Cytotoxicity screening of Thymus vulgaris L. in breast cancer: in vitro study

Nazmiye Bıtgen; Gozde Ozge Onder; Munevver Baran; Arzu Yay

doi:10.1093/toxres/tfad052

. 2023 Jun 26;12(4):584–590. doi: 10.1093/toxres/tfad052

Cytotoxicity screening of Thymus vulgaris L. in breast cancer: in vitro study

Nazmiye Bıtgen ^1,^2,^✉, Gozde Ozge Onder ^3,⁴, Munevver Baran ⁵, Arzu Yay ^6,⁷

PMCID: PMC10470352 PMID: 37663807

Abstract

Breast cancer is one of the leading causes of cancer-related deaths due to its aggressive course. There is an increasing need for alternative therapy strategies, including herbal medications, to treat the disease because of its high incidence. Medicinal plants, such as Thymus vulgaris L. (T. vulgaris), have recently attracted great interest due to the antitumor properties of their extracts. The purpose of this investigation was to ascertain whether T. vulgaris had any cytotoxic effects on two different breast cancer cell lines. MTT test was applied to evaluate the effect of T. vulgaris on cell viability. TUNEL method was used to determine its apoptotic effect. LC3 and Beclin-1 expression levels were determined by immunofluorescence staining method and its autophagic effect was evaluated. Our findings demonstrate that T. vulgaris greately lowers proliferation, both in terms of concentration and duration. Consistent with decreased proliferation, an increase in apoptotic and autophagic cell death were also observed. The migration capacity of MCF-7 and MDA-MB-231 breast cancer cells was greatly suppressed by T. vulgaris, while significantly reducing colony formation. This study is the first to look into how T. vulgaris methanol extract affects breast cancer cells. All of these findings demonstrate that T. vulgaris prevents breast cancer cells from developing a malignant phenotype. It is possible to say that the methanol extract of T. vulgaris is suitable for the treatment of breast cancer, including aggressive types. However, in vivo research should support these results.

Keywords: apoptosis, autophagy, breast cancer, migration, Thymus vulgaris

Graphical abstract

Introduction

The second most frequent malignancy in women worldwide is breast cancer.¹ It is a diverse illness with a number of subgroups that vary in terms of their clinical and histological characteristics. Breast cancer is defined as invasive and noninvasive depending on whether it has spread to the breast, lymph, or distant healthy tissues.² Also, according to the differences in their receptors, they are grouped as estrogen receptor (ER) and progesterone receptor (PR).³ The primary clinical treatment options for breast cancer are surgical resection, radiation, chemotherapy, and targeted treatments. However, the outcomes are still inadequate, and the prognosis for the patient remains bad.⁴ However, the pathophysiology and etiology of the condition remain poorly known. The goal of cancer research is to discover novel anticarcinogenic medicines that are more effective and have fewer adverse effects in order to treat breast cancer.

The MCF-7 cell line has functional estrogen and epidermal growth factor (EGF) receptors, is dependent on estrogen and EGF for growth, and is noninvasive, while MDA-MB-231 cells are a model for more aggressive, hormone-independent breast cancer. For this reason, MCF-7 and MDA-MB-231 cell lines are frequently preferred as in vitro models in cancer research.⁵ Lately, herbal products have attracted the attention of researchers in terms of both physiological and biological activities, especially for the identification of new drugs, and they have made great contributions especially in the field of medicine.⁶ Medicinal plants, which are effective in the treatment of certain illnesses (including cancer and infections), have been extensively researched in recent years.⁷ About 40–50% of cancer patients include plant-derived products and their derivatives in their diets, as they have fewer side effects, even at high doses.⁸ Our previous study showed that hesperidin was a potential therapeutic candidate for preventing the progression of breast cancer. In addition, it was shown that hesperidin could significantly stimulate the death mechanisms in ER/PR (+) MCF-7 and ER/PR (−) MDA-MB-231 breast cancer cells in a different way.⁹ Therefore, plant components may have therapeutic potential as anticancer agents.

T. vulgaris L. is one of the few traditional herbs shown to have a therapeutic effect. Recent studies with T. vulgaris indicate that this plant has antibacterial, antifungal, antioxidant, and antiinflammatory activities.¹⁰^,¹¹ Recent reports suggest that T. vulgaris prevents the spread of several malignancies, such as human cervical and squamous cell carcinomas.¹²^,¹³

Apoptosis occurs as a protective mechanism when cells are damaged by diseases or harmful agents. After receiving the signal for apoptosis, many biochemical and morphological changes are observed in the cell. The resulting DNA fragmentation can be demonstrated histochemically by the TUNEL method.¹⁴ Autophagy is a response to stress and has been frequently studied in human cancers recently. Beclin-1 and microtubule-associated protein light chain 3 (LC3) are two important key proteins involved in autophagy. While Beclin-1 mediates the initiation phase of autophagy, LC3 plays an important role in the cytoplasm of autophagosomes and autologous lysosomes.¹⁵

This study investigates whether the methanol extract of T. vulgaris has an anticarcinogenic effect on breast cancer cell lines (MCF-7 and MDA-MB-231). Although there are studies showing the anticarcinogenic effect of T. vulgaris L. essential oil on various cancer cells, including breast cancer;¹⁶^,¹⁷ in this study, the efficacy of T. vulgaris L. methanol extract in breast cancer cells was investigated not only on apoptotic cell death but also on autophagy pathways. Moreover, the role of T. vulgaris on cell proliferation, apoptosis, autophagy, cell cycle, migration, and colony formation was determined using specific markers in the study.

Materials and methods

Plant material

About, 70% methanol extract of T. vulgaris was used, and the extracts were provided by Erciyes University, Faculty of Pharmacy, Department of Pharmacognosy. The extraction process and a thorough phytochemical analysis of the extract were completed in 2019 by Gurbuz et al.¹⁸

Preparation of T. vulgaris

The methanol extract of T. vulgaris was used in this study. Dimethyl sulfoxide (DMSO) was used to dissolve T. vulgaris, and a stock solution containing 10 mg/5 mL of T. vulgaris was created and followed by filtration-based sterilizing. Then, by dilution with the medium, various concentrations of T. vulgaris needed for dose determination were created.

Cell line and culture

The American Type Culture Collection provided the human breast cancer cells MCF-7 and MDA-MB-231, respectively (ATCC HTB 22 and ATCC HTB 26, Manassas, VA, USA). Cells 10% fetal bovine serum (ThermoScientific), 1% they were cultured in DMEM with added antibiotic mixture of L-glutamine (Sigma-Aldrich), and 1% penicillin–streptomycin (Hyclone, ThermoScientific). Cells were seeded into 75 cm² flasks at 10⁶ cells/mL and passaged when they reached approximately 90% confluence.

Cell viability assay

In 96-well plates, 5×10³ cells were planted per well. By measuring mitochondrial activity with the aid of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA), the viability of the cell was assessed.¹⁹ About, 20, 50, 80, 100, 120, 100, 150, and 200 μg/mL doses were selected in the first MTT trial to determine the IC50 dose of T. vulgaris for experimental groups. In addition, 24-h and 72-h analyzes were performed to observe the change of doses over time. The dose range was changed to repeat the test until the IC50 value for cell viability was determined exactly. For this purpose, 1–10 μg/mL doses of T. vulgaris extract were applied to the cells. Absorbance values were obtained at 560 nm using a UV–Vis microplate reader.²⁰ The percent viability of cells was calculated by comparison with the control group (no application group). Four independent experiments were performed.

Cell cycle analysis

MCF-7 and MDA-MB-231 cells were cultivated in 6-well culture dishes at 5×10³ cells/cm² in standard growth media and cultured for 24 h. Cells treated with T. vulgaris at different concentrations (0, 2.5, or 5.0 μg/mL) were harvested and fixed in 70% ethanol pre-chilled at 4 °C. After being kept in a hypotonic buffer solution containing propodium iodide for 30 min at room temperature, cell counts, and proportions in G1/G0, S, and G2/M stages were measured in accordance with the product method using the flow cytometry-based Muse Cell Analyzer and the cell cycle test kit (Cell Cycle Assay Kit, Muse, Millipore).²¹

Migration assay

MCF-7 and MDA-MB-231 cells were cultivated in 6-well culture plates at 5×10³ cells/cm² in standard growth media. Wound areas were created by drawing wells with growing cells neatly along their central line using a sterile yellow tip. After the medium was aspirated, two phosphate buffered saline (PBS) washes were performed on the cells. It was added fresh medium, and zero hour photographs were taken. Following that, cells were treated with and without T. vulgaris and wound areas were photographed using an inverted microscope. The experiment was terminated when the created wound areas were covered by the migrating cells. Using Image J software, a measurement was made of the line’s width, which represents cell migration.²²

Colony formation assay

The impact of T. vulgaris on colony development was examined using a colony formation test. In 6-well plates, 5×10² cells per well from MCF-7 and MDA-MB-231 cells were seeded and treated with T. vulgaris at different concentrations (0, 2.5, and 5.0 μg/mL). Colony formations that were 14 days old were dyed with .1% crystal violet after being fixed with 4% paraformaldehyde. Colonies were counted by hand. The % vitality value was calculated and the data were evaluated statistically.²²

Terminal deoxynucleotidyl transferase dUTP nick end labeling assay

The degree of DNA fragmentation caused by apoptosis was monitored using the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test. Cells were cultured onto coverslips and treated with T. vulgaris for 24 h. At the end of the period, the cells fixed with 10% formaldehyde. Then, TUNEL test was performed in accordance with the datasheet of the kit (ApopTag Fluorescein In Situ Apoptosis Detection Kit, EMD Millipore, Darmstadt, Germany). The TUNEL reaction was triggered by the addition of a reaction mixture to the cells, which was then incubated at 37 °C for 60 min in the dark. The reaction mixture contained nucleotides and the TdT enzyme. Then stop/wash buffer was dropped into the slides. Slides were waited for 10 min at 37 °C. After PBS washing, slides were incubated with Antidigoxigenin antibody for 30 min. 3,3′-diaminobenzidine (DAPI, Roche, Basel, Switzerland) was used to stain DNA fragments. At the end, each slide was viewed by using an immunofluorescence microscope (Olympus BX51, Tokyo, Japan).²⁰

Immunofluorescence assay

An immunofluorescence assay was used for the detection of Beclin-1 and LC3 expression in both cell lines. After the cells were treated with T. vulgaris for 24 h, they were fixed in 10% formaldehyde. After PBS washing, 10% normal goat serum was dropped to the slides and waited for 20 min. Next, Beclin-1(Novus Biologicals, USA) or LC3 (Cell Signaling Technology, USA) primary antibodies were added to cells and incubated overnigth at 4 °C. PBS was used to negative control. The next day, the cells were washed in PBS before being exposed for an hour to a secondary antibody (Jackson ImmunoResearch, UK). After a washing procedure, the cells were counterstained with DAPI. The surface of the cells was covered with water-based sealer. The cells were photographed using an Olympus BX51 fluorescence microscope (Tokyo, Japan).²⁰

Statistical analyses

Each sample image was selected at random from 10 distinct microscopic fields at 400x original magnification for each group in order to identify apoptosis and autophagy in both cell lines. Using Image J software (Bethesda, USA), immunoreactivity intensities were measured. The statistical software program GraphPad Prism 9.4.1 was used to conduct data analyses. The data’s normality was assessed using the Shapiro–Wilk and Kolmogorov–Smirnov tests. The MTT variables were investigated using the one-sample t test. The Kruskal–Wallis test and one-way analysis of variance (ANOVA) were employed to evaluate the quantitative variables in more than two groups. The multiple comparisons were done using the Tukey and Bonferroni tests. P values that were less than ^*P < .05, ^*^*P < .01, and ^*^*^*P < .001 vs were regarded as statistically significant.

Results

T. vulgaris inhibits the proliferation of breast cancer cell lines

When breast cancer cells were exposed to various T. vulgaris doses, the MTT test was used to assess the impact of T. vulgaris on cell proliferation. We found that T. vulgaris treatment considerably reduced the proliferative ability of MCF-7 and MDA-MB-231 cells, with IC50 values of 5.0 μg/mL against MCF-7 cells and 5.0 μg/mL against MDA-MB-231 cells at 24 h.

T. vulgaris induces the cell cycle arrest in breast cancer cell lines

Cells are treated with T. vulgaris at various doses (0, 2.5, and 5.0 μg/mL). When the control and experimental groups were compared, it was shown that there was a statistically significant decrease in S phase in MCF-7 cells compared to the control group (^*^*^*P < .001). It was determined that T. vulgaris in the MCF-7 cell line blocked cell division in the S phase (Fig. 1A). It was demonstrated that the G0/G1 phase in MDA-MB-231 cells decreased statistically significantly when compared to the control group (^*^*^*P < .001). It was determined that T. vulgaris in the MDA-MB-231 cell line blocked cell division in the G0/G1 phase (Fig. 1B).

Fig. 1 — Effect of *T. vulgaris* on cell cycle arrest in MCF-7 (A) and MDA-MB-231 cells (B). The mean standard deviation of three separate experiments is used to represent all data. ns; nonsignificant (^*^*^*P < .001).

T. vulgaris inhibits the migration of breast cancer cell lines

By using a migration assay, we realized the effect of T. vulgaris on the migration ability of breast cancer cells. Cells were treated with T. vulgaris at different concentrations (0, 1.25, and 2.5 μg/mL). Cell migration was assessed at 24, 48, 72, and 96 h in both MCF-7 and MDA-MB-231 cells. The cells on the lower side of the filter were stabilized, colored, and counted after migration. The images were all captured at a magnification of 40. Our results showed that breast cancer cell migration was prevented by T. vulgaris in a concentration-dependent manner (Fig. 2). These data indicate that T. vulgaris has an antimetastatic effect on breast cancer cells.

Fig. 2 — The effect of *T. vulgaris* on the migration of MCF-7 (A, B) and MDA-MB-231 cells (C, D) (^*^*^*P < .001, ^*^*P < .01).

T. vulgaris decreases colony formation of breast cancer cell lines

Compared with the control groups, the number of colonies decreased significantly in both groups treated with T. vulgaris. Cells were treated with T. vulgaris at different concentrations (0, 2.5, and 5.0 μg/mL) (P < .001; Fig. 3).

Fig. 3 — The effect of different concentrations of *T. vulgaris* on colony formation in MCF-7 (a, B) and MDA-MB-231 (C, D) cells (^*^*P < .01, ^*P < .05).

T. vulgaris induces apoptosis and autophagy of MCF-7 cells

In MCF-7 cells, for the TUNEL immunoreactivity intensity, there was a statistically significant rise in the 5.0 μg group compared to the control group (P < .001). Similarly, in the 2.5 μg group compared to the control group, there was a significant increase in TUNEL immunoreactivity intensity (P = .0070). The TUNEL immunoreactivity intensity in the 5.0 μg group was statistically significantly higher than the 2.5 μg group as compared to the 2.5 μg group (P = .003) (Fig. 4 and Table 1).

Fig. 4 — Images of the MCF-7 breast cancer cell line treated with *T. vulgaris*. Apoptotic bodies released by cells in the first column could be detected under a fluorescent microscope. Beclin-1 expression was seen in the second column and LC3 expression was discernible in the third column.

Table 1.

TUNEL, Beclin-1, and LC3 expression data from the statistical analysis of MCF-7 and MDA-MB-231 breast cancer cell lines.

	Groups
MCF-7	Control	2.5 μg/mL	5.0 μg/mL	P
TUNEL	(.59 ± .12)^a	(1.20 ± .52)^b	(2.03 ± .46)^c	<0,0001
BECLIN-1	(.38 ± .12)^a	(.53 ± .09)^a	(.81 ± .18)^b	<0,0001
LC3	(2.02–3.24)^a	(1.52–3.87)^a,b	(3.64–5.13)^b	=0,0015
MDA-MB-231	Control	2.5 μg/mL	5.0 μg/mL	P
TUNEL	(1.90 ± .86)^a	(3.98 ± .76)^b	(4.76 ± .88)^b	<0,0001
BECLIN-1	(1.01 ± .42)^a	(1.33 ± .26)^a	(1.85 ± .39)^b	<0,0001
LC3	(.80–1.35)^a	(1.35–2.12)^a,b	(1.37–2.51)^b	=0,0030

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The mean ± standard deviation is used to express data. P: refers to the importance of the groups’ differences. Comparing groups using the same lowercase letters in the same row reveals similarities between them, whereas comparing groups using different lowercase letters reveals disparities.

To evaluate autophagy in MCF-7, Beclin-1 and LC3 immunoreactivity were examined. A significant Beclin-1 expression increased was determined in the 5.0 μg group rather than the control group (P < .001). It was determined that there was no statistically significant difference in the expression of Beclin-1 between the 2.5 μg group and the control group (P = .0597). Beclin-1 expression in the 5.0 g group was statistically substantially higher than the 2.5 g group when compared to the 2.5 g group (P = .003). When LC3 expression was evaluated in MCF-7, a significant LC3 increase was determined in the 5.0 μg group rather than the control group (P = .0123). When compared to the control group, LC3 expression was higher in the 2.5 g group. However, this difference was not significant (P > .9999). LC3 expression in the 5.0 g group was higher than in the 2.5 g group. However, this increase was not statistically significant (P = .624) (Fig. 4 and Table 1).

T. vulgaris induces apoptosis and autophagy of MDA-MB-231 cells

In MDA-MB-231 cells, it was determined that in the 5.0 μg group, TUNEL immunoreactivity intensity increased compared to the control group (P < .001). Compared to the control group, it was seen that there was a statistically significant rise in the 2.5 μg group’s TUNEL immunoreactivity intensities (P < .001). About, 5.0 μg group’s TUNEL immunoreactivity intensity was higher than the 2.5 μg group. However, this difference was no significant (P = .1351).

To evaluate autophagy in MDA-MB-231 cells, Beclin-1 and LC3 immunoreactivity were examined. It was determined that there was a significant Beclin-1 expression’s increase in the 5.0 μg group rather than the control group (P < .001). It was found that the 2.5 μg group’s Beclin-1 expression was higher than that of the control groups. However, there was no statistically significant rise in this (P = .1954). When compared with the 2.5 μg group, 5.0 μg group’s Beclin-1 expression was statistically significantly higher (P = .0103). It was determined that 5.0 μg group’s LC3 expression was higher than the control group in MDA-MB-231 cells (P = .0038). It was determined that there was statistically nonsignificant an increase in 2.5 μg group’s LC3 expression compared to the control group (P = .0583). When compared to the 2.5 μg group, it was identified that 5.0 μg group’s LC3 expression was higher. But this difference lacked statistical significance (P > .9999) (Fig. 5 and Table 1).

Fig. 5 — *T. vulgaris*-treated MDA-MB-231 breast cancer cell line images. The apoptotic bodies discharged by the cells in the first column could be seen. Beclin-1 expression was visible in the cells of the second column. LC3 expression could be noticed in the third column, under a fluorescent microscope.

Discussion

Cancer is an important health problem for the whole world, and many methods are used for cancer treatment today. However, due to the nonselectiveness of drugs, a high percentage of healthy cells are lost, as well as cancer cells.²³ The usage of traditional herbs is based on acquired knowledge, and because of their great efficacy and widespread availability, people have been using them for millennia. Therefore, although interest in herbal medicines has increased in recent years, the majority of current pharmaceutical drugs are derived from plants. One of the traditional herbs shown to have therapeutic potential is T. vulgaris L., which is rich in essential oil.²⁴^,²⁵ In addition to its antioxidant, antiseptic, and antimicrobial properties,²⁶^,²⁷T. vulgaris has recently been the subject of studies showing that it has anticarcinogenic properties.²⁸^,²⁹ In this study, we sought to assess T. vulgaris methanol extract’s anticancer properties on breast cancer cell lines (MCF-7 and MDA-MB-231).

Al-Menhali A. et al., in their study in 2015, investigated the anticancer activity of T. vulgaris extract from the HCT116 colorectal cancer cell line and showed that T. vulgaris extract inhibited proliferation in a concentration- and time-dependent manner. It has been stated that this decrease in cell number is due to an increase in apoptosis via the caspase 3/7 pathway.²⁴ It has also been proven that T. vulgaris has antitumor effects on human leukemia THP-1 cells³⁰ and human oral cavity squamous cell carcinoma.¹³ Our results are in line with other studies that demonstrate T. vulgaris has anticancer potential in various cell lines.

In many current treatment modalities in recent years, inducing apoptosis is considered to be the most excellent choice for cancer therapy. Because the escape of cancer cells from apoptosis causes them to show malignant potential and chemotherapeutic resistance.³¹ It has been shown that the TUNEL method is an important technique in the detection of apoptotic cells and is more advantageous than other apoptosis detection methods.³² This method is based on marking the 3′ end of the breaks in DNA with the help of an exogenic enzyme.³³ In our study, it was observed that T. vulgaris prevented the proliferation of breast cancer cell lines (MCF-7 and MDA-MB-231) by directing them to apoptosis in this way.

Adham et al. (2020) found that when they applied chloroform and ethyl acetate extracts of T. vulgaris to multiple myeloma cell lines, these extracts induced apoptosis and autophagy.³⁴ In our study, we investigated the autophagic effect of a methanol extract of T. vulgaris on breast cancer cells. Our data show that T. vulgaris induces autophagy in breast cancer cells by increasing Beclin-1 and LC-3, which are important markers for autophagy. Based on these findings, the methanol extract of T. vulgaris suppresses the proliferation of breast cancer cells by activating both apoptotic and autophagic pathways.

In cancer cells, the cell cycle is disrupted. Therefore, in the search for anticancer drugs, one of the strategies necessary to keep cancer cells under control is to inhibit the cell cycle. The effect of T. vulgaris ethanol extract on the cell cycle of T-47D breast cancer cells was evaluated, and it was discovered that it inhibited the cell cycle in the S phase.³⁵ Based on this, the effect of T. vulgaris methanol extract on cell cycle in breast cancer cell lines was investigated. Our research shows that T. vulgaris methanol extract promotes apoptosis by halting the MCF-7 cell cycle in the S phase and the MDA-MB-231 cell cycle in the G0/G1 phase. This confirms our findings that we showed by the TUNEL method that T. vulgaris leads breast cancer cells to apoptosis.

Increased migratory capacity is one of a tumor cell’s features. This study used a wound scratch assay to find out how T. vulgaris affected breast cancer cells’ ability to migrate. Al-Menhali A. et al. In their study conducted in 2015, they showed that T. vulgaris extract inhibited the migration of colorectal cancer cells.²⁴ Consistent with this study, breast cancer cells were also observed to inhibit migration when treated with T. vulgaris. This shows that T. vulgaris has an antimetastatic effect on breast cancer cells.

The effect of T. vulgaris methanol extract on the capacity of breast cancer cells to form colonies was evaluated using the colony formation assay. It has been shown that the application of thymol extract to colorectal cancer cells reduces colony formation.³⁶ Consistent with this study, we found that when we applied T. vulgaris methanol extract to breast cancer cells, it reduced colony formation of both breast cancer cells. This shows that T. vulgaris methanol extract inhibits the colony formation ability of breast cancer cells and suppresses the proliferation of cancer cells.

Conclusion

These findings serve as the foundation for additional in vitro research that will be required to clarify the antitumor mechanism of action of T. vulgaris. The present study undertakes an evaluation of the antiproliferative, cytotoxic, apoptotic, and autophagic effects, cell cycle arrest, migration, and colony formation of methanol extract obtained from T. vulgaris, with a special focus on breast cancer cells (MCF-7 and MDA-MB-231). This study is the first to investigate the effect of a methanol extract of T. vulgaris on breast cancer cells. It is possible to say that the methanol extract of T. vulgaris is a novel approach for the treatment of breast cancer, including aggressive breast cancer cells.

Acknowledgments

We thank the Erciyes University Betül Ziya Eren Genome and Stem Cell Center for enabling this study to be carried out. We also thank Perihan Gurbuz, Faculty Member of Erciyes University Faculty of Pharmacy, Department of Pharmacognosy, who prepared the 70% methanol extract of T. vulgaris used in the study.

Contributor Information

Nazmiye Bıtgen, Department of Medical Biology, Faculty of Medicine, Erciyes University, Melikgazi 38039, Kayseri, Turkey; Genome and Stem Cell Center (GENKOK), Erciyes University, Melikgazi 38039, Kayseri, Turkey.

Gozde Ozge Onder, Genome and Stem Cell Center (GENKOK), Erciyes University, Melikgazi 38039, Kayseri, Turkey; Department of Histology and Embryology, Faculty of Medicine, Erciyes University, Melikgazi 38039, Kayseri, Turkey.

Munevver Baran, Department of Pharmaceutical Basic Science, Faculty of Pharmacy, Erciyes University, Melikgazi 38039, Kayseri, Turkey.

Arzu Yay, Genome and Stem Cell Center (GENKOK), Erciyes University, Melikgazi 38039, Kayseri, Turkey; Department of Histology and Embryology, Faculty of Medicine, Erciyes University, Melikgazi 38039, Kayseri, Turkey.

Author contributions

Study design: Nazmiye Bitgen, Arzu Yay. Study conduct and data analysis: Nazmiye Bitgen, Gozde Ozge Onder, Munevver Baran. Drafting manuscript: Nazmiye Bitgen, Arzu Yay. Revising manuscript: Nazmiye Bitgen, Gozde Ozge Onder, Munevver Baran, Arzu Yay. Approving final version of manuscript: Nazmiye Bitgen, Gozde Ozge Onder, Munevver Baran, Arzu Yay.

Funding

No financial resources were used to carry out the study.

Conflict of interest statement. Bitgen N, Onder GO, Baran M, and Yay A declare that they have no conflict of interest.

Ethics approval and consent to participate

Ethics committee approval is not required as the study did not include human and/or animal studies.

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26. Lee SJ, Umano K, Shibamoto T, Lee KG. Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chem. 2005:91(1):131–137. [Google Scholar]
27. Bozin B, Mimica-Dukic N, Simin N, Anackov G. Characterization of the volatile composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. J Agric Food Chem. 2006:54(5):1822–1828. [DOI] [PubMed] [Google Scholar]
28. Kapinova A, Stefanicka P, Kubatka P, Zubor P, Uramova S, Kello M, Mojzis J, Blahutova D, Qaradakhi T, Zulli A, et al. Are plant-based functional foods better choice against cancer than single phytochemicals? A critical review of current breast cancer research. Biomed Pharmacother. 2017:96:1465–1477. [DOI] [PubMed] [Google Scholar]
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32. Negoescu A, Lorimier P, Labat-Moleur F, Drouet C, Robert C, Guillermet C, Brambilla C, Brambilla E. In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations. J Histochem Cytochem. 1996:44(9):959–968. [DOI] [PubMed] [Google Scholar]
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PERMALINK

Cytotoxicity screening of Thymus vulgaris L. in breast cancer: in vitro study

Nazmiye Bıtgen

Gozde Ozge Onder

Munevver Baran

Arzu Yay

Abstract

Graphical abstract

Introduction

Materials and methods

Plant material

Preparation of T. vulgaris

Cell line and culture

Cell viability assay

Cell cycle analysis

Migration assay

Colony formation assay

Terminal deoxynucleotidyl transferase dUTP nick end labeling assay

Immunofluorescence assay

Statistical analyses

Results

T. vulgaris inhibits the proliferation of breast cancer cell lines

T. vulgaris induces the cell cycle arrest in breast cancer cell lines

Fig. 1.

T. vulgaris inhibits the migration of breast cancer cell lines

Fig. 2.

T. vulgaris decreases colony formation of breast cancer cell lines

Fig. 3.

T. vulgaris induces apoptosis and autophagy of MCF-7 cells

Fig. 4.

Table 1.

T. vulgaris induces apoptosis and autophagy of MDA-MB-231 cells

Fig. 5.

Discussion

Conclusion

Acknowledgments

Contributor Information

Author contributions

Funding

Ethics approval and consent to participate

References

ACTIONS

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