Next Article in Journal
Evaluation of Bone Biomarkers in Renal Osteodystrophy
Previous Article in Journal
Assessing the Comparative Efficacy of Sentinel Lymph Node Detection Techniques in Vulvar Cancer: Protocol for a Systematic Review and Meta-Analysis
Previous Article in Special Issue
Cancer Patient-Derived Cell-Based Models: Applications and Challenges in Functional Precision Medicine
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Life
Volume 14
Issue 12
10.3390/life14121539
life-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

In Vitro Anticancer Effects of Aqueous Leaf Extract from Nepeta nuda L. ssp. nuda

by
Zlatina Gospodinova
1,*,
Georgi Antov
1,
Svetozar Stoichev
2 and
Miroslava Zhiponova
3,*
1
Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, “Acad. Georgi Bonchev” Str., Bl. 21, 1113 Sofia, Bulgaria
2
Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, “Acad. Georgi Bonchev” Str., Bl. 21, 1113 Sofia, Bulgaria
3
Department of Plant Physiology, Faculty of Biology, Sofia University “St. Kliment Ohridski”, 8 Dragan Tsankov Blvd., 1164 Sofia, Bulgaria
*
Authors to whom correspondence should be addressed.
Life 2024, 14(12), 1539; https://doi.org/10.3390/life14121539
Submission received: 10 October 2024 / Revised: 15 November 2024 / Accepted: 22 November 2024 / Published: 24 November 2024
(This article belongs to the Special Issue Molecular Mechanisms and Therapeutic Effects of Drugs in Cancer—2nd Edition)
Browse Figures
Versions Notes

Abstract

:
Despite significant efforts, cancer remains the second leading cause of mortality worldwide. The medicinal plant Nepeta nuda L. represents a valuable source of biologically active compounds with pharmacological activities including antioxidant, anti-inflammatory, antimicrobial, and antiviral. This study aimed to assess the antiproliferative potential and mechanisms of action of aqueous extract from the leaves of wild-grown N. nuda. Cancer cell lines, MDA-MB-231, MCF7 (breast), HT29, Colon 26 (colon), and HepG2 (liver cancer), and a non-cancerous skin cell line, BJ, were assessed for antiproliferative activity by MTT assay and observation of cell morphological alterations. The cancer cell line that was most sensitive to the extract was further studied for apoptotic alterations by Annexin V/propidium iodide staining, colony-forming assay, and qRT-PCR analysis. The results revealed that the plant extract inhibited the proliferation of all investigated cancer cell lines with the strongest cytostatic effect on Colon 26 cells with a half maximal inhibitory concentration (IC50) value of 380.2 μg/mL and a selectivity index (SI) of 3.5. The extract significantly inhibited the ability of cells to form colonies, exhibited considerable proapoptotic potential involving the participation of the CASP8 gene, and increased the expression levels of ATG3 and the BECN1 gene, which suggests a role of autophagic cell death in the antitumor action.

1. Introduction

Cancer is a leading cause of mortality globally, accountable for nearly one in six deaths [1]. According to the World Health Organization (WHO), in 2022, there were about 20 million new cases, and a considerable increase to about 35 million new cases is predicted by 2050. Breast, colon, and liver cancer are among the most commonly diagnosed cancer types in the world and take second (2,308,897 new cases or 11.6% of all cases worldwide), third (9.6%), and sixth (4.3%) places, respectively. Regarding malignant diseases-related deaths, the second position is occupied by colon cancer with 903,859 death cases (9.3% of all cancer deaths), followed by liver (7.8%) and breast cancer (6.9%).
Due to the adverse side effects of standard cancer therapy, the scientific community is increasingly turning its attention to plant-based cancer treatment options. Offering an extraordinary variety of biologically active substances with diverse functions, plant species are relatively safe sources of new specific and reliable medicinal substances.
Nepeta nuda L., also known as “hairless” catmint, is a herbaceous perennial plant that belongs to the genus Nepeta (fam. Lamiaceae). Primarily distributed in Central and Southern Europe, the Middle East, Central and Southern Asia, and parts of Africa, N. nuda is also naturalized in Northern Europe, New Zealand, and North America [2,3]. Considering the wide climatic range and altitude of distribution, the plant is a rich source of bioactive compounds, some of which are phenolic derivatives (protocatechuic, chlorogenic, rosmarinic, and ferulic acids; aesculin; apigenin and rutin), terpenoid derivatives (camphene, nerolidol, and camphor), etc. [4,5,6,7].
Thanks to its exceptional chemical composition, the medicinal herb is used not only in traditional but also in modern medicine, as lotion against snake and scorpion bites; a palliative against syphilis, diuretic, and bronchodilator; and a sedative, as well as for stomach disorders and rheumatic pains, etc. [8,9,10,11]. In addition to the applications already mentioned, numerous studies have reported the presence of anti-inflammatory, wound-healing, antiviral, antibacterial, virucidal, antiprotozoal, and other properties in various N. nuda subspecies [12,13,14,15].
The literature data on the antitumor properties of N. nuda extracts are extremely scarce [16,17], and at present, no information is available about the antineoplastic potential of N. nuda L. ssp. nuda. Antitumor properties were reported for water, ethanol, and methanol extracts from the aerial parts of N. nuda L. subsp. annua var. annua collected from Turkey, investigated through an Agrobacterium tumefaciens-induced potato disc tumor assay [16]. Another study detected the cytotoxic potential of N. nuda subsp. lydiae essential oil from Turkey against Caco-2 colon cancer cells with an LD50 (median lethal dose) value of the oil of 129.6 µg/mL [17].
For this reason, the first objective of the present work is to assess the antitumor potential of aqueous leaves extract of wild-grown N. nuda L. ssp. nuda. The extract action on five cancer cell lines originating from some of the most common and lethal cancer types: breast (MDA-MB-231, MCF7), colon (Colon 26, HT29), and liver (HepG2) cancer, is assessed to select the cell line with the highest sensitivity and selective index. Subsequently, the mechanisms of action of the N. nuda extract anticancer activity in the cancer cell line most sensitive to the extract, including cytostatic capability and proapoptotic qualities, and identification of target molecules are investigated.

2. Materials and Methods

2.1. Plant Material and Extraction Procedure

The plant N. nuda L. ssp. nuda was collected from natural habitats in the Pirin mountain, Bulgaria, during the flowering period. A voucher specimen (SO108017) was deposited in the Herbarium of Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria.
As described previously [6], the air-dried plants were stored in the dark at room temperature. The N. nuda phytochemical content has been previously investigated by UHPLC-LTQ OrbiTrap XL, 2.7.2 and UHPLC/qqqMS2 (Table S1, Figure S1). For the extraction, only leaves were ground into a powder and subjected to subsequent maceration at 60 ± 5 °C for 24 h at a ratio of 1 g dry weight to 10 mL solvent (water). The aqueous extract was filtered, and the solvent was evaporated as the frozen filtrate was lyophilized at −65 °C (Alpha 1-2 LDplus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany).

2.2. Cell Line and Cultivation Conditions

In this work, human breast cancer cell lines, MCF7 (luminal A breast cancer subtype) and MDA-MB-231 (triple negative breast cancer subtype) (American Type Culture Collection—ATCC, Manassas, VA, USA); a human colorectal carcinoma cell line, HT29 (ATCC, USA); a murine colon adenocarcinoma cell line, Colon 26 (CLS, Germany); a human liver cancer cell line, HepG2 (ATCC, USA); and a human non-cancerous skin cell line, BJ (ATCC, USA) were used. MDA-MB-231, HT29, Colon 26, HepG2, and BJ cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2 at 37 °C. For the cell line MCF7, insulin (0.01 mg/mL) was added to DMEM cell culture complete medium, containing 10% FBS.

2.3. MTT Cell Proliferation Assay

The cellular metabolic activity and cell proliferation rate were evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay [18]. Briefly, cells (MCF7, MDA-MB-231, HT29, Colon 26, HepG2, and BJ) were seeded in 96-well plates at a density of 5 × 103 cells/well and, on the next day, were treated with increasing concentrations of the N. nuda aqueous extract (10–600 μg/mL) for 72 h. As a negative control, untreated cells cultured in a medium were used. During the last 4 h of the treatment period, 20 μL MTT solution at a concentration of 5 mg/mL was added, and the samples were incubated in the dark. The formazan complex was then dissolved in 10% SDS and 0.01 M HCI, and the absorbance was measured at 570 nm on a microplate reader (Thermo Scientific Multiskan Spectrum, Waltham, MA, USA).
The percentage of cell proliferation was calculated using the following formula:
C e l l   p r o l i f e r a t i o n % = A b s o r b a n c e   t r e a t e d   s a m p l e / A b s o r b a n c e   c o n t r o l × 100
The values of the half-maximal inhibitory concentrations (IC50) of the extract for the different cell lines were calculated using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).
The degree of selectivity in the antiproliferative action of N. nuda extract was quantified by calculating the selectivity index (SI) according to the following formula:
S I = I C 50   n o n − c a n c e r o u s   c e l l   l i n e / I C 50   c a n c e r   c e l l s   l i n e

2.4. Light Microscopy of Cell Morphology Changes

The monitoring of alterations in the monolayer and morphology of MCF7, MDA-MB-231, HT29, Colon 26, HepG2, and BJ cells 72 h after treatment with N. nuda extract in a variety of concentrations was performed at magnification of 20× using an inverted microscope (Leica DMI3000 B, Leica Microsystems GmbH, Wetzlar, Germany and Carl Zeiss, Jena, Germany) equipped with a digital camera.
All the subsequent analyses were carried out on the most sensitive to the action of the extract cell line.

2.5. Colony-Formation Assay

The ability of single cells to reproduce into a colony of at least 50 cells was evaluated after seeding of cells at a density of 1 × 103 cells per well in 6-well plates, incubated overnight, and then treated with N. nuda extract for 7 days. The experimental time of 7 days was chosen in accordance with the cell line characteristics and was optimal for the formation of visible well-differentiated colonies in the untreated controls. The colonies were visualized after fixation and staining with 2% methylene blue in 50% ethanol and then were counted under a microscope.

2.6. Fluorescence Microscopy Analysis of Apoptosis

To assess the potential of N. nuda extract to induce programmed cell death (apoptosis and necrosis) in cancer cells, a fluorescence microscopic analysis was performed by staining with Annexin V/propidium iodide (PI). Cells were placed at a density of 2 × 105 per well in 6-well plates and, on the next day, were exposed to the action of the extract for 72 h. Afterward, cells were detached with a 0.25% trypsin-EDTA solution, collected, centrifugated for 5 min at 200× g, and washed twice with phosphate buffer saline (PBS). As a final step, incubation with Annexin V and PI was carried out in the dark for 15 min at room temperature, according to the instruction of the Annexin-V-FLUOS Staining Kit (Roche, Mannheim, Germany), and the samples were observed under a fluorescence microscope (Olympus BX-41, Olympus Europa GMBH, Hamburg, Germany).

2.7. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) Analysis of Gene Expression

Analysis of the expression of specific genes, with a key role in the initiation of programmed cell death and in the control of the cell cycle and cell proliferation was performed by qRT-PCR analysis. For the analysis, cells treated for 72 h with N. nuda extract were harvested by trypsinization, centrifuged for 5 min at 200× g, washed with ice-cold PBS, and centrifuged again at 250× g for 5 min at 4 °C. RNA isolation was performed with a GeneJET RNA Purification Kit (Thermo Scientific Inc., Vilnius, Lithuania), according to the manufacturer’s protocol, with on-column DNase digestion using an RNase-Free DNase Set (Qiagen, Hilden, Germany). The purity and concentration of the extracted RNA were quantified with a BioSpec-nano Spectrophotometer (Shimadzu Biotech, Kyoto, Japan). Subsequently, 1 µg total RNA from the control and each sample was subjected to first-strand complementary DNA (cDNA) synthesis using a First Strand cDNA Synthesis Kit (Thermo Scientific Inc.) and random hexamer primers.
The quantitative RT-PCR analysis was performed using a 1x Luna® Universal qPCR Master Mix (New England Biolabs Inc., Frankfurt am Main, Germany) on a PikoRealTM Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA). For amplification, initial denaturation at 95 °C for 15 min was applied, followed by 45 cycles at 95 °C—15 s, 60 °C—30 s, 72 °C—45 s, and melting curve analysis (55–95 °C, with a temperature step of 0.2 °C). Data were analyzed using PikoReal Software version 2.2 (Thermo Fisher Scientific, Waltham, MA, USA). The Pfaffl equation [19] was used to quantify the relative expression fold changes of the genes of interest (p53, BAX, CASP3, CASP8, BECN1, ATG3, MYC, and CDKN1A) relative to the endogenous control (β-actin).

2.8. Statistical Analysis

All results are presented as mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical significance between the untreated control and treated groups was determined by one-way ANOVA, followed by Dunnett’s post hoc test using GraphPad Prism 8, and the values at p < 0.05 were considered statistically significant. Correlation analysis was performed by SigmaPlot 11.0.

3. Results

3.1. Cell Proliferation Inhibitory Effect of N. nuda Extract

The anticancer properties of aqueous leaf extract from N. nuda enriched in valuable compounds ([6], Table S1, Figure S1) were studied on a panel of five cancer cell lines—breast (MDA-MB-231, MCF7), colon (Colon 26, HT29) and hepatocellular carcinoma (HepG2). The cancer cell lines were compared to the non-cancerous skin cell line BJ by MTT cell proliferation assay after 72 h treatment period in a wide range of concentrations.
The obtained results showed that the extract inhibited statistically significant growth of all of the investigated cancer cell lines with the strongest cytostatic effect on Colon 26 cells (Figure 1) with a maximal reduction of proliferation to 23.88% at 600 μg/mL. The weakest cytostatic effect of the studied extract was registered for the MCF7 breast cancer cell line. The extract exerted a slighter reduction in cell proliferation of the non-cancerous cell line BJ compared to cancer cells, and the estimated antiproliferative selectivity showed the highest selectivity index (SI), 3.5, regarding the Colon 26 cell line. The obtained values for the IC50 concentrations and selectivity index are described in Table 1. A statistical correlation was performed between the previously reported bioactive constituents (polyphenols and flavonoids), the antiradical activity of the aqueous extract [6], and the IC50 values reported in Table 1. The analysis showed strong interdependence between total polyphenols and the inhibition of Colon 26 tumor cell proliferation (Table S2).

3.2. Cell Morphological Alterations Caused by N. nuda Extract

To visualize possible changes in cell morphology and monolayer after exposure to the action of medicinal plant N. nuda, an observation under an inverted light microscope was carried out, which indicated alterations in the tumor cells, including rounding and reduction of the volume of the cells (Figure 2). Considerable decrease in the density of the cell monolayer after extract treatment were also observed in cancer cell lines.
Some distinguishable changes in cell morphology and monolayer density of non-cancerous cells BJ were found only after treatment with the highest tested concentration of plant extract of 600 μg/mL (Figure 3).
Based on the obtained results as a model system for further studies of the mechanisms of action of the N. nuda extract, colon cancer line, Colon 26, was chosen, showing the highest sensitivity towards the medicinal plant.

3.3. Clonogenic Potential of N. nuda Extract on Colon Cancer Cells

Colony-formation assay for the evaluation of the proliferative capacity of a single cell was accomplished after treatment of Colon 26 cells with the leaf extract of the plant for a period of 7 days (Figure 4).
A strong concentration-dependent anti-clonogenic effect of the plant substance was detected, while in the untreated control cells, well-formed colonies were observed. Applying the extract at a concentration of 180 µg/mL led to a reduction in the tumor cell clonogenicity to 70.63%, while the treatment of the cells with the detected IC50 concentration of the extract of 380.2 μg/mL resulted in complete inhibition of cancer cells’ colony-forming ability.

3.4. Proapoptotic Ability of N. nuda Extract

The fluorescence microscopy assay for the examination of proapoptotic properties of the medicinal plant N. nuda in relation to Colon 26 cells was carried out after staining with the dyes Annexin V and propidium iodide (PI), which allowed detecting changes in cell morphology associated with different phases of apoptosis (Figure 5).
Cancer cells were treated for 72 h with the extract and the observation under a fluorescent microscope showed induction of considerable late apoptotic events at applied concentrations of 180 (13.76% of the cells) and 380.2 μg/mL (11.26%) when compared to the untreated control (3.27%). No induction of necrosis was detected in Colon 26 cells at the applied extract concentrations.

3.5. Influence of N. nuda Extract on the Expression Level of Genes Associated with Programmed Cell Death and Cell Cycle Control

To deepen studies on the targeted molecules and mechanisms of the anticancer action of the aqueous leaf extract of N. nuda, the relative expression levels of specific genes with a key role in the process of directing cancer cells to apoptosis and autophagy and in the cell cycle control, were evaluated by quantitative RT-PCR analysis. Transcriptional analysis was performed after treatment of Colon 26 tumor cells with the plant extract for 72 h, and untreated cells incubated for the same time period were used as negative controls, as gene expression levels in the control were taken as 1. The performed transcriptional analysis revealed a statistically significant increase in the expression of CASP8, ATG3 and BECN1 genes involved in the apoptotic and autophagy pathways (Figure 6).

4. Discussion

From the medical perspective, surgery, radiation therapy, hormone therapy, immunotherapy, and chemotherapy are the most common cancer treatment options. However, these approaches are often accompanied by numerous adverse side effects, including hepatotoxicity, nephrotoxicity, low platelet count, alopecia, vomiting, fatigue, and many others. The possibility of introducing new, targeted, relatively safe, and cost-effective cancer treatment alternatives as a preferential option is increasingly desirable. As an aid, medicinal plants provide an extraordinary variety of powerful and health-beneficial mesonutrients.
Nepeta nuda is known for its strong allelopathic properties [6], serving to suppress the growth of neighboring plants in competition for resources. Therefore, it is intriguing to investigate the combination of the plant’s compounds displaying antiproliferative effects. Various studies have analyzed the antitumor potential and determined the cytotoxic values of different species of Nepeta [20,21,22,23,24,25,26,27,28], but none of them includes N. nuda L. ssp. nuda or has thoroughly analyzed the mechanisms by which the species exerts its anticancer effects.
In our work, the in vitro cytostatic potential of the aqueous leaf extract of N. nuda was evaluated by MTT assay and light microscopy observation after 72 h treatment on a wide panel of tumor cell lines, representing some of the most common and deadly cancer types, and the non-cancerous cell line BJ was used as a control. The time period of 72 h was chosen for the analyses of cell proliferation because of the different doubling time of cell lines used as a model system in the study varying from about 20 to 48 h. N. nuda extract decreased cell growth of all studied cancer cell lines and the detected cytostatic effect was most considerable on colon cancer cell line Colon 26 with an IC50 value of 380.2 μg/mL. The calculated IC50 concentrations of the extract for MDA-MB-231, MCF7, HT29, and HepG2 cells were 481.6 μg/mL, 576.1 μg/mL, 504.2 μg/mL, and 541.4 μg/mL, respectively. A significantly higher IC50 concentration value of 1346 μg/mL was established for the non-cancerous cell line BJ, pointing to selectivity in the antiproliferative action of the extract. Although the cytostatic effect of the extract does not meet the criteria for high anticancer activity established by the US National Cancer Institute [29], the substance has high selectivity for cancer cells (SI values > 2 was considered as high selectivity), which is an indication of safety even when applied in higher concentrations, and this is essential for finding novel plant-derived anti-neoplastic agents without side effects.
Based on the extract-specific sensitivity of the Colon 26 cell line and a favorable and higher selectivity index (SI = 3.5) found in the same cell line, it was selected for further investigation. Thereafter, a clonogenic test, fluorescence microscopy, and gene expression analysis were conducted to clarify some aspects and pathways in the anticancer effects of the extract in detail. Our study showed that the analyzed extract had an enviable anti-clonogenic effect against Colon 26 after 7 days of treatment. Furthermore, subsequent treatment for 72 h induced characteristic features of late apoptosis in the Colon 26 cell line, as determined by Annexin V/PI staining. The performed qRT-PCR analysis detected alterations in the relative expression of genes involved in the programmed cell death pathways of apoptosis and autophagy (upregulation of CASP8, ATG3, and BECN1).
The water solvent was shown to assure the highest extract yield from N. nuda plant material ~37% dry extract, compared to methanol (~11%), ethanol (~9%), acetone (~4%), and chloroform (~14%) [6]. At 60 °C, the highest total content of phenolics and respective antioxidant activity was measured in aqueous and methanol extracts—nearly 2 to 10 folds more than the use of other solvents. Recent advances suggest the role of natural products including phenolics in photodynamic therapy based on photosensitization (PDT) [30,31]: due to their heterocyclic structure, the phenolic antioxidants act as photosensitizers, and combined with visible light and oxygen, they generate reactive oxygen species (ROSs). High levels of ROSs disrupt cellular homeostasis, damaging lipids, proteins, and DNA, which often triggers apoptosis, necrosis, and autophagy. The balance and extent of these processes vary depending on the type and intensity of oxidative stress, making ROSs a potential target for anticancer therapies that aim to selectively kill cancer cells. In support, in our work, the phenolic antioxidants strongly correlate with the Colon 26 cancer cells’ antiproliferative activity.
The presence of numerous phytoconstituents in the investigated leaf extract of the herb [6] and, in particular, the high content of the polyphenolic compounds rosmarinic acid (RA) and ferulic acid (FA), as well as the iridoid glycoside epideoxyloganic acid, could be a cause of the established antiproliferative and anti-clonogenic effect of N. nuda. Available data confirm that RA possesses various pharmacological effects such as antioxidant, anti-inflammatory, antimutagenic, colony-inhibitory, anticancer, etc. [32,33,34,35,36,37,38]. In particular, in colon carcinoma cell lines, CT26 and HCT116, the antimetastatic, antiproliferative, anti-invasive, and anti-migratory activities of RA are mediated by the activation of AMP-activated protein kinase, induction of caspase-3, -8, and -9, as well as reduction of the MMP-2, MMP-9, cyclin D1, CDK4, ICAM-1, and integrin β1 expression [39].
In regard to FA, a considerable body of scientific evidence has also reported the immunoregulatory [40], anti-inflammatory [41], antiproliferative and apoptosis-inducing [42], anti-migratory [43], and anti-invasive [44] activities of the polyphenolic substance. Furthermore, there are data reporting that FA induces autophagy [45] and inhibits multidrug resistance (MDR) in tumor cells [46]. In the case of colon cancer, Chen et al. (2023) [47] found that FA inhibited the growth of CT26WT cells and promoted apoptosis by downregulating BCL-2 and upregulating the BAX, JNK, and ERK expressions.
Already identified in numerous flowering plants of the genus Nepeta [48,49,50,51], as well as in our previous study on N. nuda L. nuda as the metabolite with the highest amount [6], 1,5,9-epideoxyloganoic acid (1,5,9-eDLA) is known to have various properties, including antibacterial, antioxidant, anti-inflammatory, DNA-protective, and enzyme-inhibitory activities [52,53,54].
This study’s transcriptional analysis of the genes involved in the processes of programmed cell death, apoptosis and autophagy, and cell cycle control found a statistically significant alteration in CASP8, ATG3, and BECN1’s gene expression. Caspase-8 plays a central role in mediating Fas-induced apoptosis [55]. A dose-dependent increase in relative expression levels was found for CASP8 to 2.09-fold at the highest applied extract concentration of 380.2 μg/mL.
ATG3 (Autophagy-related gene 3) is essential for the autophagy process, acting as an E2 ubiquitin-like conjugating enzyme in the ATG8 conjugation system, contributing to the elongation of the phagophore. ATG3 also is involved in many physiological and pathological processes in an autophagy-dependent manner, such as tumor progression, maintaining mitochondrial homeostasis, etc. [56,57]. A 1.89-fold increase was observed in the transcriptional levels of ATG3 after the cancer cells treatment with N. nuda extract.
BECN1 (Beclin 1) plays a key role in autophagy initiation, autophagosome formation, and maturation [58]. It is found that in colon cancer the significantly lower expression of BECN1 compared with the normal colon tissue was positively associated with poor prognosis in patients [59]. In our work, upregulation of BECN1 was detected with a 1.64-fold increase in the expression levels.
The current study provides information on the very poorly studied antitumor properties of N. nuda and represents a comprehensive assessment of some mechanisms of the antitumor potential of the plant.

5. Conclusions

In this study, we demonstrated that the aqueous leaf extract of wild-grown medicinal plant N. nuda possesses antiproliferative properties against breast, colon, and liver cancer cell lines, and the strongest inhibitory effect is detected against Colon 26 cancer cells. The growth of the non-cancerous cell line BJ was affected to a considerably lower degree. Regarding Colon 26 cells, the extract significantly inhibited the ability of cells to form colonies, exhibited considerable proapoptotic potential involving the participation of the CASP8 gene, and affected the expression levels of ATG3 and the BECN1 gene, possibly involving autophagic cell death among the mechanisms underlying its antitumor action. Future research will be directed to a more detailed study on the molecular targets of the anticancer action of N. nuda extract. Biotechnological strategies, including the optimization of plant extraction conditions and the use of in vitro plant cultures, will be tested to improve the antitumor effect. Efforts will be made to identify whether a more specific phytochemical fraction would retain the bioactive potential.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/life14121539/s1, Table S1: Secondary metabolites identified by Orbitrap-MSn analysis in N. nuda leaves [6]; Table S2: Correlation between quantitative parameters of the aqueous extract from [6] and the antitumor properties (IC50; Table 1); Figure S1: Quantitative representation of metabolites in leaves of N. nuda by using UHPLC/MS2 analysis [6].

Author Contributions

Conceptualization, M.Z. and Z.G.; methodology, Z.G., G.A., S.S. and M.Z.; software, Z.G.; validation, Z.G. and G.A.; formal analysis, Z.G. and G.A.; investigation Z.G., G.A., S.S. and M.Z.; resources, M.Z. and Z.G.; data curation, Z.G. and G.A.; writing—original draft preparation, Z.G. and G.A.; writing—review and editing, M.Z. and Z.G.; visualization, Z.G.; supervision, M.Z.; project administration, M.Z.; funding acquisition, M.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Bulgarian National Science Fund, Ministry of Education and Science, Grant No. KP-06-N56/9/12.11.2021.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors would like to acknowledge Tanya Angelova, student in the Faculty of Biology, Sofia University “St. Kliment Ohridski” for the technical assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Bray, F.; Laversanne, M.; Sung, H.; Ferlay, J.; Siegel, R.L.; Soerjomataram, I.; Jemal, A. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2024, 74, 229–263. [Google Scholar] [CrossRef] [PubMed]
  2. Kokkini, S.; Babalonas, D. Morphological variation in Nepeta nuda L. Fedd. Repert. 1982, 93, 71–76. [Google Scholar] [CrossRef]
  3. Ghendov, V.; Ciocarlan, N.; Danila, D. Bioecological peculiarities of Nepeta pannonica L. under in situ and ex situ conditions. J. Bot. 2015, 2, 35–39. [Google Scholar]
  4. Aras, A.; Bursal, E.; Dogru, M. UHPLC-ESI-MS/MS analyses for quantification of phenolic compounds of Nepeta nuda subsp. Lydiae. J. Appl. Pharm. Sci. 2016, 6, 9–13. [Google Scholar] [CrossRef]
  5. Smiljković, M.; Dias, M.I.; Stojković, D.; Barros, L.; Bukvički, D.; Ferreira, I.C.F.R.; Soković, M. Characterization of phenolic compounds in tincture of edible Nepeta nuda: Development of antimicrobial mouthwash. Food Funct. 2018, 9, 5417–5425. [Google Scholar] [CrossRef]
  6. Petrova, D.; Gašić, U.; Yocheva, L.; Hinkov, A.; Yordanova, Z.; Chaneva, G.; Mantovska, D.; Paunov, M.; Ivanova, L.; Rogova, M.; et al. Catmint (Nepeta nuda L.) phylogenetics and metabolic responses in variable growth conditions. Front. Plant Sci. 2022, 13, 866777. [Google Scholar] [CrossRef]
  7. Zaharieva, A.; Rusanov, K.; Rusanova, M.; Paunov, M.; Yordanova, Z.; Mantovska, D.; Tsacheva, I.; Petrova, D.; Mishev, K.; Dobrev, P.I.; et al. Uncovering the interrelation between metabolite profiles and bioactivity of in vitro- and wild-grown Catmint (Nepeta nuda L.). Metabolites 2023, 13, 1099. [Google Scholar] [CrossRef]
  8. Arnold, N.; Bellomaria, B.; Valentini, G.; Yanniou, C.; Arnold, H.J. Anatomical and phytochemical study of Cyprus endemic Nepeta troodi Holmboe (labiatae). Plantes Médicinales Et Phytothérapie 1993, 26, 52–63. [Google Scholar]
  9. Arnold, N.; Valentini, G.; Bellomaria, B.; Arnold, H.J. Contribution to the chemical study of essential oil of Nepeta Parnassica Heldr. B Sart. ex. Boiss. Plantes Médicinales Et Phytothérapie 1993, 26, 149–157. [Google Scholar]
  10. Formisano, C.; Rigano, D.; Senatore, F. Chemical constituents and biological activities of Nepeta species. Chem. Biodivers. 2011, 8, 1783–1818. [Google Scholar] [CrossRef]
  11. Sokolov, P.D. (Ed.) Plant Resources of the USSR. Flowering Plants, Their Chemical Composition, Use. Families Hippuridaceae-Lobeliaceae; Academia Nauk SSSR: St. Petersburg, SPb, Russia, 1991; Available online: https://z-lib.io/book/16128734 (accessed on 21 November 2024). (In Russian)
  12. Alim, A.; Goze, I.; Cetin, A.; Atas, A.D.; Cetinus, S.A.; Vural, N. Chemical composition and in vitro antimicrobial and antioxidant activities of the essential oil of Nepeta nuda L. subsp. Albiflora (Boiss.) gams. Afr. J. Microbiol. Res. 2009, 3, 463–467. [Google Scholar]
  13. Kozuharova, E.; Benbassat, N.; Getov, I. Ethnobotanical records of not yet documented therapeutic effects of some popular Bulgarian medicinal plants. Emir. J. Food Agric. 2014, 26, 647–651. [Google Scholar] [CrossRef]
  14. Todorov, D.; Shishkova, K.; Dragolova, D.; Hinkov, A.; Kapchina-Toteva, V.; Shishkov, S. Antiviral activity of medicinal plant Nepeta nuda. Biotechnol. Biotechnol. Equip. 2015, 29, S39–S43. [Google Scholar] [CrossRef]
  15. Aćimović, M.; Stanković-Jeremić, J.; Cvetković, M. Phyto-pharmacological aspects of Nepeta nuda L.: A systematic review. Nat. Med. Mater. (Lek. Sirovine) 2020, 40, 75–83. [Google Scholar] [CrossRef]
  16. Yildirim, A.B.; Karakas, F.P.; Turker, A.U. In vitro antibacterial and antitumor activities of some medicinal plant extracts, growing in Turkey. Asian Pac. J. Trop. Med. 2013, 6, 616–624. [Google Scholar] [CrossRef]
  17. Kabalay, B.; Mutlu, D.; Arslan, S.; Semiz, G.; Kocabıyık, K. Chemical composition and Cytotoxicity of Nepeta nuda subsp. lydiae P. H. Davis Essential Oil Towards Colon Carcinoma. In Proceedings of the 4th International Symposium on EuroAsian Biodiversity (SEAB2018), Kiev, Ukraine, 3–6 July 2018. [Google Scholar]
  18. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
  19. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 2001, 29, e45. [Google Scholar] [CrossRef]
  20. Suschke, U.; Sporer, F.; Schneele, J.; Geiss, H.K.; Reichling, J. Antibacterial and cytotoxic activity of Nepeta cataria L., N. Cataria Var. Citriodora (Beck.) Balb. and Melissa officinalis L. essential oils. Nat. Prod. Commun. 2007, 2, 1277–1286. [Google Scholar] [CrossRef]
  21. Al-kahraman, Y.; Baloch, N.; Kakar, A.M.; Nabi, S. In vitro antimicrobial, insecticidal, antitumor, antioxidant activities and their phytochemical estimation of methanolic extract and its fractions of Nepeta praetervisa leaves. Int. J. Phytomed. 2012, 4, 531–536. [Google Scholar]
  22. Shakeri, A.; Khakdan, F.; Soheili, V.; Sahebkar, A.; Rassam, G.; Asili, J. Chemical composition, antibacterial activity, and cytotoxicity of essential oil from Nepeta ucrainica L. spp. kopetdaghensis. Ind. Crops Prod. 2014, 58, 315–321. [Google Scholar] [CrossRef]
  23. Kahkeshani, N.; Razzaghirad, Y.; Ostad, S.N.; Hadjiakhoondi, A.; Ardekani, M.R.S.; Hajimehdipoor, H.; Attar, H.; Samadi, M.; Jovel, E.; Khanavi, M. Cytotoxic, acetylcholinesterase inhibitor and antioxidant activity of Nepeta menthoides Boiss & Buhse essential oil. J. Essent. Oil-Bear. 2014, 17, 544–552. [Google Scholar] [CrossRef]
  24. Al-Oqail, M.M.; Al-Sheddi, E.S.; Siddiqui, M.A.; Musarrat, J.; Al-Khedhairy, A.A.; Farshori, N.N. Anticancer activity of chloroform extract and sub-fractions of Nepeta deflersiana on human breast and lung cancer cells: An in vitro cytotoxicity assessment. Pharmacogn. Mag. 2015, 11, S598–S605. [Google Scholar] [CrossRef] [PubMed]
  25. Orfali, R.; Siddiqui, N.A.; Alam, P.; Alhowiriny, T.A.; Al-Taweel, A.M.; Al-Yahya, S.; Majrashi, N.M.; Mehmood, R.; Khan, S.I.; Perveen, S. Biological evaluation of different extracts of aerial parts of Nepeta deflersiana and standardization of active extracts using 8-epi-7-deoxyloganic acid and ursolic acid by validated HPTLC method. Evid. Based Complement. Alternat. Med. 2018, 1, 8790769. [Google Scholar] [CrossRef] [PubMed]
  26. Zahirnia, A.; Boroomand, M.; Nasirian, H.; Soleimani-Asl, S.; Salehzadeh, A.; Dastan, D. The cytotoxicity of malathion and essential oil of Nepeta crispa (lamiales: Lamiaceae) against vertebrate and invertebrate cell lines. Pan. Afr. Med. J. 2019, 33, 285. [Google Scholar] [CrossRef]
  27. Şafak, E.K.; Karatoprak, G.Ş.; Dirmenci, T.; Duman, H.; Küçükboyacı, N. Cytotoxic effects of some Nepeta species against breast cancer cell lines and their associated phytochemical properties. Plants 2022, 11, 1427. [Google Scholar] [CrossRef]
  28. Amirzadeh, M.; Soltanian, S.; Mohamadi, N. Chemical composition, anticancer and antibacterial activity of Nepeta mahanensis essential oil. BMC Complement. Med. Ther. 2022, 22, 173. [Google Scholar] [CrossRef]
  29. Grever, M.R.; Schepartz, S.A.; Chabner, B.A. The National Cancer Institute: Cancer drug discovery and development program. Semin. Oncol. 1992, 19, 622–638. [Google Scholar]
  30. Aziz, B.; Aziz, I.; Khurshid, A.; Raoufi, E.; Esfahani, F.N.; Jalilian, Z.; Mozafari, M.R.; Taghavi, E.; Ikram, M. An overview of potential natural photosensitizers in cancer photodynamic therapy. Biomedicines 2023, 11, 224. [Google Scholar] [CrossRef]
  31. Yamali, C.; Nenni, M.; Sakarya, M.T.; Sakagami, H.; Gul, H.I. Biological activities and drug-likeness properties of phenol-based heterocyclic compounds. Pharm. Chem. J. 2024, 57, 1754–1760. [Google Scholar] [CrossRef]
  32. Hossan, S.; Rahman, S.; Bashar, A.B.M.A.; Jahan, R.; Al-Nahain, A.; Rahmatullah, M. Rosmarinic acid: A review of its anticancer action. World J. Pharm. Pharm. Sci. 2014, 3, 57–70. [Google Scholar]
  33. Alagawany, M.; Abd El-Hack, M.E.; Farag, M.R.; Gopi, M.; Karthik, K.; Malik, Y.S.; Dhama, K. Rosmarinic acid: Modes of action, medicinal values and health benefits. Anim. Health Res. Rev. 2017, 18, 167–176. [Google Scholar] [CrossRef] [PubMed]
  34. Jang, Y.G.; Hwang, K.A.; Choi, K.C. Rosmarinic acid, a component of rosemary tea, induced the cell cycle arrest and apoptosis through modulation of HDAC2 expression in prostate cancer cell lines. Nutrients 2018, 10, 1784. [Google Scholar] [CrossRef] [PubMed]
  35. Swamy, M.K.; Sinniah, U.R.; Ghasemzadeh, A. Anticancer potential of rosmarinic acid and its improved production through biotechnological interventions and functional genomics. Appl. Microbiol. Biotechnol. 2018, 102, 7775–7793. [Google Scholar] [CrossRef] [PubMed]
  36. Rahbardar, M.G.; Amin, B.; Mehri, S.; Mirnajafi-Zadeh, S.J.; Hosseinzadeh, H. Rosmarinic acid attenuates development and existing pain in a rat model of neuropathic pain: An evidence of anti-oxidative and anti-inflammatory effects. Phytomedicine 2018, 40, 59–67. [Google Scholar] [CrossRef]
  37. Anwar, S.; Shamsi, A.; Shahbaaz, M.; Queen, A.; Khan, P.; Hasan, G.M.; Islam, A.; Alajmi, M.F.; Hussain, A.; Ahmad, F.; et al. Rosmarinic acid exhibits anticancer effects via MARK4 inhibition. Sci. Rep. 2020, 10, 10300. [Google Scholar] [CrossRef]
  38. Messeha, S.S.; Zarmouh, N.O.; Asiri, A.; Soliman, K.F.A. Rosmarinic acid-induced apoptosis and cell cycle arrest in triple-negative breast cancer cells. Eur. J. Pharmacol. 2020, 885, 173419. [Google Scholar] [CrossRef]
  39. Han, Y.H.; Kee, J.Y.; Hong, S.H. Rosmarinic acid activates AMPK to inhibit metastasis of colorectal cancer. Front. Pharmacol. 2018, 9, 68. [Google Scholar] [CrossRef]
  40. Brugiolo, A.S.S.; Gouveia, A.C.C.; de Souza Alves, C.C.; de Castro E Silva, F.M.; Esteves de Oliveira, É.; Ferreira, A.P. Ferulic acid supresses Th2 immune response and prevents remodeling in ovalbumin-induced pulmonary allergy associated with inhibition of epithelial-derived cytokines. Pulm. Pharmacol. Ther. 2017, 45, 202–209. [Google Scholar] [CrossRef]
  41. Nouri, A.; Ghatreh-Samani, K.; Amini-Khoei, H.; Mohammadi, A.; Heidarian, E.; Najafi, M. Ferulic acid prevents cyclosporine-induced nephrotoxicity in rats through exerting anti-oxidant and anti-inflammatory effects via activation of Nrf2/HO-1 signaling and suppression of NF-κB/TNF-α axis. Naunyn Schmiedebergs Arch. Pharmacol. 2022, 395, 387–395. [Google Scholar] [CrossRef]
  42. Wang, T.; Gong, X.; Jiang, R.; Li, H.; Du, W.; Kuang, G. Ferulic acid inhibits proliferation and promotes apoptosis via blockage of PI3K/Akt pathway in osteosarcoma cell. Am. J. Transl. Res. 2016, 8, 968–980. [Google Scholar]
  43. Fong, Y.; Tang, C.C.; Hu, H.T.; Fang, H.Y.; Chen, B.H.; Wu, C.Y.; Yuan, S.S.; Wang, H.M.D.; Chen, Y.C.; Teng, Y.N.; et al. Inhibitory effect of trans-ferulic acid on proliferation and migration of human lung cancer cells accompanied with increased endogenous reactive oxygen species and β-catenin instability. Chin. Med. 2016, 11, 45. [Google Scholar] [CrossRef] [PubMed]
  44. Dodurga, Y.; Eroğlu, C.; Seçme, M.; Elmas, L.; Avcı, Ç.B.; Şatıroğlu-Tufan, N.L. Anti-proliferative and anti-invasive effects of ferulic acid in TT medullary thyroid cancer cells interacting with URG4/URGCP. Tumor Biol. 2016, 37, 1933–1940. [Google Scholar] [CrossRef] [PubMed]
  45. Liu, Y.; Shi, L.; Qiu, W.; Shi, Y. Ferulic acid exhibits anti-inflammatory effects by inducing autophagy and blocking NLRP3 inflammasome activation. Mol. Cell. Toxicol. 2022, 18, 509–519. [Google Scholar] [CrossRef]
  46. Muthusamy, G.; Balupillai, A.; Ramasamy, K.; Shanmugam, M.; Gunaseelan, S.; Mary, B.; Prasad, N.R. Ferulic acid reverses ABCB1-mediated paclitaxel resistance in MDR cell lines. Eur. J. Pharmacol. 2016, 786, 194–203. [Google Scholar] [CrossRef]
  47. Chen, S.; Zhao, D.; Luan, C.; Zheng, J.; Liu, W.; Feng, Z.; Luo, R.; Han, X.; Wang, D. Ferulic acid induces autophagy and apoptosis in colon cancer CT26 cells via the MAPK pathway. Molecules 2023, 28, 6014. [Google Scholar] [CrossRef]
  48. Murai, F.; Tagawa, M.; Damtoft, S.; Jensen, S.R.; Nielsen, B.J. (1R, 5R, 8S, 9S)-Deoxyloganic acid from Nepeta cataria. Chem. Pharm. Bull. (Tokyo) 1984, 32, 2809–2814. [Google Scholar] [CrossRef]
  49. Takeda, Y.; Ooiso, Y.; Masuda, T.; Honda, G.; Otsuka, H.; Sezik, E.; Yesilada, E. Iridoid and eugenol glycosides from Nepeta cadmea. Phytochemistry 1998, 49, 787–791. [Google Scholar] [CrossRef]
  50. Kökdil, G.; Yalçin, S.M.; Topçu, G. Nepetalactones and other constituents of Nepeta nuda ssp. albiflora. Turk. J. Chem. 1999, 23, 99–104. [Google Scholar]
  51. Aničić, N.; Matekalo, D.; Skorić, M.; Gašić, U.; Živković, J.N.; Dmitrović, S.; Božunović, J.; Milutinović, M.; Petrović, L.; Dimitrijević, M.; et al. Functional iridoid synthases from iridoid producing and non-producing Nepeta species (subfam. Nepetoidae, fam. Lamiaceae). Front. Plant Sci. 2024, 14, 1211453. [Google Scholar] [CrossRef]
  52. Aničić, N.; Gašić, U.; Lu, F.; Ćirić, A.; Ivanov, M.; Jevtić, B.; Dimitrijević, M.; Anđelković, B.; Skorić, M.; Živković, J.N.; et al. Antimicrobial and immunomodulating activities of two endemic Nepeta species and their major iridoids isolated from natural sources. Pharmaceuticals 2021, 14, 414. [Google Scholar] [CrossRef]
  53. Başar, Y.; Yenigün, S.; İpek, Y.; Behçet, L.; Gül, F.; Özen, T.; Demirtaş, İ. DNA protection, molecular docking, enzyme inhibition and enzyme kinetic studies of 1,5,9-epideoxyloganic acid isolated from Nepeta aristata with bio-guided fractionation. J. Biomol. Struct. Dyn. 2024, 42, 9235–9248. [Google Scholar] [CrossRef] [PubMed]
  54. Başar, Y.; Yenigün, S.; Behçet, L.; Demirtaş, İ.; Ozen, T. Antibacterial and antioxidant molecule isolated from Nepeta aristata Boiss Et Kotschy Ex Boiss plant: 1,5,9-Epideoxyloganic Acid. Int. J. Chem. Technol. 2024, 8, 27–32. [Google Scholar] [CrossRef]
  55. Boldin, M.P.; Goncharov, T.M.; Goltsev, Y.V.; Wallach, D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 1996, 85, 803–815. [Google Scholar] [CrossRef] [PubMed]
  56. Radoshevich, L.; Murrow, L.; Chen, N.; Fernandez, E.; Roy, S.; Fung, C.; Debnath, J. ATG12 conjugation to ATG3 regulates mitochondrial homeostasis and cell death. Cell 2010, 142, 590–600. [Google Scholar] [CrossRef]
  57. Fang, D.; Xie, H.; Hu, T.; Shan, H.; Li, M. Binding features and functions of ATG3. Front. Cell Dev. Biol. 2021, 9, 685625. [Google Scholar] [CrossRef]
  58. Kang, R.; Zeh, H.J.; Lotze, M.T.; Tang, D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011, 18, 571–580. [Google Scholar] [CrossRef]
  59. Hu, F.; Li, G., Huang; Hou, Z.; Yang, X.; Luo, X.; Feng, Y.; Wang, G.; Hu, J.; Cao, Z. The autophagy-independent role of BECN1 in colorectal cancer metastasis through regulating STAT3 signaling pathway activation. Cell Death Dis. 2020, 11, 304. [Google Scholar] [CrossRef]
Life 14 01539 g001
Figure 1. MTT cell proliferation assay of MCF7, MDA-MB-231, Colon 26, HT29, HepG2, and BJ cells treated with increasing concentrations of N. nuda aqueous leaf extract for 72 h. Error bars represent standard error of the mean (SEM). a, b, and c indicate significant differences from the control group by one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test (a p < 0.0001, b p < 0.001, c p < 0.01).
Figure 1. MTT cell proliferation assay of MCF7, MDA-MB-231, Colon 26, HT29, HepG2, and BJ cells treated with increasing concentrations of N. nuda aqueous leaf extract for 72 h. Error bars represent standard error of the mean (SEM). a, b, and c indicate significant differences from the control group by one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test (a p < 0.0001, b p < 0.001, c p < 0.01).
Life 14 01539 g001
Life 14 01539 g002
Figure 2. Morphological changes of Colon 26 cells after treatment for 72 h with 60, 180, 380.2, 480, and 600 μg/mL of N. nuda aqueous leaf extract compared to the untreated control. Magnification 20×.
Figure 2. Morphological changes of Colon 26 cells after treatment for 72 h with 60, 180, 380.2, 480, and 600 μg/mL of N. nuda aqueous leaf extract compared to the untreated control. Magnification 20×.
Life 14 01539 g002
Life 14 01539 g003
Figure 3. Morphological changes of BJ cells after treatment for 72 h with 480 and 600 μg/mL of N. nuda aqueous leaf extract compared to the untreated control. Magnification 10×.
Figure 3. Morphological changes of BJ cells after treatment for 72 h with 480 and 600 μg/mL of N. nuda aqueous leaf extract compared to the untreated control. Magnification 10×.
Life 14 01539 g003
Life 14 01539 g004
Figure 4. Effect of N. nuda aqueous leaf extract on clonogenicity of Colon 26 cells. (a) Effect of treatment for 7 days with 180 and 380.2 μg/mL extract on cancer cells when compared to the untreated control; (b) Quantitative evaluation of Colon 26 cells clonogenicity. Error bars represent standard error of the mean (SEM). ** p < 0.01, **** p < 0.0001 vs. the control group.
Figure 4. Effect of N. nuda aqueous leaf extract on clonogenicity of Colon 26 cells. (a) Effect of treatment for 7 days with 180 and 380.2 μg/mL extract on cancer cells when compared to the untreated control; (b) Quantitative evaluation of Colon 26 cells clonogenicity. Error bars represent standard error of the mean (SEM). ** p < 0.01, **** p < 0.0001 vs. the control group.
Life 14 01539 g004
Life 14 01539 g005
Figure 5. Proapoptotic effect of N. nuda aqueous leaf extract. (a) Fluorescent microscopy analysis of Colon 26 cells after Annexin V/PI staining for visualization of apoptotic (EA—early apoptotic; LA—late apoptotic) and necrotic cells: the untreated control and extract treated cells (180 and 380.2 μg/mL, 72 h). (b) Quantitative evaluation of apoptosis and necrosis. Error bars represent standard error of the mean (SEM). ** p < 0.01, *** p < 0.001 vs. the control group. Magnification 40×; Scale bar 50 μm.
Figure 5. Proapoptotic effect of N. nuda aqueous leaf extract. (a) Fluorescent microscopy analysis of Colon 26 cells after Annexin V/PI staining for visualization of apoptotic (EA—early apoptotic; LA—late apoptotic) and necrotic cells: the untreated control and extract treated cells (180 and 380.2 μg/mL, 72 h). (b) Quantitative evaluation of apoptosis and necrosis. Error bars represent standard error of the mean (SEM). ** p < 0.01, *** p < 0.001 vs. the control group. Magnification 40×; Scale bar 50 μm.
Life 14 01539 g005
Life 14 01539 g006
Figure 6. Relative expression levels of CASP8, ATG3 and BECN1 genes at 72 h after treatment of Colon 26 cells with N. nuda aqueous leaf extract at concentrations of 180 and 380.2 μg/mL. Black columns refer to untreated controls. Error bars represent standard error of the mean (SEM); β-actin was used as an endogenous control to normalize the expression of the genes. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the control group.
Figure 6. Relative expression levels of CASP8, ATG3 and BECN1 genes at 72 h after treatment of Colon 26 cells with N. nuda aqueous leaf extract at concentrations of 180 and 380.2 μg/mL. Black columns refer to untreated controls. Error bars represent standard error of the mean (SEM); β-actin was used as an endogenous control to normalize the expression of the genes. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the control group.
Life 14 01539 g006
Table 1. The IC50 values determined by MTT assay and selectivity index.
Table 1. The IC50 values determined by MTT assay and selectivity index.
Cell LineIC50 [μg/mL]SI
MDA-MB-231481.62.8
MCF7576.12.3
HT29504.22.7
Colon 26380.23.5
HepG2541.42.5
BJ1346
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Gospodinova, Z.; Antov, G.; Stoichev, S.; Zhiponova, M. In Vitro Anticancer Effects of Aqueous Leaf Extract from Nepeta nuda L. ssp. nuda. Life 2024, 14, 1539. https://doi.org/10.3390/life14121539

AMA Style

Gospodinova Z, Antov G, Stoichev S, Zhiponova M. In Vitro Anticancer Effects of Aqueous Leaf Extract from Nepeta nuda L. ssp. nuda. Life. 2024; 14(12):1539. https://doi.org/10.3390/life14121539

Chicago/Turabian Style

Gospodinova, Zlatina, Georgi Antov, Svetozar Stoichev, and Miroslava Zhiponova. 2024. "In Vitro Anticancer Effects of Aqueous Leaf Extract from Nepeta nuda L. ssp. nuda" Life 14, no. 12: 1539. https://doi.org/10.3390/life14121539

APA Style

Gospodinova, Z., Antov, G., Stoichev, S., & Zhiponova, M. (2024). In Vitro Anticancer Effects of Aqueous Leaf Extract from Nepeta nuda L. ssp. nuda. Life, 14(12), 1539. https://doi.org/10.3390/life14121539

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop