, A) and VEGFα (Fig.2, B) in the cell
medium by 40% and 33%, respectively, and the quantities of COX-2 and MMP2, respectively by 31.5% and
33%.
5-FU alone does not affect TNFa/COX-2, but when combined with the plant, the effect is greater than with
the plant alone or 5-FU alone (p ≤ 0.05). The most visible modulatory effect on each other is TNFα
(combined effect decreases it close to the value of RO), VEGFa (33% and 50% decrease, respectively,
compared to 5-FU and RO alone, p ≤ 0.01), and in the case of COX-2 (the reduction is about 90% for 5-FU
and 80% for RO). In the case of COX-2, a synergism phenomenon can be documented.
2.3. Regulation of PI3K/Akt pathway
To further understand the cause of decreased TNFα, VEGFa, COX-2, MMP-2 and its effect on the cell, the
downstream signaling pathway from TNFα and VEGFa to PI3K/Akt, whose dysregulation is characteristic
of cancer, was elucidated.
We demonstrated that RO acts as an inhibitor of the Pi3K/Akt pathway. Particularly, quantitative
reduction of total and phosphorylated PI3K and Akt was observed after the treatment of A549 cells with
RO extract (Fig.3, A). The combination of RO and 5-FU reduced the amount of total and phosphorylated
PI3K by about 2.5-fold compared to the control cells (p ≤ 0.01). RO alone does not affect these two forms
of PI3K, and 5-FU only affects the total amount of PI3K. RO and 5-FU reduce both phosphorylated and
total Akt amounts, and the combination can increase this effect several-fold, showing a synergistic effect
(Fig.3, B). We assumed one of the ways the effects of RO is the TNFa-PI3K-Akt cascade. Changes in the
amount and activity of NO, MDA, Arginase, and NOS participants were elucidated to understand where the
effect goes next.
2.4. Stimulation of RNS and ROS by regulation of Arginase
and NOS activity
Since in most cases COX-2 interacts with arginase and NOS 14, changing the concentration of VEGFa
with NO and NOS and TNFa with ROS and RNS was valuable and necessary to observe the activities of
arginase, NOS, and quantitative changes of NO and MDA (Fig.4).
The results showed that by reducing the amount of VEGF, the plant also inhibited the activity of NOS (p ≤
0.001), but increased the quantity of NO (p ≤ 0.05, Fig.4, B and C). Since the activity of NOS is depressed,
but the amount of nitrite ions is increased, the increase in the amount of RNS may be due to the increase

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in ROS. ROS and generated NO most likely lead to RNS generation and ROS/NO-mediated apoptosis. The
latter is conrmed by the high quantity of MDA, which is promoted by the plant (p ≤ 0.01, Fig.4, D).
Normally in cancer, activation of the PI3K/Akt pathway leads to increased ROS 15,16, and conversely,
increased ROS leads to activation of this pathway, but this phenomenon was not observed in our
experiments. In this case, the plant adjusted in such a way that by suppressing the PI3K/Akt pathway, it
increases the amount of ROS and RNS. This indicates a multi-target effect of the compounds contained
in the herb. To verify that increased RNS and ROS quantities, as well as inhibition of the PI3K/Akt
pathway, should lead to apoptosis, Caspase-3 activity was assessed and chromatin staining was
performed with Hoersch 3328 dye to observe segmentation and condensation.
2.5. Assessment of apoptosis rate by Hoechst 33258
staining
Hoechst 33258 staining allows discrimination of apoptotic and non-apoptotic cells based on
morphological changes of the nuclei (Fig.5). The nuclei in normal cells exhibited evenly dispersed and
weak uorescence, as well as smooth edges (Fig.5, A). Apoptotic cells can be distinguished by the
condensed chromatin (Fig 5, B, marked with arrows) and rough edges of their nuclei (Fig.5, C, marked
with arrows), as well as signs of nuclear fragmentation (Fig.5, D, marked with arrow). The results indicate
that incubating A549 cells with 5-FU, RO, and their combination 5-FU + RO for 24 h signicantly increased
the rate of apoptotic cells (Fig 5, E). In the control, the rate of apoptosis was 2.40 ± 0.56%. Treatment
with 5-FU or RO alone signicantly increased the rate of apoptotic cells up to 14.70 ± 1.83% and 11.30 ±
0.98%, respectively. At the same time, the combination of 5-FU + RO elevated the rate of apoptotic cells up
to 29.5 ± 4.94% (p < 0.01). Thus, the apoptosis of A549 cells induced by the combination of 5-FU + RO
was signicantly higher when compared with that of 5-FU or RO alone (p < 0.05).
Since Caspase-3 is active in the execution phase of apoptosis, we next used colorimetric assay to
determine whether it was activated following treatment with 5-FU, RO, and their combination 5-FU + RO for
24 h (Fig 5, F). The spectrophotometric analysis revealed a signicant increase in caspase-3 activity in
all treatment variants which was more pronounced in cells treated with 5-FU + RO (p < 0.01).
2.6. The interaction of potential compounds with PI3K and
Akt
The docking of the top compounds present in the RO ethanolic extract (Suppl. Table) by Autodock Vina
was performed on the binding pockets of AKT (PDB ID: 2JDO) and PI3K (PDB ID: 6 AUD) (Fig, 6 and 7).
Each crystallographic structure’s binding pocket contained a bound ligand, which was extracted and
rocked as a control. The docking results are presented in Table1. Based on the average score on two
target proteins, none of the compounds showed a better docking score (the lower, the better) than the
redocking score of the PI3K control. However, 14 out of 17 investigated compounds demonstrated better
docking scores than the redocking score of the AKT control ligand. Therefore, this shows that the
extracted compounds of interest may have a better anity toward AKT.

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Table 1
Docking results of RO on PI3K and Akt.
Compound AKT (2JDO) PI3K (6AUD) Average
PI3K-control ligand  -9.6 -9.6
Endocrocin -9.1 -9.2 -9.15
Emodin -8.8 -9.2 -9
Luteolin -8.5 -8.8 -8.65
Quertecin -8.5 -8.4 -8.45
Epicatechin-gallate -8.5 -8.3 -8.4
Eriodictol -8.3 -8.5 -8.4
Quercetin-3-D-galactoside -8.3 -8.5 -8.4
Hamamelofuranose -8.4 -8.2 -8.3
Isorhamnetin-3-O-glucoside -8 -8.5 -8.25
Catechin -7.8 -8.5 -8.15
Epicatechin -8.1 -7.8 -7.95
Apigenin-sulfate -8.1 -7.6 -7.85
Qurecetin-diglucoside -7.5 -7.8 -7.65
4-glucogallic acid -7.5 -6.9 -7.2
AKT-control ligand -7  -7
Procyanidin-dimer -7.1 -6.3 -6.7
Protocatechuic-acid -5.7 -5.6 -5.65
Hydroxybenzoic-acid -5.5 -5.5 -5.5
Understanding the absorption, distribution, metabolism, and excretion (ADME) properties of drug
candidates is essential due to their profound inuence on the pharmacokinetics, ecacy, and potential
side effects of therapeutic agents 17. For this reason, we computationally analyzed the ADME
characteristics of the 17 leading compounds using the SwissADME online service, and key features were
outlined in Table2. A variety of physical and chemical properties such as molecular weight (MW),
partition coecient (LogP), hydrogen bond acceptors (HBA), and hydrogen bond donors (HBD) were
assessed. Based on these calculations, we examined how the compounds adhere to Lipinski’s rule of 5
which is a set of rule-of-thumb guidelines in medicinal chemistry that predict oral bioavailability in drugs,
stating that a molecule will likely be an effective oral medication when it does not violate more than one
of these rules: no more than 5 hydrogen bond donors, 10 hydrogen bond acceptors, a molecular mass

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less than 500 Daltons, and a LogP not exceeding 5 18. The results illustrate that 10 out of the 17
compounds did not violate Lipinski’s rule of 5, indicating their potential to be drug candidates.
Remarkably, these 10 compounds included the top 4 ones having the best docking score (namely:
endocrocin, emodin, luteolin, and quercetin).
Table 2
ADME properties of RO extract phytochemicals.
Compounds MW HBA HBD LogP Num of Viol
Endocrocin 314 7 4 1.43 0
Emodin 270 5 3 1.87 0
Luteolin 284 6 4 1.73 0
Quercetin 299 7 5 0.17 0
Epicatechin gallate 442 10 7 1.25 1
Erodictol 280 6 4 0.84 0
Quercetin 3-D-galactoside 464 12 8 -0.25 2
Hamamelofuranose 180 6 5 -1.94 0
Isorhamnetin 3-O-glucoside 478 12 7 -0.15 2
Catechin 290 6 5 0.85 0
Epicatechin 290 6 5 0.85 0
Apigenin sulfate 364 9 3 1.28 0
Qurecetin diglucoside 607 17 11 -2.8 3
4 glucogallic acid 332 10 7 1.9 1
Procyanidin dimer 562 12 10 0.54 3
Protocatechuic acid 151 4 3 0.4 0
Hydroxybenzoic acid 134 3 2 0.72 0
Accordingly, we decided to examine further the 4 compounds mentioned above. In particular, we analyzed
the binding mode of their best docking scores for the two proteins in addition to a 3D visual inspection
(Figs.6 and 7). Based on the analysis, emodin forms hydrogen bonds with Asp293 and Lys160 amino
acids of AKT. The interactions with 9 other amino acids are hydrophobic (Fig.6, A). With PI3K, emodin
forms hydrogen bonds with Tyr867 and Asp 964. The interactions of emodin and Glu880, Val882, Trp812,
Ile831, Ile963, and Ile879 interactions are hydrophobic (Fig.7, A). In the case of endocrocin and AKT
interactions, there are hydrogen bonds with Glu230, Ala232, Lys160, and Asp293. In addition, there are
hydrophobic interactions with 8 other amino acids (Fig.6, B). With PI3K, endocrocin forms only one

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hydrogen bond, namely with Asp963. Furthermore, there are hydrophobic interactions with 12 additional
amino acids (Fig.7, B).
Luteolin forms hydrogen bonds with Lys181 and Asp293 amino acids of AKT, complemented by 8 other
hydrophobic interactions (Fig.6, C). With PI3K, luteolin forms one hydrogen bond with Tyr867 and Ala885
and two hydrogen bonds with Val882. Furthermore, there are hydrophobic interactions with 8 other amino
acids (Fig.7, C). Finally, Quercetin forms four hydrogen bonds with Asp293, Lys181, Ala232, and Glu230
of AKT. In addition, there are 8 hydrophobic interactions (Fig.6, D). With PI3K, quercetin forms hydrogen
bonds with Ser806 and Val882. This is complemented by hydrophobic interactions with 10 other amino
acids (Fig.7, D). The specic interactions between emodin, endocrocin, luteolin, and quercetin with the
target proteins AKT and PI3K highlight their potential to modulate the function of these proteins. It is
noteworthy that while all four compounds formed hydrogen bonds with key residues in AKT, the variety
and number of interactions differ, which may inuence their anity and specicity. The prevalence of
hydrophobic interactions alongside hydrogen bonds, particularly with PI3K, underscores the potential of
these compounds to anchor rmly within the binding pockets, possibly conferring stable interactions and
effective inhibition. Such differential binding patterns could translate into varying degrees of therapeutic
ecacy and selectivity among these compounds.
3. DISCUSSION
Although chemotherapy is the most commonly used treatment, it also kills normal cells, causing many
side effects. Therefore, it is urgent to develop novel alternative therapeutic strategies to overcome these
problems. Many phytochemicals have been isolated from various plants that have regulatory effects on
the targets that are considered in our study. We hypothesized that RO extract, either single or in
combination with chemotherapy compounds, may effectively modulate the immune system (TNFa/COX-
2/Arginase), inhibit angiogenesis and progression of metastasis (VEGFa/NOS/NO/MMP-2) via regulation
of PI3K/Akt signaling pathway.
During the study, the effect of RO on 5-FU-induced apoptosis in A549 cells was examined. MTT assay
showed that RO alone did not induce noticeable inhibition of growth of A549 cells. This is interesting as
according to our previous research, RO seed extract expressed strong cytotoxic activity on two tested
cancer cell lines (HT29 and MCF-7) at even 0.125 mg DW/mL concentration 12. Although RO extract did
not possess growth-inhibiting activity in A549 cells, we assumed that acting synergically could increase
the cytotoxic properties of chemotherapeutic agents like Fluorouracil. The speculations made based on
earlier studies, where RO extract when combined with NG-nitro-L-arginine methyl ester (NOS inhibitor) and
NG-hydroxy-nor-L-arginine (arginase inhibitor) increased their therapeutic effects probably by regulating
redox homeostasis 12. The experiments were done in an in
vivo
rat mammary carcinogenesis model and
based on the obtained data RO extract possessed a modulating effect on 5-FU. It is important to point out
that RO expressed a synergic effect rather than an additive as RO extract did not show any growth-
inhibiting effect at the concentration used in combined treatment. The modulating properties of RO seed
extract could have great importance, taking into account that modulation of the anticancer effects of

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chemotherapy drugs through plant extracts or derived compounds is a promising strategy to overcome
drug resistance and reduce side effects 19.
To further understand biochemical mechanisms underlying modulating properties RO on 5-FU different
biochemical parameters were explored including quantitative changes of TNFa, VEGFa, COX-2, and MMP-
2, regulation of PI3K/Akt pathway, assessment of apoptosis,
etc.
We considered the PI3K/Akt signaling
pathway taking into account that it is a major signaling pathway in various types of cancer. It controls the
hallmarks of cancer, including cell survival, angiogenesis, inammation, metastasis, and metabolism.
According to the literature, the vascular endothelial growth factor (VEGF) is the most potent stimulant of
angiogenesis and can activate NOX isoforms either directly or indirectly through PI3K/Akt induction 6.
After activation by VEGF, Akt promotes the proliferation, migration, and survival of endothelial cells, thus
affecting angiogenesis. This nding also provides lateral support for the conclusion that endothelial nitric
oxide synthase (eNOS), which controls vascular tone, is a specic substrate of Akt1 in endothelial cells 5.
The subsequent production of superoxide and hydrogen peroxide is necessary for the regulation of
transcription factors, which promote angiogenesis, including NF-κB, MMPs, COX-2, and HIF-1α. COX-2 is
up-regulated in many malignant cancers, including gastric, colon, breast, esophagus, pancreas,
hepatocellular carcinoma, and NSCLC. The overexpression of COX-2 effectively potentiates the cisplatin
and other chemotherapy drug resistance of NSCLC cells by promoting EMT. NS398, a COX-2 inhibitor,
induced apoptosis and additionally potentiated chemosensitivity to cisplatin-mediated apoptosis in
human non-small cell lung cancer by targeting the AKT 20. Studies indicate MMP-2 siRNA inhibited lung
cancer cell-induced tube formation of endothelial cells
in vitro
; the addition of recombinant human-MMP-
2 restored angiogenesis.
Our research obtained results showed that RO extracts signicantly decreased the quantities of TNFα,
VEGFa, COX-2, and MMP2 in A549 cancer cells in combination with 5-FU. Inammatory cytokines, growth
factors, and their receptors, such as TNF, TNFR, VEGF, and VEGFR, act as positive regulators to transmit
signals to mTOR through the PI3K/Akt pathway 5. PI3K/Akt signaling blocks the expression of
proapoptotic proteins reduces tissue apoptosis and increases the survival rate of cancer cells. Akt inhibits
the proapoptotic factors Bad and procaspase-9 through phosphorylation and induces the expression of
the proapoptotic factor Fas ligand. In addition, Akt activation is associated with resistance to increased
apoptosis induced by TNF. During routine chemotherapy, no treatment interval exists, allowing resistant
cells to be generated and leading to tumor regeneration. The PI3K/Akt signaling pathway is important for
the drug resistance of different types of cancer, such as lung cancer and esophageal cancer. For NSCLC
cells with high Akt expression, the use of PI3K/Akt signaling pathway inhibitors increases their cell
apoptosis induced by chemotherapy and reduces their resistance to chemotherapy 21. Therefore,
inhibition of the PI3K/Akt signaling pathway, which has been shown to regulate cancer cell apoptosis can
serve as a new direction for future research on cancer treatment 5. The importance of this work is also
that the obtained results touch on such a question as plant pro-oxidation. Increased malondialdehyde
and nitrite ions are present in the cellular environment, indicating increased ROS and RNS. According to
the literature, the latter is also regulated by Akt 6. In addition, the change in Akt activity also affects the

Page 12/28
regulation of Caspase-3 activity and therefore apoptosis. Given that cell leakage may be a factor in RNS-
and ROS-mediated apoptosis, the alteration of Caspase-3 activity was observed. The possibility of
chromatin segmentation and condensation under the effect of herb and combination was also studied by
Hoechst stain to further elucidate the stimulation of apoptosis. Hoechst staining revealed an increase in
the rate of apoptotic cells after treatment with 5-FU (40 uM) or RO alone. The combination of 5-FU + RO
synergistically evoked Caspase-3 activity, thus RO elevated the frequency of 5-FU-induced apoptosis. The
results obtained in the case of combinations of herbs and chemotherapeutic agents showed a decrease
in TNFa and VEGFa and an increase in NO and MDA quantity. The latter is indicative of ROS/RNS-
mediated cytotoxicity of herbs in the tumor microenvironment. A decrease in COX-2, Arginase, and MMP-2
was observed in the A549 under the inuence of herb extracts and combinations. The work is also
highlighted by considering the herb together with a classical chemotherapeutic compound. As a classic
chemotherapeutic compound, 5-uorouracil was used, which has a broad spectrum effect and is used in
chemotherapeutic cocktails 22. It was important to observe the herb-drug interaction and identify whether
there is a synergistic effect between this herb and 5-FU. Even though several works show the anticancer
effect of various herbs, and our
in vivo
model showed the effective use of this herb against breast cancer
in combination with L-arginine metabolic pathway inhibitors, there are few works, which revealed the
mechanisms by which this effect occurs. The work is also valuable in that, by using a multi-component
decoction of the medicinal plant, the possible protection of these compounds against PI3K and Akt
enzymes was also claried by parallel
in silico
research. The research has 3 main ndings. Elucidated the
mechanisms of the anticancer effect of an unexplored herb by looking at the TNFa/PI3K/Akt/COX-
2/ARG/NOS/ROS/RNS/Caspase-3 pathway, demonstrated herb-drug synergistic interactions affected by
different compounds, which were revealed based on
in silico
studies. These compounds had also the
greatest anity for PI3K/Akt, which may play a key role in RO extracts with promising anticancer
properties. An important nding of the work is also the fact that the quantitative images of MDA and
nitrite anions differ from our previous studies
in vivo
12. During earlier
in vivo
studies on the rat mammary
carcinogenesis model, a decrease in the amount of malondialdehyde and nitrite ions was observed in the
blood, while an increment of their quantity was detected in the cell culture. The circumstance of selective
effect is also seen here, thanks to which it is possible to deliver these active compounds to the tumor
environment itself with the use of delivery systems and to leave a point effect on the targets presented 23.
The purpurin (naturally occurring anthraquinone) could effectively kill A549 cancer cell lines and lead to
cell death, thus conforming to increased cytotoxicity, production of ROS-mediated enhancement of lipid
peroxidation, nuclear fragmentation, and apoptosis. The study demonstrates that purpurin inhibits the
phosphorylated PI3K/AKT molecules mediated cyclin-D1, thereby inducing apoptosis by observing
increased proapoptotic mediators Bax cleaved PARP, cytochrome-c, caspase-9, and caspase-3; and
decreased Bcl-2 expression in the lung cancer cell lines 24. Later we tried to elucidate the main
compounds that contribute promising anticancer properties of RO extract. In our previous research works
more than 200 phytochemicals were identied in the ethanol extract of RO ethanol extract based on LC-Q-
Orbitrap-HRMS analysis. The full list of identied compounds in RO ethanol extract is presented in earlier
work 12. Further
in silico
analyses revealed that 4 of these compounds (namely: endocrocin, emodin,

Page 13/28
luteolin, and quercetin) have a high anity for PI3K and Akt, indicating that the downregulation of the
PI3K/Akt pathway by the herbs may be responsible for their benecial effects on the quantitative
changes in the explored factors and enzymes. The results demonstrate that all 4 compounds form at
least 2 hydrogen bonds and at least 6 hydrophobic interactions with amino acids of the binding pockets
of both AKT and PI3K. The only exception is the endocrine-PI3K interaction, where there is only one
hydrogen bond. Nevertheless, this is amply compensated with an additional 12 hydrophobic interactions.
The analysis indicates strong interactions in the case of all 8 ligand-protein pairs, which have the
potential to change both proteins’ function and achieve biological modulation of physiological pathways.
These ndings imply that the unique binding patterns of these compounds may contribute to varying
therapeutic ecacies and selectivities, highlighting their promising potential for modulating the functions
of AKT and PI3K.
Literature data partially conrms the obtained results based on
in silico
studies. Particularly, luteolin, a
bioactive avone derivative present mainly in its shell, exerts breast cancer-inhibiting properties through
an anti-angiogenesis mechanism by inhibiting VEGF production and its binding with the receptor 23. In
addition, it also downregulates epithelial-mesenchymal transition markers and lowers metastatic activity.
Studies have shown that another compound quercetin reduces tumor weight by targeting VEGFR2
through the Akt/mTOR/P70S6K signaling pathway 25. Emodin, which is another selected compound
based on
in silico
experiments, inhibits cancer growth by suppressing the expression of MMP7, MMP9,
VEGF, EMT, N-cadherin, b-catenin, and Snail based on literature data. It also inhibits the Wnt/b-catenin
signaling pathway by downregulating target genes, including c-Myc, Cyclin-D1, and TCF4. According to
the literature, endocrocin is reported to have anticancer properties, although there is a lack of available
data about the possible mechanisms of its action 26. Based on
in silico
studies, we assumed that these 4
compounds could have important contributions to the overall promising anticancer properties of RO
extract. Further in vitro and in vivo evaluation of their anticancer potential combined and in different
combinations could have great importance.
In conclusion, this study revealed the potential of
R. obtusifolius
seed alcoholic extract as an adjunct
therapy in cancer treatment, specically in combination with the classical chemotherapeutic agent 5-
uorouracil. The study allowed us to nd several key insights into the mechanisms underlying the
anticancer effects of RO and its synergy with 5-FU. We extensively explored the
TNFa/VEGFa//PI3K/Akt/COX-2/ARG/NOS/ROS/RNS/Caspase-3 pathway during the study, revealing a
complex interplay of factors inuenced by RO and 5-FU. The combination of RO and 5-FU demonstrated a
synergistic effect on various cellular components. This nding suggests that RO, while not directly
inhibiting the growth of A549 cells on its own, can enhance the cytotoxic properties of 5-FU, potentially
leading to more effective cancer cell eradication. The
in silico
analysis identied specic compounds
within RO with high anities for PI3K and Akt, hinting at their potential role in mediating the observed
therapeutic effects. This computational approach deepens our understanding of the molecular
interactions involved. Notably, the study revealed the selective effects of RO on MDA and nitrite ions in
different environments. This selective action suggests the possibility of targeted drug delivery systems to

Page 14/28
achieve localized therapeutic effects while minimizing systemic side effects. Overall, this research
contributes signicantly to the eld of cancer therapeutics by unraveling the complex molecular
mechanisms underpinning the anticancer effects of RO and its synergistic relationship with 5-FU. The
detailed molecular analysis reveals that emodin, endocrocin, luteolin, and quercetin present in RO extract
exhibit distinctive interaction proles with the target proteins AKT and PI3K These ndings pave the way
for further investigations into the development of novel, targeted cancer treatment strategies that harness
the potential of medicinal plants like
R. obtusifolius
in the research.
4. Methods
4.1. Chemicals and reagents
All chemicals were purchased from Sigma-Aldrich (USA) and Abcam (UK). Antibodies against TNFa
(ab46087), VEGFa (ab193555), MMP-2 (ab92536), COX-2 (ab38898), PI3K and phosphorylated (p)-PI3K
(ab191606), as well as ELISA kits for AKT and p-AKT (ab179463) were purchased from Abcam.
4.2. Plant material
The seeds of
Rumex obtusifolius
L. were harvested from the Tavush region of Armenia (1400–1600 m
height above mean sea level) according to the protocol described before 27. Identication of plant
material was carried out at the YSU Department of Botany and Mycology by Dr. Narine Zakaryan. Plant
materials were deposited at the Herbarium of YSU, where the Voucher specimen serial number was given
(ERCB 13208). The collection of plant material complied with relevant institutional, national, and
international guidelines and legislation.
Rumex obtusifolius
L., commonly known as broad-leaved dock, is
an edible plant widely distributed and commonly found throughout Armenia. It is not on the list of
Endangered species in Armenia( https://worldrainforests.com/biodiversity/en/armenia/EN.html /
https://www.iucnredlist.org/search?query=Rumex%20obtusifolius%20&searchType=species /
https://cites.org/eng/search?search_api_fulltext=Rumex+obtusifolius+ ). The plant is not only prevalent
in natural settings but also routinely collected by local populations for culinary purposes. There is no
specic prohibition or regulatory constraint on the collection of this plant in Armenia, and it is a common
sight at local markets, where it is sold after being gathered from the wild. This widespread availability
and cultural integration into local diets supports the ethical sourcing and utilization of
Rumex
obtusifolius
for research purposes under the conditions described in our study. For our research, we
specically collected only the seeds of
Rumex obtusifolius
. This method of collection ensures minimal
impact on the natural populations of the plant, as it does not involve uprooting or damaging the plants
themselves. We ensure our research practices are sensitive to ecological and conservation concerns, even
in cases where no formal collection restrictions exist. As such, our study strictly adheres to general ethical
guidelines for botanical research, despite the lack of specic regulations surrounding the collection of
Rumex obtusifolius
in Armenia.
4.3. Plant crude extract

Page 15/28
The grounded seeds were extracted by maceration with 96% ethanol at a 10:1 solvent-to-sample ratio
(v/w). Stock solutions of 50 mg DW/mL crude ethanol extract were prepared as described earlier 28. The
percent yield was 10.60 ± 2.31%.
4.4. Cell cultures
Human lung adenocarcinoma A549 cell culture was obtained from ATCC (cat # CCL-185) and maintained
in DMEM medium supplemented with L-glutamine (2 mmol/L), sodium pyruvate (200 mg/L), fetal bovine
serum (100 mL/L), and antibiotics (100 U/mL penicillin and 100 µg/L streptomycin). Cells were grown at
37°C under a humidied atmosphere with 5% CO2 in a Biosmart (Biosan, Latvia) as described before 29.
Cultured cells were regularly examined for the presence of mycoplasma contamination using the
Universal Mycoplasma Detection Kit from ATCC (Manassas, Virginia, USA).
4.5. MTT cytotoxicity test
The MTT test was performed as described previously 30 to assess the growth inhibition of A549 cells
exposed to different concentrations of the
R. obtusifolius
extract for 4, 24, or 72 h.
4.6. ELISA of TNFa, VEGFa, COX-2, MMP-2, and Akt.
A549 cells (2 × 105) were cultured in 12-well plates and incubated for 24 h. After incubation, the cell
medium (630 𝜇L) was replaced and the cells were treated with PBS and 1% Ethanol solution (Control,
A549C), 5-FU (40𝜇M), RO (0.25mg/mL), and RO + 5- FU (0.25mg/mL + 40𝜇M) for 24 h and then the
culture medium was harvested. TNFa, VEGFa, and MMP-2 in the supernatant were quantied according
to the manufacturer's instructions. Cells from each group were collected (trypsinized, neutralized,
centrifuged), lysed on ice with Lysis buffer, collected in a centrifuge tube, and further lysed for 10 min.
After centrifugation at 13,000 × g for 10 min at 4°C, the supernatant was collected. Changes in the levels
of COX-2 and Akt were measured using ELISA kits, according to the manufacturer's instructions. Protein
concentration in cell culture medium and lysates were measured with a Bradford method. Each test
sample (70 𝜇L) was added to three different passages, which were triplicated.
4.7. Cells preparation for Arginase, NOS, and NO activity,
MDA analysis
A549 cells were seeded in 24-well (5 × 104 cells per well) plates and incubated for 24 h. After incubation,
the medium in wells (450 𝜇L) was refreshed. The cells were treated with 50 𝜇L control or test compounds
with the following nal concentrations: PBS, 1% ethanol (Control, A549C), 5-FU (40𝜇M), RO (0.25mg/mL),
and RO + 5-FU (0.25mg/mL + 40𝜇M). After 24 h incubation, the supernatant without cells was discarded.
Cells from each group were collected (trypsinized, neutralized, centrifuged), lysed on ice with Lysis buffer,
collected in a centrifuge tube, and further lysed for 10 min. The supernatant was collected after
centrifugation at 13,000 × g for 10 min at 4°C. The levels of Nitrite anions, MDA, Arginase, and NOS were
quantied according to the methods described below 12,31. Each test sample (50 𝜇L) was added to ve
different passages, which were triplicated.

Page 16/28
4.8. NO quantity measurement
NO levels in the cell culture medium were determined as nitrite anions. Griess assay was used for
measurement as described before 32. 100 𝜇L Griess reactant was added to 100 𝜇L of each sample. The
supernatants were transferred to the tubes containing pellets of cadmium and incubated at room
temperature for 12 h to convert nitrate to nitrite. The samples’ absorbance was measured at λ = 550 nm
and the NO quantity was calculated based on a standard curve prepared with NaNO2.
4.9. MDA assay
MDA quantity in the cell culture medium was determined with a colorimetric assay using the Ohkawa
thiobarbituric acid-malondialdehyde method 33.
4.10. Arginase activity
The modied Diacetyl Monoxime colorimetric method assessed the arginase activity in A549 cell lysates
34.
4.11. NOS activity
Nitric oxide synthase activity (µmol citrulline/mg protein) in A549 cell lysates was measured by the
conversion of L-arginine to L-citrulline 35. 100µl of cell lysates was added to 200 mL of reaction mixture
(50 mmol/L Tris buffer, pH 7.4, containing 10 mmol/L dithiothreitol (DTT), 10 µmol/L tetrahydrobiopterin
(THB4), 10 µg/mL calmodulin, 1 mmol/L NADPH, 4 µmol/L avin adenine dinucleotide (FAD), 4 µmol/L
avin mononucleotide (FMN), and 2 µmol/L L-arginine). The assay was carried out at 37oC, and it was
terminated with 2 mL of ice-cold stop buffer (20 mmol/L CH3COONa, pH 5.5, containing 2 mmol/L EDTA,
and 1 mmol/L L-citrulline). Assays were systematically performed with Ca2+ (1 mmol/L CaCl2) or without
Ca2+ (0 mmol/L CaCl2) to measure total versus Ca2+-independent NOS activities. The Ca2+-dependent
NOS activity was calculated as total NOS activity minus Ca2+-independent NOS activity. All assays were
performed in triplicate on aliquoted samples (to avoid freezing/thawing cycles). The results were
normalized for protein content.
4.12. Phospho-PI 3 kinase p85 + Total In-Cell ELISA assay
A549 cells (1.5x104 cells per well) were seeded in the 96-well plates treated for tissue culture. After 24 h
incubation, the cell medium (180 𝜇L) was refreshed and the cells were treated with 20𝜇L control or test
compounds with the following nal concentrations: PBS, 1% ethanol solution (Control, A549C), 5-FU
(40𝜇M), RO (0.25mg/mL), and RO + 5-FU (0.25mg/mL + 40𝜇M). The calculations during the seeding of
the cells were done in a way to reached approximately 80% conuency at xation time. After 24 h
exposure, the medium was discarded and cells were xed with 100 µL of 4% formaldehyde in PBS.
Crystal Violet was used to stain cells for normalizing readings in 450nm for Phospho-PI 3 kinase p85 +
Total. The measured OD450 readings were corrected for cell number by dividing the OD450 reading for a
given well by the OD595 reading for that well. This relative cell number was then used to normalize each

Page 17/28
reading. Total and phospho-PI 3 kinase p85 were each assayed in triplicate using the phospho- and total
PI 3 Kinase p85 antibodies included in the PI 3 Kinase Kit. Phospho-PI 3 kinase p85 and Total PI3K levels
were measured using an In-Cell ELISA kit (ab207484), according to the manufacturer's instructions.
4.13. Caspase-3/CPP32 Colorimetric assay
A549 cells (5 × 105 cells per well) were cultured in 6-well plates and incubated for 24 h. Then, the cell
medium (900 𝜇L) was refreshed and the cells were treated with 10 𝜇L of PBS + 1% ethanol solution
(control, A549C) or test compounds with the following nal concentrations: 5-FU (40 𝜇M), RO (0.25
mg/mL), and RO + 5- FU (0.25 mg/mL + 40 𝜇M). After 24 h the cells were harvested. Each test sample
(100 𝜇L) was added to three different passages, which were triplicated. Cells were resuspended in 50 µL
of chilled Cell Lysis Buffer and incubated on ice for 10 minutes. Then, cell lysate was centrifuged for 1
min (10,000 x g). After that supernatant (cytosolic extract) was transferred to a fresh tube and put on ice
for immediate assay. Fold-increase in CPP32 activity has been determined by comparing these results
with the level of the uninduced control. Optical density values were corrected taking into account the
number of cells. All steps were performed according to the protocol presented in the Caspase-3/CPP32
Colorimetric Assay Kit (K106, BioVision) instructions.
4.14. Analysis of apoptosis by Hoechst 33258 staining
The percentage of apoptotic cells was evaluated as previously described 36. A549 cells (2×105 cells/mL)
were treated with vehicle or 5-FU (40 𝜇M), RO (0.25 mg/mL), and RO + 5- FU (0.25 mg/mL + 40 𝜇M) for 24
hours, respectively. After treatment cells were washed with PBS and xed with 4 %paraformaldehyde in
PBS for 10 min. Then cells were washed twice with PBS for 5 min and stained with Hoechst 33258
reagent (10 𝜇g/mL) for 10 mins at room temperature in the dark. Then cells were washed with PBS and
analyzed under a uorescence microscope (x250 magnication) (Zeiss, Germany). The Hoechst 33258
staining allows the identication of apoptotic cells based on nuclear morphology. Cells with typical
morphological changes, such as karyopyknosis, hyperuorescence, nuclear fragmentation, and apoptotic
bodies, were considered apoptotic. All variants were examined in duplicate. For each treatment variant,
500 cells were scored and the percentage of apoptotic cells was calculated as follows: % apoptotic cells
= (the number of apoptotic cells/500 cells)*100.
4.16. Preparation of Protein structures
The crystallographic structures of PI3K and AKT were procured from the Protein Data Bank (PDB)
database (https://www.rcsb.org/), using the identiers 6AUD and 2JDO, respectively. Visualization and
preliminary assessment of these structures were performed with the PyMOL Molecular Graphics System
(Schrödinger, LLC). The retrieved crystallographic structures were subject to preprocessing, which
involved removing extraneous entities such as water molecules, ions, and other non-protein moieties
contained within the structures. Simultaneously, the ligands co-crystallized with each protein structure
were separated and retained for redocking validation experiments. The resulting streamlined protein
structures were then used for docking explorations. The extracted ligands, on the other hand, were
reserved for ensuing redocking studies as controls.

Page 18/28
4.17. Docking
Ligand docking and binding site analysis with PyMOL and Autodock/Vina were used for docking 37. The
protein and ligand structures were prepared using Autodock Tools 38. During a typical procedure, the
"exhaustiveness" parameter was calibrated to 8 and standard parameters suggested by the program
creators were used to ensure the delity of the results. The compounds were sorted based on their
binding strengths. The 2D binding mode analysis of best docking scores was performed using LigPlot +
software (EMBL-EBI).
4.18. Statistic analysis
All results are presented as means ± SEM. We analyzed the data either by one-way ANOVA or by its non-
parametric analog Kruskal-Wallis test based on the normality test performed followed by Dunn's test was
used to evaluate the statistical signicance of the TNFa, VEGFa, MMP-2, COX-2, arginase, NOS, MDA,
nitrite anions, Caspase-3, and apoptosis rate results. The signicance of the results obtained for PI3K and
Akt was assessed using two-way ANOVA and Tukey's multiple comparisons tests. Statistical analyses
were performed using GraphPad Prism 8 software (San Diego, CA, USA), and a signicance level of p <
0.05 was deemed statistically signicant.
Abbreviations
Akt, protein kinase B; ANOVA, analysis of variance;Bcl-2, B-cell lymphoma-2; COX-2, cyclooxygenase-
2;CoQ0, Coenzyme Q0; ELISA, enzyme-linked immunosorbent assay; EMT, Epithelial-mesenchymal
transition; FAD, avin adenine dinucleotide; FMN, avin mononucleotide; HBA, hydrogen bond acceptors;
HBD, hydrogen bond donors, IκBα, an inhibitor of NF-κBα;IL-6, interleukin-6; MAPK, mitogen-activated
protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; MMP-2, matrix
metalloproteinase-2;MW, molecular weight; NOXs,NADPH oxidases;NF-κB, nuclear factor-κB; NOS, nitric
oxide synthase; NSCLC, non-small cell lung cancer; PI3K, phosphoinositol-3-kinase; PG E2, prostaglandin
E2;RO,
Rumex obtusifolius
extract; ROS, reactive oxygen species; TNF-α, tumor necrosis factor alpha;
THB4, tetrahydrobiopterin; VEGF, vascular endothelial growth factor.
Declarations
5. Availability of data and materials
The data used to support the ndings of this study are included in the articles.
6.Declaration of interest
The authors declare no conicts of interest in this article.

Page 19/28
7. Authors' contributions
The study's conception and design were the results of collective contributions from all authors. The
investigations and analysis of results were carried out by MG, NA, HJ, SH, EN, GS, and TH. MG and NA
wrote the manuscript.Assessment of apoptosis rate by Hoechst 33258 staining and analysis of
apoptosis performed by TH. The docking and ADME of the top compounds present in the RO ethanolic
extract were performed by SG. NA, MG, HJ, ZK, and AM directed theproject,corrected, and edited the
manuscript. All authors participated in the revision and approval of the nal version of the manuscript.
8.Acknowledgments
Plant materials were identied by Dr. Narine Zakaryan from the Department of Botany and Mycology at
Yerevan State University (YSU).This work was supported by the Science Committee of MESCS RA through
research projects numbered 21T-1F283, 21AG-1F068, and 23LCG-1F010.
References
1. Gordaliza, M. Natural products as leads to anticancer drugs. Clinical and Translational Oncology 9,
767–776 (2007).
2. Cragg, G. M. & Pezzuto, J. M. Natural Products as a Vital Source for the Discovery of Cancer
Chemotherapeutic and Chemopreventive Agents. Medical Principles and Practice 25, 41–59 (2016).
3. Hou, X. L.
et al.
Suppression of Inammatory Responses by Dihydromyricetin, a Flavonoid from
Ampelopsis grossedentata, via Inhibiting the Activation of NF-κB and MAPK Signaling Pathways. J
Nat Prod 78, 1689–1696 (2015).
4. Yin, S. Y., Wei, W. C., Jian, F. Y. & Yang, N. S. Therapeutic applications of herbal medicines for cancer
patients.
Evidence-based Complementary and Alternative Medicine
vol. 2013 Preprint at
https://doi.org/10.1155/2013/302426 (2013).
5. He, Y.
et al.
Targeting PI3K/Akt signal transduction for cancer therapy.
Signal Transduction and
Targeted Therapy
vol. 6 Preprint at https://doi.org/10.1038/s41392-021-00828-5 (2021).
. Koundouros, N. & Poulogiannis, G. Phosphoinositide 3-Kinase/Akt signaling and redox metabolism
in cancer.
Frontiers in Oncology
vol. 8 Preprint at https://doi.org/10.3389/fonc.2018.00160 (2018).
7. Fruman, D. A.
et al.
The PI3K Pathway in Human Disease.
Cell
vol. 170 605–635 Preprint at
https://doi.org/10.1016/j.cell.2017.07.029 (2017).
. Jiang, B. H. & Liu, L. Z. Chapter 2 PI3K/PTEN Signaling in Angiogenesis and Tumorigenesis.
Advances in Cancer Research
vol. 102 19–65 Preprint at https://doi.org/10.1016/S0065-
230X(09)02002-8 (2009).
9. Pang, L.
et al.
Cox-2 Inhibition Protects against Hypoxia/Reoxygenation-Induced Cardiomyocyte
Apoptosis via Akt-Dependent Enhancement of iNOS Expression.
Oxid Med Cell Longev
2016, (2016).
10. Zhao, Y.
et al.
COX-2 is required to mediate crosstalk of ROS-dependent activation of MAPK/NF-κB
signaling with pro-inammatory response and defense-related NO enhancement during challenge of

Page 20/28
macrophage-like cell line with Giardia duodenalis. PLoS Negl Trop Dis 16, (2022).
11. Chetty, C., Lakka, S. S., Bhoopathi, P. & Rao, J. S. MMP-2 alters VEGF expression via αVβ3 integrin-
mediated PI3K/AKT signaling in A549 lung cancer cells. Int J Cancer 127, 1081–1095 (2010).
12. Ginovyan, M.
et al.
Anti-cancer effect of Rumex obtusifolius in combination with arginase/nitric
oxide synthase inhibitors via downregulation of oxidative stress, inammation, and polyamine
synthesis. Int J Biochem Cell Biol 158, 106396 (2023).
13. Choudhari, A. S., Mandave, P. C., Deshpande, M., Ranjekar, P. & Prakash, O. Phytochemicals in cancer
treatment: From preclinical studies to clinical practice. Front Pharmacol 10, 1–18 (2020).
14. Abd El-Aleem, S. A., Abdelwahab, S., AM-Sherief, H. & Sayed, A. Cellular and physiological
upregulation of inducible nitric oxide synthase, arginase, and inducible cyclooxygenase in wound
healing. J Cell Physiol 234, 23618–23632 (2019).
15. Wang, S., Yan, Y., Cheng, Z., Hu, Y. & Liu, T. Sotetsuavone suppresses invasion and metastasis in
non-small-cell lung cancer A549 cells by reversing EMT via the TNF-α/NF-κB and PI3K/AKT signaling
pathway. Cell Death Discov 4, (2018).
1. Yang, H. L.
et al.
Anti-EMT properties of CoQ0 attributed to PI3K/AKT/NFKB/MMP-9 signaling
pathway through ROS-mediated apoptosis. Journal of Experimental and Clinical Cancer Research 38,
(2019).
17. Alqahtani, S. In silico ADME-Tox modeling: progress and prospects.
Expert Opinion on Drug
Metabolism and Toxicology
vol. 13 1147–1158 Preprint at
https://doi.org/10.1080/17425255.2017.1389897 (2017).
1. Lipinski, C. A., Lombardo, F., Dominy, B. W. & Feeney, P. J.
Experimental and Computational
Approaches to Estimate Solubility and Permeability in Drug Discovery and Development q Settings
.
Advanced Drug Delivery Reviews
vol. 46 www.elsevier.com/locate/drugdeliv (2001).
19. Bazrafshani, M. S.
et al.
The prevalence and predictors of herb-drug interactions among Iranian
cancer patients during chemotherapy courses. BMC Complement Med Ther 23, 1–11 (2023).
20. Alhamed, A. S.
et al.
Blockade of store-operated calcium entry sensitizes breast cancer cells to
cisplatin therapy via modulating inammatory response. Saudi Pharmaceutical Journal 31, 245–254
(2023).
21. Sanaei, M. J., Razi, S., Pourbagheri-Sigaroodi, A. & Bashash, D. The PI3K/Akt/mTOR pathway in lung
cancer; oncogenic alterations, therapeutic opportunities, challenges, and a glance at the application
of nanoparticles.
Translational Oncology
vol. 18 Preprint at
https://doi.org/10.1016/j.tranon.2022.101364 (2022).
22. Sundaram, M. K.
et al.
Combinational Use of Phytochemicals and Chemotherapeutic Drugs Enhance
Their Therapeutic Potential on Human Cervical Cancer Cells. Int J Cancer Manag 12, (2019).
23. Parveen, A.
et al.
Phytochemicals targeting VEGF and VEGF-related multifactors as anticancer
therapy.
Journal of Clinical Medicine
vol. 8 Preprint at https://doi.org/10.3390/jcm8030350 (2019).
24. Bo, S.
et al.
Purpurin, a anthraquinone induces ROS-mediated A549 lung cancer cell apoptosis via
inhibition of PI3K/AKT and proliferation. Journal of Pharmacy and Pharmacology 73, 1101–1108

Page 21/28
(2021).
25. Lot, N.
et al.
The potential anti-cancer effects of quercetin on blood, prostate and lung cancers: An
update.
Frontiers in Immunology
vol. 14 Preprint at https://doi.org/10.3389/mmu.2023.1077531
(2023).
2. Khaliq, T., Akhter, S., Sultan, P. & Hassan, Q. P. Critical review on Rumex dentatus L. a strong
pharmacophore and the future medicine: Pharmacology, phytochemical analysis and traditional
uses. Heliyon 9, e14159 (2023).
27. Ginovyan, M., Petrosyan, M. & Trchounian, A. Antimicrobial activity of some plant materials used in
Armenian traditional medicine. BMC Complement Altern Med 17, 1–9 (2017).
2. Ginovyan, M., Hovhannisyan, S., Javrushyan, H. & Sevoyan, G. Screening revealed the strong
cytotoxic activity of Alchemilla smirnovii and Hypericum alpestre ethanol extracts on different cancer
cell lines. 10, 12–22 (2022).
29. Koss-Mikołajczyk, I., Kusznierewicz, B., Namieśnik, J. & Bartoszek, A. Juices from non-typical edible
fruits as health-promoting acidity regulators for food industry. LWT - Food Science and Technology
64, 845–852 (2015).
30. Koss-Mikołajczyk, I., Kusznierewicz, B., Wiczkowski, W., Sawicki, T. & Bartoszek, A. The comparison of
betalain composition and chosen biological activities for differently pigmented prickly pear (Opuntia
cus-indica) and beetroot (Beta vulgaris) varieties. Int J Food Sci Nutr 70, 442–452 (2019).
31. Avtandilyan, N., Javrushyan, H., Petrosyan, G. & Trchounian, A. The Involvement of Arginase and
Nitric Oxide Synthase in Breast Cancer Development: Arginase and NO Synthase as Therapeutic
Targets in Cancer.
Biomed Res Int
2018, 1–9 (2018).
32. Vodovotz, Y. Modied microassay for serum nitrite and nitrate. Biotechniques 20, 390–394 (1996).
33. Zeb, A. & Ullah, F. A Simple Spectrophotometric Method for the Determination of Thiobarbituric Acid
Reactive Substances in Fried Fast Foods. J Anal Methods Chem 1–5 (2016)
doi:10.1155/2016/9412767.
34. Avtandilyan, N., Javrushyan, H., Petrosyan, G. & Trchounian, A. The Involvement of Arginase and
Nitric Oxide Synthase in Breast Cancer Development: Arginase and NO Synthase as Therapeutic
Targets in Cancer.
Biomed Res Int
2018, (2018).
35. Combet, S., Balligand, J.-L., Lameire, N., Gon, E. & Devuyst, O.
A Specic Method for Measurement
of Nitric Oxide Synthase Enzymatic Activity in Peritoneal Biopsies
.
Kidney International
vol. 57
(2000).
3. Yang, X., Wang, Y., Luo, J., Liu, S. & Yang, Z. Protective effects of YC-1 against glutamate induced
PC12 cell apoptosis. Cell Mol Neurobiol 31, 303–311 (2011).
37. Seeliger, D. & De Groot, B. L. Ligand docking and binding site analysis with PyMOL and
Autodock/Vina. J Comput Aided Mol Des 24, 417–422 (2010).
3. Trott, O. & Olson, A. J. AutoDock Vina: Improving the speed and accuracy of docking with a new
scoring function, ecient optimization, and multithreading. J Comput Chem 31, 455–461 (2010).

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Figures
Figure 1
Growth rate of A549 cells treated with RO extract for 4, 24, and 72h (A). The growth-inhibiting effect of 5-
uorouracil separately and in combination with the none-inhibitory concentration of RO extract (0.25 mg
DW/mL) on A549 cells for 4 (B), 24 (C), and 72h (D). Results represent means ± SD from three
independent experiments; SD values did not exceed 15%.

Page 23/28
Figure 2
The inuence of
R. obtusifolius
extract alone and in combination with 5-FU on quantitative changes of
TNFa (A), COX-2 (B), VEGFa (C) and MMP-2 (D) in A549 cells. Control - A549C, 5-Fluorouracil - 5-FU
(40𝜇M),
Rumex obtusifolius
- RO (0.25mg/mL), ROFU - RO+5-FU (0.25mg/mL + 40𝜇M). Each test sample
was added to three different passages, which were triplicated (n=3, * - p≤0.05, ** - p≤0.01, ns – non-
signicant).

Page 24/28
Figure 3
Effect of RO, 5-FU, and their combination on PI3K/Akt pathway in A549 cells (A-PI3K, B - Akt). Total and
phospho-kinases were each assayed in triplicate using the phospho- and total Kinase antibodies included
in the PI 3 Kinase and Akt kits (n=3, * - p≤0.05, ** - p≤0.01, *** - p≤0.001, **** - p≤0.0001). p85-PI3K -
Phospho-PI 3 kinase p85, pS473AKT - phospho-Akt (Ser473).

Page 25/28
Figure 4
Stimulation of RNS and ROS by RO regulation of arginase (A) and NOS (B) activity, nitrite anions (C), and
MDA (D) quantity in A549 cells. Each test sample was added to ve different passages, which were
triplicated (n=5, * - p≤0.05, ** - p≤0.01, *** - p≤0.001, **** - p≤0.0001).

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Figure 5
Hoechst 33258 staining (blue) assay of apoptosis in A549 cells. Apoptotic cells are indicated with white
arrows. The scale bar is 100 μm. (A) Untreated cells (A549C) have smooth edges and dispersed
uorescence. (B and E) 5-FU induced apoptosis as can be seen by the occurrence of pyknotic cells with
condensed chromatin. (C and E) Incubation with RO elevated the number of apoptotic cells with rough
edges of nuclei. (D and E) The combined treatment of cells with 5-FU+RO resulted in the occurrence of
cells with signs of nuclei fragmentation and chromatin condensation. (E) Apoptosis rate evaluated by the
Hoechst 33258 staining, *p<0.05 - compared with the RO, **p<0.01 - compared with the control. (F) –
caspase-3 activity evaluated by the colorimetric assay, *p<0.05 - compared with the 5-FU, **p<0.01 -
compared with the control.

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Figure 6
2D binding analysis and interaction types on AKT in combination with 3D visualization. A – Emodin (a), B
– Endocrocin (b), C – Luteolin (c), D – Quercetin (d). Hydrogen bonding is indicated by green dotted lines,
while the remaining interactions are hydrophobic.

Page 28/28
Figure 7
2D binding analysis and interaction types on Pi3K in combination with 3D visualization. A – Emodin (a),
B – Endocrocin (a), C – Luteolin (c), D – Quercetin (d). Hydrogen bonding is indicated by green dotted
lines, while the remaining interactions are hydrophobic.
Supplementary Files
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Supplementarymaterial.docx