Anethole induces anti-oral cancer activity by triggering apoptosis, autophagy and oxidative stress and by modulation of multiple signaling pathways

Oral cancer is one of the major public health problems. The aim of this study was to evaluate the effects of anethole, 1-methoxy-4-[(E)-1-propenyl]-benzene, on growth and apoptosis of oral tumor cells, and to identify the signaling pathways involved in its interaction with these cancer cells. Cancer gingival cells (Ca9-22) were treated with different concentrations of anethole. Cell proliferation and cytotoxic effects were measured by MTT and LDH assays. Cell death, autophagy and oxidative stress markers were assessed by flow cytometry while cell migration was determined by a healing capacity assay. The effect of anethole on apoptotic and pro-carcinogenic signaling pathways proteins was assessed by immunoblotting. Our results showed that anethole selectively and in a dose-dependent manner decreases the cell proliferation rate, and conversely induces toxicity and apoptosis in oral cancer cells. This killing effect was mediated mainly through NF-κB, MAPKinases, Wnt, caspase 3, 9 and PARP1 pathways. Anethole showed an ability to induce autophagy, decrease reactive oxygen species (ROS) production and increased intracellular glutathione (GSH) activity. Finally, anethole treatment inhibits the expression of oncogenes (cyclin D1) and up-regulated cyclin-dependent kinase inhibitor (p21WAF1), increases the expression of p53 gene, but inhibits the epithelial-mesenchymal transition markers. These results indicate that anethole could be a potential molecule for the therapy of oral cancer.

Oral cancer is amongst the world's highest public health threat issues 1 . Ranking in 11th position as most common cancer in the world, it can cause more than 145,000 deaths a year worldwide, with higher prevalence in men compared to women 2 . It varies depending on socioeconomic and environmental conditions. Multiple factors such as alcohol, tobacco, malnutrition, viral infection and genetic aspect can play a major role in the etiology of the disease 2 . Oral cancer is a squamous carcinoma frequently identified on lips, gums, tonsils, tongue, salivary glands, back of the throat, floor of the mouth and palate, inside of cheeks and oropharynx 3 . A high level (52.7%) of oral cancer is diagnosed with stage IV of cancer development 4 . Conventional treatments for oral cancer rely heavily on surgery with or without assistance of chemo-and radiotherapy care 5 . Although they are efficient, both have significant adverse effects limiting their use 6 . Those treatments can therefore be more aggressive causing numerous unwanted side effects and can have a negative impact on non-cancer cells in mucosa tissue 7,8 . To lower these adverse effects, plant products may be used as complementary treatments in conjunction with reduced exposure to chemo-and radiotherapy. For decades, in countries such as China, India, and Egypt, traditional medicine has been making use of flavored plants as home-based treatments of many diseases including cancer 9 . Herbal ingredients were shown to inhibit proliferation of cancer cells and to reduce side effects caused by chemo-and radiotherapy treatments 10 . Traditional medicine could therefore be an effective combination or alternative treatment for cancer that may be efficient and more specific than compounds like cisplatin used in chemotherapy 11 . Indeed, many bioactive compounds derived from scented plants, like anethole, 1-methoxy-4-[(E)-1-propenyl]-benzene, have been reported to have anti-cancer properties on MCF-7 and PC-3 cell lines 12,13 . Indeed, it has been demonstrated that anethole inhibits cell growth, induces apoptosis and cytotoxicity towards cancer cell lines. However, no study has yet investigated the effects of anethole on oral squamous carcinoma and on normal cells of mucosa.
Cancer initiation and development involve signaling pathways such as the activation of caspases, MAP kinases, and Wnt 14,15 . Anticancer treatment can include one or all these signaling pathways. It was reported that identical mutations downstream of MEK/ERK proteins of EGFR pathway, particularly in KRAS and BRAF, were highly frequent in cancers 16 . Therefore, the objective of this study was to investigate the effects of anethole on the proliferation, apoptosis, autophagy and oxidative stress of Ca9-22 cells. We also assessed the effects of anethole on cell migration and modulation of pro-oncogenic (cyclin D1), apoptotic proteins, epithelial-mesenchymal transition (E-cadherin, vimentin), MAPK (ERK1/2, p38, Jnk), Wnt (β-catenin) and NF-κB signaling pathways. The different results could help understanding the anti-cancer activities of anethole which may lead to an alternative or complementary anti-oral cancer therapy.

Materials and methods
Cells and cell culture conditions. The  Effect of anethole on the morphology and LDH activity of normal and cancer gingival cells. Cells were seeded at 10 5 /well in 6-well cell culture plates, and exposed to various concentrations (0, 0.3, 3 and 30 μM) of anethole for 24 h. The cell morphology was evaluated by means of an inverted optical microscope (Eclipse TS100, Nikon, Montreal, QC, Canada) and photographed with a digital camera. Each experiment was repeated three times. The lactate dehydrogenase (LDH) activity was assessed in culture supernatants collected after cell exposure to anethole for 24 h. The LDH activity was measured using LDH (Sigma-Aldrich, Oakville, ON, Canada) as described previously 19 . Each experiment was repeated seven times.
Effect of anethole on cell proliferation. Normal and cancer gingival cells (3 × 10 5 cells/well) were seeded in 6-well plates and cultured for 24 h. Cells were then exposed or not to various concentrations of anethole for 24 h, then sued to assess the cell growth by means of MTT colorimetric assay (Sigma-Aldrich) as described by Semlali et al. 20 Briefly, each culture was supplemented with 10% of MTT (5 mg/mL) and incubated for 3 h at 37 °C. Stained cells were then lysed using 1 mL of isopropanol-HCl 0.05 M solution with agitation for 15 min. Effect of anethole on cancer gingival cell migration. Ca9-22 cells were seeded in 6-well plates and cultured to 100% confluence. A cross-shaped scratch was created on each confluent monolayer with a sterile 200 μL pipette tip. Culture medium was then refreshed, and cells were incubated with or without 3 μM of anethole at 37 °C in a humid atmosphere containing 5% CO 2 . Digital photographs of each lesion were taken with an inverted optical microscope at different times (0, 6, 12 and 24 h) post injury. Wound closure was analyzed using an image processing program to measure the distance, at 3 separate sites, between opposite edges at each time point (0, 6, 12 and 24 h). Stimulated and non-stimulated cell cultures were compared with respect to their percentage of closure. The experiment was repeated four times.
Effect of anethole on cell autophagy. To determine the in vitro effect of anethole on Ca9-22 cells autophagy, we used ICT's Autophagy assay (Red from ImmunoChemistry Technologies, Burlington, ON, Canada). After treatment or not with anethole for 24 h, the Ca9-22 cells were detached and cultured with the autophagy solution (1:50), incubated in the dark for 1 h, washed three times with the cellular assay buffer, and resuspended in 0.5 mL of the same buffer. Stained cells were analyzed by flow cytometry using a green/yellow laser. The Autophagy Probe, Red excites at 590 nm and emits at 620 nm wavelength. The experiment was repeated three times. www.nature.com/scientificreports/ Determination of ROS and GSH levels. Oxidative stress was assessed by flow cytometry using two markers, reactive oxygen species (ROS) and intracellular glutathione (GSH) (ImmunoChemistry Technologies, Burlington, ON, Canada). Firstly, cells were cultivated for 24 h with or without anethole. Second, total ROS Green was reconstituted with DMSO, diluted in the buffer, added to each sample (10 6 cells/mL), and incubated in the dark for 1 h at 37 °C. The key reagent can then be detected by flow cytometry depending on presence of ROS. Lastly, a decrease of intracellular GSH, a molecule with reducing power playing a key role against free radicals and toxins, is an indicator of the induction of apoptosis in cells stressed by oxidation. To evaluate levels of intracellular GSH, ThioBright™ Green reagent was reconstituted in DMSO, added to the cells suspension (1:200), and incubated in the dark for 30 min. After washing with PBS twice, the intensity of fluorescence was analyzed by BD Accuri C6 Flow Cytometry system (BD Bioscience) and the percentage of positive cells was calculated. Each experiment was repeated three times.
Western blotting. Gingival cancer cells were exposed to anethole (10 μM) for 24 h, then total proteins were extracted from each condition, by adding 70 μL of the lysis buffer (25 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 10% glycerol, 0.1% SDS, 0.05% sodium deoxycholate and 1% Triton X-100) to the cell pellet. This buffer was supplemented with 1:100 protease and phosphatase inhibitors (Sigma-Aldrich, Oakville, Ontario, Canada). The solution was kept at 4 °C for 1 h with shaking before being centrifuged at 13,000 rpm for 10 min to recover the supernatant. Protein concentration was determined by the Bradford assay using Coomassie Brillant Blue G-250 Dye (Bio-Rad, Mississauga, ON, Canada). Extracted proteins were subjected to Western blot analyses. Proteins (20 μg to 50 μg) were denatured at 95 °C for 5 min by adding a reducing buffer (61.5 mM Tris, 2% SDS, 10% glycerol and 100 mM dithiothreitol). Then, these were separated by SDS-PAGE with a Bio-Rad electrophoresis system. Briefly, 8% to 15% of acrylamide SDS-PAGE were used depending on protein molecular weight. After protein migration and transfer onto polyvinylidene difluoride membrane (PVDF) (Cytiva, Vancouver, BC, Canada). The membrane was incubated in a blocking buffer (5% non-fat milk in a solution of TBS 1x) with shaking for 1 h at room temperature followed by overnight incubation with appropriate primary antibod- Statistical analysis. Each experiment was performed at least three times, with experimental values expressed as means ± SD. The statistical significance of differences between values for the control (absence of anethole) and the test (presence of desired concentration of it) was determined by a percentage. The statistical significance of differences between the values was determined by using a one-way ANOVA. p-value < 0.05 was defined as significant.

Results
Anethole has selective cytotoxic effect on oral cancer cells. We have investigated the cytotoxic effect of anethole on oral cancer cells (Ca9-22), non-tumorigenic primary gingival epithelial cells and fibroblasts (GEC and GF) using four types of experiment. At first, growth and cytotoxic effect were measured by MTT and cell count assays (Fig. 1A,B). While GEC and GF cells exhibited great resistance to anethole, with an LC 50  These outcomes indicate that anethole is having differential cytotoxicity against oral cancer cell lines, but it seems less toxic against non-neoplastic gingival epithelial cells and fibroblasts. Microscopic analysis of the morphology is indicating a qualitative change in gingival cancer cells when treated with low concentration (3 µM) of anethole unlike in normal gingival cells (GEC and GF). Anethole is considerably decreasing the adherence of oral cancer cells (Ca9-22), promoting an irregular shape as well as the presence of dose-dependent granules, while it has low morphological damage with normal gingival cells (Fig. 1D). For clonogenic assay (Fig. 1E), cell viability of the Anethole treatment was significantly lower than the untreated cells (control) especially at 10 µM and 30 µM of anethole. In addition, the percentage in the S phase was significantly increasing (p < 0.005), from 2.8% with the control, to 11.2% and 6% respectively when the cells were treated at 3 µM and 30 µM. Also, the percentage of Ca9-22 cells at G2/M phase increased with anethole stimulation, from 3.3% with the control, to 12.2% with 3 µM and 5.1% at 30 µM (Fig. 2). These outcomes are suggesting that anethole could induce S and G2/M cell cycle arrest in Ca9-22 cells.

Anethole triggers apoptosis of oral cancer cells via the mitochondrial pathway. To understand
the mechanism by which anethole plays a potential anti-oral cancer role, we have investigated its apoptotic effect on Ca9-22 cells using Annexin V measurement by flow cytometry. Figure 2A shows that anethole at 30 μM induces more than 29% of cells towards apoptosis after 48 h of treatment. As for Fig. 2B, it compares a proposed model of anethole-induced cell death (early and late apoptosis) with non-treated cells after 24 h and 48 h. This data is indicating that anethole induces cytotoxicity through an apoptosis pathway. To determine the apoptotic mechanism activated by anethole, proteins were analyzed by western blot showing an increase of the Bax/Bcl-2 www.nature.com/scientificreports/ ratio, due to an increase of the Bax (pro-apoptotic) expression, and a decrease of Bcl-2 (anti-apoptotic) proteins. Our results showed that anethole is triggering cell death in oral cancer cells through induction of caspase 3 and PARP-1 active forms (Fig. 2C). Furthermore, anethole is strongly inhibiting the level of total caspase 9 but increasing the expression of caspase 9 and cytochrome C active forms after 24 h of treatment (Fig. 2C,D). The results suggest that anethole activated intrinsic pathways via caspases and PARP1.

Anethole suppresses migration/invasion abilities of oral cancer cells and mediates the anti-metastatic activity in the latter by suppressing epithelial-to-mesenchymal transition.
One of the cancer cell properties is the migration/invasion, thus treatment should contribute inhibiting cancer cell migration. In this study section, we investigate the effect of anethole on Ca9-22 cells migration after scratch. As shown in Fig. 3A,B, anethole is repressing the migration of oral cancer cells. Only 23% of the area was covered with migrating cells following treatment with 3 µM of anethole, while 100% of the scratch area was covered with migrating cells in the untreated control. Cancer metastases are closely associated with induction of epithelial-to-mesenchymal transition (EMT), which is characterized by increased cell motility, loss of cell adhesion, and repression of E-cadherin expression. In the current study, Western blotting showed that anethole at 10 µM is enhancing E-cadherin expression, an EMT epithelial marker, but attenuating that of vimentin, a mesenchymal marker (Fig. 3C). These results imply that anethole inhibits oral cell migration and blocks the epithelial-to-mesenchymal transition in oral cancer cells.   Compared to the control, the treatment at 10 μM of anethole significantly induced the overexpression of LC3B and p62 protein levels in the Ca9-22 cells (Fig. 4B).
Anethole exhibits potent anti-oral cancer properties through targeting several cancer-promoting pathways. In order to study the molecular pathway that regulates the induction of anti-oral cancer properties related to anethole, we investigated the effect of the latter on cancer-related pathways. As shown in Fig. 6, anethole has a strong inhibiting effect on the expression of cyclin D1. Furthermore, anethole increased the expressions of p53 and p21 (Fig. 6A,B). Anethole strongly inhibited the activities of MAPKases (ERK1/2, p38 and Jnk), NF-κB and Wnt pathway genes in Ca9-22 cells (Fig. 6C,D). These results indicate that anethole is an efficient inhibitor of several oral cancers related signaling pathways including MAPK/NF-κB and Wnt/βcatenin, leading to strong inhibition of cyclin D1. These data show that anethole is an efficient inhibitor for several cancer-promoting pathways.
Anethole has anti-metastatic activity to reduce expressions of MMPs and to strongly enhance that of TIMPs in oral cancer cells. The fact that NF-κB controls the expression of several pro-metastatic cytokines such as MMPs and TIMPs prompted us to investigate anethole effect on matrix metalloproteinases (MMPs) and their inhibitor proteins (TIMPs) by Luminex xMAP technology. Figure 7A shows that anethole at 30 μM can reduce the level of MMPs by 85% inhibition for MMP-7 and 82% for MMP-9 in Ca9-22 cells, whereas for metalloproteinase inhibitors (TIMPs), it promotes expression, ranging from 1.09 to 6.09-fold higher (Fig. 7B). These data indicate that MMPs and TIMPs family is closely associated with oral tumorigenesis and may be an important potential prognostic marker for oral cancer progression.

Discussion
In the current study, proliferation and apoptosis analysis provided evidence that anethole has anticancer properties in human oral squamous cell carcinoma, and could be considered as a potential therapeutic agent for oral cancer by disrupting their cell cycle, increasing cell apoptosis, and targeting MAPkases, NF-κB and Wnt pathways and the transcriptional activity of theses pathways. After treatment with 30 µM of anethole for 24 h, we are attending a high accumulation of cells in G2/M phase. The increase of the cells at the G2/M phase could be associated with apoptosis and with the level of DNA damage 22,23 . Blocking oral cancer cells at G2/M phase was www.nature.com/scientificreports/ strongly associated with the inhibition of Cdc2 dephosphorylation 24 and to that of MAPKs known to induce the cell cycle arrest and apoptosis 25 . These results are consistent with previous studies reporting that anethole was considered as an interesting anti-cancer molecule for gastric cancer treatment by reduction of adenocarcinoma www.nature.com/scientificreports/ cells, triggering apoptosis and arresting cell cycle through mitochondrial pathways and the down-regulation of an oncogene, such as cyclin D1, known to be the downstream effector of the RAS/MEK/ERK pathway 12,26 . Cyclin D1 has been found highly expressed in cancer tumors and was associated with metastasis 27 . In addition, inhibition of MEK1/2 pathway by anethole significantly destabilized several oncoproteins, such as cyclin D1, p21, and β-catenin expressions. Inhibition of cyclin D1 is critical for destroying efficiently oral cancer cells. Wnt/β-catenin is considered as the important cancer pathway involved that has been found highly active in various types, including oral cancer 28,29 . In recent study by Eun Jeong Choo et al. 30 , it has been shown that anethole exerts anti-metastatic activity on human fibrosarcoma cells by suppressing phosphorylation of AKT, extracellular signal-regulated kinase (ERK), p38, and nuclear transcription factor-kappa B (NF-κB). Furthermore, anethole exhibits potent selective anti-oral cancer properties through targeting these cancer-promoting pathways in epithelial monolayers. Inhibition of these pathways can be an excellent target for cancer therapy. In addition, MAPK and Wnt pathways activated are a marker of poor prognosis in human oral cancer, and persistent MAPK activation in it is associated with enhanced cellular and tumor growth 31,32 . The high cytotoxic effect of anethole in oral cancer cells at low concentration but in normal gingival cells, it has a marginal effect at low concentration, but this molecule appears to have some toxicity when used at high concentration. Also, our results shown that anethole is cytotoxic and triggering apoptosis and autophagy as well as inducing oxidative stress. This data was confirmed by western blot analysis, which has clearly demonstrated that anethole induces apoptosis through activation of caspases 3 and 9 as well as PARP-1 cleavage. Anethole-induced apoptosis was controlled through a mitochondrial pathway via up-regulation of Bax/Bcl-2 ratio and with increased expression of regulatory genes in the cell cycle arrest as p53 and p21 WAF-1 proteins. Same results were reported by Al-Sharif et al. 33 in 2013 while www.nature.com/scientificreports/ using another phenolic natural product for breast cancer therapy. They indicated that eugenol exhibits anti-breast cancer properties by triggering apoptosis through the induction of the intrinsic pathway, by increasing p21 WAF1 expression, by inhibiting cyclin D1 and ERK1/2, p38 and NF-κB signaling pathways that were known to play a key role in oral carcinogenesis. Our suggestion is that anethole suppresses the activation of MAPkase and induction of caspase cleavage; this is leading to the down-regulation of several MAPK targets, such as cyclin D1, p53, and caspases ones, as Bax, Bcl-2 and cytochrome C. All these proteins play key roles in oral tumorigenesis [34][35][36] . Second, we demonstrated that anethole exhibits anti-carcinogenic activities through another physiological phenomenon called autophagy, as a target for treatments of oral cancer diseases. Indeed, flow cytometry analysis indicated the activation of autophagy, an important cell death process, in oral cancer cells, in presence of anethole. In Ca9-22 cells, the basic level of autophagy seems to be very low and while treated with anethole, we assist to p53 suppression causing a significant increase of the phenomenon and leading to cell death. In this order, several previous studies have reported that autophagy was discovered in inducing G0/G1 arrest and apoptosis of cancer cells via β-catenin pathway 37 . We can suggest that autophagy may be related to the apoptosis induction by anethole treatment of oral cancer cells by increasing the Bax/Bcl-2 ratio and activating the subsequent caspases. The anti-oral cancer properties of this treatment was associated with the reduction of oxidative stress, also an important factor leading to the formation and progression of cancer 38 . Although, anethole has been known as an antioxidant and an inhibitor of lipid peroxidation, as well as having an important role in regulating activities of mTOR/PPARγ and ROS control 39 . These functions of anethole, as an antioxidant, depend on how ROS are efficiently regulated both inside and outside the cell. The facts that anethole has the ability to modulate positively, by decreasing the presence of reactive oxygen species, is another indicator of its anticancer potential. In addition, these results correlate with an increase in intracellular GSH, a mediator that plays a crucial role in cellular processes such as differentiation, proliferation and apoptosis, and indicate an inhibition of cancer progression 40 . Anethole was reported playing a key role as a protective agent against radiation-and ROS-induced cytotoxicity, also known to be an activator of the enzyme gamma-glutamylcysteine synthetase, which is involved in GSH synthesis 39,41 . It is thus able to maintain the redox status of ROS-stimulated cells by increasing the intracellular level of GSH 42 .
The metastases are directly associated with induction of EMT destruction, MAPK and Wnt pathways 43,44 . In the current study, we have demonstrated that anethole inhibits the epithelial-to-mesenchymal transition in oral cancer through blocking migration/invasion abilities of cells and suppressing the EMT via the enhancement of the epithelial marker E-cadherin, attenuating the prominent mesenchymal marker (vimentin) and reducing the MMPs secretion. It previously reported that anethole strongly inhibits the migration of prostate cancer cells by regulating the epithelial-to-mesenchymal transition and MMPs, which are considered, with their tissue inhibitors, as key factors for metastatic tumors 45 . There is accumulating evidence that MMP-9 potentiates various cancer metastasis 46,47 . It has a powerful role to destroy collagen IV and other ECM components 48 . It clearly reported by scientists that MMP-9 is a strong candidate to be a marker for different types of cancer 49,50 . TIMPs inhibitors of MMPs were also indicated to be associated with the same cancer 51 . Experimental reports demonstrated direct implication of TIMPs in the majority of hallmarks and showed their deregulation in most of the tumors 51 .

Conclusion
Our results confirm that the anethole treatment selectively induces anti-oral cancer proprieties via shifting proliferative signaling pathways and by triggering apoptosis, autophagy and oxidative stress (Fig. 8). Anethole shows potential as a natural product possessing potent therapeutic activity and show very promising effects as regards to its use in the treatment of gum cancer. Further studies in animals and humans will help in translating these results in vivo.