Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Anticancer Effect of Thymol on AGS Human Gastric Carcinoma Cells

  • Received : 2015.06.29
  • Accepted : 2015.10.04
  • Published : 2016.01.28
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Abstract

Numerous plants have been documented to contain phenolic compounds. Thymol is one among these phenolic compounds that possess a repertoire of pharmacological activities, including anti-inflammatory, anticancer, antioxidant, antibacterial, and antimicrobial effects. Despite of the plethora of affects elicited by thymol, its activity profile on gastric cancer cells is not explored. In this study, we discovered that thymol exerts anticancer effects by suppressing cell growth, inducing apoptosis, producing intracellular reactive oxygen species, depolarizing mitochondrial membrane potential, and activating the proapoptotic mitochondrial proteins Bax, cysteine aspartases (caspases), and poly ADP ribose polymerase in human gastric AGS cells. The outcomes of this study displayed that thymol, via an intrinsic mitochondrial pathway, was responsible for inducing apoptosis in gastric AGS cells. Hence, thymol might serve as a tentative agent in the future to treat cancer.

Keywords

Introduction

Gastric cancer is a major threat worldwide. It has the second leading global mortality rate, with an incidence rate of more than one million cases per year and a poor survival rate [8,34].

Many studies have revealed that the high mortality rate of gastric cancer is related to the lack of an effective therapy during the advanced stages of the disease. Many conventional therapy options have been developed for the treatment of gastric cancer, including surgery, chemotherapy, radiation therapy, and combination treatments [29]. However, these therapies produce side effects, such as immunosuppression, toxic hepatopathy, and myelosuppression, and the current chemotherapeutic drugs are not sufficiently effective [2,17]. Therefore, it is important to discover new treatments and increase the survival rate among gastric cancer patients [32].

Apoptosis is a well-organized program executed by both intrinsic and extrinsic signaling pathways [4]. It is related to distinct morphological and biochemical changes in the nucleus, cytoplasm, and plasma membrane [22,26]. Apoptosis is executed by a family of cysteine aspartases (caspases) [4], apoptotic signaling via B-cell lymphoma-2 (Bcl-2) superfamily members involving Bax, Bak, and Bid, or changes in homeostasis [16,27,33]. Caspases are the molecular executioners of apoptosis, causing the morphological and biochemical characteristics of apoptotic cell death [4]. Apoptosis plays an important role in a variety of diseases by self-regulation. Thus, it is necessary to search for new chemotherapeutic agents in cancerous cells [6].

The structural formula of thymol (2-isopropyl-5-methylphenol) is given in Fig. 1A; thymol is a major phenolic compound present in the essential oils of various plants, including Thymus vulgaris (Lamiaceae) and Carum copticum (Apiaceae) [1,7,20]. It has been reported that the plant has antioxidant, antispasmodic, antibacterial, and anti-inflammatory effects [15]. Thymol is also an active compound in the inhibition of cancer cells. Thymol and its essential oils are widely used as a general antiseptic in medical practices, agriculture, cosmetics, and the food industry [28].

Fig. 1.Structure of thymol (A) and its cell viability effects in AGS cells (B).

Thymus quinquecostatus Celak has been proven to exert strong antioxidant [15] and antitumor effects [30]; therefore, we decided to use the peptide thymol, which is the major compound in Thymus quinquecostatus Celak, to identify its effects on cancer cell lines. Despite thymol being known for its multifaceted activities, the anti-gastric carcinoma activity has not been studied.

We discovered that thymol suppressed cell growth and induced apoptosis in AGS human gastric carcinoma cells by causing morphological changes, generation of cellular reactive oxygen species (ROS), and depolarization of mitochondrial membrane potentials through the activation of Bax, cysteine aspartases (caspases), and poly ADP ribose polymerase (PARP).

 

Materials and Methods

Materials

We purchased the RPMI-1640, fetal bovine serum (FBS), and penicillin-streptomycin from Hyclone (Thermo Scientific, Logan, UT, USA), and 2-isopropyl-5-methylphenol (thymol), 2’,7’-dichlorofluorescein-diacetate (DCF-DA), 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 5,5-dimethyl-lpyrroline-N-oxide (DMPO), and Hoechst 33342 from Sigma Chemical Co. (St. Louis, MO, USA).

Antibodies for Bax and Bcl-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-Actin, PARP, cleaved caspase-7, -8, -9, anti-rabbit IgG, and anti-mouse IgG were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). The detective reagent (SuperSignal West Pico Chemiluminescent Substrate) was purchased from Thermo Scientific Inc. (Rockford, IL, USA). All other reagents used were of analytical grade.

Cell Culture and Treatments

We purchased the AGS human gastric carcinoma cells from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.05 mM L-glutamine in a humidified incubator at 37℃ with 5% CO2. The collected cells were treated with 0, 100, 200, and 400 μM of thymol. The thymol was dissolved in DMSO to concentrations of less than 0.1%. Doxorubicin (2 μM) was used as a positive control.

Cell Viability Assays

The cell viability was tested by MTT assay. The cells were distributed in 48-well microtiter plates at a density of 1 × 104 cells/well and allowed to adhere overnight. Then the cells were treated with various concentrations of thymol and doxorubicin (2 μM) and incubated for 6, 12, and 24 h, respectively. After all of the medium was removed, 200 μl of fresh medium, supplemented with 8 μl MTT stock solution (50 mg/ml), was added into each well and the cells were incubated for 3 h at 37℃. Thereafter, the intracellular formazan products were dissolved in 200 μl of DMSO by shaking for 10 min. The absorbance was measured at a wavelength of 540 nm by spectrofluorometry (Spectra-Max M2/M2e, CA, USA). Cell viability was expressed as a percentage of the control.

Hoechst 33342 Staining

AGS cells were seeded in 8-well chamber slides and incubated for 12 h. The thymol and doxorubicin (2 μM) were treated at various concentrations for 24 h. The cells were then washed twice with PBS and fixed in PBS containing 4% paraformaldehyde for 30 min at room temperature. After two additional rinses with PBS, the cells were stained with Hoechst 33342 for 20 min at room temperature in the dark. The stained nuclei were observed under a fluorescence microscope (Carl Zeiss, UY, USA).

Propidium Iodide Staining

The rate of cell death was measured by flow cytometric analyses using propidium iodide (PI) dye [5]. Seeded AGS cells were treated with different concentrations of thymol and doxorubicin (2 μM) for 24 h. After washing twice with PBS, the cells were fixed in ice-cold 100% ethanol at –20℃ overnight, and then washed twice. The resulting pellet was resuspended in PBS containing 1% PI and 0.1% RNase A at 37℃ for 30 min. Data were analyzed using a FACS Calibur flow cytometer (BD, Franklin Lakes, NJ, USA) and Cell Quest software (BD Bioscience). Samples were run using 10,000 cells per test sample.

Annexin V-FITC/PI Staining

To detect cells in early apoptotic and late apoptotic/necrotic stages, cellular DNA levels were determined using the annexin V-FITC apoptosis detection kit (BD Bioscience, CA, USA). AGS cells were seeded and exposed to various concentrations of thymol and doxorubicin (2 μM) for 24 h. The cells were washed twice with PBS and resuspended in 500 μl of 1× binding buffer at a concentration of 1 × 106 cells/ml and incubated with 5 μl of annexin V-FITC and 5 μl of PI for 15 min in the dark. The rate of cells in early apoptosis and late apoptosis/necrosis was determined by the percentage of annexin V+/PI- or annexin V+/PI+ cells. Data were analyzed by flow cytometry (BD). At least 10,000 events were evaluated using the CellQuest software (BD Biosciences).

Intracellular ROS Determination by DCF-DA

Intracellular formations of ROS were detected, as described previously, using the oxidation sensitivity dye DCF-DA as a substrate [15]. AGS cells were seeded and treated with different concentrations of thymol and doxorubicin (2 μM) for 1 h. Then 10 μM DCF-DA was added to the medium. The cells were incubated at 37℃ for 30 min under dark condition. The harvested cells were suspended in 1 ml of PBS. DCF, obtained by the oxidation of DCF-DA by ROS, is a highly fluorescent compound that can be detected by flow cytometry. The data (10,000 events/sample) were analyzed using CellQuest software (BD Biosciences).

Mitochondrial Membrane Potential Assessed by Rhodamine 123 Fluorescent Dye

To demonstrate the depolarization of the mitochondrial membrane potential (MMP), release of apoptogenic factors, and loss of oxidative phosphorylation, the fluorescent dye rhodamine 123, a cell-permeable cationic dye, was used. Seeded AGS cells were treated with various concentrations of thymol and doxorubicin (2 μM) for 24 h. Then 10 μM of rhodamine 123 was added to the medium and incubated at 37℃ for 30 min. The cells were harvested, washed twice with PBS, and then analyzed using a spectrofluorometer (BD). Events were recorded statistically (10,000 events/sample) using CellQuest software (BD Biosciences).

Western Blotting

After treatment for 24 h, the cells were washed with PBS and lysed with PRO-PREP solution on ice for 30 min. The lysates were centrifuged at 13,000 rpm for 30 min at 4℃. After insoluble fractions were removed, the supernatants were collected. Equal amounts of samples were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. After blocking with Tris-buffered saline (TBS) containing 0.1% Tween-20 (pH 7.4) in the presence of 5% (v/v) non-fat skim milk, the membranes were washed and incubated overnight with 1:1,000 diluted primary antibodies in TBS-T at 4℃. After the membranes were washed three times, secondary antibody reactions were performed with an appropriate source of antibody labeled with horseradish peroxidase for 2 h at room temperature; then, the membrane was washed three more times. The resulting protein bands were detected with an enhanced chemiluminescence advanced detection kit, using the imaging program Luminescent image analyzer (LAS-3000; Fujifilm, Tokyo, Japan). Immunoblotting for β-actin was performed as an internal control.

Immunocytochemistry

Cells were attached onto a slide glass by cytospin centrifugation for 5 min at 300 rpm using cellspin (Hanil, Seoul, Korea) and then fixed with 4% paraformaldehyde at room temperature (RT) for 20 min. Fixed cells were washed three times with PBS for 10 min and incubated with 0.2% Triton X-100 for 10 min. The cells were washed three times with PBS and blocked with 5% horse serum and rinse with PBS. Cells were incubated overnight with primary antibody cytochrome c at 4℃. For secondary reaction, cells were incubate with a rabbit-FITC secondary antibody at RT for 1 h. Cells were incubated with PI (50 μg/ml) at RT for 10 min and were observed under a confocal microscope (Olympus Fluoview, Tokyo, Japan).

Statistical Analysis

Data are expressed as the mean ± standard deviation of triplicate measurements. The values were evaluated by one-way analysis of variance (ANOVA). Significant differences (p < 0.05) between mean values of triplicates were observed in all experiments.

 

Results

Thymol Had Cytotoxic Effects on AGS Cells

Cell viability of AGS cells was decreased from 89.56 ± 0.84% to 50.75 ± 2.40%, following the increase of concentrations of thymol for 24 h (Fig. 1B). The results showed that the best exposure time was 24 h, and thymol exhibited the cytotoxic effects in AGS human gastric carcinoma cells in a dose-dependent manner.

Thymol Induced Morphological Changes and Chromatin Condensation

To determine how the morphology of thymol-treated AGS cells was changed, we stained the cells with the DNA binding dye Hoechst 33342. Non-treated cells were round-shaped and were detected at a high density, whereas the density of thymol-treated cells was decreased in a dose-dependent manner (Fig. 2). Condensed, fragmented, and bright blue nuclei were observed in thymol- and oxorubicin-treated cells, contrary to untreated cells. In addition, 400 μM thymol had similar effects to 2 μM doxorubicin used for anticancer treatments. Thus, the results showed that thymol could induce morphological changes and chromatin condensation in AGS cells.

Fig. 2.Morphological changes and induction of chromatin condensation in AGS cells by thymol.

Thymol Induced an Increase of Sub-G1 Phase

We analyzed the cell cycle regulation using PI staining by flow cytometry as shown in Fig. 3. For untreated cells, 3.05% were in the sub-diploid DNA peak (sub-G1) phase, 58.73% in the G1 phase, 14.59% in the S phase, and 22.71% in the G2 phase. Hypo-diploid DNA (sub-G1) contents in AGS cells treated with 100, 200, and 400 μM of thymol and 2 μM of doxorubicin were 7.23%, 17.64%, 42.70%, and 20.81%, respectively. The numbers of cells in the sub-G1 phase after treatment with 400 μM of thymol were more than twice the number of cells in the same phase when treated with doxorubicin and 12-fold that of the number of untreated cells in the sub-G1 phase. The increase in the peak in the sub-G1 phase signified the DNA was in the process of cleavage, which is a characteristic feature of apoptosis [13]. However, as reported by Lüpertz et al. [21], besides induction of apoptosis, the cytostatic drug doxorubicin is known to mediate cell cycle arrest in cancer cells. This means doxorubicin basically arrests the S-phase of cell cycle to exhibit apoptosis. On the other hand, thymol was not observed to show a similar pattern of mechanism and thus indicating that it has a different mode of action to exhibit apoptosis. We believe that doxorubicin is more potent and efficacious than thymol to show its anticancer effect and that was why we could see a rapid decrease in cell viability in Fig. 1 at 6, 12, and 24 h. Thus, these results showed that AGS cells treated with thymol were regulated by increasing the duration of the sub-G1 cell phase in the cell cycle.

Fig. 3.Effects of thymol on the cell cycle distribution of AGS cells.

Thymol Induced Apoptotic Cell Death

The number of thymol-induced apoptotic cells increased in early and late apoptosis, contrary to the untreated cells (Fig. 4). The percentage of early apoptosis (AV+/PI-) increased 3.12%, 8.54%, and 8.70% and the proportion of late apoptosis (AV+/PI+) increased from 12.98% to 20.23% by thymol treatment. Treatment of doxorubicin showed a significant increase in late apoptosis. As determined by FACS analysis of the cells stained for DNA contents, thymol-treated AGS cells significantly induced apoptosis. Altogether, the data showed that thymol induced cell death by apoptosis.

Fig. 4.Effects of thymol on apoptotic and non-apoptotic cell death in AGS cells.

Thymol Induced Generation of Intracellular Reactive Oxygen Species

To test the generation of ROS by thymol treatment, we used DCF-DA staining on AGS human gastric carcinoma cells. The results in Fig. 5 indicate that thymol generated intracellular ROS in AGS cells. Exposure of cells to thymol for 1 h caused 30.81%, 30.94%, and 39.76% of ROS when theconcentrations of thymol increased respectively. Specifically, the cells treated with 400 μM thymol generated ROS ofmore than 3-fold as compared with the untreated cells. The data shown suggest that thymol increased ROS production during apoptosis in AGS cells.

Fig. 5.Effects of thymol on intracellular ROS levels in AGS cells.

Thymol Induced Depolarization of Mitochondrial Membrane Potential

We assessed the loss of MMP by using the rhodamine 123 fluorescent dye and flow cytometry. As shown in Fig. 6, depolarization of the MMP was increased to 2.40%, 2.83%, and 7.89% in thymol-treated AGS cells in a dose-dependent manner. Thus, 7.89% of the cells treated with the highest concentrations of thymol showed disrupted MMP, which was 5.2-fold the disruption of MMP found in the non-treated cells. Therefore, the data indicated that thymol caused severe disruption on the MMP of AGS cells.

Fig. 6.Effects of thymol on the mitochondrial membrane potential in AGS cells.

Thymol Induced the Expression of Bcl-2 Family Members, Caspases, and PARP

As shown in Fig. 7, the expression of proapoptotic protein, Bax, was increased in a dose-dependent manner. Cells treated with 400 μM thymol showed a higher expression of Bax than the doxorubicin-treated cells (used as a positive control). However, the expression of the antiapoptotic protein, Bcl-2, was not significantly changed compared with the positive control. These data showed that thymol induced apoptosis by the activation of the proapoptotic protein Bax. In Fig. 8, thymol-treated AGS cells also activated caspase-8 in a dose-dependent manner; the cells treated with a high concentration of thymol cleaved caspase-8, -7, and -9. The final apoptotic signaling molecule, PARP, was cleaved in a dose-dependent manner. These results showed that thymol induced apoptosis by activating caspase-7, -8, -9, and by the cleavage of PARP.

Fig. 7.Effects of thymol on the expression of Bcl-2 family members in AGS cells.

Fig. 8.Effects of thymol on expression of caspases and PARP in AGS cells.

Immunocytochemical Localization of Cytochrome c

Various apoptotic stimuli could be responsible for cytochrome c release from mitochondria, which induces a series of biochemical reactions that lead to caspase activation and cell death. Bak and Bax have been categorized as the last gateway of cytochrome c release. The homooligomerization of Bax and Bax on the mitochondrial membrane is essential for cytochrome c release [12]. In this study, the overexpression of Bax by treatment with thymol induced the efflux of cytochrome c from the mitochondria and the initiation of apoptosis (Fig. 9).

Fig. 9.Immunocytochemical localization of cytochrome c in AGS cells.

 

Discussion

In a previous study, when Chang cells were treated with thymol for 24 h, no toxicity was observed at concentrations up to 100 μg/ml, which is equivalent to 665 μM [15]. In thisstudy, therefore, we determined the highest concentration to be 400 μM; the AGS cells were treated with 100, 200, and 400 μM of thymol to assess whether it has effects on the viability of the AGS human gastric cancer cell line. We used MTT cell proliferation. The results showed that thymol suppressed the cell growth in a dose-dependent manner and the optimal exposure time was 24 h (Fig. 1B). When the cells died, the morphology was changed—chromatin condensation, cleavage of DNA, cytoplasm shrinkage, membrane blebbing, and formation of apoptotic bodies [3,25] were different after cell death. Thymol inhibited the proliferation of AGS cells, as described above;the cells were stained with the DNA binding dye Hoechst 33342, to determine how the morphology of thymol-treated AGS cells was changed, and the changes were observed using a fluorescent microscope. Consequently, the nuclei were condensed, fragmented, and bright (Fig. 2). Based on the above results, we determined that thymol suppressed the growth of the cells and induced cell death in AGS cells.

The cell cycle regulation controls the growth and proliferation of normal cells; however, this control is lacking in cancer cells [10,14]. To monitor which phases were influenced by thymol in the AGS cell cycle, we analyzed the cell cycle regulation using PI staining by FACS, as shown in Fig. 3. Increasing the peak in the sub-G1 phase signified that DNA was in the process of cleavage, which is a characteristic feature of apoptosis [13]. These results indicated that thymol regulated the cell cycle by prolonging the sub-G1 cell phase in AGS cells.

Based on the previous data, we performed the Annexin V-FITC and PI staining to evaluate whether thymol induced cell death by apoptosis or necrosis. The number of thymol-induced apoptotic cells increased in early and late apoptosis, contrary to the non-treated cells (Fig. 4). As determined by FACS analysis of cells stained for DNA contents, thymol-treated AGS cells showed significant apoptosis. Taken together, the data showed that thymol induced cell death by apoptosis. The apoptosis was induced by the generation of ROS in various cancer cells, as reported previously [11,23,31]. To test the possibility of ROS generation by thymol treatment, we used DCF-DA staining (Fig. 5). The data shown suggested that thymol caused an increase of more than 3-fold in the generation of ROS, as compared with the untreated cells during apoptosis.

Apoptosis is accompanied by the collapse of mitochondrial membranes due to loss of the electrochemical gradient [22]. To assess the loss of MMP, we used rhodamine 123 fluorescence. The results showed that thymol disrupted MMP, as shown in Fig. 6. When the MMP was depolarized, cytochrome c will be released from mitochondria. Moreover, pro- and anti-apoptotic Bcl-2 family members regulate the mitochondrial membrane permeability [4]. When apoptotic signals are transduced into the cells, cysteine aspartases (caspases) are cleaved and activated [9]. Activated caspases lead to fragmented DNA in the nucleotide [19]. A crucial protein, cytochrome c, released from the mitochondria by apoptotic stimulus, activates the apoptotic protease activating factor-1 (Apaf-1) during the formation of the apoptosome, and recruites procaspase-9 [24]. Caspase-9 is cleaved, and thereby activates the executioner procaspases through the intrinsic apoptotic pathway [35]. Fas-associated proteins with death domain (FADD), adaptor proteins, assemble procaspase-8 to form death-inducing signal complexes (DISC) when the Fas ligand binds to the Fas death receptor. The formation of DISC induces apoptosis through the extrinsic pathway [18]. To determine which proteins are activated during the induction of apoptosis, we used the western blot analysis. The results showed that the proapoptotic proteins, Bax, PARP, and caspase-8, were activated by thymol treatment in a dose-dependent manner, but no significant changes on the expression of Bcl-2 were observed. Moreover, caspase-7 and -9 were cleaved with a dose of 400 μM thymol on the treated cells. In the present study, thymol-induced apoptotic cell death was proved in AGS cells. Therefore, the present study concluded that thymol has potent anticancer effects on gastric cancer cells. Thus, it is highly deserved of further study.

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  3. Pharmacological Properties and Molecular Mechanisms of Thymol: Prospects for Its Therapeutic Potential and Pharmaceutical Development vol.8, pp.None, 2017, https://doi.org/10.3389/fphar.2017.00380
  4. Anti-cancer and anti-oxidant properties of ethanolic leaf extract of Thymus vulgaris and its bio-functionalized silver nanoparticles vol.8, pp.3, 2018, https://doi.org/10.1007/s13205-018-1199-x
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